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dissertation entitled

MOLECULAR STUDIES OF ARGININE AUXOTROPHY
IN NEISSERIA GONORRHOEAE

presented by
Paul Richard Martin

has been accepted towards fulfillment
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MOLECULAR STUDIES OF ARGININE AUXOTROPHY
IN NEISSERIA GONORRHOEAE

BY

Paul Richard Martin

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology

1991

687- M97

ABSTRACT

MOLECULAR STUDIES OF ARGININE AUXOTROPHY
IN NEISSERIA GONORRHOEAE

BY

Paul Richard Martin

Neisseria gonorrhoeae is a strict human pathogen and the causative
agent of gonorrhea. The development of auxotyping media for Neisseria
led to the discovery that gonococcal strains display a diverse array of
nutritional requirements. Among the more common nutritional defects
found among clinical isolates is the requirement for arginine. Arginine
biosynthesis from glutamate requires eight enzymatic steps. The most
common defects found among gonococcal arginine auxotrophs occur at steps
five and six; the conversion of acetylornithine to ornithine, and the
carbamoylation of ornithine to form citrulline respectively. The fifth
step is catalyzed by the enzyme ornithine acetyltransferase and is
encoded by the argJ gene. The sixth is catalyzed by ornithine
transcarbamoylase (OTCase), encoded by argF. I have cloned these genes
by complementation of defined E. coli mutants, analyzed their products
in minicells, and sequenced the genes. The gonococcal argF gene encodes
a protein of 331 amino acids with a deduced molecular weight of 36,731.
This gene uses a 606 codon for translation initiation, which was
confirmed by N-terminal amino acid sequencing of the purified protein.
The gonococcal argF gene contains regions of high homology with OTCase
genes previously sequenced from E. coli and Pseudomonas aeruginosa, and
with OTCases from eukaryotic sources, with the amino acids involved in

substrate binding being particularly well conserved.

The gonococcal argJ gene is of particular interest because it is
defective in all strains tested with the multiple requirements for
arginine, hypoxanthine, and uracil (AHU strains). AHU strains are a
homogeneous group that have been associated with disseminated gonococcal
infections, and are believed to be clonally derived. AHU strains possess
an apparently intact argJ gene as seen by Southern blot. The argJ gene
encodes a protein of 406 amino acids with a deduced molecular weight of
42,879. The mutant argJ gene from an AHU strain was cloned, sequenced,
and compared to the wild type gene. The mutant gene contained a 3 base
pair deletion within a repetitive region of the gene. This deletion was
restored in a naturally occuring Arg+ revertant, thus correlating this
mutation with impaired enzyme activity. When other AHU strains were
tested they were all found to possess this deletion, whereas other ArgJ-
and wild type strains did not. These data support the theory that AHU
strains are clonally derived and distinct from other strains of N.

gonorrhoeae.

for Eve

iv

ACKNOWLEDGEMENTS

I would like to thank my graduate committee, Dr. Robert P. Hausinger,
Dr. Loren R. Snyder, Dr. Michael Thomashow, and Dr. Steven Triezenberg
for their time and counsel. I offer a special thanks to Dr. Martha Mulks
for her advice and constant support over the past five years.

I am endebted to a long list of bright young colleagues who assisted
my research efforts in so many ways. Your helpful services and engaging
discussions were greatly appreciated; I thank you for your support.

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . viii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . ix
INTRODUCTION
Literature Review . . . . . . . . . . . . . . . . . . . 1
Arginine Biosynthesis . . . . . . . . . . . . . . . 4
Gonococcal Genetics . . . . . . . . . . . . . . . . 9
Auxotyping and Serotyping . . . . . . . . . . . . . 13
History of AHU Strains . . . . . . . . . . . . . . . 15
References . . . . . . . . . . . . . . . . . . . . . 21
CHAPTER I
ARTICLE: Sequence of the argF gene encoding ornithine
transcarbamoylase from Neisseria gonorrhoeae . . . . 32
Abstract . . . . . . . . . . . . . . . . . . . . . 33
Introduction . . . . . . . . . . . . . . . . . . . 34
Materials and Methods . . . . . . . . . . . . . . . 36
Results and Discussion . . . . . . . . . . . . . . . 38
References . . . . . . . . . . . . . . . . . . . . 53
CHAPTER II
ARTICLE: Sequence analysis and complementation studies
of the argJ gene from Neisseria gonorrhoeae . . . . 56
Abstract . . . . . . . . . . . . . . . . . . . . . 57
Introduction . . . . . . . . . . . . . . . . . . . 58
Materials and Methods . . . . . . . . . . . . . . . 61
Results . . . . . . . . . . . . . . . . . . . . . . 64
Discussion . . . . . . . . . . . . . . . . . . . . . 78
References . . . . . . . . . . . . . . . . . . . . 82
CHAPTER III
ARTICLE: Molecular charaterization of the argJ mutation
in AHU strains of Neisseria gonorrhoeae . . . . . . 85
Abstract . . . . . . . . . . . . . . . . . . . . . . 86
Introduction . . . . . . . . . . . . . . . . . . . . 87

vi

Materials and Methods
Results
Discussion .
References .
SUMMARY AND CONCLUSIONS
APPENDIX

NOTE: Sequence of the argF gene encoding ornithine
transcarbamoylase from Neisseria gonorrhoeae .

vii

89
93
103
108

111

114

LIST OF TABLES

Table Page
CHAPTER I

1. Codon usage in the N. gonorrhoeae argF gene . . . . . 49

2. DNA and amino acid sequence homologies of OTCase

and ATCase genes . . . . . . . . . . . . . . . . . . 50

CHAPTER II

1. OATase assays of pRGES and deletions . . . . . . . . 67

2. Codon usage in the N. gonorrhoeae argJ gene . . . . . 73
CHAPTER III

1. Gonococcal strains and phenotypes . . . . . . . . . . 90

viii

Figure

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DOOM?“

boom

LIST OF FIGURES

INTRODUCTION
Arginine Biosynthetic Pathway
CHAPTER I

Restriction map and deletions of pRGFl .
Minicell analysis of pRGF4 . .
Nucleotide sequence of the gonococcal argF gene
and flanking sequences . .
Comparison of the predicted amino acid sequences
of the gonococcal argF, E. coli argI, and

P. aeruginosa ach genes . .

CHAPTER II

Arginine Biosynthetic Pathway .
Restriction map and deletions of pRGES .
Minicell analysis of pRGES and deletions . .
Nucleotide sequence of the gonococcal argJ gene
and flanking regions . . . .
Southern blot of N. gonorrhoeae, E. coli, and P.

aeruginosa DNA probed with the gonococcal argJ gene.

CHAPTER III

Southern blot of wild type and AHU strains of

N. gonorrhoeae probed with the argJ gene .
Chimeras of wild type and mutant argJ genes
Comparison of wild type and mutant argJ sequences.
Presence of deletion in wild type, mutant, and
revertant argJ genes . . . . . . .
Presence of deletion in AHU, ArgJ-, and ArgJ+
strains of N. gonorrhoeae . . . . . .

APPENDIX

Nucleotide sequence of the gonococcal argF gene and
flanking regions .

ix

Page

40
41

44

46

59
66
69
71

76

94
97
99
101

102

115

LITERATURE REVIEW

Neisseria gonorrhoeae is a Gram negative diplococcus that has adapted
a lifestyle intimately associated with our own. N. gonorrhoeae is an
obligate human pathogen and, as its name implies, is the causative agent
of gonorrhea, a sexually transmitted disease of major importance. The
gonococcus was first identified in 1879, when Albert Neisser found the
organism in stained smears of urethral exudates (65). This organism
continues to be a serious and widespread public health problem in this
country. In 1989, 733,151 new cases of gonorrhea were reported in the
U.S. (18), and another million cases are estimated to have gone
unreported.

Gonococcal infections occur on mucous membranes lined with
nonsquamous epithelium (53). A typical infection is initiated by
adherence of the bacteria to mucosal epithelial cells which is mediated
by the presence of pili on the surface of the gonococcus (99). After 24-
48 hours the gonococcal cells penetrate the epithelium and cause
localized cell destruction (48). This damage induces the infiltration of
polymorphonuclear leukocytes and local inflammation. This results in the
pain and purulent discharge associated with symptomatic gonococcal
infections (53).

Gonococcal infections are usually restricted to the site of
introduction, but complications can occur. Among these are ascending
gonococcal infections which in males can lead to colonization and
inflammation of secondary sites such as the epididymis or prostate gland

(53). In females ascending infection can cause pelvic inflammatory

2
disease, a serious complication that can result in infertility (39). The
gonococcus can also be invasive causing disseminated gonococcal
infection (D61) (56). D61 is characterized by fever, skin lesions and
polyarthralgia, and infrequently involves endocarditis or meningitis (56).

The gonococcus possesses a variety of outer membrane proteins that
along with lipopolysaccharide interact with the environment and allow
for attachment and invasion of the mucosal epithelium and evasion of
host immune responses. Studies of these proteins have found that they
show a high degree of antigenic diversity between strains, and that
several can generate antigenic variability within a strain (87).

Pilin is the protein component of gonococcal pili which are the
primary mediators of attachment to epithelial cells (100). Primary
cultures of clinical isolates produce almost exclusively piliated cells,
but piliation is quickly lost when subcultured (66,118,123). Non-
piliated cells show a markedly reduced ability to initiate infection
(66), substantiating the importance of pili to pathogenesis. The type of
pilin expressed in a given gonococcal cell can spontaneously change to a
structurally and antigenically different type of pilin in its progeny
(52,79). This is possible due to the presence of a family of pilin
genes located in the gonococcal chromosome, only one of which is
typically expressed at a given time (51). The "silent” pilin genes are
incomplete and lack promotors but can become expressed by homologous
recombination into the expression locus (42).

Protein I (P.I) is the major outer membrane protein of the gonococcus
and functions as a porin (33). A large number of structural varients of
P.I exist in the gonococcal population as detected by serological

reactivity. This has been exploited to develop a panel of monoclonal

3
antibodies against P.I to classify gonococcal strains into serovars, a
tool that has been very useful for epidemiological studies (9,71,110).

Protein II (P.II) is also known as opacity protein for its noticable
affect on colony morphology; cells expressing P.II develop opaque
colonies while colonies from those not expressing P.II are transparent
(122,133). P.II is believed to be involved in attachment to epithelial
cells (78) as well as intergonococcal adherence (122). Similar to pilin,
P.II is capable of phase and antigenic variation (133), though the
mechanism is quite distinct (95,121). At least 11 copies of the P.II
structural gene exist in the chromosome, but unlike pilin they all have
functional promotors and are constitutively transcribed (121). Control
of P.II expression occurs by the presence of a repeated pentameric
sequence in the 5'-end of the gene encoding leader peptide. The number
of pentameric repeats determines whether the remainder of the gene will
be in or out of frame and thus whether it will be translated into
functional P.II protein (4,95,121). The number of pentameric repeats can
change by slipped-strand mispairing during DNA replication (4,95). In
this way from none to several different P.II proteins can be expressed
at a given time.

The gonococcus produces and secretes an extacelluar enzyme that
specifically cleaves immunoglobulin A1 at the hinge region (103),
impairing the ability of the antibody to effect an immune response.
This enzyme, IgAl protease, comes in two types that differ in the
specificity of the site of cleavage of the antibody (94). Essentially
all gonococcal strains tested produced an IgAl protease, but no strain
produced both types (94). This allowed for strains to be characterized

as either type 1 or type 2 IgAl protease producers.

4
Gonococci survive poorly outside their human hosts, but they have

been successfully cultured in vitro and are actually quite nutritionally
independant. The prototrophic gonococci requires cysteine, and can use
glucose as a carbon and energy source (14). The development of defined
media by Catlin (l4) allowed for the elucidation of the nutritional
requirements of clinical isolates. She found that auxotrophs were common
and varied widely. Among the more common auxotrophs in the population

were those that required arginine.
ARGININE BIOSYNTHESIS

In bacteria, arginine is synthesized from glutamate by an eight step
pathway (25) (Fig. 1). Initially an acetyl group is transferred to
glutamate from acetyl CoA to form N-acetylglutamate. This acetyl group
effectively prevents the cyclization of the molecule that occurs in the
proline biosynthetic pathway. The acetyl group is subsequently removed
at step five when N-acetylornithine is converted to ornithine. Ornithine
is combined with carbamoyl phosphate to form citrulline in the sixth
step of the pathway and citrulline is converted to arginine in the last
two steps.

As shown in Figure 1, arginine biosynthesis is intertwined with other
other biosynthetic pathways including those for proline (glutamate is
shared), pyrimidines (carbamoyl phosphate is shared), and polyamines
(ornithine and arginine are precursors). These other pathways influence
the availability of intermediates and can affect the regulation and

activities of some arginine biosynthetic enzymes (43).

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pyrimidine, and polyamine biosynthetic pathways are

6

Two independent enzymes have evolved to catalyze the conversion of
acetylornithine to ornithine, and the enzyme an organism produces
affects the level of control of the pathway (127). E. coli and other
enteric bacteria catalyze this reaction by the linear pathway in which
acetylornithine is hydrolyzed to form ornithine and acetate using the
enzyme acetylornithinase (105,127). Bacteria of the genera Pseudomonas,
Thermus and Neisseria, as well as cyanobacteria, methanogens and lower
eukaryotes catalyze this reaction by the cyclic pathway in which the
acetyl group is transfered to glutamate by the enzyme ornithine acetyl-
transferase (OATase)(l6,28,55,86,91,127). The acetylglutamate produced
from this reaction can be channeled back into the arginine biosynthetic
pathway, bypassing step one. In organisms utilizing the linear pathway
regulation occurs through feedback inhibition by arginine on the first
enzyme of the pathway, N-acetylglutamate synthetase (127). In organisms
that use the cyclic pathway, arginine feedbacks inhibits the second
enzyme of the pathway, N-acetylglutamate 5-phosphotransferase (127). The
cyclic pathway represents the energetically more efficient pathway in
that acetyl group recycling reduces the need for input from acetyl-GOA
to times of increasing activity.

Along with feedback inhibition control of arginine biosynthesis,
repression of arginine biosynthetic gene expression has also been
reported (81). In E. coli, the genes for the eight enzymes of the linear
pathway have been designated sequentially argA-argH. The argE,C,B and
argH genes are clustered and share a common operator located between
argE and argC, while the remainder of the genes are independent and
scattered throughout the chromosome (43,46). The operator regions of

the arginine biosynthetic genes plus the genes for carbamoyl phosphate

7
synthesis (carA and carB) share a common regulatory sequence designated
the "arg box" to which the arginine repressor protein (encoded by argR)
binds under conditions of excess arginine (24,63). An arg box very
similar to that of E. coli is also found in front of the argC-A-E-B-D
gene cluster of Bacillus subtilis (116), and the B. subtilis arginine
repressor was capable of regulating E. coli erg genes in trans (116). In
Pseudomonas aeruginosa all the arginine biosynthetic genes are scattered
and only one (argF) is repressible by arginine (50,61,132). The promoter
of the P. aeruginosa argF gene does not contain sequences resembling an
arg box (62), and the regulatory mechanism of this gene has not yet been
identified. In N. gonorrhoeae the genes for arginine biosynthesis appear
to be scattered and no evidence for repression control has been found
(101,114,115,Mulks unpublished).

The gene for OATase is commonly designated argJ. This gene has been
cloned from N. gonorrhoeae, and it was found that clones carrying this
gene could complement E. coll argE and argA mutations (101). It was
proposed that the gonococcal OATase could complement an E. coli argA
mutation through the feedback pathway, though it was also possible that
the gonococcal argA gene might also have been present on the same clone
(101).

Bacillus subtilis was initially reported to produce acetylornithinase
based on enzyme assays that found a slightly higher level of this
activity than OATase activity (127). This was later supported by showing
that the ability of the cloned B. subtilis argC-A-E-B-D gene cluster to
complement E. coli argA and argE mutations could be separated (93). This
assumption has recently come under question though when the B. subtilis

argC-A~E-B-D cluster was sequenced and only one open reading frame was

8
found in the argA-E region (98). Since this region could complement E.
coli argE and argA mutations, these researchers proposed that the B.
subtilis argA-E region actually encodes only one gene, argJ, an OATase
(98). At this writing this question remained unresolved.

The arginine biosynthetic pathway has proven to be a good model for
studies of molecular evolution, and has provided several interesting
examples of gene duplication (25). In E. coli K12 there are two copies
of the argF gene, the second being designated argI (44). The argI gene
is the copy of the gene common to all E. coli strains. The argF gene is
flanked by 181 elements and is presumed to have entered the E. coli
chromosome by transposition from an unidentified source (59,139). The
argI and argF genes share 86% amino acid sequence identity, and both
genes are expressed (44,130). E. coli also possesses two copies of the
argD gene (6). The second copy, argM, shows the interesting property of
being induced, not repressed, by the arginine repressor (6), and it is
thought to be part of a cryptic arginine degradative pathway. In P.
aeruginosa there are two copies of argF (50,119). The second copy, ach,
catalyzes the reaction in the reverse direction and functions in
arginine catabolism (49). Interestingly the ach gene shows a
significantly higher sequence identity to the E. coli argI and argF
genes than does the P. aeruginosa argF gene (3,62).

Ornithine transcarbamoylase (OTCase, encoded for by argF in N.
gonorrhoeae) has been thoroughly characterized in many prokaryotic and
eukaryotic species (58,82,104,129), and the OTCase genes have been
sequenced and compared among these organisms (3,5,57,60,62,83,124,
128,130,131). These comparisons revealed that all OTCases appear to have

evolved from a common ancestor. All the OTCases studied function as

9
trimers of a peptide with a molecular weight between 33.5 and 38.5 kDa.
Amino acids involved in the binding of ornithine and carbamoyl phosphate
have been identified (58,80,82), and are conserved among the OTCases.
The amino acids involved in carbamoyl phosphate binding are also found
in a different enzyme, aspartate transcarbamoylase (ATCase)(58,80,88).
ATCase is part of the pyrimidine biosynthetic pathway (pyrB in Fig. 1).
In E. coli, pyrB and argI have sequence homology beyond those amino
acids that interact with carbamoyl phosphate, and it is thought that

pyrB may actually be the ancestor of the OTCase genes (58,130).

GONOCOCCAL GENETICS

The gonococcus possesses a single circular chromosome with a G+C base
composition of 50%. The size of the chromosome was originally estimated
to be 1,500 kb in length based on reassociation kinetics (67). Recent
studies using pulse field gel electrophoresis of large chromosomal
restriction fragments revealed that the chromosome was actually closer
to 2,219 kb long (29), approximately half the size of the E. coli
chromosome. The original underestimate in size is thought to be due to
the existance of multiple copies of genes such as pilin and P.II, and
other redundant sequences (21,22) affecting the rate of annealing.

Several plasmids have been identified in N. gonorrhoeae (107). The
most prevalent plasmid is the 2.6 MDa cryptic plasmid which was first
isolated in 1972 (38) and is found in approximately 96% of clinical
isolates (109). The entire cryptic plasmid (4,207 bp) has been sequenced
(75), and several open reading frames were correlated to proteins

expressed in E. coli minicells, but no functional role has been

10
determined for this plasmid or its products.

Another common plasmid is the 24.5 MDa conjugal plasmid (37). This
plasmid allows for the efficient transfer of itself and other smaller
plasmids between gonococcal strains (35,108) and can mobilize antibiotic
resistance plasmids to other Neisseria and to E. coli (41). The conjugal
plasmid itself has a narrow host range and is maintained poorly in other
species (41).

A group of plasmids that have been increasing in prevalence in N.
gonorrhoeae are those that carry antibiotic resistance markers (107).
These appear to have been introduced into the gonococcus either through
direct acquisition from other bacteria (e.g. fl-lactamase plasmid) (40),
or by the transposition of antibiotic resistance genes into indigenous
gonococcal plasmids (92). The appearance and spread of fi-lactamase
plasmids among gonococci has inevitably led to the decreased efficacy of
penicillin as a first line antibiotic in the treatment of gonorrhea.

Conjugation is capable of mobilizing plasmid DNA, but chromosomal
markers are not transferred by this means. Gonococci can exchange
chromosomal DNA through transformation (8). Members of the genus
Neisseria are naturally competent and can take in DNA from the
environment at any stage of growth (117). The expression of pili greatly
increases the transformability of the gonococcus (117), though pili do
not directly participate in DNA binding or uptake (84). The importance
of transformation to the gonococcus has recently been established when
it was found that recombination among pilin genes occurs predominantly
with DNA brought in from outside the cells rather than by gene
conversion, although both processes do occur (113).

Not all DNA in the environment is taken up by the gonococcus; instead

11
DNA of neisserial origin is recognized and is preferentially taken into
the cell (11,32,47). This also occurs in Haemophilus influenzae, another
naturally competent organism. In H. influenzae selective DNA uptake is
mediated by the presence of an 11 bp sequence that is present at a much
higher frequency in its DNA than in DNA from other sources (26). DNA
bearing this sequence is brought into the cell through specialized
membrane vesicles called "transformasomes" (2,64). A similar system of
DNA recognition operates in N. gonorrhoeae, though transformasomes have
not been identified (8). In the gonococcus, the uptake sequence has been
identified as a 10 bp sequence unrelated to that of H. influenzae
(36,45). The gonococcal uptake sequence has frequently been found in
inverted repeats at the ends of identified and unidentified open reading
frames (45,83). This positioning suggests that uptake sequences may
often be incorporated into transcriptional terminators. This could
explain how the uptake sequence may have been "selected", and how it can
be maintained in high copy number without disrupting coding regions
(45).

The mechanism behind this selective uptake is still largely unknown,
but recent studies have turned up some interesting leads. While looking
for gonococcal proteins that specificallly bind neisserial DNA, Dorward
and Caron found an 11 kDa peptide located in the inner membrane of the
gonococcal cell that bound DNA strongly and had a higher affinity for
DNA containing the uptake sequence than to DNA without (30). This
protein was strikingly absent in the DNA uptake deficient mutants
isolated by Biswas et a1. (7). Gonococcal inner membranes can form
membrane blebs that are released from the cell and can be incorporated

by other cells in the area (31). It was proposed that these blebs may

12
mediate chromosomal DNA transfer, functionally analogous to H.
influenzae transformasomes (30). Blebs have been shown to carry plasmids
between cells (31), but their role in transport of chromosomal DNA
remains unclear.

Besides DNA discrimination during uptake, the gonococcus possesses
other bariers to genetic exchange (8). Gonococcal DNA is heavily
methylated at cytosine residues and in some strains adenine is
methylated as well (76,77,97,125). This methylation is sequence specific
and serves at least in part to protect the DNA from endogenous
restriction endonucleases, several of which have been identified
(19,27,97). The production of methylases and restriction enzymes is not
uniform among gonococcal strains, which has been shown to limit exchange
of DNA by transformation to strains with compatible restriction and
modification systems (120). The methylated cytosines also interfere with
cloning gonococcal DNA into E. coli strains that possess the McrA or
Mch restriction enzymes (106,138). These enzymes recognize DNA
sequences with methylated cytosines but not unmethylated sequences.
Fortunately cloning strains are available that lack these enzymes.

The ability of gonococcal strains to take in chromosomal DNA by
transformation has allowed for the mapping of selectable chromosomal
markers by cotransformation (137). This technique involves transforming
a recipient strain with chromosomal DNA from a donor, selecting for one
marker (i.e. antibiotic resistance), and then testing those
transformants for one or more other selectable markers. Logically,
markers that are physically closer together on the chromosome will have
a higher cotransformation frequency than those further apart, and thus

map distances and orders can be determined. In this way a short linkage

13
map representing approximately 3% of the gonococcal chromosome was
constructed (1,137). This map consisted mostly of antibiotic resistance
markers representing genes for ribosomal proteins and cell wall
synthesis, but also included a few outer membrane protein and auxotypic

markers.

AUXOTYPING AND SEROTYPING

In 1973, Wesley Catlin developed a particularly useful defined medium
for growth of Neisseria species (14). The medium was not a minimal
medium but a complex of amino acids, vitamins, purines, pyrimidines,
minerals and metals, with glucose provided as a carbon and energy
source. The purpose of creating this type of medium was to provide a
rich growth environment that could be manipulated for the rapid and
reliable detection of auxotrophs. The initial study was in part intended
to determine the prevalence of auxotrophs among the Neisseria, so as to
determine if auxotyping would be useful for epidemiological studies.
Three Neisseria species were examined; N. meningitidis, N. lactamica,
and N. gonorrhoeae. The N. meningitidis strains tested were relatively
homogeneous, with 5 of 57 requiring cysteine but otherwise few
auxotrophs were identified. The N. lactamica strains were similarly
homogeneous in nutritional requirements. The 74 strains of N.
gonorrhoeae tested on the other hand contained a wide variety of
auxotrophs including those with requirements for hypoxanthine, uracil,
thiamine, methionine, proline, isoleucine, and arginine, forming 13
distict auxotypes.

In a follow up study, Carifo and Catlin auxotyped 325 clinical

14
isolates, and of these 75% had at least one requirement and the number
of different auxotypes was increased to 22 (13). The auxotype of an
isolate was shown to be stably maintained when transmitted to sexual
partners or different anatomical sites (13). The ease in detecting
auxotypic markers along with their inherent stability made auxotyping
very attractive to epidemiologists, and auxotyping rapidly became a
standard tool for identifying gonococcal strains.

The most common nutritional requirements detected by Carifo and
Catlin were for proline and arginine, and similar patterns were later
seen in other studies (20,54,102). Arginine auxotrophy was further
dissected by testing the ability of arginine auxotrophs to grow in the
presence of intermediates of the arginine biosynthetic pathway (16).
This study revealed that the defects were not randomly distributed
throughout the pathway but instead occurred predominantly at steps five
and six. Of the 212 arginine auxotrophs studied, 1% had a block in the
first step of the pathway (required acetylglutamate for growth), 69% had
a block at step five (required ornithine), and 30% had a block at step
six (required citrulline). Enzyme assays were done on a selection of
these strains to determine if the growth requirements correlated to
defective enzymes of the arginine biosynthetic pathway (16,114,115). It
was found that strains that required ornithine did indeed lack
detectable ornithine acetyltransferase activity (16,114), but strains
that required citrulline unexpectedly had ornithine transcarbamoylase
activity levels comparable to wild type (16,114,115). It was later shown
that the citrulline requirement of these strains was due to a defect in
carbamoylphosphate synthetase, which in part explains the dual

requirement for uracil in all citrulline auxotrophs (115).

15

Shortly after the development of auxotyping, another typing system
was developed based on serological reactivity. The first serotyping
strategies used polyclonal antibodies against formalinized whole
gonococcal cells and were able to separate gonococcal strains into three
broad groups (111,134). These groups were later divided into nine with
the development of polyclonal antibodies against purified gonococcal
porin (P.I) proteins (10). Serotyping was further refined with the
development of a serotyping system based on a set of 12 monoclonal
antibodies against P.I proteins (71). This separated gonococcal strains
into two serotypes (P.IA and P.IB), with each serotype being subdivided
into multiple serovars (23 for P.IA, 32 for P.IB). The serovar system
works well for epidemiological studies in that it recognizes the two
distinct types of P.I proteins (94), as well as detecting minor

variations within these groups for more detailed analyses.

HISTORY OF AHU STRAINS

At the same time that auxotyping media were being developed, other
labs were looking for characteristics of gonococcal strains that may
influence their ability to cause disseminated infections. It was already
known that the incidence of disseminated disease was higher in some
locations than others, and it was thought that strain specific factors
may be involved in this discrepancy. The first correlation found among
strains isolated from disseminated infections was that they had an
increased sensitivity to penicillin and tetracycline (136). The basis
for this correlation was unclear, but it was proposed that mutations

that led to increased antibiotic resistance were achieved at the cost of

16
decreased virulence (34).

The next major correlation came in 1975 when Knapp and Holmes found
that strains causing DGI in Seattle predominantly had the multiple
requirements for arginine, hypoxanthine, and uracil (AHU auxotype) (68).
This observation was followed by other reports supporting this
correlation from other parts of the U.S. and Canada (72,96,126).

The discovery that AHU strains were associated with DGI stimulated
further epidemiologic and biological investigations into these strains,
and soon a picture of a rather unusual, homogeneous and clinically
important set of strains was revealed. AHU strains were common in parts
of Europe, the United States and Canada, but were not found in the Far
East, where DGI was uncommon (20,54,68,72,96). Along with being
associated with DGI, AHU strains were also frequently isolated from men
with asymptomatic infections (23,72). The association of AHU strains
with asymptomatic infection potentially explains their success in
becoming widespread in so many areas, in that asymptomatic males are
less likely seek treatment and more likely to spread the infection than
symptomatic males.

Besides being associated with patterns of infection, AHU strains were
also shown to share a wide variety of unusual phenotypic markers. AHU
strains characteristically gave atypically small colonies when cultured
and were more sensitive to the toxic components of agar than strains of
other auxotypes (68,90). AHU strains are more sensitive to penicillin
(34,68,72,126), but were found to be more resistant to the bactericidal
affects of normal human serum than non-AHU strains (112). Another study
found that AHUs show a decreased ability to utilize lactoferrin-bound

iron than other strains, a characteristic thought to be important in

17
their slow growth and correlation with asymptomatic infection (89).

Several studies found that AHU strains comprise a very homogeneous
group. These strains have identical plasmid profiles in that they all
had the cryptic plasmid, but not the conjugal plasmid (70). AHU strains
all produce type 1 IgA protease, while type 2 is more common among other
strains (94). DNA methylation patterns were also shared by AHU strains,
with all AHU strains possessing an adenine methylase (74), not commonly
found in other strains. Serotyping studies found that AHU strains
belonged almost exclusively to 2 closely related serovars in the P.IA
serotype that are rarely found outside of the AHU auxotype (70,71). All
of these factors supported the popular hypothesis that AHU strains may
have arisen only once, with all the present day strains being clonally
derived.

The hypothesis that AHU strains may be clonally derived was first
proposed by Mayer et a1. after studying the genetic defects of different
gonococcal arginine auxotrophs (85). Genomic DNA from a variety of AHU
and Arg- non-AHU strains was tested for the ability to transform one
another to Arg+. They found that DNA from one AHU strain could not
restore any other to Arg+, but it was capable of transforming other Arg-
strains to form Arg+ recombinants. The ny- and Ura- markers from AHU
strains were also shown to be incapable of recombining. From this they
concluded that the mutations responsible for auxotrophy in AHU strains
must be identical or overlapping, and that AHU strains were likely to be
clonally derived.

Biochemical studies of AHU strains revealed that they were all
defective in converting acetylornithine to ornithine (argJ mutants)(l6).

This was shown by their ability to grow on an arginine deficient medium

18
supplemented with ornithine, but not with supplemental acetylornithine,
as well as by showing that ornithine acetyltransferase activity was lost
in these strains (114). Some AHU strains had an additional lesion at the
sixth step of the pathway and thus required citrulline for growth. It
was found that these strains were defective in carbamoylphosphate
synthetase as well as OATase.

Interest in AHU strains began with their association with DGI, but as
it turns out the association of auxotype with DGI would later be
dispelled. In 1977, Eisenstein et al. did a careful study independently
analyzing the different characteristics that had been correlated with
strains isolated from disseminated infections (34). They found that
serum resistance and penicillin sensitivity both had a positive
correlation with DGI, but the relationship of the AHU auxotype and DGI
was not significant. The reports indicating a correlation were most
likely detecting an artifact of the lack of diversity of AHU strains
and the real association of AHU strains and penicillin sensitivity (34).
Another characteristic of AHU strains that was positively correlated to
DGI was the P.IA serotype (12). Gonococci of the P.IA serotype were
repeatedly shown to be more common among DGI isolates than P.IB,
independent of auxotype (9,12,94).

The homogeneous nature of AHU strains along with their unusual
collection of phenotypic markers led several groups to search for their
origin. In a retrospective study, Catlin and Reyn auxotyped a variety of
gonococcal strains isolated between 1935 and 1948, a time window chosen
to predate the widespread use of penicillin, which was introduced in the
mid-19408 (17). In their survey, a wide variety of auxotypes were

discovered, but no uracil requiring strains were found. Two hypoxanthine

19
requiring strains were found but they were not also arginine auxotrophs.
Arginine auxotrophs were common and were found alone and with other
requirements but no AHU strains were identified. Another retrospective
study looked at several different phenotypes (plasmid content, auxotype,
serotype, protease type, and adenine methylation) in gonococcal strains
collected from 1928 - 1976 in Copenhagen, Denmark (70). This study found
that AHU strains did not appear until 1946, and then increased in
frequency through the seventies. This study had the potential of
identifying a possible ancestor of AHU strains, but no strain was found
that shared the characteristic set of phenotypes found in AHUs. The
event that created AHU strains remains a mystery, but it has been
speculated that a large portion of the chromosome may have been affected
if several characteristics were incorporated simultaneously.

The working hypothesis of today is that AHU strains appeared early in
the penicillin era, essentially complete with phenotypes that increased
their ability to cause asymptomatic and disseminated infections, and
changed very little over time (70). The appearance of a multiply
auxotrophic strain at the time of the introduction of penicillin therapy
is likely to be more than coincidental. Penicillin kills bacteria by
preventing the formation of peptide cross bridges between peptidoglycan
molecules in the cell wall, thus compromising the integrity of the
entire cell. One consequence of this mode of action is that only
actively growing cells will be killed by penicillin. Auxotrophic cells,
normally at a disadvantage compared to their prototrophic counterparts
in nutritionally depleted environments, would be more likely to survive
a shot of penicillin due to their increased likelihood of being in

stasis from nutrient starvation. This "advantage" along with their

20
propensity for causing asymptomatic infections probably contributed to
the spread of AHU strains and to their becoming the predominant
gonococcal strain in many areas (69,70,135). The incidence of recovery
of AHU strains decreased in the 1980's in several areas (69,73). The
secret of their initial success has never been fully explained, but is
likely to be due to the pleiomorphic effects of their constellation of
unique phenotypes, including arginine auxotrophy, making them
particularly well adapted to their hosts, and increasing their frequency

of transmission.

10.

ll.

12.

21

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CHAPTER I

(ARTICLE)

Sequence of the argF Gene Encoding Ornithine Transcarbamoylase
from Neisseria gonorrhoeae

Published as a note in Gene
Volume 54, pages 149-150
(See Appendix)

32

33

ABSTRACT

We report the nucleotide sequence of the gonococcal ornithine
transcarbamoylase gene (argF) and flanking sequences. This gene
contained an open reading frame of 993 nt which encoded a peptide with a
predicted molecular weight of 36,731. This was in close agreement with
the size of the gene product as determined by minicell analysis. The
translation-initiation codon used by this gene was GUG, which was
confirmed by N-terminal amino acid sequencing of the purified protein.
In the flanking regions of the gene were found 15 bp inverted repeats
containing the neisserial DNA uptake sequence, supporting the hypothesis
that these sequences may be used as transcriptional termination signals.
We compared the predicted amino acid sequence to OTCase sequences
previously determined from E. coli, Pseudomonas aeruginosa, and several
eukaryotic species, and found that highly conserved regions in the genes
from these organisms were also conserved in N. gonorrhoeae. Amino acids
known to be important for carbamoyl phosphate and ornithine binding were

also conserved in the gonococcal OTCase.

34

INTRODUCTION

Arginine biosynthesis from glutamate occurs by an eight step pathway
(6). Neisseria gonorrhoeae possesses all the enzymes required for this
pathway, yet arginine auxotrophs are commonly encountered among clinical
isolates (4). A survey of 212 arginine auxotrophs showed that blocks in
the pathway occurred almost exclusively at the fifth or sixth steps; that
is, the conversion of acetylornithine to ornithine by ornithine acetyl
transferase, and the conversion of ornithine and carbamoyl phosphate to
citrulline by ornithine transcarbamoylase (OTCase) (5). Thirty percent
of the arginine auxotrophs examined in this study required citrulline,
suggesting that these would be OTCase mutants. Subsequent studies
(30,31) showed that all of the citrulline auxotrophs screened showed
normal levels of OTCase activity, and that the requirement for
citrulline was due to a defect in carbamoyl phosphate synthesis. We have
also examined a number of citrulline auxotrophs and to date none of
these have been OTCase mutants (Mulks, unpublished data).

OTCase is the product of the argF gene in N. gonorrhoeae and other
bacteria. E. coli K-12 strains have two copies of this gene, the second
being designated argI. ArgI is actually the copy common to all E. coli
strains; argF is thought to have been acquired in E. coli K-12 by
transposition from an unidentified source (14). Pseudomonas aeruginosa
also has two OTCases, though with separate functions. The pseudomonas
ArgF enzyme is involved in arginine biosynthesis, while the Ach enzyme

is involved in arginine catabolism (25). All four of the above mentioned

35
genes have been cloned and sequenced (1,2,16,34).

The gonococcal argF gene and several other genes of the arginine
biosynthetic pathway have been cloned (22), and gonococcal OTCase has
been purified (23). The purified enzyme was shown to exist as a trimer
of identical subunits of 36,500 Mr. We report here the complete
nucleotide sequence of the argF gene from N. gonorrhoeae, and the
predicted amino acid sequence of the gonococcal ArgF enzyme. These
sequences were compared to the OTCase sequences of E. coli, P.
aeruginosa, and several eukaryotic species and the relationship of these

genes is discussed.

36

MATERIALS AND METHODS

Bacterial strains and growth conditions. E. coli GM2163 (McrA-,
Mch-, are-14, leuB6, tonA13, lach, tsx-78, supE44, galKZ, dam-6,
hisG4, rpsLl36, dam-13::Tn9, xyl-5, mtl-l, thi-l hstZ) was used as the
initial receptor of the gonococcal genomic library. E. coli N134
(argI68, thi-l, spaTl, relAl, A(gpt-lac) 5) was used for selection of
the argF clone, and for complementation experiments. E. coli D8410
(minAl, minBZ, rpsL135, xyl-7, mtl-Z, thi-l) was used for minicell
experiments. N. gonorrhoeae CD050 (Arg+,Pro-,Dam+) was the source of our
gonococcal argF clone. E. coli strains were routinely grown on LB medium
with ampicillin added to 50 ug/ml for plasmid selection when required.
N. gonorrhoeae strains were grown on GC base agar (Difco) with 1%

Kellogg's suplement (37) at 36°C under 5% CO . NEDA defined medium (4),

2
either as a broth or with 1.5% agarose (Baker), was used for auxotyping
and complementation studies.

DNA manipulations. Restriction enzymes and DNA ligase were purchased
from Boehringer Mannheim Biochemicals or Bethesda Research Laboratories.
Genomic and plasmid DNA preparations, gel elctrophoresis, and E. coli
transformation were all done by conventional methods (28).

Cloning and subcloning argF. A plasmid library of N. gonorrhoeae
strain CD050 was constructed by partially digesting genomic DNA with
Sau3A, ligating fragments from 1.5-8 kb into the BamHI site of the
plasmid vector pUCl2, and transforming this ligation reaction into E.
coli GM2163. This library was screened for the gonococcal argF gene by

transforming the pooled plasmids into E. coli N134 and selecting for the

ability to grow on NEDA plates lacking arginine. The resulting clone was

37
restriction mapped by conventional means, and subcloned into M13mpl9 for
DNA sequencing.

OTCase assays. OTCase activity was assayed in cell-free extracts by
measuring the production of citrulline from ornithine and carbamoyl
phosphate as described by Shinners and Catlin (30).

Minicell analysis. Minicells were purified by differential
centrifugation followed by filtration through a Whatman GF/F glass
filter as described by Lurquin (18). Purified minicells were incubated
in the presence of 35S-methionine and the labelled proteins were
separated on an SDS-polyacrylamide gel and visualized by
autoradiography.

DNA sequencing. Nested deletions of the argF containing insert in
M13mp19 were constructed using the Cyclone deletion system (International
Biotechnologies, Inc). These deletions were sequenced using the dideoxy
chain termination reagents and Sequenase 2.0 enzyme from U.S.
Biochemicals. The insert was sequenced completely on both strands using
the method of Sanger et a1. (29).

Protein sequencing. The ArgF protein was isolated from E. coli
minicells containing the gonococcal argF clone by band purification from
an SDS-polyacrylamide gel. The minicell proteins were separated on a
10% polyacrylamide gel and transferred to a polyvinylidene difluoride
membrane (Immobilon P, Schleicher and Schuell) using the method of
Matsudaira (20). The proteins on the membrane were stained with
Coommasie blue and the unique ArgF band was excised from the membrane.
The first 10 amino acids were determined using an Applied Biosystems
477A Protein Sequencer at the Macromolecular Structural Facility at

Michigan State University.

38

RESULTS AND DISCUSSION

Cloning and initial characterization of the gonococcal argF gene. The
cloning of the gonococcal argF gene has been previously reported
(22). We independently isolated the gene from N. gonorrhoeae
strain CD050 by constructing a genomic library of this strain in the
plasmid vector pUCl2. The library was initially transformed into E. coli
GM2163 to avoid restriction of the heavily cytosine-methylated
gonococcal DNA (24). This library was screened for the gonococcal argF
gene by transformating it into E. coli N134 and selecting for Arg+
transformants on NEDA plates lacking arginine. A plasmid with a 2.3 kb
insert complementing the argI mutation was isolated and designated
pRGFl. The presence of the argF gene was confirmed by assaying cell-free
extracts of E. coli N134 containing pRGFl for OTCase activity. By
restriction mapping and the construction of deletions, the clone was
trimmed to a 1.3 kb fragment containing the active gene in the plasmid
pRGF4 (Fig. 1). This plasmid was used for minicell analysis of the gene
product, and the insert was subcloned in both orientations into the
phage vector Ml3mpl9 for DNA sequencing.

Minicell analysis. The protein encoded by the gonococcal argF clone
was analyzed by transforming the minicell producing E. coli strain D8410
with pRGF4. Figure 2 shows that the pRGF4 containing minicells produced
a unique band of 38,500 be when compared to minicells containing only
the plasmid vector. The size of this protein correlated well with the

size of 36,731 Da predicted from DNA sequence analysis.

39

Figure 1. Restriction map and deletions of pRGFl. Plasmid deletions are
shown along with their ability to complement the argI,F E. coli strain
N134 and OTCase activity produced in E. coli N134 containing each
plasmid. OTCase activity is given as nmol citrulline produced/minute x
mg protein in cell-free extract. Deletion pRGF33 was constructed by
exonucleolytic digestion of the pRGF4 insert in M13mp19 with T4 DNA
polymerase and then subcloning back into pUCl9 to test for

complementation.

40

mvm

2.2.2
885

vmsa
“5.8.300

u-nv—

=QRI .30

 

‘i
mmco

d auswfim

QFEQQ

41

-97.4
--66.2
42.7
Fr.-
.. -31.0
-21.5
1 2 a

Figure 2. Minicell analysis of pRGF4. Minicells contain: Lane 1, pRGF4;
Lane 2, pUCl9; Lane 3, no plasmids. Molecular weight standards in kDa

are indicated along the right. The arrow indicates the ArgF protein.

42

DNA sequence analysis. The sequence of the gonococcal argF gene and
flanking regions is shown in Figure 3. This sequence contains an open
reading frame of 993 nt preceded by a 6 nt ribosome binding site
sequence complementary to the 3'-end of the gonococcal l6S rRNA (27).
This gene uses the codon GUG at position 141-143 for translation
initiation. This is the first report of a GUG start codon in N.
gonorrhoeae, but GUG start codons have been found in approximately 8% of
sequenced E. coli genes (32). The first AUG codon within the open
reading frame occurs at nt 266. Several lines of evidence show that this
is not the start codon for the gonococcal argF gene. First, previously
sequenced OTCase genes from E. coli and P. aeruginosa, when aligned with
the argF sequence of N. gonorrhoeae, initiate near the GUG codon, not
the AUG (Fig.4). Second, in the plasmid pRGF33, the GUG codon has been
deleted and the AUG codon remains, yet the plasmid does not complement
E. coli N134 (Fig.1). Third, the molecular weight of the ArgF protein as
produced in minicells (Fig.2) closely matches the predicted size of the
protein transcribed beginning at the GUG codon, but would be
significantly larger than the protein predicted if the start codon were
the first available AUG. Also, the predicted size correlated very well
to the size determined from the purified protein of 36,500 (23), but
only if the GUG initiator is utilized. The most compelling evidence of
all for the GUG initiator came from N-terminal amino acid sequence
analysis of the ArgF protein (see below).

The promotor and transcription start site could not be clearly
identified from the sequence of the argF gene since no obvious -10

(TATAAT) sequence was found upstream of the open reading frame. A

43

Figure 3. Nucleotide sequence of the gonococcal argF gene and flanking
sequences. The predicted amino acid sequence is shown below the coding
region. N-terminal amino acids that were sequenced are indicated in
italics. Inverted repeats are underlined as is the putative ribosome
binding site (RBS). Neisserial DNA uptake sequences are underlined

twice.

44

TCCCCCTCATCCCTATGGAGTAACCCATTCACCQQAATCCQGIQIQAACAACCIICAQACG

QCAIICCAACATTCCCCTAACCCTTCTTTCCCCAAACCCTGCAAATACGCCCTTCACGCCCCACATAAAQQAAACCACA

0T0
Met

CCC
Ala

CCC
Arg

CCC
Ala

ACC
Ser

CAT
Asp

CCC
01y

CCA
Ala

CCC
01y

0A0
Asp

CAC
His

OTC
Val

CTC
Val

CCA
Ala

ACC
Thr

CAA
Glu

ATT
Ile

AAC CTC
Asn Leu

TAC OTC
Tyr Leu

ATC AAA
Met Lys

TTT 0AA
Phe Glu

0AA ATC
Cln Ile

CCC ATC
Ala Ile

CTA CCC
Val Pro

CTG ACT
Leu Thr

GAO CCC
Asp Ala

GTO CCT
Val Arg

CCC CCC
Ala Ala

AAA CCT
Lys 01y

TCC CAO
Trp Gln

TCC CCC
Ser 01y

AAA CTO
Lys Val

CTA TTC
Val Phe

AAA CCC
Lys Ala

AAA
Lys
GAO
Asp
000
Cly
CTO
Val
CCC
01y
CAA
Clu
0T0
Val
ATC
Met
CCT
Arg
ATC
Ile
000
Ala
0T0
Val
CAA
Glu
AAT
Asn
000
Cly
0AA
Glu
CTA
Val

AAC
Asn

CTT
Leu

AAA
Lys

000
Ala

CAC
His

TAT
Tyr

TTC
Phe

CCC
Arg

TAC
Tyr

000
01y

AAA
Lys

CCT
01y

CCC
Arg

000
Pro

CAA
Clu

ACT
Ser

ATC
Met

ccc
Arg

CCC
Ala

AAC
Asn

GCA
Ala

Lys

000
Arg

AAC
Asn

0AA
Glu

AAC
Asn

GOA
Ala

CAA
Glu

TTC
Phe

ATC
Ile

CAA
Cln

T00
Trp

CCC
Pro

CTC
Val

CAT
His
000
Ala
ATC
Ile
000
Arg
0AA
Glu
CCC
01y
CCC
01y
CAO
His
ATC
Met
CCT
Pro
A00
Thr
ATT
Ile
CAT
Asp
CTC
Val
ATT
Ile
000
Ala
000
Ala

TTT
Phe

CAO
Clu

CCC
Ala

0A0
Asp

A00
Ser

TTC
Phe

OTC
Leu

A00
Ser

CCC
01y

CAA
Gln

CCT
Cly

CAT
His

TTC
Leu

AAA
Lys

TAC
Tyr

000
01y

CCT
Ala

OTC
Leu

TTC
Leu

0T0
Leu

CAA
Gln

ATC
Ile

CCT
Ala

ACC
Thr

000
01y

AAT
Asn

A00
Ser

00A
Ala

ACT
Thr

OTC
Leu

TTC
Phe

CAA
Clu

ATC
Ile

0T0
Leu

AAA
Lys
AAA
Lys

ATT
Ile

CCC
01y

AAA
Lys

OAC
Gln

AAC
Asn
Lys

TCC
Set
0T0
Leu
AAA
Lys
CAC
Asp
AAA
Lys

ATC
Met

ACC
Thr

GTC
Val

CCC
01y

CTT TTC
Leu Leu

GAO CCC
Asp Ala

TTT CAA
Phe Glu

000 CAT
Ala Asp

CAC A00
Asp Thr

CAA ACT
Glu Thr

CAC TTC
Glu Phe

OCT TTC
Pro Leu

OTC 0T0
Leu Leu

0A0 TTC
Asp Phe
AAA AAC
Lys Lys
AAA ACA
Lys Thr

CCA ACC
Arg Thr

000 000
Ala Arg
OTC CAA
Val Glu
CAT 000
His Phe
AAC CAA
Asn Gln
ATT TTA
Ile Leu

TCC CCC [CT CAA

Trp Pro

ATT ACC
Ile Thr

CTA TGC
Val Trp

CAT TAO
Asp Tyr

CAO T00
His Cys

TTC CCC
Phe Cly

TTC CAT
Phe Asp

CAO TGA
Asp End

Ser Glu

CTG ACC
Leu Thr

GTC ACC
Val Ser

000 CTT
Arg Val

OTC CCC
Leu Pro

CTC AAC
Leu Asn

CAO CCC
Cln Ala

ACO
Thr

CCA
Ala

TCC
Ser

TAT
Tyr

0T0
Val

CAA
Glu

ACA
Thr

ACC
Thr

000
01y

CCC
Cly

CAA
Clu

ATC
Met
A00
Thr

CCC
Ala

CCT
01y

CAA
Glu

000
Pro

000
Gly

ACC
Thr

OTC
Leu

TTA
Leu

TTC
Leu

CAA
Gln

CCC
Ala

CCA
Ala

ATT
lle

AAC
Asn

000
01y

000
Pro

TTC
Phe

0T0
Val

AAC
Asn

0AA
Glu
CCC
Arg
000
Arg
CAA
Glu
CCC
01y
GOA
Ala
ATC
Met
TTT
Phe
AAA
Lys
ATC
Ile
000
Ala
0A0
Glu
0AA
Glu
CAC
His
CAA
Clu
CCT
Arg

S/D

CAA
Glu

GAG
Clu

ACA
Thr

000
Pro

ACA
Arg

AAA
Lys

CTT
Leu

CCC
Ala

TTC
Leu

CCC
Ala

CAT
His

000
Pro

OTC
Leu

AAC
Asn

CTT
Val

ATC
Met

ATC
Ile

ATT
Ile

CCC
Arg

TCC
Ser

ATC
Met

TAT
Tyr

000
Ala

TAC
Tyr

CCC
01y

CCC
Ala

CAA
Glu

AAA
Lys

ATC
Met

CCC
Arg

ACA
Thr

CAC
His

ACC
Thr

CAO
Cln
TCT
Cys

000
Ala

TAC
Tyr

CCC
Ala

CAO
Asp
0T0
Val

ATC
Met

CCA
Ala

000
Ala
0AA
Clu
CCC
Ala
CAA
Glu
CAA
Glu

ACO
Thr

CACAACTGTGCCTCTTTAAATTCATCCGCAA

CACACAIACQC{CTCAACACCAIGTTCACACCCIATCCATATAACAAACTCCCTACACCATCTGTACGCAGTCCCGTTT

CAAAACAATCACTTTTTCTTCTTCCTCGACT

61

140

200

260

320

380

440

500

560

620

680

740

800

860

920

980

1040

1100

1167

1246

12??

45

Figure 4. Comparison of the predicted amino acid sequences of the
gonococcal argF, E. coli argI, and P. aeruginosa ach genes. Two dots
indicate identical residues, one dot represents conservative
substitutions. Amino acids implicated in carbamoyl phosphate binding or
catalysis are marked by an asterisk. The cysteine residue involved in

binding of ornithine is indicated by a.

("I

N.

.argI
.argF

.ach

.argI
.argF

.ach

.argI
.argF

.ach

.argI
.argF

.ach

.argI
.argF

.ach

.argI
.argF

.ach

46

Figure 4

* es
MSCFYHKHFLKLLDFTPAELNSLLQLAAKLKADKKSGKEEAKLTCKNIALIFEKDSTRT

eeeeeeeeeeeeee
eeeeeeeeeeeeee

MAFNMHNRNLLSLMHHSTRELRYLLDLSRDLKRAKYTGTEQQHLKRKNIALIFEKTSTRT

* *
RCSFEVAAYDQCARVTYLGPSGSQIGHKESIKDTARVLGRMYDGIQYRGXGQEIVETLAQ

eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

RCAFEVAAYDOCANVTYIDPNSSQIGHKESMKDTARVLCRMYDAIEYRGFKOEIVEELAK

* * *
YRSVPVWNGLTNEFHPTQLIEYKLTMQEHLPGKAFNEMTLVYACDARNNMGVSMLEAAAL

eeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

FAGVPVFNGLTDEYHPTQMLADVLTMREH- SDKPLHDISYAYLCDARNNMGNSLLLIGAK

TCLDLRLVAPQACWPEAALVTECRALAQQNCGNITLTEDVAKGVEGADFIYTDVWVSMGE

eeeeeeeeeeeeeeeeeeeeeee
eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

LGMDVRIAAPKALWPHDEFVAQCKKFAEESGAKLTLTEDPKEAVKGVDFVHTDVWVSMGE

e
AKQKWAERIALLAEYQVNSKMMQLTCNPEVKFLHCLPAFHDDQTTLCKKMAEEF -GLHCG

PVEAWCERIKELLPYQVNMEIMKATCNPRAKFMHCLPAFHNSETKVCKQIAEQYPNLANG

MEVTDEVFESAASIVFGQAENRMHTIKAVMVATLSK

IEVTEDVFESPYNIAFEQAENRMHTIKAILVSTLADI

59
58

60

119
118

120

179
177

179

239
237

_239

298
295

299

334
331

336

47
potential —35 (TTGACA) sequence of TTGCAA was found starting at nt 67
(Fig 3). This would put the transcription initiation site at
approximately nt 104, but this has not yet been confirmed. It seems
likely that the cloned gonococcal argF gene uses its own promotor for
expression in E. coli since equivalent levels of expression were
obtained when the gene was present in either orientation in pUC12 (data
not shown).

Gonococcal cells are naturally competent for DNA transformation
during all stages of growth, though only DNA of neisserial origin will
bind to the cells (8). This selectivity is due to the higher occurence
of the lObp sequence 5'-GCCGTCTGAA-3' in neisserial DNA than in DNA from
other sources (9). This DNA uptake sequence, in at least a 9 out of 10
bp match, is present 5 times in the argF sequence, twice as part of 15 bp
inverted repeats in the flanking regions of the gene, and once
unrepeated within the coding region. The presence of the uptake sequence
within these flanking inverted repeats supports the hypothesis of
Goodman and Scocca that the uptake sequence may be maintained in high
frequency in Neisseria by being incorporated into transcription
termination sequences, though the presence of the uptake sequence within
the coding region of the gene suggests that this is not the only means
of preserving this sequence. Downstream from the proposed argF
transcriptional terminator containing the uptake sequences, which has a
AG value of -25.6 kcal, is a second 11 bp inverted repeat (AG - -l9.2
kcal). Both sets of inverted repeats resemble rho-dependent
transcriptional terminators (11), but which, if either, of these
inverted repeats actually functions in termination of the argF gene has

not yet been determined. It should be noted that the inverted repeat

48
upstream of the argF gene (AG - -30.8 kcal) overlaps the putative -35
promoter sequence. Whether there is an actual gene upstream of argF
that utilizes this inverted repeat as a terminator, or whether
transcription of that gene might interfere with initiation of the argF
gene, is not yet known.

The codon usage data for the gonococcal argF gene is shown in
Table I. This data is in good agreement with the codon usage frequencies
compiled by West and Clark (36) for previously sequenced gonococcal
genes. Codons infrequently used in gonococcal genes were either not used
or used only once in argF with the exception of GGG (Gly) which was used
7 times. Codons infrequently used in highly expressed E. coli genes (10)
were also used no more than once in argF, again with the exception of
GGG.

N-terminal amino acid sequence analysis. N-terminal amino acid
sequencing of the ArgF protein was performed in order to confirm the
translation initiation site of the gene. The seventh amino acid could
not be determined with certainty, but the remaining amino acid sequence
data supported the conclusion that the GUG codon at nt l4l-l43 is the
initiation codon for the gonococcal argF gene.

Comparison to OTCases from other species. The nucleotide sequences of
OTCases from other bacteria (E. coli argF and argI, P. aeruginosa argF
and ach), fungi (Aspergillus niger and Saccharomyces cerevisiae), and
mammals (human, mouse, and rat) have been previously reported
(1,2,3,12,15,l6,33,34,35). The DNA sequence and predicted amino acid
sequence of the gonococcal argF gene were compared to these other

OTCases (Table II). The gonococcal argF gene showed 43.4 to 66.6% DNA

49

TABLE I a
Codon usage in the N. gonorrhoeae argF gene

 

 

used used used used

Codon Amino in Codon Amino in Codon Amino in Codon Amino in

Acid argF Acid argF Acid argF Acid argF
TTT Phe 4 TCT Ser 1 TAT Tyr 3 TGT Cys l
TTC Phe 10 TCC Ser 3 TAC Tyr 6 TGC Cys 1
TTA Leu 2 TCA Ser 0 TAA *** O TGA *** 1
TTG Leu 6 TCG Ser 1 TAG *** O TGG Trp 4
CTT Leu 3 CCT Pro 2 CAT His 4 CCT Arg 3
CTC Leu 1 CCC Pro 4 CAC His 6 CCC Arg 11
CTA Leu 0 CCA Pro 0 CAA Gln 6 CGA Arg 1
CTC Leu 15 CCG Pro 6 GAO Gln 4 CCC Arg 1
ATT Ile 8 ACT Thr 3 AAT Asn 2 ACT Ser 1
ATC Ile 9 ACC Thr 9 AAC Asn ll AGC Ser 5
ATA Ile 0 ACA Thr 4 AAA Lys 20 ACA Arg 1
ATC Met 11 ACG Thr 5 AAC Lys 1 A66 Arg 0
CTT Val 2 GCT Ala 2 CAT Asp S CCT Cly 4
GTC Val 11 GCC Ala 18 CAC Asp lO CCC Gly 14
GTA Val 4 CCA Ala 9 CAA Glu 26 GGA Gly 0
GT6 Val 5 CCC Ala 11 GAG Glu a ccc Gly 7

 

a . . . .
Total number of codons in the argF gene is 331; *** indicates stop codons.

a

50

Table 2. DNA and amino acid sequence comparison of the OTCase of
Neisseria gonorrhoeae and OTCases and ATCases from other species

% identity for each pair of sequences was calculated using the

 

3 Identity
Organism DNA
E. coli OTCase (argF) 64.0 64.
E. coli OTCase (argI) 62.5 61.
P. aeruginosa OTCase (ach) 66.6 68.
P. aeruginosa OTCase (argF) 50.1 37.
A. niger OTCase 43.7 37.
S. cerevisiae OTCase 43.4 33.
Human OTCase 48.5 39.
Mouse OTCase 47.9 40.
Rat OTCase 47.7 40.
E. coli ATCase (pyrB) 42.6 30.
S. typhimurium ATCase (pyrB) 42.1 30.
B. subtilis ATCase (pyrB) 41.8 30.

u3P'UIP‘U1GDUIP‘U1C>O\C>>

O
H-
G-

Am—

GAP

program from the University of Wisconsin Genetics Computer Group
(Devereux et al., 1984), which makes use of the algorithm of Risler

et al.,

(1988).

51
sequence identity and 33.5 to 68.0% amino acid sequence identity with
these bacterial, fungal, and mammalian OTCases, suggesting that these
genes may have all arisen from a common origin. However, the data
suggest that the gonococcal argF gene is more closely related to
P. aeruginosa ach (66.6% identity) and E. coli argF (64.0%) and argI
(62.5%) than to P. aeruginosa argF (50.1%) or the fungal or mammalian
OTCases (43.4-48.5%). These data correlate well with the results of
Itoh et al., (16) showing a closer relationship of P. aeruginosa ach to
E. coli argF and argI than to P. aeruginosa argF.

The nucleotide sequences of three bacterial aspartate trans-
carbamoylases (ATCases), which catalyze the conversion of aspartate
and carbamoyl phosphate to carbamoyl aspartate, have been reported
(13,17,21). Comparison of the DNA and predicted amino acid sequences of
these genes to that of gonococcal argF (Table II) indicates ~42%
identity of the DNA and ~30% identity of the protein sequences and
supports the conclusion of Houghton et a1. (13) that these two classes
of enzymes share a common evolutionary origin.

Figure 4 compares the predicted amino acid sequences of the
gonococcal argF, the E. coli argI, and the P. aeruginosa ach genes.
Long stretches of near identity were found throughout the length of the
sequences. The regions from amino acids 55-59 (Ser-Thr-Arg-Thr-Arg) and
from 133-136 (His-Pro-Thr-Gln) are highly conserved in all OTCases as
well as ATCases sequenced to date from either prokaryotic or eukaryotic
sources. Within and between these regions are the amino acids involved
in carbamoyl phosphate binding (13,17), and these are all present in the
gonococcal OTCase sequence (Fig. 4). Another strongly conserved region

common to OTCases occurs at amino acids 262-268 (Phe-X-His-Cys-Leu-Pro).

52
The cysteine residue in this sequence is required for ornithine binding
in OTCases, (19), is not found in ATCases, and is conserved in the
gonococcal argF protein.

Unlike E. coli and P. aeruginosa, a gonococcal strain lacking
OTCase activity has not yet been identified. Experiments are in
progress to construct defined argF mutants, which should help determine
if the lack of argF mutants among arginine auxotrophs is due to a

selective dissadvantage of this type of mutation.

53

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1.

10.

ll.

12.

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l3.

14.

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17.

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22.

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54

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CHAPTER II

(ARTICLE)

Sequence analysis and complementation studies of the
argJ gene from Neisseria gonorrhoeae

Submitted for publication in
Journal of Bacteriology

56

57

ABSTRACT

Clinical isolates of Neisseria gonorrhoeae frequently are deficient
in arginine biosynthesis. These auxotrophs often have defects in the
fifth step of the arginine biosynthetic pathway, the conversion of
acetylornithine to ornithine. This reaction is catalyzed by the enzyme
ornithine acetyltransferase which is a product of the argJ gene. We have
cloned and sequenced the gonococcal argJ gene and found that it contains
an open reading frame of 1,218 nucleotides and encodes a peptide with a
deduced Mr of 42,879. This predicted size was supported by minicell
analysis. This gene was capable of complementing E. coli argE and argA
mutations, and of transforming an ArgJ- strain of N. gonorrhoeae to
Arg+. Southern blots were able to detect bands that specifically
hybridized to the gonococcal argJ gene in genomic DNA from Pseudomonas
aeruginosa, but not Escherichia coli, a result that reflects the
divergent nature of the arginine biosynthetic pathway in these

organisms.

58

INTRODUCTION

Arginine biosynthesis in prokaryotes occurs by an eight step pathway
(6) (Fig. 1). The first four steps of the pathway involve N-acetylated
intermediates, beginning with the acetylation of glutamate and ending
with acetylornithine. N-acetylornithine is converted to ornithine in the
fifth step of the pathway, and two separate enzymes have evolved to
catalyze this reaction. In the Enterobacteriaceae, N-acetylornithine is
hydrolyzed to ornithine and acetate by the ArgE enzyme N-acetyl-
ornithinase (EC 3.1.5.16) (35). In other bacteria including the
methanogens, cyanobacteria, pseudomonads, and N. gonorrhoeae, the acetyl
group of N-acetylornithine is transferred to glutamate by the ArgJ
enzyme ornithine acetyltransferase (OATase, EC 2.3.1.35) (l9,l3,34,28).
The N-acetylglutamate produced from this reaction can be cycled back
into the arginine biosynthetic pathway, bypassing step one. N-
acetylornithinase has been purified from E. coli and appears to function
as a monomer with a Mr of 62,000 (6). In contrast, no OATase from any
source has been characterized and little is known of the physical
properties of this enzyme. While the biosynthesis of arginine by
E. coli has been extensively studied as a model of gene regulation, few
of the genes encoding the enzymes involved in this pathway have been
sequenced. Nucleotide sequence data is available for E. coli argA,
argF, argI, and carAB, and the regulatory region of the argECBH operon,
and for Pseudomonas aeruginosa argF and ach. Neither E. coli argE nor

Pseudomonas aeruginosa argJ have been sequenced.

GLUTAMATE

ACETYL COA —i (ar‘g A)

 

GLUTAMATE
OrniThine Acefgl

Transf erase (69

Y

 

 

>- ACETYLCLUTAMATE

ACETYLGLUTAMYL PHOSPHATE

ACETYLGLUTAMATE SEMIALDEHYDE

ACETYLORNITHINE

Acefglorni‘rhinase
(argE)

\’ ACETATE

V

 

ORNITHINE

CARBAMOYLPHOSPHATE ——i

CITRULLINE

ARGINOSUCCINATE

ARGININE

Figure l. Arginine biosynthetic pathway. The two alternative

pathways for the conversion of acetylornithine to ornithine

are indicated.

60

Although N. gonorrhoeae possesses all the enzymes required for
arginine biosynthesis, arginine auxotrophs are commonly encountered
among clinical isolates of this pathogen (3). In a survey of 212
arginine auxotrophs, 69% were found to have blocks at the fifth step of
the pathway (4). Gonococcal strains with combined auxotrophy for
arginine, hypoxanthine and uracil (AHU strains) are of particular
interest due to their prevalence among isolates from asymptomatic and
disseminated gonococcal infections (4,14). AHU strains have repeatedly
been shown to be a highly homogeneous group and are believed to be
clonally derived (15,18,21). All AHU strains tested were found to be
argJ mutants (28), and AHU strains were shown to have identical or
overlapping argJ mutations in that DNA from one AHU strain could not
restore any other to Arg+ in paired transformation experiments (4,18).

At least four of the genes of the arginine biosynthetic pathway
(argB, argF, argG, and argJ) plus the genes for carbamoyl phosphate
synthetase (carA and carB) have been cloned from N. gonorrhoeae (23). In
that report, Picard and Dillon found that a single lambda clone
containing the gonococcal argJ gene could complement both E. coli argE
and argA mutations. They proposed that the gonococcal OATase may be
able to complement the argA mutation through the feedback pathway,
though it was also possible that the gonococcal argA gene was present on
the same clone.

In this paper we report the cloning and sequencing of the gonococcal
argJ gene, and investigate its potential to complement E. coli argE and
argA mutations. This represents the first reported sequence of an
ornithine acetyltransferase from any source. Along with providing new

information on the properties of the OATase enzyme, this study will

61
provide a foundation for further investigations into the molecular basis
of arginine auxotrophy and on the regulation of gene expression in N.

gonorrhoeae.

MATERIALS AND METHODS

Bacterial strains. The mcrA,B E. coli strain GM2163 (McrA-, Mch-,
ara-l4, 1euB6, tonA13, lach, tsx-78, supE44, galKQ, dew-6, hisG4,
rpsLl36, dam-13::Tn9, xyl-S, mtl-l, thi-l, hstZ) was used for the
initial construction of the genomic library. The argE E. coli strain
AT2538 (thr-l, are-14, 1euB6, A(gpt-proA)62, lach, supE44, ga1K2, A-,
hisG4, rpsL31, xyl-S, mtl-l, pyrE60, argE3, thi-l) and argA E. coli
strain W3421 (argAZl, galT23, IN(rrnD-rrnE)l, A-) were used for cloning
and complementation studies. The minicell producing E.coli strain D8410
(minAl, minBZ, rpsLl35, xyl-7,mtl-2, thi-l) was used for minicell
isolation and protein labelling experiments. E. coli DHl (Arg+), E. coli
N134 (A(gpt-lac)5, relAl, spoTl, thi-l, argI68) and Pseudomonas
aeruginosa ATCC 27853 were used for Southern blots. N. gonorrhoeae
strain CD050 (Pro-) was the source of the argJ gene. N. gonorrhoeae
strain 30465 (ArgJ-,ny-,Ura-) was used as a transformation recipient.

Media and growth conditions. All E. coli strains were grown on LB
medium and, when needed, ampicillin was added to 50 ug/ml for plasmid
selection. All N. gonorrhoeae strains were routinely grown on GC base
medium (Difco) plus 1% Kellogg's supplement (39) at 36°C under 5% C02.
NEDA defined medium for gonococci (4), either as a broth or with 1.5%

agarose (Baker), was used for growth of all bacteria in auxotyping or

62
complementation experiments.

DNA manipulations. Restriction enzymes and DNA ligase were purchased
from Boehringer Mannheim Biochemicals or Bethesda Research Laboratories
and used according to the manufacturer's instructions. Genomic and
plasmid DNA preparations, gel electrophoresis, and E. coli
transformation were all performed by conventional methods (26).

Cloning and sequencing of the gonococcal argJ gene. A genomic
library of N. gonorrhoeae CDC50 was constructed by partially digesting
genomic DNA with Sau3A and ligating fragments from 1.5 - 8 kb into the
BamHI site of the plasmid vector pUC12. A recombinant plasmid containing
the gonococcal argJ gene was isolated from this library by
complementation of the argE E. coli strain AT2538. This isolate was
subcloned into Ml3mpl9, and nested deletions were constructed using the
Cyclone deletion kit from International Biotechnologies, Inc. These
deletions were sequenced using the dideoxy chain termination reagents
and Sequenase 2.0 enzyme from U.S. Biochemicals, by the method of Sanger
et al. (27). The sequence was analyzed using GENEPRO software
(Riverside Scientific Enterprises), and this data was used to determine
the probable argJ open reading frame. Second strand sequencing was done
from identified restriction sites in M13 or pUC constructs.

OATase assays. Ornithine acetyltransferase activity was assayed by
measuring the production of ornithine from acetylornithine and glutamate
using the ninhydrin reaction as described by Shinners and Catlin (28).

Gonococcal transformation. Transformation of N. gonorrhoeae strain
30465 was done as previously described (1). Positive transformation was
scored as growth of GC30465 on NEDA plates lacking arginine after 48

hrs.

63

Minicell analysis. The minicell producing E. coli strain D8410,
transformed with pUCl9 or pRGE clones, was grown to high density in one
liter LB broth cultures and minicells were harvested by differential
centrifugation followed by sucrose density gradient centrifugation as
described by Dougan and Kehoe (8). The minicells were resuspended in 1
ml of NEDA broth lacking methionine to which 50 p61 35S-methionine
(Amersham) was added and labelled at 37°C for 1 hr. Labelled proteins
were separated by SDS-PAGE and visualized by autoradiography.

Southern blots. Genomic DNA from N. gonorrhoeae CD050 and 30465,
E. coli DHl, N134, and AT2538, and P. aeruginosa ATCC 27853 was digested
to completion with either ClaI or EcoRI, separated on an 0.8% agarose
gel, and transferred to nitrocellulose using the method of Southern
(31). Blots were probed with either the entire 3.4 kb insert of pRGE5
or with the 0.6 kb HincII fragment of pRGEB labelled with digoxigenin
using the Genius kit from Boehringer Mannheim Biochemicals.
Hybridizations were performed at both low and high stringency. High
stringency conditions included hybridization at 60°C in 5X SSC followed
by washes in 0.1x SSC plus 0.1% SDS at 60°C. Low stringency included
hybridization at 50°C in 5X SSC followed by washes in 2X SSC plus 0.1%
SDS at 50°C.

Nucleotide sequence accession number. The nucleotide and amino acid
sequences for the Neisseria gonorrhoeae argJ gene have been submitted to

GenBank and assigned the accession number M65216.

64

RESULTS

Cloning of the Neisseria gonorrhoeae argJ gene. A genomic library of
N. gonorrhoeae strain CD050 DNA was constructed in pUClZ and used to
transform argE E. coli strain AT2538 to arginine prototrophy. A
recombinant plasmid, designated pRGE5, was isolated from an Arg+
transformant. Restriction enzyme analysis revealed that pRGE5 contained
a 3.4 kb insert of gonococcal DNA (Fig. 2). OATase assays of E. coli
AT2538 cells containing pRGE5 confirmed that this plasmid encoded active
OATase enzyme (Table l). Deletion mutants were constructed using AvaI,
ClaI, and HincII restriction sites identified in pRGE5; these were
tested for argE complementation (Fig. 2) and for production of OATase
(Table 1). All three deletion mutants were negative for OATase activity
and were unable to complement E. coli argE, indicating that at least
part of the gonococcal argJ gene is encoded by the 0.19 kb AvaI-ClaI
fragment deleted in all three constructs (Fig. 2).

Sequencing and identification of the argJ gene. The entire 3.4 kb
insert in pRGE5 was subcloned into Ml3mpl9 for DNA sequencing. Nested
deletions of the 3.4 kb insert were generated using the 3'-5'
exonuclease activity of T4 polymerase provided in the Cyclone deletion
kit from IBI. These deletions were sequenced and provided a 3.01 kb
stretch of continuous nucleotide sequence which when analyzed revealed
four large open reading frames (Fig. 2). It was clear that DRE 1 could
not encode ArgJ, since pRGEA could neither complement argE nor specify
production of active OATase. To determine which of the three remaining

ORFs encoded argJ, three more deletion mutants were constructed. In

65

Figure 2. Restriction map and analysis of deletions of the gonococcal
argJ gene. Restriction sites shown are; A, AvaI; B, BsmI; C, ClaI;

H, HincII; S, SacII. Arrows represent open reading frames. ArgE
complementation was tested in E. coli AT2538; ArgA complementation was
tested in E. coli W3421. Gonococcal transformation to Arg+ was tested in
strain 30465. Asterisk indicates a lO-fold decreased transformation

efficiency relative to pRGE5.

66

+++

+

53 9 3g

+

<9< m9<
SE00; HZméwEEOU

+

2x

Qm

md

ON

m._

0..

md

0.0

 

 

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.4

 

 

11

 

d

U

.—

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poem

N muswam

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eilllo

E

30%
80%
mm 50%
100%
000%
30%
30%

67

Table 1. Ornithine acetyltransferase activity

 

Strain Enzym§_flgi§§a
E. coli AT2538 (0.1

E. coli AT2538 + pRGE5 6.54

E. coli AT2538 + pRGEA <0.l

E. coli AT2538 + pRGEC <0.l

E. coli AT2538 + pRGEH <0.l

a . .
One unit of enzyme activ1ty produces one nanomole

of ornithine from glutamate + N-acetylornithine

per minute per mg protein in the enzyme sample (3).

68

pRGEl72 (Fig. 2), the nested deletion closest to the 3' end of ORF 2 was
subcloned into pUCl9; this plasmid, which contained all four ORFs
intact, retained the ability to complement E. coli AT2538. pRGEl72 was
trimmed to the BsmI site to construct pRGEB, and also to the SacII site
to construct pRGES. pRGEB contained two complete overlapping ORFs, ORF
2 and ORF 3, as well as a truncated ORF 4, and was able to complement
argE. The ability to complement E. coli argE, which requires an intact
argJ gene, was lost in pRGES, a deletion that truncates ORF 2 but does
not enter ORF 3. These data indicate that argJ is encoded by ORF 2.

Minicell analysis. Plasmid pRGE5 and its deletions were transformed
into the minicell producing E. coli strain DS410 and the protein
products of these plasmids were labelled with 35S-methionine, separated
by SDS-PAGE, and visualized by autoradiography (Fig. 3). When compared
to the pU019 vector, the plasmids containing the complete ORF 2 coded
for the production of a unique peptide with an apparent Mr of 46,000.
In pRGES this peptide was truncated to an apparent Mr of 38,000, which
is consistant with the conclusion that this peptide is encoded by the
argJ ORF, ORF 2. A smaller peptide of 23,000 Mr was also produced from
plasmids pRGE5, pRGEB, and pRGES. Although this peptide was produced
only by plasmids containing the argJ ORF, it is unlikely to represent
the intact ArgJ peptide since it appeared unaltered in pRGES, a plasmid
in which ArgJ activity was lost. We were unable to identify peptides
that correlated with either ORF 1 or ORF 4.

Nucleotide sequence analysis. Second strand sequencing was
performed to confirm the sequence of the argJ gene and its flanking
regions. The nucleotide sequence and corresponding amino acid sequence

of argJ are shown in Figure 4. The gonococcal argJ gene consists of an

69

(56.2-' -

42.7-

31.0-3 :a “0"!“

'9.“

21.5-

 

1 2 £3 4 5 6

Figure 3. Minicell analysis of pRGE5 and deletions. Minicells
contained; lane 1, pUCl9; lane 2, pRGE5; lane 3, pRGEA; lane 4, pRGEB;
lane 5, pRGES; lane 6, pRGEH. Molecular weight standards are indicated
on the left. Arrows on the right indicate peptides produced by
recombinant plasmids; 46 kDa ArgJ peptide, 38 kDa truncated ArgJ

peptide, and 23 kDa peptide.

70

Figure 4. Nucleotide sequence of the gonococcal argJ gene and flanking
regions. The predicted amino acid sequence is shown below the coding
region. Inverted repeats are underlined as is the putative ribosome-
binding site (RBS). Neisserial DNA uptake sequences are double-

underlined.

71

Figure 4

GCATACGCGGCGATCGGGCTCACGTTCAACCGCCTCCTCACCCCCCTCATTGCCCCGCTGCTTATCCCCCTTTTC

CGGTTCTCAACCCGTTTCACACCCCAIIICAACCCAIGCCGTCTCAACGCCCACACACTCGCAAQQAQAACCGTT
RES

ATCGCTGTCAACCTGACCCAAAAAACCGCCGAACAACTCCCCGACATCCACCGCATTCCCCTCTACACCCCCCAA
M A V N L T E K T A E Q L P D I D G I A L Y T A Q

CCAGGCCTGAACAACCCCGGGCATACCCACCTGACACTGATTGCCGTACCCCCCGGCAGCACCCTCGCTCCACTC
A C V K K P G H T D L T L I A V A A G S T V C A V

TTCACGACCAACCCTTTCTGTGCCCCCCCCGTCCACATCGCCAAATCCCACCTTTTCCACGAACACCCCCTCCCC
F T T N R F C A A P V H I A K S H L F D E D C V R

CCCCTCGTCATCAACACGGGCAACCCCAACCCGGGTACGCGCGCACACCCCAGAATCGATGCTTTCGCAGTCTCT
A L V I N T G N A N A G T G A Q G R I D A L A V C

CCCGCCGCCCCCCGCCAAATCGGCTGCAAACCGAACCAGGTGATGCCCTTCTCCACCGCCGTGATTCTCCAACCG
A A A A R Q I 0 C K P N Q V M P F S T C V I L E P

CTCCCCCCACACAAAATCATCCCCCCCCTCCCCAAAATCCACCCTCCCTTCTCGAACGAACCGGCACCCCCCATC
L P A D K I I A A L P K M Q P A F W N E A A R A I

ATGACCACCCACACCGTTCCCAAAGCCGCCTCCCCCCAACCCAACCTCCCCGACCAACACACCGTCCGCGCCACA
M T T D T V P K A A S R E C K V G D Q H T V R A T

CCCATTGCCAAAGCCTCCGCCATGATTCATCCCAATATCCCCACCATGCTCCGTTTCATCGCCACCGATCCOAAA
C I A K G S G M I H P N M A T M L G F I A T D A K

CTTTCCCAACCCGTCCTCCAACTCATCACCCACCAAATCGCCCACCAAACCTTCAACACCATCACCCTTCACCGC
v s Q P v L Q L a T Q E I 7A o s T F N r I r v D c

GACACCACCACCAACCACACCTTCCTCATCATCGCCACCGGCAAAAACACCCAAAGCGAAATCGACAACATCGCC
D T S T N D S F V I I A T G K N S Q S E I D N I A

CAOCCCCCTTACCCCCAACTCAAACAATTCTTCTGCAGCCTTGCCCTCCAACTCCCCCAACCCATCGTCCCCCAC
D P R Y A Q L K E L L C S L A L E L A Q A I V R D

CGCGACCCCGCCACCAACTTCATCACCCTCCCCCTCCAAAACCCCAAAACCTCCCACGAAGCCCCCCAACCCGCC
0 E G A T K F I T V R V E N A K T C D E A R Q A A

TACCCCCCGCCACGTTCCCCACTGCTCAAAACCGCCTTTTTCCOCTCCCACCCCAACCTCGCCAACCCTCTCCCC
‘ Y A A 'A R S P L V K T A F F A S D P N L G K R L A

GOCATCGCTTATCCCCACGTTCCCCACCTCCATACCGACCTCCTCCAAATCTATCTCGACCATATTTTGCTTCCC
A I G Y A D V A D L D T D L V E M Y L D D I L V A

GAACACGGCGGACCCGCCGCAACCTACACCGAACCACAAGCGCAGCCGCTCATCTCCAAGGACGAAATCACCCTC
a a c c a A A s Y T a A Q "c Q A v n s K D a I T v

CGCATCAACCTGCATCGCGCACAAGCCGCCGCCACCCTCTATACCTGCGACCTGTCCCACCCATACCTTTCCATC
R I K L H R C Q A A A T V Y T C D L S H G Y V S I

AACCCCGACTACCGTTCCTGACCCCACACGGCTTCAGACCGCATACATAAAATCCCCTCTGAACCGTCCGACAAC
N A D Y R S ***

 

 

ATACCATGACCTCCACATTCCCCCCCCCCCTCCCCCCCAAAATCCCCCAAACCCCCCGCCTGTCCCCCAAAACCA

75

150

225

300

375

450

525

600

675

750

825

900

975

1050

1125

1200

1275

1350

1425

1500

72
ORF of 1,218 bp encoding a peptide with a predicted mol wt of 42,879.
This is in excellent agreement with the Mr of 46,000 for the ArgJ enzyme
determined by minicell analysis. The argJ ORF is preceeded by a
potential ribosome binding site containing a 6 bp sequence, AAGGAG,
complementary to the 3' end of the gonococcal 16S rRNA (25). No
consensus promoter sequence of either the -35/-10 (12) or -24/-12 (33)
type was found. It is possible that this gene could be cotranscribed
from a promoter upstream of ORF 1, although the fact that pRGEB, which
has the upstream portion of ORF I deleted, can complement E. coli argE
argues against this assumption. We also found no evidence of an "arg
box" operator sequence, the binding site for the arginine repressor that
has been described in E. coli (5) and Bacillus (29), upstream of the
argJ open reading frame.

Codon usage for the gonococcal argJ gene is listed in Table 2. We
compared the codon usage pattern for this gene with reference codon
usage data for N. gonorrhoeae (37), Pseudomonas aeruginosa (38), and both
highly and weakly expressed E. coli genes (10,11). Codon usage in argJ
most closely resembled that seen in P. aeruginosa, with 19 of 21 rarely
used codons and 10 of 14 most commonly used codons found in argJ
matching the codon usage reference table for P. aeruginosa. There was a
distinct bias in the choice of nucleotide in the wobble (third) position
in degenerate codons, with 58.9% containing cytosine and 76.1%
containing either cytosine or guanine in the this position. This is
similar to the data for P. aeruginosa, where the 52.6% cytosine and
85.6% cytosine or guanine in the third position reflects an overall
guanine-plus-cytosine ratio of 67%. In contrast, the N. gonorrhoeae

reference table indicates 34.3% of codons contain cytosine and 52.1%

73

Table 2. Codon usage of the Neisseria gonorrhoeae argJ gene

Used Used Used Used
Amino in Amino in Amino in Amino in
Codon Acid argJ Codon Acid aer Codon Acid aer Codon Acid araJ

TTT Phe 1 TCT Ser 0 TAT Tyr 3 TCT Cys 2
TTC Phe 10 T00 Ser 5 TAC Tyr 6 TCC Cys 4
TTA Leu 0 TCA Ser 0 TAA *** 0 TCA *** l
TTC Leu 4 TCC Ser 6 TAG *** 0 TCC Trp l
CTT Leu 2 CCT Pro 1 CAT His 2 CCT Arg 4
CTC Leu 13 000 Pro 10 CAC His 5 CCC Arg 10
CTA Leu 0 CCA Pro 1 CAA Cln 12 CCA Arg 0
CTC Leu 11 000 Pro 3 CAC Gln. 5 CCC Arg 1
ATT Ile 6 ACT Thr 0 AAT Asn 1 ACT Ser 0
ATC Ile 21 ACC Thr 28 AAC Asn 14 ACC Ser 7
ATA Ile 0 ACA Thr 2 AAA Lys 12 ACA Arg 1
ATC Met 10 ACC Thr 4 AAC Lys 6 ACC Arg 1
CTT Val 6 CCT Ala 2 CAT Asp 4 CCT Cly 4
CTC Val 15 etc Ala 47 CAC Asp 24 etc Gly 20
CTA Val 1 CCA Ala 9 CAA Glu 17 CCA 01y 3
CTC Val 7 CCC Ala 8 GAO Glu 1 CCC 01y 2

 

74
contain cytosine or guanine in the third position, which would be
expected of an organism with a 49.5% guanine-plus-cytosine ratio.

The 10 bp neisserial DNA uptake sequence, 5'-GCCGTCTGAA-3', (9)
occurs four times in this sequence, but only as part of a larger 12 bp
sequence, 5'-ATGCCGTCTGAA-3', in closely spaced inverted repeats in the
flanking regions of the gene and not in the coding region. The
downstream inverted repeats could form a stable stem and loop structure
with a AC of -23.4 kcal/mol that may act as a transcriptional
terminator. The upstream inverted repeats could also form a stable stem
and loop structure with a AC of -22.4 kcal/mol. This structure follows
the unidentified ORF l upstream of the argJ ORF, and overlaps the region
where a promoter for argJ would be expected to be located.

We reviewed 18 different N. gonorrhoeae DNA sequences available in
GenBank release 68, June/1991. Of these, 12 contained at least one copy
of the gonococcal uptake sequence, either singly or in inverted repeats.
When we aligned these uptake sequences and their flanking sequences, we
found that 16 of 22 copies (12 inverted repeats and 10 single copies) of
the 10 bp uptake sequence also contained the 5'-AT extension. This
occurred more frequently in the inverted repeats than in single copies,
suggesting that this extension may contribute to the proposed terminator
function of these repeats. We also found this 12 bp sequence in three
N. meningitidis genes, both in single copies within the genes and in
inverted repeats at the end of the open reading frames.

Analysis of the predicted ArgJ protein. The argJ open reading frame
encodes a protein with a predicted molecular weight of 42,879 and an
isoelectric point of 5.18. A hydrophobicity plot, using the algorithm

of Kyte and Doolittle (16), indicated no pronounced hydrophobic regions

75
in the argJ gene product, suggesting that this protein is soluble rather
than membrane-bound. We could find no recognizable signal peptide
sequence at the N-terminus (36), suggesting that this protein is not
secreted. We therefore conclude that ArgJ is a soluble cytoplasmic
protein, as would be expected for an enzyme involved in amino acid
biosynthesis.

Southern blots. Genomic DNA from E. coli and P. aeruginosa were
tested for homology to the cloned gonococcal argJ gene by Southern blot
hybridization (Fig. 5). Under high stringency conditions, neither E.
coli nor P. aeruginosa DNA hybridized with either the 0.6 kb (Fig. 5c)
or the 3.4 kb (data not shown) gonococcal argJ probes. Under low
stringency conditions, specifically hybridizing bands were apparent
above background in N. gonorrhoeae and P. aeruginosa lanes, but not E.
coli lanes, with both the 0.6 kb and 3.4 kb argJ probes (Fig. 5b, 5a).

Homology to other sequenced genes. Comparison of the argJ
nucleotide and predicted amino acid sequences with the GenEMBL
database, using the FASTA and TFASTA programs of the University of
Wisconsin Genetics Computer Group software (7), yielded no listed DNA
or protein sequences with significant homology to the gonococcal argJ
gene.

We carefully compared the gonococcal argJ gene and its product to
E. coli argA (2), Neisseria gonorrhoeae argF (l7), and to a gene from
Leptospira biflexa which complements argE in E. coli (40), using a
variety of nucleotide and amino acid sequence analysis and protein
structure analysis programs, and found no significant sequence or
structural similarity between argJ and any of these genes. We were

also able to compare our sequence with the proposed sequence of the

76

 

ii? :

a T

      

1.0-,

at»-
%

 

 

Figure 5. Southern blot of gonococcal, E. coli, and P. aeruginosa DNA
probed with the gonococcal argJ gene. DNA size markers are indicated in
kilobases. Genomic DNA used were from; Lane 1 - N. gonorrhoeae 30465,
Lane 2 - N. gonorrhoeae CD050, Lane 3 - E. coli DHI, Lane 4 - E. coli
AT2538, Lane 5 - E. coli N134, Lane 6 - P. aeruginosa ATCC 27853
digested with EcoRI, Lane 6a - P. aeruginosa ATCC 27853 digested with
ClaI. Panel A: EcoRI digested DNA probed with the 3.4 kb insert from
pRGE5 under low stringency (see methods). Panels B & C: ClaI digested
genomic DNA (except lanes 6) probed with the 0.6 kb HincII fragment from
pRGEB. Panel B: low stringency conditions, Panel 0: high stringency
conditions. Solid circles mark P. aeruginosa bands that hybridize to the
gonococcal probes under low stringency. Asterisks mark N. gonorrhoeae
bands that hybridize under high stringency.

77
putative argJ gene from Bacillus subtilis (22). Significant identity
was found between the predicted amino acid sequences of these two genes,
which included a region with 23 of 26 identical amino acid residues
(88.5% homology). We also used this conserved 26 amino acid sequence,
and a larger 84 amino acid conserved sequence containing this region, to
search the GenBank and EMBL databases, but found no other published
sequences with significant homology to this sequence.

E. coli complementation and N. gonorrhoeae transformation. The
original pRGE5 clone and subsequent deletions were tested for their
ability to complement argE and argA mutations in E. coli, and for the
ability to transform the argJ N. gonorrhoeae strain 30465 to Arg+
(Fig. 2). These experiments demonstrated that the argE and argA
complementing activities were inseparable, and dependent on the presence
of an intact argJ ORF. In contrast, transformation of GC 30465 to Arg+
was not dependent on an intact argJ ORF. Both plasmid pRGES, which
lacks the argJ gene downstream of the SacII site, and plasmid pRGEC,
which lacks the 5' end of the argJ gene, were able to transform GC
30465 to Arg+, while plasmids pRGEA and pRGEH could not. These data
imply that the lesion in the argJ gene of 00 30465 lies at least
partially within the 0.39 kb ClaI - HincII fragment in the center of
the argJ ORF and entirely within the 0.63 kb ClaI - SacII fragment

(Fig. 2).

78

DISCUSSION

In this paper we report the cloning and complete nucleotide sequence
of the Neisseria gonorrhoeae argJ gene, which encodes ornithine
acetyltransferase, the enzyme that catalyzes the conversion of
acetylornithine plus glutamate to ornithine plus acetylglutamate at the
fifth step of the arginine biosynthesis pathway. The argJ clone was
identified by its ability to complement an E. coli argE mutant, and its
identity was confirmed by demonstrating that this clone encoded OATase
activity and that it could transform a gonococcal argJ mutant to Arg+.

We sequenced most of the 3.4 kb insert in the original argJ clone,
and found four potential open reading frames in this sequence. The ORF
encoding argJ was identified by construction of deletion mutants and
analysis of their ability to produce active OATase and to complement
E. coli argE mutants. The gonococcal argJ gene contained an open
reading frame of 1,218 bp which encoded a peptide with a predicted
molecular weight of 42,879. There is a good ribosomal binding site in
the appropriate position upstream from the putative start codon.
However, we were unable to identify a sequence homologous to either the
-35/-10 or -24/-12 E. coli consensus promoters. This was also the case
when we studied the gonococcal argF gene (17). While E. coli-type
promoters have been found associated with some neisserial genes, such as
the opaEl and IgAl protease genes (32,24), little is known about
promoter structure in Neisseria spp., particularly for housekeeping
genes such as those involved in arginine biosynthesis.

In both E. coli and Bacillus subtilis, many of the genes involved in

79
arginine biosynthesis are repressed by arginine (6, 30). In E. coli,
this repression occurs when the arginine repressor protein binds to the
"arg box" operator sequence overlapping the promoter of arginine
repressible genes (5). One or two copies of the arg box consensus
sequence have been found upstream of several E. coli arginine
biosythetic genes (5), as well as in the B. subtilis argC promoter (29).
We found no evidence of an arg box sequence upstream of the gonococcal
argJ gene. Previous studies in Catlin's laboratory (28), as well as
preliminary studies in this lab have found no evidence of repression of
the gonococcal argJ or argF genes by arginine.

The N. gonorrhoeae argJ ORF is closely flanked by inverted repeats
of the neisserial DNA uptake sequence. In their study that elucidated
the uptake sequence (9), Goodman and Scocca found that this sequence was
often present within inverted repeats at the end of undefined ORFs and
proposed that the uptake sequence may also be involved in transcription
termination. The presence of the uptake sequence in inverted repeats at
the end of the argJ ORF supports this hypothesis. This inverted repeat
was also found upstream of the argJ ORF. Whether this sequence is
involved in termination of a gene upstream of argJ, or in regulation of
argJ expression, has not yet been determined. An interesting
observation of the uptake sequences in the argJ gene is that they
occur only within the longer 12 bp sequence, 5'-AIGCCGTCTGAA-3'. This
12 bp extended sequence is also present in 16 of the 22 occurrences of
the uptake sequence that we analyzed in 12 different gonococcal genes,
as well as in 3 meningococcal genes, and is more frequently observed in
copies of the uptake sequence found as inverted repeats at the end of

open reading frames than in single copies of the sequence. These data

80
suggest that the extended uptake sequence may contribute to a more
stable stem-and-loop terminator structure.

This is the first ornithine acetyltransferase sequence to be
reported from any source. The sequence of a gene complementing an E.
coli argE mutation from the spirochete Leptospira biflexa has been
previously reported, but the nature of the enzyme activity encoded by
that gene, was not defined (40). We compared this sequence with the
gonococcal argJ gene and found no significant similarity at either the
DNA or the predicted amino acid sequence level, suggesting that these
two genes are unrelated. We were also able to compare our sequence to
the putative argJ gene from Bacillus subtilis (22). Significant
identity was found between the predicted amino acid sequences of these
two genes. This comparison revealed that these genes appear to be
distantly related with several regions being highly conserved.

Picard and Dillon (23) found that their gonococcal argJ lambda clone
could complement both E. coli argE and argA mutations. They proposed
that this may be the result of the gonococcal ArgJ enzyme producing the
required acetylglutamate for the pathway, but due to the size of their
clone, they could not rule out the possibility that the gonococcal argA
gene might also be present. Our findings strongly support the former
theory. We have shown that argA complementation is dependent on having
an intact argJ gene, and that no extra DNA outside the argJ gene is
required for argA complementation. In addition, we have isolated a
clone which complements argA in E. coli without complementing argE.
This clone likely contains the gonococcal argA gene and has no homology
to pRGE5 when compared by restriction mapping (20).

Our cloned argJ gene was capable of transforming the gonococcal AHU

81
strain 30465 to Arg+. These transformants remained Ura-, ny- as
expected since previous cotransformation studies have shown that these
markers are not closely linked (1,18). These previous studies also
reported that naturally occuring Arg+ revertants of AHU strains do
arise, but at a very low frequency. The occurrence of revertants makes it
unlikely that the argJ gene has been deleted in AHU strains. In this
study it was found that N. gonorrhoeae 30465 contains an apparently
intact argJ gene as seen on Southern blot. The ability of deletions of
the argJ gene to transform 30465 to Arg+ revealed that the argJ mutation
in this strain is apparently located between the internal ClaI and SacII
sites. Experiments are in progress to determine the nature of the argJ
mutation in AHU strains of N. gonorrhoeae. These experiments will lay
the groundwork for future studies on arginine biosynthesis and on the

regulation of housekeeping genes in Neisseria gonorrhoeae.

ACKNOWLEDGEMENTS
This research was supported by Public Health Service grant Al-21264

from the National Institute of Allergy and Infectious Diseases.

82

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170:4548-4554.

CHAPTER III

(ARTICLE)

Molecular characterization of the argJ mutation in
AHU strains of Neisseria gonorrhoeae

Submitted for publication in
Infection and Immunity

85

86

ABSTRACT

Arginine auxotrophs are commonly encountered among clinical isolates
of Neisseria gonorrhoeae. Arginine auxotrophs which also require
hypoxanthine and uracil (AHU strains) comprise a unique set of strains
that are highly homogeneous and are believed to be clonally derived. The
Arg- phenotype of these strains is due to a lesion in the argJ gene
encoding ornithine acetyl-transferase. We have cloned the mutant argJ
gene from an AHU strain, and compared the sequence of this gene to the
wild type argJ gene. The mutant gene contained a 3 bp deletion within a
repetitive region of the argJ gene. This mutation was restored to the
wild type sequence in a naturally occuring Arg+ revertant of the AHU
strain. This deletion was detected in a wide variety of other AHU
strains, but not in other ArgJ- strains or in ArgJ+ strains, supporting

the hypothesis that AHU strains are clonally derived.

87

INTRODUCTION

Auxotyping, the classification of strains by nutritional
requirements, has been used to identify isolates of Neisseria
gonorrhoeae since the development of defined media for Neisseria by
Catlin (5). The auxotype of a gonococcal strain has proven to be a
stable, easily identifiable marker, and auxotyping has been used
effectively to follow strains throughout a population (4,11,14).

An early epidemiological study comparing auxotype to pattern of
gonococcal infection by Knapp et al. (11) found that strains with
requirements for arginine, hypoxanthine, and uracil (AHU strains) were
predominant among isolates from disseminated gonococcal infections (DGI)
in Seattle. This observation prompted further studies of AHU strains
which led to the discovery that AHU strains are quite homogeneous and
distinctly different from other gonococcal strains. Besides sharing
auxotypic markers, AHU strains predominantly belong to two closely
related Protein I serovars (13), are resistant to the bactericidal
action of normal human serum (25,14) and show increased sensitivity to
penicillin (ll,l4,20,25). They produce type I IgAl protease and Dam
methylase, both of which are uncommon among gonococci of other auxotypes
(21,16). The correlation of AHU strains with DGI has been shown to be an
artifact of their homogeneity, with P1 serotype, serum resistance and
penicillin sensitivity showing a higher correlation with DGI than
auxotype (3,10,2) .

Genetic analysis of AHU strains revealed that the genes involved in

88
the AHU auxotype, serum resistance, and penicillin sensitivity are
independent and not closely linked (6,19,l). These studies also found
that the Arg- lesions in AHU strains are identical or overlapping, in
that DNA from one AHU strain could not transform any other to Arg+ (l9),
and that this lesion is different from that of Arg- strains not of the
AHU auxotype. These observations led to the hypothesis that AHU strains
are clonally derived.

Biochemical analysis of AHU strains revealed that the Arg- phenotype
of these strains was at least partially due to the inability to convert
N-acetylornithine to ornithine (7). This reaction is catalyzed by the
enzyme ornithine acetyltransferase, encoded by the gene argJ, which
converts acetylornithine and glutamate to ornithine and acetylglutamate.
The N. gonorrhoeae argJ gene has been cloned by complementation of an E.
coli argE mutant (23,18) and has been sequenced (18). The gonococcal
argJ gene contains an open reading frame of 1,218 bp and encodes a
protein of 42,879 Mr' The cloned gene was capable of transforming an AHU
strain to Arg+. The gonococcal argJ gene was also able to complement an
E. coli argA mutant (18,23), which presumably occurs by feedback of the
acetylglutamate produced by the ArgJ enzyme back into the arginine
biosynthetic pathway.

In this paper we report the cloning of the argJ gene from an AHU
strain, and identify a mutation in this gene that correlates to the Arg-
phenotype. We also show that this mutation is common to other AHU

strains, but not Arg+ strains or ArgJ- strains that are not ny-, Ura-.

89

MATERIALS AND METHODS

Bacterial strains and growth conditions. E. coli AT2538 (argE3, chi-1,
thr-l, ara-14, 1euB6, A(gpt-proA)62, lach, supE44, galKQ, A-,hisG4,
rpsL31, xyl-5, mtl-l, pyrE60), and W3421 (argAZl, ga1T23, A-, IN(rmD-
rrnE)l) were used for complementation studies, E. coli GM2163 (McrA-,
Mch-, ara-14, 1euB6, TonAl3, Lach, tsx-78, supE44, ga1K2, A-, dam-6,
hisG4, rpsLl36, dam-l3:Tn9, xyl-5, mtl-l, chi-1, hstZ) was used as the
initial recipient in the cloning of the mutant gonococcal argJ gene. N.
gonorrhoeae strains used in this study are listed in Table 1. All E.
coli strains were grown on LB medium; when needed ampicillin was added
to 50 ug/ml for plasmid selection. N. gonorrhoeae strains were grown on
00 base (Difco) plus 1% Kellogg's supplement (29) at 36°C under 5% 002.
NEDA defined medium for gonococci (4) with 1.5% agarose (Baker) was used
for growth of all bacteria in auxotyping or complementation experiments.

DNA manipulations. Restriction enzymes, DNA ligase, and alkaline
phosphatase were purchased from Boehringer Mannheim Biochemicals (BMB)
or Bethesda Research Laboratories and used according to manufacturers
recomendations. Genomic and plasmid DNA preparations, gel
electrophoresis, and E. coli transformations were all done by
conventional methods (17). DNA probes were labelled with either
digoxygenin using the Genius kit from BMB, or with 32P-dCTP using the

random priming kit from BMB. Southern blots were done by the method of

Southern (27).

90

Table 1. Gonococcal strains and phenotypes.

 

Strain Auxotypea Serovar Sourceb

l. CD050 Pro IBl Fla.

2. NRL30465 AHU (ArgO) IAl Japan

3. NRL905 AHU (ArgO) IAl *

4. CD022 AHU (ArgO) IAl Fla.

5. CD06 AHU (ArgC) IAl Iowa

6. CD054 AHU Ser (ArgO) 182 Iowa

7. CD0208 AHU (ArgO) IB2 Ohio

8. CD01 ArgO I324 Fla.

9. CD070 ArgO IBl N.J.
10. CD087 ArgO IB2 Iowa
11. CD034 ArgA Pro Leu Met IBl Iowa
12. CD09 Pro IA3 Mass.
13. CD099 Pro IAl Virginia
14. MS-ll WT IB9 *

15. NRL9396 AHU IAl Copen.

 

a Auxotype: WT - prototrophic; Arg - requires arginine;

ArgC - arginine requirement can be satisfied by citrulline;
ArgO - arginine requirement can be satisfied by citrulline

or ornithine; ArgA - arginine requirement can be satisfied
by citrulline, ornithine or acetylglutamate; AHU - requires
arginine, hypoxanthine and uracil; Pro - requires proline;
Ser - requires serine; Leu - requires leucine, Met - requires

methionine.

Source: Copen. - Copenhagen, Denmark; others include Florida,
Iowa, Massachusetts, New Jersey, Ohio, and Virginia states.

Asterisk indicates source location not available.

91

Cloning of the mutant argJ gene. Genomic DNA from N. gonorrhoeae
strain NRL30465 was completely digested with Sau3A and fragments from
3.2 to 3.6 kb were hand purified from a 0.8% agarose gel. These were
ligated into the BamHI site of pUCl9 and transformed into E. coli
GM2163. Transformants were screened for plasmids containing the argJ
gene by probing colony blots with the 0.8 kb ClaI fragment of the wild
type gonococcal argJ gene using methods described by Maniatis et al (17).

Enzyme assays. Ornithine acetyltransferase assays were done on cell-
free extracts using the ninhydrin reaction to measure the production of
ornithine from acetylornithine and glutamate as described by Shinners
and Catlin (26).

Construction of chimeric argJ clones. Chimeras of the wild type and
mutant argJ genes were constructed by exchanging restriction fragments
between the wild type clone (pRGE5) and the mutant clone (pREMl).
Restriction endonuclease ClaI was used to release a fragment containing
the 5' end of the argJ gene, and SacII and HindIII were used to release
a fragment containing 3' end of the gene. These fragments were separated
from the remainder of the plasmid in an 0.8% agarose gel, and the
fragments and remaining plasmids were band purified. Chimeras were
constructed by ligating the fragments back into the plasmid from which
they did not originate. Chimeric clones were tested for proper insertion
and ArgE complementing activity.

DNA sequencing. The mutant argJ gene was subcloned into Ml3mpl8 and
M13mp19 and sequenced on both strands in the region shown to contain the
argJ mutation. Subclones were constructed from identified restriction
sites. Sequencing was done using the Sequenase enzyme from U.S.

Biochemicals, by the method of Sanger et al. (24).

92

Isolation of Arg+ revertant. N. gonorrhoeae strain NRL30465 was grown
16-18 hr on GC base plates and resuspended to >109 cfu/ml in NEDA broth.
100 pl of this suspension was spread onto multiple NEDA plates lacking
arginine and incubated at 36°C. Revertant colonies were isolated after
48 hr and streaked for isolation on 00 base. Revertants were auxotyped
prior to further use.

Detection of 3 bp deletion. Gonococcal genomic DNA was digested to
completion with Hian and the reaction was stopped by adding 0.8 volumes
of Sequenase stop buffer (U.S. Biochemicals). The digested DNA was
denatured at 80°C for 3 minutes and loaded onto an 8% polyacrylamide
sequencing gel. 35S—labelled sequencing reactions were used as size
markers. The gel was run at 60 W for 2.5 hr, disassembled, and lifted
onto 3MM paper. A 7.5 x 15 cm piece of Nytran (Schleicher and Schuell),
presoaked in transfer buffer (25 mM sodium phosphate, pH 6.0), was
placed over the region of the gel to be transferred. This area was cut
away from the remainder of the gel, sandwiched between transfer buffer
soaked 3MM paper, and placed into a western blotting chamber (Hoefer
Scientific) filled with transfer buffer. The DNA was transferred to the
nytran membrane at 40 V (1.5 amp) for 1 hr. The membrane was allowed to
air dry and then baked under vacuum at 80°C for 2 hr. This blot was
prehybridized and probed with the 0.6 kb HincII fragment of the wild
type argJ gene by conventional means (17). Hybridization and washings

were done at a reduced stringency (56°C) to increase sensitivity.

93

RESULTS

Initial characterization of the argJ gene in AHU strains. Genomic DNA
from 6 different AHU strains of N. gonorrhoeae were checked for the
presence of the argJ gene by comparing Southern blots of these strains
to the Arg+ strain CD050 (Fig. 1). In blots probed with the internal
0.6kb HincII fragment of pRGE5, the AHU strains possessed a 3.4 kb band
apparently identical to the wild type when digested with Sau3A. Digests
with other enzymes (ClaI, Aval) also revealed no differences in
restriction pattern in the argJ gene between AHU strains and CD050 (data
not shown).

Cloning of the argJ gene from an AHU strain. We cloned the mutant argJ
gene from the AHU N. gonorrhoeae strain NRL30465 by digesting genomic
DNA from this strain with Sau3A, band purifying fragments of
approximately 3.4 kb, and cloning these fragments into the BamHI site of
pUCl9. Recombinant plasmids containing the argJ gene were identified by
probing colony blots with the 0.8 kb ClaI fragment from the wild type
argJ gene previously cloned in this laboratory. A recombinant plasmid
with a 3.4 kb insert was isolated in this way and designated pREMl. The
mutant argJ clone appeared identical to the wild type clone pRGE5 by
restriction mapping, but did not show ArgJ activity either by enzyme
assay or by ability to complement E. coli AT2538. The mutant argJ clone
was also unable to complement the argA E. coli strain W3421, while the

wild type clone could complement this strain.

94

12J2-

(i1-
451‘

313-

2.0-

1 2 3 ‘4 5 E5 7

Figure 1. Southern blot of wild type and AHU strains of N. gonorrhoeae

probed with the argJ gene. Lane 1, CD050; Lane 2, NRL30465; Lane 3,

NRL9396; Lane 4, CD06; Lane 5, CD022; Lane 6, CD054; Lane 7, CD0208.

95

Analysis of chimeric argJ genes. Chimeras of the wild type and mutant
gonococcal argJ genes were constructed and tested for the ability to
complement the argE E. coli strain AT2538 (Fig. 2). When the 5'-end of
the mutant argJ gene upstream of the ClaI site (pRGMlS) or the 3'-end of
the mutant gene downstream of the SacII site (pRGSMS) was substituted
into the wild type clone, argE complementing activity was maintained.
These results suggest that these regions of the mutant gene do not
contain mutations that impair ArgJ activity, and that the argJ lesion is
located between the internal ClaI and SacII sites.

Identification of the argJ mutation. The mutant argJ gene was
subcloned into Ml3mp18 and Ml3mpl9 and the region between the Clal and
SacII sites was sequenced (Fig. 3). When compared to the wild type
sequence, only one difference was found: a 3 bp deletion just downstream
of the ClaI site. This deletion occurred within a region in which the
sequence -GCC- is repeated four times in the wild type gene. This
deletion would leave the remainder of the mutant gene in frame with
respect to the wild type, and would result in the loss of one alanine
from the predicted amino acid sequence.

In order to confirm the importance of this deletion to ArgJ activity,
a spontaneous Arg+ revertant of the AHU gonococcal strain NRL30465 was
isolated by plating approximately 109 cfu of N. gonorrhoeae NRL30465
onto NEDA plates lacking arginine, and checking for the appearance of
colonies after 48 hr. A single Arg+ colony was recovered. This isolate
remained ny- and Ura-. Genomic DNA was extracted from this revertant,
and the argJ gene was cloned into pUCl9 by the same methods used to
clone the mutant argJ gene. The revertant clone allowed growth of E.

coli AT2538 on Arg- NEDA equally as well as the wild type argJ clone.

96

Figure 2. Chimeras of wild type and mutant argJ genes. Ability to
complement the argE E. coli strain AT2538 is indicated. Narrow lines
represent DNA that originated from the wild type argJ clone; boxed lines
represent DNA from the mutant argJ clone. Restriction sites indicated

are; A, AvaI; C, Clal; H, HincII; S, SacII.

97

+

 

=80 09m

9.

HZm<<w._n_<<OU

0.0

md

0.0

m._

0..

m0

0.0

 

 

 

 

 

 

 

c-IHI

 

 

dhn

 

 

 

 

 

—
—
cu—

 

090

N muswfim

—

220%
802%
m2m0%

202%
920%

000mm

98

Figure 3. Comparison of the wild type and mutant argJ sequences.

The predicted amino acid sequence is shown below the DNA sequence. The
DNA sequenced in the mutant argJ gene and shown to be identical to the
wild type sequence is capitalized. The 3 bp deletion of the mutant gene

is boxed. The Hian sites flanking this deletion are indicated.

99

atggctgtcaacctgaccgaaaaaaccgccgaacaactgcccgacatcgacggcattgccctctacaccgcccaa
M A V N L T E K T A E Q L P D I D G I A L Y T A Q

gcaggcgtgaagaagcccgggcataccgacctgacactgattgccgtagccgccggcagcaccgtcggtgcagtc
A C V K K P G H T D L T L I A V A A G S T V C A V

ttcacgaccaaccgtttctgtgccgcgcccgtccacatcgccaaatcgcaccttttcgacgaagacggcgtgcgc
F T T N R F C A A P V H I A K S H L F D E D C V R
. . . . . Hinflzglal . .
gccctcgtcatcaacacgggcaacgccaacgcgggtacgggcgcacagggcagaATCGATGCTTTGGCAGTGTGT
A L V I N T G N A N A G T G A Q G R I D A L A V C
. . . . . . 313:1 .

GCCGC .fi CCCGGCAAATCGGCTGCAAACCGAACCAGGTGATGCCCTTCTCCACCGCCGTGATTCTCGAACCG
A A A R Q I G C K P N Q V M P F S T G V I L E P

CTCCCCCCACACAAAATCATCCCCCCCCTCCCCAAAATGCACCCTCCCTTCTCGAACGAACCCCCACCCCCCATC
L P A D K I I A A L P K M Q P A F W N E A A R A I

ATGACCAOCCACACCGTTCCCAAACCCGCCTCCCCCGAACGCAACCTCGCCGACCAACACACCCTCCCCCCCACA
M T T D T V P K A A S R E C K V C D Q H T V R A T

CCCATTCCCAAACCCTCGGGCATGATTCATCCCAATATCGCCACCATCCTCCCTTTCATCCCCACCCATCCCAAA
G I A K G S G M I H P N M A T M L G F I A T D A K

CTTTCCCAACCCGTCCTCOAACTCATGACCCAGCAAATCCCCCACCAAACCTTCAACACCATCACCCTTCACCGC
V S Q P V L Q L M T Q E I A D E T F N T I T V D G

CACACCACCACCAACCACACCTTCCTCATCATCCCCACCCCCAAAAACACCOAAACCCAAATCCACAACATCCCC
D T S T N D S F V I I A T G K N S Q S E I D N I A

CAOCCCCCTTACCCCCAACTCAAACAATTGTTGTCCAGCCTTGCGCTCCAACTCCCCCAACCCATCGTCCCCCAC
D P R Y A Q L K E L L C S L A L E L A Q A I V R D

CGCGAGGGCGCCACCAAGTTCATCACCGTCCGCCTCGAAAACGCCAAAACCTGCGACGAAGCCCGCCAACCCGCC
C E C A T K F I T V R V ,E N A K T _0 D E A R Q A A
TACGCCGCGGcacgttcgccactggtcaaaaccgcctttttcgcctccgaccccaacctcggcaagcgtctcgcc
Y A A A R S P L V K T A F F A S D P N L C K R L A

gccatcggttatgccgacgttgccgacctcgataccgacctcgtggaaatgtatctcgacgatattttggttgcc
A I G Y A D V A D L D T D L V E M Y L D D I L V A

gaacacggcggacgcgccgcaagetacaccgaagcacaagggcaggcggtgatgtcgaaggacgaaatcaccgtc
E H C C R A A S Y T E A Q C Q A V M S K D E I T V

cgcatcaagctgcatcgcggacaagccgccgccaccgtctatacctgcgacctgtcgcacggatacgtttccatc
R I K L H R C Q A A A T V Y T C D L S H C Y V S I

aacgccgactaccgttcctga
N A D Y R S ***

75

150

225

300

375

450

525

600

675

750

825

900

975
1050
1125
1200

1221

100
The revertant gene was subcloned into M13mpl9 and sequenced over the
region containing the 3 bp deletion (Fig. 4). The wild type sequence was
restored in the revertant, providing a strong correlation between ArgJ+
phenotype and absence of a 3 bp deletion in this part of the gonococcal
argJ gene.

Prevalence of 3 bp deletion among AHU strains. Previous studies
showing that AHU strains could not be transformed to Arg+ by genomic DNA
from other AHU strains suggested that the argJ mutation in these strains
was identical or overlapping (19). To test this hypothesis, a variety of
AHU strains, ArgJ- non-AHU strains, and Arg+ strains were screened for
the presence or absence of the 3 bp deletion described above. The
deletion occurs within a Hian restriction fragment which is 85 bp in
the wild type gene, but only 82 bp in the mutant (Fig. 3). This
difference in restriction fragment length was used to screen gonococcal
strains for the deletion. Hian digests of genomic DNA were separated on
an 8% polyacrylamide sequencing gel and fragments in the range of
approximately 75-100 nt were electrotransferred to Nytran. This blot was
probed with the 32P-dCTP labelled 0.6 kb HincII fragment of the wild
type argJ gene, and hybridizing bands were visualized by autoradiography
(Fig. 5). All AHU strains tested possessed a Hian fragment of 82 nt,
while all wild type strains had an 85 nt band. The ArgJ- non-AHU strains
tested had either an 85 nt fragment, or no Hinfl fragment in the 75-100

nt range, indicating that the argJ lesion of these strains was not

identical to that in AHU strains.

101

 

care «are cars 3

Figure 4. Presence of deletion in wild type, mutant, and revertant argJ

genes. The 3 bp sequence -GCC- is repeated 4 times in the wild type

(WT) and revertant (Arg+ HU) genes, but is present only 3 times in the

argJ mutant (AHU). This repeated region is indicated by brackets.

102

 

AHU argJ- argJ+
85-.‘ ’— — fl *
82' .‘T —‘ -""—"
1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 5. Presence of deletion in AHU, ArJ-, and ArgJ+ strains of
Neisseria gonorrhoeae. Hian digested gonococcal genomic DNA was
separated on an 8% sequencing gel and probed with the 32P-labelled 0.6
kb HincII fragment of the wild type argJ gene. Hybridizing bands were
either 85 or 82 nucleotides in length. Lanes 1 and 2 contain DNA from
the wild type strain (CD050) and AHU strain (NRL30465) in which the
sizes of the Hian fragments were predetermined by DNA sequencing. DNA

from AHU strains, ArgJ- strains, and ArgJ+ strains are indicated. Lane

numbers correspond to strain numbers given in Table l.

103

DISCUSSION

The development of auxotyping media for Neisseria in the early 1970's
allowed for the discovery of the unusually invariant set of strains with
multiple requirements for arginine, hypoxanthine and uracil.
Retrospective studies of N. gonorrhoeae clinical isolates revealed that
AHU strains did not appear until after the introduction of penicillin
therapy in the mid 1940's (8,12). Since that time AHU strains have been
spread widely and have become the predominant auxotype recovered in some
locations (12,14). Though defined by auxotype, these strains also share
other characteristics including PI serotype, serum resistance, and
penicillin sensitivity, traits that are positively correlated to
occurrence of disseminated gonococcal infection (3,10).

Genetic analysis of AHU strains revealed that the mutation responsible
for the Arg- phenotype of these strains was identical (19), while
biochemical analysis found that this mutation affected the argJ gene
(7). In this study we have defined the argJ mutation in AHU strains at
the molecular level.

The cloning and sequencing of the wild type argJ gene have been
previously reported (18,23). We used this cloned gene to probe Southern
blots of genomic DNA from AHU strains and found that AHU strains possess
an apparently intact argJ gene with a restriction pattern comparable to
the wild type gene. This information was used to clone the mutant argJ
gene from the AHU strain NRL30465 on an identical restriction fragment
as our wild type clone. The mutant clone appeared identical to the wild

type by restriction mapping, but had no detectable ArgJ activity. The

104
ability to complement an argA mutation in E. coli was also lost in the
mutant argJ clone. This supports our previous study in which argA
complementation was found to be dependent on ArgJ activity and was not a
separate function on our wild type argJ clone (18).

The lesion in the mutant argJ gene was identified by first narrowing
down the location of the lesion by constructing chimeras of the mutant
and wild type genes. This localized the lesion to a 630 bp region
between the internal ClaI and SacII sites. This region was completely
sequenced and revealed only one difference between the mutant and wild
type genes: a 3 bp deletion in the mutant gene. This deletion occurred
within a region of the argJ gene in which the 3 bp sequence GCC is
repeated several times.

The importance of this 3 bp deletion was analyzed by examining this
region in a naturally occurring Arg+ revertant of NRL30465. It would be
expected that the restoration of even a small deletion to the wild type
sequence would be an excedingly rare event. Previous studies have shown
that other AHU strains revert to Arg+ at the rate of approximately 3 x
10.9 per cell per generation (19). We were also able to obtain an Arg+
revertant of NRL30465, and the argJ gene was cloned from this revertant.
The revertant argJ clone showed ArgJ activity equivalent to the wild
type, and when sequenced revealed that the 3 bp deletion of the mutant
gene had reverted to the wild type sequence. It seems unlikely that this
type of reversion event would have occurred if the 3 bp deletion had not
been a part of tandem direct repeats of this deleted sequence. The
repeated nature of this sequence may allow for an occasional slippage of
the DNA polymerase during replication, resulting in an insertion or

deletion of the 3 bp repeat. A similar mechanism has been proposed to

105
explain the phase variation of expresion of the gonococcal opacity
proteins (28,22). In that instance there is a 5 bp coding repeat in the
5' end of the gene encoding the leader peptide. The number of 5 bp
tandem repeats in this region determines whether the rest of the gene
will be in or out of frame, thus controlling expression at the
translational level. The insertion or deletion of the coding repeat of
the opacity genes occurs much more frequently than that of the mutant
argJ gene. This is most likely due to the higher copy number of the
repeats in the opacity genes (7-28 copies in the different opa genes),
though differences in the length and base composition of the repeat may
also influence the probability of slipped-strand mispairing events.

The 3 bp deletion in the mutant argJ gene would not affect the reading
frame of the gene downstream of the deletion, but it would result in
the loss of one alanine residue from the predicted amino acid sequence.
The assumption that a minor change in the coding region of the gene is
responsible for the lack of ArgJ activity was supported by several
pieces of data. First, when the products of the mutant argJ gene were
labelled with 35S-methionine in E. coli minicells and compared to the
wild type clone, the mutant clone produced an apparently identical 46
kDa protein (data not shown). This implies that the promotor was still
functional, and the reading frame was not substantially disrupted in the
mutant clone. Second, the construction of chimeras of the mutant and
wild type argJ genes revealed that both the promotor end and the 3'-end
of the mutant gene could be substituted into the wild type clone without
impairing ArgJ activity (Fig. 2). Chimeras which contained the central
portion of the mutant gene between the internal ClaI and SacII sites

remained ArgJ-. This, along with sequencing data of the mutant and

106
revertant argJ genes, support the conclusion that the 3 bp deletion is
responsible for the Arg- phenotype of the AHU strain NRL30465.

The 3 bp deletion would result in the net loss of one alanine residue
from the predicted amino acid sequence in the mutant protein. The amino
acid sequence was analyzed using the Plotstructure program of the
sequence analysis package from the University of Wisconsin Genetics
Study Group (9). The deletion is predicted to occur within an alpha
helical region of the protein which is followed by a turn. This region
is flanked by cysteine residues (Fig. 3), making it possible that the
deletion affects disulfide bond formation.

The fact that the mutant argJ gene contained a small deletion when
compared to the wild type gene should allow the detection of this
mutation by differences in restriction digest patterns. We used this
approach to screen genomic DNA from a variety of AHU and non-AHU strains
for the presence of this deletion. To detect a 3 bp deletion a
restriction enzyme was chosen that closely flanked the site of the
deletion (Hian, Fig. 3), and digests were separated on gels capable of
distinguishing the 85 and 82 nt bands produced. When these digests were
blotted and probed with a fragment of the argJ gene, all AHU strains
tested were found to possess a 3 bp deletion in this area (82 nt band)
while all ArgJ+ strains tested were intact (85 nt band, Fig. 5). The
ArgJ- strains that were not ny- or Ura- either had an 85 nt band or no
detectable band in the size range of 75 - 100 nt. These strains probably
possess lesions in the argJ gene different from that of AHU strains and
not identifiable by this methodology.

This experiment revealed that the 3 bp deletion truly exists in the

gonococcal chromosome of strain NRL30465 and was not simply an artifact of

107
cloning. An identical or very similar deletion was also found in a
variety of other AHU strains from different sources and of differing
serovars. Though we did not screen all the AHU strains in our collection
in this way, our sampling does support the theory that AHU strains arose
from a single clone. This also provided further evidence that the 3 bp

deletion is real and important to the Arg- phenotype of AHU strains.

ACKNOWLEDGMENTS

We thank Christina Bolger for her enthusiastic technical assistance.
This research was supported by Public Health Service grant Al-21264 from

the National Institute of Allergy and Infectious Disease.

10.

ll.

12.

108

REFERENCES

. Atkinson, B.A., and M.H. Mulks. 1988. Linkage of genetic markers in

AHU strains of Neisseria gonorrhoeae, p. 223-230. In J.T. Poolman er
al. (ed.), Gonococci and Meningococci. Academic Publishers,
Dordrecht.

. Brunham, R.C., F. Plummet, L. Slaney, F. Rand, and W. DeWitt. 1985.

Correlation of auxotype and protein I type with expression of
disease due to Neisseria gonorrhoeae. J. Infect. Dis. 152:339-343.

. Cannon, J.G., T.M. Buchanan, and P.P. Sparling. 1983. Confirmation

of association of protein I serotype of Neisseria gonorrhoeae with
ability to cause disseminated infection. Infect. Immun. 40:816-819.

. Carifo, K., and B.W. Catlin. 1973. Neisseria gonorrhoeae auxotyping:

differentiation of clinical isolates based on growth responses on
chemically defined media. Appl. Microbiol. 26:223-230.

. Catlin, B.W. 1973. Nutritional profiles of Neisseria gonorrhoeae,

Neisseria meningitidis, and Neisseria lactamica in chemically
defined media and the use of growth requirements for gonococcal
typing. J. Infect. Dis. 128:178-194

. Catlin, B.W. 1974. Genetic transformation of biosynthetically

defective Neisseria gonorrhoeae clinical isolates. J. Bacteriol.
120:203-209.

. Catlin, B.W., and E.H. Nash. 1978. Arginine biosynthesis in

gonococci isolated from patients, p. 1-8. In G.F. Brooks et al.
(ed.), Immunobiology of Neisseria gonorrhoeae. American Society for
Microbiology, Washington, D.C.

. Catlin, B.W., and A. Reyn. 1982. Neisseria gonorrhoeae isolated from

disseminated and localised infections in pre-penicillin era. Br. J.
Vener. Dis. 58:158-165.

. Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive

set of sequence analysis programs for the VAX. Nucleic Acids Res.
12:387-395.

Eisenstein, B.I., T.J. Lee, and P.P. Sparling. 1977. Penicillin and
serum resistance are independent attributes of strains of Neisseria

gonorrhoeae causing disseminated gonococcal infection. Infect.
Immun. 15:834-841.

Knapp, J.S., and K.K. Holmes. 1975. Correlation of auxotype and
disseminated infection among clinical isolates of Neisseria
gonorrhoeae. J. Infect. Dis. 132:204-208.

Knapp, J.S., M. Mulks, I. Lind, H.B. Short, and V.L. Clark. 1985.
Evolution of gonococcal populations in Copenhagen, Denmark, 1928-
1979, p. 82-88. In G.K. Schoolnik, G.F. Brooks, 3. Falkow, et al.
(ed.), The pathogenic neisseria. American Society for Microbiology,

l3.

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Washington, D.C.

Knapp, J.S., M.R. Tam, R.C. Nowinski, K.K. Holmes, and E.G.
Sandstrom. 1984. Serological classification of Neisseria gonorrhoeae
with use of monoclonal antibodies to gonococcal outer membrane
protein I. J. Infect. Dis. 150:44-48.

Knapp. J.S., C. Thornsberry, G.A. Schoolnik, P.J. Wiesner, K.K.
Holmes, and the Cooperative Study Group. 1978. Phenotypic and
epidemiologic correlates of auxotype in Neisseria gonorrhoeae. J.
Infect. Dis. 150:44-48.

Kohl, P.K., J.S. Knapp, H. Hofmann, K. Gruender, D. Petzoldt, M.R.
Tams, and K.K. Holmes. 1986. Epidemiological analysis of Neisseria
gonorrhoeae in the Federal Republic of Germany by auxotyping and
serological classification using monoclonal antibodies. Genitourin.
Med. 62:145-150.

Kolodkin, A.B., V.L. Clark, F.C. Tenover, and F.E. Young. 1982. High
correlation of the presence of methyladenine in Neisseria
gonorrhoeae DNA with the AHU auxotype. Infect. Immun. 36:586-590.

Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982. Molecular
Cloning: a laboratory manual. Cold Springs Harbor Laboratory, Cold
Spring Harbor, New York.

Martin, P.R., and M.H. Mulks. 1991. Sequence analysis and
complementation studies of the argJ gene of Neisseria gonorrhoeae.
Manuscript submitted.

Mayer, L.W., G.K. Schoolnik, and S. Falkow. 1977. Genetic studies on
Neisseria gonorrhoeae from disseminated gonococcal infections.
Infect. Immun. 18:165-172.

Merello, J.A., S.A. Lerner, and M. Bohnhoff. 1976. Characteristics
of atypical Neisseria gonorrhoeae from disseminated and localized
infections. Infect. Immun. 13:1510-1516.

Mulks, M.H., and J.S. Knapp. 1987. Immunoglobulin AI protease types
of Neisseria gonorrhoeae and their relationship to auxotype and
serovar. Infect. Immun. 55:931-936.

Murphy, G.L., T.D. Connell, D.S. Barritt, M. Koomey, and J.G.
Cannon. 1989. Phase variation of gonococcal protein II: regulation
of gene expression by slipped-strand mispairing of a repetitive DNA
sequence. Cell 56:539-547.

Picard, P.J., and J.R. Dillon. 1989. Cloning and organization of
seven arginine biosythetic genes from Neisseria gonorrhoeae. J.
Bacteriol. 171:1644-1651.

Sanger, F., S. Nicklen, and A.R. Coulson. 1977. DNA sequence with
chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-
5467.

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110

Schoolnik, G.K., T.M. Buchanan, and K.K. Holmes. 1976. Gonococci
causing disseminated gonococcal infection are resistant to the
bactericidal action of normal human serum. J. Clin. Invest. 58:1163-
1173.

Shinners, E.N., and B.W. Catlin. 1978. Arginine biosynthesis in
Neisseria gonorrhoeae: enzymes catalyzing the formation of ornithine
and citrulline. J. Bacteriol. 136:131-135.

Southern, R.M. 1975. Selection of specific sequences among DNA
fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517.

Stern, A., M. Brown, P. Nickel, and T.P. Meyer. 1986. Opacity genes
in Neisseria gonorrhoeae: control of phase and antigenic variation.
Cell 47:61-71.

White, L.A., and D.S. Kellogg. Jr. 1965. Neisseria gonorrhoeae
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counterstain method. Appl. Microbiol. 13:171-174.

lll
SUMMARY AND CONCLUSIONS

In N. gonorrhoeae, the arginine biosynthetic pathway has been of
interest primarily due to the prevalence of naturally occuring arginine
auxotrophs. Initial epidemiological studies found that arginine
auxotrophs were common and occurred singly or in combination with other
requirements. These studies uncovered an unusual set of strains with
multiple requirements for arginine, hypoxanthine, and uracil (AHU
strains). These strains shared a variety of unusual phenotypes and were
associated with asymptomatic and disseminated gonococcal infections.

These epidemiological studies spurred further investigations into
arginine biosynthesis in N. gonorrhoeae. It was found that defects in
arginine biosynthesis were not scattered throughout the eight step
pathway, but occurred predominantly at steps five or six, the conversion
of acetylornithine to ornithine and the combination of ornithine with
carbamoylphosphate to form citrulline respectively. AHU strains all had
defects in the fifth step of the pathway (ArgJ mutants). These defects
were presumed to be identical due to their inability to recombine.
Strains with defects at step six (citrulline auxotrophs) were shown to
have defects in carbamoylphosphate synthetase (encoded by carA and carB),
and not ornithine carbamoyltransferase (encoded by argF). A naturally
occuring argF mutant has not yet been identified.

We have further examined arginine auxotrophy in N. gonorrhoeae by
cloning and sequencing the argJ and argF genes, and identifying the argJ
lesion common to AHU strains. The gonococcal argF gene was quite similar

to the argF genes previously sequenced from E. coli and P. aeruginosa

112
with regions involved in ornithine and carbamoylphosphate binding being
particularly well conserved. This gene was unusual in that it used a GUG
initiation codon, which had been not previously reported in gonococcal
genes. An internal deletion mutant of the cloned argF gene has been
constructed, and attempts were made to introduce this mutation back into
the gonococcus, but they have not yet been successful. This of course
does not prove that argF mutations are lethal to the gonococcus, but
this possibility will remain until an argF mutant can be isolated.

The gonococcal argJ gene was the first ornithine acetyltransferase
gene to be sequenced from any source. We were able to compare our
sequence to a preliminary sequence of a putative argJ gene from Bacillus
subtilis, and distinct regions of high homology were found. As this
enzyme has not yet been purified or characterized we cannot report if
these conserved regions contain residues important to substrate binding
or catalysis, though these sequences will be of predictive value for
future biochemical investigations of this enzyme.

Perhaps the most interesting outcome of our molecular investigations
of arginine auxotrophy in N. gonorrhoeae was the elucidation of the
lesion in the argJ gene of AHU strains. These strains had previously
been proposed to be clonally derived, and our study strongly supports
this theory. All AHU strains tested in our study contained a 3 bp
deletion within a repetitive region of the argJ gene. This deletion was
not found in Arg+ strains or in ArgJ- strains that were not AHUs. The
most likely explanation for these findings is that the AHU strains
tested inherited the deletion from a common ancestor, and that the other
ArgJ- strains were independently derived.

The fact that this deletion occurred within tandem direct repeats of

113

the deleted sequence gives a clue to how this mutation may have
originated. This direct repeat arrangement can result in slipped-strand
mispairing during DNA synthesis, leading to the insertion or deletion of
the repeated unit. This model was supported by finding that a naturally
occurring Arg+ revertant of an AHU strain had regained this deleted
sequence. This same model has been used to explain the phase variation
of expression of gonococcal P.II genes.

The 3 bp deletion in the gonococcal argJ gene would result in the
net loss of one alanine residue from the predicted amino acid sequence.
This implies that this alanine residue may be directly involved in the
catalytic properties of the enzyme; or the loss of one amino acid in this
region may alter the folding of the molecule and thus impair its
enzymatic activity. This data should be quite useful to future studies

on the characterization of the ornithine acetyltransferase enzyme.

APPENDIX

114

Gene. 94 (N90) [JV—HO
Elsevicr

GENE 03705

l39

Sequence of the argf gene encoding ornithine transcarbamoylase from Neisseria gonorrhoeae

(Arginine biosynthesis: DNA uptake sequence: start codon: sequence homology: recombinant DNA)

Paul R. Martin. John W. Cooperider and Martha H. Vlulks

Department of Microbiologv and Public Health. Michigan State L’nt't-em'ty. East Lansing. MI 48824 (U. S .A.)

Received by CR. Hutchinson: I May 1990
Accepted: ll June I990

 

SUMMARY

The gonococcal argF gene encoding ornithine transcarbamoylase (OTCase) contains an open reading frame of 993
nucleOtides which starts with a GUG codon and encodes a peptide with a deduced M, of 36 73 1. We compared the predicted
amino acid (aa) sequence to OTCase sequences previously determined for Escherichia coli and Pseudomonas aentgitma and
found that highly conserved regions in the genes from these organisms were also conserved in Neisseria gommhoeae. including

those as known to be important for carbamoyl phosphate and ornithine binding. In the flanking regions of the gene were . .

found l5-bp inverted repeats that may serve as transcriptional termination signals. and which contain the neisserial

DNA-uptake sequence.

 

Arginine biosynthesis from glutamate occurs by an eight-
step pathway (Cunin et al.. 1986). The sixth step of the
pathway involves the conversion of ornithine and carba-
moyl phosphate to citrulline and is catalyzed by the enzyme
OTCase. Arginine auxotrophs are commonly encountered
in N. gottorrhoeue. comprising 40-60", of clinical isolates
(Catlin and Nash. 1973: Picard and Dillon. I989). While
30". of these auxotrophs require citrulline for growth. the
lesion in these strains is in carbamoyl phosphate synthetase.
not OTCase (Catlin and Nash. I978). No strains of
.V. gonorrhoeae deficient in OTCase production have been
identified. .

The cloning of the argF gene from N. gonorrhoeae has
been previously reported (Picard and Dillon. 1989). We
independently cloned this gene in the plasmid pUClZ by
complementation in E. coli. confirmed that the clone

 

Correspondence to: Dr. M.H. Mulks. Department of Microbiology and
Public Health. Michigan State University. East Lansing. MI 48824
(U.S.A.) Tel. (5|?)355-65l5: Fax (5 ”USE-3957.

Abbreviations: as. amino acid(s): bp. base pains): kb. ktlobasds) or
low bp; nt. nucleotide-(s); ORF. open reading frame; OTCase. orntthine
transcarhantoy lase.

M's-I l I‘D 90 SM 50 O I‘M!) Elscucr Science Puhhshcrs B \' tBtomcdtcal Dmuom

produced active OTCase using the procedure of Catlin and
Nash (1978). and sequenced the gene (both strands) in
Mlepl9 (Fig. l). The gonococcal argF gene contains a
993-m ORF initiating at nt 141 with a GUG codon. and
coding for a 36 731-Da peptide. This was supported by

. minicell analysis and direct N-terminal as sequence de-

termination showing that the GUG start codon was being
utilized. The codon usage data for the argF gene are in good
agreement with the data for previously sequenced gono-
coccal genes (West and Clark. 1989).

Comparison of the predicted at and aa sequences of the
gonococcal OTCase revealed that this gene is more closely
related to the E. coli OTCases encoded by mg!“ and argI
(64.0 and 62.5". DNA and 64.0 and 61.6'. an identity.
respectively) (Bencini et al.. 1983; Van Vliet et aL. 1984)
and the P. aeruginosa catabolic OTCase (uch. 66.6“;
DNA and 68°. an identity) (Baur et al.. 1987) than to the
P. aemgirwsa anabolic OTCase (argF. 50.l'. DNA and
375", aa identity) (ltoh etal.. 1983). The gonococcal
OTCase also had significant DNA (HA-43.5“.) and aa
(33.5-40.5",,) identity with eukaryotic OTCases from
fungi. rodents. and humans (Huygen ct al.. I987). Those an
involved in interactions with carbamoyl phosphate

115

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Fig. I. Thentsequeneeofthegonoooecalargfgeneandflankingregions.
ThepredictedaasequenoeisshownbelowthecodingregionN-terminal
aathatweresequericedareinitaliealnvertedrepeatsareunderlinedas
is the putative ribosome-binding site (RBS). Neisserial DNA uptake
sequences are double-underlined. The a identical in flood argI.
P.aenigrhos¢are3.andfl.gonarhoeaeargfareunderlined.1'heaa
implicated in wt..-,! r" r‘ ‘ binding or catalysis and ornithine
bindingaredouble—rmderlinedThesequeneedatahavebeensubrnitted
toGenBankandassignedthemessionnunsberMJlflo.

 

(Houghton et al.. 1984) and ornithine (Marshall and
Cohen. I980) were all conserved in the gonococcal OTCase
(Fig. I).

Gonococcal cells are naturally competent for DNA
transformation during all stages of growth. though only
DNA of neisserial origin will bind to the cells (Daugherty
et al.. I979). This selectivity is due to the higher occurrence
of the IO-bp sequence. S'oGCCGTCTGAA-3'. in neis-
serial DNA than in DNA from other sources (Goodman
and Scocca. 1988). This DNA-uptake sequence. with no
more than one mismatch. is present five times in the argF
sequence: twice as part of IS-bp inverted repeats in the
flanking regions of the gene. and once within the coding
region. The presence of the uptake sequence within these
flanking inverted repeats supports the hypothesis of
Goodman and Scocca (I983) that the uptake sequence may

be maintained in high frequency in Neisseria in part by being
incorporated in transcription termination sequences.

ACKNOWLEDGEMENTS

This research was supported by Public Health Service
grant AI-2126-l from the National Institute of Allergy and
Infectious Diseases.

REFERENCES

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quaternary structure of the catabolic ornithine carbamoylotransferase
born Pseudomonas aerugt'nosa. Eur. J. Biochem. I66 (I987) III-I I7.

Icncini. D.A.. Houghton. LE. Hoover. T.A.. Folterrnann. K.K. Wild.
J.R. and O'Donovan. G.A.: The DNA sequence ofotgl from E. ecu
K-IZ. Nucleic Acids Res. II (I983) 8509-85l8.

Catlin. B.W. and Nash. EH.: Arginine biosynthesis in gonococci isolated
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Houghton. I .E.. Bencini. D.A.. O’Donovan. G.A. and Wild. J.R.: Pretein
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