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2008

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PROGRESS TOWARD DEVELOPMENT OF AN IN VI VO
SYSTEM TO ASSAY SIGNAL RECOGNITION PARTICLE
FUNCTION IN NEISSERIA GONORRHOEAE

presented by

 

KEVIN DAVID BRUCE SMITH

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6/07 p:lCIRC/DateDue.indd-p.1

PROGRESS TOWARD DEVELOPMENT OF AN IN VIVO SYSTEM TO ASSAY
SIGNAL RECOGNITION PARTICLE FUNCTION IN NEISSERIA GONORRHOEAE

By

Kevin David Bruce Smith

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Microbiology and Molecular Genetics

2008

ABSTRACT

PROGRESS TOWARD DEVELOPMENT OF AN IN V1 V0 SYSTEM TO ASSAY
SIGNAL RECOGNITION PARTICLE FUNCTION IN NEISSERIA GONORRHOEAE

By
Kevin David Bruce Smith
Assembly of inner membrane proteins (IMPs) presents a unique challenge for bacteria.
The presence of multiple, hydrophobic transmembrane domains renders such proteins
highly susceptible to formation of insoluble inclusions within the cytoplasm.
Cotranslational export via the signal recognition particle (SRP) pathway evades this
difficulty by delivering the peptide to the membrane as it is translated. Previous work has
demonstrated a novel feature of the SRP pathway in Neisseria gonorrhoeae, specifically
the sequence-specific binding of DNA by the IM-associated SRP receptor, PilA, which
stimulates the GTPase activity necessary for its activity in SRP-mediated protein export.
This work describes attempts to develop an in vivo protein targeting assay system to

elucidate a role for DNA in SRP-mediated protein transport.

ACKNOWLEDGEMENTS

Heartfelt gratitude goes out to the many people who have aided me over the years.
First and foremost to my parents, without whose love and support I would not be the
person I am today. To my wife, Shendra, for brightening my days. To Alexia
Karanikas, Jim Tumbrink, Daniel Wood, and Kevin Nuttall for their invaluable
assistance. Many thanks are due to the ALAZZ, for preserving my sanity, as well as
to my faithful dog, Slagathor, for her mindless devotion. Finally, I would like to thank
my advisor, Dr. Cindy Grove Arvidson, for the countless hours she has dedicated to

guiding and instructing me over the past few years.

iii

TABLE OF CONTENTS

LIST OF FIGURES ........................................................................................................ v
INTRODUCTION
Neisseria gonorrhoeae ............................................................................................ 1
Gonococcal Antibiotic Resistance ........................................................................... 2
Multidrug Efflux Pumps .......................................................................................... 3
Protein Secretion by Gram-Negative Bacteria ........................................................ 5
The General Secretory Pathway .............................................................................. 5
The Twin Arginine Transport Pathway ................................................................... 7
The Signal Recognition Particle Pathway ............................................................... 8
Overview of SRP composition ................................................................................ 9
Signal Peptide Recognition .................................................................................... 15
Binding of the Signal Peptide ................................................................................ 17
Translation Arrest ................................................................................................... 19
Transfer of the Polypeptide to the Translocon ....................................................... 22
Interaction of F tsY with the IM ............................................................................. 22
Interaction between FtsY and Ffl'l .......................................................................... 24
Overview of SRP Regulation ................................................................................. 26
Effects of Bacterial SRP Deficiency ...................................................................... 26
The Gonococcal PilA Protein ................................................................................. 27
CHAPTER 1 - Construction and Characterization of the Conditional pilA Strain
Inducible Expression of pilA in Strain MP5 ........................................................... 30
Effects of PilA Repression on strain MP5 .............................................................. 33
CHAPTER 2 — Construction and Characterization of the mtrD-phoA Fusion Strain
Construction of Strain PT2 ..................................................................................... 40
Effects of PilA Expression of mtrD-phoA Expression ........................................... 40
Effects of Altered Expression of PilA upon MtrD-PhoA Fusion Localization ...... 42
Assessment of AP as a Measure of Protein Localization ....................................... 42
CONCLUSIONS AND PERSPECTIVES ................................................................... 49
WORKS CITED ........................................................................................................... 55

iv

LIST OF FIGURES

Figure 1. SRP Targeting of IM Proteins ..................................................................... 14
Figure 2. GTP Hydrolysis by Ffl‘l and FtsY ................................................................ 25
Figure 3. Construction of the Conditional pilA Strain MP5 ........................................ 31
Figure 4. Induction of PilA in Strain MP5 by IPTG ................................................... 32
Figure 5. Effects of IPTG on pilA transcription in N. gonorrhoeae MP5 ................... 34
Figure 6. Growth of M81] and MP5 in Presence and Absence of IPTG ................... 35
Figure 7. Effects of Limiting PilA on sensitivity to Mtr Substrates ........................... 37
Figure 8. Western blot analysis of pilin and PilA in cytoplasmic fractions of MP5
grown in PilA-depleting and overproducing cconditions .......................... 39
Figure 9. AP Activity of Gonococcal Strains .............................................................. 41
Figure 10. Effect of pilA Induction on Transcription of an mtrD-phoA Fusion .......... 43
Figure 11. Localization of MtrD-PhoA Fusion Under Varied SRP Conditions ......... 44
Figure 12. Effect of SRP Levels on AP Activity ........................................................ 45

Figure 13. Growth Defects as a Result of SRP Deficiency in E. coli N4156::Pa,a-
ftsY. ............................................................................................................. 47

Figure 14. Effect of ftsY Induction on MtrD-PhoA (AP) Activity in N4156zsz-
ftsY ............................................................................................................... 48

Introduction

Neisseria gonorrhoeae. Neisseria gonorrhoeae, (gonococcus, or GC) is a Gram-
negative, obligate human pathogen and the etiologic agent of the sexually transmitted
infection (STI) Gonorrhea (52). It is the second most reported bacterial STI in the United
States, with a yearly incidence of 115.6 cases per 100,000 people (3). Neisseria
gonorrhoeae typically colonizes the urogenital epithelium, and is also capable of
infecting the conjunctiva, which can be serious for infants born to infected mothers.
Gonococcal infection is frequently asymptomatic, especially in women, and can result in
severe complications, including ectopic pregnancy, infertility, and pelvic inflammatory
disease (121). Epidemiological studies have demonstrated a correlation between HIV
acquisition and gonococcal infection (152). It is thought that lesions in the urogenital
tract caused by ulcerative STIs contribute to crossover of virus particles from the
bloodstream to the urogenital mucosa, increasing the risk of transmission (85). Urethritis
has been demonstrated to significantly increase the level of HIV in semen, giving
credence to this hypothesis (26).

Several factors contribute to the gonococcal virulence, including the gonococcal
type IV pilus (97, 99). The pilus mediates initial attachment to host epithelial cells, and
has been demonstrated to be essential for gonococcal virulence (76-78). Attachment of
the pilus to the epithelial cell has been demonstrate to induce signal cascades in both the
host cell and the bacterium (67). In the host cell, pilus binding to host cell surface protein
CD46, a widely distributed complement regulatory factor, results in an increase of

cytosolic [Ca2+] (73). Phosphatidyl inositol-3 kinase activation within the host cell has

also been demonstrated to stimulate the activity of the gonococcal PilT protein,
responsible for extension and retraction of the type IV pilus, resulting in the formation of
GC microcolonies on the surface of the host cell (88).

The genes encoding the pilus are specially arranged to allow for recombinatorial
variation of the major pilin subunit (28, 79, 134). Pilin is transcribed from the pilE, or
expression, locus. Elsewhere on the gonococcal chromosome are 19 silent loci (pilS),
which consist of sequences DNA encoding different pilin forms but lacking
transcriptional and translational machinery. PilS loci include homologous sequences
allowing for recombination into the expression locus (99). Recombination between the
limited regions of homology among the expressed and silent loci result in a high-
frequency of antigenic variation of the pilus, necessitating a fresh round of processing
and recognition by the host immune system (97). Although pili are essential for
attachment to and invasion of epithelial cells, it has been shown that the pilE loci undergo
modification during transcytosis, such that gonococci in the subepithelial space do not
express fully-formed pili (67).

Gonococcal Antibiotic Resistance. Of particular interest in recent years has been the
trend of increasing resistance of the gonococcus to the antibiotics with which it is treated
(72). The Centers for Disease Control and Prevention (CDC) maintain a multi-site
surveillance program to monitor levels of antibiotic resistance among clinical GC isolates
in the US [GISP, (113)]. One reason for the current level of surveillance is what appears
to be an increase in the speed with which the organism acquires resistance to frontline
treatment methods. Treatment of gonorrhea with tetracycline was largely abandoned in

1985, due to widespread resistance. This was followed in 1987 by the recommendation

that penicillin no longer be used. Parts of Asia are now reporting that more than 60% of
isolates are resistant to quinolones. F luoroquinolone resistance has been rising sharply in
Hawaii and the Western US in recent years, resulting in the abandonment of these drugs
as the primary treatment for gonorrhea in Hawaii (2, 4). In 2007, the CDC recommended
that ciprofloxacin no longer be used as a frontline treatment for gonorrhea in the US (6).
The current primary drugs for treatment of gonorrhea are third generation cephalosporins
- cettriaxone or cefixime. However, resistance to azithromycin and cefixime are
currently on the rise. For these reasons, significant efforts are being made to understand
the molecular mechanisms underlying gonococcal resistance to antibiotics, and how the
organism acquires them.

Multidrug Efflux Pumps. There are several mechanisms by which microorganisms
resist the action of antibiotics including: modification of the site of action, modification
of the antibiotic, and efflux of the antibiotic from the cell. Gram-negative bacteria, such
as GC, tend to be resistant to lipophilic and amphiphilic agents, allowing them to survive
in environments toxic to other organisms. This activity is generally attributed to the
action of multidrug efflux pumps (110). The gonococcus has several such pumps,
mediating resistance to a broad range of antimicrobial agents.

The mtr efflux pump is known for its ability to remove structurally diverse,
hydrophobic agents (HA’s) from the cell. It has been demonstrated to mediate resistance
to several antibiotics, such as penicillin, bile salts, ethidium bromide, crystal violet, and
antimicrobial peptides (145). First identified based on homology to the AcrAB and
MexAB pumps of Escherichia coli and Pseudomonas aeruginosa, respectively, the

structural genes of the mtr pump, mtrC, mtrD, and mtrE, are transcribed as a single

operon. (5 7). Immediately upstream of the structural operon and transcribed divergently
is gene for the mtr repressor, MtrR. Inactivation of mtrR results in overexpression‘of the
mtr pump components, resulting in an increase in HA resistance (113). Inactivation of
mtrC and mtrD result in hypersusceptibility to the action of HAS (56, 113, 144). The mtr
pump has been demonstrated to play a key role in survival of GC in the presence of toxic
fecal lipids both in vitro and in an murine model of infection (70, 113).

MtrD, the inner membrane (1M) component of the pump, shows similarity to the
Resistance/Nodulation/Division family of transporters (58). MtrE localizes to the outer
membrane (OM), where it forms the efflux pore (32). MtrC is a membrane fusion protein
responsible for periplasmic linkage of MtrD and MtrE (57). The mtr repressor, MtrR,
binds to a DNA sequence in the region between mtrR and mtrCDE (90). The promoters
for mtrR and mtrC are both divergent and overlapping, allowing for coregulation of the
repressor and structural genes. Recently, it has been demonstrated that the mtr system
can be induced in the presence of sub-lethal concentrations of HA’s (124). The regulator
responsible has been identified and named MtrA.

Mtr has also been implicated in efflux via other pump systems. The F arAB pump,
responsible for efflux of antimicrobial fatty acids, requires MtrE to mediate its interaction
with the OM (86). Another gonococcal efflux pump, MacAB, has been shown to be
coordinately regulated with mtr. GC deficient in macAB demonstrate increased levels of
mtrCDE expression (126). The ability of GC to express and regulate efflux pumps
specific to a broad range of antimicrobial compounds enables it to survive in the mucosa]

environment, and to deal with the various defensive molecules therein.

Protein Secretion by Gram-Negative Bacteria. Newly-synthesized proteins of Gram-
negative bacteria can be localized to any of five compartments: the cytoplasm, IM,
periplasm, OM, or extracellular milieu. Systems of protein transport in these organisms
are characterized by the means with which the nascent polypeptide is translocated across
the two membranes. Some proteins transit both membranes in a single step, such as the
Type I, or ATP-Binding Cassette (ABC) and Type III pathways, while others are
transferred across each membrane separately. Common to all mechanisms is the
necessity for export of the preprotein from the cytoplasm, which is primarily mediated by
one of three export systems: The Twin-Arginine Translocation (TAT) pathway, the
General Secretory Pathway (GSP or See), and the Signal Recognition Particle (SRP)
pathway.
The General Secretory Pathway. The GSP transports preproteins across the IM in an
unfolded state (25, 34, 46, 71). GSP-transported proteins can be further targeted to the
periplasm, outer membrane, or extracellular milieu. Nascent polypeptides are targeted to
the GSP based on recognition of signal peptides by the peptidyl-prolyl cis-trans
isomerase known as Trigger Factor (TF), which binds with high affinity to N-terminal
signal peptides as they exit the translating ribosome (80). Binding of TF to a signal
peptide prevents binding of the SRP recognition complex, thus the two pathways are in
direct competition for partitioning of preprotein secretion (16).

The GSP signal sequence consists of a short, hydrophobic peptide at the N-
terrninus of the preprotein . The GSP recognizes signal sequences based on their
hydrophobicity and structure. GSP signal peptides usually consist of a highly

hydrophobic a-helical N-terminal segment of approximately 10-15 residues, known as

the H domain, followed by a more flexible segment known as the C domain . The two
regions are frequently separated by a helix-destabilizing amino acid, giving the typical
signal peptide a helix-break-helix structure . Signal recognition is flexible, in that
deficiency in one portion of the sequence can be compensated for by strength in another
segment . The GSP signal sequence remains inserted in the membrane following
translocation, and frequently contains a cleavage site in its C-terminal portion, allowing
for release of the preprotein to the periplasm for further transport (112).

As the preprotein is translated, it is maintained in a semi-unfolded state by the
binding of the Sec chaperone, SecB (45). SecB is organized as a homotetrarner,
functioning as a dimer of dimers (155). Structural analysis has identified binding pockets
for hydrophobic or aromatic regions of the preprotein (27). Binding of these regions
serves to prevent their self-aggregation in the hydrophilic cytoplasm. It is thought that
the preprotein can be wound around the outside of SecB, as a further mode of binding
and maintenance. Its ability to bind hydrophobic or aromatic residues rapidly and with
high affinity allows SecB to act as a generalized chaperone in cells deficient in one or
more other chaperones (141).

Following SecB binding and transit to the IM, the preprotein is translocated
across the membrane through the Sec translocon, composed of the integral IM proteins
SecY, SecE, and SecG (23). The translocon forms a funnel-shaped pore approximately
12-20 angstroms in diameter at its narrowest point (17). This is sufficient for passage of
a-helical portions of preproteins, but does not allow for passage of proteins with fixed
tertiary structure. Translocon pores tend to oligomerize in the presence of ribosomes or

the SecA protein. It is possible that this oligomerization contributes to localization of

secretion machinery to specific regions of the IM, or assembly of larger pores to mediate
passage of larger, more structured proteins across the [M (129).

Several accessory proteins are involved in [M transit, most notably SecA and
YidC. YidC mediates lateral transfer of signal peptides and transmembrane domains
(TMDs) from the translocon to the membrane (83, 84). SecA is an ATP-driven motor
protein, which uses a DEAD motif similar to that of DNA and RNA helicases to convert
energy from ATP hydrolysis into the mechanical force necessary to drive translocation
across the membrane (81). SecA binds to the 1M as well as the translocon (24, 62, 89),
and this binding is strongest in the presence of anionic phospholipids, which may
contribute to localization.

The Twin Arginine Transport Pathway. The Twin Arginine Transport (or Tat)
pathway mediates transfer of fully-folded proteins (123). Although Tat secretion systems
are not universally conserved, they have been identified in organisms from all three
domains (36). The Tat system was first identified in a screen for mutants deficient in
secretion of proteins to the chloroplast thylakoid lumen (146). Tat-mediated secretion of
proteins to the thylakoid lumen was determined to be dependent on ApH for energy
(135). Transport of a single protein has been described as requiring the transfer of as
many as 30,000 protons (8). Proteins transported by this pathway were demonstrated to
be folded during translocation, and often including bound cofactors (21). Secretion of
proteins in their folded state was demonstrated by experiments based on the transport of
Dihydrofolate Reductase (DHFR) into mitochondria (43). DHFR binds to the cofactor
Methotrexate MTX), resulting in DHFR assuming a tightly folded, protease-resistant

conformation. In the absence of a fimctional Tat pathway, the Sec system, which

transports unfolded proteins, is unable to transport DHFR into the mitochondrion. It was
also demonstrated that DHFR retained its protease resistant state following transport,
indicating that MTX remained bound throughout the process (66).

The bacterial Tat system was first identified in 1998 as a Sec-independent
mechanism for transport of proteins with cleavable N-terminal signal sequence
containing a characteristic twin-arginine motif (151). The Tat genes are arranged on two
transcriptional units, the tatABCD operon and the separately transcribed tatE gene.
Mutations in tatB or tatC are non-permissive for Tat transport, while mutations in tatA
result in a minimal transport phenotype (21). TatA and TatB are small proteins, 9.6 and
18.4 kDa, respectively, each containing a single trans—membrane domain (TMD). TatC is
larger, 28.9 kDa, with four TMDs (54). Although Gram-negative bacteria usually encode
tatA, tatB, and tatC, Gram-positive Tat systems often consist of only MM and tatC,
indicating that TatB is not essential in all organisms (69). Whether this is due to an
ability of TatA to complement the function of TatB in these organisms, or whether the
activity of TatB is not necessary remains a matter of debate.

The Signal Recognition Particle Pathway. The Signal Recognition Particle Pathway
was first identified as a mechanism by which eukaryotic cells could co-translationally
insert nascent polypeptides into the endoplasmic reticulum (ER) for export from the cell
(150). The eukaryotic SRP specifically binds hydrophobic, N-terminal signal sequences
of nascent polypeptides as they emerge from the ribosomal exit channel. Translation is
paused while the ribonucleoprotein (RNP) complex thus formed transits to the ER
membrane. The SRP Receptor (SR) assists in transfer of the polypeptide to the

membrane-integral insertase, at which point translation of the nascent polypeptide

resumes concomitant with insertion into the membrane. The SRP and SR then dissociate
to repeat the process. Genomic analysis has identified a components of putative SRP
pathway in organisms from a diverse range of taxa (75). Such evolutionary conservation
across all Domains of life indicates the indispensable nature of this pathway. Another
indicator of the importance of the SRP is that loss-of-function mutations in SRP genes are
lethal (11). Recent advances in molecular and structural techniques have yielded
significant insight into the molecular interactions necessary for the function of this
critical pathway.

Overview of SRP composition. Although the SRP pathway varies among species,
essential activities are conserved. Conservation of function is reflected in the similarity
of subunit conservation among taxa. The eukaryotic SRP has a more complex subunit
structure than those of bacteria and archaea, which may be considered to contain a more
minimal SRP.

The eukaryotic SRP is comprised of six proteins and a 78 RNA molecule. As
demonstrated by Gundelfinger et al, the RNA molecule serves as a structure for SRP
assembly (55). Cleavage of the SRP by micrococcal nuclease results in a single cleavage
event, yielding two separate SRP domains but not inhibiting their function. Further
cleavage into smaller domains resulted in non-functional SRPs. The two domains are
designated the Alu domain, which consists of the proteins SRP9 and SRP14 as well as the
3’ and 5’ ends of the RNA, and the S domain, which consists of the remainder of the
RNA molecule in complex with the SRP19, SRP54, SRP68, and SRP72 proteins. Further
investigation has elucidated the separate roles of these domains as pausing of translation

and binding of the signal peptide (specifically by SRP54), respectively (see below).

The eukaryotic SRP utilizes a mechanism known as two-compartment assembly
(94, 128) to properly assemble the SRP protein subunits on the RNA scaffold. The first
obstacle to SRP assembly is that while the RNA in synthesized in the nucleus, translation
of the SRP proteins takes place in the cytoplasm (148). More recent study has
determined that, with the exception of SRP54, the SRP proteins are imported into the
nucleus to the nucleolus, where they are bound to the 7S RNA (116). The complex thus
formed is exported to the cytoplasm and subsequently binds SRP54, to form the mature
SRP.

Work by Kuglstatter et a1 (82) has shed further light on the assembly sequence.
Binding of SRP19 to the 7S RNA aligns portions of helices 6 and 8 for their association
with SRP54. Upon SRP54 binding, an asymmetric loop in helix 6 collapses, the four
nucleotides of the long loop interacting with the SRP54 M domain, allowing the two
adenines of the short loop to flip outward and interact with the backbone of helix 8. This
interaction stabilizes the proteins and the RNA, acting as a molecular latch, holding the
ribonucleoprotein complex in the correct, active conformation.

Premature binding of SRP54 to the complex has been demonstrated to inhibit the
binding of SRP19 (94). Interaction of SRP19 with a pre-formed SRP54-7S RNA
complex induces misfolding in two RNA-binding loops of SRP19, preventing its binding
to the complex and rendering the SRP inactive. Thus, compartrnentalization of assembly
serves as a mechanism to ensure that any SRP in the cytoplasm is fully formed and
active. Improperly assembled SRP may be capable of binding a signal peptide, but
malfunctions at some downstream portion of the pathway. In such a case, not only would

the nascent peptide likely fail to reach its targeted destination, the cell must remove the

10

mistransported IMP. It is noteworthy that prokaryotic SRPs do not rely on a two-
compartment method of assembly—however, due to the fact that they require fewer
protein subunits, it is likely that spontaneous complex formation is sufficient.

The eukaryotic SR is composed of two proteins, SRor and SRB, both of which are
GTPases (53, 138). SRB is an integral ER membrane protein, responsible for association
of SRor, and through it the SRP, with the ER membrane and the translocation channel.
Although the SRB GTPase activity and interaction with the translocation channel are
essential for SRP function, no deleterious effects result from removal of its single TM
domain (111). These results indicate that anchoring of SRa to the membrane by SRB is
mediated either by surface-surface interaction between SRB and the membrane or by
direct interaction of SRB with the translocon.

Although they are located within eukaryotic cells, chloroplasts encode their own
SRP pathway [Reviewed in (30, 35, 42)]. The chloroplast SRP (cpSRP) is unique in that
it is best known for post-translational transport, and that it is the only SRP yet identified
as lacking an RNA component. Although the cpSRP does co-translationally transport
several chloroplast-encoded proteins to the thylakoid membrane, the bulk of its activity is
the post—translational insertion of nuclear-encoded light harvesting complex (LHCP)
proteins. Recognition of SRP substrates is mediated by the cpSRP54 protein,
homologous to SRP54, and the thylakoid membrane-associated receptor, chtsY, which
links the SRP to the membrane insertase Alb3, is homologous to SRa. Another novel
aspect of the cpSRP is the cpSRP43 protein. It appears to be required only for the post-
translational activity of the cpSRP. cpSRP43 has no known enzymatic activity, but is

believed to regulate the GTPase activity of cpSRP54.

11

The Archaeal SRP features a 78 RNA molecule similar to that found in
eukaryotes (139). Archaea encode SRP19, which in eukaryotes plays a pivotal role in
SRP assembly, as well as SRP54, which mediates peptide binding. Archaeal SRP19
binds at loop 6 of the 7S RNA, enhancing interaction of SRP54 and the RNA molecule
(139). Although it appears to play a role in the Archaeal SRP, the SRP19 subunit
appears to be dispensable in archaea (156). In vitro studies have demonstrated that
SRP54 and the 7S RNA can assemble in the absence of SRP19, and an SRP19 knockout
in Haloferax volcanii shows no deleterious effects from its absence. It remains to be
determined whether SRP19 plays some as yet unidentified role within the cell.

Bacillus subtilis also builds its SRP around a 78 RNA molecule, which is a
significant departure from the majority of eubacterial SRP systems (106). Although the
B. subtilis SRP RNA includes an Alu domain, the SRP9 and SRP14 proteins are absent.
Their function is complemented by a histone-like protein, Hbsu. Hbsu was first
identified as a 10kDa protein that co-segregated with the SRP54 homolog, th (Fifty-four
homolog) and 7S RNA after sucrose gradient centrifugation. Subsequent structural
analysis demonstrated weak sequence similarity to SRP9 and 14, as well as a highly-
conserved RNA-binding saddle structure composed of a characteristic a-B-B-B-a folding
motif. The inner surface of the saddle is enriched in positively-charged residues,
stabilizing interactions with the phosphate backbone of the 7S RNA. Nakamura et al
demonstrated that although a deficiency in the 7S RNA could be complemented with
either human 78 RNA or the 4.58 RNA of E. coli, which lacks the Alu domain, the Hbsu

protein is essential for viability (104, 106). It remains to be determined whether this is

12

due to a requirement for its function in SRP-mediated protein transport, or whether Hbsu
plays some other key role in the cell.

The E. coli SRP [reviewed in (91)] has been the focus of extensive study in recent
years. As with most bacterial systems, it appears to include only essential components: a
homolog of SRP54, th, a 4.58 RNA, similar to the eukaryotic S domain, and the SRa
homolog, FtsY. Ribes et al demonstrated that depletion of 4.5S RNA molecule could be
functionally complemented by expression of 7S RNA with no deleterious effects on the
cell (122). The structure of the th protein is similar to that of SRP54, consisting of an
N-terrninal M (Methionine-rich) domain, responsible for binding of the signal peptide; an
N domain, composed of a characteristic four-helix motif thought to regulate nucleotide
occupancy of the C-terminal G domain, which includes the GTP binding and hydrolysis
active sites (15, 19, 51, 127, 158). FtsY, the SRP receptor, shows strong similarity to the
SRor N and G domains, which demonstrate twofold symmetry with the th N and G. The
N-terminal A domain, however, is quite different from the SRor N terminus. Although it
is found in both the cytoplasm and at the IM (143), the A domain of FtsY is enriched in
acidic residues which likely play a role in its ability to interact with the membrane (33).

Although SRP pathways are composed of different sets of subunits, it is clear that
their fiinction remains much the same. The RNA S-domain and SRP54 protein remain
highly conserved across domains, implying that their function in signal peptide binding is
universally vital to cell function. The same holds true for the SRP receptor, for which
membrane association and interaction with the protein translocation channel are essential

for polypeptide transport (10). This is perhaps a consequence of the nature of the

13

Ribosome

Translation Initiation
Signal Peptide binding

RNP Complex

   
 

Signal
Peptide

 

Translocation to IM

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Figure 1. SRP Targeting of IM proteins in E. coli. Nascent polypeptides with N-
terrninal signal sequences are recognized and bound by the the Signal Recognition
Particle (SRP) composed of the Fflr protein and a 4.5S RNA molecule. The
ribonucleoprotein (RNP) thus formed translocates to the IM, where the SRP Receptor
(SR), FtsY, binds the SRP. The nascent polypeptide is transferred to the SecYEG
translocon, which mediates insertion of the protein into the IM concomitant with
translation. Pm and FtsY hydrolyze GTP to GDP, the complex dissociates, and the cycle

begins again.

proteins transported through the SRP. IMPs comprised of multiple TM domains would
be prone to spontaneous folding and aggregation should the hydrophobic TM domains
become exposed to the aqueous environment of the cytoplasm. As will be discussed
below, ample evidence exists to demonstrate the difficulties incurred upon the cell by
IMP mistransport—usually resulting in cytoplasmic accumulation of MP3 (115, 133).
Poritz et al demonstrated that E coli responds to SRP deficiency with the induction of a
modified heat shock response, inducing cytoplasmic proteases to degrade the
accumulated MP5 (117). Such a response reduces the amount of IMP accumulated in
the cytoplasm, but does not affect insertion of MP5, many of which are critical for
viability.

Signal Peptide Recognition. A characteristic common to both the GSP and the SRP is
the recognition of N-terminal, hydrophobic signal sequences as they emerge from the
ribosome. The question thus arises of exactly how partitioning between the two
pathways occurs. One factor that likely aids in pathway discrimination is the degree of
hydrophobicity of the signal sequence. A binding and crosslinking study involving a
series of signal sequence mutants of varying hydrophobicity demonstrated an almost
linear correlation between hydrophobic character and degree of SRP utilization, with
more hydrophobic signal sequences more dependent on SRP (96). To further
characterize the import of signal sequence binding, Lee et a1 (87) demonstrated that
replacement of the signal sequence of periplasmic maltose-binding protein (MBP) or
OmpA (porin), an integral OM protein, with that of the integral IM protein Ach was
sufficient to redirect translocation of those proteins to the SRP pathway. Adams et a] (7)

suggest that the relatively higher hydrophobicity of TM domains may serve as a measure

15

to ensure that the SRP sequesters IMPs, regardless of potential initial GSP pathway
involvement.

The GSP-directed signal sequence consists of a tripartite structure characterized
by a 9-12 residue stretch of hydrophobic amino acids that form an a-helix (147). It was
determined that the replacement of a helix-breaking glycine residue with a helix-
promoting Ala, Cys, or Leu would modify the PhoE signal sequence sufficiently to direct
transport from the GSP to the SRP (7). The ability of a signal sequence with a structure
similar to that of a TM domain to direct SRP transport is reinforced by the demonstration
that SRP could bind to an exposed TM domain, and that overexpression of hydrophobic
proteins overwhelms the SRP (96).

Another aspect of the signal sequence that may contribute to binding by the SRP
is the presence of basic amino acids flanking the hydrophobic region (114). It was
determined that replacing the OmpA signal sequence with one of similar hydrophobicity
but of higher basic content was sufficient to redirect OmpA to the SRP. Decreases in
either the hydrophobicity or number of basic amino acids resulted in a corresponding
decreases in utilization of the SRP. The authors suggest that the basic amino acids could
serve to stabilize interactions between the signal peptide and the SRP RNA by forming
salt bridges.

The flexibility of SRP recognition is demonstrated by the finding that hemoglobin
protease (pr), an autotransporter protein secreted by E. coli, is targeted to the [M via the
SRP (136). The pr signal sequence consists of a hydrophobic core region flanked by a
region of basic amino acids and an unusually long N-terminal extension. Inclusion of

basic amino acids is sufficient to direct a mildly hydrophobic signal sequence to the SRP,

16

indicating that the basic amino acids play a role in SRP targeting It was determined that
under conditions of SRP depletion, pr transport shifts to the GSP, although GSP
transport in this case is less efficient than the SRP. The pr signal sequence is near the
threshold of discrimination between the two pathways, allowing for shift from one to the
other during conditions of distress. It is likely that the function of the GSP in partial
complementation of SRP transport in this case is due to the general structure of
autotransporters, in which TM domains are arranged in an amphipathic B-barrel (63).
The fact that most autotransporters are secreted via the GSP supports this argument.
Binding of the Signal Peptide. Signal peptide binding is carried out by the S domain of
the SRP, specifically by the th M domain. The high density of Met residues in this
region are thought to play a role in interaction with the signal peptide. Structural studies
have shown that the M domain forms a surface-exposed, hydrophobic pocket for peptide
binding, flanked by a “finger loop” structure, which serves to clamp the peptide in place
(15). Cross-linking studies (40) have identified a site on the ribosome with which the
SRP54 protein interacts. Crosslinking to riboproteins L23a and L35, both of which flank
the peptide exit site, indicate that SRP54 associates with the ribosome, positioned to bind
nascent polypeptides with SRP-directed signal sequences. It has been further
demonstrated that association with SR induces a conformational change, such that SRP54
no longer crosslinks with L23a—in effect distancing SRP54 from the exit channel, likely
a prerequisite for docking of the ribosome and transfer of the nascent polypeptide to the
translocation channel (40).

As has already been discussed, there are similarities between signal sequences

recognized by the GSP and SRP. There is evidence to suggest that partitioning between

17

the two pathways is based in large part upon direct competition for binding of the nascent
signal as it exits the ribosome. TF, which stabilizes GSP-targeted polypeptides for post-
translational transport has also been shown to associate with riboprotein L23 (29, 44). A
nascent polypeptide, as it leaves the ribosomal exit site, is thus presented to the two
recognition systems, in the form of TF and SRP54. TF appears to dock with the
polypeptide exit site at a slightly more distal location than SRP54, which could indicate
that the SRP has primacy of place at the exit site, but could also be an artifact of the
differing architecture of the two proteins with respect to their direct interaction with the
ribosome. This supports previous binding data in which it was determined that the
OmpA signal sequence is bound exclusively by TF, and the MtlA signal sequence is
likewise exclusively bound by SRP (16). The apparent spatial competition for access to
the signal peptide, coupled with the exclusivity of binding, strongly suggests that the two
recognition factors compete for access to the signal peptide, with the stronger-binding
factor inhibiting binding of the weaker, ensuring targeting of the nascent peptide to the
pathway for which its signal directs. Recent Cryo-Electron Microscopy and Forster
Resonance Energy Transfer data support this hypothesis, clearly showing SRP and TF
closely flanking the exit channel, positioned such that each has access to the emergent
signal sequence (93).

The matter of competition for binding may have been firrther complicated by
findings that structural information regarding the nascent chain is transmitted through the
conformation of the ribosomal exit channel. Differential folding within the ribosome of
the TM segments of integral membrane proteins, as opposed to secreted proteins, has

been demonstrated to affect ribosome interactions with the Sec translocon (154). Signal

18

peptides have been shown to induce specific conformational changes in the exit channel,
resulting in surface rearrangements of L23 which could reposition trigger factor and SRP
in such a way as to favor binding of one factor or the other. SRP has been demonstrated
to have a significantly higher affinity for actively-translating ribosomes, as opposed to
those that are resting (47). This increase in affinity occurs prior to the emergence of the
nascent peptide from the ribosome, indicating that this interaction results from
translation-specific structural changes at or near the exit channel. Direct interaction of
the nascent polypeptide and exit channel has been demonstrated in a study of the SecM
protein in E. coli (107). SecM, the secretion monitor protein, regulates expression of
secA. SecM undergoes translation arrest in the absence of a firnctioning translocon to
“pull” the nascent protein through the ribosomal exit channel. Arrest occurs near the C-
tenninus of the protein, such that arrested SecM polypeptides are exposed in the
cytoplasm, where they can induce expression of secA, increasing the bacterium’s ability
to export proteins from the cytoplasm. This arrest can be rescued by mutations in
riboprotein L25 or in the 23S rRNA, which are located at the narrowest point in the exit
channel. It remains to be determined exactly to what extent nascent polypeptides interact
with the ribosomal exit channel during elongation, and how those interactions affect
downstream events such as membrane targeting.

Translation Arrest. Translation arrest by the eukaryotic SRP was first described as a
mechanism by which the complete synthesis of ER proteins could be delayed in order to
better facilitate insertion to or through the membrane. Eventual release of arrest was
known to be mediated by a salt-extractable factor later identified as SR, a protein that

couples the arrested ribosome-SRP to the transport channel (98, 149). Cryo-EM has

19

revealed that the SRP Alu domain associates with the ribosome in such a way as to block
the EF binding site, thus pausing translation (61). The relative lack of conservation of the
RNA Alu domain and SRP9/14 imply that translation arrest is not essential for SRP
function. Early attempts to address this question were carried out using a cell-free system
composed of mammalian SRP incapable of inducing arrest (118, 153). The absence of
translation arrest led to minimal defects in protein transport, suggesting that arrest may
not be truly vital for proper transport. A more recent study examined the effects of arrest
deficiencies in yeast (95). Yeast cells expressing a truncated SRP14 were demonstrated
to be deficient in arrest of translation, resulting in deficiencies in protein translocation.
These contradictory results may be results of different systems’ reliance on this particular
activity.

The B. subtilis SRP utilizes a 7 S RNA and the histone-like protein Hbsu in SRP-
mediated protein transport (105). It seems likely that this fimctions in a similar manner to
the eukaryotic Alu domain, but there is as yet no direct evidence that translation is
actually arrested. The lack of an Alu domain in E. coli suggests that the prokaryotic SRP
is incapable of inducing a pause in translation. Several studies have shed light on the
ability of the E. coli SRP to induce translational arrest. Nakatogawa et al (107)
demonstrated translational arrest for the SecM protein, but it is unknown whether this
arrest is specific to that protein-ribosome interaction, or whether this activity extends to
other SRP-targeted proteins.

Using an affinity-tagged th M domain, Avdeeva et al demonstrated the presence
of a significant pool of truncated Fflr in association with 4.5S RNA and ribosomes (14).

They concluded that the accumulation of such complexes must be the result of

20

translational arrest induced by the presence of M domain bound to the signal peptide. A
study using a cell-free, reconstituted SRP was unable to detect any slowing of translation
upon addition of SRP (120). The possibility exists that the contradictory nature of these
results stems from their greatly differing methodologies.

Perhaps a more pertinent question is whether translational pause is actually
required in bacteria. Support for the necessity of translational arrest can be found in the
work of Schierle et al, who identify thioredoxin as an excellent reporter for SRP-
mediated transport due to its incredibly rapid cytoplasmic folding, which results in
jamming of the Sec translocon during post-translational transport (130). Several factors,
such as the smaller size of the bacterial cell, may combine to make translational arrest
unnecessary. Several studies have suggested that following a successful round of SRP-
mediated co-translational transport, the ribosomes remain associated with the membrane,
ready for another round of targeting (20, 64, 65, 132). Indeed, such a mechanism would
make a great deal of sense, obviating the need not only for translational arrest, but
perhaps predisposing those particular ribosomes toward SRP binding of nascent
polypeptides by ensuring a high local concentration of SRP. The localization of SRP
components could take the place of translation arrest in prokaryotes, ensuring that IMPs
are properly transported with little opportunity for cytoplasmic accumulation. A
contributing factor to the continued difficulty in studying this particular interaction is the
relative speeds of protein synthesis and translocation (119). As translocation takes place
much more rapidly than synthesis, it is difficult to determine whether a delay in
translocation is due to a pause in translation or merely an artifact of the limiting step in

the pathway.

21

Transfer of the polypeptide to the translocon. Both the GSP and SRP utilize the Sec
translocon for transport of the nascent polypeptide across the [M (102, 143). SecA has
been identified as a requirement for co-translational transport in E coli (108). Fusion of
the signal peptide of the SRP-targeted MtlA protein to the GSP-transported OmpA
protein resulted in targeting of OmpA to the SRP. Translocation of the protein did not
occur in the absence of SecA, however, indicating that both SRP and SecA are necessary
for co-translational transport, and that they function sequentially in peptide transfer and
insertion. A photo crosslinking study determined that TM domains of FtsQ, an [M
protein involved in cell division, are inserted into the membrane in the vicinity of SecY
and lipids, then progressing upon elongation to associate with the Sec insertase YidC and
lipids (142). This study confirmed the role of the membrane insertase YidC in IMP
biogenesis, receiving the nascent polypeptide from the Sec translocon and mediating
lateral insertion into the membrane. Further evidence for the action of SecYEG and
YidC was obtained in a study of the emergence of the leader peptidase, Lep, from the
ribosome (48). The nascent polypeptide was demonstrated to interact with YidC and
SecY as the peptide length reached approximately 50 amino acids, at which point the
signal sequence was not yet fully exposed. This implies close association of the
ribosome and translocon prior to protein synthesis, evidence perhaps for translation arrest
or localization of SRP components.

Interaction of FtsY with the IM. GTP hydrolysis is absolutely essential to proper SRP
function, but it is currently unknown what role hydrolysis plays in protein transport. GTP
hydrolysis could contribute to initiation energy needed to re-start the ribosome, or to

power translocation of the nascent polypeptide across the membrane. More recent

22

evidence has accumulated to suggest that the role of GTP hydrolysis is to regulate the
SRP pathway, ensuring unidirectionality of transport. Early analysis of the SRP GTPases
(68, 103) demonstrated high rates of binding of GTP and dissociation of GDP, indicating
that the GTP-bound state would likely predominate.

Further work on FtsY, the E coli SRor homolog, demonstrated that its GTPase
activity is intimately linked with its association with both the 1M and the Sec translocon.
Zelazny et al demonstrated that interaction of FtsY with the membrane is essential to SRP
function (31). A truncated FtsY lacking the A domain was insufficient to complement
depletion of FtsY (157). Addition of an unrelated TM domain to the FtsY NG domain
restored viability. Further study determined that association of FtsY with the membrane
was mediated by the A and N domains (100), and that this binding was most favored in
conditions of low pH and low salt, indicating that electrostatic interactions are the
primary mediator of binding (33). The high concentration of H+ in acidic conditions
likely counteracts the largely negative charge of the FtsY A domain. Binding was also
determined to be independent of the nucleotide occupancy of FtsY, and did not affect the
affinity of the protein for GTP. The rate of GTP hydrolysis, however, increased
significantly in the presence of anionic phospholipids. In 2001, Millman et al determined
that FtsY associates with phosphatidylethanolamine and a trypsin-sensitive component of
the membrane (101). Increased GTPase activity of the SR in the presence of the
membrane and translocon indicates that the GTPase function is specific to the interaction
of the SRP and Sec translocon, but does not provide insight into the role played by

hydrolysis.

23

Angelini et al have further characterized the link between FtsY GTP hydrolysis
and the Sec translocon, as well as its role in SRP-mediated protein transport (9). It was
demonstrated that blocking of the GTPase function of FtsY, using either a non-
hydrolysable GTP analog or by mutating the active site such that GTP could be bound,
but not hydrolyzed, resulted in stabilization of the membrane-associated form of FtsY, as
measured by an increase in resistance to alkaline carbonate extraction. It was also
determined that this stabilization could be disrupted by mutation of SecY, altering
surface-exposed loop C5. A similar mutation disrupting interaction between SecY and
SecA did not stabilize FtsY at the membrane, signifying that the interaction is
independent of active transport. A third mutant in which SecY was absent likewise
conferred no added stability (9). These results show that FtsY in its GTP-bound state
preferentially associates with both the 1M and the SecYEG translocon, stabilizing the
receptor-translocation channel complex in the form signifying readiness to receive a
nascent polypeptide for translocation.

Interaction between FtsY and F fh. One of the most striking features of the SRP is the
unique mechanism by which the Fth and FtsY GTPases function. Two independent
studies in 2004 revealed crystal structures describing the interaction between the two
GTPases (41, 49). A crystal structure of the SRP-SR complex obtained in the presence of
the non-hydrolysable GTP analog GMPPCP revealed that the N domain, consisting of
four a-helices, changes its angle relative to the G domain upon nucleotide binding. The
G domain consists of a GTPase core similar to that of the small GTPase Ras, with an
added B-or-B-or fold termed the “Insertion Box Domain” (IBD), which includes a portion

of the active site. Nucleotide binding alters the angle of N relative to G, bringing the two

24

th

GTP
ch bhdhg /
N—G Rearrangement . \

   
 

Figure 2. GTP hydrolysis by th and FtsY. Binding of GTP induces conformational
changes between the N and G domains of both GTPases, bringing the binding site on the
N domain into closer association with the active site on the G domain. Dimerization of
F fh and FtsY closes the complex around the two GTP molecules, allowing for coordinate

regulation of enzyme activity. The complex dissociates following hydrolysis.

25

domains closer together. Dimerization of the SRP GTPases allows for rearrangement of
the two IBDs, fully closing the binding site, and bringing the final catalytic amino acids
into contact with the bound substrates. It was also determined that there is extensive
interaction between the two GTP molecules, particularly the y—phosphates, with the active
sites. Upon hydrolysis of the GTP molecules, these stabilizing interactions are disrupted,
resulting in dissociation of the complex due to electrostatic forces.

Overview of SRP Regulation. An increasing body of data suggests that GTP hydrolysis
regulates the formation of the SRP-SR complex as well as its dissociation. Association
of FtsY with the IM and translocon results in stabilization of the SR in its GTP—bound
state. Fflr, also in its GTP-bound state, is docked at riboprotein L23, where it recognizes
and binds an SRP-targeted signal sequence. Translation may or may not halt while the
ribosome-nascent chain-SRP complex transits to the membrane. Dimerization of Fflr and
FtsY acts as a molecular “latch,” firmly associating the entire complex with the
translocon and inducing a conformational change in Fflr, weakening its association with
the ribosome and signal peptide. The peptide is passed to the translocon, which with the
aid of SecA and YidC begins inserting the IMP into the membrane. Once peptide
transfer has occurred, the SRP GTPases hydrolyze their bound substrates, forcing the
SRP-SR complex to dissociate. The GTPases readily acquire fresh GTP molecules, and
the sequence is ready to begin anew.

Effects of bacterial SRP deficiency. An E. coli strain conditionally expressing FtsY
under control of the tightly-regulated arabinose promoter, N4156::Pm-ftsY, has been
instrumental in the measurement of the effects of SRP deficiency in bacteria (92).

Growth defects, including decreased grth rate and a filamentous phenotype, are

26

evident fewer than eight hours after repression of ftsY. Protease accessibility assays,
which differentiate cytoplasmic proteins from those that have been exported, indicate that
SRP-transported proteins accumulate in the cytoplasm under conditions of SRP
depletion. It was also determined that overexpression of F tsY can be just as damaging to
the cell as underexpression, presumably by titrating the other SRP components and the
translocon (92).

Conditions of SRP deficiency have been shown to result in cytoplasmic
accumulation of mistransported IMPs. This accumulation is enhanced in the absence of
the cytoplasmic proteases Lon and Clp (18). It was also determined that mutants lacking
these proteases were more susceptible to SRP deficiency, as measured in an efficiency of
plating assay, indicating that proteolytic degradation of mislocalized MP3 is essential for
survival of the bacterium (18).

The lethality of SRP deficiency has been used to identify putative SRP-
transported proteins (37). Overexpression of an SRP-transported protein under
conditions of SRP limitation results in lethality. An E. coli strain conditionally
expressing Ffl‘l was used to screen plasmid library of gonococcal genes. Those plasmids
which could not grow under mild repression of flh were analyzed for the presence of
putative SRP substrates. Each of the nine severely-affected clones thus identified
contained at least one gene encoding a putative IM protein. Further analysis of one of
these clones identified an SRP-dependent gonococcal homolog of the ZipA protein,
which in E. coli plays a role in cell division (37).

The Gonococcal PilA Protein.(5). PilA was first identified in a screen for

transcriptional regulators of piIE (137). Due to weak sequence similarity, pilA and a

27

neighboring locus, pilB, were thought to encode a two-component regulatory system
modulating expression of the major pilus subunit. Further experiments demonstrated that
purified PilA could selectively bind to specific segments of DNA including the piIE
promoter region (Pm-,5) (12). Selectivity of binding was determined in an electrophoretic
mobility shift assay, in which 32P-labeled PM; DNA was shown to be bound by PilA in
the presence of excess, unlabeled control DNA. Binding of labeled PpflE DNA was
inhibited by incubation in the presence of excess unlabeled Ppug. Serial truncations were
used to map the site of PilA binding on the DNA, identifying two regions, from -183 to -
125 and from -55 to +36 with respect to the start of transcription, that are essential for
binding.

Analysis of the sequence of PilA resulted in the identification of a GTPase
domain similar to the NG domains of Fflr and FtsY of E. coli (13) . Measurements of
expression of a pilE-lacZ fusion indicated that the presence or absence of PilA had no
effect on transcription from the pilE promoter, directly contradicting earlier findings (11).
It was firrther determined that overexpression of PilA in E. coli results in cytoplasmic
accumulation of pre-B-lactamase, indicative of defective SRP transport. PilA was
demonstrated to be able to complement FtsY deficiency in E. coli both in vivo and in
vitro (11). The inability of a GTPase-inactive PilA mutant to recover a FtsY deficiency
indicates that the function of PilA is dependent on GTP hydrolysis. For these reasons,
PilA was identified as the gonococcal homolog of FtsY, the [NI-associated SRP receptor
(11).

Further investigation of the binding of Pp”),- DNA binding by PilA results in a

significant increase in GTPase activity (50). No stimulation of the th GTPase is

28

observed in the presence of Ppilg, nor does the GTPase activity of th appear to change
based on the DNA-bound or —unbound status of PilA. Further investigation has
identified several other DNA loci capable of stimulating the GTPase activity of PilA
(unpublished data). These loci are all found at the 5’ end of genes encoding putative SRP
substrates, suggesting that DNA binding by PilA may play a role in SRP-mediated

protein transport.

29

Chapter 1: Construction and Validation of the pilA conditional mutant

The ability of the gonococcus to efficiently translate and insert most IMPs into the IM is
dependent on the function of the SRP. In order to facilitate study of protein transport
through this pathway, and because of the lethality of pilA knockouts, it was necessary to
construct a conditional pilA mutant. As shown in Figure 3, the construct was assembled
in E. coli on a low copy number plasmid containing a tac promoter driven copy of pilA
(12). As N. gonorrhoeae has no lac operon or Lac repressor, it was also necessary to
include laclg, which expresses high levels of the lac repressor. An erythromycin
resistance cassette (ermC) was included to allow for selection following transformation
into N. gonorrhoeae. Inclusion of a portion of pilB provides the necessary homologous
sequences to allow for recombination of the construct into the gonococcal chromosome.

Erythromycin-resistant transformants were screened for Kanamycin sensitivity to
ensure that the cloning vector had not integrated in its entirety. PCR amplificiation of the
DNA region between PilA and PilB yielded a 4kb band, indicative of the presence of
[(1619 and ermC, as expected for the conditional construct. No lkb band was evident,
indicating that the wildtype piIA-pilB locus had been disrupted.

Inducible Expression of pilA in Strain MP5. To verify the function of the
conditional piIA construct in strain MP5, we used immunoblot analysis to measure the
amount of PilA protein produced by cells grown in the presence and absence of the

inducer IPTG. The result of this experiment (Figure 4) shows that abundant PilA is

produced in the presence of SOOuM IPTG.

30

Ptac

I pilB’ ”I I ermC II lac1£J I IF pilA I
pChRePIE6 ‘3‘ 2..
\“‘.‘: Pp!“ ::;,/

I p213 ~. Ir MA I

MS11 Chromosome

 

 

 

 

 

 

I pr‘lB I I ermC II lacIQ I III. pilA I
MP5 Chromosome

Figure 3. Construction of the conditional pilA strain MP5. The high copy
number plasmid pTPA5 (12) encoding a Ptac-driven pilA, was modified to include the lac
repressor gene, lac1Q, and ermC as a selectable marker. This construct was inserted into a
low copy number plasmid encoding an intact pilB gene, to allow for recombination into
the GC genome. The resulting plasmid, pChRePIE6, was linearized and transformed into

N. gonorrhoeae strain MS11 (131), selecting for erythromycin resistance.

31

MS11 MP5

PilA Std 0 50 500 O 50 500 IPTG (pM)

 

 

Figure 4. Induction of PM in strain MP5 by IPTG. N. gonorrhoeae MS11 and MP5
were grown for 6 hours in GCB supplemented with 0, 50, or 500 uM IPTG. Cells were
pelleted and lysed by boiling in SDS-PAGE buffer. Proteins were separated by SDS-
PAGE on a 12.5% polyacrylamide gel, electrophoretically transferred to a nitrocellulose
membrane, and immunoblotted using a polyclonal antibody against PilA (12). lug of

purified PilA was included as a positive control.

32

Since endogenous levels of pilA were not readily detectable by immunoblot, we used
semi-quantitative RT-PCR to assess expression of the Ptac-driven pilA in the absence of
IPTG. In the absence of IPTG, less pilA RNA was detected in the conditional strain than
in the wildtype, indicating that the promoter, although leaky, does repress transcription to
levels lower than the wildtype level (Figure 5). Image densitometry analysis of the gels
showed that pilA expression was decreased to 66% of the wildtype level. From this
analysis, it was also possible to determine an IPTG concentration that results in
expression similar to wildtype. From these data we concluded that media containing
SuM IPTG resulted in roughly wildtype levels of pilA in MP5.
Effects of PilA Repression on strain MP5. As shown above, pilA expression is not
completely repressed in the absence of IPTG in strain MP5. To determine whether this
low—level expression is sufficient to maintain proper function of the SRP, we assessed the
function by measuring grth of the conditional mutant in liquid medium in the absence
of IPTG. As shown in Figure 6, no difference in grth rate was observed for MP5
grown in the presence or absence of IPTG, nor was grth different between MP5 and
MS] 1. Similar results were observed for gonococci grown on solid medium. Serial
passage of MP5 on GC agar plates without IPTG was possible with no obvious defects in
growth, indicating that sufficient amounts of PilA are maintained to allow for at least
minimal SRP function.

In order to assess SRP function in the pilA conditional strain in vivo, we chose to
examine function of the Mtr efflux pump. The rationale for this was two-fold. First, a
well-characterized substrate of the SRP in E. coli is Ach (109), a homolog of N.

gonorrhoeae MtrD (56), the IM component of the Mtr efflux pump. We hypothesized

that MtrD would be SRP-dependent in N. gonorrhoeae and that export of MtrD, (and

33

5 5 O 50 500 IPTG (pM)

 

Figure 5. Effects of IPTG on pilA transcription in N. gonorrhoeae MP5.
M811 and MP5 were grown in GCB supplemented with varying amounts of IPTG. Total
RNA was isolated, DNase treated, and reverse transcribed. pilA cDNA was PCR

amplified for 20 cycles and electrophoresed on agarose gels stained with EtBR.

34

12 -...-__._- . 1--.- cm__,u_¥_._-_-__,uz _. ,s_,.-_, B _ aw... Dc ru1_n.-,z-.-u. _

 

 

 

 

 

 

 

 

 

 

Figure 6. Growth of M81] and MP5 in presence and absence of IPTG. N

gonorrhoeae MS11 and MP5 were grown overnight on GC agar plates with or without 5
pM IPTG, then transferred to GC broth of identical IPTG concentration. Absorbance

readings were taken every two hours.

35

therefore Mtr function) would correlate with levels of PilA in the cell. Second, previous
work in our lab has shown that PilA binds DNA in a sequence-specific manner (Arvidson
et al, 1999). Subsequent experiments have revealed that PilA binds to two regions in the
mtrCDE locus (D. Wood, A. Karanikas and CG. Arvidson, unpublished). The Mtr efflux
pump has been shown to protect gonococci from varied hydrophobic agents including
crystal violet, bile salts, and erythromycin (5 7). Our hypothesis is that the amount of HA
effluxed by the Mtr pump is proportional to the number of pumps assembled at the
membrane, specifically, the amount of MtrD in the IM. It would be expected that the
ability of gonococci to survive in the presence of HAS could be decreased under SRP-
limited conditions. To test this, we used a modified version of the agar-dilution minimum
inhibitory concentration (MIC) assay to examine SRP effects on Mtr function (1). This
assay measures susceptibility to antimicrobial compounds as a function of the minimum
concentration of a given agent to completely inhibit grth of the strain assayed. In our
assays, strains were pre-conditioned by 18-20 hours growth on media with or without 5
uM IPTG. Approximately 104 colony forming units (CFU) were spotted onto GC agar
plates containing the same concentration of IPTG, and varied amounts of HA, and then
streaked for isolation. MIC values were determined as the minimum HA concentration
sufficient to completely inhibit growth of the bacteria. Figure 7 shows the MICs
determined for both strains M811 and MP5 in the presence or absence of 5 uM IPTG.
MP5 was demonstrably more susceptible to chloramphenicol, ethidium bromide, bile
salts, and ciprofloxacin. Chloramphenicol, EtBr, and bile salts are effluxed by the Mtr

pump (57), and ciprofloxacin is effluxed by the NorM efflux pump (125). There was no

36

Ethidium Bromide Ciprofloxacin Bile Salts

 

 

 

 

 

 

 

 

 

     

 

 

 

 

 

 

 

 

14o -. 140 a ,, ..... .. 140 .
120 120 - ' 120
o 100 - o - o
g g I g100
E 80' 2 '2 80'
E 60 B 3 50 _
° 40 - “5 ‘8
°\o Q a 40
20 ~ °\ e\
20
0 -
0 .
M611 W5
Penicillin Chloramphenicol
140 140
120 - 120
O 0
a. a.
:3 :3 a + IPTG I- IPTG
‘H Int-4
O Q
,\° .\°

 

 

 

 

MS11 MP5

 

Figure 7. Effects of limiting lPiIA on sensitivity to Mtr substrates. MS11 and MP5
were grown overnight on agar containing 0 or 5 uM IPTG, as well as varied amounts of
the compound listed. Values represent the least amount of antimicrobial agent needed to
completely inhibit bacterial growth. Data were normalized to percent of MIC for M811

in the presence of SuM IPTG. Error bars are included for those conditions in which all

three replicates were not identical

37

apparent difference in susceptibility to penicillin, which is also a substrate of the Mtr
pump.

As the pilE promoter was the first DNA shown to be bound by PilA (12), we next
assessed whether PilA deficiency as a result of growth in the absence of IPTG would
result in accumulation of mistransported pilin protein in the cytoplasm. Strains M811
and MP5 were grown in the presence of varying concentrations of IPTG and fractionated
by sonication. Soluble fractions were run on SDS-PAGE and immunoblotted using a
monoclonal antibody against pilin. The results, shown in Figure 8, show that no
accumulation of pilin protein was detected in the cytoplasm. Nor was any decrease noted

in the membrane fractions (data not shown).

38

Pilin M311 0 5 20 50 500

‘— PilE

<— PilA

Figure 8. Western blot analysis of pilin and PilA in cytoplasmic fractions of MP5
grown in PilA-depleting and overproducing cconditions. MS11 and MP5 were grown
overnight on GC agar containing varied amounts of IPTG and fractionated by sonication.
Soluble (cytoplasmic) fractions were separated by SDS-Page and immunoblotted using

monoclonal antibodies against pilin and polyclonal antibodies against PilA.

39

Chapter 2: Construction and Analysis of an mtrD-phoA reporter.

To assess the ability of the SRP to properly mediate the insertion of IMPs, it was
necessary to develop a means to quantify the amount of a given SRP-targeted protein
following export. To this end, we constructed a reporter by fusing a promoterless, signal
sequence-free phoA gene in frame to the 5’ region of mtrD, which encodes a putative
SRP-dependent protein. The rationale for this is that the phoA-encoded enzyme, PhoA
(alkaline phosphatase/AP) is only active when it is exported out of the bacterial
cytoplasm (refs). The mtrD-phoA fusion, with a linked selectable marker, was inserted at
a neutral site of the GC chromosome so as not to disrupt expression of the wild-type mtr
operon. Presence of the construct was confirmed by PCR amplification of a DNA
fragment including the junction between mtrD and phoA. Genomic DNA from this strain
was then back-crossed into M81 1, to yield the strain MT2, which was used in subsequent
experiments. The fusion was also transformed into two additional N. gonorrhoeae strains,
MP5 (IPTG-inducible pilA), and M1366 (38), which contains a transposon insertion in
mtrR, which encodes a transcriptional repressor of the mtrCDE operon (59), to yield
strains PT2 and M1366T2, respectively. Function of the AP fusion was assessed based on
development of blue color in response to growth on media containing X-phos. AP
function was then quantified using the pNPP assay (Pierce). AP activity of each strain
was measured following overnight growth on GC agar or GC agar with 5 uM IPTG
added (strains MP5 and PT2) (Figure 9).

Effects of PiIA expression on mtrD-phoA transcription. Variation in Mtr function

could conceivably occur at the level of mtr transcription, translation, MtrD export, or

40

90
so -~—~ ~—~ e w ~~~ ” +
7o ._ --
60 A f + i ma
50 —— __, ~w~e~ #W_
40 +- -r ~~~++ -
30 ~m
20 — —
10

 

 

 

 

 

 

Activity

 

 

 

 

 

 

 

 

 

 

 

 

 

M81 1 MP5 M1366 MT 2 PT2 M1 366T2
Strain

 

 

Figure 9. AP activity of gonococcal strains. Gonococci were grown overnight on GCB
agar plates (with 5 pM IPTG for MP5, PT2) swabbed into 500ul PBS, and assayed for AP
activity using the pNPP assay substrate from Pierce. Units of activity are umol pNPP

hydrolyzed min'l ug protein".

41

assembly of the efflux pump. To rule out potential effects on transcription, we used a
semiquantitative RT-PCR approach. The results of these experiments (Figure 10) show
that the amount of mtrD-phoA transcript does not vary in response to IPTG
concentrations between 0 and 50 ug/ml.
Effects of altered expression of PilA upon MtrD-PhoA fusion localization. To
determine whether the mtrD-phoA fusion is SRP-dependent for export from the
cytoplasm, we assessed MtrD-PhoA localization in the conditional pilA strain grown with
varying concentrations of inducer. Western blot analysis was used to determine the
amount of MtrD-PhoA present in subcellular fractions of PT2 grown in the presence or
absence of IPTG. Figure 11 shows the results of this experiment, from which we
concluded that localization of MtrD-PhoA was not dependent on the amount of PilA
produced by the cell.

A more sensitive measure of localization is to quantify AP activity in samples. As
AP is only active following transit to the periplasm, AP activity assays of whole-cell
suspensions are informative as to the total amount of fission successfully inserted into the
IM. As shown in Figure 12., overnight growth in media containing 0, 5, or 500 uM IPTG
resulted in no difference in AP activity.
Assessment of AP as a measure of protein localization. PilA is the gonococcal
homolog of FtsY, and it has been shown that PilA can replace E. coli FtsY both in vivo
and in vitro (11). Therefore, we hypothesized that the mtrD-phoA fusion would be SRP-
dependent in E. coli. To test this, the mtrD-phoA plasmid pWGT2 was introduced into
E. coli strain N4156szzftsY, which conditionally expresses ftsY (92). In E. coli

N41 56szzfts Y, expression of ftsY is under control of the tightly-repressed arabinose

42

MS11 PT2
No RT DNA 0 5 50 O 5 50

n g g n mtrD-phoA
pilA

m 163

Figure 10. Effect of pilA induction on transcription of an mtrD-phaA fusion.

 
  

Bacteria were grown for 6 hours in GCB supplemented with 0, 5, or 50 uM IPTG. RNA
was isolated from the cells using the TRIzol reagent (Invitrogen), DNase treated, and
reverse transcribed. Gene-specific primers were used to PCR amplify cDNA
corresponding to the junction of mtrD and phoA in the fusion construct. Products were

separated on a 1.2% agarose gel and visualized by staining with ethidium bromide.

43

MT2 PT2

o s 500 o s 500 [IPTG] (PM)
Soluble
Membrane

 

 

PiIA

Figure 11. Localization of MtrD-PhoA fusion under varied SRP conditions.
GC strains MT2 and MP5 were grown overnight on GC agar plates containing 0, 5, or
500 uM IPTG. Bacteria were collected and either lysed directly or sonicated to separate
the soluble fraction from the insoluble membranes. Proteins were separated by
electrophoresis on 12.5% SDS-polyacrylamide gels, transferred to nitrocellulose
membranes and immunoblotted using a monoclonal antibody to PhoA (Sigma, St. Louis

MO.)

44

N N o:
.0 .U‘ .0
o o 0

AP Activity
.7.
O

 

 

 

10.0
5.0
0.0 e
o 5 500 o 5 500 [IPTG] (uM)
MT2 PT2

Figure 12. Effect of SRP levels on AP activity. GC strains MT2 and PT2 were grown
overnight on GC agar plates containing 0, 5, or 500pM IPTG, collected, and assayed for
AP activity. Units of activity are umol pNPP hydrolyzed min'l pg protein".

Representative experiment.

45

promoter (74). Growth defects and protein mislocalization result when this strain is
grown in the absence of arabinose (92)

These strains were grown in the presence and absence of arabinose and growth was
assessed by measurement of the absorbance at 600 nm at two h intervals over an eight h
time period. At t = 8 h, cells were harvested and assayed for AP activity. As shown in
Figure. 13, N4156::Pm-ftsY pWGT2 grew more slowly in the absence of arabinose,
indicative of SRP malfunction, as expected. The doubling times for N4156::Pm-ftsY and
N4156::Pm-ftsY pWGT2 grown in the presence of arabinose were 1.52 and 1.59 hours,
respectively. In the absence of arabinose, N4156::Pm-ftsY pWGT2 had a doubling time
of 1.82 hours. In both intact and cells lysed with chloroform and SDS, increased activity
was detected in the absence of arabinose (Figure 14). For both strains, lysis of the cells
resulted in a slight increase of detected AP activity. Such an increase is consistent with

detection, of minimal quantities of un-exported AP.

46

 

 

3.000 - -. -_..--W---c-- _.__

2.500 Iv, i

2.000 ,_-

1.500 , . F——-

A600

1.000 ~77

0.500 WL_W ,

 

 

 

 

0.000

 

Time (h)

Figure 13. Growth defects as a result of SRP deficiency in E. coli N4156::P.,.-fisY.
Bacteria were grown in minimal medium M9 with or without 0.2% arabinose. Aliquots
were removed every two hours and A600 measured. 6 - N4156::Para-ftsY. I -

N4156::Para-fisYpWGT2 + arabinose. o - N4156::Para-ftsY pWGT2 — arabinose.

47

90

 

 

80
70
60 1
‘ I + Arabin0Se i

50 .
% it] -Arabir£seia

AP Activity

40

30

20

 

 

 

 

 

 

 

 

Intact Lysed

Figure 14. Effect of ftsY depletion on MtrD-PhoA activity in N4156::Pm-ftsY
pWGT2. Bacteria were grown for 8 h in minimal M9 medium with or without 0.2%
arabinose. Cells were pelletted, resuspended in PBS, and assayed for AP activity. Units

of activity are umol pNPP hydrolyzed min"1 pg protein'l.

48

Conclusions and Perspectives

The goal of this work was to develop a means to assess SRP function in vivo in N.
gonorrhoeae. The hypothesis was that a strain conditionally expressing pilA would be
dependent on the inducer for SRP-dependent protein export. In order to reduce SRP
function, pilA transcription was reduced by growing the conditional pilA strain, MP5, in
media lacking the inducer IPTG. Decreased transcription should lead to a reduction in
the pool of available PilA, and subsequent reduction in SRP transport.

In our experiments, the tac promoter driving pilA in MP5 is not completely
repressed in the absence of IPTG. Semi-quantitative RT-PCR analysis shows that
detectable levels of transcript remain afier 8 hours growth in the absence of IPTG. As
prokaryotic mRNAs are short-lived, it is likely that the remaining signal is due to
persistent, low-level transcription,

Strain MP5 was constructed to measure the effects of SRP depletion and as a
background in which to assess SRP dependence of a MtrD-PhoA reporter in GC. To do
this, it is necessary first to demonstrate that manipulation of the tac-driven pilA gene—
either by induction or repression—results in measurable defects in SRP-mediated protein
transport. The first measure of SRP function was cell viability. Passage of MP5 on GC
agar with no IPTG showed no significant changes in number or size of colonies, nor any
visible indication of a growth defect. In liquid culture, similar results were observed in
that no decrease in growth rate was observed in the absence of IPTG. To assess the
ability of pilA repression to affect IMP transport, we adopted a single protein approach,

whereby the transport of a specific protein through the SRP was measured in both the

49

presence and absence of IPTG. IPTG-dependent alterations of transport, either by
decrease of the amount of protein detected at the membrane, or by aggregation of mis-
transported protein in the cytoplasm, would indicate perturbation of the SRP pathway.
The first protein tested was pilin, the major subunit of the gonococcal type IV pilus. The
identification of pilin as a potential substrate for SRP transport was based on previous
reports showing that PilA binds to DNA immediately upstream of pilE (12).
Mistransport of pilin under conditions of SRP limitation would serve as confirmation of a
role of the SRP in pilin transport, and would suggest that the conditional pilA in strain
MP5 can be repressed sufficiently to induce transport defects. However, no difference in
PilE transport was observed in the absence of IPTG (Figure 8).

MtrD is the gonococcal homolog of Ach, which has been shown to be SRP-
dependent in E. coli (92). Function of MtrD as an integral part of the mtr multidrug efflux
pump should correlate with its transport to the IM. SRP deficiencies resulting in a
decrease of MtrD in the IM would be expected to result in a concurrent decrease in the
number of functional efflux pumps available to mediate resistance to hydrophobic agents,
and therefore a decrease in the MIC for such agents. To test this, we measured the MIC
of several HAS, some known to be Mtr substrates and some that were not, in both M811
and MP5 in the presence and absence of IPTG. For each HA tested, the MIC of strain
MP5 decreased following 18 h growth in the absence of IPTG. For strain MS11, there
should be little or no change in MIC in the presence or absence of IPTG. IPTG-
dependent MIC decreases were observed in strain MSll for penicillin, ciprofloxacin, and
bile salts. Only for penicillin is the decrease in MS11 proportional to that in MP5. This

could be evidence of one of the drawbacks to the agar dilution method. Penicillin

50

dilutions over a smaller interval between 0.5 ug/ml and 0.75 ug/ml might more accurately
quantify susceptibility of the two strains to this agent. Further experimentation including
more HAS, including agents effluxed by the related FarAB and NorM pumps as well as
agents known to be unaffected by Mtr, will be necessary to determine whether the
observed results are specific to SRP status.

The relative levels of the SRP components and targets are known to be critical for
function and ultimately cell viability (140). Therefore, we asked whether overproduction
of PilA affected growth of gonococci. Immunoblot analysis clearly demonstrates that
significant excess PilA is produced in the presence of 500 uM IPTG (Figure 4).
However, viability of MP5 at 500 nM IPTG, tested out to two serial passages, was not
appreciably affected.

Due to the inability to conclusively detect a decrease in PilA below
wildtype levels, coupled with the lack of a strong phenotype indicative of disruption of
SRP function, we must consider the possibility that the conditional pilA strain MP5 does
not repress pilA sufficiently to induce SRP malfunction. Several methods are available to
enhance repression of this system. First, it would be beneficial to insert multiple tandem
copies of the tac operator upstream of the conditional pilA. This would provide multiple
binding sites for the lac repressor, increasing the probability that pilA expression would
be inhibited in the absence of [PT G. It is important to ensure that LacI is being produced
within the bacterium. RT—PCR analysis of pilA expression indicates a decrease in
transcription in the absence of IPTG, which has been attributed to the action of LacI, but
confirmation of [ac] expression could be easily obtained using a similar RT-PCR

approach.

51

Alkaline phosphatase is an ideal reporter for transport across the IM because it
only becomes enzymatically active once it is in the periplasm (22). Experiments
measuring the AP activity in intact cells will detect only AP that has been properly
exported from the cytoplasm. Fusion of the AP reporter to a periplasmic domain of the
C-terminal portion of an SRP transported protein allows for measurement of the ability of
the SRP to mediate protein export. As N. gonorrhoeae has no endogenous alkaline
phosphatase activity, all AP activity measured in the fusion-bearing derivatives can be
attributed to the fusion itself. The MtrD-PhoA fusion protein was first detected by growth
on agar plates containing X—phos, which produces a blue color in colonies producing
alkaline phosphatase. The mtrD-phoA fusion strain, MT2 was very light blue on GC-X-
phos agar. However, the product of X-phos cleavage by AP is reportedly toxic to
gonococci, thus we next quantified AP activity using pNPP, a colorimetric AP substrate.
The advantage of this assay is that it is adaptable to both liquid culture and plate-grown
bacteria and can be easily scaled to accomodate large numbers of samples. An initial
experiment using this assay was conducted to measure AP activity for newly-constructed
N. gonorrhoeae strains MT2, PT2, and M1366T2. As no AP activity is detected in the
parent strains MS11, MPS, and M1366, it can be assumed that all activity measured in
subsequent experiments is specific to the MtrD-PhoA fusion.

Transformation of the mtrD-phoA reporter into a AmtrR background (39) allowed
for verification of the fimction of the promoter upstream of mtrD, Pm”, which is known to
be repressed by MtrR (56, 57, 60). The 1.4-fold increase of AP activity in the absence of

mtrR is indicative that derepression of the operon is resulting in greater expression of the

52

reporter. Thus, the mtr promoter region appears to be intact and functional, which will be
important for studies of the effects of PilA binding to this region. In order to rule out
effects of SRP imbalance on mtrD-phoA transcription, RT-PCR was used to assess the
transcription of mtrD-phoA under varied levels of IPTG in strain PT2, which
conditionally expresses pilA. No change was observed (Figure 10), confirming that the
construct is uniformly expressed and transcription is not affected by PilA.

Measurement of AP activity in the presence of varied IPTG concentrations
showed no transport defect under conditions of under- or overexpression of pilA.
Because SRP deficiency could have pleitropic effects on the cell, it is possible that these
assays are not detecting only the properly-transported AP. Another possibility is that the
reporter is being transported by other means in the absence of a fully-functional SRP.
We also used of this assay to measure AP export in E. coli N4156::Pm-ftsY to examine
MtrD-PhoA export in this heterologous system. The lack of detectable defects in
transport of the reporter in this system could suggest differences between the E. coli and
N. gonorrhoeae SRP systems and/or the dependence of MtrD on the SRP for export.

To assess MtrD’-‘PhoA protein production independent of enzymatic activity,
cellular fractions were immunoblotted using an antibody against PhoA. The results of
these experiments again showed no change in expression or transport of the MtrD-PhoA
fusion in response to IPTG concentrations (Figure 11). The maintenance of a consistent
amount of total fusion protein correlated with the RT—PCR results, indicating that SRP
status has no effect on the translation of the mtrD-phoA fusion.

The fact that strain MP5 can survive serial passage in the absence of IPTG, and

the relatively moderate effects on MICs under these conditions indicate that the decrease

53

in the amount properly transported protein at the membrane is unlikely to be significant.
The observation that Western blots on these fractions did not detect a change is therefore
not sufficient to say that the change does not exist. It is clear from these results that
future studies are still necessary to address the role of the SRP in gonococcal protein
export.

This work describes the initial stages in the development of a system for study of
SRP-mediated protein transport in Neisseria gonorrhoeae. In its current state, this
system lacks the power to induce and measure defects in SRP function. Areas for
improvement have been identified, both in the conditional pilA strain and in the mtrD-
phoA reporter. Future studies incorporating these enhancements should serve to establish
a system with the potential use of investigating the potential role of DNA in SRP-

mediated protein transport.

54

WORKS CITED

55

Works Cited

CDC. Sexually Transmitted Disease Surveillance 2004 Supplement: Gonococcal
Isolate Surveillance Project (GISP) Annual Report - 2004. Atlanta, Georgia: US.
Department of Health and Human Services, December 2005.

Centers for Disease Control and Prevention, F luoroquinolone-resistance in
Neisseria gonorrhoeae, Hawaii, 1999, and decreased susceptibility to
azithromycin in N gonorrhoeae, Missouri, 1999, MMWR Morb Mortal Wkly Rep
49 (2000), pp. 833—837.

Centers for Disease Control and Prevention. Sexually Transmitted Disease
Surveillance 2005 Supplement, Gonococcal Isolate Surveillance Project (GISP)
Annual Report 2005. Atlanta, GA: US. Department of Health and Human
Services, Centers for Disease Control and Prevention, January 2007.

2002. Increases in Fluoroquinolone-Resistant Neisseria gonorrhoeae--Hawaii and
California, 2001. JAMA 288:2961-2963.

, posting date. STADEN DNA sequence analysis package. [Online.]

2007. Update to CDC's Sexually Transmitted Diseases Treatment Guidelines,

2006: Fluoroquinolones No Longer Recommended for Treatment of Gonococcal
Infections. MMWR 56:332-336.

Adams, H., P. A. Scotti, H. De Cock, J. Luirink, and J. Tommassen. 2002.
The presence of a helix breaker in the hydrophobic core of signal sequences of
secretory proteins prevents recognition by the signal-recognition particle in
Escherichia coli. European Journal Of Biochemistry / FEBS 269:5564-5571.

Alder, N. N., and S. M. Theg. 2003. Energetics of Protein Transport across
Biological Membranes: A Study of the Thylakoid [Delta]pH-Dependent/cpTat
Pathway. Cell 112:231-242.

Angelini, S., D. Boy, E. Schiltz, and H.-G. Koch. 2006. Membrane binding of
the bacterial signal recognition particle receptor involves two distinct binding
sites. J. Cell Biol. 174:715-724.

56

10.

ll.

12.

13.

14.

15.

l6.

17.

18.

19.

Angelini, S., S. Deitermann, and H.—G. Koch. 2005. FtsY, the bacterial signal-
recognition particle receptor, interacts functionally and physically with the
SecYEG translocon. EMBO Reports 6:476—48 l.

Arvidson, C. G., T. Powers, P. Walter, and M. So. 1999. Neisseria
gonorrhoeae PilA is an FtsY homolog. J. Bacteriol. 181 :731-739.

Arvidson, C. G., and M. So. 1995. Interaction of the Neisseria gonorrhoeae PilA

protein with the pilE promoter involves multiple sites on the DNA. J. Bacteriol.
177 :2497-504.

Arvidson, C. G., and M. So. 1995. The Neisseria transcriptional regulator PilA
has a GTPase activity. J. Biol. Chem. 270:26045-8.

Avdeeva, O. N., A. G. Myasnikov, P. V. Sergiev, A. A. Bogdanov, R.
Brimacombe, and O. A. Dontsova. 2002. Construction of the 'minimal' SRP that

interacts with the translating ribosome but not with specific membrane receptors
in Escherichia coli. FEBS Letters 514:70-73.

Batey, R. T., R. P. Rambo, L. Lucast, B. Rha, and J. A. Doudna. 2000. Crystal
structure of the ribonucleoprotein core of the signal recognition particle. Science
287 :1232-1239.

Beck, K., L.-F. Wu, J. Brunner, and M. Muller. 2000. Discrimination between
SRP- and SecA/SecB-dependent substrates involves selective recognition of
nascent chains by SRP and trigger factor. EMBO J 19:134-143.

Berg, B. v. d., W. M. Clemons, I. Collinson, Y. Modis, E. Hartmann, S. C.
Harrison, and T. A. Rapoport. 2004. X-ray structure of a protein-conducting
channel. 427:36-44.

Bernstein, H. D., and J. B. Hyndman. 2001. Physiological Basis for
Conservation of the Signal Recognition Particle Targeting Pathway in Escherichia
coli. J. Bacteriol. 183:2187-2197.

Bernstein, H. D., D. Zopf, D. M. Freymann, and P. Walter. 1993. Functional
substitution of the signal recognition particle 54-kDa subunit by its Escherichia
coli homolog. Proc Natl Acad Sci U S A 90:5229-5233.

57

20.

21.

22.

23.

24.

25.

26.

27.

28.

Bibi, E., A. A. Herskovits, E. S. Bochkareva, and A. Zelazny. 2001. Putative
integral membrane SRP receptors. Trends in Biochemical Sciences 26:15-16.

Bogsch, E. G., F. Sargent, N. R. Stanley, B. C. Berks, C. Robinson, and T.
Palmer. 1998. An Essential Component of a Novel Bacterial Protein Export

System with Homologues in Plastids and Mitochondria. J. Biol. Chem.
273:18003-18006.

Brickman, E., and J. Beckwith. 1975. Analysis of the regulation of Escherichia
coli alkaline phosphatase synthesis using deletions and [phi]80 transducing
phages. Journal of Molecular Biology 96:307-316.

Brundage, L., J. P. Hendrick, E. Schiebel, A. J. M. Driessen, and W.
Wickner. 1990. The purified E. coli integral membrane protein SecY/E is

sufficient for reconstitution of SecA-dependent precursor protein translocation.
Cell 62:649-657.

Cabelli, R., K. Dolan, L. Qian, and D. Oliver. 1991. Characterization of
membrane-associated and soluble states of SecA protein from wild-type and
SecA51(TS) mutant strains of Escherichia coli. J. Biol. Chem. 266:24420-24427.

Cianciotto, N. P. 2005. Type II secretion: a protein secretion system for all
seasons. Trends in Microbiology 13:581-588.

Cohen, M. S., I. F. Hoffman, R. A. Royce, P. Kazembe, J. R. Dyer, C. C.
Daly, D. Zimba, P. L. Vernazza, M. Maida, S. A. Fiscus, and J. J. J. Eron.
1997. Reduction of concentration of HIV -1 in semen after treatment of urethritis:

implications for prevention of sexual transmission of HIV -1. AIDSCAP Malawi
Research Group. Lancet 349: 1 868-1873.

Crane, J. M., Y. Suo, A. A. Lilly, C. Mao, W. L. Hubhell, and L. L. Randall.
2006. Sites of Interaction of a Precursor Polypeptide on the Export Chaperone

SecB Mapped by Site-directed Spin Labeling. Journal of Molecular Biology
363:63-74.

Criss, A. K., K. A. Kline, and H. S. Seifert. 2005. The frequency and rate of
pilin antigenic variation in Neisseria gonorrhoeae. Molecular Microbiology
58:5 10-5 19.

58

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

Crooke E, Guthrie B, Lecker S, Lill R, and W. W. 1988. ProOmpA is
stabilized for membrane translocation by either purified E. coli trigger factor or
canine signal recognition particle. Cell 54: 1003-1 1.

Dalbey, R. E., and C. Robinson. 1999. Protein translocation into and across the
bacterial plasma membrane and the plant thylakoid membrane. Trends in
Biochemical Sciences 24:17-22.

de Leeuw, E., D. Poland, 0. Mol, I. Sinning, C. M. ten Hagen-Jongman, B.
Oudega, and J. Luirink. 1997. Membrane association of F tsY, the E. coli SRP
receptor. FEBS Letters 416:225-229.

Delahay, R., B. Robertson, J. Balthazar, W. Shafer, and C. Ison. 1997.
Involvement of the gonococcal MtrE protein in the resistance of Neisseria
gonorrhoeae to toxic hydrophobic agents. Microbiology 143:2127-2133.

deLeeuw, E., K. teKaat, C. Moser, G. Menestrina, R. Demel, B. Dekruijff, B.
Oudega, J. Luirink, and I. Sinning. 2000. Anionic phospholipids are involved
in membrane association of F tsY and stimulate its GTPase activity. EMBO J
19:53 1-541 .

Desvaux, M., N. J. Parham, A. Scott-Tucker, and I. R. Henderson. 2004. The
general secretory pathway: a general misnomer? Trends in Microbiology 12:306-
309.

Di Cola, A., E. Klostermann, and C. Robinson. 2005. The complexity of
pathways for protein import into thylakoids: it's not easy being green. Biochem
Soc Trans 33:1024-7.

Dilks, K., R. W. Rose, E. Hartmann, and M. Pohlschroder. 2003. Prokaryotic
Utilization of the Twin-Arginine Translocation Pathway: a Genomic Survey. J.
Bacteriol. 185: 1478-1483.

Du, Y., and C. G. Arvidson. 2003. Identification of ZipA, an SRP-targeted
protein from Neisseria gonorrhoeae. J Bacteriol 185:2122-2130.

Du, Y., and C. G. Arvidson. 2006. RpoH mediates the expression of some, but
not all genes induced in Neisseria gonorrhoeae adherent to epithelial cells. Infect.

- Immun. 74:2767-2776.

59

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

Du, Y., J. Lenz, and C. G. Arvidson. 2005. Global gene expression and the role
of sigma factors in Neisseria gonorrhoeae in interactions with epithelial cells.
Infect Immun 73:4834-4845.

Edwards, J. L., and M. A. Apicella. 2002. The role of lipooligosaccharide in
Neisseria gonorrhoeae pathogenesis of cervical epithelia: lipid A serves as a C3
acceptor molecule. Cell. Microbiol. 4:585-598.

Egea, P. F., S.-o. Shan, J. Napetschnig, D. F. Savage, P. Walter, and R. M.
Stroud. 2004. Substrate twinning activates the signal recognition particle and its
receptor. 427 :21 5-221 .

Eichacker, L. A., and R. Henry. 2001. Function of a chloroplast SRP in
thylakoid protein export. Biochimica et Biophysica Acta (BBA) - Molecular Cell
Research 1541:120-134.

Eilers, M., and G. Schatz. 1986. Binding of a specific ligand inhibits import of a
purified precursor protein into mitochondria. 322:228-232.

Eisner, G., H.-G. Koch, K. Beck, J. Brunner, and M. Muller. 2003. Ligand
crowding at a nascent signal sequence. J. Cell Biol. 163:35-44.

Fekkes, P., and A. J. M. Driessen. 1999. Protein targeting to the bacterial
cytoplasmic membrane. Microbiol and Mol Biol Rev 63:161-173.

Filloux, A. 2004. The underlying mechanisms of type H protein secretion.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1694:163-179.

Flanagan, J. J., J.-C. Chen, Y. Miao, Y. Shao, J. Lin, P. E. Bock, and A. E.
Johnson. 2003. Signal Recognition Particle Binds to Ribosome-bound Signal

Sequences with Fluorescence-detected Subnanomolar Affinity That Does Not
Diminish as the Nascent Chain Lengthens. J. Biol. Chem. 278:18628-18637.

Focia, P. J., I. V. Shepotinovskaya, J. A. Seidler, and D. M. Freymann. 2004.
Heterodimeric GTPase Core of the SRP Targeting Complex. Science 303:373-
377.

Focia, P. J., I. V. Shepotinovskaya, J. A. Seidler, and D. M. Freymann. 2004.
Heterodimeric GTPase core of the SRP targeting complex. Science 303:373-377.

60

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

Frasz, C., and C. G. Arvidson. 2003. A role for both DNA a_n_d_ RNA in GTP
hydrolysis by the Neisseria gonorrhoeae signal recognition particle receptor. J
Bacteriol 185:801-808.

Freymann, D. M., R. J. Keenan, R. M. Stroud, and P. Walter. 1999.
Functional changes in the structure of the SRP GTPase on binding GDP and
Mg2+ GDP. Nature Struct Biol 6:793-801.

Ghosh, P. 2004. Process of Protein Transport by the Type III Secretion System.
Microbiol. Mol. Biol. Rev. 68:771-795.

Gilmore, R., G. Blobel, and P. Walter. 1982. Protein translocation across the
endoplasmic reticultnn. 1. Detection in the microsomal membrane of a receptor
for the signal recognition particle. J. Cell Biol. 95:463-469.

Gouffi, K., C.-L. Santini, and L.-F. Wu. 2002. Topology determination and
fimctional analysis of the Escherichia coli TatC protein. FEBS Letters 525:65-70.

Gundelfinger, E. D., E. Krause, M. Melli, and B. Dobberstein. 1983. The
organization of the 7SL RNA in the signal recognition particle. Nucleic Acids
Research 11:7363-7374.

Hagman, K., C. Lucas, J. Balthazar, L. Snyder, M. Nilles, R. Judd, and W.
Shafer. 1997. The MtrD protein of Neisseria gonorrhoeae is a member of the

resistance/nodulation/division protein family constituting part of an efflux system.
Microbiology 143:21 17-2125.

Hagman, K., W. Pan, B. Spratt, J. Balthazar, R. Judd, and W. Shafer. 1995.
Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is
modulated by the mtrRCDE efflux system. Microbiology 141 :61 1-622.

Hagman, K. E., C. E. Lucas, J. T. Balthazar, L. A. Snyder, M. L. Nilles, R. C.
Judd, and W. M. Shafer. 1997. The MtrD protein of Neisseria gonorrhoeae is a

member of the resistance/nodulation/division protein family constituting part of
an efflux system. Microbiology 143:2117-2125.

Hagman, K. E., W. P. Pan, B. Spratt, J. T. Balthazar, R. C. Judd, and W. M.
Shafer. 1995. Resistance of Neisseria gonorrhoeae to antimicrobial agents is
modulated by the mtrRCDE efflux system. Microbiology 141:611-622.

61

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

Hagman, K. E., and W. M. Shafer. 1995. Transcriptional control of the mtr
efflux system of Neisseria gonorrhoeae. J Bacteriol 177:4162-4165.

Halic, M., T. Becker, M. R. Pool, C. M. T. Spahn, R. A. Grassucci, J. Frank,
and R. Beckmann. 2004. Structure of the signal recognition particle interacting
with the elongation-arrested ribosome. 427 :808-814.

Hartl, F.-U., S. Lecker, E. Schiebel, J. P. Hendrick, and W. Wickner. 1990.
The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to
the E. coli plasma membrane. Cell 63:269-279.

Henderson, 1. R., F. Navarro-Garcia, and J. P. Nataro. 1998. The great escape:
structure and function of the autotransporter proteins. Trends in Microbiology
6:370-3 78.

Herskovits, A. A., and E. Bibi. 2000. Association of Escherichia coli ribosomes
with the inner membrane requires the signal recognition particle receptor but is
independent of the signal recognition particle. Proc Natl Acad Sci U S A 97:4621-
4626.

Herskovits, A. A., E. Shimoni, A. Minsky, and E. Bibi. 2002. Accumulation of
endoplasmic membranes and novel membrane-bound ribosome-signal recognition

particle receptor complexes in Escherichia coli. The Journal Of Cell Biology
159:403-410.

Hynds, P. J., D. Robinson, and C. Robinson. 1998. The Sec-independent Twin-
arginine Translocation System Can Transport Both Tightly Folded and Malfolded
Proteins across the Thylakoid Membrane. J. Biol. Chem. 273:34868-34874.

Ilver, D., H. Kallstrom, S. Normark, and A. B. Jonsson. 1998. Transcellular
passage of Neisseria gonorrhoeae involves pilus phase variation. Infect Irnmun
66:469-473.

Jagath, J. R., M. V. Rodnina, G. Lentzeu, and W. Wintermeyer. 1998.
Interaction of guanine nucleotides with the signal recognition particle from
Escherichia coli. Biochemistry 37: 1 5408-1 5413.

Jain, S., P. van Ulsen, I. Benz, M. A. Schmidt, R. Fernandez, J. Tommassen,
and M. B. Goldberg. 2006. Polar Localization of the Autotransporter Family of
Large Bacterial Virulence Proteins. J. Bacteriol. 188:4841-4850.

62

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

Jerse, A. E., N. D. Sharma, A. N. Simms, E. T. Crow, L. A. Snyder, and W.
M. Shafer. 2003. A Gonococcal Efflux Pump System Enhances Bacterial
Survival in a Female Mouse Model of Genital Tract Infection. Infect. Immun.
71 :5576-5582.

Johnson, T. L., J. Abendroth, W. G. J. Hol, and M. Sandkvist. 2006. Type II
secretion: fi'om structure to function. FEMS Microbiology Letters 255:175-186.

Jorgensen, J. H., S. A. Crawford, and K. R. Fiebelkorn. 2005. Susceptibility of
Neisseria meningitidis to 16 Antimicrobial Agents and Characterization of
Resistance Mechanisms Affecting Some Agents. J. Clin. Microbiol. 43:3162-
3171.

Kallstrom, H., M. S. Islam, P.-O. Berggren, and A.-B. Jonsson. 1998. Cell
Signaling by the Type IV Pili of Pathogenic Neisseria. J. Biol. Chem. 273:21777-
21782.

Kaplan, D. A., L. Greenfield, T. Boone, and G. Wilcox. 1978. Hybrid plasmids
containing the araBAD genes of Escherichia coli B/r. Gene 3:177-189.

Keenan, R. J., D. M. Freymann, R. M. Stroud, and P. Walter. 2001. The
signal recognition particle. Annu. Rev. Biochem. 70:755-775.

Kellogg, D., 1. Cohen, L. Norins, A. Schroeter, and G. Reising. 1968. Neisseria
gonorrhoeae. II. Colonial variation and pathogenicity during 35 months in vitro.
J. Bacteriol. 96:596-605.

Kellogg, D. 8., Jr., 1. R. Cohen, L. C. Norins, A. L. Schroeter, and G. Reising.
1968. Neisseria gonorrhoeae II. Colonial Variation and Pathogenicity During 35
Months In Vitro. J. Bacteriol. 96:596-605.

Kellogg, D. S., J r., W. L. Peacock, Jr., W. E. Deacon, L. Brown, and C. I.
Pirkle. 1963. NEISSERIA GONORRHOEAE I.: Virulence Genetically Linked to
Clonal Variation. J. Bacteriol. 85:1274-1279.

Kline, K. A., A. K. Criss, A. Wallace, and H. S. Seifert. 2007. Transposon
Mutagenesis Identifies Sites Upstream of the Neisseria gonorrhoeae pilE Gene
That Modulate Pilin Antigenic Variation. J. Bacteriol. 189:3462-3470.

63

80.

81.

82.

83.

84.

85.

86.

87.

88.

Koch, H.-G., T. Hengelage, C. Neumann-Haefelin, J. MacFarlane, H. K.
Hoffschulte, K.-L. Schimz, B. Mechler, and M. Muller. 1999. In vitro studies
with purified components reveal signal recognition particle (SRP) and SecA/SecB
as constituents of two independent protein-targeting pathways of Escherichia coli.
Mol Biol Cell 10:2163-2173.

Koonin, E. V., and A. E. Gorbalenya. 1992. Autogenous translation regulation
by Escherichia coli ATPase SecA may be mediated by an intrinsic RNA helicase
activity of this protein. FEBS Letters 298:6-8.

Kuglstatter, A., C. Oubridge, and K. Nagai. 2002. Induced structural changes
of 7SL RNA during the assembly of human signal recognition particle. 9:740-
744.

Kuhn, R. E. D. a. A. 2004. YidC family members are involved in the membrane
insertion, lateral integration, folding, and assembly of membrane proteins. J Cell
Biol 166:769-774.

Laan, M. v. d., N. P. Nouwen, and A. J. Driessen. 2005. YidC - an evolutionary
conserved device for the assembly of energy-transducing membrane protein
complexes. Current Opinion in Microbiology 8: 1 82-187.

Laga, M., A. Manoka, M. Kivuvu, B. Malele, M. Tuliza, N. Nzila, J. Goeman,
F. Behets, V. Batter, M. Alary, W. L. Heyward, R. W. Ryder, and P. Piot.
1993. Non-ulcerative sexually transmitted diseases as risk factors for HIV-1
transmission in women: results from a cohort study. AIDS 7 :95-102.

Lee, E. H., and W. M. Shafer. 1999. The farAB—encoded efflux pump mediates
resistance of gonococci to long-chained antibacterial fatty acids. Mol Microbiol
33:839-845.

Lee, H. C., and H. D. Bernstein. 2001. The targeting pathway of Escherichia
coli presecretory and integral membrane proteins is specified by the
hydrophobicity of the targeting signal. Proc Natl Acad Sci USA 98:3471-3476.

Lee, S. W., D. L. Higashi, A. Snyder, A. J. Merz, L. Potter, and M. So. 2005.
PilT is required for PI(3,4,5)P3-mediated crosstalk between Neisseria
gonorrhoeae and epithelial cells. Cellular Microbiology 7: 1271 -1284.

64

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

Lill, R., W. Dowhan, and W. Wickner. 1990. The ATPase activity of secA is
regulated by acidic phospholipids, secY, and the leader and mature domains of
precursor proteins. Cell 60:271-280.

Lucas, C. E., J. T. Balthazar, K. E. Hagman, and W. M. Shafer. 1997. The
MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of
Neisseria gonorrhoeae. J Bacteriol 179:4123-4128.

Luirink, J., G. v. Heijne, E. Houhen, and J.-W. d. Gier. 2005. BIOGENESIS
OF INNER MEMBRANE PROTEINS IN ESCHERICHIA COLI. Annual
Review of Microbiology 59:329-355.

Luirink, J., C. M. ten Hagen-Jongman, C. C. van der Weijden, B. Oudega, S.
High, B. Dobberstein, and R. Kusters. 1994. An alternative protein targeting
pathway in Escherichia coli: studies on the role of FtsY. EMBO J 13:2289-2296.

Mainprize, I. L., D. R. Beniac, E. Falkovskaia, R. M. Cleverley, L. M.
Gierasch, F. P. Ottensmeyer, and D. W. Andrews. 2006. The Structure of

Escherichia coli Signal Recognition Particle Revealed by Scanning Transmission
Electron Microscopy. Mol. Biol. Cell 17:5063-5074.

Maity, T. S., C. W. Leonard, M. A. Rose, H. M. Fried, and K. M. Weeks.
2006. Compartmentalization Directs Assembly of the Signal Recognition Particle.
Biochemistry 45: 14955-14964.

Mason N, Ciufo LF, and B. JD. 2000. Elongation arrest is a physiologically
important function of signal recognition particle. EMBO J 19:4164-74.

McNeil, G., and M. Virji. 1997. Phenotypic variants of meningococci and their
potential in phagocytic interactions: the influence of opacity proteins, pili, PilC
and surface sialic acids. Microb Pathog 22:295-304.

Mehr, I. J., and H. S. Seifert. 1998. Differential roles of homologous
recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA
transformation and DNA repair. Molecular Microbiology 30:697-710.

Meyer, D. 1., E. Krause, and B. Dobberstein. 1982. Secretory protein
translocation across membranes--the role of the ‘docking protein/'. 297:647-650.

65

99.

1. 00.

101.

102.

103.

104.

105.

106.

107.

Meyer, T. F., E. Billyard, R. Haas, S. Storzbach, and M. So. 1984. Pilus genes
of Neisseria gonorrheae: chromosomal organization and DNA sequence. Proc.
Natl. Acad. Sci. USA 81:6110-4.

Millman, J. S., and D. W. Andrews. 1999. A site-specific, membrane-dependent
cleavage event defines the membrane binding domain of FtsY. J Biol Chem
274:33227-33234.

Millman, J. S., H.-Y. Qi, F. Vulcu, H. D. Bernstein, and D. W. Andrews.
2001. F tsY binds to the Escherichia coli inner membrane via interactions with

phosphatidylethanolamine and membrane proteins. J. Biol. Chem. 276:25982-
25989.

Mitra, K., J. Frank, and A. Driessen. 2006. C0- and post-translational
translocation through the protein-conducting channel: analogous mechanisms at
work? 13:957-964.

Moser, C., 0. Mol, R. Goody, and I. Sinning. 1997. The signal recognition
particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built
into the GTPase domain. Proc Natl Acad Sci USA 94:11339-11344.

Nakamura, K., Y. Imai, A. Nakamura, and K. Yamane. 1992. Small
cytoplasmic RNA of Bacillus subtilis: functional relationship with human signal
recognition particle 7S RNA and Escherichia coli 4.58 RNA. J Bacteriol
174:2185-92.

Nakamura, K., S. Yahagi, T. Yamazaki, and K. Yamane. 1999. Bacillus
subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle
that can bind the Alu domain of small cytoplasmic RNA. J Biol Chem 274:13569-
13576.

Nakamura, K., S.-i. Yahagi, T. Yamazaki, and K. Yamane. 1999. Bacillus
subtilis Histone-like Protein, HBsu, Is an Integral Component of a SRP-like
Particle That Can Bind the Alu Domain of Small Cytoplasmic RNA. J. Biol.
Chem. 274:13569-13576.

Nakatogawa H, and I. K. 2002. The ribosomal exit tunnel functions as a
discriminating gate. Cell 108:629-36.

66

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

Neumann-Haefelin, C., U. Schafer, M. Muller, and H.-G. Koch. 2000. SRP-
dependent co-translational targeting and SecA-dependent translocation analyzed
as individual steps in the export of a bacterial protein. EMBO J 19:6419-6426.

Newitt, J. A., N. D. Ulbrandt, and H. D. Bernstein. 1999. The structure of
multiple polypeptide domains determines the signal recognition particle

requirement of Escherichia coli inner membrane proteins. J Bacteriol 181:4561-
4567.

Nikaido, H. 1996. Multidrug efflux pumps of gram-negative bacteria. J.
Bacteriol. 178:5853-5859.

Ogg, S. C., W. P. Barz, and P. Walter. 1998. A Functional GTPase Domain, but
not its Transmembrane Domain, is Required for Function of the SRP Receptor
beta -subunit. J. Cell Biol. 142:341-354.

Paetzel, M., R. E. Dalbey, and N. C. J. Strynadka. 1998. Crystal structure of a
bacterial signal peptidase in complex with a [beta]-lactam inhibitor. 396: 186-190.

Pan W, S. B. 1994. Regulation of the permeability of the gonococcal cell
envelope by the mtr system. Mol Microbiol 11:769-75.

Peterson, J. H., C. A. Woolhead, and H. D. Bernstein. 2003. Basic amino acids
in a distinct subset of signal peptides promote interaction with the signal
recognition particle. J Biol Chem 278:46155-46162.

Phillips, G. J., and T. J. Silhavy. 1992. The E. coliflh gene is necessary for
viability and efficient protein transport. Nature 359:744-746.

Politz, J. C., S. Yarovoi, S. M. Kilroy, K. Gowda, C. Zwieb, and T. Pederson.
2000. Signal recognition particle components in the nucleolus. PNAS 97 :55-60.

Poritz, M. A., H. D. Bernstein, K. Strub, D. Zopf, H. Wilhelm, and P. Walter.
1990. An E. coli ribonucleoprotein containing 4.5S RNA resembles mammalian
signal recognition particle. Science 250:1 1 11-1 117.

Prehn S, Wiedmann M, Rapoport TA, and Z. C. 1987. Protein translocation
across wheat germ microsomal membranes requires an SRP-like component.
EMBO J 6:2093-2097.

67

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

Pugsley, A. P. 1993. The complete general secretory pathway in gram—negative
bacteria. Microbiol Rev 57:50-108.

Raine A, Ullers R, Pavlov M, Luirink J, Wikberg JE, and E. M. 2003.
Targeting and insertion of heterologous membrane proteins in E. coli. Biochimie
85:659-68.

Reiser, R. C., and W. M. McCormack. 1994. Pelvic Inflammatory Disease. N
Engl J Med 330:1689-a—.

Ribes, V., K. Romisch, A. Giner, B. Dobberstein, and D. Tollervey. 1990. E.
coli 4.58 RNA is part of a ribonucleoprotein particle that has properties related to
signal recognition particle. Cell 63:591-600.

Robinson, C., and A. Bolhuis. 2004. Tat-dependent protein targeting in
prokaryotes and chloroplasts. Biochimica et Biophysica Acta (BBA) - Molecular
Cell Research 1694:135-147.

Rouquette, C., J. B. Harmon, and W. M. Shafer. 1999. Induction of the
mtrCDE-encoded efflux pump system of Neisseria gonorrhoeae requires MtrA, an
AraC-like protein. Molecular Microbiology 33:651-658.

Rouquette-Loughlin, C., S. A. Dunham, M. Kuhn, J. T. Balthazar, and W. M.
Shafer. 2003. The NorM efflux pump of Neisseria gonorrhoeae and Neisseria

meningitidis recognizes antimicrobial cationic compounds. J Bacteriol 185:1101-
1106.

Rouquette-Loughlin, C. E., J. T. Balthazar, and W. M. Shafer. 2005.
Characterization of the MacA-MacB efflux system in Neisseria gonorrhoeae. J.
Antimicrob. Chemother. 56:856-860.

Samuelsson, T., M. Olsson, P. M. Witkstrom, and B. R. Johannson. 1995. The
GTPase activity of the Escherichia coli Fflr protein is important for normal
growth. Biochim. Biophys. Acta. 1267 :83-91.

Sauer-Eriksson, A. E., and T. Hainzl. 2003. S-domain assembly of the signal
recognition particle. Current Opinion in Structural Biology 13:64-70.

68

129.

130.

131.

132.

133.

134.

135.

136.

137.

Scheuring, J., N. Braun, L. Nothdurft, M. Stumpf, A. K. J. Veenendaal, S.
Kol, C. van der Does, A. J. M. Driessen, and S. Weinkauf. 2005. The
Oligomeric Distribution of SecYEG is Altered by SecA and Translocation
Ligands. Journal of Molecular Biology 354:258-271.

Schierle, C. F., M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J.
Beckwith. 2003. The DsbA signal sequence directs efficient, cotranslational
export of passenger proteins to the Escherichia coli periplasm via the signal
recognition particle pathway. J Bacteriol 185:5706-5713.

Sega], E., E. Billyard, M. So, S. Storzbach, and T. F. Meyer. 1985. Role of
chromosomal rearrangement in N. gonorrhoeae pilus phase variation. Cell
40:293-300.

Seiser, R. M., and C. V. Nicchitta. 2000. The fate of membrane-bound
ribosomes following the termination of protein synthesis. The Journal Of
Biological Chemistry 275:33820-33827.

Seluanov, A., and E. Bibi. 1997. F tsY, the prokaryotic signal recognition particle
receptor homologue, is essential for biogenesis of membrane proteins. J Biol
Chem 272:2053-2055.

Serkin, C. D., and H. S. Seifert. 1998. Frequency of Pilin Antigenic Variation in
Neisseria gonorrhoeae. J. Bacteriol. 180: 1955-1958.

Settles, A. M., A. Yonetani, A. Baron, D. R. Bush, K. Cline, and R.
Martienssen. 1997. Sec-Independent Protein Translocation by the Maize Hcf106
Protein. Science 278: 1467-1470.

Sijbrandi, R., M. L. Urbanus, C. M. ten Hagen-Jongman, H. D. Bernstein, B.
Oudega, B. R. Otto, and J. Luirink. 2003. Signal recognition particle (SRP)-
mediated targeting and Sec-dependent translocation of an extracellular
Escherichia coli protein. The Journal Of Biological Chemistry 278:4654-4659.

Taha, M. K., M. So, H. S. Seifert, E. Billyard, and C. Marchal. 1988. Pilin
expression in Neisseria gonorrhoeae is under both positive and negative
transcriptional control. EMBO J 7 :4367-78.

69

138.

139.

140.

141.

142.

143.

144.

145.

146.

Tajima, S., L. Lauffer, V. Rath, and P. Walter. 1986. The signal recognition
particle receptor is a complex that contains two distinct polypeptide chains. J. Cell
Biol. 103:1167-1178.

Tozik, 1., Q. Huang, C. Zwieb, and J. Eichler. 2002. Reconstitution of the
signal recognition particle of the halophilic archaeon Haloferax volcanii. Nucl.
Acids Res. 30:4166-4175.

Ulbrandt, N. D., J. A. Newitt, and H. D. Bernstein. 1997. The E. coli signal
recognition particle is required for the insertion of a subset of inner membrane
proteins. Cell 88: 1 87-196.

Ullers, R. S., J. Luirink, N. Harms, F. Schwager, C. Georgopoulos, and P.
Genevaux. 2004. SecB is a bona fide generalized chaperone in Escherichia coli.
Proceedings of the National Academy of Sciences 101:7583-7588.

Urbanus, M. L., Pier A. Scotti, Linda Frtiderberg,l Annika seam Jan-
Willem L. de Gier,1 Josef Brunner,2 James C. Samuelson,3 Ross E. Dalbey,3
Bauke Oudega, and J oen Luirinka. 2001. Sec-dependent membrane protein
insertion: sequential interaction of nascent FtsQ with SecY and YidC. EMBO
Reports 2:524—529.

Valent, Q. A., P. A. Scotti, S. High, J.-W. L. deGier, G. von Heijne, G.
Lentzeu, W. Wintermeyer, B. Oudega, and J. Luirink. 1998. The Escherichia

coli SRP and SecB targeting pathways converge at the translocon. EMBO J
17:2504-2512.

Veal, W., A. Yellen, J. Balthazar, W. Pan, B. Spratt, and W. Shafer. 1998.
Loss-of-fimction mutations in the mtr efflux system of Neisseria gonorrhoeae.
Microbiology 144:621-627.

Veal, W. L., R. A. Nicholas, and W. M. Shafer. 2002. Overexpression of the
MtrC-MtrD-MtrE Efflux Pump Due to an mtrR Mutation Is Required for

Chromosomally Mediated Penicillin Resistance in Neisseria gonorrhoeae. J.
Bacteriol. 184:5619-5624.

Voelker, R., and A. Barkan. 1995. Two nuclear mutations disrupt distinct
pathways for targeting proteins to the chloroplast thylakoid. EMBO J 14:3905—
3914.

70

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

157.

von Heijne, G. 1985. Signal sequences : The limits of variation. Journal of
Molecular Biology 184:99-105.

Walter, P., and G. Blobel. 1983. Disassembly and reconstitution of signal
recognition particle. Cell 34:525-533.

Walter, P., and G. Blobel. 1981. Translocation of proteins across the
endoplasmic reticulum III. Signal recognition protein (SRP) causes signal
sequence-dependent and site- specific arrest of chain elongation that is released
by microsomal membrane. J. Cell Biol. 91 :557-561.

Walter, P., and V. R. Lingappa. 1986. Mechanism of Protein Translocation
Across the Endoplasmic Reticulum Membrane. Annual Review of Cell Biology
2 :499-5 16.

Weiner, J. H., P. T. Bilous, G. M. Shaw, S. P. Lubitz, L. Frost, G. H. Thomas,
J. A. Cole, and R. J. Turner. 1998. A Novel and Ubiquitous System for

Membrane Targeting and Secretion of Cofactor-Containing Proteins. Cell 93:93-
101.

Weir, S. S., P. J. Feldblum, R. E. Roddy, and L. Zekeng. 1994. Gonorrhea as a
risk factor for HIV acquisition [see comments]. Aids 8:1605-8.

Wolin, S., and P. Walter. 1989. Signal recognition particle mediates a transient
elongation arrest of preprolactin in reticulocyte lysate. J. Cell Biol. 109:2617-
2622.

Woolhead, C. A., P. J. McCormick, and A. E. Johnson. 2004. Nascent
Membrane and Secretory Proteins Differ in FRET-Detected Folding Far inside the
Ribosome and in Their Exposure to Ribosomal Proteins. Cell 116:725-736.

Xu, Z., J. D. Knafels, and K. Yoshino. 2000. Crystal structure of the bacterial
protein export chaperone SecB. 7:1 172-1177.

Yurist, S., I. Dahan, and J. Eichler. 2007. SRP19 Is a Dispensable Component
of the Signal Recognition Particle in Archaea. J. Bacteriol. 189:276-279.

Zelazny, A., A. Seluanov, A. Cooper, and E. Bibi. 1997 . The NG domain of the
prokaryotic signal recognition particle receptor, FtsY, is fully functional when

71

fused to an unrelated integral membrane polypeptide. Proc Natl Acad Sci U S A
94:6025-6029.

158. Zheng, N., and L. M. Gierasch. 1997. Domain interactions in E. coli SRP:
stabilization of M domain by RNA is required for effective signal sequence
modulation of NG domain. Mol. Cell 1:79-87.

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