CURRENT MICROBIOLOGY Vol. 21 (1990), pp. 341-347

Current Microbiology © Springer-Verlag New York Inc. 1990

Molecular Cloning of the EnvC Gene of Escherichia coli Jfirgen Robert Klein, Bernhard Henrich, and Roland Plapp Department of Biology, Division of Microbiology, University of Kaiserslautern, Kaiserslautern, FRG

Abstract. DNA fragments complementing the envC mutation could be isolated by cloning chromosomal DNA in the vector pUH84. When the frequencies of transformation and the frequencies of restoring the envC + phenotype were compared, the high copy number hybrid plasmids complemented with a frequency of 10 -5. After subcloning the e n v C - c o m p l e m e n t i n g DNA fragment into the low copy number plasmid pLG339, efficient complementation was achieved by spontaneous integration of the IS2 element of Escherichia coli. By nucleotide sequence analysis, a potential promoter, a ribosome-binding site, and an unidentified reading frame were detected in the respective DNA fragment.

The cell cycle of Escherichia coli is a process that requires the participation of a large number of genes. Many mutations have been isolated and classified, according to their phenotype, into nine classes [7]. The gene envC is thought to be required for septum formation during the process of cell division [28]. Since nearly all cell division genes are essential, the corresponding mutants are only conditionally defective. The only known envC mutant strain PM61 could be isolated from the parental strain P678 as a nonconditional mutant [28], and the e n v C gene was mapped at min 81 of the Escherichia coli chromosome. The defect in cell separation of strain PM61 results in the formation of filaments consisting of 10-80 individual cells. At normal septation sites, successive initiations of septa can be observed, but only a minor portion is successfully completed [28]. In the bacterial cell cycle the formation of a division septum between two segregated chromosomes is a central event, and many genes involved in the division process ofE. coli have been identified [19]. Like other mutants that are involved in the E. coli cell cycle, the e n v C mutant, besides the defect in cell separation, shows a number of phenotypic anomalies. The mutation in the e n v C gene of E. coli is likely to be responsible for a number of phenotypic alterations. The phenotype associated with the e n v C mutation includes increased sensitivity against hydrophobic and hydrophilic antibiotics, detergents, lysozyme, and against the dye crystal violet, leakiness, and alterations in phospholipid composition and metabolism [16, 23, 24, 29, 33, 34]. Thus, e n v C

was considered to be a pleiotropic mutation. Comparison of the cell envelope of mutant strain PM61 and parental strain P678 did not reveal any difference in the protein patterns of the cytoplasmic or outer membranes, nor could a difference in the composition of the lipopotysaccharides be detected [3, 34]. To contribute to the determination of the function of the e n v C gene product, we cloned a DNA fragment complementing the e n v C mutation. The selectable phenotype, sensitivity against crystal violet, was chosen to determine e n v C + transformants and therefore was defined as the e n v C phenotype. Materials and Methods Bacterial strains and plasmids. All strains used in this study were Escherichia coil K12 and are listed in Table 1. The e n v C - strain

PM61 was obtained from J. Starka (Marsaille, France), and the genetic and phenotypic markers could be verified with the exception of the anomalous filamentous growth, originally described to be temperature independent, which could be observed only at incubation temperatures above 42°C. The plasmids used were pUH84, a vector suitable for positive selection of cloned DNA fragments [13]; pLG339, a low copy number vector [35]; and the vector pUCI8 [38]. Construction of a recA- derivative of PM61. In order to avoid recombination events during cloning experiments, we crossed a recA56 allele mutation into the e n v C - strain PM61 by conjugation with strain JC10240, using the method of Csonka et al. [6]. Cloning of the D N A complementing the envC- phenotype. A library ofE. coli chromosomal DNA was constructed by inserting 7- to 20-kitobase (kb) fragments from a Sau3A partial digest into

Address reprint requests to: Dr. Jiirgen Robert Klein, Fachbereich Biologie, Abteilung Mikrobiologie, Universit/it Kaiserslautern, D675 Kaiserslautern, FRG.

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CURRENT MICROBIOLOGYVol. 21 (1990)

Table 1. Bacterial strains Strain P678 PM61 PM6110 MM294

Genotype F - thr leu thi malA xyl lacY gal rpsL as pP678 but envC

as PM61 but recA srl::Tnl0 F - endA1 hsdR17(rk- mk ") supE44 thil lambdaJM109 recA endAl gyrA96 hsdR17(rk- mk +) rel A1 lambda supE44 thi del(lac proAB) IF' traD36 proAB lacIq Z del M15] GM48 thr |eu thi lacY galK gaiT ara tonA tsx dam dcm supE44 JC10240 Hfr PO45 srlC300::Tnl0 recA56 thr300 ilv318 rpsE300 CK103 ara malT cysE::Tn5 mtl2 recA1 [F'ts mtl secB]

A

80

Source and reference J. Starka J. Starka [29] This study B. Bachman [9, 21] Pharmacia [38]

--

;ct

--

fdhA*

/

/

pLCll-7 pLC15-48 . . . . . . .

\

pLC19-11 - - 81-pLC15-29 . . . . . . . . . .

--

82

cysE gpsA rfaC. D. P * gadRS pclA * j.

--

pyrE

--

IpoT gltS

cysE secB

\

/

Tn10

I

/ /"

F" ts mr! secB

J. Kok [38] B. Bachman [6l J. Beckwith [17]

the BamHI site of the lysis gene E of qbXt74 in pUH84. For prevention of a loss of cloned genes during propagation of the library in E. coli, the ligation mixture was directly transformed into the envC mutant strain PM61. Because of the positive selection for insert DNA provided by the E-gene of pUH84 [13], each viable transformant selected with ampicitlin contained a recombinant plasmid. Plasmids complementing the envC mutation were selected with crystal violett at a concentration of 10/xg/ml.

Recombinant DNA techniques. Restriction enzymes and other nucleic acid-modifyingenzymes were purchased from Boehringer Mannheim GmbH, or Pharmacia LKB GmbH (Freiburg) and were used as recommended by the manufacturers. The isolation of plasmid DNA and the recovery of DNA fragments from gels were performed according to the methods of Maniatis et al. [20]. M13 DNA was isolated according to Messing and Vierra [22].

Generation of deletions and DNA sequencing. DNA was sequenced by the dideoxy method [32] with strain JM109 as a host for Mt3mpl8 and M13mpl9 derivatives [38]. M13 subclones carrying overlapping deletions in the respective inserts of chromosomal DNA were generated by the exonuclease III method of Henikoff [12]. Nucleotide sequence analysis. Sequence data were analyzed with the Microgenie software package (Beckman Instruments Inc., Palo Alto, California, USA) [27].

Results A t t e m p t s w e r e m a d e to r e s t o r e t h e e n v C + p h e n o t y p e in t h e m u t a n t s t r a i n PM61 w i t h r e s p e c t to its hypersensitivity against crystal violet.

Complementation of the envC mutation by an F' factor. A c c o r d i n g t o t h e g e n e t i c m a p o f t h e E s c h e richia c h r o m o s o m e g i v e n b y B a c h m a n [1], t h e e n v C

Fig. 1. (A) Part of the linkage map ofEscherichia coti [1], containing the envC gene locus. The map positions of some plasmids of the Clarke-Carbon bank, are indicated according to the gene protein index of E. coli K-12 [26]. An asterisk indicates that the position with respect to the nearby markers is not known. (B) The F'factor F'ts mtl secB [17] spanning the genes mtt, cysE, and secB and conferring the envC* phenotype.

g e n e is l o c a t e d b e t w e e n m i n 80 a n d 81. H o w e v e r , the p r e c i s e p o s i t i o n w i t h r e s p e c t to t h e a d j a c e n t genes has not been determined. The envC mutation was complemented by introduction of the F' factor F ' t s mtl s e c B [17], w h i c h is k n o w n to c o v e r t h e m a p p o s i t i o n o f t h e e n v C g e n e (Fig. I).

Screening of plasmids of the Clarke-Carbon bank. The four plasmids pLCll-7, pLC15-48, pLCI9-11, a n d p L C 1 5 - 2 9 o f the C l a r k e - C a r b o n b a n k [5], w h i c h a r e k n o w n to m a p n e a r t h e e n v C l o c u s [26] (Fig. 1), w e r e i n t r o d u c e d i n t o t h e s t r a i n PM61 a n d t e s t e d f o r complementation of the envC phenotype. Colicin El-resistant transformants harboring the plasmids were plated on LB medium containing crystal violet. All p l a s m i d s f a i l e d to c o m p l e m e n t t h e s e n s i v i t y o f t h e s t r a i n a g a i n s t this d y e .

Cloning of chromosomal fragments in pUH84 and identification of a hybridplasmid that complements the envC mutation. C h r o m o s o m a l D N A of E. colt w t w i t h a s i z e r a n g i n g f r o m 7 t o 20 k b w a s l i g a t e d i n t o the B a m H I r e s t r i c t i o n site o f t h e v e c t o r p U H 8 4 . T h e l i g a t i o n m i x t u r e w a s u s e d to t r a n s f o r m s t r a i n MM294, which was selected for ampicillin resist a n c e . P l a s m i d D N A w a s i s o l a t e d f r o m 1500 t r a n s f o r m a n t c l o n e s , a n d o n e p l a s m i d c o m p l e m e n t i n g the e n v C m u t a t i o n o f PM61 c o u l d b e i s o l a t e d , T h i s p l a s mid was called pJK1411, and PM61(pJK1411) was a b l e to g r o w o n m e d i u m c o n t a i n i n g c r y s t a l v i o l e t (10 /~g/ml). E x a m i n i n g t h e p r o p e r t i e s o f p J K 1 4 1 1 , w e c a l c u l a t e d t h a t o n l y o n e cell o u t o f 105 t r a n s f o r m a n t s showed the desired resistance against the dye used

J.R. Klein et al.: Cloning of envC gene of E. coli for selection. This f r e q u e n c y of c o m p l e m e n t a t i o n lies in the range o f the f r e q u e n c y o f s p o n t a n e o u s mutations in E. coli, but it is significantly higher than the f r e q u e n c y by which PM61 shows spontaneous resistance against crystal violet. This f r e q u e n c y ranges b e t w e e n 108 and 109. Isolation of the plasmid from crystal violet-resistant cells and reintroduction into the mutant strain did not change the complementation frequency.

343

o EcoRI

c

2000 I ClaI Clal*

I

Nucleotide sequence analysis of the insertion in pJK129 and the respective region of pJK128. F o r determination o f the nature o f the D N A fragment

8000

Hindlll

[6p]

.gall ~ o

pJKl411

I

II~--

pJK~4~2

I,,

L~--

pJK1413

L~---

pJKl414

II

L~---

pJK1415

II

~---

pJK1416

KmR

Localization of the e n v C complementing gene. The plasmid pJK1411, containing a c h r o m o s o m a l insert of about 8.3 kb, was used to construct a series of plasmids (Fig. 2). The genetic determinants conferring crystal violet resistance to the host cell could be localized on an E c o R I - H i n d I I I fragment of plasmid pJKl413. This plasmid c o m p l e m e n t e d the e n v C p h e n o t y p e with the same low frequency as pJK1411. The high c o p y n u m b e r of the plasmid was suspected to be reason for this low f r e q u e n c y o f c o m p l e m e n t a tion. T h e r e f o r e the c o m p l e m e n t i n g D N A fragment was subcloned into the vector pLG339, present in six to eight copies p e r c h r o m o s o m e [35]. A 5.3-kb fragment o f p J K l 4 1 3 generated by partial E c o R I and H i n d l I I restriction and filling in the ends was ligated into the H i n c I I site of pLG339. T r a n s f o r m a n t s , selected with k a n a m y c i n and containing the desired hybrid plasmid pJK128, were tested for c o m p l e m e n tation of the e n v C - p h e n o t y p e . Again a low frequency o f c o m p l e m e n t a t i o n was observed. Only one crystal violet-resistant colony arose from 5 × 106 plated plasmids harboring PM61 cells. Surprisingly, plasmids isolated f r o m these crystal violet-resistant transformants were a p p r o x i m a t e l y 1.3 kb larger in size than the original plasmid pJK128. After reintroduction of this enlarged plasmid, called p J K 129, into PM61, all o f the k a n a m y c i n - r e s i s t a n t transformants also showed resistance against crystal violet. The increase in plasmid size, which coincided with the acquisition of e n v C c o m p l e m e n t i n g activity, was fully reproducible and could be o b s e r v e d in PM61 as well as in PM61 I0, the r e c A - derivative of the e n v C mutant strain. Restriction analysis of ten independently isolated e n v C c o m p l e m e n t i n g plasmids revealed identical H i n c I I restriction patterns. The differences in the restriction patterns of pJK128 and pJK129 allowed the location of a 1.3 kb D N A insertion in pJK129, which in all cases had been inserted in the same orientation.

CI~I EioRl BFHI

I

--J--]

0oo0

4000 I

r

--T-7

G

~

H

,,1,,, IS2

i

II

I

I i

I ~

l J

pJK128

l

p~K129

Fig. 2. Restriction maps of derivatives of the plasmid pJKI411. Plasmids A-F are derivatives of the high copy number vector pUH84 carrying DNA fragments inserted into the polylinker region located in the gene E of the bacteriophage d~X174. Plasmids G and H are derivatives of the low copy number vector pLG339. The plasmids pJK1411 and pJK1413 complement the envC mutation with a low frequency, pJK129 fully complements the envC phenotype, and all other plasmids do not. (A) pJKl411 is the originally isolated hybridplasmid which complements the envC mutation with a frequency of about 10 5 (B) pJK1412 resulted from pJK1411 after treatment with EcoRI and religation. (C) pJK1413 is pJKt411 from which a 2,4 kb HindIII fragment was deleted. (D) To construct pJKl414, a 1.45 kb BamHI fragment carrying the kanamycin resistance gene from Tn903 [25] was isolated from pUC4K [36] and inserted into the unique BamHI site of pJKl413. (E) pJK1415 arose from pJK1413 by deletion of a 0.45 kb BgliI fragment. (F) To construct pJKl416, a 3 kb DNA fragment was isolated after partial ClaI and HindIII restriction of pJKl413. After filling in the ends with PolIK, this fragment was inserted into the HincII site of pUH84. (G) pJK1413 was digested with HindIII and partially digested with EcoRI. The resulting 5.7 kb fragment was isolated and, after filling in the ends, was used to generate pJK128 by ligation into the HincII site within the tetracycline resistance gene of the vector pLG339 [35]. (H) pJK 129 was reproducibly obtained by selection of PM61(pJK 128) for resistance against crystal violet. It contains a 1.3 kb IS2 insertion, indicated by the solid bar. Sequence analysis allowed identification of an overlap of two dam-methylation sites with the ClaI* site, therefore the enzyme restricts only if the DNA is isolated from dam-cells.

and the m e c h a n i s m that leads to c o m p l e m e n t a t i o n o f the e n v C p h e n o t y p e by plasmid pJK129, p a r t s o f the plasmids pJK128 and pJK129 w e r e sequenced. The sequencing strategy is p r e s e n t e d in Fig. 3. C o m paring the two s e q u e n c e s allowed the identification of a 1331 bp insert in pJK129, which turned out to be identical with the insertion e l e m e n t IS2 o f E . coli. Our sequence fully confirms the revised nucleotide sequence of IS2 reported by R o n e c k e r and R a k [30]. The IS2 insertion was a c c o m p a n i e d b y a doubling of

344

CURRENT MICROBIOLOGY Vol. 21 (1990)

Hincll

]

Hindl

H~ncll

I

I

pJK128

Hin¢II

I

pLG339

pJK12q

IS2 sequence

.... % ~--t

Hini ......... [ ~ ell . i([B. . Hin.cll . . ~1S2 C/or'L HincU[CEllOI EcoRl[ I I C] [ I ~} IH;ncl[ ° l t M/nell ,gglll BamHI~E¢oR

r E F

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I

I

i

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0

SOD

[000

....

Cla f"

J C/of"

]500

2000

Hincll H/nell

pJK~2,

2500

1

pJK134

[bp]

I 0

Fig. 3. Sequencing strategy. Restriction analysis of plasmids pJK128 and pJK129 allowed the localization of the integration site of a yet unidentified 1,3 kb DNA fragment into the 1 kb HincII fragment of pJK 128. Sequencing of this fragment and comparison with the nucleotide sequences of the 2.38 kb HincII and the 1.2.5 HindIII-ClaI fragments of pJK129 allowed the identification of the IS2 sequence in pJK 129. The restriction site ClaI* is protected if the DNA is not isolated from a dam- strain.

m2(,) ......................... -35

EcoRI Clm

H;~cfl

J

I 1000

t ................~......~... 2000 ~000

~nc

I I

| 4000

I {

] 5000

I

,~000 [bp]

Fig. 5. Restriction maps of DNA inserts of plasmid pJK129 and its derivatives pJK131 and pJK134. The asymmetric position of the HindIII site in the IS2 sequence allowed the identification of the orientation of IS2 in independent isolates of plasmid pJK129. In order to delete a DNA fragment lying upstream of the potential promoter (P), pJK129 was cut with HindIII, and the ends were filled in with PolIK. To construct pJK131, a fragment generated by subsequent restriction with Sphl was isolated and cloned into HincII and SphI digested pLG339. The deletion of an SphI-BglII fragment (marked with a delta) yielded plasmid pJK134, which has lost the ability to complement the envC mutation.

~;,TA,~,CAO;,TO TCTOC-MATA T AGbGGCAAA " ,,, I 2>so TCbAGTAC~A TAGCGAACTG TTGACat.. [

TATTTTTCTT

t POTENTIAL PROMOTER

TAtAa

TCTGCGAGTT AALCGCGTTGC C T T T T T G G G T Hl,'~Ii

Tg

cat

': 2410 AAATAACGCG

POTENTIAl. RIBOSOME BINDING SITE

2 4.6 C

C T T T T G G T T T T T T G A G G A A T A G T A A T G A c G AAACAT.,C,.Am r" G G T T T T T C C T 3 auUCCUCC

a c ....

161RNA SEQUENCE

UNIDENTIFIED READING FRAME

25'~0 CCTGCCCTCC TTTATTCTGA TCTCCGCGGC TTTAATCGCC GGTTGTAACG 2560

ATAAGGGAGA AGAGAAAGCT CACGTCGGTG

AACCGCAGGT TACCGTTCAT

ATTGTAAAAA CGGCCCCGTT AGAAGTTAAG

AC']'GAATTAC C A G G C C G C A C

CAATGCTTAT CGTATAGCCG AAGTTCGCCC

ACAGGTTAGC GGGATCGTAC 2710 TGCAAGCAGG CCAGTCCCTG

2610 2660

TGAATCGCAA TTTCACTGAA GGCAGCGATG T A C C A G A T C G A T :r Clol

Fig. 4. A p o t e n t i a l e n v C p r o m o t e r c o n s t i t u t e d b y s e q u e n c e s o f IS2 a n d c h r o m o s o m a l D N A . T h e p r o m o t e r p r e c e d e s a p o t e n t i a l

ribosome binding site and an unidentified reading frame. The consensus sequence of E. coli promoters [ 11] is given below the sequence of the promoter. The sequence upstream of the IS2 insertion is not given, owing to the fact that no gene structure like promoter site or open reading frame could be identified.

the p e n t a m e r G T A G G at the site of integration (Fig. 4). Various reports o f g e n e activation that were associated with IS2 integration h a v e b e e n published, and in several cases the fusion of IS2 D N A in orientation II [8] and c h r o m o s o m a l D N A created a powerful p r o m o t e r activity [4, 14, 15, 31]. The sequence anal-

ysis of the IS2 integration site in pJK129 m a d e it possible to identify a potential p r o m o t e r , which agrees well with the c o n s e n s u s sequence for E. coli p r o m o t e r s [11] (Fig. 4). The IS2 e l e m e n t in orientation II contributes the - 35 site, and the 5 bp integration site serves as a good - 10 site. A p p r o x i m a t e l y 70 nucleotides d o w n s t r e a m of this hypothetical promoter, a potential r i b o s o m e binding site can be located. This site, at a distance of 6 bp, p r e c e d e s an A T G start c o d o n that belongs to an unidentified reading frame extending to the end of the established sequence.

Construction of pJK131 and pJK134. The sequence analysis suggested that the gene responsible for complementation o f the e n v C mutation might be located d o w n s t r e a m o f the hypothetical IS2 fusion promoter. T h e r e f o r e , it should be possible to delete D N A segments located u p s t r e a m o f this p r o m o t e r , without affecting the envC c o m p l e m e n t i n g activity. This was confirmed by subcloning a HindIII-SphI fragment f r o m pJK129 in pLG339. T h e resulting plasmid pJK131 (Fig. 5) still conferred resistance to crystal violet to e a c h transformant. Efforts to clone the isolated HindIII-SphI f r a g m e n t f r o m pJK129 also into the high c o p y n u m b e r v e c t o r pUC18 [38], h o w e v e r , were not successful.

J.R. Klein et at.: Cloning of envC gene of E. coli

For determination of the extension of the envC complementing gene, a BglII-SphI fragment of pJK131 was deleted. Although this construction, yielding plasmid pJK134, eliminated only 350 bp of the chromosomal insert (Fig. 5), the resulting transformants showed the envC- phenotype. These data indicate that approximately 4.5 kb of chromosomal DNA located downstream of the potential IS2 fusion promoter in pJK 131 are essential for complementation of the envC mutation. Expression of tetracycline resistance. The envC mutant showed a delayed expression of tetracycline resistance. After transformation of strain PM61 with plasmid pBR322, the resistance to ampicillin and tetracycline did not appear simultaneously. Instead, as shown in Fig. 6A, the expression of tetracycline resistance lagged behind the expression of ampicillin resistance by more than 70 min. When the experiment was performed with the strain PM61(pJK129), a significantly shorter tetracycline expression time could be measured (Fig. 6B). This suggests that plasmid pJK129 contributes a function that is essential to the fast expression of tetracycline resistance. Since the proteins mediating tetracycline resistance are associated with the cytoplasmic membrane [18], the envC mutation may interfere either with the integration or with the proper functions of these proteins in the membrane. Influence of the cloned envC gene on cell morphology. In contrast to the envC mutant strains PM61 or PM6110, which show chain formation and filamentous growth at elevated growth temperatures (42°-45°C), no signs of anomalous cell morphology could be observed in the presence of plasmids pJK 129 or pJK 131, respectively.

Discussion Because of the numerous phenotypic anomalies of the envC mutant, it can not be decided whether the envC mutation as a pleiotropic mutation is responsible for all its attributes or whether several mutations confer the "envC phenotype'. On the basis of our studies with the different plasmids constructed in this work, a minimum of 4.5 kb chromosomal DNA is required to complement the envC mutation, as defined by the crystal violet sensitivity phenotype. Several experiments indicated that overexpression of the envC gene had an inhibitory effect on cell growth. This might be a reason for the failure to

345

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Fig. 6. Expression of the tetracycline resistance genes encoded on plasmid pBR322. Competent cells of PM61 and PM61(pJKI29) were prepared and transformed according to the RbCt method of Hanahan [10]. The number of transformants that grew in the presence of 100/~g/ml ampicillin after the standard recovering time of 1 h at 37°C were taken as 100%. Transformants were plated on LB medium supplemented with ampicillin (100 t~g/mL) or ampicillin (100 ~g/ml) and tetracycline (12.5 p~g/ml respectively. (A) Competent cells of PM61 transformed with plasmid pBR322. The expression of tetracycline resistance lagged behind the expression of ampicillin resistance. (B) In strain PM61 (pJK129), complemented in its envC phenotype, the time necessary for expression of tetracycline resistance was significantly shortened after transformation with pBR322.

complement the envC mutation with high copy number plasmids of the Clarke and Carbon genebank. The nature of the envC mutation. Because spontaneous envC revertants can be observed, the mutation in PM61 can not be a deletion, but the relatively low reversion frequency of 108-109 indicates that mutations in two distinct genes may be responsible for the envC phenotype. This would agree well with the calculation that the insertion of pJK131 has a coding capacity of about 4.5 kb, sufficient for a rather large gene product or for several distinct proteins. The IS2 insertion. A complete map of the nonrandom

346 IS2 l o c a t i o n s in the E. coli g e n o m e has b e e n published [2]. Since this m a p c o n t a i n s no IS2 insertion site n e a r the e n v C locus at min 81, the IS2 insertion site found in pJK129 s e e m s not to be a natural locus. IS2 s e q u e n c e s can c a u s e a d j a c e n t genes to be overe x p r e s s e d [15]. A s the IS2 insertion was o b s e r v e d only a f t e r s u b c l o n i n g o f the e n v C - c o m p l e m e n t i n g D N A f r a g m e n t into a low c o p y n u m b e r p l a s m i d , the p r o t e i n p r o d u c t ( s ) e n c o d e d by this D N A fragment m a y h a v e lethal effects if p r e s e n t in high a m o u n t s . A l t h o u g h the i n s e r t i o n of the IS2 e l e m e n t generated a p o t e n t i a l p r o m o t e r , originally the e n v C gene might h a v e b e e n c l o n e d t o g e t h e r with the authentic p r o m o t e r in the high c o p y n u m b e r p l a s m i d p U H 8 4 . Due to a p o s s i b l y lethal effect of e n v C h y p e r e x p r e s sion, a m u t a t i o n , affecting or p r e v e n t i n g e n v C exp r e s s i o n , might have b e e n selected. The low effic i e n c y of e n v C c o m p l e m e n t a t i o n e x h i b i t e d by the resulting p l a s m i d could then be e x p l a i n e d by the n e c e s s i t y for a s e c o n d m u t a t i o n in the host cell, which p a r t l y c o m p e n s a t e s the affection of e n v C expression.

The mechanism of envC- complementation. At this time no d a t a c o n c e r n i n g the e n v C gene p r o d u c t are a v a i l a b l e . O n e o f the m o s t intriguing o b s e r v a t i o n s is that e x p r e s s i o n o f the c l o n e d e n v C gene has an effect on the p e r m e a b i l i t y of the cell wall. In n o r m a l E. coli cells, the o u t e r m e m b r a n e s e r v e s as a p e r m e a b i l i t y barrier, which p r o t e c t s the cell, e s p e c i a l l y against h y d r o p h o b i c c o m p o u n d s . In PM61 this b a r r i e r is d i s t u r b e d , resulting in h y p e r s e n s i t i v i t y against several a n t i b i o t i c s and d y e s . R e s i s t a n c e to these comp o u n d s can be r e c o n s t i t u t e d by use of e n v C c o m p l e menting p l a s m i d s like pJK129 and pJK131. As can be inferred f r o m the d e l a y e d e x p r e s s i o n of t e t r a c y cline r e s i s t a n c e , not only the o u t e r m e m b r a n e seems to be affected by the e n v C m u t a t i o n , but also the c y t o p l a s m i c m e m b r a n e . H o w e v e r , we can not decide w h e t h e r a gene p r o d u c t e n c o d e d by the plasmids pJK129 or pJK131 d i r e c t l y alters the m e m b r a n e s o r w h e t h e r it has an indirect effect on m e m b r a n e c o m p o s i t i o n or function. R a t h e r c o m p l e x m e c h a n i s m s are to be e x p e c t e d to c h a n g e the cell m o r p h o l o g y f r o m chains and filaments to n o r m a l rods. H o w e v e r , the o b s e r v a t i o n s that no differences in the m e m b r a n e p r o t e i n p a t t e r n s of PM61 and P678 c o u l d be d e t e c t e d [24, 29] a n d that the e n v C gene s e e m s to act lethally if p r e s e n t in high c o p y n u m b e r s s u p p o r t a m o r e i n d i r e c t m e c h a n i s m of a l t e r a t i o n of the cell e n v e l o p e by the e n v C p r o d u c t . A m o r e detailed i n v e s t i g a t i o n of the m e c h a n i s m by which the E n v C p r o t e i n influences the p r o p e r t i e s and c o m p o s i -

CURRENT MICROBIOLOGYVol. 21 (1990)

tion of the cell e n v e l o p e might h a v e to await the e l u c i d a t i o n o f the c o m p l e t e p r i m a r y s t r u c t u r e o f the e n v C gene and its e v e n t u a l regulation.

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