Mol Gen Genet (1992) 232:145-153

MGG

© Springer-Verlag 1992

Characterization of the F plasmid bifunctional conjugation gene, traG Neville Firth* and Ron Skurray* Department of Microbiology,Monash University, Clayton, Victoria 3168, Australia Received May 31, 1991

Summary. The Escherichia coli F plasmid gene, traG, is required for two stages of the conjugation process: pilus biosynthesis and mating aggregate stabilization. The nucleotide sequence of traG has been determined and the topology of its product in the cytoplasmic membrane analysed using protease accessibility experiments. Complementation analysis employing plasmid deletions revealed a correlation between an N-terminal periplasmic segment of the protein product (TraGp) and its pilus assembly activity. Production of an anti-TraGp antiserum has facilitated the detection of TraGp*, a possible internal cleavage product of TraGp. Although its function is unknown, TraGp* is located in the periplasm and has been shown to possess sequences required for aggregate stabilization, The detection of TraGp* raises the possibility that the two functions of traG are carried out by separate products. Key words: F pilus biosynthesis - Mating aggregate stabilization - Conjugation - Protein processing - Bifunctional protein

Introduction The transfer system of the Escherichia coli F plasmid serves as the paradigm of bacterial conjugation since the underlying genetics and basic stages of the process are well characterized (for review see Ippen-Ihler and Minkley 1986; Willetts and Skurray 1987). All of the F plasmid-encoded genes that are essential for conjugation are clustered in the 33 kb transfer (tra) region. To date, 23 tra genes have been shown to be involved in the mechanism, while the roles of several other genes identified in the region have yet to be elucidated. Genes involved in the prevention of homosexual mating and * Present address: School of BiologicalSciences,University of Sydney, New South Wales 2006, Australia Offprint requests to: R. Skurray

lethal zygosis, regulation and conjugal D N A metabolism have been identified. In addition, the products of at least 14 other tra genes are directly involved in the biosynthesis of the F pilus, which is a filamentous surface appendage required for preliminary donor-recipient cell interactions. Of these only the pilus subunit, F pilin, encoded as a precursor by the traA gene (Minkley et al. 1976; Frost et al. 1984), and the product of the traQ gene, TraQp, which is implicated in the processing of prepropilin (Laine et al. 1985), have defined functions in pilus assembly. Despite the vast accumulation of information concerning F conjugation, many aspects of the process remain unclear. In particular, the mechanistic details of the processes leading up to and then facilitating the union of donor and recipient cells in a form proficient for DNA transfer are poorly understood; however, the product of the traG gene (TraGp) has been demonstrated to play a crucial role in donor-recipient cell interactions. Complementation analyses have indicated that the product of the traG gene is bifunctional, as it is required for two distinct stages in the conjugation process (Achtman et al. 1972, 1978). Together with a number of other tra region products, TraGp is required for the assembly of the F pilus, which is involved in initial donor-recipient cell contacts (Achtman et al. 1971, 1972). After the mating cells establish wall-to-wall contact, possibly via retraction of the pilus, TraGp and the product of the traN gene are needed to convert these unstable contacts to a conjugationally proficient form that is resistant to shear forces (Manning et al. 1981). Studies utilizing cloned fragments of tra region D N A have shown that traG spans the E c o R I site between E c o R I fragments 1 (fl) and 17 (1"17), the portion of the gene encoded by f17 being dispensable for piliation but essential for DNA transfer (Achtman et al. 1978). Contrary to earlier suggestions (Laine et al. 1985; IppenIhler and Minkley 1986), it has been found that TraGp is not responsible for the N-terminal acetylation of F pilin (C. Hamilton and K. Ippen-Ihler, personal communication; our unpublished data). Polypeptide analysis

146 has indicated that traG encodes a polypeptide of approximately 100 kDa (Willetts and Maule 1980; Moore et al. 1981) which fractionates with the inner membrane (Manning et al. 1981 ; Moore et al. 1981). It is intriguing that TraGp, which is localized in the inner membrane, is involved in processes occurring presumably close to or at the cell surface. To gain an insight into the nature of TraGp, we have determined the nucleotide sequence of the traG gene. Analysis of the deduced amino acid sequence of the traG product and investigations into its membrane topology suggest that while TraGp spans the inner membrane, the majority of the protein may actually reside in the periplasm. Furthermore, the two functions of TraGp have been correlated with specific regions of the protein using complementation experiments. Production of an anti-TraGp antiserum has facilitated the identification of a second, smaller traG-derived product, TraGp*. Materials and methods

Bacterial strains, plasmids and growth conditions. The E. coli K-12 strains used in this study were JM101 (YanischPerron et al. 1985), CSR603 (Sancar et al. 1979), HB101 (Boyer and Roulland-Dussoix 1969), JC3272 (Achtman etal. 1971), ED2196 (Gasson and Willetts 1977), MC1022 (Casadaban and Cohen 1980) and HL202 (Poustka etal. 1984). Plasmids used were JCFL0 (Flac+), JCFL24 (Flac + traG24), JCFL86 (Flac + traG86) (Achtman etal. 1971), JCFL101 (Flac + traGlOl) and JCFLI06 (Flac+ traGl06) (Achtman et al. 1972). General culture conditions used were 37°C in Luria broth or on Luria agar plates (Miller 1972). Ampicillin was used as required at a concentration of 50 gg/ ml. Recombinant DNA techniques. Restriction endonuclease digests were performed in TA buffer (O'Farrell et al. 1980). S1 nuclease, D N A polymerase I Klenow fragment, exonuclease Bal31 and T4 D N A ligase were used as recommended by the manufacturer (Pharmacia, Amersham, New England Biolabs and Promega). Methods for the transformation of E. coli, isolation of D N A and agarose gel electrophoresis have been described (Maniatis et al. 1982). Nucleotide sequence determination and data analysis, traG region D N A for cloning into M13 sequencing vectors (Yanisch-Perron et al. 1985) was isolated from pRS1076 (L. Ham and Ron Skurray, unpublished) and pSH17 (Achtman et al. 1978). Deletion derivatives were subsequently generated using specific restriction sites, exonuclease Ba131 (Maniatis et al. 1982) and DNaseI/Mn 2+ (Lin et al. 1985). M13 templates were sequenced by the dideoxynucleotide chain termination method of Sanger et al. (1977). Both strands were originally sequenced using the Klenow fragment of E. coli DNA polymerase I (Amersham). This sequence was subsequently confirmed by re-sequencing with T7 polymerase and dITP as outlined by the manufacturer's recommendations in

the Sequenase kit (United States Biochemical). All restriction sites were crossed. Sequence data were stored and analysed using the computer programmes of Staden (1986) and Gamier et al. (1978) as modified by A. Kyne (Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia). Functional assays. Strains to be used in complementation tests were constructed by transforming the relevant plasmid into a strain harbouring the Flac + derivative to be complemented. Flac + traG amber mutants were first transferred by conjugation from their suppressing hosts to the non-suppressing strains ED2196 or HL202. Phage fl, MS2 and T7 spot tests were performed as described by Achtman et al. (1971). All phage sensitivity spot tests employed to assay for complementation between nonpiliated Flac+ traG mutants and recombinant traG deletion derivatives were performed in the recA strain HL202 (Poustka et al. 1984) with the exception of those with plasmid JCFL106, where the cultures used were also tested for transfer deficiency to confirm the absence of recombination. Conjugal transfer efficiencies were measured using the method of Achtman et al. (1971). To confirm that the transfer observed resulted from complementation rather than recombination, exconjugant progeny were tested for their inability to act as donors. Expression of polypeptides in maxicells. The method of Sancar et al. (1979), with modifications (Ray and Skurray 1983), was used for labelling plasmid-encoded polypeptides with [35S]methionine ( > 1200 Ci/mmol, Amersham) in derivatives of the maxicell-producing strain CSR603. Prior to labelling, samples were induced with isopropyl/~-D-thiogalactoside (IPTG) at a final concentration of 333 gM. Radiolabelled polypeptides were fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli 1970) using 10 or 15% (w/v) acrylamide slab gels, and detected by fluorography using sodium salicylate as the scintillant (Chamberlain 1979). Proteinase K accessibility experiments. Radiolabelled maxicells were exposed to lysozyme and proteinase K essentially as described by Forst et al. (1987). Samples were treated with proteinase K at a final concentration of 450 ~tg/ml for 30 rain at room temperature. After the addition of phenylmethylsulfonyl fluoride (Sigma) to terminate digestion, trichloroacetic acid was added to give a final concentration of 10% (v/v) and polypeptides were recovered by centrifugation. Samples were washed in acetone before being resuspended in sample buffer. Antisera production. Glutathione agarose (Sigma)-bound glutathione-S-transferase (GST)-'TraGp' fusion protein, purified as described by Smith and Johnson (1988), was emulsified with Freund's incomplete adjuvant and used to immunize female New Zealand White rabbits. Rabbits were injected intradermally with 0.1 ml of the emulsion at approximately 10 sites. Five weeks after immunization the rabbits were boosted with a similar dose and subsequently bled and the serum recovered after a further 5 weeks. The immune sera were preadsorbed

147 for 1 h at room temperature against French press lysates of HB101 and ED2196 immobilized on nitrocellulose. Anti-GST antibodies were removed from the antisera by preadsorbing against purified GST immobilized on glutathione agarose. Western blot analysis. Suspensions of strains containing approximately 4 × 10 s cells were boiled in sample buffer and their proteins separated using SDS-PAGE. Chloroform shock (Ames et al. 1984) was used to release periplasmic proteins from E. coli cells. Transfer and Western analysis of proteins was performed by the method of Towbin et al. (1979). Blots were immunostained using the method of Chapman et al. (1987), except that reactions with primary antibody were performed by incubating for 18 h at room temperature. Anti-TraGp antisera was used at a dilution of 1:300. Goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) was used as the secondary antibody at a 1:2000 dilution.

Results

Nucleotide sequence of the traG gene

The 3182 bp sequence of the traG-encoding region of plasmid F, from the PstI site at 86.03 F to the EcoRI site at 89.21 F, is shown in Fig. 1. Nucleotide 1 of the sequence reported here (Fig. 1) corresponds to nucleotide 1668 of Fig. 2 in Ham et al. (1989b). One long open reading frame (ORF) was detected in the sequence, starting at position 102 and ending at nucleotide 3071 (Fig. 1). This ORF spans the EcoRI site at the junction of restriction fragments fl and f17 (88.45 F; Fig. 1) which is consistent with the notion that it corresponds to the traG gene. No potential A U G initiation codon preceded by a consensus ribosome-binding site (Shine and Dalgarno 1974) is apparent in the region that, given the molecular weight of the traG-encoded polypeptide observed previously (Willetts and Maule 1980; Moore et al. 1981), would be expected to contain the translation start signal for this ORF. However, a G U G codon, representing the second most abundant initiation codon in E. coli, which is preceded by a good Shine-Dalgarno sequence, is present at position 258 (Fig. 1). A polypeptide of 938 amino acids with a calculated molecular weight of 102 kDa would result if translation were to initiate at this codon, a size correlating well with that determined for TraGp using SDS-PAGE (Willetts and Maule 1980; Moore et al. 1981 ; see also Figs. 3 and 4). An incomplete ORF corresponding to the 3' end of the trail gene is located immediately upstream of the traG gene. Our resequencing of the region 84.70-86.03 F (S.M. Loh, N. Firth and R. Skurray, unpublished data) has demonstrated that the reported nucleotide sequence of trail (Ham et al. 1989b) contains two spurious bases: we find that the T previously reported as nucleotide 1592 and the A previously reported as nucleotide 1590 are not actually present. Removal of these two bases extends the trail open reading frame by 99 codons, such that the stop codon of trail overlaps the proposed start co-

don of traG (Fig. 1). The molecular weight of TraHp calculated from the deduced amino acid sequence is therefore 50.2 kDa, and 47.8 kDa if processing occurs at the proposed signal peptidase cleavage site of Ham et al. (1989b). These values are in good agreement with the observed electrophoretic mobility of the trail product (Ray et al. 1986). The distal end of EeoRI fragment 17 of F encodes the 5' sequences of the surface exclusion gene traS. The sequence of the traS region determined in this study (Fig. 1) is identical to the first 179 nucleotides of the traS sequence reported by Jalajakumari et al. (1987), as corrected by Ham et al. (1989a). A detailed analysis of the traS promoter has been reported elsewhere (Ham et al. 1989a). Analysis of the traG product

Examination of the hydropathy profile (Engelman et al. 1986) of the deduced traG product indicates that it consists of largely hydrophilic regions punctuated by three long hydrophobic stretches, labelled I, II and III (Figs. 1 and 2); each of the hydrophobic segments is predicted to be membrane-spanning (Engelman et al. 1986). The two internal hydrophobic segments (II and III) are of a length such that each might span a membrane more than once. Thus the hydropathy profile of TraGp supports previous cell fractionation data indicating that TraGp is located in the inner membrane (Manning et al. 1981 ; Moore et al. 1981).

Mapping of the TraGp functional domains

Complementation analyses using Flac + traG mutants have indicated that TraGp is bifunctional, as mutations in the traG gene fall into one of two complementation classes (Achtman et al. 1971, 1972); those that abolish pilus assembly and hence conjugal transfer, and those that prevent transfer but do allow the synthesis of seemingly normal pill (Achtman et al. 1978). It is believed that donor cells carrying the second type of mutation are unable to form stable contacts with recipient cells once the pilus has brought the cells into direct association (Manning et al. 1981). Such mutants are therefore defined as being defective in mating aggregate stabilization. It has been shown that the traG sequences encoded by EcoRI fragment 17 of F are necessary for aggregate stabilization but are not essential for the pilus assemblyrelated activities of the gene (Achtman et al. 1978). To study further the extent of TraGp needed to facilitate piliation, a series of deletion derivatives was constructed for use in complementation experiments with non-piliated Flac + traG mutant plasmids. pRS1662 was constructed by ligation of the PstI-EcoRI fragment that encompasses the region comprising 86.03-88.45 F into the vector pGEX-1 (Smith and Johnson 1988). In this construct, transcription of traG is directed by the tac promoter of the vector. The lacIq gene encoded by pGEX-1 stringently represses the tac promoter in the absence of an inducer such as IPTG. Fur-

'traH L O Y I Q E L I Q Q A R A M V A T G N Y D E A V I G H I N D N M N D A T R Q I A CTGCAGTACATTCAGGAGCTGATACAGCAGGCACGGGCGATGGTGGCCACGGGAAATTATGACG~GCGGTTATCGGGCATATT~CGAC~CA~G~TGATGCCACCCGGCAGATTGCG P~CI (86.03F)

A F O S Q V Q V Q Q D A L L V V D R O M S Y M R Q Q L S A R M L S R Y Q N N y H GCGTTTCAGTCACAGGTGCAGGTACAGCAGGATGCGCTGCTGGTTGTCGATCGTCAGATGAGCTACATGCGTCAGCAGCTTTCCGCCCGCATGCTCAGTCGTTACCAG~C~CTATcAC

120

240

sp~ F

G

G

S

T

LI *'t~GSta~~ IM N e V Y V I A G G E W L R N N L N A I A A F M G T W T W D S I E K TTCGGAGGGAGCACGCTGTG~TG~GTTTATGTGATTGCCGGTGGTGAGTGGTTGCGG~T~CCTG~CGCCATTGCCGCCTTTATGGG~CCTGGACGTGGGATTCCATTG~u~

360

~ ~ ~ I\ ~ ~ \ ~ \ ~ \ ~ \ ~ \ ~ \ } I A L T L S V L A V A V M W V O R H N V M D L L G W V A V F V L I S L L V N V R TTGCGCTCACATTGTCTGTTcTCGCGGTGGCCGT~TGTGGGTACAGCGGCAC~CGTGATGGATTTGCTGGGCTGGGTGGCCGTGTTTGTGCTTATCAGCCTGCTGGTT~TGTCCGCA

480

T S V Q I I D N S D L V K V H R V D N V P V G L A M P L S L T T R I G H A M V A CATCGGTGCAGATTATTGAT~CAGTGACCTGGTCAAAGTTCACCGGGTGGAT~TGTGCCGGTCGGTCTGGCGATGCCACTTTCACTGACGACCCGTATCGGGCATGC~TGGTGGCCA SphI

600

S Y E M I F T O P D S V T Y S K T G M L F G A N L I V K S T D F L S R N P E I I GTTACGAGATGATCTTCACGC~CcGGAcAGcGTCACCTACAGC~CGGGGATGCTGTTCGGGGCG~TCTGATTGTG~GCACCGATTTCCTGTCCCGG~TCCGGA~TCATcA

720

N L F O D Y V O N C V L G D I Y L N H K Y T L E D L M A S A D P Y T L I F S R P ATCTGTTCCAGGACTACGTCCAG~CTGCGTGCTGGGTGATATTTACCTG~TCATAAATACACGCTGG~GACCTGATGGCCTCCGCTGACCCCTACACGCTGATTTTTTCCCGCCCCA

840

S P ~ R G V Y D N N N N F I T C K D A S V T L K D R L N L D T K T G G K T W H Y GCCCGCTACGGGGCGTTTATGAC~C~T~T~TTTCAT~CTTGT~GGATGCGT~GGTCA~GCTGAAAGACAGG~TG~TCTCGATACA~GACGGGAGGC~GACCTGGCATTATT

960

Y V Q O I F G G R P D P D L L F R Q L V S D S Y S Y F Y G S S Q S A S Q I M R Q ATGTGCAGCAGATATTTGGCGGCAGACCGGACCCGGACCTGTTATTCAGAC~CTGGTCAGTGACAGTTACAGTTATTTCTACGGCT~CAGTCAGTCTGCcAGCCAGATTATGCGCCAGA

1080

N V T M N A L K E G I T S N A A R N G D T A S L V S L A T T S S M E K Q R L A H ACGTCACCATG~TGCCCTG~GAG~GTATCAc~AGT~TGCAGCCCGT~CGGTGACACCGCCAGCCTGGT~GTCTGGCCACCACGTCATCGATGGAG~C~CGTCTGGCACATG ClaI

1200

~%~%~%~%%~%~%~%~%%~%~%~%~%%~%~%%%%~%~%~%%~%%~

~

%

~

V S I G H V T M R ~ L P M V O T I L T G I A I G I F P L L I L A A V F N ~ L T L TCTCCATTGGTCATGT~CTATGCG~CCTGCcGATGGTCCAGACTATCCTGACGGGGATTGCCATTGGTATATTCCCGCTCCTGATAcTGGCGGCTGTCTTT~C~GcTGACATTGT

1320

S V L K G Y V F A L M W L O T W P L L Y A I L N S A M T F Y A K Q N G A P V V L CTGTATT~GGGGTATGTGTTTGCCCTGATGTGGCTGCAGACCTGGCCGTTGTTGTATGCCATTCTG~CAGTGCCATGACATTCTATGCG~CAG~TGGTGCGCCGGTCGTGTTGT PstI ~ ~ ~ % % ~ % % ~ ~ ~ ~ III ~ ~ S E L S Q I O L K Y S N L A S T A G Y L S A M I P P L S W M M V K G L G A G F S CTG~CTCTCTCAGATACAGCTG~TACT~T~CCTGGCCTCCACTGCCGGGTATCTTTCAGCCATGAT~CCCCCGTTGTCATGGATGATGGT~GGCCTTGGCGCAGGTTTTTcCA ~%~%%~%~%~%~%%%%%%~%% ~ -~ -i~+~ S V Y S H F A S S S I S P T A S A - - A G S V V D G N Y S Y G N M Q T E N V N G F S GCGTGTACAGCCACTTCGCCTCTTCATcTATCAGTCC~CGGCCAGTGCAGCAGGCAGTGTGGTTGACGGT~TTACTCCTACGGC~CATGCAGA~GG~CGTG~CGGCTTCAGCT

1680

W S T N S T T S F G O M M Y O T G S G A T A T Q T R D G N M V M D A S G A M S R GGAGCACC~CAGCACCACGTCGTTTGGTCAGATGATGTACCAGACCGGCAGTGGCGC~CCGCCACACAGACCCGTGACGGT~TATGGTGATGGATGC~GCGGAGCGATGTCCCGGT

1800

L P V G I N A T R Q I A A A Q Q E M A R E A S N R A E S A L H G F S S S I A S A TACcGGTCGGTATC~TGC~CGCGTCAGATTGCAGCGGCAC~CAGG~TGGCCCGGGAGGCGTCG~CAGAGCCG~GTGCCCTGCATGGTTTCAGCAGCAGTATTGCCAGTGCCT S~I

1920

1440

1560

W N T L S Q F G S N R G S S D S V T G G A D S T M S A O D S M M A S R M R S A V GG~CACACTCAGTCAGTTTGG~T~T~CCGGGGGAGCAGTGACTCTGTCACTGGTGGTGCTGACAGCACGATGAGTGCTCAGGACTCCATGATGGCCAGCCGTATGCGCAGTGCAGTGG 2040 E S Y A K A H N I S N E O A T R E L A S R S T N A S L G L Y G D A Y A K G H L G ~GCTATGCG~GGCGCAT~TATCAGT~TGAGCAGGC~GAGAG~CTGGCCTCGAG~GTAC~TGCGTCGCTTGGCCTTTACGGTGATGCTTATGCT~GGGCATTTAGGCA

2160

I S V L G N G G G V G L O A G A K A S I D G S D L D S H E A S S G S R A S H D A T~GTGTATTGGGT~CGGTGGAGGGGTTGGT~TCCAGGCCGGAGCT~GGC~GCATTGATGGcAGTGATCTTGACTCACATG~GCCAGTAGTGGTTCACGGGCCAGCCATGATGCTC

2280

R H D I D A R A T Q D F K E A S D Y F T S R K V S E S G S H T D N N A D S R V D GTCATGATATCGATGCCAGAGCTA~AC~GACTTT~G~GCCAGCGATTACTTTACCAGCCGC~GTCAGTG~TCCGGCAGCCATACTGAc~T~TGCTGATTCCCGTGTGGATC ClaI

2400

Q L S A A L N S A K Q S Y D Q Y T T N M T R S H E Y A E M A S R T E S M S G Q M AGTTGTCTGCAGCCCTG~TTCAGCG~CAGAGTTACGACCAGTACAcGACG~TATGACCCGCAGCCATG~TATGCTG~TGGCATCCCGTACTGAGAGTATGAGTGGGCAGATGA PstI Eu~ (88.45F)

2520

S E D L S ~ O F A O y V M K N A P O D V E A I L T N T S S P E I A E R R R A M A GT~GATCTGTCGC~CAGTTTGCACAGTATGT~ATG~CGCACCGCAGGATGTTG~GCTATTCTGAC~TACCA~TTCGCC~GA~ATTGCAG~CGCCGTC~GGCTATGGCGT

2640

W S ~ V Q E ~ V Q P G V D N T W R E S R R D I G K G M E S V P S G G G S Q D I I GGTCTTTTGTGCAGG~CAGGTACAGCCTGGTGTTGAT~CAC~TGGCGTG~TCCCGTAGAGATATAGGT~GG~TGGAGAGCGTACCTTCGGGTGGTGGCAGCCAGGATATTATTG

2760

A D H O G H O A I I E O R T O D S N I R N D V K M Q V D N M V T E Y R G N I G D CTGATCATCAGGGACATCAGGCCATTATTGAGC~G~CGCAGGACAGT~TATCCGT~TGATGT~CATCAGGTTGAT~TATGGTCACAG~TATCGAGGT~TATCGGAGATA

2880

s~zx

T O N S

I

R G E E N

I V K G O Y S E L Q N H H K T

E A L T Q N N K Y N E E K L A

~ E R I P G A D ~ K E L L E K A K S Y Q H K ~ * QQ AGG~G~TACCTGGTGCTGATAGT~CG~GAGCTTTTAGAG~GC~GAGTTACCAGCAT~G~T~TCAT~CATGTTGGGTAGGGTATGGAGAGACCATGATTACAC~CA

3120

I I S S E L ~ V L K K H I D S G D I R I ~TTATCAGTAGTGAGTTGG~GTTTT~G~CATATTGACTCAGGAGATATTAG~TTC E=O~

3182

Fig. 1. Nucleotide sequence of the traG gene; also shown is the 3' end of trail (trail') and the 5' end of traS. The deduced amino acid sequences of the gene products are shown in single letter codes. Putative Shine-Dalgarno sequences are underlined and stop codons indicated by an asterisk. Recognition sequences for the restriction endonucleases BglII, ClaI, EcoRI, PstI, Sinai and SphI are indicated. The extents of potential membrane spanning segments I, II and III are indicated by hatched boxes above the TraGp amino acid se-

(89.21F)

quence. The potential cleavage site in TraGp is represented by a vertical arrow. Also indicated are the amino acids that correspond to the - 3, - 1 and + 1 positions of the signal peptidase I cleavage site consensus sequence (von Heijne 1984). Nucleotide 1 corresponds to 86.03 F on the 100 kb F plasmid map (Willetts and Skurray 1987) and to nucleotide 1668 of the sequence presented in H a m et al. (1989b). This sequence has been submitted to E M B L and GenBank databases and assigned the accession number M59763

149 Table 1. Complementation of Flac + traG mutants for conjugational transfer by pRS1670 ~