National Institute of Genetics, Mishima, Shizuoka-ken 411-8540, Japan1
Department of Bioengineering, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan2
Author for correspondence: A. Nishimura. Tel: +81 559 81 6827. Fax: +81 559 81 6826. e-mail: anishimu{at}lab.nig.ac.jp
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ABSTRACT |
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Keywords: kdsA mutants of E. coli, KDO biosynthesis, membrane structure, cell division, lipopolysaccharide synthesis
Abbreviations: DAPI, 4',6-diamino-2-phenyl-indole; KDO, 3-deoxy-D-manno-octulosonic acid
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INTRODUCTION |
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The outer membrane of Gram-negative bacteria is composed principally of lipopolysaccharide (LPS) and phospholipids. LPS comprises approximately 30% of the outer membrane by gross weight (Smit et al., 1975 ). LPS, comprising hydrophobic lipid A, the hydrophilic core oligosaccharide chain and 3-deoxy-D-manno-octulosonic acid (KDO), connecting lipid A to the oligosaccharide chain (Raetz et al., 1985
; Rietschel, 1984
), is important in determining outer-membrane barrier function (Leive, 1974
; Nikaido & Vaara, 1985
).
KDO is synthesized by the condensation of D-arabinose-5-phosphate and phosphoenolpyruvate, followed by dephosphorylation. The first step of the reaction is catalysed by KDO 8-phosphate synthetase, encoded by the kdsA gene in both Salmonella typhimurium (Rick et al., 1977 ) and E. coli (Ray, 1980
). E. coli kdsA, however, was first identified as a gene complementing the kdsA mutation of Salmonella (Woisetschläger & Högenauer, 1987
), although no similar mutants have yet been identified in E. coli. Mutations in kdsA of Salmonella typhimurium cause the accumulation of lipid A in the periplasm, also resulting in the disappearance of LPS from the outer membrane (Rick & Osborn, 1977
; Osborn et al., 1980
). LPS is an essential membrane component (Strohmaier et al., 1995
) and only conditional lethal mutants in KDO biosynthesis can be isolated in Salmonella (Lehmann et al., 1977
; Rick et al., 1977
; Rick & Young, 1982
). The biosynthesis of LPS is growth-phase-regulated at the transcriptional level in E. coli (Strohmaier et al., 1995
); no evidence, however, has uncovered a relationship between LPS biosynthesis and cell division. In this study, we isolated temperature-sensitive mutants of kdsA and demonstrated that membrane instability resulting from the defect in KDO biosynthesis affected the FtsZ-ring formation.
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METHODS |
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Fluorescence microscopy.
To observe cells and nucleoids by microscopy, cells were stained by 4',6-diamino-2-phenyl-indole (DAPI) as described by Hiraga et al. (1989) . Localization of FtsZ was visualized by immunofluorescence microscopy, as described by Hiraga et al. (1998)
using a rabbit anti-FtsZ antibody, kindly provided by Dr J. Lutkenhaus (Addinall et al., 1996
), as the first antibody and Cy3-labelled anti-rabbit IgG antibody as a second antibody.
Site-directed mutagenesis.
We constructed a plasmid by site-directed mutagenesis substituting a G and A for T696 and G699 of kdsA, respectively, carried by pTN18H. We designed four primers to prepare PCR products containing either the 5' or 3' half of kdsA using pTN18H as the template. The sequence of the forward primer to amplify the 5' half region of kdsA was 5'-TCATCGATAAGCTTTAATGCGGTAG-3', homologous to the HindIII flanking region of pTN18H. The sequence of the reverse primer was 5'-AGCCCCGCTAGCCCTACC-3', the complementary sequence from G690 to T707 of kdsA containing the T696G and G699A substitutions. The resulting PCR products contained both the 2 base substitutions and an additional NheI site, 5'-GCTAGC-3'. To amplify the 3' region of kdsA, the forward primer sequence 5'-GCGGTAGGGCTAGCGGGGC-3' was used to create 2 base substitutions and an NheI site. The reverse primer 5'-CGCGCGAGGCAGCTGCGGTAA-3' contained the PvuII flanking region of pBR322, the parent vector of pTN18H. The PCR product, containing the 5' half region, was cut by NheI and HindIII; the 3' half region was cut by NheI and PvuII. Both were ligated to the 2 kb HindIIIPvuII fragment of pBR322 concurrently. The resulting plasmid, pTN18HX, was used for the complementation test of the fts mutants.
Quantitative analysis of KDO.
Quantitative analysis of KDO was performed as described by Karkhanis et al. (1978) with the following modifications. Cultures growing exponentially (0·2 OD600) in L-broth at 30 °C were diluted 1- to 150-fold with fresh medium [for JE10830/p(kdsA+) 10 µM IPTG was added] and incubated at 41 °C for 0·5, 1, 2 and 3 h, respectively. At each time point, OD600 was measured, the values of which were corrected for the dilution factors and are shown relative to the value of the 0 h sample. This makes it possible to give the same nutritional condition for each point of analysis. Cells were harvested from 12 ml of each culture and washed twice with 0·5 ml 10 mM HEPES (pH 7·4) by centrifuging at 3500 r.p.m. for 10 min. Cells were broken with an Ultrasonic Cleaner USC-1 (Pasorina) for 30 s in an Eppendorf tube. A 10 µl aliquot of the sample was utilized to quantify the total proteins. We added 60 µl 0·9 M H2SO4 to the remaining sample and boiled in a water bath for 40 min to separate the KDO from LPS. Samples were allowed to cool to ambient temperature, then centrifuged at 15000 r.p.m. for 5 min. A 0·25 ml aliquot of the supernatant was transferred to a new tube, 125 µl 40 mM HIO4 was then added and incubated at room temperature for 20 min to form KDO-COOH. After 5 min incubation with 125 µl 2·6% NaAsO2 in 0·5 M HCl, 250 µl 0·6% TBA was added. Samples were then boiled for 15 min. Following the addition of 500 µl undiluted DMSO, the OD548 was measured. Purified KDO (Sigma) was used as a standard.
Construction of plasmids.
A 2·3 kb DNA fragment containing intact msbA was amplified from genomic DNA of the wild-type strain MG1655 by PCR using a pair of primers: 5'-CAGGCAACATTGCTTCAGGATCCTC-3' and 5'-CAGCCTTACCACCGGATCCCCGCGA-3'. Amplified DNA was digested with BamHI and then cloned at the BamHI site on the vector plasmid pMW118 (Nippon Gene Co.). One of the resulting plasmids, in which the msbA gene is located downstream of the lac promoter on the vector plasmid in the same direction, was named as pMsbA.
Western blotting.
Western blotting was performed as described by Sambrook et al. (1989) . Pelleted cells were resuspended in 100 µl SDS sample buffer to give 1 OD600 unit, then boiled for 3 min. Ten microlitres of each sample was electrophoresed on a 10% SDS polyacrylamide gel, transferred to membranes and immunostained using an anti-FtsZ polyclonal antibody. The anti-FtsZ polyclonal antibody was kindly provided by Dr J. Lutkenhaus. Western blots were visualized using an ECL Western blotting kit (Amersham). The resultant membranes were exposed to X-ray films.
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RESULTS |
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We examined the effects of the fts830 mutation on cell division and growth. Cultures of both JE10830 and the parent bacterium, PA3092, growing exponentially at 30 °C, were diluted to 5x107 cells ml-1 and incubated at 41 °C. We monitored cell numbers utilizing a Coulter counter and cell growth as OD600. The OD600 of JE10830 bacteria continued to increase after the temperature shift, although the increase was lower than that of the wild-type strain. Cell division, however, stopped completely 2 h after the temperature shift to 41 °C (Fig. 1). DAPI staining demonstrated that chromosomal replication and segregation appeared normal for at least 3 h at 41 °C (Fig. 2
). We examined the six remaining strains similarly, using the same medium, LB containing 0·5% NaCl. JE10446, JE11171, and JE11241 demonstrated similar phenotypes to JE10830 (data not shown). JE10705 and JE11212 appeared to have a leaky phenotype since cell number per OD600 after 3 h incubation at 41 °C was about three times more than that of JE10830. Cells, however, stopped dividing completely at 41 °C when grown in LB medium without NaCl. Many cell division mutants recover cell division when the growth medium has an NaCl concentration higher than a critical concentration specific to a mutation due to osmotic effects (Reeves et al., 1970
; Ricard & Hirota, 1973
). The critical concentration of NaCl for these two strains may be lower than 0·5%.
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To clone the wild-type fts830 gene, we constructed the plasmids pTN18 and pTN23 by insertion of the 4·8 kb SspI segment of pLC13-27 into the EcoRV site within pBR322 in both directions. As shown in Fig. 3(a), both plasmids complemented the fts830 mutation. After testing subclones containing various deletions for their ability to complement fts830, we determined that a 1·5 kb HindIIIPvuII segment, carried by the pTN18H plasmid, which lacks 3·3 kb of the original SspI fragment, complemented fts830. Sequencing analysis demonstrated that this segment contained both kdsA and ORF-X, on the opposite strand. ORF-X may encode a protein composed of 315 aa. The six remaining fts mutants were also complemented by the pTN18H plasmid.
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We found recently that the ts20 mutant, defective in cell division at the restrictive temperature (Nagai & Tamura, 1972 ), carried the same allele as fts705.
The kdsA mutations affect KDO production and sensitivity to hydrophobic materials
LPS serves as a barrier against hydrophobic materials and mutations involved in LPS synthesis result in an increased sensitivity to hydrophobic materials (Hancock & Reeves, 1976 ). To examine membrane stability, we compared the sensitivity of both the mutants and the wild-type strain to hydrophobic drugs. Exponentially growing cultures of both JE10830 and the parent, PA3092, were diluted and plated on LB medium containing variable concentrations of novobiocin, a hydrophobic antibiotic inhibitor of the gyrB protein (Drlica, 1984
). Incubation was carried out at 36 °C, the maximum temperature at which the mutants were able to form colonies. The colony-forming ability of JE10830 decreased inversely with increasing concentrations of novobiocin; growth of the parent strain was not affected (Fig. 5
). We also examined the effects of other substances on mutant growth. Cultures of the JE10830 mutant and the parent strain were diluted to 5x105 cells ml-1, and 1 µl was spotted onto LB agar containing varying concentrations of substances that destabilize, including SDS, eosin Y, EDTA and methylene blue. These cultures were incubated at 36 °C for 36 h. JE10830 was highly sensitive to eosin Y and SDS; the minimum concentration of eosin Y and SDS inhibiting colony-forming ability at 36 °C in JE10830 was 0·1% and 1%, respectively, whilst that in PA3092 was more than 0·5% and 4%, respectively. The mutant was less sensitive to EDTA than the wild-type strain; JE10830 was sensitive up to 10 mM EDTA while PA3092 was sensitive up to 8 mM. On the contrary, the colony-forming ability of JE10830 was enhanced by the addition of methylene blue; JE10830 colony size increased with the addition of 0·01% methylene blue whilst PA3092 colony size decreased compared to the control at 36 °C. Although JE10830 did not grow on LB medium without NaCl at 36 °C, the addition of methylene blue restored growth under these conditions. Sensitivity to hydrophilic materials, such as kanamycin, was not altered by the mutation. These results suggest that the kdsA mutation may cause instability in membrane structures, possibly affecting gene expression.
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The kdsA mutations affect FtsZ-ring formation but not the production of FtsZ
The seven mutants examined in this study did not demonstrate constriction at the potential division site (Fig. 2), suggesting cell division was inhibited at an early step. We therefore analysed the effect of the fts830 mutation on FtsZ-ring formation by immuno-fluorescence microscopy using an anti-FtsZ antibody. As shown in Fig. 8
, although the cell length of JE10830 continued to increase after temperature shift, the FtsZ ring increased marginally. After 3 h incubation at 41 °C, 65% of the cells contained only one FtsZ ring at one end of the filaments and the remaining 35% did not have an FtsZ ring. The ftsI mutant, RP41, contained FtsZ rings, forming at almost all the potential division sites under the same experimental conditions. Therefore, the absence of FtsZ rings in JE10830 filaments is specific to the mutation.
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DISCUSSION |
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We concluded that instability of the outer membrane, due to a defect in the synthesis of complete LPS molecules, affects cell division. Complete LPS molecules may be required for some essential physiological function of the outer membrane or assembly of a functional outer membrane. Rick & Osborn (1977) also demonstrated in S. typhimurium that a defect in KDO synthesis causes pleiotropic effects on growth, LPS synthesis and accumulation of lipid A precursor in the isolated cytoplasmic membrane (Osborn et al., 1980
). A defect in htrB, encoding KDO-dependent lauroyltransferase which acts after KDO addition during lipid A biosynthesis (Clementz et al., 1996
), also inhibits cell division, producing filamentous cells at the non-permissive temperature, and inhibition of cell division of the htrB mutant is suppressed by msbA which may encode an ATP-dependent translocator (Karow & Georgopoulos, 1993
). It is proposed that accumulation of lipid A precursors is toxic to cell growth (Rick & Young, 1982
) and that enhanced export of these precursors mediated by extra copies of msbA may permit cell growth (Zhou et al., 1998
). The defect in cell division of JE10830 is also suppressed by the presence of extra copies of msbA. The defect in colony-forming ability, however, was not suppressed at all. Moreover, a mutant of lpxA, functioning at the first stage in lipid A biosynthesis to affect LPS synthesis (Raetz, 1986
), also inhibits cell division in E. coli, producing short filamentous cells, although the growth was very poor at the restricted temperature. Our results, therefore, demonstrate that the inhibition of cell division by the kdsA mutation is not caused by the accumulation of lipid A in the inner membrane. It seems more likely that the presence (or indeed the transport) of the lipid A precursor to the outer membrane may be essential for the stability of this membrane and that its instability in turn affects cell division.
The loss of LPS during KDO deficiency destabilizes the membrane structure, presumably causing large abnormalities in both the localization of membrane proteins and cell division. We observed a deficiency of FtsZ rings in the JE10830 filaments, although the levels of FtsZ were not affected by the mutation. The FtsZ ring, however, is constructed onto the inner membrane, maintaining a position at the leading edge of the invaginating septum (Bi & Lutkenhaus, 1991 ; Sun et al., 1998
) and the protein(s) governing this cell division at the outer membrane have not been found as yet. The mechanism whereby the instability of the outer membrane results in the loss of FtsZ-ring formation, therefore, remains to be established.
One of the possible explanations is that the instability of the outer membrane might affect the transcription of a cell division gene(s), required for FtsZ-ring formation. The outer membrane functions to communicate information received from the external environment into the cell. Therefore, membrane instability is expected to affect the transcription of genes, including those involved in cell division. We analysed the effect on membrane stability by examining the sensitivity of mutants to various hydrophobic materials. The results demonstrated that although the mutants were more susceptible to various hydrophobic materials, some hydrophobic materials restored kdsA mutant growth, indicating that the instability of the outer membrane creates the altered cellular responses to the environment. Null mutations in either htrB or msbB also enable growth on four times the concentration of deoxycholate relative to wild-type bacteria (Karow & Georgopoulos, 1993 ). Destabilization of the outer membrane may result in the expression of a subset of genes overcoming the sensitivity to the hydrophobic materials.
LPS comprises approximately 30% of the outer membrane in gross weight (Leive, 1974 ). Therefore, decreases in LPS may destabilize the outer membrane and may expose phospholipids to the external environment. Phospholipids are integral in the regulation of cell cycle, and interruptions of phospholipid synthesis inhibit both the initiation of chromosome replication and cell division (Norris, 1989
). In addition, the physical nature of phospholipids changes dramatically with various growth conditions (Tilcock, 1986
). Phospholipids are also important in the transfer of signals from the extracellular environment to the replication and transcription machinery (Sekimizu, 1994
). Therefore, loss of LPS may change the physical nature of phospholipids due to exposure to their surroundings, altering the expression of various genes. In S. typhimurium, the rate of OmpA synthesis is proposed to be activated by the kdsA mutation (Rick et al., 1983
, 1984
), although the mechanism of this activation is not well understood.
All six mutations analysed in this article concerned non-polar amino acids of which four were replaced by polar ones. Mutations substituting these amino acids inhibited cell division severely, except fts1167. Recently, the crystal structure of KdsA was determined by Radaev et al. (2000) . The unit cell of the crystal consists of a homotetramer of KdsA. The six mutation sites are not localized in space, but are scattered throughout the three-dimensional structure: two sites, Leu 118 (fts705) and Ala 227 (fts1212), were located on the monomermonomer interfaces making contacts between the same residues, LeuLeu and AlaAla, of different subunits, and thus might affect the stability of tetramer complexes. Ala 203 (fts446) is close to His 202, which is one of the binding sites of phosphoenolpyruvate (Radaev et al., 2000
), and thus might disturb catalytic activity of KdsA. Gly 73 (fts830 and fts1241), sitting at the start of an
-helix, has a positive phi-angle, which is allowed for Gly but may not be adequate for other amino acids such as Asp. Ala 14 (fts1167) and Ala 234 (fts1171) are close to each other in space, suggesting similar effects if any, whereas we cannot see any definite reason for instability caused by mutations at these sites. These data suggest that the reduction of catalytic activity of KdsA might result from the instability of three-dimensional structures at high temperature, rather than any direct effects on the active centre of the enzyme.
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ACKNOWLEDGEMENTS |
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Received 25 May 2001;
revised 7 August 2001;
accepted 25 September 2001.