APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Dec. 1987, p. 2733-2738 Vol. 53, No. 120099-2240/87/122733-06$02.00/0Copyright © 1987, American Society for Microbiology

Genetic and Physical Characterization of proBA Genes of theMarine Bacterium Vibrio parahaemolyticus

ATIN R. DATTA,t* RACHEL OSTROFF,t AND ANTHONY M. MACQUILLAN

Department of Microbiology, University of Maryland, College Park, Maryland 20742

Received 29 June 1987/Accepted 11 September 1987

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine thisrelationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesisgenes were cloned in various plasmids. Some genetic and structural properties of those genes were examined.Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB butnot proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V.parahaemolyticus. -y-8 insertion mutagenesis of this subcloned fragment indicated that proB and proA genes ofV. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, whichmap closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V.parahaemolyticus.

Reduced rates of growth resulting from increases in exter-nal osmolarity occur to a variable extent in a wide array ofmicroorganisms. Elevated levels of proline and glutamate inthe intracellular pools, associated with increased osmolarityof the medium, have been reported for a number of organ-isms (19, 20, 23, 25). Moreover, exogenous proline has beenfound to partially overcome the growth inhibition imposedby moderate increases in medium osmolarity for Salmonellaspp. (4, 5, 8), Klebsiella pneumoniae (21), and Escherichiacoli (unpublished data). The potential importance of in-creased proline pools for cellular mechanisms which allevi-ate high osmotic stress was heightened by the isolation of aproline-overproducing mutant of Salmonella typhimuriumwith increased salt (NaCI) tolerance (8). These studies havesparked a renewed interest in the biosynthesis of proline.The pathway comprises four reactions (9), of which three, inE. coli and S. typhimurium, are catalyzed by proA, proB,and proC gene products (15). The remaining step, third in thesequence, the cyclization of glutamate semialdehyde to1-pyrroline-5-carboxylate, is spontaneous (9).Marine bacteria, by virtue of their association with a saline

environment, present an opportunity to study the mechanismofone type of osmotolerance, i.e., that of halotolerance. Vibrioparahaemolyticus is a moderately halophilic marine bacterium.It exhibits moderate halotolerance which is greater in rich thanin synthetic media (18). The association of increased prolinelevels and halotolerance mentioned above has led us to inves-tigate the proline biosynthetic genes of V. parahaemolyticus.This report describes the cloning of a 3.1-kilobase (kb) segmentof V. parahaemolyticus DNA which contains proA and proBgenes. We also describe some genetic and physical character-istics of these genes and compare them with those of thefunctionally similar loci in E. coli.

MATERIALS AND METHODS

Bacterial strains, plasmids, and bacteriophage. All thebacterial strains and plasmids used in this work, with their

* Corresponding author.t Present address: Division of Microbiology, Food and Drug

Administration, Washington, DC 20204.: Present address: Department of Microbiology, University of

Colorado Health Science Center, Denver, CO 80262.

relevant genotypes and phenotypes, are listed in Table 1.Bacteriophage TC45 was provided by J. Tommassen.Media and culture conditions. Bacterial cultures were

routinely grown in LB broth and LB agar (10). Plasmid-containing strains were always grown with antibiotics. An-tibiotic concentrations (in micrograms per milliliter) were asfollows: tetracycline, 15; chloramphenicol, 20; ampicillinand kanamycin, 25 each; spectinomycin and rifampin, 100each. Minimal medium was M9 (26) supplemented with 0.4%carbon source and gelled with 1.5% agar. Additional supple-ments were 0.1 M NaCl for growth of V. parahaemolyticusstrains and nutritional requirements for E. coli and V.parahaemolyticus strains (thiamine hydrochloride [vitaminB1], 1 ,ug/ml; proline, 50 ,ug/ml; and other amino acids, 20,ug/ml each).

Genetic techniques. E. coli K-12 strains were transformedwith plasmid DNA by the method of Cohen et al. (7) withminor modifications. Plasmids were transferred into V.parahaemolyticus strains by a triparental mating procedure(10) in which AD24 was always used as a source of helperplasmid pRK2013. Sensitivity to phage TC45 to check for thephoE gene (29) was tested for by spotting various dilutionson a bacterial lawn on LB agar plates. The sensitivity toazaguanine or thioguanine for the gpt gene was tested asdescribed elsewhere (16).Recombinant DNA techniques. Plasmid DNA was ex-

tracted from overnight cultures by the method of Birnboimand Doly (3). For large-scale preparation of pCVD301 deriv-atives, this method was scaled up and then followed by twoCsCI-ethidium bromide equilibrium density gradient centrif-ugations. For large-scale extraction of ColEl derivatives,the overnight cultures were amplified with chloramphenicol(6). The DNA was purified by CsCI-ethidium bromide equi-librium centrifugation (11). Chromosomal DNA was isolatedby the method of Priefer et al. (27) with minor modifications.Conditions for restriction endonuclease digestion and liga-tion ofDNA were those specified by the suppliers. For DNAhybridization, probe DNA was labeled with [32P]dATP byusing a nick translation kit (Bethesda Research Laborato-ries, Inc.). Chromosomal and plasmid DNA were digestedwith PstI, and fragments were separated on a 0.8% agarosegel. DNA transfer and filter hybridization were performed bya modification of the method of Southern (28) with

2733

on April 15, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant geotype or SourcephenotypeE. coli K-12HB101

CGSC 4288

AD37

N418N485

CE1194

X340

X342

G-6

G-8

G-9

G-li

G-12

G-13

AD24RR5AD79AD89AD100AD107AD120AD123AD129AD130AD131AD132AD133AD134AD135AD136AD137AD138AD139

V. parahaemolyticusAD12AD47AD48AD49AD50AD51AD52AD53AD54AD55AD56

leuB6 proA2 lac YI galK2xyl-5 mtl-l rB mBstrR-recAJ3 supE44 A-

A (gpt-lac)5 thi-I mal-24spcAJ2 X- F' 128

A (gpt-lac)5 thi-J mal-24spcAJ2 X-

HfrH thi rpsLthr-J leu-6 thi-I lacYlgalK2 ara-14 xyl-5 mtl-lproA2 his-4 argE3 str-31 tsx-33 supE44recA13 F' 8 (F' gal)

F- bgl phoS21 thr leuproA2 his thi argElac Yl galk xyl rpsL

proB28 metBI relAlspcTl X-

proC29 metBI relAlspcTl X-

F- proA leu thr thi lacrpsL

F- proA leu thr thi lacrpsL

F- proA leu thr thi lacrpsL

F- proB leu thr thi lacrpsL

F- proB leu thr thi lacrpsL

F- proB leu thr thi lacrpsL

HB1O1(pRK2013)HB1O1(pAD5)HB101(pAD79)HB1O1(pAD89)AD37(pCVD301)HB1O1(pAD107)HB1O1(pAD120)N485(pAD120)AD37(pAD120::y-8) 4AD37(pAD120::-y-8) 5AD37(pAD120::-y-8) 6AD37(pAD120::y-8) 9AD37(pAD120::y-8) 10AD37(pAD120::-y-8) 11AD37(pAD120::-y-8) 13AD37(pAD120::-y-8) 14AD37(pAD120::y-8) 15HB101(pAD120::y-8) 9HB1O1(pAD120::-y-8) 15

Prototroph KP- RifbPro- AD12Pro- AD12Pro- AD12Pro- AD12Pro- AD12Pro- AD12Pro- AD12Pro- AD12Pro- AD12Pro- AD12

D. Morris

B. Bachmann

A. Datta

NIHaNIH

TABLE 1-ContinuedRelevant geotype or Source

Strain or plasmid phenotypphenotype

PlasmidspBR322 Tetr Ampr, A Wortman

nonconjugativepBR325 Tetr Ampr Camr, S. Chakroborty

nonconjugativepCVD301 Tetr, nonconjugative A. DattapAD5 Tetr Pro' pCVD301 This workpAD89 Tetr Ampr Pro' pBR325 This workpAD107 Tetr Pro' pBR322 This workpAD120 Tetr Pro' pCVD301 This workpRK2013 Kanr, conjugative J. Kaper

'NIH, National Institutes of Health Culture Collection.bKP-, Kanagawa phenomenon negative.

J. Tommassen

GeneScreen (New England Nuclear Corp.) as the mem-D. Stein brane. Hybridization at 65°C was followed by washings at

65°C to achieve stringent conditions. All other techniquesD. Stein were performed by the method of Maniatis et al. (24).L. Csonka Isolation of Pro- mutants of V. parahaemolyticus. V.

parahaemolyticus Pro- mutants (AD47 through AD56) wereL. Csonka isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutagen-

esis (1) followed by two steps for enrichment. First, theL. Csonka mutagenized culture was grown in glucose minimal medium

containing proline (50 p.g/ml), so that only Pro- mutants andL. Csonka prototrophs survived (17). Second, this culture was sub-

jected to a nalidixic acid enrichment procedure (30), since V.L. Csonka parahaemolyticus is resistant to different penicillins. TheL. Csonka detailed protocol was as follows.A single colony of AD12 from thiosulfate citrate-bileJ. Kaper salts-sucrose agar was suspended in LB broth and grown atThis work 37°C to a concentration of about 2 x 108 to 5 x 108 cells perThis work ml. The culture was centrifuged, and the pellet was washedThis work twice with citrate buffer (1) and suspended in one-fifthA. Datta volume of citrate buffer. N-Methyl-N'-nitro-N-nitrosoguani-This work dine (2 mg/ml in citrate buffer) was added to give a finalThis work concentration of 50 p.g/ml, and the culture was incubatedThis work with aeration for 30 min at 37°C. After this period, theThis work mutagenized culture (98% killing) was centrifuged, washedThis work twice with saline, diluted 1:20 in M9-glucose medium (con-This work taining proline and 0.25 M NaCl), and incubated overnight atThis work 37°C with aeration. This culture was then centrifuged,This work washed twice with saline, diluted (1 ml of culture to 9 ml ofThis work medium) in M9-glucose-NaCl (0.25 M), and incubated atThis work 37°C for 60 min with aeration. Nalidixic acid was then addedThis work (20 ,ug/ml, final concentration), and the culture was incu-This work bated with shaking overnight at 37°C. The culture was thenThis work

centrifuged, washed twice with saline, and plated onto LBagar. Single colonies from these LB agar plates were then

A. Datta checked for their ability to grow on M9-glucose-NaCl (0.25This work M) agar with and without proline. Among 250 coloniesThis work checked, 4.8% were Pro-. Ten Pro- mutants selected forThis work further study gave frequencies of reversion to Pro' betweenThis work 10-6 and 10-9, indicating that the mutations must be singleThis work point.This work Isolation of -y-B insertions in V. parahaemolyticus proBAThis work genes. Insertion element -y-8 (TnJOOO), which is present in theThis work F plasmid (13), was used to mutagenize cloned proBA genesThis work of V. parahaemolyticus by the method of Guyer (14). For

this procedure, plasmid pAD120 was introduced into E. coliContinued N485 by transformation. The presence of F' 8(F' Gal) was

monitored by the Gal' phenotype, and the presence ofpAD120 was monitored by the Tetr and Pro' phenotypes.

2734 DATTA ET AL.

on April 15, 2020 by guest

http://aem.asm

.org/D

ownloaded from

V. PARAHAEMOLYTICUS proBA GENES 2735

Several single colonies of AD123 (donor) were grown in LBbroth to midexponential phase. AD37 (recipient) was grownin LB broth with aeration to midexponential phase. Thedonor and recipient cultures were mixed in 1:1 proportionsand incubated at 37°C for 60 min, after which, cultures werevortexed and appropriate dilutions were plated onto LB agarcontaining tetracycline and spectinomycin and were incu-bated at 37°C. Single colonies obtained from these matingswere checked for the Pro phenotype on M9-glucose-B1plates with and without proline. Between 5 and 10% of theTetr Spcr colonies were Pro-. One Pro- colony from eachmating was streak purified and kept for further study (AD129to AD137). Since AD37 has a deletion of the proBA genes, itwas expected that insertion of -y-8 either in the proB or in theproA donor gene would make transconjugants Pro-. Thiswas confirmed by restriction enzyme analyses of individualplasmid DNA (pAD120::-y-8) and by complementation stud-ies. To map the -y-8 insertions, pAD120::-y-8 DNA sampleswere subjected to single and double digestion by SmaI andKpnI restriction enzymes. Double digestion was achieved byfirst digesting with SmaI and then digesting the same samplewith KpnI without purifying the DNA. The -y-8 sequence hasan internal SmaI site and a KpnI site very close to the 8 end(13). By finding out which SmaI fragment was cleaved byKpnI and by measuring the sizes of the restriction fragmentsgenerated by these two enzymes, the position and orienta-tion of -y-8 were determined.

Chemicals. Restriction endonucleases and T4 DNA ligasewere purchased from New England BioLabs, Inc., andBethesda Research Laboratories. Calf intestine alkalinephosphatase was from Boehringer Mannheim Biochemicals.All other chemicals were pure reagent grade.

RESULTS

Isolation of proline genes of V. parahaemolyticus. A V.parahaemolyticus gene bank (kindly provided by Jim Kaper)in cosmid pCVD301 in E. coli HB101 was screened for V.parahaemolyticus proline genes by transferring about 960Tetr colonies to M9-glucose-B1-leucine-tetracycline plateswith and without proline. After 24 h of incubation at 37°C,four colonies grew on plates without proline. These colonieswere streak purified, and a plasmid (pAD5) isolated from oneof the colonies (RR5) was used for further investigations. Toconfirm that this plasmid had V. parahaemolyticus DNAwhich could complement pro mutations in E. coli and tofurther characterize this DNA, the plasmid was introducedinto E. coli strains (X340, X342, and AD37) with known promutations. Complementation data showed that pAD5 con-tained sufficient genetic information to restore the prolineprotrophy of E. coli in proA and proB but not in proCmutants. Since pAD5 restored the wild-type phenotype(Pro') of a proBA deletion mutant, AD37, it seems logical toassume that this cloned DNA provides proBA gene functionsby complementation rather than by extragenic suppression.Plasmid pAD5 also complemented pro mutations in all buttwo (AD51 and AD56) of the pro V. parahaemolyticusstrains listed in Table 1.

Subcloning pro genes of V. parahaemolyticus. Restrictionenzyme analyses revealed (data not shown) that the plasmidpAD5, about 48 kb in size, contained about 27 kb of insertDNA with two internal EcoRI sites. To subclone the V.parahaemolyticus pro genes, pAD5 and pBR325 DNA wereeach digested with EcoRI for 60 min at 37°C. These DNAsamples were then incubated at 75°C for 10 min to inactivatethe EcoRI (11), mixed, and ligated overnight with T4 DNA

ligase. This ligated DNA was then used to transform HB101cells. Of about 475 Tetr Ampr colonies, 11 were Pro' Cams,and the rest were Pro- Cams. The Pro' Cams colonies ofHB101 were streak purified, and one strain (AD89) contain-ing pAD89 was kept for further analysis. Figure 1 shows apartial restriction map of pAD89, which was about 19.5 kb insize and contained insert DNA of about 13.5 kb. When thisplasmid was introduced into AD37, all the Tetr transform-ants were Pro', indicating that the 13.5-kb EcoRI fragmentof V. parahaemolyticus had both proB and proA genes. Tofurther reduce the size of the insert DNA containing V.parahaemolyticus pro genes, pAD89 DNA was completelydigested with PstI, and the fragments were separated on a0.6% agarose gel. From this gel, the 3.1-kb band waselectroeluted, precipitated with ethanol, and ligated withpBR322 DNA which had been digested with PstI, treatedwith calf intestine alkaline phosphatase, and purified byphenol extraction. The ligated DNA mixture was used totransform HB101. Several Tetr Pro' colonies were isolated.Plasmid DNA from one of these colonies (AD107) waspurified and was shown to complement the pro deletionmutation of AD37. Figure 1 shows the restriction map of thisplasmid, pAD107, in which the distribution of PstI and SalIsites was quite similar to that in the proBA genes of E. coli(22). However, when the 3.1-kb PstI fragment of pAD107was used as a probe for a Southern hybridization withPstI-digested chromosomal DNA, hybridization occurred

Sm

'Pv

FIG. 1. Cloning of V. parahaemolyticus proBA genes. The thickline represents V. parahaemolyticus DNA. Bars indicate restrictionenzyme cleavage sites. B, BamHI; E, EcoRI; H, HindlIl; Bg, BglII;P, PstI; Pv, PvuII; S, Sall; Sm, SmaI; *, relative position notdetermined; CIP, calf intestine alkaline phosphatase.

VOL. 53, 1987

on April 15, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

with V. parahaemolyticus DNA but not with E. coli DNA(data not shown).Mapping the proBA genes of V. parahaemolyticus. Our

subcloning experiments and complementation tests indi-cated that the 3.1-kb PstI fragment of pAD107 contained V.parahaemolyticus genes proA and proB. To map thesegenes, the 3.1-kb PstI fragment was cloned into the PstI siteof pCVD301 (Fig. 2), and the resultant plasmid, pAD120(Fig. 2), was subjected to y-8 transposon mutagenesis. Nineindependently isolated Pro- mutants of pAD120 weremapped by single and double digestion with SmaI and withSmaI plus KpnI, respectively. The -y-8 transposon map wasobtained from M. S. Guyer (13). Figure 2 shows the posi-tions of the -y-8 insertions in pAD120. Each plasmid DNAwas then introduced into several known pro mutants of E.coli, i.e., strains G-6, G-8, and G-9, which carry proA;strains G-11, G-12, and G-13, which carry proB; and AD37,which is deleted for proA and for proB.The results (Table 2) revealed that plasmids pAD120::-y-8

4, 5, 6, 11, and 15 complemented proB but not proA strainsand therefore carried proB+ and proA. Plasmids pAD120::-y-8 9, 10, 13, and 14 complemented proA but not proB strainsand therefore carried proB and proA+. The data also indi-cated that the proB gene lies within the 1.2-kb DNA segmentproximal to the BglII site of pAD120, whereas the proA geneis in the 1.3-kb segment adjacent to the proB gene (Fig. 2).None of these mutations showed a polar effect, i.e., bothproA and proB, suggesting that in V. parahaemolyticus theproA and proB genes might be transcribed from differentpromoters and therefore function as separate operons.

Characterization of V. parahaemolyticus Pro- mutants. Tofurther characterize the V. parahaemolyticus Pro- mutants(AD47 through AD56), we introduced the plasmids pAD120(proB+ proA+), pAD120: :y-8 9 (proBproA+), and pAD120: :y-8 15 (proB+ proA) into all the mutants, with HB101 hosts ofthese plasmids as donors; pCVD301 was also introduced asa control. Complementation data from the resultant mero-diploids showed that pAD120 complementation with Pro-mutants was identical to that described earlier for pAD5(Table 3). Plasmid pAD120::y-8 9 complemented strainsAD48 and AD53, indicating that they had a defect in theproA gene, while AD47, AD49, AD50, AD52, AD54, andAD55 showed complementation with pAD120::y-8 15 andtherefore had mutations in the proB gene. Mutants AD51 andAD56, which did not show complementation with eitherpAD120 or pADS, might have mutations in proC or someother gene.

Sm SmpCVD301(21.5kb)

n*. eLPv s

Sm SmpAD 120 LJT(24.6kb) P S

pAD120:r-8 Sm(30.3kb)

Bg BgEBSPSBE

Bg Ig1

TABLE 2. Complementation analysis of pAD120::y-8 with E:coli proB or proA mutants

Pro phenotype of merodiploidsa in strains:Plasmid G-6, G-8, G-9 G-11, G-12, G-13 AD37

(proA) (proB) A(Iac-gpt)

pAD120 + + +pAD120::-y-8 - +

(4, 5, 6, 11, 15)pAD120::y-8 + -

(9, 10, 13, 14)a Plasmids were introduced into these strains by transformation.

Localization of gpt+ and phoE+ genes of V. parahaemo-lyticus. On the E. coli chromosome, the gpt and phoE genesmap very closely to one side of the proBA gene cluster (2).The gpt gene codes for a guanine-hypoxanthine phosphori-bosyltransferase (16). E. coli strains carrying mutations inthe gpt gene show resistance to the guanine analogsazaguanine and thioguanine, because these analogs cannotbe converted to their respective nucleotides to exert theirtoxic effects (16). The phoE gene, which codes for a porin forphosphates and other anions, also serves as a receptor forphage TC45 (29). To determine whether V. parahaemoly-ticus also has these two genes closely located to the proBAgenes, the following experiments were conducted. PlasmidpAD5 and several derivatives were introduced into E. coliAD37 to check 8-azaguanine and 6-thioguanine sensitivityand into E. coli CE1194 to check TC45 sensitivity. InCE1194, the proA2 mutation is in reality a proA phoE gptdeletion (29). The transformants were checked for Gpt+ andPho- phenotypes. Results of these experiments (data notshown) were incorporated in Fig. 1. Both gpt and phoE werepresent in pAD5 and pAD89 but absent from pAD107.Subcloning experiments with different fragments of pAD89(data not shown) indicated that gpt and phoE were locatedon opposite sides of the proBA cluster. PstI cuts the E. coliphoE gene (22). Therefore, the absence of phoE activity inpAD107 might be due to our generation of the V.parahaemolyticus DNA with PstI.

DISCUSSION

Thus far, the proline biosynthesis system of V. parahae-molyticus has shown a number of genetic similarities tothose of E. coli and S. typhimurium. The proA and proBgenes were adjacent. The complementation of different proA

Sm--a I

Pv

BgEBSPBg Bg IzqS

-.0-.

Pv - Pv

-',le

,- -9 Pv 1t5 '3I BS 13

pro B

PSB E

ISm

I - ,.

Pv Pv "- "

"I-.

4 S Pv 11 .li71 - -

Sm

55 6 Pv

pro A

FIG. 2. Mapping ofproBA genes of V. parahaemolyticus. Positions of -y-8 insertions in the 3.1-kb fragment in plasmid pAD120 are shownin the bottom drawing. Thick lines indicate V. parahaemolyticus DNA. Numbers represent y-8 insertion isolate numbers. Arrowheadsindicate the orientations of ends 8 of -y-8 (13). Symbols of restriction enzymes are as given in the legend to Fig. 1.

2736 DATTA ET AL.

I

on April 15, 2020 by guest

http://aem.asm

.org/D

ownloaded from

V. PARAHAEMOL YTICUS proBA GENES 2737

TABLE 3. Complementation of V. parahaemolyticus Pro-mutants with different plasmids

Pro phenotype of merodiploidsa containing plasmid:

Strain pCVD301 pAD120 pAD120::-y-8 (15) pAD120::y-b (9)(proB+A') (proA) (proB)

AD47 - + +AD48 - + - +AD49 - + +AD50 - + +AD51 - - -AD52 - + +AD53 - + - +AD54 - + +AD55 - + +AD56 - - -

a Plasmids were introduced by the triparental mating procedure (10).

and proB mutations in E. coli indicated that V. parahaemo-lyticus proBA gene products correspond functionally toglutamate kinase and glutamate semialdehyde dehydro-genase (15). The absence of complementation of an E. coliproC mutation suggested that the proC-complementing geneof V. parahaemolyticus lies well removed from the proBAgenes, as in E. coli (2). In S. typhimurium, the proBA andproC loci are also far apart (22). Additionally, the limitednumber of mapped restriction endonuclease sites in thecloned 3.1-kb DNA fragment from V. parahaemolyticusmatches quite well with the sites found in proBA genes in E.coli. These similarities in E. coli, S. typhimurium, and V.parahaemolyticus point to a degree of conservation in theproline biosynthetic pathways in these organisms. The prox-imity of gpt+ and phoE+ genes to the proBA genes in V.parahaemolyticus would seem to extend the region of pos-sible chromosome conservation for this organism and E.coli. However, the lack of cross-hybridization between theV. parahaemolyticus 3.1-kb DNA probe and E. coli DNAtempers the degree of similarity.A possible difference between the proA and proB genes in

V. parahaemolyticus, S. typhimurium, and E. coli concernsthe question of promoters. In S. typhimurium, the proBAgenes are reported to form an operon with a single promoter,and the direction of transcription is from proB to proA (22).In E. coli, although Mahan and Csonka (22) found no polareffects with different insertion mutations in the proA andproB genes, their results did not preclude the possibility thattranscription was initiated from promoters present in thetransposons. Recently Deutch et al. (12) published thecomplete nucleotide sequence of the proBA region of E. coliK-12 and have shown that there is only one possible pro-moter sequence (-10 sequence and -35 sequence) beforethe proB gene, but none between the proB and proA genes.Their results strongly indicate that E. coli proBA genes alsoform an operon and that the direction of transcription is fromproB to proA. Our results on -y-8 mutagenesis suggest thusfar that in V. parahaemolyticus, the proBA genes might betranscribed from separate promoters. Digestion of pAD89with Sall and of pAD107 with PvuII, followed by religationof the DNA and transformation of various Pro- mutants ofE. coli, resulted in the generation of a pAD89 derivativewhich is ProB- ProA+ Tets Ampr and of a pAD107 deriva-tive which is ProB+ ProA- Ampr (data not shown). Thesedata could support the idea that proB and proA genes aretranscribed from independent promoters. However, conclu-sive evidence can be obtained only from more geneticanalyses and sequence data.

Mechanisms which underlie the halotolerance of bacterialcells have long been a matter of interest. The recent studiesof enteric organisms focusing on the link between prolinepools and halotolerance led us to examine proline biosyn-thesis in V. parahaemolyticus. Our cloning and geneticstudies put us in a position to conduct a number of compar-ative growth rate experiments with E. coli and V. parahae-molyticus containing exchanged proA and proB genes and toobserve any relationship between enhanced halotoleranceand endogenous or exogenous levels of proline. However,since proline metabolism and glutamate metabolism areclosely intertwined (9), it may be that proline influences onhalotolerance in V. parahaemolyticus, if they exist, are onlyindirect and that adjustments of glutamate pools are ofgreater importance. Reports some years ago of higherintracellular levels of glutamate in V. parahaemolyticus (25)and in Beneckea harveyi (23) at higher external salt concen-trations suggest that more extensive investigation of thatarea might also be rewarding.

ACKNOWLEDGMENT

This work was supported by Office of Naval Research contractN00014-82-K-0636, P0002.

LITERATURE CITED1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. Optimal

conditions for mutagenesis by N-methyl-N-nitro-N-nitrosogu-anidine in Escherichia coli K-12. Biochem. Biophys. Res.Commun. 18:788-795.

2. Bachmann, B. J. 1983. Linkage map of Escherichia coli K-12,edition 7. Microbiol. Rev. 47:180-230.

3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

4. Christian, J. H. B. 1955. The water relations of growth andrespirations of Salmonella oranienburg at 30°C. Aust. J. Biol.Sci. 8:490-497.

5. Christian, J. H. B. 1955. The influence of nutrition on the waterrelations of Salmonella oranienburg. Aust. J. Biol. Sci. 8:75-82.

6. Clewell, D. B. 1972. Nature of Col E1 plasmid replication inEscherichia coli in the presence of chloramphenicol. J. Bacte-riol. 110:667-676.

7. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchromo-somal antibiotic resistance in bacteria: genetic transformation ofEscherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA69:2110-2114.

8. Csonka, L. N. 1981. Proline overproduction results in enhancedosmotolerance in Salmonella typhimurium. Mol. Gen. Genet.182:82-86.

9. Csonka, L. N., and A. Baich. 1983. Proline biosynthesis, p.35-51. In K. Herrnann and R. L. Somerville (ed.), Amino acidbiosynthesis and genetic regulation. Addison-Wesley PublishingCo., Reading, Mass.

10. Datta, A. R., J. B. Kaper, and A. M. MacQuillan. 1984. Shuttlecloning vectors for the marine bacterium Vibrio parahaemo-lyticus. J. Bacteriol. 160:808-811.

11. Davis, R. W., D. Botstein, and J. R. Roth (ed.). 1980. Advancedbacterial genetics. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

12. Deutch, A. H., K. E. Rushlow, and C. J. Smith. 1984. Analysisof the Escherichia coli proBA locus by DNA and proteinsequencing. Nucleic Acids Res. 12:6337-6354.

13. Guyer, M. S. 1978. The y-8 sequence of F is an insertionsequence. J. Mol. Biol. 126:347-365.

14. Guyer, M. S. 1983. Uses of transposon y-8 in the analysis ofcloned genes. Methods Enzymol. 101:362-369.

15. Hayzer, D. J., and T. Lensinger. 1980. The gene-enzyme rela-tionship of proline biosynthesis in Escherichia coli. J. Gen.Microbiol. 118:287-293.

VOL. 53, 1987

on April 15, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

16. Holden, J. A., P. D. Harriman, and J. D. Wall. 1976. Esche-richia coli mutants deficient in guanine-xanthine phosphori-bosyltransferase. J. Bacteriol. 126:1141-1148.

17. laccarino, M., and P. Berg. 1971. Isoleucine auxotrophy as a

consequence of a mutationally altered isoleucyl-transfer ribonu-cleic acid synthetase. J. Bacteriol. 105:527-537.

18. Joseph, S. W., R. R. Colwell, and J. B. Kaper. 1983. Vibrioparahaemolyticus and related halophilic vibrios. Critical Rev.Microbiol. 10:77-124.

19. Koujima, I., H. Hayashi, K. Tomochika, A. Okabe, and Y.Kanemnasa. 1978. Adaptational change in proline and watercontent of Staphylococcus aureus after alteration of environ-mental salt concentration. Appl. Environ. Microbiol. 35:467-470.

20. LeRudulier, D., A. R. Strom, A. M. Dandekar, L. T. Smith, andR. C. Valentine. 1984. Molecular biology of osmoregulation.Science 224:1064-1068.

21. LeRudulier, D., S. S. Yang, and L. N. Csonka. 1982. Nitrogenfixation in Klebsiella pneumoniae during osmotic stress: effectof exogenous proline overproducing plasmid. Biochim.Biophys. Acta 119:273-283.

22. Mahan, M. J., and L. N. Csonka. 1983. Genetic analysis of theproBA genes of Salmonella typhimurium: physical and geneticanalyses of the cloned proB+A+ genes of Escherichia coli and ofa mutant allele that confers proline overproduction and en-

hanced osmotolerance. J. Bacteriol. 156:1249-1262.23. Makemson, J. C., and J. W. Hastings. 1979. Glutamate functions

in osmoregulation in a marine bacterium. Appl. Environ. Mi-crobiol. 38:178-180.

24. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

25. Measures, J. C. 1975. Role of amino acids in osmoregulation ofnon-halophilic bacteria. Nature (London) 237:398-400.

26. Miller, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

27. Priefer, U., R. Simon, and A. Puhler. 1984. Cloning withcosmids. In A. Puhler and K. N. Timmis (ed.), Advancedmolecular genetics. Springer-Verlag KG, Berlin.

28. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

29. Tommassen, J., P. Overdudin, B. Lugtenberg, and H. Bergmans.1982. Cloning of phoE, the structural gene for the Escherichiacoli phosphate limitation-inducible outer membrane pore pro-tein. J. Bacteriol. 149:668-672.

30. Weiner, R. M., M. J. Voll, and T. M. Cook. 1974. Nalidixic acidfor enrichment of auxotrophs in cultures of Salmonella typhi-murium. Appl. Microbiol. 28:579-581.

2738 DATTA ET AL.

on April 15, 2020 by guest

http://aem.asm

.org/D

ownloaded from