The SOS promoter dinH is essential for ftsK transcription during cell division

Robert Dorazi1 and Susan J. Dewar1

Department of Biological Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK1

Author for correspondence: Susan J. Dewar. Tel: +44 131 451 3457. Fax: +44 131 451 3009. e-mail: s.j.dewar{at}hw.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The formation of the Escherichia coli division septum has been well characterized and the majority of the genes involved have been shown to map to the dcw cluster. One exception is ftsK, which lies at 20 minutes, immediately downstream of the global response regulatory gene, lrp. The promoter for ftsK has not yet been assigned. Here, it is reported that ftsK is transcribed from two promoters; the first is located within the lrp reading frame and is dispensable whilst the second is essential and corresponds to dinH, previously characterized as an SOS promoter regulated by LexA. ftsK is the first essential gene to be described that is controlled by an SOS-inducible promoter. A possible mechanism by which dinH may be activated in recA minus strains, or in strains with uncleavable LexA, is discussed.

Keywords: cell division, FtsK, dinH, LexA, transcription


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Polymerization of the tubulin-like GTPase FtsZ into a cytokinetic ring at midcell has been identified as one of the earliest steps in the formation of the bacterial division septum (Bi & Lutkenhaus, 1991 ). The mature septation complex comprises the FtsZ ring associated with an ordered complement of accessory division proteins, including the recently characterized FtsK. Immunofluorescence studies have provided detailed evidence of the order in which the division proteins are recruited to the division site. Thus, ZipA and FtsA target the Z-ring shortly after its formation, with FtsK localizing immediately after this (Addinall & Lutkenhaus, 1996 ; Addinall et al., 1997 ). ftsK does not belong to the dcw cluster at 2 minutes on the Escherichia coli chromosome (Vicente et al., 1998 ), but maps to 20 minutes, next to the leucine-responsive regulatory gene lrp.

The ftsK gene encodes a 1329 amino acid protein that is essential for septation (Begg et al., 1995 ; Wang & Lutkenhaus, 1998 ). Sequence analysis suggests that the gene encodes a tripartite protein with strong sequence similarity in its C terminus to a family of DNA-translocating proteins that includes Bacillus SpoIIIE. Significantly, only the N-terminal hydrophobic domain (the first 202 amino acids) is essential for cell division (Diez et al., 1997 ; Yu et al., 1998a ; Draper et al., 1998 ), whilst a role in chromosome partition and the resolution of chromosome dimers has recently been suggested for the C terminus (Liu et al., 1998 ; Yu et al., 1998b ; Steiner et al., 1999 ; Recchia et al., 1999 ). The N-terminal domain is predicted to contain four membrane-spanning regions, as does SpoIIIE, which are likely to localize FtsK to the division site (Begg et al., 1995 ). Localization of FtsK to the septum is dependent on the prior localization of FtsZ and FtsA, but not on localization of FtsI or FtsQ nor, by inference, FtsN (Yu et al., 1998a ). Cells depleted in FtsK form smooth-sided filaments, which in combination with a rodA mutation exhibit the swollen, partially constricted filaments typical of division block that occurs after formation of the Z-ring (Begg & Donachie, 1985 ).

Whilst the ftsK promoter has not yet been identified, an SOS-inducible promoter designated dinH (damage inducible; Lewis et al., 1992 ) has been located within the 134 bp upstream gap between ftsK and the global regulator gene lrp. Transcription of the SOS regulon is controlled by the LexA repressor (Little & Mount, 1982 ), which binds with high affinity to a consensus operator, the LexA box (Berg & Von Hippel, 1987 ), located within or very close to the promoter. RecA is activated in the presence of ssDNA (Anderson & Kowalczykowski, 1998 ) and greatly enhances LexA self-cleavage (Slilaty & Little, 1987 ) to allow high level expression of the SOS proteins. As DNA damage is repaired, LexA autodigestion stops and the system returns to a quiescent state in which the SOS promoters are repressed.

The SOS response has been shown to induce dinH promoter activity and FtsK expression is increased in a recA+lexA+-dependent manner during the SOS response (Wang & Lutkenhaus, 1998 ). We present experimental evidence that dinH is the primary ftsK promoter during normal cell growth. Primer extension, RT-PCR and site-directed mutagenesis confirmed the location of the primary ftsK promoter (P1ftsK, previously designated dinH) and indicated the presence of a second promoter, P2ftsK, within lrp. Transcriptional fusions demonstrated that transcription of ftsK from P1ftsK is recA-independent and occurs in the absence of DNA damage, whilst complementation data indicated that dinH is essential for ftsK transcription during the division process.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
E. coli DH5{alpha} (recA1; Low, 1968 ), was used to harbour all fusion vectors; E. coli TG1 is recA+ . E. coli C600/44/pcn, obtained from K. Begg, was constructed by transferring the temperature-sensitive ftsK44 mutation from TOE44 (AB2497 ftsK44; Begg et al., 1995 ) and the plasmid-copy-number mutation (pcn) into the C600 background. Strains were grown overnight in Luria–Bertani (LB) medium at 37 °C with the exception of C600/44/pcn, which was grown at 30 °C. Where necessary, media were supplemented with ampicillin (100 µg ml-1) and X-Gal (40 µg ml-1). Assays for ß-galactosidase were performed and the specific activity was calculated as described by Miller (1972 ). Comparison of promoter activities showed negligible fluctuations between constructs prepared in different experiments and the data presented are means of three different experiments from two separate isolates. Complementation tests were carried out on L broth (no salt) plates supplemented with 50 µg thymine ml-1 and appropriate antibiotics. The lrp coding sequence of the strain CV1008 (Haney et al., 1992 ) was amplified, cloned into pUC18 and sequenced by Cambridge BioScience.

Construction of transcriptional fusions.
The reporter vector pDDZ100 was constructed by fusing lacZ in-frame with the first 38 amino acids of the promoterless chloramphenicol acetyl transferase (CAT) gene in pKK232-8 (Brosius, 1984 ). The vector retains translational stops in all three reading frames between the polycloning site and the lacZ gene, ensuring that cloned inserts are in transcriptional fusion with lacZ. Whilst transcription of lacZ is dependent on the introduction of a promoter within a cloned insert, it is translated from its own RBS, independent of the insert.

Construction of ftsK reporter vectors for complementation studies.
The parental plasmid pDDK20 was constructed using oligonucleotides P1BamHI and Frag1P2SalI to amplify the upstream regulatory region and the first 337 codons of ftsK. Plasmids with upstream deletions were derived by restriction digestion at internal sites.

Site-directed mutagenesis.
Site-directed mutagenesis of ftsK was carried out using the Pfu protocol described by Stratagene. Oligonucleotides {Delta}-10 P1 and {Delta}-10 P2 deleted the dinH -10 consensus and generated pDDK30 and pDDK31, whilst oligonucleotides -35 P1 and -35 P2 modified the -35 sequence to generate pDDK35 and pDDK36. Constructs generated by site-directed mutagenesis were sequenced by Cambridge BioScience to verify the accuracy of the procedure. Mutated inserts were excised from the vector and recloned into pDDZ100 to ensure that secondary mutations in lacZ were not responsible for the changes observed in their activity.

RT-PCR and primer extension analyses.
Total RNA was extracted from DH5{alpha}, treated with DNase I, precipitated after extraction with phenol/chloroform and resuspended in ultra-pure water. RNA was incubated at 60 °C for 1 h, then, with the radiolabelled oligonucleotides, for 20 min at room temperature. Primer extensions were performed at 48 °C for 45 min in the presence of 1 unit AMV reverse transcriptase. Extension products were resolved on an 8% denaturing polyacrylamide gel alongside {phi}X174/HinfI size standards.

RT-PCR was performed on cDNA synthesized from total DH5{alpha} mRNA. Forward primers were RT0, RT1, RT2, RT3 and RT4. Reverse primers were EXT1, EXT2 and EXT3 (Table 1, Fig. 2). PCR was also carried out on mRNA preparations to ensure the absence of contaminating chromosomal DNA and on E. coli TG1 DNA to confirm the amplification conditions.


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Table 1. Primers used

 


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Fig. 2. Localization of the non-essential promoter P2ftsK. (a) Position of RT-PCR and primer extension oligonucleotides used. The linear map depicts the ftsK gene and its regulatory region (not drawn to scale). The position of each primer is indicated by an arrow and the location of the 3' end of each primer is given relative to the ftsK start codon. A band of around 45 bp is also present in the RT-PCR products. Since a similar-sized product is found with other primers of the same length but which initiate at different positions, these bands are likely to represent primer concatemers. (b) Table showing the primer combinations used in RT-PCR reactions. Plus or minus symbols indicate the presence or absence, respectively, of the predicted PCR product. (c) Primer extension of mRNA extracted from DH5{alpha} transformed with pDDZ3. {phi}X174/HinfI size standard (lane 1) and the extension product (lane 2) were run on an 8% denaturing polyacrylamide gel. The position of the transcript is indicated by an arrow with the estimated size of the transcript given above. (d) Diagram showing part of the sequence of pDDZ3. lrp sequence is shown in lower case, the plasmid sequence is capitalized, and the HindIII fusion site is in bold. The start site of the transcript lies between the nucleotides marked with an asterisk (boxed). The consensus -10 and -35 sequences for the P2ftsK promoter are indicated by lines above the sequence and the EXT8 binding site is shown by an arrow.

 
Gel shift assay.
A 160 bp DNA fragment carrying the LexA-2 box was prepared using P1BamHI labelled with [{gamma}-32P]ATP and P4HindIII. The PCR fragment was digested with BglII to release the end-labelled fragment. Reaction mixtures (20 µl each) containing approximately 100 ng labelled probe, excess competitor DNA (PCR-generated fragments lacking a recognizable LexA binding site) and E. coli TG1 crude extract were incubated with or without anti-LexA antibodies (100 ng) at room temperature for 20 min in binding buffer (50 mM Tris/HCl, 5 mM EDTA, 5% glycerol, 0·2 mg BSA ml-1). Protein–DNA complexes were separated in native polyacrylamide gels. Gels were dried and subsequently exposed to X-ray film.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of a plasmid complementing ftsK44
Previous work (Begg et al., 1995 ) has demonstrated that, in addition to the first 1170 bp of ftsK, a 750 bp fragment of DNA upstream of the gene is required to complement the ftsK44 chromosomal mutation. The 750 bp sequence includes a short non-coding sequence upstream of the lrp gene, the lrp gene itself, and a further 134 bp non-coding region immediately upstream of the ftsK reading frame (Fig. 1). Deletion of the first 154 bases of this sequence (the non-coding region upstream of lrp and the first 30 bp of lrp) abolishes complementation of the ftsK44 mutant, whilst deletion of most of lrp has no effect on complementation. From these results, it was concluded that the 154 bp AccI–BglII fragment upstream of lrp was required for transcription of ftsK and that the two genes were likely to be co-transcribed.



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Fig. 1. ftsK/lacZ complementation vectors. The 5' end of ftsK and its upstream DNA was cloned in pDDZ100 to give pDDK20. Deletions of pDDK20 generated the derivatives shown. The P1ftsK promoter (dinH) is shown as a striped rectangle. The lacZ gene is shown as a rectangle shaded with squares. The ß-galactosidase activities (‘ß-Gal’; Miller units) of pDDK and pDDZ vectors shown were standardized against the pDDK21 control (set to zero units), and the ability of each construct to complement the C600/44pcn mutant [ftsK(ts)] (‘Comp’) is also indicated. NA, Not applicable.

 
These preliminary conclusions were not fully supported by data from other studies. For example, it has been reported that a transposon insertion in lrp had no effect on cell division (Haney et al., 1992 ); also, the lrp promoter has been shown to lie 177 bp upstream of the AccI–BglII fragment. To examine the patterns of expression that might be expected in vivo, we investigated transcription from plasmids containing part of the ftsK coding sequence and varying amounts of upstream DNA.

A plasmid (pDDK20) was constructed that contained 1011 bp of the ftsK ORF and 750 bases of upstream DNA, sufficient to complement ftsK44 and provide an in vivo assay for functional FtsK expression (Fig. 1). Translational stops present in the vector between the end of ftsK and the lacZ cassette ensure independent translation of the gene products. This plasmid exhibits a ß-galactosidase activity of 2600 Miller units. The DNA in pDDK20 was deleted between the AccI site and a SexAI site 750 and 50 bp, respectively, upstream of ftsK to allow the ftsK coding sequence to be tested in isolation. ß-Galactosidase activity from this plasmid (pDDK21) was just 100 units and, crucially, this construct does not complement the ftsK44 mutant. The basal activity of pDDK21 was subsequently subtracted from the values obtained from the other pDDK derivatives to standardize their activities.

Location of a dispensable promoter (P2ftsK) for ftsK inside lrp
RT-PCR of chromosomal RNA was used to identify mRNA species transcribed from the AccI/ftsK' sequence (Fig. 2). Primers EXT1, EXT2 and EXT3 (Fig. 2a), generate the predicted PCR products when used in combination with forward primers RT0, 1, 2 and 3 (Fig. 2b), indicating that a transcript initiates from sequences upstream of RT0, beyond the MluI site. In contrast, no product was generated using RT4. The mRNA therefore must initiate from a position between RT0 and RT4 in lrp, less than 40 bp upstream of the MluI site. The RT-PCR results also indicate that the AccI/BglII fragment does not contain a promoter and that lrp and ftsK are not cotranscribed. Primer extension analysis was performed on mRNA extracted from DH5{alpha} cells transformed with plasmid pDDZ3 (Fig. 1) containing the BglII/MluI sequence to allow enrichment of P2-specific mRNA. The primer EXT8 (Fig. 2d) generates a cDNA of between 85 and 90 bases in length (Fig. 2c). This locates the start of the mRNA to between 18 and 23 bases from the MluI site, in keeping with the RT-PCR results. The presence of well-conserved -35 and -10 sequences upstream of the predicted mRNA start suggests that this is the location of the P2ftsK promoter (Fig. 2d).

A LexA-controlled promoter regulates expression of ftsK
The above results identify a promoter for ftsK within lrp, but previous work (Begg et al., 1995 ) has demonstrated that the BglII/MluI region that would contain this promoter can be deleted without affecting ftsK transcription. Primer extension was therefore used on total cellular mRNA to reveal whether shorter transcripts, obscured in the RT-PCR analysis, were initiated within the sequences upstream of ftsK. When annealed at the ftsK initiation codon (Fig. 3b), the primer EXT1 generates a single cDNA product of between 93 and 96 bp in length (Fig. 3a). The 5' end of this mRNA delimits the inducible promoter that transcribes ftsK, P1ftsK. Unexpectedly, this locates the 5' end of the P1ftsK transcript to within 12 bp of the consensus -10 sequence previously defined for the SOS-inducible promoter, dinH. Although the transcription studies were carried out in a rec minus background, which precludes SOS-induced transcription from PdinH, we still considered the possibility that transcription of ftsK might initiate from PdinH and that PdinH and P1ftsK represent the same sequence.



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Fig. 3. Localization of the primary ftsK promoter. (a) Primer extension of total DH5{alpha} mRNA incubated with EXT1 end-labelled with [35S]ATP{gamma}S by standard procedures. The extension product (lane 1) and {phi}X174/HinfI size standard (lane 2) were run on an 8% denaturing polyacrylamide gel. The position of the signal corresponding to the ftsK transcript is indicated by an arrow. (b) The approximate start of the mRNA, calculated from the primer extension result, lies between the nucleotides indicated by asterisks. The arrow shows the position of the EXT1 primer. The -10 and -35 consensus sequences for the dinH promoter are indicated by lines above the sequence and the LexA operator is boxed. The ftsK coding sequence is capitalized.

 
We constructed a set of plasmids modified within the dinH promoter (Fig. 4). Deletion of the dinH -10 sequence (pDDK30) resulted in a 90% reduction in transcription of lacZ compared to the unmodified sequence in pDDK20 (2600±266 Miller units; Fig. 1). The remaining 275 units of activity in pDDK30 were contributed primarily from P2ftsK, as deletion of P2ftsK by BglII/MluI digestion of the plasmid reduced transcription to just 47 units (pDDK31). Modification of three nucleotides within the dinH -35 sequence caused a dramatic reduction in transcription (pDDK35 and pDDK36). None of the derivatives complemented C600/44/pcn. These results provided strong evidence that the dinH promoter is essential for transcription of the ftsK gene during unperturbed cell growth.



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Fig. 4. Site-directed mutagenesis of the dinH promoter consensus. The nucleotide sequence of the dinH region is illustrated. The -10 and -35 consensus sequences are capitalized and the LexA operator is boxed. Site-directed mutagenesis was used to modify three bases within the -35 sequence (shown in bold), and to delete the -10 region (indicated). The position of the modified sequences are shown on the plasmid derivatives by a filled triangle (-10 deletion) and by an open rectangle (modified -35). The lacZ gene is shown as a rectangle shaded with squares. ß-Galactosidase activities and complementation data are given as in Fig. 1.

 
To evaluate the contribution of the two putative promoter regions P1 and P2 in ftsK transcription shown by pDDK20, we constructed pDDK22 (deleted for P2) and pDDK24 (deleted for P1). The 1845 units of transcription in pDDK22 derive solely from P1 and represent 70% of ftsK transcription. The 755 unit (30%) reduction in ß-galactosidase activity from pDDK22 can be attributed to P2 as the BglII/MluI sequence in isolation gives a ß-galactosidase activity of 1041 (pDDZ3). In the absence of P1, transcription from P2 in pDDK24 is reduced to levels that are insufficient to complement the C600/44/pcn strain (102 units) whilst both pDDK20 and pDDK22 complement this mutant. The construct pDDK23 (Fig. 4) lacks both P1 and P2 and does not express lacZ to assayable levels or complement C600/44/pcn, providing additional confirmation that there is no promoter within the AccI/BglII fragment. These data show that P1 (dinH) is the principal ftsK promoter. P2 does not transcribe ftsK to significant levels except in the presence of P1; consistent with the BglII/MluI sequence being dispensable, as noted by Begg et al. (1995 ).

The AccI/BglII region is essential for ftsK transcription and binds LexA
Expression of genes controlled by LexA has a genetic dependence on recA function, yet our observations show transcription of ftsK from dinH in a recA minus background. Moreover, the existence of non-cleavable LexA mutants such as LexA3 indicates that activation of dinH occurs by a mechanism other than rec-induced LexA cleavage. This is not without precedent; Dri & Moreau (1994 ) reported that transcription of LexA-regulated genes could be increased without LexA cleavage by inducing changes in the intracellular pH.

Despite the absence of a promoter within the AccI/BglII DNA sequence [confirmed by RT-PCR and the absence of detectable levels of ß-galactosidase activity in pDDZ5 (Fig. 1)], deletion of this region results in the abolition of transcription from the downstream promoters (Fig. 4, pDDK26). This was unexpected and suggests that there may be a regulatory element present within the AccI/BglII sequence.

Closer examination of the AccI/BglII region reveals a 19 bp motif, 5'-CTG AAcAgTcATgTTT CAG-3' (uppercase letters represent bases which show identity with the LexA operator consensus; bold letters represent the invariable palindrome found within the LexA operator sequence), from -671 to -689 bp that shows similarity to the consensus sequence of a LexA operator. We will refer to this sequence as LexA-2, and the LexA box associated with dinH as LexA-1. As the homology to the consensus LexA operator is only partial, we carried out gel shifts to assay directly for LexA binding. The 160 bp AccI/BglII sequence containing the LexA-2 site was radiolabelled and incubated with E. coli TG1 protein extract, then subjected to electrophoresis in a 6·5% polyacrylamide gel. Fig. 5(a) shows the retardation of the LexA-2 operator sequence (lane 2) compared to the mobility of the free probe (lane 1). The shift is also shown on electrophoresis in 5% polyacrylamide (Fig. 5c, lane 5) where the retardation can be seen more clearly. The presence of LexA within the primary retardation complex was confirmed by carrying out the binding reactions in the presence of anti-LexA antibodies. The specific interaction of anti-LexA antibody with the DNA–LexA complex results in the further retardation or ‘supershift’ seen in lanes 3 and 4. Western analysis of the gel shifts confirmed the presence of LexA in both the primary and secondary retarded complexes. No comparable retardation was observed when the binding reactions were performed with a control protein extract prepared from a lexA null mutant strain (data not shown). These results demonstrate that the LexA-2 operator-like sequence can be recognized and bound by LexA.



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Fig. 5. Gel shift assays showing LexA binding to the AccI–BglII fragment. (a, b) The labelled AccI/BglII PCR fragment was incubated with E. coli TG1 crude extract in the absence (lane 2) or in the presence (lanes 3 and 4) of 100 ng anti-LexA antibodies. The complexes were separated in a 6·5% polyacrylamide gel. Lane 1 is the control, free DNA probe (P) without extract or antibodies. C1 indicates the primary complex of DNA–LexA whilst C2 indicates the secondary (supershift) complex of DNA–LexA–anti-LexA. When the ratio of TG1 extract to competitor DNA is increased (lane 4), only C2 is detected. (c) The primary shift is best seen on a 5% polyacrylamide gel (lanes 5 and 6). The presence or absence of protein extract and anti-LexA antibodies is indicated by plus and/or minus symbols under each lane.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The results of this study demonstrate that the cell division gene ftsK is transcribed from two promoters. P2ftsK is dispensable under our experimental conditions, and is located within the lrp coding sequence, whilst P1ftsK is essential and corresponds to dinH, an SOS-inducible promoter located upstream of ftsK (Lewis et al., 1992 ). The intracellular concentration of FtsK increases on induction of the SOS response (Wang & Lutkenhaus, 1998 ), and it has been suggested that this may be linked with its proposed role in chromosome partition under these conditions (Liu et al., 1998 ; Yu et al. 1998b ). This study establishes that dinH (P1ftsK) is not activated exclusively in response to SOS stimuli, but that it is also active during unperturbed growth in cells that lack RecA protein. As a consequence, ftsK is the first essential gene to be identified that is under the direct control of an SOS promoter.

The LexA repressor controls transcription of the genes of the SOS regulon. LexA binds with high affinity to a consensus operator, the LexA box, located within or very close to the promoter of each SOS gene (Berg & Von Hippel, 1987 ). Once activated, RecA enhances LexA self-cleavage to prompt high level expression of the SOS proteins (Slilaty & Little, 1987 ). As DNA damage is repaired, LexA autodigestion stops and the system returns to a quiescent state in which the SOS promoters are repressed. In this study, gene expression was monitored by assaying for ß-galactosidase in a DH5{alpha} rec minus background, which does not support a mechanism for LexA-mediated regulation of ftsK. Whilst it is possible that basal transcription from LexA-repressed dinH may supply sufficient ftsK to support cell division, an alternative explanation is that ftsK is regulated by a RecA-independent mechanism.

Plasmid pDDK26 carries the dinH promoter and produces only 13 units of ß-galactosidase, about 0·5% of the level produced by pDDK20 and insufficient to allow complementation of C600/44/pcn. If basal transcription of ftsK was being initiated from dinH, it might reasonably be expected that this construct would exhibit enzyme levels comparable to pDDK20. The very low enzyme levels associated with this plasmid are more likely to reflect that LexA binds with high affinity to the dinH promoter, in keeping with the observation that LexA-1 is highly homologous to the sfiA LexA operator, which is known to bind LexA very strongly (Preobrajenskaya et al., 1994 ). Furthermore, strains containing uncleavable LexA do not exhibit the expected filamentation phenotype, suggesting that even when LexA is irreversibly bound to its operators, ftsK transcription is unaffected and cell division proceeds normally. Rec-independent regulation, whilst novel, fits better with the experimental observations. Closer examination of the AccI/BglII region reveals a sequence within it that shows similarity to the LexA consensus operator motif and that this putative operator (LexA-2) is able to bind LexA in vitro. This suggests a potentially straightforward mechanism that would account for the requirement of the promoterless AccI/BglII region in the Rec-independent regulation of ftsK. According to this model, LexA would ordinarily repress ftsK expression by binding its cognate operator at dinH, transient displacement of LexA from LexA-1 to LexA-2, in response to an uncharacterized cellular signal, would free dinH and allow transcription of ftsK in response to the cell’s requirement for FtsK protein. The poorer homology of LexA-2 to the consensus binding motif might reflect the lower differential binding affinity of the site for LexA and ensure that the repressor binds preferentially to LexA-1 at dinH.

A second promoter, P2ftsK, lies in the lrp coding sequence and is able to transcribe ftsK. In the absence of SOS induction, transcription from P2ftsK alone is insufficient to support cell division in C600/44/pcn. P2 transcripts were only detected by RT-PCR or with primer extension with mRNA extracted from a strain transformed with a multicopy plasmid containing the lrp coding sequence. This suggests that P2 mRNA is either expressed at a very low level or that it has a rapid turnover. It seems likely that this promoter is dispensable, although it is not clear if it might have a role under other growth conditions; its presence within the lrp ORF may be of importance in lrp expression studies.

In the original isolation of ftsK (Begg et al., 1995 ), the promoter for ftsK was tentatively located within the AccI/BglII sequence upstream of lrp, suggesting that lrp and ftsK might be cotranscribed. However, this was challenged by the existence of strain CV1008 which, despite having transposon Tn10 inserted within the lrp coding region, had no apparent cell growth defect (Haney et al., 1992 ). We used PCR to accurately locate the position of the transposon within CV1008 and found, unexpectedly, that it does not lie within lrp, but is located upstream of it, close to the lrp promoter. Sequencing revealed that a 49 bp insertion sequence corresponding to part of the IS10-L element of the transposon remains within the lrp reading frame, (the insertion introduces a stop codon at the 78th codon). It is not possible to determine when this second transposition event occurred, but since ftsK synthesis is likely to have been restricted with the transposon in its original location, transposed derivatives might be expected to have a selective advantage. The transposition event may have occurred shortly after its initial isolation since the phenotype of the strain, lrp minus, would appear unchanged. The 49 bp CV1008 insertion and the 300 bp deletion in pPDDK22 show that the LexA-2 box can be relocated relative to dinH/LexA-1 without significantly altering ftsK expression. However, there is an upper limit to the distance over which the two LexA sites are functional; complementation is lost when LexA-2 is positioned 3 kb from dinH/LexA-1 (data not shown). The loss of the transposon from lrp in CV1008 may reflect this. The insertion of an intact Tn10 in lrp would impair ftsK transcription and allow selection of a derivative in which the LexA boxes are within functional distance. Additionally, the LexA-2 box does not function in trans since a chromosomal copy of LexA-2 does not compensate for the absence of the region on a plasmid.

Finally, these results may have implications for the regulation of the genes in the dcw cell division cluster. Three LexA-binding sites have been identified within the first three genes at the 5' end of the gene cluster; two lie upstream of mraW whilst the third lies close to the promoter for ftsL (Ishino et al., 1989 ; Vicente et al., 1998 ; Gomez, 1991 ). The arrangement of SOS box I and SOS box II lying upstream of the mraW initiation site is of particular note, since it resembles the arrangement seen in the ftsK regulatory region.

This report demonstrates that LexA has an important and unexpected general role in cellular regulation. Our results support a model for the regulation of ftsK by LexA in a rec-independent manner. Future experiments will characterize the molecular basis of the regulation and examine the significance of LexA control with respect to cell division gene expression in normal growth conditions, and on SOS induction.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr Ken Begg for providing the C600/44/pcn mutant strain and for critical reading of the manuscript. The work was supported by the Biotechnology and Biological Sciences Research Council, UK.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Addinall, S. G. & Lutkenhaus, J. (1996). FtsA is localized to the septum in an FtsZ-dependent manner. J Bacteriol 178, 7167-7172.[Abstract]

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Brosius, J. (1984). Plasmid vectors for the selection of promoters. Gene 27, 151-160.[Medline]

Diez, A. A., Farewell, A., Nanmark, U. & Nystrom, T. (1997). A mutation in the ftsK gene of Escherichia coli affects cell–cell separation, stationary-phase survival, stress adaptation, and expression of the gene encoding the stress protein UspA. J Bacteriol 179, 5878-5883.[Abstract]

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Received 12 April 2000; revised 7 July 2000; accepted 21 July 2000.



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