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
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ABSTRACT |
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Keywords: cell division, FtsK, dinH, LexA, transcription
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INTRODUCTION |
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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.
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METHODS |
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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 -10 P1 and
-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, 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
X174/HinfI size standards.
RT-PCR was performed on cDNA synthesized from total DH5 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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RESULTS |
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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
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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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 DNALexA 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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DISCUSSION |
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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
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 cells 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 April 2000;
revised 7 July 2000;
accepted 21 July 2000.
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