Role of tol genes in cloacin DF13 susceptibility of Escherichia coli K-12 strains expressing the cloacin DF13-aerobactin receptor IutA.

IutA is the outer membrane protein receptor for ferric aerobactin and the bacteriocin cloacin DF13. Although the same receptor is shared, ferric aerobactin transport across the outer membrane in Escherichia coli is TonB dependent, whereas cloacin DF13 transport is not. We have recently observed that tolQ is required for cloacin DF13 susceptibility (J.A. Thomas and M.A. Valvano, FEMS Microbiol. Lett. 91:107-112, 1992). In this study, we demonstrate that the genes tolQ, tolR, and tolA, but not tolB, tolC, and ompF, are required for the internalization of cloacin DF13 and they are not involved in the transport of ferric aerobactin.     

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.

The TolQ, TolR, TolA, TolB, and Pal proteins appear to function in maintaining the integrity of the outer membrane, as well as facilitating the uptake of the group A colicins and the DNA of the infecting filamentous bacteriophages. Sequence data showed that these genes are clustered in a 6-kb segment of DNA with the gene order orf1 tolQ tolR tolA tolB pal orf2 (a newly identified open reading frame encoding a 29-kD9 protein). Like those containing orf1, bacteria containing an insertion mutation in this gene showed no obvious phenotype. Analysis of beta-galactosidase activity from fusion constructs in which the lac operon was fused to various genes in the cluster showed that the genes in this region constitute two separate operons: orf1 tolQRA and tolB pal orf2. In the orf1 tolQRA operon, translation of MR was dependent on translation of the upstream tolQ region. Consistent with this result, no functional ribosome-binding site for TolR synthesis was detected.     

Identification by genetic suppression of Escherichia coli TolB residues important for TolB-Pal interaction

The Tol-Pal system of Escherichia coli is involved in maintaining outer membrane stability. Mutations in tolQ, tolR, tolA, tolB, or pal genes result in sensitivity to bile salts and the leakage of periplasmic proteins. Moreover, some of the tol genes are necessary for the entry of group A colicins and the DNA of filamentous bacteriophages. TolQ, TolR, and TolA are located in the cytoplasmic membrane where they interact with each other via their transmembrane domains. TolB and Pal form a periplasmic complex near the outer membrane. We used suppressor genetics to identify the regions important for the interaction between TolB and Pal. Intragenic suppressor mutations were characterized in a domain of Pal that was shown to be involved in interactions with TolB and peptidoglycan. Extragenic suppressor mutations were located in tolB gene. The C-terminal region of TolB predicted to adopt a beta-propeller structure was shown to be responsible for the interaction of the protein with Pal. Unexpectedly, none of the suppressor mutations was able to restore a correct association between Pal and peptidoglycan, suggesting that interactions between Pal and other components such as TolB may also be important for outer membrane stability.     

Characterization of an OprL null mutant of Pseudomonas putida.

A Pseudomonas putida oprL null mutant was generated with reverse genetics by using an in vitro-truncated oprL::xylE construct and in vivo allelic exchange. The nature of the mutation introduced in P. putida was confirmed by Southern blotting. Western blots (immunoblots) of peptidoglycan-associated proteins revealed that the OprL protein was not made in the mutant strain, whereas it was detectable as a 19-kDa band in protein preparations of the wild-type strain. The P. putida oprL, mutant exhibited altered cell morphology as revealed by electron microscopy and was more sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA than the wild-type strain. The oprL gene was conserved in a wide variety of the Pseudomonas strains belonging to rRNA group I, which suggests that this gene is important for the maintenance of the cell envelope and cell morphology in this group of microorganisms.     

Cell envelope mutants of Pseudomonas putida: physiological characterization and analysis of their ability to survive in soil

To generate mutants with altered lipopolysaccharides (LPS) of the wild-type Pseudomonas putida KT2442, we used the mini-Tn 5luxAB -Km transposon. A mutant was found among luminescent colonies and selected as a negative clone in enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 7.3B, which recognizes the O-antigen of P. putida LPS. The DNA region of the LPS mutant interrupted by the minitransposon insertion was cloned and sequenced. Comparison of the deduced amino acid sequence with protein sequence databases showed similarity to the O-antigen polymerase (Wzy) of Salmonella enterica (muenchen). The wild-type gene was rescued by polymerase chain reaction (PCR), cloned into a broad-host-range plasmid and used to carry out complementation assays. The cloned gene was able to restore the wild-type phenotype of the P. putida wzy mutant. We constructed an isogenic mutant of the luminescent wzy mutant to which an oprL mutation was transferred by homologous recombination with an oprL :: xylE cassette. The wzy mutants of P. putida were more sensitive to SDS, deoxycholate and EDTA than the corresponding parental strains. We analysed the ability of wzy , oprL and wzy oprL mutants of P. putida to colonize soil. In comparison with the wild-type strain, the ability of single mutants to colonize soil decreased; this characteristic was more evident for the double mutant, especially at high temperatures.     

CTXphi infection of Vibrio cholerae requires the tolQRA gene products

CTXphi is a lysogenic filamentous bacteriophage that encodes cholera toxin. Filamentous phages that infect Escherichia coli require both a pilus and the products of tolQRA in order to enter host cells. We have previously shown that toxin-coregulated pilus (TCP), a type IV pilus that is an essential Vibrio cholerae intestinal colonization factor, serves as a receptor for CTXphi. To test whether CTXphi also depends upon tol gene products to infect V. cholerae, we identified and inactivated the V. cholerae tolQRAB orthologues. The predicted amino acid sequences of V. cholerae TolQ, TolR, TolA, and TolB showed significant similarity to the corresponding E. coli sequences. V. cholerae strains with insertion mutations in tolQ, tolR, or tolA were reduced in their efficiency of CTXphi uptake by 4 orders of magnitude, whereas a strain with an insertion mutation in tolB showed no reduction in CTXphi entry. We could detect CTXphi infection of TCP(-) V. cholerae, albeit at very low frequencies. However, strains with mutations in both tcpA and either tolQ, tolR, or tolA were completely resistant to CTXphi infection. Thus, CTXphi, like the E. coli filamentous phages, uses both a pilus and TolQRA to enter its host. This suggests that the pathway for filamentous phage entry into cells is conserved between host bacterial species.     

Energy-Dependent Conformational Change in the TolA Protein of Escherichia coli Involves Its N-Terminal Domain, TolQ, and TolR

TolQ, TolR, and TolA inner membrane proteins of Escherichia coli are involved in maintaining the stability of the outer membrane. They share homology with the ExbB, ExbD, and TonB proteins, respectively. The last is involved in energy transduction between the inner and the outer membrane, and its conformation has been shown to depend on the presence of the proton motive force (PMF), ExbB, and ExbD. Using limited proteolysis experiments, we investigated whether the conformation of TolA was also affected by the PMF. We found that dissipation of the PMF by uncouplers led to the formation of a proteinase K digestion fragment of TolA not seen when uncouplers are omitted. This fragment was also detected in Delta tolQ, Delta tolR, and tolA(H22P) mutants but, in contrast to the parental strain, was also seen in the absence of uncouplers. We repeated those experiments in outer membrane mutants such as lpp, pal, and Delta rfa mutants: the behavior of TolA in lpp mutants was similar to that observed with the parental strain. However, the proteinase K-resistant fragment was never detected in the Delta rfa mutant. Altogether, these results suggest that TolA is able to undergo a PMF-dependent change of conformation. This change requires TolQ, TolR, and a functional TolA N-terminal domain. The potential role of this energy-dependent process in the stability of the outer membrane is discussed.     

Surface expression of O-specific lipopolysaccharide in Escherichia coli requires the function of the TolA protein

We investigated the involvement of Tol proteins in the surface expression of lipopolysaccharide (LPS). tolQ, -R, -A and -B mutants of Escherichia coli K-12, which do not form a complete LPS-containing O antigen, were transformed with the O7+ cosmid pJHCV32. The tolA and tolQ mutants showed reduced O7 LPS expression compared with the respective isogenic parent strains. No changes in O7 LPS expression were found in the other tol mutants. The O7-deficient phenotype in the tolQ and tolA mutants was complemented with a plasmid encoding the tolQRA operon, but not with a similar plasmid containing a frameshift mutation inactivating tolA. Therefore, the reduction in O7 LPS was attributed to the lack of a functional tolA gene, caused either by a direct mutation of this gene or by a polar effect on tolA gene expression exerted by the tolQ mutation. Reduced surface expression of O7 LPS was not caused by changes in lipid A-core structure or downregulation of the O7 LPS promoter. However, an abnormal accumulation of radiolabelled mannose was detected in the plasma membrane. As mannose is a sugar unique to the O7 subunit, this result suggested the presence of accumulated O7 LPS biosynthesis intermediates. Attempts to construct a tolA mutant in the E. coli O7 wild-type strain VW187 were unsuccessful, suggesting that this mutation is lethal. In contrast, a polar tolQ mutation affecting tolA expression in VW187 caused slow growth rate and serum sensitivity in addition to reduced O7 LPS production. VW187 tolQ cells showed an elongated morphology and became permeable to the membrane-impermeable dye propidium iodide. All these phenotypes were corrected upon complementation with cloned tol genes but were not restored by complementation with the tolQRA operon containing the frameshift mutation in tolA. Our results demonstrate that the TolA protein plays a critical role in the surface expression of O antigen subunits by an as yet uncharacterized involvement in the processing of O antigen.     

Low-frequency infection of F- bacteria by transducing particles of filamentous bacteriophages.

Filamentous particles containing single-stranded plasmid and bacteriophage DNA are able to infect F- Escherichia coli at frequencies of approximately 10(-6). This infection is dependent on an intact particle and requires the products of the tolQ, tolR, and tolA genes of the bacteria. The addition of CaCl2 can increase the frequency about 100-fold, presumably by increasing the concentration of particles at the bacterial surface.     

Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli.

Mutations in fii or tolA of the fii-tolA-tolB gene cluster at 17 min on the Escherichia coli map render cells tolerant to high concentrations of the E colicins and do not allow the DNA of infecting single-stranded filamentous bacteriophages to enter the bacterial cytoplasm. The nucleotide sequence of a 1,854-base-pair DNA fragment carrying the fii region was determined. This sequence predicts three open reading frames sequentially coding for proteins of 134, 230, and 142 amino acids, followed by the potential start of the tolA gene. Oligonucleotide mutagenesis of each open reading frame and maxicell analysis demonstrated that all open reading frames are expressed in vivo. Sequence analysis of mutant fii genes identified the 230-amino acid protein as the fii gene product. Chromosomal insertion mutations were constructed in each of the two remaining open reading frames. The phenotype resulting from an insertion of the chloramphenicol gene into the gene coding for the 142-amino acid protein is identical to that of mutations in fii and tolA. This gene is located between fii and tolA, and we propose the designation of tolQRA for this cluster in which tolQ is the former fii gene and tolR is the new open reading frame. The protein products of this gene cluster play an important role in the transport of large molecules such as the E colicins and filamentous phage DNA into the bacterium.     

Organisation and evolution of the tol-pal gene cluster

The genomic context and phylogenetic distribution of the tol-pal gene cluster and homologues to its various components have been investigated. The structure of this operon is well conserved across the gram negative bacteria, and the machine encoded by these genes probably evolved with the appearance of gram negative bacteria. Since the evolutionary appearance of the operon some species appear to have lost the genes. These bacteria seem to fall into two classes, namely obligate intracellular parasites and bacteria that produce large numbers of outer membrane vesicles. The evolution of the alphabeta and gamma proteobacteria was accompanied by the association of an additional gene (ybgC) with the operon. Several coincidences of genomic context argue for an important role of the tol-pal operon in cell envelope maintenance. Genes homologous to tolQ and tolR proved to be very widespread being found throughout the eubacteria, and one example in the archea, this distribution argues for an ancient origin of these genes. The genomic context of these genes often suggests a role in micronutrient uptake. Interestingly in all the cases examined the tolQ and tolR genes or their homologues appear to be present as a pair, with a potential for a tight translational regulation.     

Genome-wide screens: novel mechanisms in colicin import and cytotoxicity

Only two new genes ( fkpA and lepB ) have been identified to be required for colicin cytotoxicity in the last 25 years. Genome-wide screening using the 'Keio collection' to test sensitivity to colicins (col) A, B, D, E1, E2, E3, E7 and N from groups A and B, allowed identification of novel genes affecting cytotoxicity and provided new information on mechanisms of action. The requirement of lipopolysaccharide for colN cytotoxicity resides specifically in the lipopolysaccharide inner-core and first glucose. ColA cytotoxicity is dependent on gmhB and rffT genes, which function in the biosynthesis of lipopolysaccharide and enterobacterial common antigen. Of the tol genes that function in the cytoplasmic membrane translocon, colE1 requires tolA and tolR but not tolQ for activity. Peptidoglycan-associated lipoprotein, which interacts with the Tol network, is not required for cytotoxicity of group A colicins. Except for TolQRA, no cytoplasmic membrane protein is essential for cytotoxicity of group A colicins, implying that TolQRA provides the sole pathway for their insertion into/through the cytoplasmic membrane. The periplasmic protease that cleaves between the receptor and catalytic domains of colE7 was not identified, implying either that the responsible gene is essential for cell viability, or that more than one gene product has the necessary proteolysis function.     

Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.     

Mutational analysis of the TolA C-terminal domain of Escherichia coli and genetic evidence for an interaction between TolA and TolB

The Tol proteins are involved in the outer membrane stability of gram-negative bacteria. The C-terminal domain of TolA was mutagenized to identify residues important for its functions. The isolation of suppressor mutants of tolA mutations in the tolB gene confirmed an interaction between TolAIII and the N-terminal domain of TolB.     

The TOL Plasmid pWW0 xylN Gene Product from Pseudomonas putida Is Involved in m-Xylene Uptake

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli. To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type and xylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylN mutant TOL plasmid was not. The apparent K(s) value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.