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THE MODE OF ACTION OF PESTICIN
presented by

Donna Mae Ferber

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THE MODE OF ACTION OF PESTICIN

By

Donna Mae Ferber

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1980

ABSTRACT

THE MODE OF ACTION OF PESTICIN
By

Donna Mae Ferber

Pesticin is a bacteriocin produced by wild-type isolates of

Yersinia pestis, the causative agent of bubonic plague. Homogeneous

 

preparations of pesticin neither significantly inhibited the net
synthesis of deoxyribonucleic acid, ribonucleic acid, or protein in

Escherichia coli ¢ nor caused detectable degradation of deoxyribo-

 

nucleic acid jg_yjyg, However, incorporation of cell wall-specific
radioactive label into trichloroacetic acid-insoluble material by
growing cells was inhibited by pesticin which also promoted release
of such radioactivity. As judged by chromatography of double-
labeled murein and pesticin-dependent release of reducing sugars

but not 4-hydroxyacetamido sugars, pesticin exhibited N-acetylglucos-
amidase activity. Hydrolysis of mureinlipoprotein occurred over a
broad pH range with an optimum of 4.7.

Pesticin-resistant mutants were isolated from E. 5911 ¢, a
universal colicin indicator strain. Several pesticin-resistant
mutants showed cross-resistance to one or more of the group B
(tgggedependent) colicins, but not to those colicins of group A.
Mutants isolated as pesticin-resistant fell into five phenotypic

classes: Psr' (pesticin-resistant), Ebe' (excretes enterochelin,

Donna Mae Ferber

insensitive to all group B colicins), Ebe' (resistant to colicins
B, D, G, and M), TonB' (deficient in coupling membrane energy to
outer membrane receptors, resistant to all group B colicins), and
TonB/S4' (like TonB but also resistant 'U: colicin S4). Accordingly,
pesticin is closely related to the colicins of group B, as judged
by utilization of a similar TonB-dependent receptor system for

adsorption to sensitive cells.

ACKNOWLEDGMENTS

I wish to express my gratitude to R. R. Brubaker for his
strong support over the years, and to my committee members for
their sage guidance: L. R. Snyder, R. N. Band, R. J. Patterson,
and J. A. Breznak. In addition, I thank H. L. Sadoff, R. N.
Costilow, R. L. Uffen, and L. F. Velicer for the use of equipment

essential to my research.

ii

TABLE OF CONTENTS

 

Page
LIST OF TABLES . . . . . . . . . . . . . . . . iv
LIST OF FIGURES . . . . . . . . . . . . . . . v
INTRODUCTION . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . 3
Virulence Determinants of Y. pestis 3
Pesticin 7
Bacteriocins of Gram-Negative Bacteria 9
The Gram- -Negative Cell Envelope . l2
REFERENCES 15
ARTICLE 1: MODE OF ACTION OF PESTICIN:
N-ACETYLGLUCOSAMINIDASE ACTIVITY . . . . . . 21
ARTICLE 2: MUTATIONS IN ESCHERICHIA COLI ¢ PROMOTING
TOLERANCE AND RESISTANCE T0 PESTICIN AND
COLICINS. . . . . . . . . . 29

SUMMARY . . . . . . . . . . . . . . . . . . 52

iii

Table

LIST OF TABLES

LITERATURE REVIEW

Established virulence determinants in Yersinia pestis .

 

ARTICLE I

Organisms yielding mureinlipoprotein sensitive to
pesticin-dependent hydrolysis . .

ARTICLE 2

Titers of colicins and organisms used for their
production . . . . . . .

Number of isolates of mutant phenotypes of E. coli ¢
recovered by selection with coliphage T5 or various
bacteriocins . . . . . . . . . .

Lethality of colicins and pesticin tested against type

mutants of E. coli ¢ selected for insensitivity to
bacteriocins or inability to propagate coliphage T5

iv

Page

26

35

42

43

LIST OF FIGURES

Figure Page

ARTICLE I

l. Optical density of pesticin- -treated cells of E. co oli
¢L during incorporation of [14C]hypoxanthine Tnto
DNA and RNA . . . . . . . . . . . . . . 24

 

2. Percent radioactivity remaining in TCA-insoluble
material of E. coli ¢ previously labeled with
[ 4C]thym1'd1’ne . . . . . . . . . . . . . 24

3. Net incorporation of [14C]diaminopimelic acid into
TCA-insoluble material of E. coli ¢LD . . . . . 25

4. Degradation of cell wall material in E. coli ¢LD
previously labeled with [14C]diaminomeelic acid. . 25

5. Sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis of double- labeled mureinlipoprotein
purified from E. coli ¢LD after partial hydrolysis
by pesticin . . . . 25

6. Determination of optimum pH for hydrolysis of ,
mureinlipoprotein by pesticin . . . . . . . . 26

7. Course of hydrolysis of mureinlipoprotein by
pesticin . . . . . . . . . . . . . . . 26

8. Potential cleavage sites in_§. coli mureinlipo-
protein . . . . . . . . . . . . . . . 27

ARTICLE 2

l. Elution of colicins from cell extracts of E. coli
CA. 7 and E. coli CA. 18 from DEAE-cellulose . . . 39

 

INTRODUCTION

Pesticin is a bacteriocin produced by wild-type isolates of

Yersinia pestis, the bacterium which causes bubonic plague. The

 

ability to produce pesticin is correlated with virulence; nonpesticino-
genic mutants are avirulent, perhaps due to the concomitant loss of
fibrinolysin and coagulase activities. Certain strains of Yersinia
pseudotuberculosis, Yersinia enteroclitica, Escherichia coli, and
nonpesticinogenic Y, pg§31§_are sensitive to this bacteriocin.

Conflicting reports about the mode of action of pesticin have
appeared in the literature. One group suggested that pesticin has a
mechanism of action similar to that of colicin E2, an endonuclease
which promotes degradation of deoxyribonucleic acid (DNA). They
based their conclusion on experiments, conducted with partially puri-
fied preparations of pesticin, in which they saw immediate cessation
of DNA synthesis followed by partial inhibition of protein synthesis
and degradation of ribonucleic acid (RNA). A second group reported
that homogeneous preparations of pesticin effected the conversion of
sensitive bacterial cells to osmotically stable spheroplast-like
structures, and that cells so treated were capable of at least a six-
fold increase in cell mass. The purpose of this research was to
define precisely the mode of action of this bacteriocin.

Pesticin did not inhibit the net synthesis of DNA, RNA, and
protein, nor did it cause apparent degradation of DNA or RNA. There

I

was, however, partial inhibition of net cell wall synthesis, and
pesticin did promote a significant loss of previously labelled wall
material from actively growing cells. Further study revealed that
pesticin catalyzed the hydrolysis of purified mureinlipoprotein. Its
enzymatic specificity is that of an N-acetylglucosaminidase. These
results are consistent with, and can account for the morphological
change of, pesticin-treated cells.

This dissertation consists of three parts: the first is a
literature review covering virulence determinants of 1..pg§§i§.
pesticin, colicins, and the cell envelope as it relates to cell shape,
the second part is a published manuscript describing the enzymatic
mode of action of pesticin, and the third section is a manuscript
submitted for publication which discusses the patterns of cross-

resistance to pesticin and colicins among mutants of Escherichia

 

coli ¢-

LITERATURE REVIEW

All known members of the genus Yersinia (genus XI of the
family Enterobacteriaceae) are pathogenic. The most recently
described species is 1. ruckeri sp. nov. It is one of the recognized
causes of the redmouth disease syndrome in rainbow trout (24), but
little is known about its pathogenic mechanisms. The other three
species are pathogenic for mammals, and are classified as facultative
intracellular parasites (ll). Yersinia enterocolitica, which is trans-
mitted via contaminated food and water (33, 49), is usually avirulent
in laboratory animals and causes acute terminal ileitis in infected
humans (51). Yersinia pseudotuberculosis is also transmitted orally;
it produces severe disease in rodents and mild mesenteric lymphadenitis
in man (18). The most extensively studied member of the genus is Y,
pestis, the etiological agent of bubonic plague (55). Yersinia
pestis is more limited in its metabolic potential than 1, ggtgrgf
colitica or I, pseudotuberculosis, presumably because the protected
and enriched nature of its ecological niche (mammalian host and flea
vector) obviates the need to compete with saprophytes in nutrient-

deficient environments.

Virulence Determinants of Y: pestis
Five mutable phenotypes are associated with the ability of

1, pestis to produce fatal infections in experimental animals. Loss

3

of any one of these determinants leads to a reduction in virulence;

the following information is summarized in Table l.

Era. The Fraction 1 antigen is an envelope glycoprotein.
Produced only during growth at 37°C, it confers resistence to phago-
cytosis (11). Although I, pestj§_is classified as a facultative
intracellular parasite and can survive within macrophages, it is
killed in neUtrophils and monocytes (4T). Fra' 1, pestis isolated
have reduced virulence for guinea pigs but not for mice. Possession
of this antigen is evidently not required for human infection since
there is a report of Fra' I, pgstj§_having been isolated from a fatal

case of human plague (71).

Eur, Purine prototrophy is a second requirement for
virulence in I, pestis (l4). Strains unable to synthesize either
adenine or guanine are avirulent while mutants capable of utilizing
hypoxanthine or inosine (metabolic block prior to inosine monophos-

phate)exhibit only small reductions in virulence (10, ll).

.911. Calcium-dependent Y, pg§t1§_(Cal+) requires concentra-
tions of calcium similar to those of mammalian extracellular fluid
(2.5 mM) for sustained growth at 37°C, but not at 26°C (44). Cultiva-
tion of Cal+ organisms at 37°C in the absence of Ca2+ results in the
expression of the V and W virulence antigens (16) and the onset of
bacteriostasis. Physiological events associated with bacteriostasis
include slow turnoff of DNA synthesis, cessation of ribosomal RNA

synthesis, decrease in ribonucleotide triphosphate pool sizes, and

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a drop in the adenylate energy charge from 0.85 to 0.59 (72). Cal"
mutants are avirulent and lack the V and W antigens except for a
rare phenotype (VW+, Cal') which arises during selection for mutants

resistant to high concentrations of streptomycin (12).

figmy Cells of wild-type Y, pestis, when plated on special
media (40, 64) at 26°C, adsorb low molecular weight chromatophores
(e.g., hemin, Congo red dye) to produce pigmented colonies (Pgm+).
Pgm' isolates are avirulent in guinea pigs and mice unless iron at a
dose sufficient to saturate serum transferrin has been injected (l4).
It had been proposed that the pigmentation phenomenon reflects a
mechanism for the uptake of iron in iron-deficient environments;
however recent work by Perry and Brubaker shows that Pgm+ and Pgm'
strains at 37°C are equally capable of using hemin or ferric iron as

the sole source of iron (53).

Est. Most isolates of Y, pestis produce the bacteriocin
pesticin (3). All pesticinogenic strains also produce coagulase and
fibrinolysin (l5). The coordinate loss of these three activities
results in the loss of virulence when experimental animals are
challenged by intraperitoneal or subcutaneous injection, but not by
the intravenous route. As is the case with Pgm' strains, virulence

is restored by injecting the animals with iron (14).

Potential determinants. The murine toxin appears to inhibit

 

the reception of beta-adrenergic agonists in mice and rats, but its
role in virulence has not been defined (8). The high levels of

catalase produced by Y, pestis might contribute to intracellular

survival and virulence by inhibiting host HZOZ-dependent killing
processes (17). The absence of aspartase in 1. pesti§_markedly
reduces its ability to catabolize dicarboxylic amino acids, perhaps
minimizing the impact on the nitrogen and energy metabolism of the
host cell (22). Other factors which may contribute to virulence are

endotoxin and hydrolytic enzymes (ll).

Pesticin
Ben-Gurion and Hertman were the first to demonstrate bacterio-
cin production in 1, pestis (3). They showed that pesticin was a
thermolabile protein whose synthesis was inducible by ultraviolet
light (3). The range of antibacterial activity of pesticin is
somewhat broader than that of most bacteriocins. Sensitive

organisms include serotype I strains of 1, pseudotuberculosis (l3),

 

certain isolates of Y, enterocolitica (37), and a few strains of

 

Escherichia coli, among them the universal colicin indicator strain

 

¢ (T3, 60). Nonpesticinogenic mutants of Y. pestj§_which retain the
Pgm+ phenotype are also sensitive to pesticin (9). Hemin and ferric
iron inhibit pesticin activity; calcium ions and ethylene-
diaminetetraacetic acid enhance its lethal effect (l3).

The genetics of pesticin production have not been elaborated.
As mentioned above, all pesticinogenic strains also produce coagulase
and fibrinolysin, activities which can be physically separated from
pesticin (2). Investigation of the interrelationships of these three
activities has been hampered by the lack of selection procedures.

Kol'tsova and co-workers have demonstrated the transfer of

pesticinogeny by fi'-R factor-mediated conjugation. Since there was
no apparent transfer of nutritional markers from donor to recipient,
they suggest that the genes for pesticin, coagulase and fibrinolysin
reside on a plasmid (43). However, Little and Brubaker were unable
to demonstrate a plasmid in Y, pestis by ethidium bromide-desium
chloride density gradient centrifugation (46).

Pesticin, a 65,000 dalton polypeptide, has been purified to
electrophoretic homogeneity (36, 37). Like certain colicins which
are deficient in cysteine (35), pesticin can exist in different con-
formational states. Three immunologically identical forms have been
separated by chromatography on calcium hydroxylapatite, the a and B
conformers have equal biological activity while y-pesticin is inactive
(36).

Inconsistent reports have been published concerning the
effects of pesticin on sensitive organisms. Elgat and Ben-Gurion
showed an abrupt cessation of DNA synthesis followed by an inhibition
of protein synthesis and degradation of RNA; they suggested that
pesticin has a mode of action similar to that of colicin E2 (23).
Subsequent work by Hall and Brubaker demonstrated the conversion of
sensitive cells to osmotically stable spheroplast-like structures by
pesticin. These cells lost viability immediately, but were able to
accumulate at least a six-fold increase in cell mass (29). The
latter study was done with homogeneous preparations of pesticin, and
the possibility is extant that the crude preparations used in the

former contained more than one antibacterial activity.

Bacteriocins of Gram-Negative Bacteria

Like low molecular weight antibiotics, bacteriocins kill
sensitive cells by specifically disrupting vital cellular processes
or structures. However, bacteriocins differ from antibiotics in that
they are proteins ranging in molecular weight from l8,000 for purified
colicin M (6) to 92,000 for colicin D (65). Furthermore, the range
of bacteriocin activity is limited to organisms of the same species
as the producing species or to closely related species. Gram-
negative organisms which have been reported to produce bacteriocins
include the genera Pseudomonas, Neisseria, Alcaligenes, Rhizobium,
Yersinia, Hafnia, Paracolobacterium, Erwinia, Enterobacter, Klebsiella,

Serratia, Proteus, Providencia, Citrobacter, and Escherichia (sum-

 

 

marized in reference 57).
Bacteriocinogeny appears to be a widespread phenomenon,

especially among members of the Enterobacteriaceae, but the ecological

 

role played by bacteriocins is not clear. There is evidence though,
which suggests that in some cases genes encoding virulence determi-
nants reside on plasmids carrying genes for bacteriocins. One such
instance is the genetic linkage of colicin Ib production and mucosal
adherence in an enteropathogenic strain of E. coli (70). Another
case is the apparent association of colicin V production and enhanced
lethality for chickens of invasive isolates of g, ggli_(6l). Simi-
larly, there is a strong correlation between the production of pesti-
cin and plague coagulase and fibrinolysin in Y. pestis (2).

Colicins, those bacteriocins produced by g, £911, have been

the subject of intensive study in the last two decades. Although

10

they can be divided into two mutually exclusive groups according to
their receptor sites (19, 20), their modes of action differ widely

within each group, and there is some overlap between the groups.

Mechanisms of colicin action. Protein synthesis is specifi-

 

cally inhibited by colicins E3 and D and by cloacin DFl3. The pre-
cise mode of action of colicin D has not been established (65).
Colicin E3 and cloacin DFl3 are both endonucleases which inactivate
the 305 ribosomal subunits by cleaving off a fragment of approxi-
mately 50 nucleotides in length from the 5' terminus of the 168
ribosomal RNA molecule (4, 35, 59).

Treatment of sensitive cells with colicin E2 resulted in the
inhibition of DNA synthesis and degradation of pre-existing DNA (52).
Ig_yjtrg_studies of colicin E2 showed that it is an endonuclease
(58); presumably in_yivg_the fragmentation of the chromosome is
followed by cellular nucleases.

Colicins A, El, Ia, Ib, K, and $8, and marcescin JF246, and
evidently colicin B all interact with the cytoplasmic membrane to
disrupt energy metabolism (25, 35, 42, 45, 52, 54). General effects
of these colicins are immediate inhibition of macromolecular synthesis
(52), motility, active transport (25), decrease in the intracellular
adenosine triphosphate pool size (54), and efflux of potassium ions
(54). Intensive studies of colicins El and K suggest that their
primary effect is de-energizing the cytoplasmic membrane (54), and
that the other effects are secondary. Similar results were obtained

for colicins Ia, Ib, and A (42, 45). When sensitive cells were

11

treated with colicin M, extensive lysis ensued unless isotonic

sucrose was present, whereupon spheroplasts were formed (6).

Colicin receptor sites. Colicins are divided into two groups

 

according to the receptor system involved. Group B is composed of
colicins B, D, G, H, Ia, Ib, M, Q, and Sl; uptake of these colicins
required specific receptor proteins on the cell surface in addition
to the tgnB gene function (20), which was also essential for all high
affinity iron transport processes (26). The tgnB gene product is
believed to couple energy to the cytoplasmic membrane with receptor
systems of the outer membrane (32). Mutants lacking this function
were resistant to bacteriophages Tl and ¢80 and all the colicins of
group B (20).

The fguB gene product is an 8lK outer membrane protein whose
synthesis is induced during growth in iron-deficient media. This
protein serves as the receptor for ferri-enterochelin and colicins B
and D (30). Colicins Ia and ID and Sl recognize the product of the
fguA gene, another iron stress outer membrane protein of molecular
weight 74K (56), but which plays no known role in iron transport (62).
Colicin M, ferri-ferrichrome, and bacteriophages Tl, T5, and ¢80 use
the tgnA protein as a receptor site (47, 68). Also an iron stress
protein of the outer membrane, the tgnA protein has a molecular
weight of 78K (7).

Colicins A, El, E2, E3, K, L, N, S4, and X make up group A
(19). The lethal binding of group A colicin is energy-dependent (35),
but it is independent of the £938 function (19). Bacteriophage BF23

12

and colicins El, E2, and E3 require a functional big gene product, a
60K outer membrane receptor for vitamin B12 (21, 28, 69). The ts;
gene product is a 27K outer membrane pore protein involved in the
uptake of nucleosides, bacteriophage T6, and colicin K (31, 48).

There are also several tolerance phenotypes which confer insensitivity
to colicins of group A. These mutations often result in changes in
the proteins of the outer membrane and defects in the barrier function
as evidenced by enhanced dye and surfactant sensitivity and altered

antibiotic resistance (19).

The Gram-Negative Cell Envelope

The cell envelope of gram-negative bacteria includes the
outer membrane, the murein sacculus, and the cytoplasmic membrane.
As a whole, the envelope provides a physical and chemical barrier
between the cell and its environment, mechanical stability, and a
unique milieu for enzyme systems.

In gram-negative organisms the murein sacculus contributes a
small percentage of the cell wall by weight. It is thought to be a
monolayer, in contrast to the thick, highly cross-linked peptidoglycan
layer of gram-positive bacteria (1). Its relatively small amount is
nonetheless important in the maintenance of cell shape and integrity.
Spheroplasts are gram-negative bacteria with defective murein; they
may be formed by inhibiting peptidoglycan synthesis or by treating
with lysozyme in the presence of chelating agents. These round cells
are osmotically fragile, suggesting that the outer membrane has also

been affected.

13

Work with penicillin binding proteins and studies of murein
hydrolase activities in synchronized cultures have permitted consider-
able understanding of the control of cell shape and cell division at
the level of murein synthesis.

Research conducted with B-lactam antibiotics has elucidated
the roles of distinct penicillin proteins in cell division, cell
elongation, and the maintenance of rod shape in E, 5911, Penicillin
binding protein 1 is the transpeptidase which is responsible for cross-
1inking new wall material to the murein sacculus. Inhibition of this
enzyme, required for all wall synthesis, results in cellular lysis
(63). When both penicillin binding proteins 2 and 3 are inhibited,
defective murein is synthesized and cells so treated are osmotically
stable, but exhibit aberrant morphology (63). Penicillin binding
protein 3 is the D-alanine carboxypeptidase which acts transiently at
the time of cell division to change thedirection of wall growth at the
potential division sites (63). Selective inhibition of this enzyme
results in filamentous cells whose murein is more highly cross-linked
than normal (63). In contrast, penicillin binding protein 2, which is
thought to be expressed during cell elongation, orients wall growth
to maintain a cylindrical sacculus. When this function is blocked,
cells become ovoid in shape (63).

Associated with the murein in both free and covalently bound
forms is a lipoprotein of molecular weight 7500 (39). This molecule
is bound to about one in every ten diaminopimelic acid residues in
the muropeptide (5). Inouye (38) has proposed a model which states

that the free and bound forms of the lipoprotein associate to form

14

tubular channels through the outer membrane, and to anchor the outer
membrane to the murein (5). The isolation and characterization of
mutants lacking the lipoprotein has shown that it is not an essential
envelope component, but that it does play a role in maintaining the
integrity of the outer membrane (34).

Composed of lip0polysaccharide, phospholipids, and proteins,
the outer membrane is an extremely hydrophobic entity. It provides
the primary molecular sieve-type barrier for uncharged hydrophilic
molecules, and excludes molecules of molecular weight greater than
about 900 (50). There are hydrophilic pores which traverse the outer
membrane, facilitating the uptake of essential nutrients and ions.
Outer membrane porin proteins b and e of E. coli K-12 function in
the uptake of nucleotides while pores fbrmed by proteins b and c
are involved in the uptake of copper, glucose, methionine, leucine,
and histidine (67). The t§x_protein facilitates uptake of adenosine
and functions as a receptor for colicin K and bacteriophage T6
(31, 48).

In addition there are located on the cell surface specific
receptors required for the transport of other nutrients from the
environment. Among the best studied are those involved in iron
transport, which were discussed in the previous section. Studies of
colicin and bacteriophage resistance patterns have yielded consider-
able knowledge about functional interactions in the envelope of

E. coli.

 

10.

11.

12.

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Bayer, M. E. 1974. Ultrastructure and organization of the
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Beesley, E. D., R. R. Brubaker, W. A. Janssen, and M. J.
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Ben-Gurion, R. and I. Hertman. 1958. Bacteriocin-like material
produced by Pasteurella pestis. J. Gen. Microbiol. 19: 289-297.

 

Bowman, C. M., J. E. Dahlberg, T. Ikemura, J. Konisky, and

M. Nomura. 1971. Specific inactivation of 16$ ribosomal RNA
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Braun, V. and K. Rehn. 1969. Chemical characterization,

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Braun, V., K. Schaller, and M. R. Wabl. 1974. Isolation,
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Braun, V. and H. Wolff. 1973. Characterization of the
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Brubaker, R. R. 1969. Mutation rate to nonpigmentation in
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Burrows, T. W., J. M. F. Farrel, and W. A. Gillet. 1964. The
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20

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ARTICLE 1

MODE OF ACTION OF PESTICIN:
N-ACETYLGLUCOSAMINIDASE ACTIVITY

By

Donna M. Ferber
and
Robert R. Brubaker

Appears In:

Journal of Bacteriology

 

Volume 139, Number 2, 1979

21

JOURNAL or Bacnmotocv. Aug. 1979. p. 495—501
0021-9193/79/08-0495/0730200/0

Vol. 139. No. 2

Mode of Action of Pesticin: N-Acetylglucosaminidase

Activity‘l'

DONNA M. FERBERi: AND ROBERT R. BRUBAKER‘

Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824

Received for publication 5 June 1979

Homogeneous preparations of pesticin, a bacteriocin produced by Yersinia
pestis, neither significantly inhibited net synthesis of deoxyribonucleic acid,
ribonucleic acid, or protein in Escherichia coli 4; nor caused detectable degrada-
tion of deoxyribonucleic acid in vivo. Accordingly, its mode of action does not
resemble that of colicin E2 as suggested by others. However, incorporation of cell
wall-specific label ([“C]diaminopimelic acid) into trichloroacetic acid-insoluble
material of growing cells was inhibited by pesticin which also promoted release of
such radioactivity from both resting cells and purified mureinlipoprotein. Sodium
dodeycl sulfate-polyacrylamide gel electrophoresis of reaction mixtures containing
appropriately labeled mureinlipoprotein showed that [3H]N-acetylglucosamine
comigrated either with ["C]diaminopimelic acid in the murein peptide or with
[“C]i301eucine of the Braun lipoprotein. As judged by these findings and pesticin-
dependent release of reducing equivalents but not 4-hydroxy-2-acetamido sugars,
the bacteriocin possesses N-acetylglucosaminidase activity. Hydrolysis of murein-
lipoprotein occurred over a broad pH, with an optimum of 4.7. Mureinlipoproteins
from a variety of pesticin-sensitive and -resistant organisms were hydrolyzed by
the bacteriocin, indicating that its antibacterial specificity resides at the level of

absorption.

 

Wild-type cells of Yersinia pestis produce a
bacteriocin, termed pesticin (4), that is active
against the colicin indicator strain 41, but not K-
12, of Escherichia coli (8, 38), serotype I strains
of Yersinia pseudotuberculosis (8, 11), and cer-
tain isolates of Yersinia enterocolitica (26). Pes-
ticin is also lethal for those nonpesticinogenic
mutants of Y. pestis that possess an as yet
uncharacterized surface structure that nonspe-
cifically binds certain planar low-molecular-
weight chromatophores such as hemin (28) or
the dye Congo red (39). The possibility exists
that this structure also serves as an absorption
site for pesticin (7). The bacteriocin is a mono-
meric 63,000-dalton polypeptide that exists in
two biologically active conformer states (25).
Demonstration of conjugal R-factor-mediated
transfer of pesticinogeny (29) and observation
that nonpesticinogenic mutants lack distant in-
vasive enzymes (3, 10) suggest that the structural
gene for pesticin resides on a plasmid.

Lethal mechanisms catalyzed by bacteriocins
have received considerable attention. Although
colicins could be classified into two groups ac-
cording to the nature of their receptor sites (12,

TArticle no. 8961 from the Michigan Agriculture Experi-
ment Station, East Lansing. MI 48824.

1 Present address: Department of Biology, University of
South Carolina, Columbia, SC 29208.

13), these bacteriocins attack widely different
biochemical targets. For example, colicin E2 pro-
motes rapid degradation of DNA in vivo (33)
and exhibits endonuclease activity in vitro (35).
Protein synthesis is inhibited by colicins D (40)
and E3; the latter cleaves the 168 RNA molecule
of the 30S ribosomal subunit (5, 37). Colicins A,
B, E1, Ia, Ib, and K disrupt various aspects of
energy metabolism by interacting with specific

_ components of the cytoplasmic membrane (22).

In contrast, sensitive bacteria treated with coli-
cin M undergo lysis unless the medium contains
sufficient sucrose to permit formation of spher-
oplasts (6).

Elgat and Ben-Gurion (14) suggested that pes-
ticin exhibits a mode of action similar to that of
colicin E2 as judged by cessation of DNA syn-
thesis, degradation of RNA, and inhibition of
protein synthesis in E. coli 4). However, Hall and
Brubaker (21) subsequently showed that pesti-
cin-treated cells were capable of a residual six-
fold increase in mass while undergoing conver-
sion to osmotically stable spheroplast-like struc-
tures. This observation, which suggests a mode
of action more like that of colicin M than colicin
E2, prompted a reinvestigation of the mode of
action of pesticin. In this report we show that
pesticin has no primary effect on net synthesis
of DNA, RNA, and protein but that it does

22

FERBER AND BRUBAKER

interfere with cell wall biosynthesis. We further
demonstrate that pesticin is a lysozyme that
catalyzes in vitro hydrolysis of the B-1,4 bond
between N-acetylglucosamine and N—acetylmu-
ramic acid in the glycan backbone of mureinli-
poprotein.

(Preliminary aspects of this work were pre-
sented at the Third International Symposium
on Yersinia, Montreal, Canada and Saranac
Lake, New York, 25 to 29 September 1977 and
at the 78th Annual Meeting of the American
Society of Microbiology, Las Vegas, Nevada, 15
to 19 May, 1978).

MATERIALS AND METHODS

Bacteria. Y. pestis A1122 was used to prepare
pesticin, which was routinely assayed with Y. pseu-
dotuberculosis PBI/O by the double agar layer
method as previously described (9). From E. coli is
(met pur pan), a lysine auxotroph (strain (6L) was
isolated after mutagenesis with UV light and selection
with penicillin. A diaminopimelic acid auxotroph
(strain ¢LD) was then obtained by replicate plating
after mutagenesis with N-methyl-N-nitro-N-nitroso-
guanidine (1). All other strains were obtained from the
departmental stock culture collection.

Media and cultivation. Cells of E. coli is or its
derivatives were routinely grown in the morpholino-
propane sulfonic acid-buffered synthetic medium of
Neidhardt et al. (32) supplemented with L-methionine
(0.5 mM), hypoxanthine (0.5 mM), and calcium pan-
thothenate (5.0 pM); (DL + meso)-diaminopimelic acid
(0.25 mM) and L-lysine (0.5 mM) were added for
cultivation of strain ¢LD. Concentrations of hypoxan-
thine, diaminopimelic acid, and lysine were halved
when the corresponding radioactive compounds were
included in the medium. Bacteria used for preparation
of unlabeled murein or mureinlipoprotein were grown
in heart infusion broth (Difco Laboratories, Detroit,
Mich). All cultures were aerated at 200 rpm in Erlen-
meyer flasks (10%, vol/vol) at 37°C on a model 676
gyratory water bath shaker (New Brunswick Scientific
Co., New Brunswick, N.J.). Optical density of cultures,
appropriately diluted in 0.033 M potassium phosphate
buffer, pH 7.0, was determined at 620 nm with a model
2000 spectrophotometer (Gilford Instrument Labora-
tories. lnc., Oberlin, Ohio).

Pesticin. Homogenous pesticin was prepared as
previously described (25, 26). The a-conformer was
used in all experiments.

Radioisotopes and reagents. Amersham Corp.
(Arlington Heights, Ill.) supplied [8-"C]hypoxanthine,
N-acetyl-D-[1-3H]glucosamine, and (01. + meso)-2,6-
diamino-[I,7-"C]pimelic acid. L-[4,5-"’H(N)]lysine and
L-[ U“C]isoleucine were purchased from New England
Nuclear Corp. (Boston, Mass) All other chemicals
were of the highest purity commercially available.

Net synthesis of macromolecules. Cells of E.
coli ¢L or ¢LD were grown with appropriate radioac-
tive precursors (all 0.05 uCi/pmol) for 10 generations
and then inoculated into identical medium with or
without added pesticin. Samples taken for determi-
nation of radioactivity in total nucleic acids, protein,

23

J. BACTERIOL.

or murein were added to an equal volume of 10%
trichloroacetic acid, chilled for 30 min, and then fil-
tered through a 0.45-jim-pore membrane filter (Milli-
pore Corp., New Bedford, Mass). Alkaline hydrolysis
(34) to solubilize RNA preceded precipitation with
trichloroacetic acid when radioactivity in DNA was
determined; that in RNA was taken as the difference
between a parallel sample processed for total nucleic
acids. After drying, radioactivity on membranes was
determined as previously described (2) for sufficient
time to record at least 10’ counts above background.

Degradation of DNA in vivo. Cells of E. coli 41
were labeled with [3H]thymidine in preparation for
determination of endonuclease activity of pesticin and
crude colicin E2 essentially as described by Beck and
Brubaker (2). Colicin E2 was obtained by induction of
E. coli 52(E2) with mitocycin C (23).

Solubilization of cell wall material in vivo.
Exponentially growing cells of E. coli ¢LD were incu-
bated for l h with 0.1 mM diaminopimelic acid (I jiCi/
umol), washed twice by centrifugation (10,000 x g for
10 min), and resuspended in medium containing un-
labeled diaminopimelic acid (0.5 mM). After further
incubation for 30 min, the culture was divided, pesticin
was added to a subculture, and samples were processed
for determination of trichloroacetic acid-insoluble ra-
dioactivity as described above.

Preparation of mureinlipoproteins. Double-la-

beled mureinlipoproteins were prepared from E. coli
¢I.D by the procedure of Inouye et al. (27). Organisms
were grown with carrier-free [3H]N-acetylglucosamine
(1 jiCi/ml) either 0.10 mM [“deiaminopimelic acid
(0.4 pCi/pmol) or 0.1 mM L-isoleucine (0.2 uCi/pmol)
plus unlabeled L-valine (0.1 mM). Results of control
experiments showed that these precursors were not
catabolyzed. At the final step of purification, the sam-
ples were resuspended at a concentration of approxi-
mately 0.5 mg/ml of distilled water for subsequent
assay.
Polyacrylamide gel electrophoresis. Gels of 0.5
by 16 cm (7.5% acrylamide, 0.2% N,N’-methylene-bis-
acrylamide, and 0.5% sodium dodecyl sulfate) were
prepared by using the buffer system of Fairbanks et
al. (15). Samples were electrophoresed at 6 mA/gel
until the dye front had migrated to within 1 cm of the
bottom. The gels were fractionated with a Gilson gel
fractionator (Gilson Medical Electronics. Inc.. Middle-
ton, Wis.). Radioactivity was determined by liquid
scintillation counting after addition of 3a70B scintil-
lation cocktail (Research Products International
Corp., Elk Grove Village, lll.).

Characterization of lysozyme activity. The fol-
lowing buffers were used to determine the pH optimal
for pesticin-dependent lysozyme activity: glycine-hy-
drochloride (pH 2.2, 2.8), sodium acetate (pH 3.6, 4.0,
4.3, 4.7, 5.0, 5.5), sodium 2-(N-morpholinolethane sul-
fonate (pH 6.0), sodium morpholinopropane sulfonate
(pH 6.5, 7.0, 7.5) and Tris-hydrochloride (pH 8.0. 9.0).
The reaction mixture consisted of 0.25 mg of murein-
lipoprotein from E. coli 41 and 3,300 U of pesticin per
ml of 0.01 M buffer. In subsequent studies all deter-
minations were performed in 0.01 M sodium acetate
buffer, pH 4.7. Reactions were terminated by heating
for 5 min in a boiling-water bath. Undigested controls
were prepared identically except that boiling pro-

VOL. 139, 1979

ceeded immediately after addition of pesticin.

After incubation, any insoluble material was re-
moved by centrifugation for 5 min in a Microfuge B
(Beckman Instrument Co., Downers Grove, Ill.). Sol-
uble reducing equivalents were assayed by a modifi-
cation (19) of the ferricyanide method of Park and
Johnson. Release of soluble-free amino groups was
measured with fluorodinitrobenzene ( 19). The Mor-
gan-Elson assay, which is specific for 4-hydroxy-2-ac-
etamido hexoses, was used to measure liberation of N-
acetylglucosamine at the nonreducing termini of sol-
ubilized mureinlipoprotein fragments; samples were
treated with acetic anhydride before assay (19). Bor-
ohydride reduction was performed (19), and the reac-
tion products were analyzed by chromatography as
described by Tipper and Strominger (41).

RESULTS

Macromolecular synthesis and degrada-
tion of DNA. Pesticin-treated cells of E. coli 41
underwent conversion to osmotically stable
spheroplast-like structures identical to those il-
lustrated for sensitive yersiniae (21). The in-
crease in cell mass accompanying this conversion
reflected balanced synthesis of DNA, RNA, and
protein as judged by net incorporation of radio-
active precursors into trichloroacetic acid-insol-
uble material at rates similar to those of un-
treated controls (Fig. 1). DNA of E. coli it was
labeled by growth with radioactive thymidine
before washing and addition of pesticin or a
crude preparation of colicin E2. No significant
hydrolysis of DNA occurred with pesticin,
whereas approximately 85% of the label was
solubilized in a parallel culture treated with the
colicin (Fig. 2).

 

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HOURS OF INCUBAT ION

FIG. 1. Optical density of control (C) and pesticin-
treated (200 U/ml) (0) cells of E. coli 41L during
incorporation of radioactivity from ["Clhypoxan-
thine (.4) into deoxyribonucleic acid (0, control; 0,
pesticin-treated) and RNA (0, control, 6, pesticin-
treated); incorporation of [’Hllysine (B) into protein
(0, control; 0, pesticin-treated).

24

MODE 0!" ACTION OF PESTICIN

IOO

 

80

60

 

40..

20-

REMAINING RADIOACTIVITY (°/o)

 

o l 2 31‘
HOURS

FIG. 2. Percent radioactivity remaining in trichlo-
roacetic acid-insoluble material of E. coli ¢ previ-
ously labeled with [“Cjthymidine (0, untreated con-
trol; O, 200 U ofpesticin per ml; 0, 200 U of colicin
E2 per ml).

Cell wall synthesis and degradation. The
aberrant morphology of pesticin-treated cells
suggested that the bacteriocin might catalyze
hydrolysis of murein or inhibit its biosynthesis.
To test these possibilities, net murein synthesis
was measured by incorporation of radioactive
diaminopimelic acid into trichloroacetic acid-in-
soluble material of E. coli oLD in the presence
and absence of pesticin. Both the rate and extent
of murein synthesis were decreased by pesticin
(Fig. 3).

Cell wall stability was examined by comparing
loss of trichloroacetic acid-insoluble radioactiv-
ity from pesticin-treated and control cells pre-
viously labeled with diaminopimelic acid. If pes-
ticin exerted its effects only by inhibiting cell
wall biosynthesis, treated and control cells would
be expected to lose incorporated isotope at es-
sentially the same rate. However, release of ra-
dioactive diaminopimelic acid in the presence of
pesticin was significantly greater than that in its
absence (Fig. 4). These findings suggested that
pesticin possesses enzymatic activity directed
against murein.

N-acetylglucosaminidase activity. Pesti-
cin was able to catalyze, without addition of

FERBER AND BRUBAKER

 

 

 

 

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)— -1
>- :n
t: — ' >
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92 p _ -4
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P1 1 1 l 4 1‘
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HOURS

FIG. 3. Net incorporation of [“deiaminopimelic
acid into trichloroacetic acid-insoluble material of
E. coli ¢LD; optical density (U) and radioactivity
(O) of untreated cells and optical density (I) and
radioactivity (O) in cells treated with pesticin (200
U/ml).

 

 

 

 

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O l 2 3
HOURS

FIG. 4. Degradation of cell wall material in E. coli
¢LD previously labeled with [“Cldiaminopimelic
acid; percent trichloroacetic acid-insoluble radioac-
tivity remaining in control (0) and pesticin-treated
(200 U/ml) cells (C).

cofactors, hydrolytic cleavage of purified mu-
reinlipoprotein in vitro. Analysis of reaction
products (not centrifuged) from double-labeled
mureinlipoproteins by sodium dodecyl sulfate-

25

J. BACTERIOL.

polyacrylamide gel electrophoresis revealed that
pesticin promoted release of fragments of het-
erogeneous size. [3H]N-acetylglucosamine in the
glycan backbone of murein comigrated with
["C]diaminopimelic acid in the peptide side-
chain (Fig. 5). Similar results were obtained
when murein labeled with [3H]N-acetylglucosa-
mine and ["C]isoleucine (in the Braun lipopro-
tein) was partially digested with pesticin and
then electrophoresed. Samples not treated with
pesticin barely penetrated the gels during elec-
trophoresis. These results indicate that pesticin
is a glycanase rather than an amidase.

Using solubilization of reducing equivalents as
an assay for glycanase activity, we found that
pesticin was active over a broad pH range with
an optimum at pH 4.7 (Fig. 6). By using this pH,
samples from a reaction mixture were withdrawn
at intervals and assayed for release of soluble
reducing equivalents, free amino groups, and 4-
hydroxy-Z-acetamido hexoses (Fig. 7). The latter
were not detected even after 8 h of incubation
when the reaction was complete. Analysis of
soluble dinitrophenyl derivatives from the reac-
tion mixture revealed that the reactive free
amino groups were those of hexosamines that
had been deacetylated during processing. The
above data indicate that pesticin is an N-acetyl-
glucosaminidase; this was confirmed by perform-
ing borohydride reduction and observing the
disappearance of glucosamine from hydrolyzed

 

 

m V v w r T Y T—"_T:
I ”IT” "on I
”Of 0\
i3 ‘
‘ 500,, ‘
o t . t
.33 I
" 400‘. ' .
g i 9 a
3 ° 300 .5
9 3 .’
;‘ zoo. I.
a e.
o_ b C
' m» if . i 1'
g c (TEA 7! l
. V. . 1
F W v I 1
o 2"W.
_ "L --___4 A l I L l , s.
O D 20 30 fi 50 60 TO .0

3L“ “I!

FIG. 5. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (see text) of double-labeled mu-
rein Iipoprotein purified from E. coli oLD after partial
hydrolysis by pesticin. The reaction mixture of 3.300
U of pesticin and 250 pg of mureinlipoprotein in 0.2
ml of 10 mM sodium phosphate buffer 01h 7.1) was
incubated for 3 h at 37° C; radioactivity was measured
as [’I-IIN-acetylglucosamine (0. pesticin omitted: O.
pesticin added) and as [“Cldiaminopimelic acid
(0, pesticin omitted; O, pesticin added).

Von 139, 1979

 

1001
80
60
40.‘
20.
O_lllll -1
123456 8910
pH

FIG. 6. Determination of optimum pH for hydrol-
ysis of mureinlipoprotein by pesticin. The extent of
hydrolysis was measured as the release of reducing
equivalents after incubation for I h; values were
plotted as the percent of maximum activity (200 nmol/
ml).

‘rfiij

PERCENT ACTIVITY

 

 

 

 

«1+

 

 

 

 

400 1 T 123—7—

3. —1

\ 300 #4
(I)

t 2°°I - . fixes-

g 100., -

O 1 1 14‘} 1

O l 2 6

HOURS

FIG. 7. Course of hydrolysis of mureinlipoprotein
by pesticin; soluble reducing equivalents (O) and free
amino groups (0).

samples as determined by thin-layer chromatog-
raphy.

Substrate specificity. Murein preparations
from all of the gram-negative organisms tested,
regardless of their in vivo sensitivity, were de-
graded by pesticin in vitro (Table 1). These
mureins are of the A17 type (containing m-dia-
minopimelic acid with directly cross-linked pep-
tides [36]). Pesticin was capable of at least par-
tial hydrolysis of mureins from several gram-
positive species, none of which is sensitive to
pesticin in vivo (Table 1). Murein types repre-
sented were A17, A301 (containing lysine cross-
linked by short peptide bridges [36]), and A40:
(containing lysine cross-linked by L-aspartyl res-
idues [36]).

DISCUSSION

Before this study, Hall and Brubaker (21)
showed that the pesticin-dependent conversion
of E. coli «11 to osmotically stable spheroplast-
like structures was associated with at least a
sixfold increase in cell mass. Results reported

26

MODE OF ACTION OF PESTICIN

here showed that this increase reflected bal-
anced accumulation of DNA, RNA, and protein
at rates similar to those that occurred in un-
treated cells. This observation is inconsistent
with the suggestion of Elgat and Ben-Gurion
(14), based on inhibition of net DNA and protein
synthesis with degradation of RNA, that pesticin
possesses a mode of action similar to that of
colicin E2. In fact, our attempts to demonstrate
pesticin-dependent degradation of DNA in vivo,
an activity characteristic of colicin E2 ( 33), were

‘ not successful. We can provide no explanation

for these discrepancies except that Elgat and
Ben-Gurion used a preparation of crude pesticin,
whereas that employed in this study had been
purified to electrophoretic homogeneity. Since
pesticin did not exhibit colicin E2-like activity,
a search was initiated for an alternative mode of
action.

Hall and Brubaker (21) noted that pesticin
promoted bacterial killing in the absence of net
synthesis and therefore suggested that the at-
tendant morphological change was caused by
the bacteriocin per se rather than by induction
or modification of some normal host cell activity.
Upon finding that incorporation of diaminopi-
melic acid was reduced in treated cells, the pos-
sibility arose that pesticin might act by partially

TABLE 1. Organisms yielding mureinlipoprotein
sensitive to pesticin-dependent hydrolysis"

 

 

Or - s Murein Lfithality
amsm , o est:-
8 type (Find
E. coli K-12 A17 0
E. coli «11 A17 +
E. coli CA42 A17 +
Serratia marcescens BS 303 A17 0
Salmonella typhimurium LT2 A17 0
Proteus mirabilis D799 A17 0
Pseudomonas aeruginosa BU A17 0
277

Pseudomonas fluorescens LD A17 0
Y. enterocolitica 33114 A17 +
Y. enterocolitica Becht 51 A17 0
Y. pseudotuberculosis PBl/O A17 +
Y. pseudotuberculosis 7 A17 0
Y. pestis A1122 A17 0
Y. pestis Al2(Pgm‘) A17 +
Y. pestis AlZIPgm“) A17 0
Bacillus licheniformis A17" 0
Micrococcus roseus A3n O
Staphylococcus epiderm idis A311 0
Streptococcus lactis A40 0

 

" Assayed as described in the text.

“ Obtained from the departmental stock culture col-
lection.

" Reported in reference 36.

" Determined by the double agar layer method (9).

' D-Glutamyl residues are partially amidated (36).

FERBER AND BRUBAKER

inhibiting synthesis of new murein or as a non-
lytic murein hydrolase. The latter was
strengthened by discovery that significantly
more diaminopimelic acid was released from
previously labeled cells treated with pesticin
than from untreated controls where murein hy-
drolysis would be limited to reactions involved
in normal cell division (20, 31).

Characterization of pesticin activity in vitro
with purified mureinlipoprotein as substrate re-
vealed that pesticin catalyzed the hydrolysis of
the B-l,4 bond between N-acetylglucosamine
and N-acetylmuramic acid in the glycan back-
bone. This N-acetylglucosaminidase activity is,
of course, distinct from that of bacteriophage T4
and egg white lysozymes which are N-acetyl-
muramidases capable of cleaving the bond be-
tween C1 of N-acetylmuramic acid and C4 of N-
acetylglucosamine (17, 30). Pesticin also differs
from nonlytic bacteriophage T7 lysozyme which
hydrolyzes the bond between N-acetylmuramic
acid and L-alanine of the peptide sidechain (27).
A comparison of these specificities is shown in
Fig. 8. However, N-acetylglucosaminidase activ-
ity was reported as a component of the autolytic
enzyme system of Bacillus subtilis where it may
play a role in the maintenance of the proper
orientation for wall growth (16). E. coli possesses
a similar transglycosylase activity which may
function in local rearrangements in murein and
the termination of glycan chains (24). Presum-
ably the cumulative effects of pesticin-depend-
ent hydrolysis of old murein and partial inhibi-
tion of glycan strand elongation lead to the
eventual death of sensitive bacteria as evidence
by their inability to form visible colonies on agar
or to grow indefinitely in liquid medium.

LIPOPROTEIN
DAP
D-OLU
L-ALA
-- - NAG -NAM-NAG -NAM-NAG- NAM -NAG- --
L-‘iL‘A\A | l
o-sLu B C
DAP- D-ALA
D-ALA DAP
D-(éLU
L-ALA
--- NAM-NAG-NAM-NAG-NAM-NAG---
FIG. 8. Potential cleavage sites in E. coli murein-

lipoprotein for (A) bacteriophage T7 lysozyme, (B)
pesticin, and (C) bacteriophage T4 and egg white

lysozymes.

27

J. Bacraniou

Pesticin was active against mureinlipoproteins
prepared from a variety of bacteria, including
organisms that are resistant to the bacteriocin.
This observation indicates that sensitivity to
pesticin resides at the level of specific absorp-
tion. Of interest was the finding that one class of
pesticin-resistant mutants of E. coli ¢ was also
resistant to colicins B, D, I, and SI (8). These
are members of colicin group B which are known
to require a functional tonB product for the
occurrence of lethality (12). Further study of the
physiological role of this product, which is re-
quired for all known high affinity processes of
iron transport (18), may provide an explanation
for the inhibitory effect of iron on the biological
activity of pesticin (8, 26).

ACKNOWLEDGMENTS

We thank Janet M. Fowler for excellent technical assist-
ance in this work.

This research was supported by Public Health Service
grant Al 13590 from the National Institute of Allergy and
Infectious Diseases. D.M.F. was a Public Health Service preo
doctoral trainee supported by grant GM 01911 from the Na-
tional Institute of General Medical Sciences.

LITERATURE CITED

1. Adelberg. E. A., M. Mandel. and G. C. C. Chen. 1965.
Optimal conditions for mutagenesia by N-methyl-N‘-
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2. Beck. D. J., and R. R. Brubaker. 1973. Effect of cis-
platinum (II) diamminodichloride on wild type and
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19-26.

4. BenoGurion. R., and J. Hartman. 1958. Bacteriocin-like
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5. Bowman. C. M., J. E. Dahlberg, T. Ikemura, J. Kon-
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6. Braun. V., K. Schaller, and M. R. Wabl. 1974. Isolation.
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7. Brubaker. R. R. 1970. Mutation rate to nonpigmentation
in Pasteurella pestis. J. Bacteriol. 98:1404-1406.

8. Brubaker. R. R., and M. J. Surgalla. 1961. Pesticins. l.
Pesticin-bacterium intenelationships. and environmen-
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9. Brubaker, R. R., and M. J. Surgalla. 1962. Pesticins.
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545.

10. Brubaker. R. R., M. J. Surgalla. and E. D. Beesley.
1965. Pesticinogeny and bacterial virulence. Zentralhl.
Bakteriol. Parasitenkd. lnfektionskr. Hyg. Abt. l. Orig.
196:302-312.

11. Burrows. T. W., and L. D. Bacon. 1960. V and W
antigens in strains of Pasteurella pseudt)lub(’rt'ltfu.~t.~'.
Br. J. Exp. Pathol. 41:38-44.

12. Davies, J. K., and P. Reeves. 1975. Genetics of resist
ance to colicins in Escherichia cult K-12: cross-resist-
ance among colicins of group B. J. Bat-term]. 123:9“-
l0].

28

VOL. 139, 1979

13. Davies. J. K., and P. Reeves. 1975. Genetics of resist- 28.

ance to colicins in Escherichia coli K-l2: cross-resist-
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117. 29.

14. Elgat. M., and R. Ben-Gurian. 1969. Made of action of
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15. Fairbanks, G., T. L. Steck. and D. I". H. Wallace. 1971. 30.

Electrophoretic analysis of the major polypeptides of
the human erythrocyte membrane. Biochemistry 10:

2606-2617. 31.

16. Fan, D. P.. and M. M. Beckman. 1972. New centrifuga-
tion technique for isolating enzymes from large cell
structures: isolation and characterization of two Bacil-

lus subtilis autolysins. J. Bacteriol. 109:1258—1265. 32.

17. Fleming. A. 1922. On a remarkable bacteriolytic element
found in tissues and secretions. Proc. R. Soc. London

Ser. B 93:306-317. 33.

18. Frost. G. E.. and H. Rosenberg. 1975. Relationship

between the tonB locus and iron transport in Esche- 34.

richia coli. J. Bacterial. 124:704-712.
19. Ghuysen, J.-M.. D. J. Tipper. and J. L. Strominger.
1966. Enzymes that degrade bacterial cell walls. Meth-

ods Enzymol. 8:685-699. 35,

20. Haltenbeck. R., and W. Measer. 1977. Activity of mu-
rein hydrolases in synchronized cultures of Escherichia

coli. J. Bacteriol. 129:1239-1244. 36.

21. Hall. P. J., and R. R. Brubaker. 1978. Pesticindepend-
ent generation of osmotically stable spheroplast-lilte

structures. J. Bacteriol. 136:786-789. 37.

22. Hardy. K. G. 1975. Colicinogeny and related phenomena.
Bacterial. Rev. 38:464-515.

23. Henchman, H. R., and D. R. Helinakl. 1967. Purifica- 38.

tion and characterization of colicin E2 and colicin E3.
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24. Holtje. J. V., D. Mirelman. N. Sharon, and U. 39.

Schwarz. 1975. Novel type of murein transglycosylase
in Escherichia coli. J. Bacterial. 124:1067-1076.

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26. Ha. P. C., G. C. H. Yang. and R. R. Brubaker. 1972. 41.

Specificity, induction, and absorption of pesticin. J.
Bacterial. 112:212-219.

27. Inouye. M., N. Arheim, and R. Sternglanz. 1973. Bac-
teriophage T7 lysosome is an N-acetylmuramyl-L-ala-
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MODE OF ACTION OF PESTICIN

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tion of Pasteurella pestis on a defined medium contain-
ing haemin. Br. J. Exp. Pathol. 37:570-576.

Kol'tsova, E. G.. Y. G. Suchkov, and S. A. Lebedeva.
1973. Transmission of a bacteriocinogenic factor in Pa..—
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Mirelman, D.. G. Kleppe. and H. B. Jensen. 1975.
Studies on the specificity of action of bacteriophage T4
lysozyme. Eur. J. Biochem. 55:369-373.

Mirelman. D., Y. Yashano-Gan, and U. Schwartz.
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48-56.

ARTICLE 2

MUTATIONS IN ESCHERICHIA COLI ¢ PROMOTING

 

TOLERANCE AND RESISTANCE T0
PESTICIN AND COLICINS

By

Donna M. Ferber
and
Robert R. Brubaker

Submitted To:

Journal of Bacteriology

29

ABSTRACT

Eleven distinct phenotypes of Escherichia coli ¢ were

 

obtained by selection for insensitivity to pesticin, group B colicins,
or coliphage T5. Eight of these isolates resembled resistant
receptor (FeuA', FeuB-, and TonA-) or tolerant (TonB-, Ebe', Ebe-,
Ivt', and Cmt') mutants described in pesticin-insensitive E. ggli
K-12. The three remaining phenotypes were peculiar to E, gglj_¢;

of these, one resembled TonB' but was also tolerant of the group A
colicin S4 (TonB/S4') and the remainder exhibited specific resistance
to colicin S4 (Sfr’) or pesticin (Psr'). All receptor mutants
excepting Psr' isolates remained sensitive to pesticin. In contrast,
TonB/S4', TonB', Ebe', and Ebe' mutants were highly tolerant of
this bacteriocin. The significance of these findings is discussed

with respect to transport of iron in yersiniae.

3O

INTRODUCTION

As defined by patterns of cross-resistance in Escherichia

 

_ggli K-l2, lethality caused by colicins B, D, G, H, Ia, Ib, M, Q,
Sl, and V is known to depend upon envelope structures normally
required for high-affinity transport of iron. Accordingly, these
colicins are classified within group B as opposed to those of group
A (colicins A, El, E2, E3, K, L, N, 54, and X) which enter sensitive
cells via membrane components involved in accumulation of nutrients
other than iron (9, 10).

Resistance caused by inability to absorb colicin B or D
(jgug) occurs upon mutational loss of outer membrane receptors for
enterochelin, an endogenous phenOlate siderophore or iron-carrier
(8, 15). Similarly, resistance to colicin M and coliphages ¢80, Tl,
and T5 (593g) is associated with loss of a distinct receptor for
exogenous hydroxamate siderophores (17, 30). Colicins Ia, Ib, and
51 do not absorb to jgufl_mutants which lack a 74,000 dalton outer
membrane protein (16); although induced during iron privation, the
physiological role of this receptor has not been resolved (29). The
phenomenon of tolerance occurs upon mutational loss of functions
required fbr penetration of absorbed colicin molecules to internal
target sites. In E. ggli_K-l2, tog§_isolates are tolerant to all
group B colicins and unable to propagate coliphages ¢80 and Tl (9).
Due to evident inability to energize outer membrane sites, these

3]

32

mutants lack all known high-affinity mechanisms of iron transport
including the siderophore-mediated systems already noted and that
utilizing exogenous citrate as an iron-carrier (T3, 18, 23). Other
tolerant mutants remain sensitive to coliphages ¢80 and Tl; these
include gx§§_(all group B colicins), £599 (colicins B, D, G, H, and
M), gm; (colicin M):.i!£ (colicins Ia, Ib, Q, and V), and_ggg
(colicins Q and V) (1). Like tgfl§_mutants,lgxb§_and‘gxbg isolates
are defective in Uptake of enterochelin (9).

Produced by Yersinia pestis, pesticin is a 63,000 dalton
monomeric peptide (19) possessing lethal N-acetylglucosaminidase
activity (ll). The bacteriocin is active against non-pesticinogenic
mutants of 1. pg§31§_which retain the ability to absorb certain
exogenous pigments (2) including hemin (Pgm+) (2l), serotype Ia and
Ib strains of Yersinia pseudotuberculosis (6), a few tested serotype
3 and 8 strains of Yersinia enterocolitica (20, unpublished), and
certain isolates of E. 9911 including the universal colicin-indicator
strain o of Gratia (l4), but not K-lZ (3). Probably the first
reports demonstrating a relationship between bacteriocin activity
and iron metabolism were those describing the inhibitory effects of
Fe+3, hemin, and an hydroxamate siderophore on the lethality of
pesticin; apparent pesticin-resistant mutants of E. ggli_¢ were also
found in this study to have lost sensitivity to colicins B, D, Ia,
and Sl (3, 5). The purpose of the present investigation was to

define the nature of mutations in E. coli resulting in resistance

 

and tolerance to pesticin. The results provide useful information

33

regarding the nature of known analogous mutations in yersiniae
including that to Pgm’ in 1. pestis which results in avirulence
(22).

MATERIALS AND METHODS

Bacterial strains. E, coli ¢ (14) was used as an indicator

 

of all bacteriocins. Colicin-producer organisms are shown in

Table l. Yersinia pestis A1122 was grown as a source of pesticin.

 

Mggia. Mutants insensitive to colicins and pesticin were
selected on nutrient agar (Difco, Detroit, Michigan) and blood agar
base (BBL, Cockeysville, Maryland), respectively. The latter was
used to determine sensitivity of cells to all bacteriocins and
coliphages. The defined solid medium (less added iron) of Neidhardt
et al., (24) supplemented as previously described (ll) was used to
determine nutritional requirements associated with mutations to
bacteriocin-insensitivity and excretion of enterochelin. Colicins
were produced in a medium (400 ml per 2 liter Erlenmeyer flask) con-
sisting of 3% Type A NZ Amine (Sheffield Humko Chemical, Lyndhurst,
New Jersey), 0.025 M KZHP04, 0.01 M citric acid, 0.0l M potassium

gluconate, 0.1 mM FeClz, and 0.01 mM MnCl brought to pH 7 with 10

2
N NaOH.

Preparation of bacteriocins. Pesticin was purified to
homogeneity as previously described (19, 20). Colicinogenic cells
were aerated at 37°C on a model R-25 reciprocal shaker (New Brunswick
Scientific Co., New Brunswick, New Jersey) until mid-logarithmic
growth was achieved. After receiving mitomycin C (0.4 pg per ml)

34

35

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.> cvuwpouu

.z cwuw_oun

.x stoppage
UFO, auwcmcmeu .a >.z A .<o w_ou mesuwtm;Umu
mo_ x w cowtmumeu .a em mpg emamwu m_meP;m
no, x m cavememtu .a Pm Pa wwuaon.mppmmwsm
amo_ x N coweacoeu .a z.m m_ .<u _,ou awsuweogumm
wee. x N cuwtmumtu .a x.¥ mmmx P_ou mwgupemgomm
mo, x e covemuatu .a mH mm .<u P.ou m_;uwtm;umu
co. x e cowemumtu .a a we .<o P.0u accuwemgumm
mo_ x N xuom .o mm mm .<u wpou m_;umgm;umm
cop x e cu_emuweu .a mu Ne .<u w_ou avguwtm30mm
mo_ cowewumcu .a Pu Spa teamwu appamwcm
mo_ x o __mepuoa .o a mm .<u F_ou mensweagumw
cop x N mgwngmumg .m m nacu< wpou mpguwngumm
mo. cu_emumtu .a < _m .<u wwncaaee empumnOpru
prwh mogaom vmuzvoga Emwcmmgo

ewowpou

 

.cowuuzuoga mecu com com: msmwcmmco ucm mcwuwpou mo mgmuwhuu.F m4m<k

36

the organisms were aerated for 2 h and then collected by centrifuga-
tion at 4°C (11,000 x g for 45 min); all subsequent steps were per-
formed in the cold. When antibacterial activity resided primarily

in the resulting supernatant fluids (colicins A, B, El, E2, E3, K,
and $1), the latter were brought to 90% saturation with solid
(NH4)ZSO4 and, after equilibration for 30 min, precipitated

material was sedimented (14,000 x g for 45 min) and dialyzed over-
night against 5.0 mM potassium phosphate buffer, pH 7.0 (phosphate
buffer). When the majority of activity was intracellular (colicins
D, G, Ia, M, S4, and V), the organisms were suspended in 0.05 M tris-
(hydroxymethyl)aminomethane-HCl, pH 7.8, and broken by treatment for
1 min with an ultrasonic probe (MSE Ltd., London, England) except

for colicin M where disruption was prolonged at 15 sec intervals for
20 min as suggested by Braun et al. (1). Cellular debris was removed
by centrifugation (10,000 x g for 15 min) and the resulting extracts
were dialyzed overnight against phosphate buffer.

Since E, gglj_strains K235, CA. 18, and CA. 7 produce two or
more colicins, crude preparations as described above were chromato-
graphed on DEAE cellulose exactly as described fbr purification of
pesticin (19). The major antibacterial fraction recovered from
strain K235 was termed colicin K. Profiles obtained for colicins M
and V are shown in Results. Titers of bacteriocins, determined by
dilution (4), are shown in Table 1; 1 unit is defined as the
reciprocal of the highest dilution yielding detectable inhibition of

growth of wild type E. coli o.

37

Isolation and characterization of mutants. To ensure that

 

all mutants of E. 5911 o arose independently, separate cultures of

1 ml were inoculated with about 10 organisms. When the population
reached about 107 cells, the cultures were mixed with 3 m1 of warm
nutrient agar. These preparations received 400 units of CHC13-
sterilized colicin M or 107 coliphage and were then poured over the
surface of sterile nutrient agar plates. Alternatively, the prepara-
tions were used without addition as overlayers on lawns of CHC13-
killed bacteriocin-producer bacteria. After incubation fbr 36 to 48
h, a single mutant per overlayer was isolated and purified.

The triple agar layer procedure (10) was used to determine
if bacteriocin insensitivity was caused by tolerance or resistance.
Sensitivity to coliphages was assayed by cross-streaking and
inability to accumulate iron or transport enterochelin was estimated
qualitatively by observation of size and surrounding color of
colonies streaked on solid defined medium. In all cases, colony
size was normal and no detectable enterochelin was excreted by

organisms grown on defined medium plus 20 uM FeC13.

RESULTS

Preparation of colicins. Cell-free extracts of mitomycin C-

 

induced E, £911_CA 7 contained at least two colicins as judged by
chromatography on DEAE cellulose (Figure 1A). The first eluted
activity was identified as colicin M on the basis of its inability
to absorb on DEAE cellulose (l), evident lysozyme-like mode of
action (1), and significantly reduced ability to kill known EQQA
mutants (30). The second activity, which appeared after application
of a NaCl gradient, was termed colicin V although this fraction
possibly contained an additional colicin with identical antibacterial
spectrum reported to occur in strain CA. 7 (9). The elution of anti-
bacterial activities obtained from an extract of g..ggli CA. 18

(Col 8, M) is shown in Figure 18; the first bacteriocin recovered

was colicin M.

Preliminary characterization of mutants. Sensitivity of
362 mutant clones of E. £911 4 to pesticin, l3 colicins, and
coliphages ¢80, T1, and T5 was determined qualitatively as were the
abilities to form colonies on defined medium lacking added iron and
to excrete enterochelin. Results of this survey were used to
classify the mutants according to phenotype by criteria previously
established for g, ggli_K-12 (9). Eight corresponding phenotypes
were observed in E, £911.¢ which also yielded mutants insensitive to

38

39

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41

pesticin alone (Psr') plus two additional unique phenotypes (TonB/

S4- and Sfr') described below. The number of clones of each pheno-
type recovered after selection with coliphages or various bacteriocins
is shown in Table 2.

Since all mutants of a given class were remarkably similar
regardless of their origin, only a single type stock of each pheno-
type was preserved for further study. All such mutants were as
sensitive as the parent to tested colicins of group A except TonB/

S4' and Sfr- isolates which were not killed by colicin S4. Sensi—
tivity of the ll type mutants to coliphages and bacteriocins is

defined in Table 3.

Mutations to tolerance. As shown by use of the triple agar

 

layer test, TonB/S4' and TonB' cells were tolerant to pesticin and
colicins B, D, 6, la, M, 51, and V. Both mutants propagated coli-
phage T5, but not ¢80 or Tl. Distinctions were a slight sensitivity
of the TonB' isolate to massive challenge with colicin 8 (5,000 units)
and high sensitivity (8 units) to colicin S4. The TonB/S4' mutant
was tolerant to at least 20,000 and 8,000 units of colicin B and S4,
respectively. All 68 TonB/S4' and 23 TonB' clones formed very small
colonies when plated on defined medium lacking added iron; after
growth for 2 to 3 days the agar surrounding these colonies acquired

a marked orange-purple cast due to the presence of excreted entero-
chelin. L-Tryptophan was necessary fOr growth of 12% of the TonB/S4'

isolates but was not required by any TonB' mutant.

42

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44

Also tolerant to pesticin were isolates exhibiting patterns
of tolerance similar to those described for Ebe' and Ebe' mutants
of E, ggli_K-12. Distinctions between these phenotypes were partial
resistance of Ebe' cells to coliphages ¢80 and T1 plus low levels
of tolerance to colicins Ia and $1. In contrast, Ebe' cells
possessed significantly higher levels of tolerance to colicins B and
D. Both mutants formed colonies approaching normal size on defined
medium and excreted less enterochelin than was observed for TonB/S4-
and TonB' isolates. Mutants identical to Ivt' and Cmt' isolates of
E, ggli_K-12 remained sensitive to pesticin, grew normally on

defined medium, and failed to accumulate visible enterochelin.

Mutations to resistance. Psr' organisms, comprising 42% of

 

all clones selected with pesticin, were resistant to the bacteriocin
as shown by use of the triple agar layer test. These mutants
retained sensitivity to all tested coliphages and colicins. Psr-
cells formed colonies of normal size on defined medium and did not
release detectable enterochelin. Typical TonA', FeuA', and FeuB'
receptor mutants were isolated which were killed by pesticin. Normal
growth of TonA' and FeuA' cells on defined medium was noted whereas
excretion of enterochelin by somewhat smaller colonies of FeuB'
mutants was generally evident. Although an attempt to select TonB/S4'
mutants with colicin S4 was successful, the majority of isolates
recovered in this determination proved to be resistant rather than

tolerant to this colicin as judged by results of the triple agar

layer test. These Sfr' isolates remained sensitive to all tested

45

coliphages and other bacteriocins used in this study and exhibited

no readily apparent lesion in iron metabolism.

DISCUSSION

Although colicin M was reported by Fredericq (12) to be pro-
duced by E, 9911 CA. 7, this activity was not detected in a subsequent
study by Davies and Reeves (9). We observed that significant colicin
M was produced by mitomycin C-induced cells of this strain. Titers
of other colicins prepared for this study were generally similar to
those obtained by Pugsley and Reeves (27).

We isolated four distinct mutants of_E._ggli 6 which exhibited
patterns of multiple tolerance to colicins known to absorb to differ-
ent receptors. The similarity of three of these mutants to pheno-
types described in E. ggli_K-12 (9) prompted their assignment as
TonB', Ebe', and Ebe'. The fourth phenotype, termed TonB/S4',
exhibited a unique tolerance to the group A colicin S4. However,
only TonB/S4' clones were 352, a gene known to undergo spontaneous
delection with linked EggE_at high frequency (7). Accordingly, only
the TonB/S4' phenotype of E, gglj_¢ may be entirely analogous to
EgnE_mutants as described for E, 5911 K-12. Further study will be
necessary to determine if the differences between these four tolerant
phenotypes reflect distinct alterations of the same gene or loss of
separate genes. 0f more immediate interest was the discovery that
cells of these four organisms, as opposed to Ivt' and Cmt' mutants,
were also highly tolerant to pesticin. In contrast, isolates indis-

tinguishable from TonA', FeuA', and FeuB' receptor mutants of E. coli

 

46

47

K-12 retained sensitivity to pesticin as did Sfr' isolates which
evidently lack receptors for colicin S4. Furthermore, pesticin-
resistant Psr' mutants remained sensitive to tested coliphages and
colicins. This finding indicates that the pesticin receptor was not
shared by these activities. Attempts are currently in progress to
locate this receptor in E. 9911 o and yersiniae by electrophoresis
of outer membrane preparations and to determine if its synthesis
is regulated by iron.

It is established that the outcome of many bacterial infec-
tions is dependent upon the availability of iron in the host (31)
and that, at least in certain enteric species, the expression of
virulence requires the presence of a siderophore-mediated process of
Fe+3 uptake (28, 32). Iron transport in yersiniae remains an enigma
since these organisms failed to produce detectable siderophores
under conditions of iron-privation although Fe+3-starved cells
accumulated the cation by some as yet undefined cell-bound process
(25). If pesticin can select EQgE_and ps5 mutants 0f.§-.£Qli a,
it might also serve the same purpose in yersiniae. Such mutants,
especially the former, might be of reduced virulence. In fact, Pgm'
mutants of’l, pg551§_may represent one of these genotypes. This
hypothesis is made attractive by the fact that such isolates are
avirulent and unable to absorb exogenous hemin, a compound capable

of serving as a sole source of iron for yersiniae (25).

ACKNOWLEDGMENTS

This investigation was supported by grant AI 13590 from the
National Institute of Allergy and Infectious Diseases; the manuscript
was assigned Article No. by the Michigan Agricultural Experiment

Station.

48

10.

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Brubaker, R. R. 1970. Mutation rate to nonpigmentation in
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Brubaker, R. R. and M. J. Surgalla. 1961. Pesticins. I.
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Brubaker, R. R. and M. J. Surgalla. 1962. Pesticins. II.
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Brubaker, R. R., M. J. Surgalla, and E. D. Beesley. 1965.
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J. K. Davies and P. Reeves. 1975. Genetics of resistance to
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Hancock, F. E. W. and V. Braun. 1976. The colicin I receptor
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Hantke, K. and V. Braun. 1975. Membrane receptor dependent
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Hantke, K. and V. Braun. 1978. Functional interaction of the
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Hu, P. C. and R. R. Brubaker. 1974. Characterization of
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Hu, P. C., G. C. H. Yang, and R. R. Brubaker. 1972.
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Jackson, S. and T. W. Burrows. 1956. The virulence enhancing
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Pugsley, A. P. and P. Reeves. 1976. Characterization of group

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SUMMARY

Pesticin, the bacteriocin produced by Yersinia pestis, does

 

not have a mode of action like that of colicin E2, as was previously
reported. Preparations of pesticin, purified to electrophoretic
homogeneity, did not inhibit the net synthesis of deoxyribonucleic
acid, ribonucleic acid or protein. Consistent with the observation
that treatment of sensitive cells with pesticin results in their
conversion to osmotically stable, spheroplast-like structures, some
inhibition of peptidoglycan synthesis was noted in pesticin-treated
cells. Moreover, pesticin promotes the solubilization of peptido-

glycan both 13_vivo and 13_vitro. Pesticin was found to have a broad

 

pH optimum centered around 4.7. A nonlytic lysozyme, pesticin was
characterized as an N-acetylglucosaminidase; hydrolysis of the glycan
backbone of murein was such that the reducing termini of the frag-
ments was identifiable as N-acetylglucosamine.

Pesticin hydrolyzed a wide variety of murein and murein-
lipoprotein preparations from sensitive as well as resistant bacteria,
suggesting that the antibacterial specificity depends on specific
receptor interactions on the target bacterial cell surface. To
approach the question of receptor site, a series of pesticin- and

colicin-resistant mutants of Escherichia coli were isolated and

 

characterized. The results showed that killing of sensitive cells by
pesticin was dependent on a functional tonB gene product.

52

53

Pesticin-colicin cross resistance patterns also revealed a close

relationship between pesticin and the colicins of Group B.

3056

   

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