The [beta]-lactamases of the marine genus Photobacterium : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

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Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

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THE B-LACTAMASES OF THE MARINE GENUS PHOTOBACTERIUM

A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE IN M ! CROBIOLOGY AT MASSEY UNIVERSITY

SUSAN ELIZABETH LAMB

1981

• f . ' • if

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ABSTRACT

All naturally occurring isolates of all five species of the marine genus Photobac.tvr.,i_um have B-lac tamase ac ti vi ty. In

this study, the B-lactamase from the laboratory strain

P.

£eiogna;th,i_ 206 is fully characterised. The enzyme is constitutive and is released from the cell by osmotic shocking techniques, suggesting a periplasmic location.

The enzyme is maximally active at pH 6.2 and has over 90% maximum activity at pH 7.0. The hydrolytic activity of the

B-lactamase is independent of zinc ion presence. On the basis of substrate profile and inhibition studies the

B-lactamase is classified as a Richmond and Sykes Class II enzyme. Crypticity tests indicated that there is no outer membrane permeability barrier to B-lactam substrates.

l l .

Comparative substrate profiles performed with the B-lactamases from three strains of each species of Photobac.tvr.,i_um, indicate that the enzymes from strains of P. £eiogn.ath.l, P. angU/2tum and P. phOJ.:iphOJteum are active only on penicillins, whereas those

from P. f/.,6c.hvr..l and P. £oge.l a 1 so hydro lyze cepha los porins.

This division is in agreement with an imminent taxonomic change for the latter two species. Analytical iso-electric focusing of the B-lac tamases from 45 Photobac.tvi.-w.m strains resulted in pI values which were not necessarily species specific and there was little correlation between the pI of the B-lactamase and its substrate profile, excepting the enzymes from P. angU/2tum and P. £.ogei .

.AL though plasmid DNA is present in many Photobac.tvi.lwn strains, conjugative transfer of B-lactamase activity from six

different Photoba.c.tvr.,i_um donors to a res tric tionles s E-6c.hvr.ic.h.la.

c.ofi mutant was not observed. All attempts to 'cure' the bacteria of B-lactamase activity with five different curing agents, were also unsuccessful. A chromosomal location for the Bla+ gene is postulated.

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DEDICATION

I would like to dedicate this thesis to my parents, Dr and Mrs R.F. Lamb, without whose limitless financial support and unceasing encouragement this work would not have been possible.

111 .

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ACKNOWLEDGEMENTS

I wish to express my gratitude to Massey University and the Department of Microbiology and Genetics for allowing me to carry out post-graduate studies.

I would especially like to thank Professor D.F. Bacon

(Head of Department) for all efforts made on my behalf, for always being readily available whenever a problem needed discussing and for his kindly advice.

I am very indebted to my supervisor, Dr Kathy Smith, whose help, guidance, knowledge, advice, understanding and

perseverance were of untold value.

Very special appreciation goes to Dr Eric Terzaghi for the voluntary expenditure of his valuable time and for his very helpful comments and advice.

Grateful thanks go to my sister Jan, for the selfless hours spent typing the first copy and to Mrs Veronica Lobb for

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her excellent typing of the final copy. Special thanks also to Audrey Larsen for her superb presentation of the figures throughout this work, to the Central Photographic Unit, Massey University, for the reproduction of photographs, and

to Mr Don Patrick for the fine job of binding the thesis.

I would also like to particulary acknowledge and thank the following people: -

The staff of the Department of Microbiology and Genetics for technical assistance, friendship and interesting conversations which made the laboratory working hours much pleasanter.

Special thanks in this respect, go to Bronwyn Dymock.

Dr P. Berquist and D. Lane, Department of Cell Biology, Auckland University, for kindly sending me the restriction-

less E1.>c.hVT..ic.h..w.. c.ou mutant.

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Dr M. Matthew, Glaxo Laboratories, London, for th

e

v

ery

generous gift of nitroc

e

tin and for her useful working notes pertaining to iso- electric focusing.

Dr R. Jolly, Department Veterinary Pathology and Public Health, Massey University, for his generosity in allowing me to use the analytical iso-electric focusing equipment.

Drs M. Hardman and J. McIntosh, Department of Ch

emistry,

Biochemistry and Biophysics, Massey University, for their advice and comments on biochemical problems encount

e

red.

The staff of Massey University Library for obtaining interloan requrests and for their help

.

v.

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TABLE OF CONTENTS

DEDICATION

ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES

SECTION A: INTRODUCTION 1. The genus Photoba.c.t:.Vtium

1.1 The classification of Photoba.c.tVt.w.m 1,2 The ecology of Photoba.c.tvz..w.m strains 2. B-Lactamase

2.1 Definition of B-lactamase 2.2 Nomenclature

2.3 Classification of B-lactamase

2.4 Distribution of 8-lactamases among bacterial genera

2.5

PAGE

lV

V l l

X

X l l l

1 1 1 5

6 6 6 10

15 Physiology and expression of B-lactarnases 16

(a) Location of B-lactamase in the bacterial

cell 16

(b) The permeability barrier in Gram-negative

bacteria 17

(c) Production of B-lactamases in the cell 18 2.6 Genetic determination of 8-lactamases 20 2.7 The clinical importance of B-lactamases 22

3. Aims of this research 24

Cont'd ..

V l .

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TABLE OF CONTENTS CONTINUED SECTION B: MATERIALS

1.

Bact

e

rial strains 2. Reagents and m

edia

2

.1

Reagents 2.2 Ma

rine media

2.3 Terrestrial media 3. Antibiotics

SECTION C: :METHODS

V l l .

PAGE 25 25 31

31 35

37

38

39 1. Cultivation and ma

int

enance of bacterial stra

in

s 39

2

.

Crude enzyme preparations 42

2

.

1 Sonication

2.2 Osmotic shock treatment 3. Assay of

B-l

ac

tamase

activity

42 42 42

3.1 Standard iodometric assay 42

3.2 Detection of B-lac

t

amase activity in so

lid

med

ia

47

4. Substrate profile 5.

Inhibition studies

6.

Induction

of B

-l

ac

tamase

7

.

Analytica

l

iso-electric focusing 8. Slab ge

l

El ectrophoresis

9

.

Gene

tic

determination of B

-la

ctamase 9.1 Plasmid curing

9

.2

Conjugation 9.3 Transformation

Cont'd

.

48 49

so so

52

53 53 54

57

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VlJ.J..

TABLE OF CONTENTS CONTINUED SECTION D: RESULTS

PAGE

61

1. Factors affecting

B

-lactamase activity and assay

61

1.1 Factors affecting i odomet ric assay 61

(a) Eff

ec

t of t

empe

rature 61

(b) Eff

ec

t of pH 61

1.2 Rep ro duc ibilit y of th

e

iodome tri

c

assay

64

1.3 Stability of the B -l actamase

66

(a) Effect of c

entrifuga

tion t

emperature

and r

e

susp

ending med

ia

66

(b) Effect of z1nc 1ons on

B

-l

actamase 68

2. Physiology and expression of

B

-lactamase 2.1

B

-L ac t amase producti

on during ba

cterial

gro

wth

2.2 Inducibility of the

B

-lactamas

e

from

P • .f.e.-<.o g na;th-<. 2 0 6

2 .3 Cellular l ocation of

B

-la ctamase (a) Test fo r

ex

trac

e

llular

e

nzyme (b) Cell-bound

B

-la ctamase

( i ) Modifications to th

e

osmotic

68

71 82 82 84

shock method

84

( i i )

B

-Lactamas

e

r

e

leas

e

d by osmotic

shock methods

85

(iii) Concentration of cells t

o

increase

B

-lactamase yield

89

(c) Permeability barri

e

r of th

e ce

ll wall to

B

-l actams

89

3. Classification of B -la

c

tamases 92 3.1 Substrate profile of the

B

-lactama se from

P . .f.e.-<.o g na;th-<. 2 0 6 9 4

3

.

2 Inhibiti on s tudi es with the B -lactamase

from P .

.f.e.-<.ogna,th-<.

206 94

Cont'd ..

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ix.

TABLE OF CO

NTEN

TS CONTINUED

PAGE SECTION D: CONTINUED

4. The B-lactamases produced by other

Pho:toba.c.:tVL,i.um

strains

98

4.1 Substrate profiles of 16 independent isolates

of

Pho.:tobac.:tVL,<.um

9 8

4.2 Analytical iso-electric focusing of

B-lactamases 100

(a) Enzyme preparations 101

(b) Stainin

g

of iso-electric focusin

g

gels

for

B

-lactamase activity 101 (c) Iso-electric focusing results 105 (d) Slab gel electrophoresis 108 5. The genetic determination of B-lactamases

5.1 Plasmid curing experiments

110 111 116 116 117 117 120 5.2 Transfer of

Bla+

genes by conjugation

(a) Bacterial strains (b) Selecti\e media

(c) Conjugation results

5.3 Transformation of

Bla+

genes

SECTION E: DISCUSSION 129

1.

2.

3.

4.

Factors affecting B-lactamase assay and activity 129 Production of

B

-lactamase during bacterial growth 132 Induction of B-lactamase

Cellular location of B-lactamase

133 135 5. The permeability barrier to

s

-lactams 136 6. Classification of the B-lactamase from

P • .te.io g n.a;thi 2 0 6 1 3 7

7. Comparisons of the B-lactamases of

Pho:tobac.:tVL,[um

strains 139

8. Genetic determination 143

SECTION F: CONCLUSION 145

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TABLE I

LIST OF TABLES

The dis t ingu is h ing properties of

Photoba.c.tVl.,{.LJJi1

species

II B

-

Lactam structures

a b

III IV

Penicillins Cephalosporins

Classes of B

-

lactamases

Photoba.c.tVt,<.um

strains

V B

-

Lactamase activity present at different temperatures of assay

V

I

Reproducibility of the assay with differ

ent

levels of B-lactamase activity

VII Effect of temperature of centrifugation of bacterial cells and resuspension of the cells in different media, on cell-associated

B

-

lactamase activity

VIII B

-

lactamase activity in the presenc

e

of zinc ions

IX Effect of growth

-

inhibitory B-lactam

concen

t

rations on the growth of

P • .f..e.,i_ogna;th,i_

206 cells

X

Extrace

ll

ular B-lactamase from

P .l..e.,i.ogn.a.t.h,i.

206 XI Release of

B-

lactarnase during osmotic shock

and comparison of 'shocking

'

solutions

x.

PAGE

4

7 8

11

26

-

30

62

65

67

69

81 83

87

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xi.

LIST OF TABLES CONTINUED

TABLE PAGE

XII

XIII

XIV

xv

XVI

XVII

B-Lactamase activity release from P. leiogna;thi 206 by osmotic shock methods

a B-Lactamase activity released from P.leiogna;thi 206 cells

b B-Lactamase activity and viable count of P. leiogna;thi 206 cells following each stage of the osmotic shock procedure

c Effect of concentrating

P.

leiogna;th<. 206 cells during the osmotic shock procedure on enzyme

88

yields 90

The B-lactamase crypticity factors of several

independent isolates of Photoba.c.tvi..<.Lw1 species 93

Substrate profile of the B-lactamase from

P. leiogna;thi 2 0 6 9 5

Inhibition of the S-lactamase from P.leiogna;thi

206 by four compounds 97

Substrate profiles of the B-lactamases from

independent isolates of Photoba.c.te.Jt-i.um species 99 Levels of B-lactarnase activity present in

crude enzyme preparations 102

XVIII Hydrolysis of the chromogenic cephalosporin, nitrocefin, by the B-lactamases fromPhoto-

ba.c.tviium ₁₀₄

XIX Observations with the trial agar overlay

staining technique after iso-electric focusing 106

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LIST OF TABLES CONTINUED

TABLE PAGE

XX Growth of P. le..i..ogna.:th..i.. 206 in the presence of curing agents

XXI Plate de tee t ion of P. le...i..ogn.a;th..i.. 2 06 S-lactamase activity following curing treatments

XXII

XXIII

a

b XXIV

a b

XXV a b

XXVI a

Mitomycin C-treated P. le...i..ogna.:th-<- 206 selectively plated for wild-type S-lactamase activity

levels

Single cell resistances to S-lactams used in selective plates

Benzylpenicillin Arnpicillin

Results of conjugation experiments Cross of donor P.le...i..ogna.:th..i.. 206 and recipient strain A43

Cross of donor P.le...i..ogna.:th-<- 206 and recipient E .c.ol..i.. PB1395

Characteristics of 10 independent exconjugant clones

From the cross of strain 534 and A43 From the cross of strain 534 and E. c.ol..i..

PB1395

113

114

115

118 119

121 122

123 124 Results of further conjugant experiments

Crosses with Pho:toba.c.:tvr...i..um donors and recipient strain P

9smr 125

b Crosses with Pho:tobac.:tvr...i..um donors and recipient

E.c.ou PB1395 126

xii.

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LIST 0~ FIGGRES

FIGURE PAGE

1 B-Lactamase hydrolysis of B-lactams

a B-Lactamase action on a penicillin compound 9 b B-lactamase action on a cephalosporin

compound 9

2 B-Lactamase groupings proposed by Sykes and

Matthew (1976) 14

3 a b

Growth curves of Photobacterium strains Spectronic 20 OD at 525 nm versus dry

weight of cells

Klett optical density versus mg dry weight of cells

4 Optical density at 490 nm versus titration volume of sodium thiosulphate

5 Effect of pH on the activity of B-lactamase from P . .te..{_ogna;th.{. 206

40 41

46

63

6 B-Lactamase production during bacterial growth 70

7 Growth of P • .te..{.ogna;th.{. 206 1n the prescence of B-lactams

a b

C

d

Benzylpenicillin Ampicillin

Methicillin Cephaloridine

8 B-Lactamase production by P . .te..{_ogna,th.{_ 206

a b

C

d

1n the presence of B-lactams Benzylpenicillin

Ampicillin Methicillin Cephaloridine

73 74

75

76

77 78

79 80

X l l l ,

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XlV.

LIST OF FIGURES CONTINUED

FIGURE PAGE

9 Effect of lmM EDTA on viability of P • .f.e.J.ogna.t.hJ. 206 cells

10 Sample photograph of iso-electrically focused B-lactamases, stained with the agar overlay

86

gel method 107

11 The iso-electric points of B-lactamases

produced by strains of Photoba.c.tvr.,i.,um 109