![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Department of Biochemistry, University of Oxford, Oxford, United Kingdom
Received 7 April 2004/ Accepted 1 June 2004
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In bacteria, the peptidoglycan layer principally determines cell shape. Peptidoglycan consists of glycan strands cross-linked by peptide side chains. The peptidoglycan subunits, disaccharide peptides, are assembled in the cytoplasm and linked to the preexisting peptidoglycan layer in the periplasm by transglycosylase and transpeptidation reactions. In E. coli two distinct phases of peptidoglycan synthesis occur. In cells undergoing septation the transpeptidase FtsI, in concert with the integral membrane protein FtsW, mediates the insertion of peptidoglycan that forms the new poles of the daughter cells (6). In elongating cells the transpeptidase penicillin-binding protein 2 (PBP2) and RodA function in the peptidoglycan-driven extension of the longitudinal axis (3).
Cell shape mutants typically possess mutations in genetic loci that encode proteins involved in peptidoglycan synthesis. The effects of mreB deletion in E. coli and MreB depletion in B. subtilis suggest a role for MreB in peptidoglycan synthesis (11, 12). However, these studies have also been used to suggest that MreB may define rod-shaped morphology, possibly by forming a structural brace (5), while additional studies suggest that MreB may function in chromosome segregation (12, 24). Thus, the role performed by MreB is at present uncertain and may vary between species.
Rhodobacter sphaeroides is an 
-subgroup, purple nonsulfur photosynthetic bacterium which displays different cellular morphologies under different growth conditions (2). Aerobically grown cells are rod shaped, while photoheterotrophically grown cells are coccobacillus shaped. The extent of the coccobacillus morphology is dependent on the available light, with low light levels producing a more pronounced spherical shape. Associated with the various cell shapes is an alteration in the structure of the cytoplasmic membrane. In aerobically grown cells the cytoplasmic membrane is smooth, whereas in photoheterotrophically grown cells the cytoplasmic membrane invaginates, forming the support for the photosynthetic apparatus. The extent of the invagination is dependent on the available light, with low light levels producing a very extensively invaginated cytoplasmic membrane.
The aims of this study were to determine the cellular requirement for MreB and its role in rod- and coccobacillus-shaped R. sphaeroides cells.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
was used for all molecular cloning, strain S17-1 
pir was used for conjugal transfer into R. sphaeroides, and strain M15 pREP4 was used for protein expression. E. coli strains were cultured in Luria-Bertani medium at 37°C with shaking. Antibiotics were used at 25 µg ml–1 for kanamycin, nalidixic acid, and streptomycin and at 100 µg ml–1 for ampicillin.
|
Molecular genetic techniques. All cloning steps were performed as described by Sambrook and Russell (18). Sequencing-quality DNA was prepared using the WizardPlus kit (Promega), sequenced by the University of Oxford Biochemistry sequencing service, and analyzed with the GCG software package (University of Wisconsin). All primers were supplied by Genosys Biotechnologies Inc.
Cell scoring.
Images were acquired of wild-type cells grown aerobically or photoheterotrophically, at different light intensities, by differential interference contrast (DIC) microscopy using a Nikon TE200 microscope with a cooled charge-coupled device (CCD) camera (Hamamatsu). Cells were scored as newly divided (septation clearly just completed), elongating (length extension occurring, no septum invagination visible), septation initiated (length extended, septum invagination beginning), or septation nearing completion (septum deeply invaginated, daughter cells clearly forming). Following classification the cell lengths and cell widths were measured using SimplePCI image analysis software (Digital Pixel). A total of 
25 cells were analyzed, from 5 fields of view, for each cell cycle stage. The images were processed with SimplePCI image analysis software (Digital Pixel).
For scoring green fluorescent protein (GFP)-MreB-expressing cells, images were acquired as outlined above. For wild-type populations the scoring system was identical to that described above. For GFP-MreB-expressing populations, cells were scored as normal (cell length and cell width similar or identical to wild-type cells), mildly abnormal (cell length and/or cell width varying in comparison to wild type), or severely abnormal (cell length and/or cell width varying extensively in comparison to wild type). The normal, mildly abnormal, or severely abnormal cells were grouped separately into the outlined cell cycle stage classifications. Following classification the cell lengths and cell widths were measured using SimplePCI image analysis software (Digital Pixel). For the wild-type cells, a total of 25 cells were analyzed, from 5 fields of view, for each cell cycle stage. For GFP-MreB-expressing populations a total of 25 normal, mildly abnormal, or severely abnormal cells were analyzed, from 10 fields of view, for each cell cycle stage. The images were processed with SimplePCI image analysis software (Digital Pixel).
Protein expression construct. mreB was amplified by PCR using primers that omitted the start codon and included 5' BamHI and 3' HindIII sites. The PCR product was ligated into pQE80 (QIAGEN) to produce pMLP1. The construct was sequenced to ensure the coding sequence contained no errors.
Protein purification and antibody production. His-tagged MreB was expressed in E. coli M15 pREP4 cells containing pMLP1. Purification was performed as described previously (14). A rabbit antibody was raised against purified MreB (Eurogentec).
Cell fractionation. Cell fractionation was performed as described previously (31) with the following modifications. A 100-ml culture of aerobically grown R. sphaeroides at an optical density at 700 nm of 0.6 was harvested by centrifugation. The cells were spheroplasted by resuspension in 1 ml of 0.15 M Tris-HCl (pH 8.0) containing 0.5 M sucrose, 1 mg of lysozyme, and 10 mM EDTA. The spheroplasts were harvested by centrifugation, gently lysed by the addition of 5 ml of water, and kept on ice for 3 h with gentle mixing every 30 min. Centrifugation at 25,000 x g for 30 min removed cell debris, and centrifugation at 100,000 x g for 120 min pelleted the cell membranes. The cytoplasmic fraction was decanted and stored. The cell membrane pellet was resuspended in water, in a volume equivalent to that of the cytoplasmic fraction, and stored.
Immunoblotting. Samples of membrane and cytoplasmic fractions were mixed with an equal volume of 2x sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer. Samples were run on a 12% polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Bio-Rad) by electroblotting. The polyvinylidene difluoride membranes were blocked overnight at room temperature. MreB antibodies were added at a 1:2,000 dilution in blocking solution and incubated at room temperature for 1 h. Anti-rabbit-horseradish peroxidase conjugate (DAKO) was added at a 1:1,000 dilution in blocking solution, and the membrane was incubated as above. Membranes were washed with phosphate-buffered saline (PBS) and 0.1% (vol/vol) Tween. An enhanced chemiluminescence kit (Amersham) was used for detection.
Deletion constructs.
An mreB in-frame deletion construct was generated by amplifying, by PCR, a 0.5-kb region immediately upstream of mreB with primers that encompassed the start codon and included 5' EcoRI and 3' BamHI sites. A 0.5-kb region that included the seven 3' codons and the downstream flanking DNA of mreB was amplified by PCR using primers that included 5' BamHI and 3' HindIII sites. The first PCR product was ligated into pK18mobsacB to produce pPKS1. The second PCR product was ligated into pPKS1 to generate the final construct, pPKS2. An mreB insertional inactivation construct was generated by excising the omega cartridge from pHP45
with BamHI and cloning this fragment into BamHI-cut pPKS2 to produce pPKS2. Constructs were sequenced to ensure that upstream and downstream regions were in frame and contained no errors. The constructs were introduced into R. sphaeroides by allelic exchange as described previously (8, 19).
Fixation and permeabilization. Cells were fixed and permeabilized as described previously (9) with the following modifications. A 0.5-ml volume of bacterial culture was mixed with an equal volume of concentrated fixative solution. Cells cultured in succinate medium were fixed with a final concentration of 1.3% (vol/vol) paraformaldehyde and 0.02% (vol/vol) glutaraldehyde in PBS (pH 7.4) for 15 min at room temperature and for 30 min on ice. The fixed bacteria were washed three times in PBS at room temperature and then resuspended in 200 µl of glucose-Tris-EDTA. A freshly prepared lysozyme solution, in PBS, was added to a final concentration of 2 mg ml–1. Samples (25 µl) were immediately distributed onto wells of a multiwell microscope slide (Hendley-Essex) which had been treated with 0.01% (wt/vol) poly-L-lysine (Sigma). After 2 min the liquid was aspirated from the wells, which were then allowed to dry for 4 min. A 25-µl volume of PBS was added to each well for 1 min and then removed by aspiration. A 25-µl volume of blocking solution (5% bovine serum albumin in PBS) was added to each well, and the slides were incubated for 30 min at room temperature.
Immunofluorescence staining. Immunofluorescence staining was performed as described previously (9) with the following modifications. Cells were incubated with a 1:10,000 dilution in blocking solution of rabbit polyclonal anti-MreB antibodies overnight at 4°C. The wells were washed 20 times with blocking solution. Cells were then incubated with a 3:1,000 dilution in blocking solution of fluorescein isothiocyanate-anti-rabbit antibodies (Sigma) for 3 h at room temperature in the dark. The cells were washed 10 times with blocking solution and 10 times with PBS. Slides were mounted with 2 µl of Vectashield antifade (Vector Laboratories).
Construction of a gfp-mreB fusion strain. A 0.5-kb region immediately upstream of mreB was amplified by PCR using primers that omitted the start codon and included a 5' EcoRI site and a 3' sequence complementary to the beginning of egfp. The 0.8-kb egfp was amplified by PCR using primers that omitted the stop codon and included a 5' sequence complementary to the 15-bp region immediately upstream of mreB and a 3' XbaI site. These PCR products were used as templates for further PCR, using the 5' primer from the mreB upstream PCR and the 3' primer from the egfp PCR, to generate a 1.3-kb fragment. The PCR product, upstream mreB fused to egfp, was ligated into pK18mobsacB to produce pBS1. A 0.5-kb region immediately downstream of the mreB start codon was amplified by PCR using primers that included 5' XbaI and 3' HindIII sites. This PCR product was ligated into pBS1 to generate the final construct pBS2. The construct was sequenced to ensure that upstream and downstream regions were in frame and contained no errors. The construct was inserted into R. sphaeroides by allelic exchange as described previously (8, 19).
Fluorescence analysis. For immunofluorescence microscopy, DIC and fluorescence images were acquired using a Nikon TE200 microscope with a GFP filter set and recorded with a cooled CCD camera (Hamamatsu). Z-series were captured with a custom-built Piezo driven (Physik Instruments) mechanical stage. The images were processed with SimplePCI image analysis software (Digital Pixel).
For GFP-MreB localization in log phase, cultures were embedded in 1.2% agarose on microscope slides. Phase-contrast and fluorescence images were acquired using a Zeiss Axiovert 135TV microscope with a fluorescein isothiocyanate filter set and recorded with a cooled CCD camera (Digital Pixel Advanced Imaging Systems). The images were processed with image analysis software (METAMORPH 6.1).
For GFP-MreB localization through the cell cycle, log-phase cultures were embedded in 0.8% succinate agarose on microscope slides. DIC and fluorescence images were acquired as described for immunofluorescence microscopy at 20-min intervals. The images were processed with SimplePCI image analysis software (Digital Pixel).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The cell width remained fairly constant (1.14 to 1.20 µm), while the cell length changed significantly under different growth conditions (Table 2). The longest cell lengths were observed in aerobic cells. The shortest cell lengths were seen in photoheterotrophic cells grown under low light intensities, with intermediate cell lengths observed in photoheterotrophic cells grown under much higher light intensities. These data demonstrate that the previously observed differences in R. sphaeroides morphologies grown under different conditions (2) result from an alteration in cell length rather than a modification of the cell width.
|
|
R. sphaeroides MreC, MreD, PBP2, and RodA share 27, 13, 36, and 45% identity with the E. coli proteins, respectively, and 28, 11, 23, and 28% identity with the B. subtilis proteins, respectively. Hydropathy profiles (TMHMM and TMPred) predicted the presence of one N-terminal transmembrane -helix in both MreC and PBP2 and multiple transmembrane -helices in both MreD and RodA. The levels of identity and predicted membrane topologies of the R. sphaeroides proteins are similar to those previously observed between the bacterial paradigms and other bacterial species (1).
MreB is a cytoplasmic protein. The cellular localization of MreB was determined by cell fractionation and Western blotting. MreB localizes just below the cytoplasmic membrane in E. coli and B. subtilis (11, 20). MreB may either associate with the cytoplasmic membrane through inherent properties of the protein or localization may depend upon additional proteins. R. sphaeroides spheroplasts were gently lysed, and the membrane and cytoplasmic fractions were subjected to Western blotting.
R. sphaeroides MreB localized to the cytoplasmic fraction (data not shown), confirming the in silico prediction of MreB as a soluble, cytoplasmic protein. This suggests MreB is only weakly associated with the cytoplasmic membrane.
MreB appears essential for R. sphaeroides viability. The cellular requirement for MreB, and also the morphological defects associated with MreB loss, varies among bacterial species. In E. coli MreB is nonessential, as deletion of the gene produces a rounded morphology (12). In contrast, MreB appears essential for B. subtilis and Caulobacter crescentus viability, as MreB depletion produces abnormal morphologies and these aberrant cells eventually lyse (1, 7, 11). Given the more coccoid shape of photoheterotrophically grown R. sphaeroides, it seemed possible that these cells might not require MreB. Photoheterotrophic cells also show extensive invaginations of the cytoplasmic membrane and so might be unable to maintain a subcellular MreB structure. mreB deletion studies were attempted in R. sphaeroides grown aerobically or photoheterotrophically.
Attempts at mutating R. sphaeroides mreB by both in-frame deletion and insertional inactivation, under either aerobic or photoheterotrophic growth conditions, proved unsuccessful. These strategies have been routinely used to mutate nonessential genes in this bacterium (16), suggesting that MreB is essential in R. sphaeroides under both aerobic and photoheterotrophic growth conditions.
MreB depletion analysis in R. sphaeroides or creation of an mreB merodiploid strain was not possible due to the lack of an inducible expression system in this bacterium.
MreB localization in aerobic and photoheterotrophic cells. MreB localization varies among bacterial species. In E. coli MreB localizes in a helical configuration that spans the longitudinal axis of the cell and as a transverse band at mid-cell (20). In B. subtilis MreB localizes in a helical configuration mainly at mid-cell (11). As the cell shape and cytoplasmic membrane structure of R. sphaeroides changes under different growth conditions, immunofluorescence studies were carried out on R. sphaeroides cells grown aerobically and photoheterotrophically to determine MreB localization under conditions where the cell shape and the structure of the cytoplasmic membrane change.
In aerobically grown cells MreB localized predominantly as a transverse band or as opposing foci (Fig. 2A, row a). In addition patterns suggestive of a helical configuration, consisting of a transverse band and an adjacent focus, were observed in some cells (Fig. 2A, row a). In newly formed cells, opposing foci and transverse bands were observed slanting across the cell (Fig. 2A, set I in row b). In elongating cells transverse bands were observed at mid-cell (Fig. 2A, set II in row b). In cells beginning septation, opposing foci were observed at the site of septation (Fig. 2A, set III in row b). The localization at mid-cell appeared to break down before the completion of septation (Fig. 2A, set IV in row b), the localization at mid-cell being lost and reforming at mid-cell of the daughter cell. The localization pattern observed in R. sphaeroides suggests that MreB undergoes dynamic subcellular rearrangements during the cell cycle and that the protein may function at mid-cell with a possible role in early septation.
|
MreB localization was similar in R. sphaeroides cells grown photoheterotrophically under either high or low light intensities. At low light intensities the cytoplasmic membrane of R. sphaeroides invaginates extensively to maximize the light-capturing and photosynthetic capacity of the photosynthesis apparatus (2). The MreB localization in cells grown under low light intensities (Fig. 2C) was similar to that found in both photoheterotrophic cells grown under much higher light intensities and aerobic cells. The similar pattern of fluorescence suggests MreB performs the same role under these different growth conditions and again suggests that MreB does not form a subcellular structure defining R. sphaeroides cell length. Furthermore, MreB localization appears to be independent of the extensive cytoplasmic membrane rearrangements and consequent reorganization of the cytoplasm that occurs when cells are grown under low light conditions.
MreB forms a ring. Three-dimensional (3D) reconstructions of MreB in both E. coli and B. subtilis showed that the protein forms a helix in these bacteria (11, 20). 2D immunofluorescence localization of MreB in R. sphaeroides suggested that the protein forms a ring and/or a possible helix and, therefore, 3D reconstruction was carried out to establish which structure was formed.
3D reconstructions showed that the MreB subcellular structure was a ring (Fig. 3A). Images of an aerobic cell, including an MreB transverse band, were acquired (Fig. 3A, row a), and subsequently a z-series was captured. Deconvolution of these fluorescent images revealed a single focus at both the beginning and end of the z-series (Fig. 3A, row b) and two foci in the middle (Fig. 3A, row b). 3D reconstruction of the deconvolved z-series revealed an MreB ring (Fig. 3A, row c; see supplemental data).
|
MreB localization in filamentous cells. The antibiotic cephalexin inhibits FtsI, the septum-specific transpeptidase, resulting in the formation of filaments as the cells are unable to divide. In filamentous cells septation complexes based upon the essential septation protein FtsZ form at mid-cell and at other putative septation sites. Localization to these septation sites has previously been used to suggest protein involvement in septation.
In filamentous R. sphaeroides cells, MreB localized to both mid-cell and putative septation sites (Fig. 4). Transverse bands were observed at mid-cell (Fig. 4) with apparent helical configurations but, as above, probably represented a slanted ring and a disconnected focus (Fig. 4A) and transverse bands (Fig. 4B) also seen alongside this mid-cell localization. The localization to mid-cell and putative septation sites presumably reflect the assembly of septation complexes prior to, and following, the addition of cephalexin, respectively. This suggests that MreB in R. sphaeroides may be part of the septation complex.
|
Populations of GFP-MreB-expressing cells contained both normal- and abnormal-shaped cells (Fig. 5). Normal-shaped cells showed at worst a modest increase in cell width (
9%) in comparison to wild type. These cells accounted for 49% of the population. Abnormal-shaped cells varied in their degree of abnormality. In mildly abnormal cells, the cell width increased (20%) in comparison to wild type. In severely abnormal cells, both the cell width (33%) and cell length (23%) increased in comparison to wild type. These changes were observed at all stages of the cell cycle. A few severely abnormal cells showed a decrease in cell length (12%) in comparison to wild type (data not shown). The peptidoglycan layer ultimately governs the cell shape of bacteria. Thus, the abnormal morphologies in GFP-MreB-expressing populations suggest that MreB may be involved in the cytoplasmic control of peptidoglycan synthesis.
|
|
Wild-type and GFP-MreB cells bulged at mid-cell in the presence of amdinocillin (Fig. 7). GFP-MreB was observed at these swollen sites as transverse bands or opposing foci close to the cell periphery. The maintenance of GFP-MreB localization in cells with aberrant morphologies again suggests that the MreB subcellular structure does not define R. sphaeroides morphology but may have a role in septation or peptidoglycan insertion.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The requirement for MreB varies among bacterial species. In E. coli mreB deletion produces spherical bacteria (12), whereas in S. coelicolor the gene cannot be deleted (1) and in B. subtilis and in C. crescentus MreB depletion causes cells to swell and lyse (7, 11). R. sphaeroides MreB also appears to be essential, as we were unable to delete or insertionally inactivate mreB using techniques standard for this species. This was supported by the abnormal cell shapes observed in some cells in which mreB was replaced with gfp-mreB and the reduced growth rate of this strain. The different requirement for MreB between the gram-negative E. coli and gram-positive B. subtilis and S. coelicolor had been attributed to differences in the cellular envelopes of these species. However, as MreB also appears to be essential in the gram-negative R. sphaeroides and C. crescentus, the underlying principle governing MreB requirement in bacteria is therefore uncertain.
R. sphaeroides MreB localized predominantly as a transverse band at mid-cell, and 3D reconstruction showed that it formed a ring. 2D immunofluorescence images also showed foci in some cells that could reflect a helical structure, but 3D reconstruction showed that these foci represented a slanted ring and disconnected foci rather than a helix. The disconnected foci may represent past or future sites of MreB localization; the protein may be in transit to its mid-cell destination. The localization and subcellular structure of MreB appears to vary between bacterial species. In E. coli and C. crescentus MreB localizes in both a helical configuration that spans the longitudinal axis of the cell and as a transverse band at mid-cell (7, 20). In B. subtilis MreB localizes as a helix, mainly at mid-cell (11). The mid-cell MreB localization is conserved among both gram-negative and gram-positive bacteria, suggesting that, although the subcellular structure may vary, MreB performs a similar role at this site in all bacteria possessing this protein.
R. sphaeroides MreB localization appears characteristic of a septation protein. MreB localized at mid-cell in elongating cells and remained at this site, i.e., the site of septation, as septation began. Furthermore, the protein formed a ring at mid-cell, and the localization pattern in filamentous cells was characteristic of septation proteins. MreB localization at mid-cell has also been observed in E. coli, B. subtilis, and C. crescentus (7, 11, 20); furthermore, in C. crescentus this localization depends upon the tubulin homolog FtsZ (6, 7). Early in the cell cycle, FtsZ localizes to mid-cell and forms a ring at the future site of septation termed the Z-ring. Proteins of the division complex assemble in a specific sequence to the Z-ring, forming the so-called divisome, which is capable of cytokinesis. MreB may therefore be part of the divisome. The putative role for the bacterial actin homolog MreB at the site of septation extends the functional relationship between prokaryotic MreB and eukaryotic actin by including a shared role in cytokinesis in their respective kingdoms.
In R. sphaeroides the mre locus was found to overlap with the mrd locus, and the partially functional GFP-MreB fusion resulted in abnormalities in cell shape. Thus, in R. sphaeroides, MreB may function in the cytoplasmic control of the peptidoglycan-synthetic complexes that ultimately govern cell shape. In E. coli mreB deletion produces spherical cells, i.e., there is no extension of the longitudinal axis (12), and mre mutations are associated with altered sensitivities to the PBP2-specific amdinocillin (29). In B. subtilis and in C. crescentus MreB depletion produces abnormal cell shapes (11) similar to the morphologies created by PBP inhibition (4), and in C. crescentus PBP2 localization is dependent upon MreB (7). The accumulated experimental data suggest that MreB plays a role in the cytoplasmic control of the synthetic complexes that form the peptidoglycan layer, which ultimately governs cell shape.
In B. subtilis the MreB localization and depletion studies led to the proposal that the protein also forms a subcellular structure that also helps define the rod cell shape (11). In R. sphaeroides MreB localization was similar in both long aerobic cells and comparatively short photoheterotrophic cells and was predominantly localized at mid-cell. These data, combined with the potentially essential nature of the gene and the abnormal cellular morphologies seen in some cells expressing a GFP-MreB fusion, suggest MreB may function at mid-cell to regulate the synthesis and deposition of peptidoglycan, governing cell shape by controlling synthesis of the cell wall rather than by forming a static subcellular structure defining rod-shaped cells. Furthermore, the similar localization in both aerobic and photoheterotrophic cells, possessing different patterns of membrane invagination, suggests that the cytoplasmic membrane must be demarcated with specific regions prevented from forming invaginations to allow for housekeeping cellular functions.
The precise role of MreB remains uncertain. In R. sphaeroides, the experimental data are suggestive of an involvement in both early septation and peptidoglycan synthesis. The result associating MreB with peptidoglycan formation, i.e., the abnormal shapes in some cells expressing GFP-MreB, suggests that the principle function of MreB is at sites of peptidoglycan insertion. Consequently, the localization at mid-cell may reflect the fact that this region is the main site of peptidoglycan synthesis rather than suggesting a specific role for MreB in septation.
|
ACKNOWLEDGMENTS |
|---|
S. L. Porter, A. C. Martin, J. E. Errington, R. Carballido-López, and M. Leaver are thanked for help and suggestions, and M. de Pedro is thanked for the gift of amdinocillin.
|
FOOTNOTES |
|---|
Supplemental material for this article may be found at http://jb.asm.org/
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |
