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Journal of Bacteriology, November 2008, p. 7298-7301, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00551-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Involvement of an Essential Gene, mviN, in Murein Synthesis in Escherichia coli ^,

Azusa Inoue,¹ Yoshimitsu Murata,² Hiroshi Takahashi,² Naoko Tsuji,¹ Shingo Fujisaki,² and Jun-ichi Kato^1*

Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan,¹ Department of Biomolecular Science, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan²

Received 22 April 2008/ Accepted 4 August 2008

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We isolated a temperature-sensitive mutant with a mutation in mviN, an essential gene in Escherichia coli. At the nonpermissive temperature, mviN mutant cells swelled and burst. An intermediate in murein synthesis, polyprenyl diphosphate-N-acetylmuramic acid-(pentapeptide)-N-acetyl-glucosamine, accumulated in mutant cells. These results indicated that MviN is involved in murein synthesis.

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The mviN gene was first identified in Salmonella enterica serovar Typhimurium as a gene in a chromosomal region required for virulence in a mouse model of typhoidlike disease (1, 3). One of the putative virulence genes in this region was designated mviS (mouse virulence Salmonella). Subsequently, mviS was identified as fliA, a gene that encodes a sigma factor necessary for expression of the flg operon, which includes 14 genes that are essential for flagellum synthesis (2, 3, 5, 14). Thus, although the original terminology has been maintained, there is no direct evidence that mviN is involved in virulence, and its function has not been fully elucidated.

In our lab, we are carrying out systematic construction of Escherichia coli long-range chromosomal deletion mutants (7, 12). Previously, we reported that the chromosomal region containing mviN is essential for cell growth. Viable mutants having a deletion of this chromosomal region were isolated only in the presence of a mini-F plasmid carrying the rimJ-yceH-mviM-mviN locus (12). To determine whether mviM and mviN are essential genes, we constructed mviM::Cm and mviN::Cm disruptants in the presence or absence of the complementing rimJ-yceH-mviM-mviN mini-F plasmid (see the supplemental material for details). The deletion mutants were used as donors in P1 phage-mediated transduction, and the gene disruptions were introduced into strains that contained either the complementing mini-F plasmid or a vector control. Chloramphenicol-resistant mviM deletion mutants were obtained in the presence or absence of the complementing mini-F plasmid. However, chloramphenicol-resistant mviN deletion mutants could grow only in the presence of the complementing plasmid and not in the presence of the empty control vector. These results indicated that mviN, but not mviM, is essential for growth.

To further characterize the function of mviN, we isolated a temperature-sensitive (TS) mviN mutant, designated mviN^ts555, by plasmid shuffling, which is a kind of localized mutagenesis performed using a mini-F plasmid (9, 10, 11). The growth of the TS mutant at the nonpermissive temperature was restored by pBAD-mviN, which confirmed that mviN^ts555 is an mviN mutant (see the supplemental material for details). Sequence analysis of mviN^ts555 revealed a deletion in the mviN upstream region (base pairs 1,125,808 to 1,127,003 of the E. coli genome), which included the 3' region of yceH, mviM, and a predicted promoter of the mviN gene (based on the E. coli chromosome database PEC [http://www.shigen.nig.ac.jp/ecoli/pec/index.jsp]). We did not find any mutations in the coding region of mviN, which suggests that the TS phenotype is due to decreased mviN expression (Fig. 1).

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FIG. 1. Sequence of the mviNts555 TS mutation. mviNts555 mutants had a deletion that extended from the NruI site of yceH (nucleotide 429 of the open reading frame) to the upstream region of mviN (nucleotide –57 relative to the initiation codon at nucleotide 1). Lowercase letters indicate open reading frame sequences. The termination codon of yceH and the initiation codon of mviN are underlined. The underlined uppercase letters indicate the promoter of mviN.

When we examined the mutant cells using microscopy after incubation at the nonpermissive temperature, the cells appeared to be swollen, and many cells had burst to form ghosts (Fig. 2B). These results suggest that there was a defect in the rigidity of the cell wall. To determine whether the defect involved impaired peptidoglycan synthesis, we introduced a multicopy plasmid carrying the ispU (rth) gene, which encodes an undecaprenyl diphosphate synthase involved in peptidoglycan synthesis, into mviN TS mutants (10). Expression of ispU suppressed the growth defect of mviN^ts555 cells at 42°C, indicating that mviN is involved in peptidoglycan synthesis, but it did not do this in the presence of the vector control.

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FIG. 2. Cell morphology of mviNts555. (A) Wild-type mviN+ strain [strain MG1655 mviN::Sm recA::Tn10/mini-F (Apr)-mviN+]; (B) TS mviNts555 mutant [strain MG1655 mviN::Sm recA::Tn10/mini-F (Apr)-mviNts555]. Cells were incubated at 42°C for 2 h and observed without fixation by using phase-contrast microscopy. Magnification, x100. The arrows indicate ghosts.

We next examined the levels of cell wall biosynthesis intermediates in mviN mutants. Recently, Guan et al. reported that cell wall biosynthesis lipid intermediates, including polyprenyl diphosphate-N-acetylmuramic acid—(pentapeptide)-N-acetylglucosamines (a mixture of decaprenyl and undecaprenyl derivatives), were extracted specifically from an acidified mixture into chloroform (6). We labeled mutant and wild-type cells with [³H]diaminopimelic acid (DAP), which is incorporated predominantly into the cell wall (see the supplemental material for details) and then measured the levels of radioactivity in various fractions. The incorporation of radioactive DAP into the lipid intermediates of mutant cells was markedly greater than the incorporation of radioactive DAP into the lipid intermediates of wild-type cells (Table 1). The ratio of lipid intermediates to the total compounds was four times higher in mutant cells than in wild-type cells, while the ratio of radioactivity in the cell wall fraction was lower in mutant cells than in wild-type cells. We next cultivated cells in the presence of [¹⁴C]isopentenyl diphosphate (IPP) to label the polyprenyl moiety of the lipid intermediates (see the supplemental material for details). Because IPP is not incorporated into untreated growing cells, [¹⁴C]IPP was added to lyophilized cells, and then the cells were recultivated. Radiolabeled polyprenyl phosphate derivatives were extracted from neutral and acidified mixtures with chloroform, and polyprenyl phosphates and polyprenyl diphosphates that were extracted from neutral mixtures were separated by ion-exchange chromatography (Table 2). The ratio of the radioactivity of lipid intermediates to the total radioactivity for all the polyprenyl phosphate derivatives was higher in mutant cells than in wild-type cells. Thus, based on the results of two separate labeling experiments, in which we labeled pentapeptide moieties by using [³H]DAP and polyprenyl moieties by using [¹⁴C]IPP, the lipid intermediates accumulated at higher levels in mutant cells than in wild-type cells. These results indicated that the mviN gene is involved in metabolism of the lipid intermediates of peptidoglycan synthesis.

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TABLE 1. Incorporation of [3H]DAP into the cell wall and cell wall synthesis intermediatesa

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TABLE 2. Incorporation of [14C]IPP into polyprenyl phosphate derivativesa

What is the function of MviN in cells? We analyzed the putative amino acid sequence of MviN with Pfam and SOSUI and found that the protein has 14 transmembrane domains, which indicates that MviN is an integral membrane protein. The protein also appeared to be a member of the "MviN MATE (multi-antimicrobial extrusion)-like superfamily," which is comprised of integral membrane proteins. Members of the MATE family have been shown to function as drug/sodium antiporters (15). These proteins have also been shown to mediate resistance to a wide range of cationic dyes, fluoroquinolones, aminoglycosides, and other structurally diverse antibodies and drugs (15). MATE proteins have also been implicated in the production of polysaccharides, such as RfbX (Wzx) (4) and WzxE (17), which have been implicated in E. coli O-antigen biosynthesis; Bacillus subtilis SpoVB, which is involved in spore cortex biosynthesis (16, 19); and eukaryotic RFT1, which is required for translocation of the dolichyl diphosphate-Man₅GlcNAc₂ intermediate, an oligosaccharide complex used in protein glycosylation, from the cytosolic side of the endoplasmic reticulum membrane to the lumen during the biosynthesis of dolichyl diphosphate-Glc₃Man₉GlcNAc₂ (8). These results suggest that MviN may be involved in the transmembrane transport of peptidoglycan precursors across the inner membrane. This transport process was recently reconstituted in vitro (18), which should enable further characterization of the function of MviN. Alternatively, MviN may be indirectly involved in peptidoglycan synthesis. For example, it may play a role in folding or localization of other proteins involved in peptidoglycan synthesis because the precursors have been shown to accumulate by a wide variety of treatments that inhibit peptidoglycan synthesis or that block cell division (13). Further analyses are necessary to clarify its specific role.

 
This work was supported by KAKENHI (grant-in-aid for scientific research on priority areas "Systems Genomics" from the Ministry of Education, Culture, Sports, Science and Technology of Japan).

 
* Corresponding author. Mailing address: Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan. Phone: 81-42-677-2569. Fax: 81-42-677-2569. E-mail: jkato{at}tmu.ac.jp

Published ahead of print on 15 August 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, November 2008, p. 7298-7301, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00551-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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• Fay, A., Dworkin, J. (2009). Bacillus subtilis Homologs of MviN (MurJ), the Putative Escherichia coli Lipid II Flippase, Are Not Essential for Growth. J. Bacteriol. 191: 6020-6028 [Abstract] [Full Text]  
• Ruiz, N. (2009). Streptococcus pyogenes YtgP (Spy_0390) Complements Escherichia coli Strains Depleted of the Putative Peptidoglycan Flippase MurJ. Antimicrob. Agents Chemother. 53: 3604-3605 [Full Text]  
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