BACTERIOCINOGENY IN PROTEUS VULGARIS
by
Heleen Liezel C o e t z e e
(otto ■ 0 /
Submitted in partial fulfilment of the requirements for the degree of
M„Sc„
in the Faculty of S c i e n c e , University of Pretoria,
Pretoria. December 1967
ACKNOWLEDGEMENTS
I would like to convey my thanks to Prof. J„N. C o e t z e e , Professor in Microbiology and head of the Department of
Microbiology, for the inspiration and guidance which w a s r e c e i v e d from him during the duration of t h i s s t u d y ,
A word of thanks t o o , to Dr. H , C . de Klerk for many hours of a s s i s t a n c e e s p e c i a l l y in connection with the electron microscope s t u d i e s , and to D r . I , J . Mare and Dr„ 0 , W , Prozesky for their friendly a d v i c e a t a l l times „
The chemical a n a l y s i s w a s made p o s s i b l e by the c o operation of Dr, J.A. Smit, and invaluable t e c h n i c a l a s s i s t a n c e with the electron microscope w a s given by Mr. N . Hugo, - both of the Life S c i e n c e s Division of the South African Atomic Energy Board. D r . C . R . J a n s e n , h e a d of t h i s department, i s thanked for the u s e of c e r t a i n a p p a r a t u s and c h e m i c a l s .
Very s p e c i a l thanks to my parents for their unfailing encouragement and support „
C O N T E N T S ACKNOWLEDGEMENTS SUMMARY . SAMEVATTING o & • ■ o « a * « • • • • c e * •
CHAPTER I . REVIEW OF LITERATURE • • • 6 9 • Introduction Nomenclature . « • e • • • Distribution of b a c t e r i o c i n o g e n i c s t r a i n s « • Nature of b a c t e r i o c i n s • • • • • e Bacteriocinogenic factors . . . . . . Production of b a c t e r i o c i n s . e 9 • 6
Mode of action of bact e r i o c l n s . . Chemical nature of b a c t e r i o c i n s References e a « • • e o c • • o
CHAPTER I I . INCIDENCE OF BACTERIOCINOGENY IN PROTEUS VULGARIS > j • • • Introduction Methods Media • • e • • * • • V e Q • • e ■ 4 • Bacterial c u l t u r e s • • • • Detection • « • • Ultraviolet irradiation Page (i) (v) (vii) 1 1 2 3 10 11 16 18 21 24 36 36 37 37 39 40 40 T r a n s m i s s i b i l i t y / . . .
Transmissibility i . . 42 KesuiTS c«c . . . . . . « . . • »• 4^
D i s c u s s i o n . . . 43 References . . . . . . . . . . . . 50
CHAPTER III. PURIFICATION OF PROTEUS VULGARIS.
BACTERIOCINS . . . . . . . . . . . . 54 Introduction . . . . . . . . . . s«,, 54 Methods . . . . . . . . . . . . , . . 56 Production , . . . . . . . . . . . 56 Ultraviolet irradiation . . . . . . 57 Concentration . . . 58 Purification . . . 58 rvG S \JLX L 5 • • • • • • ^ • o • « « • » • OO D i s c u s s i o n . . . 60 References „ . K 65
CHAPTER IV. ELECTRON MICROSCOPY OF PROTEUS
VULGARIS BACTERIOCINS . . . . . . 70 Introduction . , 70 Methods . . . . . . 72 Electron microscopy . . . . . . 72 Contraction of phage t a i l - l i k e structures . . . . . . . . . 74 Adsorption of phage t a i l - l i k e structures . . . . . „ . . . 74 D i e n e s / . . .
Page Dienes phenomenon
Results 0 o o * k
D i s c u s s i o n
References a • 4 •
CHAPTER V. PROPERTIES OF PROTEUS VULGARIS BACTERIOCINS . . . . . . ... Introduction Methods # # # 6 # # Agar e l e c t r o p h o r e s i s . . . Diffusibility . . . , . . Action of trypsin Heat s e n s i t i v i t y Protein e s t i m a t i o n , . . Nucleic Acid estimation
RNA estimation , . . DNA estimation , . . Carbohydrate estimation Results D i s c u s s i o n References c • « « « • « « e « • » • * • « • « • • « e •
CHAPTER VI. GENERAL DISCUSSION
• Ho 75 • o • 75 • • • 87 • « •
as
89 • « • 94 # • # 94 • • • 97 • ti o 97 « « • 98 ♦ • 0 98 • «» • 98 • C • 98 • # • 99 « 0 * 99 • • • 100 • • • 101 « « 4 101 a • • 105 • • • 106 • ♦ # 109S U M M A R Y
One hundred and eighteen different s t r a i n s of Proteus v u l -garls were i n v e s t i g a t e d for b a c t e r i o c i n o g e n y . These P . vulgaris s t r a i n s were a l s o used a s i n d i c a t o r s . Seventy of the 118 s t r a i n s produced zones of inhibition when c r o s s - s t r e a k e d with the other P . vulgaris s t r a i n s . S i x t y - s e v e n of the s t r a i n s had a n o n - t r a n s missible killing effect on one or more of the indicator organisms and subcultures of a r e a s of inhibition to broth a l s o failed to show growth. Thirty of the 67 b a c t e r i o c i n s with different spectra of a c t i v i t y were further i n v e s t i g a t e d , Individual b a c t e r i o c i n s killed from five to
87 of the P . vulgaris i n d i c a t o r s . Nine b a c t e r i o c i n s had similar h o s t ranges w h e r e a s the h o s t ranges of the remaining 58 b a c t e r i o c i n s differed. All t h e s e s t r a i n s a s well a s the n o n - b a c t e r i o c i n o g e n i c Proteus s t r a i n s d i s p l a y e d a D i e n e s demarcation line between their s w a r m s . When t e s t e d a g a i n s t a number of gram-negative b a c t e r i a the b a c t e r i o c i n s only inhibited P . vulgaris and P> mlrabllls s t r a i n s and had no effect on s t r a i n s of the family E n t e r o b a c t e r i a c e a e .
Broth c u l t u r e s of b a c t e r i o c i n o g e n i c s t r a i n s are inducible by ultraviolet light and yield b a c t e r i o c i n t i t r e s of about 1 / 1 0 0 . Activity c a n be concentrated with 40% ammonium sulphate and i s s e d i m e n
t a b l e by high speed centrifugation„ The b a c t e r i o c i n s were e l e c t r o -phoretically immobile and diffused through a g a r . Activity could b e destroyed after 20 min. at a temperature of 60 and a l s o by the a c t i o n
of t r y p s i n . Chemical a n a l y s i s showed the bacteriociyis to c o n s i s t of protein and to contain no DNA.
Electron microscopy of all 30 preparations revealed similar phage t a i l - l i k e structures with a c o n t r a c t i l e sheath round a hollow c o r e . The p a r t i c l e s resemble some pyocins and a l s o the tail of a Proteus vulgaris transducing p h a g e . In two preparations a few p h a g e - l i k e p a r t i c l e s which resemble other Proteus phages were a l s o s e e n . Bacteriocin a c t i v i t y was always a s s o c i a t e d with u n c o n -trqcted s h e a t h s and triggered t a i l s do not adsorb to s u s c e p t i b l e o r g a n i s m s . It is concluded that the t a i l - l i k e structures are the p r o d u c t s of defective l y s o g e n y . The high i n c i d e n c e of defective lysogeny may be accounted for by the s e l e c t i o n of g e n e s which im part a s e l e c t i v e advantage to the host and which were originally acquired through transduction or l y s o g e n i c c o n v e r s i o n .
S A M E V A T T I N G
Een h o n d e r d - e n - a g t i e n verskillende r a s s e van Proteus v u l garis i s ondersoek vir die produksie van b a k t e r i o s i e n e . Hierdie P . v u l g a r i s - r a s s e is ook gebruik a s indikator-organisme s . Sewen-tig van die 118 r a s s e het i n h i b i s i e - a r e a s opgelewer na dwarsstreping met die ander P„ vulgaris-ras.se. Sewe e n - s e s t i g van die r a s s e
het 'n nie-oordraagbare dodende effek op e e n of meer van die indikator-organismes gehad en subkulture van i n h i b i s i e - a r e a s het ook geen groei in boeljon getoon n i e . S e w e - e n - s e s t i g van die b a k t e r i o s i e n e met verskillende aktiwiteitspektrums is verder o n d e r s o e k . Individuele b a k t e r i o s i e n e het van vyf tot 87 van die P . v u l g a r i s - inkikators g e d o o d . Nege bakteriosiene het e e n d e r s e g a s h e e r r e e k s e gehad, terwyl die g a s h e e r r e e k s e van die oorblywende 58 b a k t e r i o s i e n e van mekaar verskil h e t . Al hierdie r a s s e sowel a s die n i e -b a k t e r i o s i n o g e n i e s e Proteus-stamme het 'n ..Dieneslyn" vertoon t u s s e n hul s w e r m s . Toe die b a k t e r i o s i e n e teen 'n a a n t a l gram-negatiewe bakterieS g e t o e t s i s , het die b a k t e r i o s i e n e s l e g s r a s s e van P . vulgaris en P . mirabilis gelhhibeer en geen inhiberende effek op r a s s e van die familie E n t e r o b a c t e r i a c e a e getoon nie
Boeljonkulture van bakteriosienproduserende r a s s e is i n d u s e e r b a a r deur middel van ultraviolet lig en lewer b a k t e r i o s i e n -t i -t e r s van ongeveer 1 / 1 0 0 . Ak-tiwi-tei-t kon g e k o n s e n -t r e e r word me-t 40% ammoniumsulfaat, en was sedimenteerbaar met
sentrifugering. Die b a k t e r i o s i e n e w a s elektroforeties onbeweeglik, kon deur agar diffundeer, en onderwerp aan 'n temperatuur van 60 i s hulle na 20 minute v e r n i e t i g . Chemiese ontleding het getoon dat die b a k t e r i o s i e n e uit proteihes b e s t a a n en geen DNA bevat n i e .
Elektronmikroskopie van al 30 preparate het e e n d e r s e f a a g -stertagtige strukture met saamtrekbare skede rondom 'n hoi kern, aan die lig g e b r i n g . Die d e e l t j i e s lyk soos sommige piosiene en ook soos die stert van 'n Proteus v u l g a r i s - t r a n s d u s e r e n d e f a a g . In twee van die preparate i s ook 'n paar faagagtige d e e l t j i e s waarge ~ neem wat soos ander Proteus-fage l y k . Bakteriosien~aktiwiteit w a s altyd g e a s s o s i e e r met n i e - s a a m g e t r e k t e s k e d e s . Saamgetrekte sterte adsorbeer nie aan gevoelige organismes n i e . Die slotsom i s d a t die stertagtige strukture die produkte i s van defektiewe l i s o g e n i e . Die hoe" voorkomssyfer van defektiewe l i s o g e n i e mag moontlik toegeskryf word a a n die s e l e k s i e van gene wat die g a s h e e r begunstig en wat oorspronklik verkry i s deur t r a n s d u k s i e of l i s o -g e n i e s e o m s k e p p i n -g .
REVIEW OF LITERATURE Page INTRODUCTION 1 Nomenclature „ . . = . . . 2 Distribution of bacteriocinogenic s t r a i n s 3 Nature of b a c t e r i o c i n s . „ „ . . „ . . . 10 Bacteriocinogenic factors , . , „ . . 11 Production of b a c t e r i o c i n s . . . 16 Mode of action of b a c t e r i o c i n s . . . 18 Chemical nature of b a c t e r i o c i n s . . . 21 REFERENCES . . . , » . . , , 24
CHAPTER I
R E V I E W OF L I T E R A T U R E
INTRODUCTION
One of the first b a c t e r i a studied for i t s a n t i b a c t e r i a l properties was Pseudomonas aeruginosa (Emmerich & Low, 1899. See Topley & W i l s o n , 1964). The a n t i b a c t e r i a l s u b s t a n c e s found were named p y o c y a n a s e (Emmerich & Low, 1899. See Topley & W i l s o n , 1964), pyocyanin (Ehrismann, 1934), a n d j ^ h y d r p x y
-phenazine (Schoental, 1941), and are a c t i v e a g a i n s t a large variety of g r a m - p o s i t i v e and g r a m - n e g a t i v e b a c i l l i .
Gratia in 1925 observed inhibition of Escherichia c o l i £ by E. cpli V (Gratia, 1925). The inhibitory s u b s t a n c e , to which the name c o l i c i n w a s later given by Gratia and Fredericq in 1946, diffuses through agar and through c e l l o p h a n e membranes, c a n be precipitated with q c e t o n e , and i s r e s i s t a n t to heat and the a c t i o n of chloroform (Adams, 1959). Jacob et a l . (1953) defined the term b q c t e r i o c i n a s a p r o t e i n - l i k e s u b s t a n c e , the b i o s y n t h e s i s of which i s lethal for the producing organism and no multiplication of the b a c t e r i o c i n o c c u r s . Most b a c t e r i o c i n s act only on c e r t a i n s t r a i n s
related to the producer s p e c i e s although some b a c t e r i o c i n s a l s o act on a limited number of strains of related s p e c i e s . The action of b a c t e r i o c i n s i s dependent on the presence of specific receptors on the surface of s u s c e p t i b l e c e l l s . Bacteriophage-like structures Which kill b a c t e r i a but do not multiply, comply with t h i s operational definition and in recent years such objects liberated by b a c t e r i a have been linked with b a c t e r i o c i n o g e n y .
Antagonism between one s p e c i e s and another and even between members of the same s p e c i e s occurs throughout n a t u r e , and the term ' a n t i b i o s i s ' which m e a n s , l i t e r a l l y , ' a g a i n s t l i f e ' , was introduced by Vuillemin in 1889 (Barber & Garrod, 1963). The term antibiotic i s g e n e r a l l y used to mean an a n t i b a c t e r i a l s u b s t a n c e derived from a living s o u r c e .
Nomenclature
A derivation of the Linnaean specific name of the producing organism is given to its b a c t e r i o c i n , e . g . the b a c t e r i o c i n s of
Escherichla coli and Pseudomonas pyocyanea are called c o l i c i n s and pyocins r e s p e c t i v e l y (Bradley & Dewar, 1966).
Distribution of bacterloclnogenlc s t r a i n s .
The b a c t e r i o c i n s were recognised a s being different from a n t i b i o t i c s and a s more b a c t e r i o c i n s were discovered they were a c c e p t e d a s a d i s t i n c t p l a s s of biological compounds
C o l i c i n s were the first b a c t e r i o c i n s to be studied in d e t a i l . Fredericq attempted to group the c o l i c i n s according to their spectrum of a c t i v i t y on wild s t r a i n s of Escherichia coli and a l s o on mutants r e s i s t a n t to one or other c o l l c i n and demonstrated that c o l i c i n s adsorh to specific receptor s i t e s on the c e l l w a l l . With the use of t h e s e criteria he grouped c o l i c i n s into 17 t y p e s (Fredericq, 1948). Hamon & Peron (1963a)refer to 23 t y p e s . The 17 t y p e s of Fredericq are
A, B, C , D , E, F , G , H , I, J, K, V, Sj , S2, Sg, S ^ and Sg. This c l a s s i f i c a t i o n i s further complicated by frequent a p p e a r a n c e s of
s t r a i n s which produce more than one c o l i c i n and of mutants r e s i s t a n t t o more than one c p l i c i n t y p e . Thus the six c o l i c i n t y p e s E, F , J, S „ , S_, and S , were l a t e r grouped into a new type E by the judicious use of indicator s t r a i n s . Within each s p e c i e s there is a l s o a t e n d e n c y to produce only c e r t a i n c o l i c i n t y p e s . Thus E . freundil s t r a i n s only produce type A; E. c o l i s t r a i n s produce t y p e s B, C , D , E, F , G , H , I or V; J and K are produced by Paracolqbactrum spp; Shigella s p p , produce S] , S_, S_, S . , and S,. while I , K or B are produced
by Salmonella s t r a i n s (Reeves, 1965),
The a l v e i c i n s , a r i z o n a c i n s , pneumocins and aerocins which are produced by Hafnia s p p . , Pgracolobactrum a r i z o n a e , Klebsiella
s p p . and Aerobqcter a e r o g e n e s r e s p e c t i v e l y , were discovered by Hamon & Peron (1963a). The a l v e i c i n s act on Hafnia s p p . , E c o l i s t r a i n s and some Shigella paradysenteriae s t r a i n s while the a r i z o n a c i n s show activity on some s t r a i n s of P . arizonae and E. c o l i . The a c t i v i t y of the pneumocins i s generally limited to the other s t r a i n s
of Klebsiella and no a c t i v i t y c a n be found on any of the colicin i n d i c a t o r s but o c c a s i o n a l l y on s t r a i n s of Aerobacter a e r o g e n e s . Entero-b a c t e r c l o a c a e and KleEntero-bsiella are the only s t r a i n s except for A. a e r o g e n e s on which the aerocins a c t .
The c a r a t o v o r i c i n s , produced by s t r a i n s of Erwinia s p p . a l s o act on E. coli s t r a i n s a s well a s on s t r a i n s of Pseudomonas f l u o r e s c e n s , on two Serratia s p p . and on Xanthomonas s p p . and have a range of a c t i v i t y on s t r a i n s of E. c p l i , Xanthomonas and Erwinia
(Hamon & Peron, 1961a). The study of the b a c t e r i o c i n s of Serratia s p p . - the m a r c e s c i n s - show them to be b a c t e r i c i d a l for s t r a i n s °* Serratia, E. coli and Erwinia, and it a p p e a r s from t h i s work on m a r c e s c i n s that Serratia s t r a i n s c a n produce two t y p e s of b a c t e r i o c i n s , one that i s similar to the c o l i c i n s and one that c a n only be c l a s s i f i e d a s a marcescin (Hamon & Peron, 1961b).
Strains of Pasteurella p e s t i s are found to produce an a n t i r
biotic which i s active on s t r a i n s of P . pseudotuberculosis (Ben-Gurion & Hertman, 195 8). This antibiotic i s named p e s t i c i n I . Brubaker
& Surgalla (1961) discovered a second p e s t i c i n (II) which i s produced
by all the s t r a i n s of P. p s e u d o t u b e r c u l o s i s and P. p e s t i s t e s t e d . There e x i s t s a c l o s e antigenic r e l a t i o n s h i p between P. pseudotuberr c u l o s i s type II and the Salmonella B group, and between P. p s e u d o t u b e r c u l o s i s type IV and the Salmonella D g r o u p . Knapp (1960)
suggested that this antigenic r e l a t i o n s h i p between t h e s e two organisms lends support to the idea that P . p s e u d o t u b e r c u l o s i s may be related to t h e E n t e r o b a c t e r i a c e a e , A P. p e s t i s phage which had been adapted to P . p s e u d o t u b e r c u l o s i s w a s a l s o found to l y s e c e r t a i n strains of Salmonella and S h i g e l l a . This fact a l s o s u g g e s t s (Lazarus & G u n n i s o n , 1947) an antigenic r e l a t i o n s h i p between strains of Salmo n e l l a and Shigella and P . p s e u d o t u b e r c u l o s i s a s the action of b a c -t e r i o p h a g e s i s limi-ted -to c l o s e l y rela-ted s p e c i e s .
Hamon, Veron & Peron (1961b) discovered a b a c t e r i o c i n
produced by Pseudomonas fluorescens namely fluocin. All the s t r a i n s which produce fluocins have different a c t i v i t y spectra on the P . fluo r e s c e n s i n d i c a t o r s . Only one of the fluocins i s b a c t e r i c i d a l for any of the s t r a i n s of P . pyocyanea t e s t e d .
Bacillus megaterium s t r a i n s produce b a c t e r i o c i n s which are named megacins (Ivanovics & Nagy, 1958). Only some B. a n t h r a c i s
and B. subtilis strains are s e n s i t i v e to the m e g a c i n s . Holland (1963) described some of the properties of a new b a c t e r i o c i n , megacin C , which several s t r a i n s of B, megaterium p r o d u c e .
Bacteriocins active a g a i n s t several c u l t u r e s of the same s p e c i e s are produced by Listeria monocytogenes; t h e s e b a c t e r i o c i n s are named monocins and can be c l a s s i f i e d into t y p e s A and B on the b a s i s of c r o s s - r e s i s t a n c e . The monocins have no effect on the
Gram-negative b a c t e r i a or on strains of Streptococcus but they do act on strains of Staphylococcus and Bacillus (Hamon & Peron, 1962).
The first c e r e c i n was discovered by McCloy (1951) in a study of Bacillus p h a g e s . They are produced by s t r a i n s of Bacillus c e r e us and do not act on any known indicator s t r a i n s for the b a c t e r i o c i n s of Gram-negative b a c t e r i a (Hamon & Peron, 1963b).
Various s p e c i e s of Streptococcus of the e n t e r o c o c c u s group produce b a c t e r i o c i n s , namely the e n t e r o c o c c i n s (Brock, Peacher & Pierson, 1963). The e n t e r o c o c c i n s are c l a s s i f i e d into five t y p e s on the b a s i s of their s e n s i t i v i t y to proteolytic e n z y m e s , h e a t , chloroform, and their a c t i v i t y s p e c t r a . Depending on the t y p e s of e n t e r o c o c c i n , they act on s t r a i n s of S. z y m o g e n e s , S. f a e c a l i s , S. faecium and S. l i q u e f a c i e n s .
Fredericq (1946) w a s the first to study b a c t e r i o c i n s from
s t r a i n s of S t a p h y l o c o c c u s . Hamon & Peron (1963d), a l s o studying the s t a p h y l o c o c c i n s , discovered t h a t , apart from the strains of
Staphylococcus and Bacillus which Fredericq had found to be s e n s i t i v e to t h e s e b a c t e r i o c i n s , s t r a i n s of Listeria and Corynebacterium are a l s o s e n s i t i v e to s t a p h y l o c o c c i n s .
In 1954 Jacob described a new a n t i b a c t e r i a l principle p r o duced by strain 10 of Pseudomonas pyocyanea which differed in i t s properties from the other known a n t i b i o t i c s such a s pyocyanin
(Ehrismann, 1934) and p y o c y a n a s e (Emmerich & L6w, 1899. See Topley & W i l s o n , 1964), This s u b s t a n c e , which he named pyocin, is mainly a c t i v e on other s t r a i n s of P. pyocyanea and h a s a similar specific b a c t e r i c i d a l a c t i v i t y typical of b a c t e r i o c i n s .
C r a d o c k - W a t s o n (1965) discovered b a c t e r i o c i n s in s t r a i n s of Proteus h a u s e r i . These b a c t e r i o c i n s are only a c t i v e on P. hquseri s t r a i n s and do not inhibit any other s t r a i n s of the family E n t e r o b a c -t e r i a c e a e . C o e -t z e e (1967) described m o r g a n i c i n s , produced by P . morganii, a s well a s b a c t e r i o c i n s produced by Providence s t r a i n s . No e x t r a s p e c i e s a c t i v i t y c a n be demonstrated for the morganicins or the b a c t e r i o c i n s produced by Providence, except for Providence Strain NCTC 9190 which h a s an inhibitory effect on a P . rettgeri s t r a i n .
Strains of Alcaligenes f a e c a l i s are found to produce b a c t e -riocins (Mar'e & C o e t z e e , 1964). They are inhibitory for several s t r a i n s of A. f a e c a l i s a s well a s s t r a i n s of E s c h e r i c h i a , S h i g e l l a , Serratia, Staphylococci and P r o t e u s .
De Klerk & C o e t z e e (1961) discovered that one heteroferment a heteroferment i v e and e l e v e n homofermenheterofermentaheterofermentive sheterofermentrains of heterofermenthe family Lacheterofermentobac -t e r i a c e a e produce b a c -t e r i o c i n s . The ac-tion of -t h e s e b a c -t e r i o c i n s i s found to be r e s t r i c t e d to c e r t a i n members of the same family,
Ryan, Fried & Mukai (1955) and Mukai (1960) reported that upon irradiation with ultraviolet l i g h t , strain 15 of Escherichia coli would l y s e and r e l e a s e an a n t i b a c t e r i a l a g e n t . Since no b a c t e r i o -phages were d e t e c t e d in the l y s a t e , t h e y considered the agent to be a colicino In order to further e l u c i d a t e the properties of c o l i c i n 1 5 , Endo et a l . (1965); Sandoval, Reilly & Tandler (1965) and Mennigmann
(1965a) found that the b a c t e r i c i d a l a c t i v i t y could be concentrated by high speed centrifugation. Electron microscopy of t h i s fraction
showed it to c o n s i s t of s m a l l - h e a d e d p h a g e - l i k e p a r t i c l e s , and the Quantity of p h a g e - l i k e bodies seems to correlate with the degree of biological a c t i v i t y .
In an i n v e s t i g a t i o n of the biochemical nature of the pyocin produced by Pseudomonas aeruginosa strain R, Kageyama & Egami
(1962) and Kageyama (1964) found that the pyocin appeared similar
to rod-like particles when examined in the electron microscope, and Ishii, Nishi & Egami (1965) could demonstrate at least two structu ral components of this pyocin which resemble the headless contractile tails of bacteriophages.
Bradley & Dewar (1966) studied the morphology of bacterio-cins with the electron microscope and ascribed the colicin H activity of Escherichia coli A 10 to phage-like particles similar to colicin 1 5 . They also proved that three other pyocinogenic strains of P.aerugi-nosg liberate structures similar to those of strain R, and that a monocin liberated by a strain of Listeria monocytogenes consists of phage-like particles. Takeya et a l . (1967) showed that pyocin 28 consisted of cross-striated rods about 1000A in length and another pyocin produced by P.aeruginosa strain C was described by Higerd, Baechler & Berk (1967).
In several Bacillus spp, a few phage-like particles, which are apparently physiologically identical to bacteriocins, but have not been classified as such were isolated by Seaman, Tarmy & Marmur (1964) and Stickler, Tucker & Kay (1965). These were described as defective temperate bacteriophages which are able to lyse sensitive cells but unable to multiply intracellularly.
Taubeneck (1963), while testing a number of Proteus strains and their stable L-forms for lysogeny, discovered that Proteus
b i l l s strain 52 l i b e r a t e s t a l l - l i k e s t r u c t u r e s , which contract upon ad<-sorption, and kill some P. mirabilis and P . v u l g a r i s s t r a i n s . In an i n v e s t i g a t i o n of microtubules in two s t r a i n s of P . m i r a b i l i s , Van I t e r s o n , Hoeniger & Nijman van Zanten (1967) demonstrated that both s t r a i n s , when induced by mitomycin C , produce phage t a i l - l i k e s t r u c tures similar to the pyocins described by Ishii et a l . (1965).
Nature of Bacteriocins
Bacteriocins comprise a group of varied antibiotic s u b s t a n c e s , which differ in numerous c h a r a c t e r i s t i c s . Mennigmann (1965b) d e v i s e d a l i s t of different criteria t o c h a r a c t e r i z e a n t i b a c t e r i a l a g e n t s and to differentiate b a c t e r i o c i n s from b a c t e r i o p h a g e s and defective b a c t e r i o phages : - .
1 . Production of a n t i b a c t e r i a l agent with ultraviolet irradiation, mitomycin C treatment or thymine d e p r i v a t i o n .
2 . Antibacterial a c t i v i t y limited to the number of related s t r a i n s .
3 . Loss of a n t i b a c t e r i a l activity on h e a t i n g .
4 . Loss of a n t i b a c t e r i a l a c t i v i t y on treatment with t r y p s i n . 5 . No t r a n s m i s s i b i l i t y of the a n t i b a c t e r i a l a c t i v i t y .
6 . Lysis of b a c t e r i a l culture on i n d u c t i o n .
7 . No a n t i b a c t e r i a l activity left in the supernatant fluid after high speed centrifugation.
Numbers 1 to 3 above indicate the p r e s e n c e of an a n t i b a c terial a g e n t , N o s . 4 and 5 favour the a n t i b a c t e r i a l agent a s being a b a c t e r i o c i n and N o s . 6 and 7 favour the a n t i b a c t e r i a l agent a s being a bacteriophage or defective b a c t e r i o p h a g e ,
Fredericq (1948, 1957) used the following criteria to d i s tinguish different c o l i c i n s from each other :
-1 . Extent and specificity of the a c t i v i t y spectrum. 2 . Specificity of r e s i s t a n t m u t a n t s . 3 . Extent of diffusibility in a g a r . 4 . Temperature s e n s i t i v i t y . 5 . Sensitivity to proteolytic e n z y m e s . 6 . Electrophoretic mobility, Bacteripcinogenic factors
The ability to produce a c o l i c i n i s a s t a b l e heritable property which i s governed by genetic determinants c a l l e d colicinogenic
f a c t o r s .
Fredericq (1954) discovered that some c o l i c i n o g e n i c s t r a i n s
of Escherlchia coli and Shigella, when grown in broth with n o n - c o l i cinogenic s t r a i n s , transmitted their colicinogenic property to the n o n -colicinogenic c e l l s . He worked with E. coll K..,,, which w a s c o l i cinogenic for c o l i c i n E , and showed that only the F s t r a i n s transmit colicinogeny while no transfer of c o l i c i n E occurs in c r o s s e s with F* c e l l s .
Alfoldi, J a c o b , Wollman & Maze (1958) and Clowes (1963) were able to demonstrate that c o l i c i n E, w a s not integrated into the
i
b a c t e r i a l chromosome, Hfr s t r a i n s with differing ' o r i g i n e s ' and orientations of transfer were used a s donors of c o l i c i n E . By i n t e r rupted mating experiments t h e y showed that there w a s a c o n s t a n t time of entry of the colicin E, factor from e a c h Hfr donor.
Ozeki & Stocker (1958) were able s u c c e s s f u l l y to transmit c o l i c i n C_ (classified a s type E) to Salmonella typhimurium and Escherlchia coli s t r a i n s by t r a n s d u c t i o n with phage PLT 22 and phage
P . The frequency of t r a n s d u c t i o n was the same a s that found for transduction with other markers (Adams, 1959).
O z e k i , Stocker & Smith (1962) i n v e s t i g a t e d the t r a n s m i s s i o n of colicinogeny between s t r a i n s of Salmonella typhimurium. They worked with n o n - c o l i c i n o g e n i c S* typhimurium strain LT2 and different
s t r a i n s of Escherichia coli which were c o l i c i n o g e n i c for c o l i c i n E1 , B or K and a Shigella sonnei strain which produced c o l i c i n s I and E .
It w a s found that the five c o l i c i n s are transferred from the E. coli and S. sonnei s t r a i n s into the S. typhimurium strain and each c o l i c i n o -genie factor c a u s e s the production of a c o l i c i n which i s i n d i s t i n g u i s h able from the original colicin transferred from the donor organism. These different colicinogenic s t r a i n s of S. typhimurium LT2 were mated with different n o n - c o l i c i n o g e n i c strains of S. typhimurium and it w a s found that c o l i c i n s I and B are transmitted by singly c o l i c i n o genic S. typhimurium s t r a i n s w h e r e a s c o l i c i n E and K are n o t . How ever s t r a i n s LT2 carrying either c o l i c i n I or c o l i c i n B in addition to c o l i c i n E , c o l i c i n K and c o l i c i n E , transmit b o t h .
The colicin I and B f a c t o r s , a s d i s t i n c t from the other c o l i cin f a c t o r s , were found to b e h a v e like s e x f a c t o r s . O z e k i , Stocker & Smith (1962) found that c o l i c i n I and B promote conjugation of c e l l s through which the transfer of non-infective c o l i c i n f a c t o r s , such a s c o l i c i n E and E , o c c u r .
The phenomenon of epidemic spread among n o n - c o l i c i n o g e n i c Strains w a s observed by Stocker, Smith & Ozeki (1963) in c o l i c i n I and B. They suggested that many or all the b a c t e r i a newly infected b y either of t h e s e factors became 'effective d o n o r s ' and that t h i s
property of high frequency transfer of colicinogeny w a s maintained by the newly converted c e l l s for a few g e n e r a t i o n s . Thereafter, only a small proportion of colicin I b a c t e r i a c a n denote c o l i c i n I , b e c a u s e
the donor ability becomes r e p r e s s e d in the same way a s the function of other newly introduced structural g e n e s , such a s in A - l y s o g e n i c c e l l s (Pardee, Jacob & Monod, 1959).
Meynell & Lawn (1967) described a new type of s e x pili in the conjugational transfer of c o l i c i n factor lb by Salmonella typhimurium. These l b pili differ from other known pili in that they are morphologi c a l l y d i s t i n c t from common pili and other sex pili such a s t h o s e d e t e r mined by the F factor. They do not adsorb any of the F specific; phages but do adsorb the I specific phage (Lawn, M e y n e l l , Meynell & D a t t a , 1967). Meynell & Lawn (1967) found that there i s a
correlation b e t w e e n the incidence of c e l l s with l b pili and the ability to donate c o l i c i n l b . The donor ability w a s a l s o permanently dimin ished by a combination of repeated high speed blending and periods for regeneration of p i l i . As a result of t h e s e f i n d i n g s , Meynell & Lawn (1967) s u g g e s t that the l b pili play a role in the transfer of c o l i c i n l b during conjugation.
Under the conditions of partial thymine d e p r i v a t i o n , Clowes (1965) found it p o s s i b l e to eliminate c o l i c i n s with high efficiency from t h y m i n e l e s s s t r a i n s a s well a s the elimination of F factors from F c e l l s and of RTF f a c t o r s , but found no elimination of the i n t e grated F s e x factors (Hfr f a c t o r s ) .
Except for c o l i c i n s I and B, the c o l i c i n o g e n i c factors are
plasmids which r e p l i c a t e in p h a s e with the b a c t e r i a l chromosome without killing the c e l l s . When a non-colicinogenic cell becomes c o l i c i n o g e n i c , the c o l i c i n a l s o confers immunity to that colicinogenic c e l l . Nomura & Maeda (1965) suggested that the immunity is due t o a change in some component or s t r u c t u r e , perhaps in the cell mem b r a n e s , which i s important in the t r a n s m i s s i o n of the specific stimulus which e v e n t u a l l y affects the target in the s e n s i t i v e c e l l s .
P a s t e u r e l l a p e s t i s differs from P . p s e u d o t u b e r c u l o s i s in that it c o n t a i n s the fibrinolytic factor (F), the c o a g u l a s e factor (C) and is n o n - m o t i l e . Brubaker, Surgalla & Beesley (1965) noted that the production of F and C i s correlated with the production of p e s t i c i n I determinant (PI). They found that the three structural g e n e s for the three a c t i v i t i e s are linked and situated on an extrachromosomal d e t e r minant. These workers (Brubaker, Beesley & Surgalla, 1965) a l s o observed t h a t a n o n - p e s t i c i n o g e n i c strain of P . p e s t i s r e s e m b l e s t h a t of a w i l d - t y p e P . p s e u d o t u b e r c u l o s i s . They s u g g e s t that mutational e v e n t s take p l a c e in P . p e s t i s to convert it to a form which r e s e m b l e s P . p s e u d o t u b e r c u l o s i s and that conversion of P . p s e u d o t u b e r c u l o s i s to a form which r e s e m b l e s P. p e s t i s could c o n c e i v a b l y occur upon donation of the PI determinant b y P . p e s t i s .
Production of Bacteriocins
According to Jacob et a l . (195 3) one of the criteria which determines a colicin i s its lethal b i o s y n t h e s i s - colicin production involves the death of the bacterium without l y s i s „ Although b a c teriocinogenic s t r a i n s p o s s e s s the genetic ability to produce b a c t e r i o -c i n , they do not do so under all -c o n d i t i o n s .
The discovery by J a c o b , Siminovitch & Wollman (1952) that c o l i c i n ML-E, produced by Escherichia c o l i ML, could be i n duced by ultraviolet l i g h t , led Fredericq (1954), and later Hamon & Lewe (1955) to u s e t h i s method of induction for the production of c o l i c i n s in different s t r a i n s of E. c o l i .
The h y p o t h e s i s t h a t all the c o l i c i n produced by a c o l i c i n o -genic c u l t u r e , either spontaneously or after induction by ultraviolet light irradiation, was s y n t h e s i s e d and r e l e a s e d by a portion of the c o l i c i n o g e n i c c e l l s and that t h e s e c e l l s were s u b s e q u e n t l y n o n - v i a b l e , led O z e k i , Stocker & Margerie (1959) to study the k i n e t i c s of c o l i c i n production. They used a Salmonella typhimurium strain which had been made c o l i c i n o g e n i c for colicin E?. When a mixture of the colicinogenic strain and the s e n s i t i v e strain w a s m a d e , and i n c o r porated into a soft agar l a y e r , they found that tiny clear s p o t s were formed in the lawn of o r g a n i s m s . Each of the c l e a r spots originated from a single bacterium and they proposed the term lacunae for t h e s e
inhibitory a r e a s . They were a l s o able to demonstrate by micromani-pulative i s o l a t i o n that the production of colicin was a lethal event and the c e l l s which r e l e a s e d the c o l i c i n s were n o n v i a b l e . In n o n -l y s o g e n i c s t r a i n s of Escherichia c o -l i , there is a continuous r e -l e a s e of c o l i c i n . In s t r a i n s that are bacteriocinogenic a s well a s l y s o g e n i c , the r e l e a s e i s at the time of l y s i s (Reeves, 1965).
lijima (1962) found that by adding chloramphenicol after induction, no c o l i c i n production w a s d e t e c t e d and suggested a s a r e s u l t of t h e s e findings that c o l i c i n production w a s a de novo s y n t h e s i s . Ben-Gurion (1965) a l s o showed that the addition of fluoroura-c i l and thymine after irradiation prevented the produfluoroura-ction of fluoroura-c o l i fluoroura-c i n and suggested that t h i s r e s u l t indicated a de novo s y n t h e s i s of RNA which w a s n e c e s s a r y for the production of c o l i c i n in c o l i c i n o g e n i c c e l l s .
I k e d a , Kageyama & Egami (1964) found that the pyocin p r o duced by Pseudomonas aeruginosa strain R was s y n t h e s i s e d de novo only after i n d u c t i o n . They d e t e c t e d no DNA s y n t h e s i s after i n d u c tion and the r e l e a s e of the pyocin w a s concomitant with c e l l l y s i s .
Megacinogenic strain 216 d o e s not normally excrete megacin (Ivanovics, 1962), but t h i s strain proves to be high inducible when exposed to ultraviolet light i r r a d i a t i o n . Megacin production was found not to be a s s o c i a t e d with normal multiplication of the c e l l s
but w a s a lethal b i o s y n t h e s i s when induced by ultraviolet irradiation«
The production of s t a p h y l o c o c c i n s could only be d e m o n s t r a ted on solid m e d i a . No s t a p h y l o c o c c i n s could be d e t e c t e d in fluid medium (Lachowicz, 1965).
Hertman & Ben-Gurion (1958) found that the production of p e s t i c i n I w a s inducible with ultraviolet i r r a d i a t i o n . Pesticin I was excreted into the surrounding medium without l y s i s of the producing o r g a n i s m s .
Mode of action of b a c t e r i o c i n s
Fredericq (1952) showed that c o l i c i n K could be adsorbed out of a solution by s e n s i t i v e b a c t e r i a and Hamori & Peron (1960) were able to prove that t h i s adsorption w a s s p e c i f i c . Their work showed that six different c o l i c i n s and five different pyocins could be adsorbed by s e n s i t i v e s t r a i n s but not by r e s i s t a n t m u t a n t s . Data produced by Holland (1962) showed that megacin 216 was a d sorbed to the s e n s i t i v e strain Bacillus megaterium 207M.
The k i n e t i c s of killing by b a c t e r i o c i n s w a s first studied by J a c o b , Siminovitch & Wollman (1952) with the u s e of c o l i c i n ML-E. Their r e s u l t s a s well a s t h o s e for pyocin ClO (Jacob, 1954) s u g g e s t e d that one b a c t e r i o c i n particle killed one b a c t e r i u m . Kageyama,
Ikeda & Egami (1964) were a l s o able to confirm the r e s u l t s of Jacob (1954) with a pyocin produced by Pseudomonas aeruginosa strain R.
Nomura & Maeda (1965) proposed a model whereby the attachment of a single c o l i c i n particle to a receptor site on the cell wall c a u s e s an irreversible change in the receptor s i t e . This change in the receptor site is transmitted to the s e n s i t i v e target within the c e l l , presumably along the c e l l membranes, which l e a d s to the death of the c e l l .
Nomura & Nakamura (1962) and Nomura (1963) found that high multiplicities of colicin K inhibits the oxidative phosphorilation system of the c e l l , which l e a d s first to the inhibition of DNA s y n t h e s i s and then to the inhibition of RNA and protein s y n t h e s i s , a s well a s the inhibition of the a c t i v e transport of potassium through the c e l l s u r f a c e . They a l s o found that the reproduction of virulent phage T w a s inhibited when c o l i c i n K w a s added soon after infection but that it d o e s not
induce the development of (\ in l y s o g e n i c c e l l s ,
Colicin E i n d u c e s the degradation of DNA in the c e l l and
t h i s degradation i s dependent on the multiplicity of c o l i c i n E (Nomura, 1963). No degradation of RNA was d e t e c t e d . Colicin E induces the development of $ in l y s o g e n i c Escherichia coll c e l l s , and h a s no effect on the oxidative phosphorilation system or on the a c t i v e t r a n s port of p o t a s s i u m .
Colicin E inhibits protein s y n t h e s i s but not DNA or RNA s y n t h e s i s (Nomura, 1963). The inhibition of protein s y n t h e s i s by colicin E~ was studied by Konisky & Nomura (1967) and it was found that some specific alteration of the ribosome l e a d s to an inactivation of the specific transfer RNA binding function. Colicin E does not degrade RNA, nor does it induce ^ in l y s o g e n i c c e l l s .
The inhibition of macromolecular s y n t h e s i s and phage growth in both c o l i c i n K treated c e l l s , and in c o l i c i n E treated c e l l s w a s reversed by the treatment with t r y p s i n . The killing action of colicin E w a s only poorly r e v e r s i b l e by treatment with t r y p s i n .
This may be due to the irreversible breakdown of DNA (Nomura, 1963). Fredericq (1958) suggested that the r e v e r s a l of the killing action of c o l i c i n s by trypsin treatment w a s due to the d i g e s t i o n of the attached colicin p a r t i c l e , s i n c e c o l i c i n s are s e n s i t i v e to t r y p s i n .
The pyocin produced by Pseudomonas pyocyanae C10 which w a s discovered and studied by Jacob (1954) c a u s e d the respiration of the b a c t e r i a l s u s p e n s i o n to d e c r e a s e g r a d u a l l y . Bacteria that have adsorbed the p y o c i n , do not multiply and e v e n t u a l l y become non - v i a b l e .
The megacins act on the c e l l membrane of s e n s i t i v e c e l l s (Ivanovics, Alfttldi & N a g y , 1959). Within 10 to 15 minutes there i s a marked drop in respiration and the cell c o n t e n t s s t a r t to leak o u t .
In the c a s e of megacin C (Holland, 1963) there i s a breakdown of the DNA of the s e n s i t i v e c e l l s . RNA and protein s y n t h e s i s a l s o
s t o p , but no degradation of RNA t a k e s p l a c e .
Studies by Puck & Lee (1955) showed that c e l l l e a k a g e , induced by T„ bacteriophage in the course of normal infection, slows down within a few minutes * This effect on c e l l permeability pro duced by viable phage i s reversed by a "sealing r e a c t i o n " induced by the phage after infection.
Phage t a i l - l i k e structures p o s s i b l y a c t in the same way a s phage g h o s t s . The mechanism of killing by g h o s t s i s different from that of viable phage in that the phage g h o s t s lack the cell wall
repair mechanism (Terzi, 1967). Phage g h o s t s puncture the c e l l wall and c a u s e c e l l leakage which eventually l e a d s to c e l l death
(Herriot, 1951).
Chemical nature of b a c t e r i o c i n s
Bacteriocins are set apart from the other a n t i b i o t i c s by their s i z e . Goebel & Barry (195 8) and Amano, Goebel & M i l l e r -Smidth (195 8) discovered that c o l i c i n K was a s s o c i a t e d with the O somatic antigen of Escherichia coli K . The O somatic antigen is a lipocarbohydrate protein complex, and on d i s s o c i a t i o n of the complex two components were found. The first component i s rich
in protein and h a s a b a c t e r i c i d a l activity ten times greater than the colicin i t s e l f . The second component is a lipopolysaccharide which has the properties of an endotoxin and is devoid of colicin a c t i v i t y .
Ribi et a l . (1964) reported that extraction of gram-negative b a c t e r i a l cell w a l l s with aqueous phenol y i e l d s a toxic macromolecu-lar complex which is named endotoxin and which c o n s i s t s of protein, lipid and p o l y s a c c h a r i d e . The polysaccharide of the endotoxin i s the O somatic antigen of the b a c t e r i a l c e l l wall and it was found that the protein c a n be eliminated from the lipopolysaccharide protein complex without l o s s of t o x i c i t y . It h a s been s u g g e s t e d that the toxicity of the lipopolysaccharide endotoxin r e s i d e s primarily in the lipid and that the polysaccharide moiety merely functions a s a carrier
(Westphal & LUderitz, 1954).
Hutton & Goebel (1962) showed that c o l i c i n V was a l i p o -carbohydrate complex which i s a s s o c i a t e d with O antigen of the producing strain Escherichia coli K . An unidentified colicin
(Ntiske, Htisel, Venner & Zinner, 1957) produced by E. coli S . G . 710 w a s a l s o found to contain protein lipid and c a r b o h y d r a t e . Moreover t h i s colicin was similar to the O somatic antigen of the parent s t r a i n ,
Chemical a n a l y s i s of colicin A (Barry, Everhart, Abbott & Graham, 1965) showed it to c o n s i s t mainly of protein and to contain
no DNA. No r e l a t i o n s h i p w a s found between c o l i c i n A and the specific O and H antigens present on the surface of strain E s c h e r i -chia coli CA , which produces t h i s c o l i c i n .
Purified colicin F ( E j , obtained from Escherichia coli CA , contains a high percentage of protein but l a c k s the lipid component
(Reeves, 1963). It i s suggested that c o l i c i n F d o e s not form part of the O somatic antigen of the parent o r g a n i s m .
Keene (1966) studied the chemical a n a l y s i s of colicin I produced by a strain of Escherichia c o l i . It w a s found to be a lipo-carbohydrate protein c o m p l e x .
Megacin 216 (Holland, 1961) in c o n t r a s t to colicin K, w a s i s o l a t e d free from any lipocarbohydrate c o m p l e x . It was found to be a chemically pure protein m o l e c u l e .
Studies on the pyocin produced by Pseudomonas aeruginosa strain R (Kageyama, 1964) showed it to be a p r o t e i n , which c o n s i s t s of 20 amino a c i d s and a negligible amount of s u g a r s .
De Klerk & Smit (1967) i n v e s t i g a t e d the properties of a b a c -teriocin produced by Lactobacillus fermenti. This b a c t e r i o c i n was found to be a lipocarbohydrate protein with small amounts of h e x o
-samine and p h o s p h o r u s . The protein fraction was found to contain 16 amino a c i d s .
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CHAPTER II
INCIDENCE OF BACTERIOCINOGENY IN PROTEUS VULGARIS Page INTRODUCTION 36 METHODS . . . . . . . . . . 37 Media . . . . . . . . . . , . . . . 37 Bacterial c u l t u r e s 39 Detection . , . . . . . . . 40 Ultraviolet irradiation . . . . . . . . « . . . 40 T r a n s m i s s i b i l i t y . . . . . . . . . . , 42 RESULTS tf . . . . . . 42 DISCUSSION 43 REFERENCES 50
CHAPTER II
I N C I D E N C E O F BAC T E R I O C I N O G E N Y IN P R O T E U S V U L G A R I S
INTRODUCTION
The d i s c o v e r y by Lwoff, Siminovitch & Kjelgaard (1950) that the mutagenic a g e n t , ultraviolet l i g h t , c a u s e s mass l y s i s of l y s o g e n i c c e l l s with the concomitant production of phage p a r t i c l e s by almost all the c e l l s led Fredericq (1954) to u s e t h i s method of induction on colicinogenic s t r a i n s . Fredericq (1954) found that c o l i c i n s , like b a c t e r i o p h a g e s , were i n d u c i b l e , but in c o n t r a s t to b a c t e r i o p h a g e s did not l y s e the producing s t r a i n . Under t h e s e experimental conditions only a few c e l l s produced c o l i c i n , while the effect of ultraviolet light was to induce a large proportion of c e l l s (about 50%) to produce c o l i c i n .
Mitomycin C , and antibiotic isolated from Streptomyces c a e s p i t o s u s , w a s found to inhibit s e l e c t i v e l y the s y n t h e s i s of DNA in Escherichia coli (Shiba, Terawaki, Taguchi & Kawamata, 1959). Iijima (1962) was the first to u s e mitomycin C s u c c e s s f u l l y on E_. coli K 30 for the induction of colicin K production.
The induction of b a c t e r i o c i n production may a l s o be brought about by temperature s e n s i t i v e mutants of a bacteriocinogenic s t r a i n . Kohiyama & Nomura (1965) found that they could induce c o l i c i n E in a temperature s e n s i t i v e mutant of Escherichia coli K 12 strain 162 .
Thymine deprivation a l s o induces b a c t e r i o c i n production, This first became known when Mennigmann (1964) found that a thymine auxotrodib strain of Escherichia c o l l , colicinogenic for c o l i c i n 1 5 , c a n be induced when the c e l l s are deprived of t h y m i n e .
The method used to indicate the p r e s e n c e of a b a c t e r i o c i n in a bacterial culture w a s originally d e v i s e d by Fredericq (1948). Bacteriocinogenic c u l t u r e s were streaked on agar p l a t e s and overlayed with a top layer seeded with the s e n s i t i v e indicator organism. This method for the d e t e c t i o n of b a c t e r i o c i n s w a s later simplified by
Abbott & Shannon (1958), and i s the method used in t h i s study for the d e t e c t i o n of b a c t e r i o c i n s .
An attempt w a s made to demonstrate the production of b a c t e r i o c i n s by different s t r a i n s of Proteus vulgaris on solid medium.
METHODS
Media
(a) Liquid media
1. Nutrient Broth: Difco nutrient broth powder, 16 g m . ; N a C l , 10 g m . ; Oxoid l a b - l e m c o broth powder. 16 g m . ; Difco tryptose broth, 52 gm. D i s s o l v e t h e s e s u b s t a n c e s in 2 l i t r e s d i s tilled w a t e r , steam for 45 min. Add 2ml. N - C a C L s o l u t i o n . Adjust the pH to 7 . 4 by adding 4% NaOH s o l u t i o n . Bottle and s t e r i l i z e in the a u t o c l a v e .
2 . Dlfco Brain-heart infusion broth: Bottled in 50 m l . and 100 m l . q u a n t i t i e s .
3 . Difco MacConkey Broth.
(b) Solid media
1 . Difco MacConkey a g a r . 2 . Difco S S - a g a r .
3 . Nutrient agar made up a s follows :
(i) Meat e x t r a c t : 2 l b s . minced lean meat - add 2000 m l . tapwater - l e a v e overnight at 4 . Filter through c h e e s e c l o t h , Steam 1 h r . Filter through filter p a p e r . Steam 1 h r . Leave overnight.
(ii) Add Ocean Gold a g a r , 30 gm. to the 2000 m l . meat e x t r a c t , Steam until d i s s o l v e d . Add Dlfco P e p t o n e , 20 gm.,; N a C l , 10 gm. Adjust pH to 7 . 4 by adding 4% NaOH s o l u t i o n . Add 14 m l . of a 3.5% Na CO s o l u t i o n . Steam for j hr, Filter through c h e e s e c l o t h . Autoclave,
Bacterial Cultures
One hundred and e i g h t e e n strains of Proteus vulgaris were l o c a l l y i s o l a t e d during 1966 and i n v e s t i g a t e d for b a c t e r i o c i n o g e n y . Nine b a c t e r i o c i n o g e n i c s t r a i n s were s e l e c t e d and t e s t e d a g a i n s t a variety of g r a m - n e g a t i v e organisms (Table I ) .
Strains were maintained at 4 . Cultures were incubated at 2 5 ° . TABLE I N o . indicator s t r a i n s Proteus mirabilis 44 P., morganii 13 P . rettgeri 15 Providence 9 Escherichia coli 18 Salmonella s p p9 6 Salmonella t y p h o s a 2 Shigella s p p . 14 Alcaligenes f a e c a l i s 7 Serratia m a r c e s c e n s 7
Table lo Various gram-negative organisms used a s indica tor s t r a i n s for nine b a c t e r i o c i n o g e n i c s t r a i n s s e l e c t e d at random.
Detection
A modification of the method of Abbott & Shannon (1958) was u s e d . In preparation for the t e s t for bacteriocinogenic a c t i v i t y , each of the 118 P. vulgarls s t r a i n s w a s inoculated in nutrient broth and incubated overnight. Each culture was then inoculated a c r o s s an SS-agar plate to give a confluent streak of growth. The p l a t e s were incubated for 7 h r . The organisms on the p l a t e s were killed by exposure to chloroform. For t h i s purpose a d i s c of filter paper w a s placed in the lid of a Petri dish and saturated with chloroform. The portion containing the medium w a s replaced and the plate left inverted for \ h r . The growth w a s scraped to one end with the edge of a c l e a n slide and then removed together with a small portion of a g a r . Overnight broth c u l t u r e s of the organisms to be t e s t e d for s e n s i t i v i t y to the b a c t e r i o c i n s were then inoculated a c r o s s the plate at right a n g l e s to the position formerly occupied by the primary s t r e a k . The p l a t e s were incubated for 16 h r , (Plate I ) .
Ultraviolet light Irradiation
The method of d e t e c t i o n of b a c t e r i o c i n s a s d e s c r i b e d above w a s repeated with p l a t e s that were d u p l i c a t e s of t h o s e used in the foregoing experiment. These p l a t e s were irradiated for 4 min. with the use of a 30-W Hanovia sterllamp (wavelength 2537 A) from a d i s t a n c e of 25 c m . The p l a t e s were incubated for 16 h r . in the
PLATE I
Proteus vulgaris s t r a i n s 11 and 35 c r o s s - s t r e a k e d according to the method of Abbott & Shannon (195 8) with P. vulgaris indicator organisms 4 and 10. Areas of inhibition in the confluent growth of the indicator organisms indicate s e n s i tivity to b a c t e r i o c i n ,
dark to prevent photoreactivation (Kelner, 1949; Newcombe, 1955) and thereafter treated in the same way as described above.
Transmisslbility
Serial transmlssibility of the killing effect was tested by cutting out a small piece of the area of inhibition with a sterile wire loop. This was then transferred to broth. The broth was sterilized with a few drops of chloroform. After the chloroform had been bubbled off, dilutions of the suspension were spotted on a lawn of the indica tor organism. Nine strains of Proteus vulgarls which produce b a c -teriocins were selected and treated in the same way as described above, except that the suspensions were spotted on lawns of differ ent organisms of the family Enterobacteriaceae.
The bactericidal property of the bacteriocins was tested by subculture in broth of clear areas of inhibition.
RESULTS
Incidence
Seventy of the strains produced areas of inhibition on one or more of the Proteus vulgarls c r o s s - s t r e a k s . These bacteriocins inhibited from five to 87 P. vulgarls indicators. Nine bacteriocins
