Unité de Microbiologie et Génétique, UMR 5122 CNRS-INSA-UCBL, Université Claude Bernard Lyon I, bât. André Lwoff, 69622 Villeurbanne Cedex, France
Correspondence
Jean Claude Lazzaroni
lazzaron{at}biomserv.univ-lyon1.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession number for the nucleotide sequence reported in this paper, corresponding to a 6967 bp fragment, is AJ297885.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The transcriptional organization of the E. coli tol-pal genes has been characterized. The genes ybgC (orf1), tolQ, tolR, tolA and tolB, and pal and ybgF (orf2) form two operons (Muller & Webster, 1997); a large ybgCybgF transcript has also been postulated (Vianney et al., 1996
). ybgC and ybgF encode proteins of unknown function located in the cytoplasm and the periplasm, respectively (Clavel et al., 1996
; Sun & Webster, 1987
). Inactivation of these two ORFs induces no obvious phenotype in E. coli. In contrast, mutations in the tol-pal genes cause the disruption of outer-membrane integrity, which is evidenced by several phenotypes, including release of periplasmic content, sensitivity to bile salts and other chemical compounds, formation of outer-membrane blebs at the cell surface, and overproduction of colanic acid (Bernadac et al., 1998
; Clavel et al., 1996
; Vianney et al., 1994
). The tol-pal genes are also necessary for proper functioning of some uptake systems at the level of the cytoplasmic membrane (Llamas et al., 2003a
). In E. coli, group A colicins and filamentous-phage DNA use the Tol proteins for their translocation across the cell envelope (Bouveret et al., 1998
; Webster, 1991
). The existence of tol-pal genes has now been established in several Gram-negative bacteria (Bowe et al., 1998
; Dennis et al., 1996
; Heilpern & Waldor, 2000
; Llamas et al., 2000
; Prouty et al., 2002
; Youderian et al., 2003
). There is also evidence that some of the tol-pal genes are involved in the pathogenesis of E. coli (Hellman et al., 2002
), Haemophilus ducreyi (Fortney et al., 2000
), Salmonella enterica (Bowe et al., 1998
) and Vibrio cholerae (Heilpern & Waldor, 2000
). However, the way that the tol-pal genes are involved in the pathogenesis of these bacteria is not well documented, except in V. cholerae, where tolQRA mutants show defects in the uptake of ctx
DNA, which encodes cholera toxin (Heilpern & Waldor, 2000
), and in E. coli, where Pal initiates inflammation in sepsis, in synergy with lipopolysaccharide (Liang et al., 2005
). The enterobacterium Erwinia chrysanthemi may be a good alternative to study the involvement of the Tol-Pal proteins in pathogenicity. It is responsible for soft rot in many plants, including vegetable and ornamental species. It colonizes parenchymatous tissues by degrading the plant cell wall by means of a battery of pectinolytic enzymes. The oligosaccharides originating from pectin degradation are used as a carbon source by the bacterium (Hugouvieux-Cotte-Pattat et al., 2001
).
Analysis of the role of the tol-pal genes in the pathogenesis of such phytopathogenic species may be easier than in animal pathogens and could reveal additional properties of the Tol-pal proteins that may be difficult to observe in the E. coli K-12 laboratory strains. In addition, complementation studies and the use of hybrid proteins between the two bacteria may help in the understanding of some unresolved features of the Tol-Pal system. In a first attempt, the Er. chrysanthemi tol-pal genes were cloned and sequenced, and mutants in most genes were isolated and characterized.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Transfer of RP4 derivative plasmids by mating.
pULB110, a kanamycin-sensitive derivative of RP4 : : mini-Mu, was used to generate R-prime derivatives containing bacterial DNA (van Gijsegem et al., 1985). Mating between the recipient E. coli strain JC11305 and Er. chrysanthemi donor strains carrying plasmids was performed by spreading 0·2 ml of overnight cultures of the strains on M63 plates and incubating for 5 h at 30 °C. Bacteria were resuspended in 1 ml of M63 medium and spread on selective media.
Cloning and sequencing of Er. chrysanthemi tol-pal genes.
Most techniques were performed as described by Sambrook et al. (1989). For Southern blot analysis, DNA probes were prepared using the DIG High prime DNA labelling and detection starter kit (Roche). Recombinant plasmids were introduced into E. coli after a CaCl2 treatment or into Er. chrysanthemi by electroporation at room temperature. Nucleotide sequencing was performed by Genome Express SA (Grenoble, France).
Construction of uidAkan insertions and marker exchange recombination.
The ybgC, tolQ, tolA, tolB, pal and ybgF genes were individually subcloned into pBR328 in order to generate plasmids with unique restriction sites in each gene. Two methods were used for marker exchange recombination. In the first one, the uidAkan cassette of pN496 was introduced in the following unique sites: MscI for ybgC, HindIII for tolQ and tolA, HpaI for tolB, StuI for pal and EcoRV for ybgF (Fig. 1). In this cassette, the promoter region of uidA was absent, allowing the generation of a transcriptional fusion between the gene containing the insert and uidA. Plasmids containing the genes inactivated by the insertion were introduced into Er. chrysanthemi cells by electroporation. Integration of the insertions into the Er. chrysanthemi chromosome by marker exchange recombination was favoured by prolonged culture in low-phosphate medium in the presence of kanamycin (Roeder & Collmer, 1985
). Colonies recovered on kanamycin LB plates were analysed by replica plating on ampicillin LB plates to confirm the loss of the plasmid vector (referred to as method 1). In the second method, the uidAkan cassette was inserted in the NheI, SnaBI, HpaI and StuI sites of tolQ, tolA, tolB and pal, respectively. Fragments overlapping the insertion were cloned into the SmaI or NotI sites of pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision (Link et al., 1997
). After electroporation, integration of the plasmid into the chromosome was selected by growth at 43 °C in the presence of kanamycin, then integrates were resolved at 30 °C and plasmid loss was selected in the presence of 10 % sucrose. The colonies were then screened for sucrose resistance and the loss of chloramphenicol resistance by replica plating. For both protocols, the correct inactivation of the genes was controlled by PCR amplification of the chromosomal DNA, using the first primer in the uidA gene and the second primer in the tol gene. A control was also carried out to check for the absence of an intact gene.
|
Phenotype analysis of mutants.
Sensitivity towards antimicrobial agents was tested on LB plates by replica plating the strains on rich media containing various amounts of the tested agent. To test strain virulence, 107 cells were inoculated in chicory leaves after scarification. After 24 h of incubation at 30 °C, the length of the rotted region was measured. Potato tubers were inoculated as previously described (Lojkowska et al., 1995). A hole was made into the tuber parenchyma and 107 cells were used for inoculation. After 2472 h incubation, tubers were sliced vertically through the infection point, and the weight of decayed tissue was taken as a measure of disease severity. All the macerated tissue was collected and used for bacterial counting. For each bacterium, 500 mg of macerated tissue was resuspended in 500 µl M63 medium, treated with 50 µl toluene, and vortexed thoroughly. After centrifugation (12 000 g for 2 min), the supernatant was used to determine
-galactosidase (which reflected the colony number, see Methods, Enzyme assays, below) and pectate lyase activities.
Microscopy.
Bacterial cells were directly observed after growth in LB medium, LB medium supplemented with 10 % sucrose or in chicory leaves. This allowed us to estimate the morphology of the cells. Bacteria were also recorded using a camera to estimate their motility.
For transmission electron microscopy, cells were grown on LB plates or LB plates supplemented with 10 % sucrose, recovered with a loop on a Parafilm sheet, and then fixed with osmium tetrachloride vapours. They were resuspended in 0·1 M sodium cacodylate, and then stained with 1 % sodium silicotungstate or uranyl acetate.
Enzyme assays.
Pectate lyase activity was measured in 0·1 M Tris, pH 8·5, 0·1 mM CaCl2, 0·05 % polygalacturonate, by following an increase in A230 from the cleavage of polygalacturonate. The assay for -galactosidase activity has been described elsewhere (Miller, 1992
). Because the cell morphology of the tol-pal mutants was heterogeneous, the OD600 did not necessarily reflect the amount of bacteria. Thus, we calculated the relative natural
-galactosidase activity of Er. chrysanthemi based on colony number, which was determined by cell enumeration. Accordingly, the pectate lyase activity of each strain was divided by its
-galactosidase activity to standardize the enzyme activity (referred to as relative units' in the text). Cell enumeration was determined by plate count.
Western blot analyses.
Cells (3x108) in the mid-exponential phase of growth were centrifuged, resuspended in loading buffer and boiled. Samples were separated by SDS-PAGE [12 % polyacrylamide, (Laemmli, 1970)] and transferred for 2 h onto a nitrocellulose membrane by using a semi-dry blotter. Immunoblots were developed with the BM chemiluminescence blotting substrate (Roche). Polyclonal antibodies raised against E. coli TolA, TolB, Pal and YbgF proteins have been previously described (Clavel et al., 1998
) and were used to detect the corresponding Er. chrysanthemi proteins.
Nucleotide sequence accession number.
The nucleotide sequence reported here, corresponding to a 6967 bp fragment, has been deposited at EMBL under the accession number AJ297885 (EMBL/genebank version AJ297885.1 GI:16116629).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning and sequencing of Er. chrysanthemi tol-pal genes
Plasmid pMC2242, containing an 8·5 kb EcoRI fragment of pR'16 cloned into pJEL250, complemented the cholate-sensitivity phenotype of the tolBpal mutation of JC11305. DNA sequence analysis revealed that it contained the 'ybgCtolQRABpalybgF region (Fig. 1
). To clone the region upstream of ybgC, an EcoRISnaBI fragment from pMC2242 containing the ybgCtolQRA genes was used as a DNA probe for Southern analysis of pR'16 digested with HindIII. A 4·3 kb HindIII fragment was isolated and cloned into pBR328 to give pMC2244 (Fig. 1
). Finally, a BamHINheI fragment from pMC2244 was introduced into pMC2242 to give pMC2256. Further sequencing showed that it contained the entire ybgC sequence and its upstream region. The 6967 bp fragment cloned into pMC2256 is presented in Fig. 1(A)
. Upstream of ybgC, homologues of genes cydB (truncated), ybgT and ybgE of E. coli were identified. The region downstream from ybgF showed high similarity to lysT, indicating that the gene order in the tol-pal region of Er. chrysanthemi is the same as that found in E. coli. Unlike in E. coli, no potential transcription terminator could be identified in the ybgEybgC intergenic region. A potential rho-dependent terminator is situated downstream from ybgF [
G=30·4 kcal mol1 (127 kJ mol1)], a characteristic also found in Pseudomonas putida and E. coli (Llamas et al., 2003b
; Vianney et al., 1996
). The Tol-Pal proteins of Er. chrysanthemi and E. coli displayed a high degree of similarity (Fig. 1B
), except for TolA, which was the least-conserved protein. This is mainly due to its central domain, in which three stretches of seven amino acids are lacking in Er. chrysanthemi (Fig. 1C
). This region is organized in an
-helical structure that crosses the periplasmic space (Derouiche et al., 1999
; Levengood et al., 1991
).
Gene inactivation
The first method used for gene inactivation allowed us to obtain mutants ybgC1, tolQ1, tolA1 and ybgF1, containing a uidAkan cassette in the MscI, HindIII (for tolQ1 and tolA1), and EcoRV sites, respectively. The correct insertion and orientation of the cassette in the chromosome were confirmed by PCR. In the course of our experiments, we observed that the tolQ1 mutation was highly unstable; this mutation could not be retained. Moreover, attempts to inactivate the chromosomal alleles of the tolB and pal genes using this method were unsuccessful. We hypothesized that prolonged culturing in low-phosphate medium could be lethal for some of the tol-pal mutants. Hence, we used the second method (see Methods), which allowed growth in rich medium. Er. chrysanthemi was transformed with pKO3 derivatives containing tolQ, tolA, tolB and pal inactivated by the uidAkan cassette in the NheI, SnaBI, HpaI and StuI sites, respectively (Fig. 1A). To our surprise, the colonies selected on LB plates supplemented with 10 % sucrose grew very slowly on LB plates without sucrose. Therefore, we retained colonies which were able to grow on plates supplemented with sucrose and kanamycin but unable to grow on rich media supplemented with sucrose and chloramphenicol (loss of the pKO3 vector), or cholate (tol phenotype). Using this technique, we were able to obtain the mutants tolQ2, tolA2, tolB1 and pal1 (Table 1
). The orientation of the uidAkan cassette was investigated and shown to generate a transcriptional fusion between the inactivated gene and uidA in all but the tolA2 mutant. The poor growth of the tolB and pal mutants in the absence of sucrose provides a good explanation for our inability to isolate these mutants using the first method. The tolA1 mutant first isolated lacked only the seven C-terminal amino acid residues of TolA1, and comparison of the tolA1 and tolA2 mutants suggested that tolA1 retained a partial functionality. This could be due to the presence of higher amounts of TolB and Pal in the tolA1 mutant (Fig. 2
). The instability of the tolQ1 mutant could also be explained by its poor ability to grow on LB plates.
|
Er. chrysanthemi tol-pal mutants are impaired in cell motility and sensitive to ionic strength and osmolarity
The wild-type strain, as well as the ybgC1, tolA1 or ybgF1 mutants, was able to grow on LB and M63 plates. In contrast, the tolQ2, tolA2, tolB1 and pal1 mutants did not form colonies on LB or M63 plates, except in the presence of 10 % sucrose. They also grew very slowly in LB liquid medium and when they were patched on LB plates. The role of sucrose was unexpected. Its addition affects the medium osmolarity, but it can also act as a membrane and protein stabilizer in adverse conditions (Crowe et al., 1988; Leslie et al., 1995
; Molina-Hoppner et al., 2004
). To discriminate between these two effects, the mutants were grown on LB plates supplemented with sucrose, various amounts of sugars, NaCl or the osmoprotectant glycine betaine. The tolQ2, tolA2, tolB1 and pal1 mutants recovered a normal growth when sucrose was replaced by 10 % glucose, galactose (two sugars metabolized by Er. chrysanthemi), maltose or lactose (two sugars not metabolized by Er. chrysanthemi), but not glycerol. Addition of the osmoprotectant glycine betaine (110 mM) also allowed growth of the mutants. In contrast, the tolQ2, tolA2, tolB1 and pal1 mutants did not grow when NaCl was added to LB plates at concentrations between 0·1 and 0·3 M. The presence of NaCl in the medium affects both osmolarity and ionic strength. Therefore, we concluded that the mutants were sensitive to high ionic strength and low osmolarity.
The motility of the mutants was tested on swarm plates, as described in Methods. After 20 h growth at 30 °C, the swarm diameter was 43·0±3·4 mm for the wild-type strain. The motility of the ybgF1 mutant was not significantly affected, that of the ybgC1 tolQ2, tolA1 and tolA2 mutants was strongly reduced, while tolB1 and pal1 mutants did not move at all (Table 2). Addition of 10 % sucrose to the swarm agar plates partially restored the motility of the mutants (Table 2
).
|
Morphological characterization of the Er. chrysanthemi tol-pal mutants
The cells were examined by electron microscopy after negative staining of the cells (Fig. 3). After the growth of patches on LB plates, the wild-type strain was rod-shaped with a mean size of 2·1x0·65 µm and presented long flagella (Fig. 3A
). Under the same conditions, the tolQ2 mutant presented an altered morphology with short twisted filaments (Fig. 3B
). This could be due to the presence of square poles at one end of the cells, where the poles tended to grow to one side rather than at the middle of the bacteria (Fig. 3C
). Bacteria had a few, short flagella. The tolA2 mutant also formed short filaments and had a few, short flagella (Fig. 3D
). The septum was not always at the middle of dividing bacteria, leading to cells of heterogeneous sizes (Fig. 3E
). Vesicles could be observed, even at the septum (Fig. 3D
). The tolB1 mutant also formed short filaments, but had no flagella (Fig. 3F
). The size of the cells and the position of the septa were highly irregular. The pal1 mutant lacked flagella (Fig. 3G
). Vesicles appeared at the cell surface as well as very large envelope excrescences.
|
Sensitivity to antimicrobial agents
The sensitivity of the mutants towards various antimicrobial agents was analysed (Table 3). All the mutants were more sensitive to sodium cholate than the wild-type strain. In E. coli, only tolQRAB pal mutants show this phenotype (Vianney et al., 1996
). The sensitivity of the ybgC1 mutant to sodium cholate can be explained by the polarity of the insertion. Consistent with this hypothesis, this phenotype could be complemented by the addition of a multicopy plasmid carrying the Er. chrysanthemi ybgCtolQRA cluster, but not when the plasmid contained only ybgC (data not shown). The ybgF1 mutant was sensitive to cholate, but resistant to the other chemical compounds tested. This phenotype could be complemented by providing the tolBpalybgF genes in trans (data not shown). The other mutants were sensitive to SDS and carbonyl cyanide m-chlorophenylhydrazone (CCCP), but resistant to vancomycin (data not shown). The tolB1 and pal1 mutants were more sensitive to chemical compounds than the tolQ and tolA mutants. However, with the exception of SDS, most differences in sensitivity were moderate.
|
Virulence and survival in plant tissues of the ybgC, tolQ, tolA, tolB, pal and ybgF mutants
The tolQ, tolA, tolB and pal mutants were strongly affected for virulence, since inoculation with these strains led to poor tissue maceration on chicory leaves (Fig. 4) or potato tubers (data not shown). Global pectate lyase activity (relative units) was strongly decreased in the macerated tissues of all the tol-pal mutants (ranging from 0·0008 to 0·008, while the activity of the wild-type strain was 0·02), decreased by two-fold in the ybgC1 mutant, but increased to 0·03 in the ybgF1 strain (data not shown). Wild-type and mutant strains were recovered from macerated tissue and observed under a phase-contrast microscope: their motility and morphology was analogous to that observed when the same strains were grown on 10 % sucrose LB plates (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Er. chrysanthemi, inactivation of tolQ, tolA, tolB or pal is deleterious, since mutants in these genes grow very slowly on LB plates. They can grow well only when the medium is supplemented with 10 % sugars (sucrose, glucose, maltose or lactose) or with the osmoprotectant glycine betaine, but not in the presence of glycerol or 0·10·3 M NaCl. Glycine betaine and sugars are nonionic compatible solutes that can be accumulated by de novo synthesis or transport without interfering with vital cellular processes. These compounds not only confer protection against high osmolarity, but also allow protein protection (Poolman & Glaasker, 1998). The addition of sugars or betaine to the growth medium probably contributes to the osmoprotection of some envelope components. Consistent with this hypothesis, all the tol-pal mutants showed increased resistance to chemical compounds in the presence of sucrose (data not shown), and the tolQ and tolA mutants were more motile under such conditions (Table 2
). The addition of sugars may also contribute to the maintenance of a turgor pressure compatible with cell viability for the Er. chrysanthemi tol-pal mutants. As in P. putida (Llamas et al., 2000
), we observed that the tolB and pal mutations led to more severe phenotypes than those observed with tolQ and tolA mutants. Attempts to construct tol-pal mutants in some bacteria have been unsuccessful, and could be explained by their poor growth in classical rich media, as observed for Er. chrysanthemi tol-pal mutants (Dennis et al., 1996
; Spinola et al., 1996
).
Our electron microscopy observations provide a good explanation for the impairment of cell motility of the tol-pal mutants. The tolB and pal mutants lack flagella, while tolQ and tolA mutants have fewer and shorter flagella, a phenotype which suggests an alteration of flagella synthesis, polymerization and/or stability.
The Er. chrysanthemi tol-pal mutants have an altered cell morphology: some of the cells do not correctly localize the septum and the poles during division. A phenotype of filamentation has also been observed in tol-pal mutants of P. putida (Llamas et al., 2000), E. coli (Meury & Devilliers, 1999
) and V. cholerae (Heilpern & Waldor, 2000
), and the incorrect positioning of the septa has also been reported (Meury & Devilliers, 1999
).
Er. chrysanthemi tol-pal mutants showed a reduced virulence on chicory leaves and potato tubers. Altered virulence of tolB and pal mutants has been reported in animal pathogens. In H. ducreyi, expression of Pal is required for virulence in a human model (Fortney et al., 2000). In E. coli, Pal is involved in Gram-negative sepsis (Hellman et al., 2002
; Liang et al., 2005
). In S. enterica, tolB mutants are attenuated in a mouse typhoid model of infection (Bowe et al., 1998
). Although tolB mutants cross the gut, they are unable to cause fatal infection. This has been attributed to their inability to survive within macrophages and resist the bactericidal effects of non-immune serum. In the same screening, the authors also identified the mdoB gene as essential for fatal infection (Bowe et al., 1998
). opg (or mdo) mutants have been associated with a lack of virulence in phythopathogenic bacteria such as Pseudomonas syringae and Er. chrysanthemi (Loubens et al., 1993
; Mukhopadhyay et al., 1988
; Page et al., 2001
). Unlike the tol-pal mutants, the opgGH mutants show a complete loss of virulence, even on potato tubers, but present some phenotypes similar to those observed for the tol-pal mutants, namely reduced motility and pectate lyase production (Page et al., 2001
). Another common trait between Opg and Tol-Pal is the activation of the Rcs phosphorelay in the opg and tol mutants (Clavel et al., 1996
; Ebel et al., 1997
). Rcs is associated with virulence in an increasing number of bacteria, including Erwinia amylovora (Bereswill & Geider, 1997
) and S. enterica (Cano et al., 2002
; Dominguez-Bernal et al., 2004
). In E. coli, the Rcs phosphorelay activates the genes necessary for capsule synthesis (Stout & Gottesman, 1990
), and ftsZ and ftsA involved in cell division (Carballes et al., 1999
), and represses the genes involved in flagella synthesis (Francez-Charlot et al., 2003
). As in E. coli, some of the phenotypes observed in the Er. chrysanthemi tol-pal mutants, such as the defects in motility and cell division, could result from an activation of the Rcs phosphorelay. Therefore, the impaired virulence of the tol-pal mutants probably results from the combination of a defect in cell envelope integrity and an activation of regulatory networks, such as the Rcs phosphorelay.
E. coli tol-pal genes were unable to complement the corresponding Er. chrysanthemi mutations. Conversely, the Er. chrysanthemi tol-pal genes complemented the E. coli tol-pal mutations, despite the fact that the TolA protein was shorter in Er. chrysanthemi [as in Erwinia carotovora (data not shown)]. The possibility that Er. chrysanthemi tol-pal genes perform additional essential functions should be considered. This hypothesis can be assessed by the construction and analysis of selected hybrid Tol-Pal proteins, as most of the functional domains of the Tol-Pal proteins have been identified in E. coli. Another particularity of the tol-pal gene cluster in Er. chrysanthemi is the phenotype of the ybgF mutant in this bacterium. The Er. chrysanthemi ybgF mutant is sensitive to sodium cholate, a phenotype that has not been described in other species. Another intriguing property of this ybgF mutant is its apparent increase in pectate lyase activity. Further examination of these phenotypes could help to clarify the role of YbgF in the Tol-Pal system.
This work further demonstrates that the Tol-Pal proteins are critical cell envelope components necessary for bacterial virulence. The low growth rate of the Er. chrysanthemi tol-pal mutants, their impaired viability, and their reduced motility and pectinase production are probably sufficient to explain the reduced virulence. It also provides further evidence that the loss of Tol-Pal functions can be deleterious for some bacterial species.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bernadac, A., Gavioli, M., Lazzaroni, J. C., Raina, S. & Lloubes, R. (1998). Escherichia coli tol-pal mutants form outer membrane vesicles. J Bacteriol 180, 48724878.
Bouveret, E., Rigal, A., Lazdunski, C. & Benedetti, H. (1998). Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli. Mol Microbiol 27, 143157.[CrossRef][Medline]
Bowe, F., Lipps, C. J., Tsolis, R. M., Groisman, E., Heffron, F. & Kusters, J. G. (1998). At least four percent of the Salmonella typhimurium genome is required for fatal infection of mice. Infect Immun 66, 33723377.
Cano, D. A., Dominguez-Bernal, G., Tierrez, A., Garcia-Del Portillo, F. & Casadesus, J. (2002). Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA. Genetics 162, 15131523.
Carballes, F., Bertrand, C., Bouche, J. P. & Cam, K. (1999). Regulation of Escherichia coli cell division genes ftsA and ftsZ by the two-component system rcsC-rcsB. Mol Microbiol 34, 442450.[CrossRef][Medline]
Cascales, E., Lloubes, R. & Sturgis, J. N. (2001). The TolQ-TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA-MotB. Mol Microbiol 42, 795807.[CrossRef][Medline]
Cascales, E., Bernadac, A., Gavioli, M., Lazzaroni, J. C. & Lloubes, R. (2002). Pal lipoprotein of Escherichia coli plays a major role in outer membrane integrity. J Bacteriol 184, 754759.
Clavel, T., Lazzaroni, J. C., Vianney, A. & Portalier, R. (1996). Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis. Mol Microbiol 19, 1925.[CrossRef][Medline]
Clavel, T., Germon, P., Vianney, A., Portalier, R. & Lazzaroni, J. C. (1998). TolB protein of Escherichia coli K-12 interacts with the outer membrane peptidoglycan-associated proteins Pal, Lpp and OmpA. Mol Microbiol 29, 359367.[CrossRef][Medline]
Crowe, J. H., Crowe, L. M., Carpenter, J. F., Rudolph, A. S., Wistrom, C. A., Spargo, B. J. & Anchordoguy, T. J. (1988). Interactions of sugars with membranes. Biochim Biophys Acta 947, 367384.[Medline]
Dennis, J. J., Lafontaine, E. R. & Sokol, P. A. (1996). Identification and characterization of the tolQRA genes of Pseudomonas aeruginosa. J Bacteriol 178, 70597068.
Derouiche, R., Benedetti, H., Lazzaroni, J. C., Lazdunski, C. & Lloubes, R. (1995). Protein complex within Escherichia coli inner membrane. TolA N-terminal domain interacts with TolQ and TolR proteins. J Biol Chem 270, 1107811084.
Derouiche, R., Lloubes, R., Sasso, S., Bouteille, H., Oughideni, R., Lazdunski, C. & Loret, E. (1999). Circular dichroism and molecular modeling of the E. coli TolA periplasmic domains. Biospectroscopy 5, 189198.[CrossRef][Medline]
Dominguez-Bernal, G., Pucciarelli, M. G., Ramos-Morales, F., Garcia-Quintanilla, M., Cano, D. A., Casadesus, J. & Garcia-del Portillo, F. (2004). Repression of the RcsC-YojN-RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence. Mol Microbiol 53, 14371449.[CrossRef][Medline]
Dubuisson, J. F., Vianney, A. & Lazzaroni, J. C. (2002). Mutational analysis of the TolA C-terminal domain of Escherichia coli and genetic evidence for an interaction between TolA and TolB. J Bacteriol 184, 46204625.
Ebel, W., Vaughn, G. J., Peters, H. K., 3rd & Trempy, J. E. (1997). Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide in Escherichia coli. J Bacteriol 179, 68586861.
Fortney, K. R., Young, R. S., Bauer, M. E., Katz, B. P., Hood, A. F., Munson, R. S., Jr & Spinola, S. M. (2000). Expression of peptidoglycan-associated lipoprotein is required for virulence in the human model of Haemophilus ducreyi infection. Infect Immun 68, 64416448.
Francez-Charlot, A., Laugel, B., Van Gemert, A., Dubarry, N., Wiorowski, F., Castanie-Cornet, M. P., Gutierrez, C. & Cam, K. (2003). RcsCDB His-Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli. Mol Microbiol 49, 823832.[CrossRef][Medline]
Germon, P., Ray, M. C., Vianney, A. & Lazzaroni, J. C. (2001). Energy-dependent conformational change in the TolA protein of Escherichia coli involves its N-terminal domain, TolQ, and TolR. J Bacteriol 183, 41104114.
Heilpern, A. J. & Waldor, M. K. (2000). CTXphi infection of Vibrio cholerae requires the tolQRA gene products. J Bacteriol 182, 17391747.
Hellman, J., Roberts, J. D., Jr, Tehan, M. M., Allaire, J. E. & Warren, H. S. (2002). Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in Gram-negative sepsis and causes inflammation and death in mice. J Biol Chem 277, 1427414280.
Hugouvieux-Cotte-Pattat, N., Blot, N. & Reverchon, S. (2001). Identification of TogMNAB, an ABC transporter which mediates the uptake of pectic oligomers in Erwinia chrysanthemi 3937. Mol Microbiol 41, 11131123.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lazzaroni, J. C., Vianney, A., Popot, J. L., Benedetti, H., Samatey, F., Lazdunski, C., Portalier, R. & Geli, V. (1995). Transmembrane alpha-helix interactions are required for the functional assembly of the Escherichia coli Tol complex. J Mol Biol 246, 17.[CrossRef][Medline]
Lazzaroni, J. C., Germon, P., Ray, M. C. & Vianney, A. (1999). The Tol proteins of Escherichia coli and their involvement in the uptake of biomolecules and outer membrane stability. FEMS Microbiol Lett 177, 191197.[CrossRef][Medline]
Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H. & Crowe, L. M. (1995). Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol 61, 35923597.
Levengood, S. K., Beyer, W. F., Jr & Webster, R. E. (1991). TolA: a membrane protein involved in colicin uptake contains an extended helical region. Proc Natl Acad Sci U S A 88, 59395943.
Liang, M. D., Bagchi, A., Warren, H. S. & 7 other authors (2005). Bacterial peptidoglycan-associated lipoprotein: a naturally occurring toll-like receptor 2 agonist that is shed into serum and has synergy with lipopolysaccharide. J Infect Dis 191, 939948.[CrossRef][Medline]
Link, A. J., Phillips, D. & Church, G. M. (1997). Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J Bacteriol 179, 62286237.
Llamas, M. A., Ramos, J. L. & Rodriguez-Herva, J. J. (2000). Mutations in each of the tol genes of Pseudomonas putida reveal that they are critical for maintenance of outer membrane stability. J Bacteriol 182, 47644772.
Llamas, M. A., Rodriguez-Herva, J. J., Hancock, R. E., Bitter, W., Tommassen, J. & Ramos, J. L. (2003a). Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane. J Bacteriol 185, 47074716.
Llamas, M. A., Ramos, J. L. & Rodriguez-Herva, J. J. (2003b). Transcriptional organization of the Pseudomonas putida tol-oprL genes. J Bacteriol 185, 184195.
Lojkowska, E., Masclaux, C., Boccara, M., Robert-Baudouy, J. & Hugouvieux-Cotte-Pattat, N. (1995). Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937. Mol Microbiol 16, 11831195.[Medline]
Loubens, I., Debarbieux, L., Bohin, A., Lacroix, J. M. & Bohin, J. P. (1993). Homology between a genetic locus (mdoA) involved in the osmoregulated biosynthesis of periplasmic glucans in Escherichia coli and a genetic locus (hrpM) controlling pathogenicity of Pseudomonas syringae. Mol Microbiol 10, 329340.[Medline]
Meury, J. & Devilliers, G. (1999). Impairment of cell division in tolA mutants of Escherichia coli at low and high medium osmolarities. Biol Cell 91, 6775.[CrossRef][Medline]
Miller, J. H. (1992). A Short Course in Bacterial Genetics: a Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Molina-Hoppner, A., Doster, W., Vogel, R. F. & Ganzle, M. G. (2004). Protective effect of sucrose and sodium chloride for Lactococcus lactis during sublethal and lethal high-pressure treatments. Appl Environ Microbiol 70, 20132020.
Mukhopadhyay, P., Williams, J. & Mills, D. (1988). Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae. J Bacteriol 170, 54795488.[Medline]
Muller, M. M. & Webster, R. E. (1997). Characterization of the tol-pal and cyd region of Escherichia coli K-12: transcript analysis and identification of two new proteins encoded by the cyd operon. J Bacteriol 179, 20772080.
Page, F., Altabe, S., Hugouvieux-Cotte-Pattat, N., Lacroix, J. M., Robert-Baudouy, J. & Bohin, J. P. (2001). Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity. J Bacteriol 183, 31343141.
Poolman, B. & Glaasker, E. (1998). Regulation of compatible solute accumulation in bacteria. Mol Microbiol 29, 397407.[CrossRef][Medline]
Prouty, A. M., Van Velkinburgh, J. C. & Gunn, J. S. (2002). Salmonella enterica serovar Typhimurium resistance to bile: identification and characterization of the tolQRA cluster. J Bacteriol 184, 12701276.
Ray, M. C., Germon, P., Vianney, A., Portalier, R. & Lazzaroni, J. C. (2000). Identification by genetic suppression of Escherichia coli TolB residues important for TolB-Pal interaction. J Bacteriol 182, 821824.
Roeder, D. L. & Collmer, A. (1985). Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi. J Bacteriol 164, 5156.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Soberon, X., Covarrubias, L. & Bolivar, F. (1980). Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene 9, 287305.[Medline]
Spinola, S. M., Hiltke, T. J., Fortney, K. & Shanks, K. L. (1996). The conserved 18 000-molecular-weight outer membrane protein of Haemophilus ducreyi has homology to PAL. Infect Immun 64, 19501955.[Abstract]
Stout, V. & Gottesman, S. (1990). RcsB and RcsC: a two-component regulator of capsule synthesis in Escherichia coli. J Bacteriol 172, 659669.[Medline]
Sun, T. P. & Webster, R. E. (1987). Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli. J Bacteriol 169, 26672674.[Medline]
Valentin-Hansen, P., Albrechtsen, B. & Love Larsen, J. E. (1986). DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor. EMBO J 5, 20152021.[Abstract]
van Gijsegem, F., Toussaint, A. & Schoonejans, E. (1985). In vivo cloning of the pectate lyase and cellulase genes of Erwinia chrysanthemi. EMBO J 4, 787792.
Vianney, A., Lewin, T. M., Beyer, W. F., Jr, Lazzaroni, J. C., Portalier, R. & Webster, R. E. (1994). Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity. J Bacteriol 176, 822829.[Abstract]
Vianney, A., Muller, M. M., Clavel, T., Lazzaroni, J. C., Portalier, R. & Webster, R. E. (1996). Characterization of the tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein. J Bacteriol 178, 40314038.
Walburger, A., Lazdunski, C. & Corda, Y. (2002). The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB. Mol Microbiol 44, 695708.[CrossRef][Medline]
Webster, R. E. (1991). The tol gene products and the import of macromolecules into Escherichia coli. Mol Microbiol 5, 10051011.[Medline]
Youderian, P., Burke, N., White, D. & Hartzell, P. (2003). Identification of genes required for adventurous gliding motility in Myxococcus xanthus with the transposable element mariner. Mol Microbiol 49, 555570.[CrossRef][Medline]
Received 30 May 2005;
revised 11 July 2005;
accepted 25 July 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |