1 Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA
2 Department of Biological Sciences, Duquesne University, Pittsburgh, PA 15282, USA
Correspondence
Nancy Trun
trun{at}duq.edu
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ABSTRACT |
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Present address: Bioinformatics Research Centre, Department of Computing Science, University of Glasgow, Glasgow G12 8QQ, UK.
Present address: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA.
Present address: Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA.
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INTRODUCTION |
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We have previously shown that overexpression of cspE, in conjunction with the two open reading frames that flank cspE (crcA and crcB), protects cells from the DNA decondensing agent camphor (Harrington & Trun, 1997; Hiraga et al., 1989
), and suppresses the chromosome instability and growth defects seen in mukB mutants (Hu, 1996
; Yamanaka et al., 1994b
). From our original studies, all three genes (crcA, cspE and crcB) are required for these phenotypes and the genes must be overexpressed at high levels (Hu, 1996
). Suppression of the mukB deletion is also seen in mutants defective in topA (Sawitzke & Austin, 2000
). Suppression of
mukB by topA, like suppression of
mukB by crcA, cspE and crcB overexpression, reverses both the Ts defect and the anucleate cell production phenotypes. It is thought that suppression by topA mutants is the result of an increase in negative supercoiling.
In this paper, we examine the phenotypic consequences of deleting or overproducing crcA, cspE and crcB (AEB). We have combined the deletion and the overproducing plasmids with mutations in the genes for the three classes of proteins implicated in chromosome folding. Class I are small DNA-binding and bending proteins such as HU, HNS, IHF and Fis that co-purify with the nucleoid (HU, HNS and Fis) (Murphy & Zimmerman, 1997) or can substitute for HU in some reactions (IHF) (Nash, 1990
). Class II is MukB (see above). Class III proteins are the enzymes that affect supercoiling, gyrase and topoisimerase IV (topo IV). Topo IV is a supercoil-relaxing enzyme needed for partitioning of daughter chromosomes (Adams et al., 1992
; Kato et al., 1992
; Peng & Marians, 1993
; Zechiedrich & Cozzarelli, 1995
). The goal of these experiments is to use what is known about the in vivo roles of the condensing proteins to determine if CrcA, CspE and CrcB share any of these properties.
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METHODS |
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Cloning of crcA, cspE, crcB or cspEcrcB into pBAD18-Kan.
The appropriate gene complete with its own predicted ShineDalgarno sequence was amplified by PCR. Incorporated into the primers were a KpnI site on the 5' end of the gene and an XbaI site on the 3' end of the gene (cspE, crcB, cspEcrcB). The PCR fragments, as well as purified pBAD33 vector DNA, were digested with KpnI and XbaI according to the manufacturer's direction (New England Biolabs). The PCR fragment was mixed with the vector, ligated and transformed. Potential clones were checked for the presence of the correct insert by restriction enzyme analysis, followed by DNA sequencing. The PCR fragments were first cloned into pBAD33 utilizing the KpnI and XbaI sites. Subsequently, they were moved into pBAD18-Kan by digesting the appropriate pBAD33 clone with MluI and SphI, isolating the fragment containing the gene of interest and subcloning into pBAD18-Kan. The potential pBAD18-Kan subclones were also checked by restriction site analysis followed by DNA sequencing. Because crcA contains an internal KpnI site, an EcoRI site was substituted for KpnI for the crcA clone and crcA was ligated directly into pBAD18-Kan. The primers used were: crcA, 5'-NNNNGGGAATTCGTAGCTTTGCTATGCTAGTAGTAG-3' and 5'-NNNNCCTCTAGAGCTATTGATTTTAAAGAAGTTAC-3'; cspE, primer 1 5'-NNNNNNNNGGGGTACCCATGTAAAGGTAATTTTGATGTCTA-3' and primer 2 5'-NNNNNNNNGCTCTAGACACTGGCATTCTGGCTGT-3'; crcB, primer 3 5'-NNNNNNNNGGGGTACCACAGCCAGAATGCCAGTG-3' and primer 4 5'-NNNNNNNNGCTCTAGATTTAACCCACTGCATCAG-3'; cspE, crcB primer 1 and primer 4. Extra bases (N) were added to the 5' ends of the primers to ensure that the enzymes would digest the DNA efficiently.
Isolation of specialized transducing phages carrying crcA, cspE and crcB.
Approximately 12 000 plaques from an E. coli library in the vector
DE3 (Katayama et al., 1988
) were screened by plaque blot (Sambrook et al., 1989
) for the presence of crcA, cspE and crcB. Twenty-six potential phages were identified, purified and reprobed. To determine which of the phages carried a given gene, we performed PCR reactions with three sets of primers, one for each gene (see above). Of the original 26 phages, 21 contain all three genes, three contain cspE and crcB, one carries only crcB and one carries only cspE (data not shown). A single phage,
MDG18, which contains the three genes on a 2·1 kb chromosomal fragment, was used for all subsequent genetics. The cI857 mutation was crossed onto
MDG18 and a Kanr marker was moved onto
MDG cI857 by growing the phage on a strain containing the minitransposon mkan (Kleckner et al., 1991
), forming the phage
MDG18K.
Crossing the deletions into the chromosome.
The deletions were first crossed from either pMDG4 or pNAB1 to MDG18K and subsequently crossed from the phage to the chromosome as described by Maurizi et al. (1985)
. The markers on the phage (cI857 and Kanr) and the marker for the deletion (cat-19) were used to follow the movement of the genes in the crosses. To ensure that no additional mutations were induced in these crosses to compensate for the loss of AEB, a miniTn10 50 % linked to the deletions was isolated and used to move the deletions non-selectively into NT3 (a haploid for AEB) or NT3 (pNT2) (a merodiploid for AEB). The AEB deletion can be transduced into the haploid 46 % linked to the miniTn10 (60 Camr transductants/130 total Tetr transductants) and into the merodiploid 51 % linked (118 Camr/233 Tetr transductants). These numbers indicate that the AEB genes are not essential.
Southern blots.
Southern blots of the chromosomal deletions were carried out as previously described by Sambrook et al. (1989). Chromosomal DNA was isolated using the Wizard genomics DNA isolation kit (Promega). 32P-labelled pNT2 was used as the probe.
Determination of supercoiling levels.
All strains used in this assay were made recA56 by P1 transduction. pBR322 or pNT2 was transformed into the recA56 derivatives by electroporation and strains containing plasmid monomers were identified by preparing plasmid DNA on random transformants and screening by agarose gel electrophoresis. Strains containing plasmid monomers were grown overnight in LB medium with ampicillin (50 µg ml-1) and plasmid DNA was prepared. Plasmid DNA was electrophoresed on a 0·8 % agarose gel with 10 µg chloroquine ml-1 in 0·5x TPE buffer (45 mM Tris pH 7·2, 0·87 mM Na2EDTA). Gels were electrophoresed for 18 h at 4 °C at 30 V with recirculating buffer, stained with SYBR Green I (Molecular Probes) and photographed.
Assaying for camphor resistance.
Plate assays for camphor resistance were carried out as described previously (Trun & Gottesman, 1990). Liquid assays for camphor resistance were carried out as follows. Five millilitres of LB was inoculated with a single colony and incubated overnight at 37 °C. The culture was diluted 100-fold into 25 ml LB and grown to OD600 0·15 at 37 °C. Either 0·10 g or 0·15 g camphor (as indicated) was added to each flask and 1 ml aliquots of cells were removed every 30 min for viable cell counts.
Other assays.
UV sensitivity was assayed as described by Li & Waters (1998) and
-galactosidase was assayed as described by Zhou & Gottesman (1998)
. Staining, fixation of cells, photography and processing of the film were carried out as described by Hu (1996)
. Suppression of the temperature-dependent growth defect of parC and parE Ts mutants was examined by switching the temperature of actively growing cells from 30 °C to 42 °C and measuring the number of viable cells at 1 h intervals for 5 h.
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RESULTS |
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Overexpression of AEB+ affects other phenotypes of gyrase and topo IV mutants
Strains with Ts mutations in gyrA, gyrB, parC and parE exhibit sensitivity to nalidixic acid. Overproduction of AEB+ in gyrase or topo IV mutants reduces this sensitivity to nalidixic acid (Table 2). This effect can also be seen with norfloxicin (data not shown).
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parC1215 and parE10 Ts mutants, when grown at the permissive temperature, exhibit a defect in nucleoid morphology that is exemplified by cells that are 23 times longer than the parental cells and contain one large centrally located nucleoid mass (Fig. 2, Ib and Ic). Cells lacking DNA are also more abundant than in the parental controls. Combining the Ts mutations with a chromosomal deletion of AEB (see below) exacerbates the nucleoid morphology defects. The cells filament more extensively (Fig. 2
, IIb and IIc) and the nucleoids occupy the entire length of the filaments. Overproduction of AEB from pNT2 corrects these defects and the cells appear normal in size and nucleoid morphology (Fig. 2
, IIIb and IIIc). gyrA and gyrB ts mutants do not exhibit a nucleoid morphology defect and cannot be scored in this assay.
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Camphor resistance in mutants defective in chromosome condensation
We tested the camphor resistance of mutations in chromosome condensing proteins in both the presence and absence of the AEB+ overexpressing plasmid (Table 3). The gyrase and topo IV Ts mutants were tested at 30 °C and the hns, hupA, fis and ihfA insertion-deletion mutants were tested at 37 °C. The gyrB202,221(Ts), parC1215(Ts) and parE10(Ts) mutants are 1001000 times more sensitive to camphor than parental cells, whereas the gyrA43(Ts) mutant shows the same level of sensitivity as the parental strain. Overexpression of AEB+ in the wild-type at 30 °C results in a 20-fold increase in camphor resistance. As compared to wild-type cells, overexpression of AEB+ in the gyrase or topo IV Ts mutants results in 20018 000-fold increase in camphor resistance.
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Overexpression of AEB protects hupAB strains from UV irradiation
Li & Waters (1998) have shown that strains lacking hupA or both hupA and hupB are UV sensitive due to a defect in homologous recombination. pNT2 (AEB+) or pBR322 were introduced into the double mutant
hupA
hupB (OS336) and UV sensitivity was measured. As can be seen in Fig. 5
, pNT2 protects the
hupA : : Kan
hupB : : Cam strain from UV by about 100-fold. The amount of killing in the
hupAB pNT2 strain is equivalent to that seen by Li & Waters (1998)
in a wild-type strain, indicating that the protection by AEB overexpression is very close to 100 %. Because HU deletions are UV sensitive, we tested our deletions of AEB and determined that they are not UV sensitive by themselves (data not shown).
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The transcription of the rcsA gene is silenced 12·9-fold by HNS (Sledjeski & Gottesman, 1995). Overexpression of AEB results in slightly higher (2·2-fold)
-galactosidase activity than that of cells containing the vector, pBR322 (Table 4
). This effect may be partially dependent on HNS because activation by AEB is lower (1·4-fold) in an hns mutant strain (Table 4
). DsrA is a small RNA that antagonizes the effect of HNS when overproduced (Sledjeski & Gottesman, 1995
) but is not required for AEB activation of rcsAlacZ (Table 4
). The transcription of the lamB gene is activated 2·5-fold by HNS (Johansson et al., 1998
) and the hns gene itself is repressed fourfold by HNS (Dersch et al., 1993
). Overexpression of AEB results in a very slight repression (0·6-fold, Table 4
) of lamB and a slight activation of hns (1·7-fold, Table 4
). We also examined the behaviour of an additional HNS-repressed fusion, proU : : lacZ, an HNS-activated fusion, malE : : lacZ and a fusion that does not respond to HNS, dsrA : : lacZ. Overexpression of AEB has no effect on proU : : lacZ, an effect in the same direction as HNS on malE : : lacZ and unlike HNS, a slight activation of dsrA : : lacZ (Table 4
). Thus, regulatory effects from overexpression of AEB are much smaller in magnitude than those seen for HNS and the patterns of gene expression are different for AEB and HNS.
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DISCUSSION |
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A model for CspE's role in condensing DNA
The results listed above suggest a very simple model for how CspE functions in DNA condensation, namely that CspE stabilizes supercoiled DNA molecules and holds them in a compacted configuration. In this scenario, overexpression of cspE would increase supercoiling levels of resident plasmids in both parental cells and cells defective in supercoiling levels (gyrase, HU or Fis mutants) by stabilizing the population of supercoiled DNA molecules. Stabilizing of the supercoiled DNA molecules by CspE would allow the cell to maintain normal supercoiling levels with less-active supercoiling enzymes. Treating cells with camphor results in decondensed nucleoids. The increase in camphor resistance in gyrB, parC and parE Ts mutants overexpressing AEB as well as suppression of the nucleoid morphology defects in parC and parE Ts mutants overexpressing AEB could also be explained by CspE stabilizing supercoiled DNA.
Suppression of gyrase, topo IV and mukB mutations by overexpression of AEB
Overexpression of AEB will completely suppress the production of DNA-less cells seen in mukB mutations (Yamanaka et al., 1994a). As we have shown here, overexpression of AEB will also efficiently suppress some of the defects associated with gyrase and topo IV Ts mutants. While overexpression of AEB will quite efficiently suppress many of these mutants' defects, it will not completely compensate for the loss of any of these essential proteins: all of the mutants do not form colonies at the higher temperatures. We think that this is because AEB do not possess the enzymic activities of MukB, gyrase or topo IV. Rather, overexpression of AEB, or more likely cspE and crcB (see below), enhances the efficiency of MukB, gyrase or topo IV. mukB mutations are also suppressed by topA mutations (Sawitzke & Austin, 2000
). In this case, the suppression has been documented to be the result of an increase in negative supercoiling. If overexpression of CspE stabilizes supercoiled DNA, this could explain the suppression of mukB mutations.
The small DNA-binding proteins
For the small DNA-binding and bending proteins HNS, HU, IHF and Fis, we see effects of overexpression of AEB in some but not all assays. Overexpressing AEB partially compensates for Fis- for resistance to camphor and plasmid supercoiling. For HU, there is compensation by overexpression of AEB for camphor resistance, plasmid supercoiling levels and the defect in homologous recombination as assayed by UV sensitivity. Overexpression of AEB does not consistently suppress defects in any one of these small DNA-binding proteins; however, the defects most often suppressed by overexpressing AEB are those associated with mutations in HU.
Gene regulation by CspE
We originally tested several HNS-regulated genes to determine if they are also regulated by overexpressing AEB. Two facts emerged from these data. First, the regulation patterns by overexpressing AEB are different from the regulation patterns exhibited by HNS. Second, the magnitude of regulation by AEB is very small at best. We suspect that this level of regulation could be accounted for by changing the condensation state of the chromosome and not through classical mechanisms of activation or repression. This result prompted us to search for other genes that are regulated by CspE to determine if all gene regulation by CspE is twofold or less. Much to our surprise, we have found several genes that are regulated 40-fold or more by overexpression of cspE (A. Kolesar, A. Pryzbylski & N. Trun, unpublished). Further studies using rcsA, as well as these newly identified genes, will shed light on the mechanism(s) of regulation by CspE.
Deletion of crcA, cspE and crcB
Not surprisingly, deleting crcA, cspE and crcB is not lethal. E. coli K-12 contains nine csp genes and it has been shown that only deletion of multiple csp genes results in noticeable growth defects (Graumann et al., 1997; Xia & Inouye, 2001
). What is interesting about the deletion of AEB is that cells carrying the deletion are hypersensitive to camphor, suggesting that their nucleoids are more sensitive to decondensation than wild-type nucleoids.
What are the roles of CrcA and CrcB?
CrcA from Salmonella typhimurium has been to shown to be an unusual outer-membrane enzyme involved in lipid A biosynthesis (Bishop et al., 2000). One phenotype of the crcA homologue (pagP) is that it makes cells more resistant to certain antimicrobial peptides (Guo et al., 1998
). Our original idea for how overexpression of crcA contributed to the camphor-resistance phenotype was that it provided some resistance to camphor that is independent of overexpression of cspE and crcB. The fact that overexpression of crcA by itself does not confer any camphor resistance on cells indicates that this cannot be the explanation. At least two other possibilities exist. Given that CrcA is a membrane protein and CrcB is predicted to be a membrane protein, perhaps there is an interaction between these two proteins that accounts for the 1000-fold increase in camphor resistance when AEB are overexpressed and the 100-fold increase when only EB are overexpressed. Alternatively, it is possible that expression levels of EB are higher when all three genes are cloned into pBR322 and expressed from their native promoters. Further investigation will distinguish between the possibilities.
It is clear from our data that overexpressing crcB amplifies the phenotypes of cspE mutants. Overexpressing cspE confers a 10-fold increase in camphor resistance and accounts for part of the gene regulation of rcsA (1·7-fold). Overexpressing crcB does not increase resistance to camphor nor does it have any effect on the regulation of rcsA. However, when cspE and crcB are overexpressed together, with the DNA sequence between the two genes being the same as in the chromosome, camphor resistance increases to 100-fold and gene regulation increases to 2·1-fold. All of the gene regulation seen with overexpression of AEB is also seen with overexpression of just EB. The two assays, camphor resistance and regulation of rcsA, do not have the same requirement for overexpression of crcA.
Our data suggest that CspE and CrcB work together to carry out their cellular function(s). One very interesting aspect of this is that CspE is a soluble cytoplasmic protein with nucleic-acid-binding capabilities and CrcB is a protein with four long predicted hydrophobic regions suggesting that it may be a membrane protein. If CrcB is indeed a membrane protein that interacts with CspE, then these two proteins may provide a way to anchor the chromosome to the membrane.
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ACKNOWLEDGEMENTS |
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Received 21 March 2003;
revised 16 May 2003;
accepted 19 May 2003.
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