of broad host range plasmid RK2 - BioMedSearch

.=) 1992 Oxford University Press

Nucleic Acids Research, Vol. 20, No. 15 3939-3944

Crosstalk between plasmid vegetative replication and conjugative transfer: repression of the trfA operon by trbA of broad host range plasmid RK2 Grazyna Jagura-Burdzy, Farhat Khanim, Christopher A.Smith and Christopher M.Thomas* School of Biological Sciences, University of Birmingham, PO Box 363, Birmingham B15 2TT, UK Received May 14, 1992; Revised and Accepted July 3, 1992

ABSTRACT Previous deletion and complementation analysis has indicated that the region between trfA and kilBi (trbB) encodes trans-acting factor, designated trbA, required for conjugative transfer of broad host range plasmid RK2. In analysing the nucleotide sequence of this region we have discovered a gene encoding a 12 kDa polypeptide. The predicted amino acid sequence of this protein shows similarity at its C-terminal to KorA from the central control operon of RK2 and at its N-terminal to immunity repressor protein from phage c105 of Bacillus subtilis as well as the Sin protein of B.subtilis which regulates alternate developmental processes including sporulation, motility and competence. We show that TrbA represses transcription of both trfA (vegetative replication) and kilBi (trbB) (required for conjugative transfer and whose product has similarity to ComG, required for competence of B.subtilis) and may help to coordinate expression of both sets of functions. This region has similarities to some temperate bacteriophage immunity regions in modulating divergent transcription required for alternative means of propagation.

EMBL accession

no.

X66021

is that the trb cistrons encoded in the Tra2 region (to distinguish them from the tra cistrons of the Tral region) are transcribed divergently from promoters located near trfAp. We have recently shown that kilBI,(9) located at the trfA proximal end of the Tra2 region referred to also as trbB (8), is required for transfer (10,11). An insertional mutant in which kilBI is inactivated can be complemented by kilBI in trans (11). However, a deletion mutant in which the tac promoter is placed upstream of a fragment which starts in kilBI (trbB) required not only kilBI but also the sequences as far back as the trfAp for complementation to give a Tra+ phenotype (8). This suggested the existence of a second trans-acting function required for transfer, encoded upstream of kilBI (trbB) which was tentatively designated trbA (8). Here we present the sequence of the trbA region and evidence that in addition to its role in transfer it encodes a regulatory protein which represses transcription of trfA and kilBI (trbB) and in this way controls both vegetative replication and conjugative transfer. ,

MATERIALS AND METHODS Bacterial strains, plasmids and growth conditions E.coli K-12 strains used were NEM259 (met, trpR, supE, supF,

hsdR-M+S+) (12) and CSR603, a recAl, phr-1 derivative of INTRODUCTION Broad host range IncP plasmid RK2 (Fig. IA), like many other bacterial plasmids, is capable of two modes of propagation: vegetative replication which results in duplication of the plasmid content in a controlled way in proportion to cell growth (3) and transfer replication which results in a copy of the plasmid being transferred to a recipient cell independently of the growth of the donor bacterium, by a process similar to rolling circle replication (4). These can be regarded as alternative life styles as the lytic and lysogenic cycles are alternatives for temperate bacteriophage. The ways that these modes of propagation are controlled and the possibility that they may be coordinated is of considerable interest. Vegetative replication of RK2 depends on the product of the trfA gene (5,6,7). Deletion analysis has recently shown that the Tra2 block of functions required for conjugative transfer starts at the trfAp region at kb coordinate 18.2 (8) and the implication *

To whom correspondence should be addressed

AB1886 (thr-J, leu-6, thi-J, lacY], galK2, ara-14, xyl-5, mtl-i, proA2, his4, argE3, str-31, tsx-33, sup-37, uvrA6) (13). Plasmids used are listed in Table 1. Bacteria were grown in L Broth or M9Caa medium (17), L-agar was used to propagate strains. All cultures were grown with shaking at 37°C. Antibiotic resistance was selected by addition of benzyl penicillin (Pn, sodium salt at 100 Ag/ml in liquid medium and 300 ,ug/ml in solid medium); kanamycin (Km, 50 4g/ml); chloramphenicol (Cm, 25 pAgIml); and tetracycline (Tc, 25 isg/ml). In vitro manipulation and analysis of DNA and RNA Plasmid DNA was prepared on both small and large scales by the alkaline SDS method (18) and also where necessary by CsCl/Ethidium bromide equilibrium density gradient centrifugation. DNA manipulations were carried out by standard techniques (19) using enzymes according to the manufacturers' instructions. The nucleotide sequencing was performed by both

3940 Nucleic Acids Research, Vol. 20, No. 15 the dideoxy chain termination method (20) and the chemical modification and cleavage method (21). The nucleotide sequences were analysed using the UWGCG programs (University of Wisconsin Genetics Computer Group, Madison, WI, U.S.A. ;22). Data base searching was carried out with the DAP programme developed by the Biocomputing Research Unit of the Molecular Biology Department (University of Edinburgh).

Plasmid pGBT43 was obtained by deletion of BamHI fragment containing the kfiA promoter from previously described pGBT40 (16). Colonies containing plasmids with a promoter directing expression of xylE were identified by their yellow colour after spraying with 0.1 M catechol solution.

Assay of catechol 2,3-oxygenase activity The level of xylE expression was determined by enzymatic assays in logarithmically growing bacteria, using a standard method (23). Protein concentration was assayed by the biuret method (24). One unit of catechol 2,3 oxygenase activity is defined as the amount of enzyme necessary to convert 1 mmol of substrate to product in 1 min under standard conditions.

DNA sequence of the trbA region Figure lB shows the genetic organization in the trfA to kilBI (trbB) region of RK2 and the extent of the DNA sequences previously reported for these regions (7,1 1). There is a gap of about 400 bp between these two regions. The nucleotide sequence of the unpublished segment with flanking regions is presented in Figure 2. The G+C content of this segment is 56% which is similar to, although slightly lower than the surrounding regions (trfA operon is 59.4% while the kilBI (trbB) region is 58%). Analysis of the possible ORFs encoded by each strand revealed one likely ORF which would be transcribed away from trfA

Analysis of proteins The polypeptides were separated by standard SDS-PAGE (25). Plasmids were introduced into CSR603 strain by transformation and their products were visualized in the maxicell system (13) with previously described modifications (6). Cloning using Polymerase Chain Reaction (PCR) PCR reactions (26) were performed in a programmable heating block (Hybaid) starting with 5 min denaturation at 96°C followed by 25 rounds of temperature cycling (96°C for 15 sec, 55°C for 30 sec and 72°C for 90 sec) and a final 5 min step at 72°C. 10 pmol of each primer, 50 ng of template and lu of Taq polymerase were used in a total volume of 50 jil with the reaction buffer as recommended by the manufacterer (NBL). Amplified fragments were electrophoresed, purified with Gene Clean according to the manufacturer's instructions, digested with appropriate restriction endonucleases for 2 h, precipitated and ligated with adequate vectors. The identity of the inserted fragment was confirmed by DNA sequencing. Cloning of repressor genes. Two putative trbA ORF's of 366 nt and 312 nt (Fig. 1) were amplified on pMMV1 15.1 template using pairs of synthetic primers described in legend to Fig.2. Upstream primers included NdeI recognition site (CATATG), in such a way that ATG is a start codon and it replaces GTG for the earlier start of trbA. The stop codon in both constructs was followed by a HindIlI recognition site. The PCR fragments were joined to the NdeI to Hindm fragment of pMS47OD8, which contains laclQ, tacp and the T7 gene 10 ribosome binding site located at optimal distance upstream of the ATG in the NdeI site. The korA ORF of 330 nt was amplified on pCT294.5 template with a pair of primers containing EcoRI and Sall recognition sites respectively. The PCR product was treated with EcoRI and Sall and inserted into pGBT30 downstream of the tac promoter and its SD sequence. Cloning of DNA fragments carrying promoter regions. Promoter fragments were amplified on pMMV 1 15. 1 template for wild type trfA and kilBI (trbB) promoters and pVl107. 1 for trfA promoterdown mutation using pairs of synthetic primers (Fig. 1 & Fig.2) with BamHI recognition sites at their ends. Primer 2 (Fig.2) introduced C instead of G at position 6 creating an additional EcoRI site, the presence of which allowed the orientation of the inserted fragment to be easily determined. All promoter fragments were introduced into BamHI-linearized and dephosphorylated promoter probe vector pGBT43 upstream of xylE cassette.

RESULTS

trbA ORF

80

kiBIp

8 631

trfAP{ cOAOB

OB

(trbBp)

trfAp

Sn

trfA B

17

trb

trbA

san

trbA

kilBI (trbB)

18 o-

kb

A

Tral

Figure 1. Genetic organization of the trfA to kilBI (trbB) region of RK2. A. Map of RK2 (1) showing the main phenotypic markers, the known transposable elements (solid segments), the replication functions: oriV and trfA, the partitioning functions

par/mrs, the main genes of the killkor regulons and the blocks involved in conjugative transfer (hatched segments). B. Expanded map the trfA to kilBI (trbB) region of RK2 from coordinates 16.4 to 20.4. The regions whose sequence has already been published are stippled. san is a gene encoding a 116aa polypeptide with similarity to single strand DNA binding protein (SSB) (la). The trfAp whose transcription start point (tsp) has been determined (2) is shown as a solid circle. Proposed locations of the trbA and kilBI (trbB) promoters are shown as open circles. Operators on which the repressors KorA and KorB act are indicated as solid bars. ORFs are open blocks. C. Location of PCR-amplified fragments (solid bars) from the trfA to ki/BI region. The numbers correspond to the pGBT plasmids from Table 1 which contain the cloned fragments.

Nucleic Acids Research, Vol. 20, No. 15 3941

towards kilBI (trbB). On the basis of the data described here, which was made available to Erich Lanka this ORF was designated trbA to avoid the necessity of reclassifying ORFs in subsequent publications (8). Previous analysis of this region indicated that there is weak transcription in this direction coming from the trfA promoter region (27) which may be responsible for expression of this ORF. We have confirmed that this transcription originates in the region located between bases 1 and 241 (Fig.2). Detailed analysis of the trbA promoter region will be published elsewhere. There are no obvious transcriptional terminators (29) in this region suggesting that trbA may be translated from a polycistronic mRNA. The codon usage in the trbA frame is consistent with that found in other RK2 genes. There are two possible starts within this ORF, both of which are shown. As described below (see 'Overproduction of the trbA product') the normal polypeptide product originates from the earlier start, 5' AGGGGnt-GTG. While this contains a less common start codon and an atypical Shine-Dalgarno sequence placed at a greater than optimal distance upstream, the arrangement is almost identical to the start we identified for kilBI (trbB) (11). We compared the predicted primary sequence of this ORF with proteins in the available data bases. This revealed that the Cterminus has considerable similarity to the C-terminus of the KorA protein of RK2 (30) and IncPfl R751 (L. Johnston, Z. Marham & C. Thomas, unpublished) (Fig. 3). This motif is rich in basic amino acids and is partly responsible for the high predicted pl (9.12) of TrbA. The motif also occurs in the predicted product of the ORF which precedes korCof RK2 (31). However in this case the sequence LPpGFAwVdAVLPaHQAFIaRKWaAsAKaK (conserved amino acids are shown in upper case letters) is located in the body of the polypeptide, not at the C-terminus. The similarity to KorA does not include the region containing the proposed helix-3 turn-a helix (HTH) motif of KorA (32). However, closer to its N-terminus TrbA shows a high degree ,

a

of similarity to the proposed HTH motifs of transcriptional repressor proteins which function in Bacillus subtilis: the Sin protein which causes inhibition of sporulation (33,34) and the phage immunity repressor from 4105 as well as the predicted product of the ORF3 of the c/105 immunity region (35) (Figure 3). This similarity suggested that TrbA may be a DNA binding protein which activates or represses transcription of RK2 genes. A possible leucine zipper has been identified in Sin (34) and this is reasonably well conserved in TrbA, thus providing the possible basis for dimerization (Fig. 3)

The trbA region encodes a repressor of transcription To test the hypothesis that trbA encodes a transcriptional repressor we

first chose as possible targets the promoters in close proximity

the new gene: trfA and kilBI (trbB) promoters. We have constructed plasmids carrying these promoters on the fragments shown in Fig. 1 and 2 linked to xylE giving pGBT58 (trfAp-xylE) and pGBT63 (kilBIp-xylE) (Fig. 2). Plasmids pVI106.1 and pVII07.1 which have 35bp insertions in the trbA ORF were placed in trans to pGBT58 and pGBT63 (Fig. 2 for sites of insertions). An isogenic plasmid, pVI104.1 which has an insertion downstream of trbA and pDS3 the vector on which the pVI plasmids are based were used as controls. The results indicated that when the trbA ORF was intact both the trfA and kilBI (trbB) to

promoters were repressed 2.5 to 3 fold.

Overproduction of the trbA product The trbA ORF contains two possible translational starts which would generate products of 121 amino acids (TrbA) and 103 amino acids (TrbA') (Fig. 2). Synthetic primers and PCR technique were used to generate DNA fragments containing the trbA and trbA' ORFs, which then were placed downstream of T7 gene 10 SD sequence under control of tac promoter. After IPTG induction plasmid pGBT79 (trbA') gave rise to overproduction of a single polypeptide with Mr = 11,000 while pGBT80 (trbA) over-produced a single polypeptide with Mr =

Table 1. Plasmids used in this study. Plasmid

Size

Replicon

Selective marker

Other relevant properties

Reference

P1SA pMBI pMBI pMB1 pMBI P15A P15A P1SA pMBI pMB1

CmRTcR pnR PnR PnR

none korAincC

PnR

lacdq tacp

14 7 15 15 7 6 6 6 16 This paper This paper This paper This paper This paper This paper This paper This paper This paper

(kb) pDS3 pCT294.5 pMMV115.1

pMMV811

pMS47MV 8 pVI104.1 pVI106.1 pVI107.1 pGBT30 pGBT37

pGBT43 pGBT58 pGBT59 pGBT63 pGBT70 pGBT79 pGBT80 pGBT81

2.5 7.4 18.4 7.45 5.35 10.2 10.2 10.2 6.05 6.38 10.1 10.35 10.35 10.60 10.35 4.28 4.33 10.25

pSC1o0 pSClO0 pSClo0 pSC1Ol pSC1o0 pMBI pMBl

pSC101

CmRTcR

CmRTcR CmRTcR PnR PnR KmR KmR KmR KmR KmR PnR PnR KmR

trfA-kilBI [19.7::TnA]l tacp-korB

trfA2-trbA[18.73::TnA1]

trfA2-trbA[18.61::TnA]I

trfA 2-trbA[18.59::TnA]'

lacdq tacp tacp-korA promoter probe xylE

trfAp-xylE (241 bp)3 trbAp-xylE (241 bp)3 kilBIp-xylE (520 bp)3 trfAp4-xylE (241 bp)3 tacp-trbA' (312 bp) tacp-trbA (366 bp)3 trfAp-xylE (143 bp)3

1. This notation indicates that the plasmid contains a 35 bp insertion at the RK2 coordinate shown (see Fig. 1), resulting from transposition of TnJ723 into the trfA-trbA-kiIbl region, followed by deletion of the transposon DNA between the EcoRI sites which lie 15 bp from each end of the Tnl 723. Thus 30 bp of Tnl723 remains plus the 5 bp duplication generated by Tn insertion. 2. The trfA promoter in this plasmid contains the T to C transition in the -10 region (Figure 2) which results in approximately 10-fold decrease in promoter strength. 3. The numbers in brackets indicate the length of the PCR products amplified from tfA-kilbl DNA.

3942 Nucleic Acids Research, Vol. 20, No. 15 1

GAATTGCCATGACGTACCTCGGTGTCACGGGTAAGATTACCGATAAACTGGAACTGATTA CTTAACGGTACTGCATGGAGCCACAGTGCCCATTCTAATGGCTATTTGACCTTGACTAAT erAsnGlyHisArgValGluThrAspArgThrLeuAsnGlyIlePheGlnPheGlfnAsfnH

KorARK2 KorA751 TrbARK2

OA

61

TGGCTCATATCGAAAGTCTCCTTGAGAAAGGAGACTCTAGTTTAGCTAAACATTGGTTCC ACCGAGTATAGCTTTCAGAGGAACTCTTTCCTCTGAGATCAAATCGATTTGTAACCAAGG isSerMet San

121

SD OB.

.J tsp

120

C -10

GCTGTCAAGAACTTTAGCGGCTAAAATTTTGCGGGCCGCGACCAAAGGTGCGAGGGGCGG

180

TrbA MetTyrAsnGnI lnePhePheThrAsnIleLeuArgLeuLeuAspGluArgG

CTTCCGCTGTGTACAACCAGATATTTTTCACCAACATCCTTCGTCTGCTCGATGAGCGGG GAAGGCGACACATGTTGGTCTATAAAAAGTGGTTGTAGGAAGCAGACGAGCTACTCGCCC

2408

TrbA'

lyMetThrLysHisGluLeuSerGluArgAlaGlyValSerIleSerPheLeuSerAspL GCATGACGAAACATGAGCTGTCGGAGAGGGCAGGGGTTTCAATTTCGTTTTTATCAGACT CGTACTGCTTTGTACTCGACAGCCTCTCCCGTCCCCAAAGTTAAAGCAAAAATAGTCTGA

38 300

39 301

euThrAsnGlyLysAlaAsnProSerLeuLysValMetGluAlaIleAlaAspAlaLeuG TAACCAACGGTAAGGCCAACCCCTCGTTGAAGGTGATGGAGGCCATTGCCGACGCCCTGG ATTGGTTGCCATTCCGG¶ITGGGGAGCAACTTCCACTACCTCCGGTAACGGCTGCGGGACC

58 360

59 361

luThrProLeuProLeuLeuLeuGluSerThrAspLeuAspArgG1uAlaLeuAlaGluI AAACTCCCCTACCTCTTCTCCTGGAGTCCACCGACCTTGACCGCGAGGCACTCGCGGAGA

78 420

TrTGAGGGGATGGAGAAGAGGACCTCAGGTGGCTGGAACTGGCGCTCCGTGAGCGCCTCT 107.1

LRLLDERGMT KAIRKERKLT KEKRKEKHLK KQYRKEKGYS

51 VLVDGKPQAT VLVDGRPQAE MEAIADALET LEAVAGALGI LSRIAILINL LEKVSAVLDV

FATSLGLTRG FVTSLGLTKG PLPLLLESTD QVSAIVGEET ELNVKMYEIQ SVHTLLDEKH

106.1

leAlaGlyHisProPheLysSerSerValProProGlyTyrGluArgIleSerValValL TTGCGGGTCATCCTTTCAGcAGCAGCGTGCCGCCCGGATACGAACGCATCAGTGTGGTTT

KorARK2

KorA751 TrbARK2 PhilOSRep

PhilO5ORF3

Sin

19 241

79 421

PhilO5Rep

Phi 050RF3 Sin

MKKRLTEAQ FQTAIKGLEI GQQTIDIARG

........... ..........

MYNQIFFTN ... MTVGQPI MLDGKKGAL I .... MIGQ

SD

CG4ACAGTT,CTTGAAATCGCCGATTTTAAAACGCCCGGCGCTGGTTTCCACGCTCCCCGCC -35 1 181

1 50 ....... MKKRLTESQ FQEAIQGLEV GQQTIEIARG

60

98 480

AACGCCCAGTAGGAAAGTTCTCGTCGCACGGCGGGCCTATGCTTGCGTAGTCACACCAAA 99 481

euProSerHisLysAlaPheleValLysysLTrpGlyAspspAThrArgLysLysLeuA TGCCGTCACATAAGGCGTTTATCGTAAAGAAATGGGGCGACGACACCCGAAAAAAGCTGC

118 540

119 541

104.1 OB rgGlyArgLeuEnd GTGGAAGGCTCTGACGCCAAGGGTTAGGGCTTGCACTTCCTTCTTTAGCCGCTAAAACGG

121 600

KorARK2 KorA751 TrbARK2 PhilOSRep Phi1O5ORF3 Sin

T

KHELSERAGV QVQLAEKANL NTEKAKALGM LS VA&EQG

SISFLSDLT. SRSYLADIER SRTYLSEIE. AKSYLSSIER

NGKANPSLKV D.RYNPSLST NGRYLPSTKT

NLOTNPSIOF

100 AAFED E GYARVTAVLP AAAGE.QLtE GFERVTAVLP GHPFKSSVPGYERISWVLP SKEEKDIAKR MEEIRKDLEK AGTCRRQAL . .......... ETEYDGQLD EWEKLVRDAM TSGVSKKQFR

AVSQAVHRVW AVSQAVSRVW LDREALAEI LIKEEQAEYN VVEEGGYDRA

101 150 EHQAYIVRKW EADAKKX QET KR EHQAFIVKKW EADAKRIKQEP KS SHKAFIVKKW GDDT RG RL SDGLSFSGEP MSQEAVESLM EAMEHIVRQT QRINKKYTPK KYRNDDQE

EFLDYQKWRK AQKEE

Figure 3. Comparison of the predicted TrbA sequence with other repressor proteins: KorA of RK2 (30) and R75 1 (L. Johnston, Z. Marham and C. Thomas, unpublished); immunity repressor and ORF3 of k105 (34) and Sin protein of B.subtilis (32). Blocks indicate regions where aligned proteins show similarity to TrbA. T flanked by solid blocks indicate HTH motifs. The residues of TrbA which correspond to the proposed leucine zipper of Sin (33) are underlined.

CACCTTCCGAGACTGCGGTTCCCAATCCCGAACGTGAAGGAAGAAATCGGCGATTTTGCC

A.±

Figure 2. DNA sequence of the trbA region including previously published flanking regions. The sequence was determined on both strands by the method of Maxam and Gilbert (21) from a variety of restriction sites, and more recently confirmed by the Sanger method (20) with synthetic primers which were also used for PCR. The translated ORFs for trbA (including the two possible starts) and san are shown, preceded by their predicted SD sequences. The tifAp -10, -35, tsp (2) are indicated as well as the trfAp -10 mutation which is present in pGBT70 (28). The 5 bp duplications generated by the transposon insertions in pVIl104. 1, pVI 106. 1 and pVI107. 1 are shown. For the PCR-generated fragments shown in Fig. lC primers were as follows. For fragment 58 the primers correspond to bases 240-220 (primer 1) and 1 -24 with base 6 changed to C creating an EcoRI site (primer 2). For fragment 81 primer 3 (bases 99 to 117) was used in addition to primer 1. For fragment 63 one primer corresponded to bases 395 to 414 (this Fig.) while the other primer corresponded to bases 360 to 341 in the hilBI sequence previously published (11) creating a fragment of 520bp. For fragment 79 the primers corresponded to bases 242 to 260 (primer 4) and 554 to 538 (primer 5) while for fragment 80 primer 6 (bases 189 to 212) was used in addition to primer 5.

12,500 (Fig. 4) suggesting that the ATG start is not utilized when upstream sequences are present. N-terminal sequencing of the longer product after purification (to be described elsewhere) confirmed that TrbA starts with the sequence MetTyrAsnGlnIlePhePheThrAsnlleLeuArg. Transcriptional repression by the overproducing plasmids The effect of TrbA produced from pGBT79 and pGBT80 on kilBI (trbB) and trfA transcription was analysed. The results show that TrbA produced from pGBT80 but not from pGBT79 represses both kilBI and trfA promoters very strongly (Table 2). This is consistent with the idea that the proposed HTH motif identified in the N-terminal of TrbA may be important for DNA binding. To allow comparison of the strength of the repression effect we included plasmids overproducing KorA and KorB in these experiments. As expected kilBIp (trbBp) is repressed strongly by KorB but in contrast to the previous results (27) a small

Figure 4. Visualization of TrbA protein. Cultures were diluted 50-fold into fresh L Broth and grown for 3 hr in the presence (+) or absence (-) of 1 mM IPTG.

reduction in activity is also seen with KorA. The KorB operator, a sequence identical to half of the proposed operator for KorA, OA, this weak repression may be due to an excess of KorA since the promoter under test here is on a low copy number plasmid (relevant to RK2 copy number) while the repressor is encoded on a high copy number plasmid. In the previous studies the situation was reversed with respect to plasmid copy number and korA was autoregulated (28) rather than being under tacp control. The repression of kilBp (trbBp) by TrbA is comparable to the effect of KorB on this promoter. The trfAp was repressed strongly by KorA, KorB and TrbA. The effect of TrbA was extremely marked even when its level was not boosted by induction of tacp. The regulation by lacI on tacp is known to be leaky so low level of expression is to be expected even in the absence of IPTG. This strong repression suggests that trfAp is more susceptible to TrbA than is kilBIp (trbBp). In an attempt to localize target sequence for TrbA in trfA promoter we studied the effect of deleting the DNA downstream of the -10 sequence. The resulting 143 bp fragment (produced by PCR using primers 1 and 3, Fig. 2) cloned in pGBT81 plasmid shows the same pattern of regulation as pGBT58 (Table 2). We also included in this study pGBT70 which is isogenic with

OB, contains

Nucleic Acids Research, Vol. 20, No. 15 3943 Table 2. Regulation of transcription by different repressors of the kilBI (trbB) and trA promoters.

Regulatory plasmid (RK2 locus present) pGBT30 (none) [XylE units]4 pGBT37 (tacp-korA) pMMV811 (tacp-korB)

pGBT80 (tacp-trbA)

pGBT79 (tacp-trbA')

IPTG2 +

+ + + +

pGBT63 kilBlp-xylE

Promoter probe pGBT58 trfAp-xylE

1.00 [3.30] 0.85 0.38 0.80 0.04 0.27 0.03 0.74 0.68

1.00 [3.12] 0.22 0.02 1.00 0.09 0.02