Bactericidal efficiency and mode of action: a comparative study of photochemistry and photocatalysis.

In order to compare the disinfection potential of photocatalysis and photochemistry, the effects of these two processes on bacteria in water were investigated under exposure to UV-A and UV-C. The well-known bacterial model Escherichia coli (E. coli) was used as the experimental organism. Radiation exposure was produced with an HPK 125 W lamp and the standard TiO(2) Degussa P-25 was used as the photocatalyst. Firstly, the impact of photocatalysis and photochemistry on the cultivability of bacterial cells was investigated. UV-A radiation resulted in low deleterious effects on bacterial cultivability but generated colonies of size smaller than average. UV-C photocatalysis demonstrated a greater efficiency than UV-A photocatalysis in altering bacterial cultivability. From a cultivability point of view only, UV-C radiation appeared to be the most deleterious treatment. A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit was then used to assess the modifications in bacterial membrane permeability. UV-A radiation did not induce any alterations in bacterial permeability for 420 min of exposure whereas only a few minutes of exposure to UV-C radiation, with the same total radiance intensity, induced total loss of permeability. Moreover, after 20 and 60 min of exposure to UV-C and UV-A photocatalysis respectively, all bacteria lost their membrane integrity, suggesting that the bacterial envelope is the primary target of reactive oxygen species (ROS) generated at the surface of TiO(2) photocatalyst. These results were further confirmed by the formation of malondialdehyde (MDA) during the photocatalytic inactivation of bacterial cells and suggest that destruction of the cell envelope is a key step in the bactericidal action of photocatalysis. The oxidation of bacterial membrane lipids was also correlated with the monitoring of carboxylic acids, which can be considered as representatives of lipid peroxidation by-products. Finally, damages to bacterial morphology induced by UV-C photocatalysis and photochemistry were investigated through Scanning electron microscopy (SEM). Bacterial cells were observed on microscopy pictures at exposure durations corresponding to a loss of cultivability. After 90 min of exposure to UV-C radiation, bacterial cells showed little alteration of their outer membrane whereas they suffered deep deleterious damages under UV-C photocatalysis exposure.

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.

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.

The Tol proteins of Escherichia coli and their involvement in the uptake of biomolecules and outer membrane stability.

The Tol proteins of Escherichia coli are involved in outer membrane stability. They are also required for the uptake of the group A colicins and the translocation of filamentous phage DNA into the cytoplasm. The tol-pal genes constitute two operons in the E. coli genome, orfltolQRA and tolBpalorf2. The TolQ TolR TolA proteins form a complex in the cytoplasmic membrane, while TolB and Pal interact near the outer membrane. Most of the amino acid residues of TolA, TolB, TolR and Pal are localized in the periplasm. Recent advances in the knowledge of interactions of Tol-Pal proteins with other envelope components, or with group A colicins, are presented, together with current hypotheses about the role of the Tol proteins in outer membrane stability.

Mutational analysis of the Escherichia coli K-12 TolA N-terminal region and characterization of its TolQ-interacting domain by genetic suppression.

The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of some tolA mutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.

Escherichia coli tol-pal mutants form outer membrane vesicles.

Mutations in the tol-pal genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents. Other outer membrane defective strains such as tolC, lpp, and rfa mutations are also altered in their outer membrane permeability. In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of the tol-pal genes formed outer membrane vesicles after growth in standard liquid or solid media. This phenotype was not observed in tolC and rfaD cells in the same conditions. A tolA deletion in three different Escherichia coli strains was shown to lead to elevated amounts of vesicles. These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins. The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface. Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction of tol strains. The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.

TolB protein of Escherichia coli K-12 interacts with the outer membrane peptidoglycan-associated proteins Pal, Lpp and OmpA.

The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. Transmembrane domains of TolQ, TolR and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. TolB and the central domain of TolA interact in vitro with the outer membrane porins. In this study, both genetic and biochemical analyses were carried out to analyse the links between TolB, Pal and other components of the cell envelope. It was shown that TolB could be cross-linked in vivo with Pal, OmpA and Lpp, while Pal was associated with TolB and OmpA. The isolation of pal and tolB mutants disrupting some interactions between these proteins represents at first approach to characterizing the residues contributing to the interactions. We propose that TolB and Pal are part of a multiprotein complex that links the peptidoglycan to the outer membrane. The Tol-Pal proteins might form transenvelope complexes that bring the two membranes into close proximity and help some outer membrane components to reach their final destination.

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.

Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis.

The tolQRABpal cluster of Escherichia coli K-12 encodes proteins involved in the maintenance of cell-envelope integrity. In addition, tol/pal mutations result in a mucoid colony phenotype at low temperature. The synthesis of capsular polysaccharides by the cps genes is controlled by the positive regulator RcsA and the two-component RcsC/RcsB system. It was shown that the mucoid phenotype of the tol/pal mutants was due to an rcsCB-dependent activation of the cps genes. Furthermore, we have identified a mutation in the rcsC gene that decreased the activity of a tolA-lac operon fusion independently of RcsA and partially independently of RcsB activators. The corresponding rcsC338 mutation resulted in a Glu to Lys substitution at residue 338 of RcsC. This mutation induced mucoidy even at high temperature. We propose that RcsC modulates the phosphorylated forms of RcsB and an uncharacterized regulatory protein involved in the control of the tolQRA genes in an opposite manner. Moreover, our findings strengthen the previous suggestion that RcsC senses some alterations in the cell surface such as those induced by tol, pal or rfa mutations, and activates capsule synthesis to protect the cell against deleterious agents.

Protein complex within Escherichia coli inner membrane. TolA N-terminal domain interacts with TolQ and TolR proteins.

The TolA, TolB, TolQ, and TolR proteins are involved in maintaining the integrity of the Escherichia coli outer membrane and in the import of group A colicins and filamentous phage DNA. TolA, TolQ, and TolR are localized in the inner membrane while TolB is periplasmic, although a small amount of membrane-associated TolB is always found. In vivo cross-linking experiments with formaldehyde were performed in order to determine the proteins interacting with TolA. In wild-type strains, two specific complexes of 65 and 71 kDa, comprising TolA, were identified. These complexes were absent in a tolQ strain, while only the 65-kDa complex was absent in a tolR strain. When the tol strains were transformed with plasmids encoding TolR or TolQ, the specific complexes were restored. Moreover, immunoprecipitation experiments with the antiserum directed against TolA indicated that TolQ and TolR were co-immunoprecipitated with TolA after cross-linking. These data demonstrate that TolA interacts directly with TolR and TolQ. Two truncated TolA proteins devoid of either the C-terminal or the central domains of the protein were subjected to in vivo cross-linking. Since these two TolA derivatives still formed specific complexes with TolR derivatives still formed specific complexes with TolR and TolQ, we concluded that the TolA N-terminal domain interacted with these proteins.

Transmembrane alpha-helix interactions are required for the functional assembly of the Escherichia coli Tol complex.

TolQ, TolR and TolA are membrane proteins involved in maintaining the structure of Escherichia coli cell envelope. TolQ and TolR span the inner membrane with three and with one alpha-helical segments, respectively. The tolQ925 mutation (A177V), located in the third putative transmembrane helix of TolQ (TolQ-III), induces cell sensitivity to bile salts and tolerance towards colicin A but not colicin E1, unlike a null tolQ mutation, which induces tolerance to all group A colicins. Since TolQ is required for colicin A and E1 uptake, in contrast to TolR, which is necessary only for colicin A, we hypothesized that the tolQ925 mutation might affect an interaction between TolQ and TolR. We therefore searched for suppressor mutations in TolR that would restore cell envelope integrity and colicin A sensitivity to the tolQ925 mutant. Five different tolR alleles were isolated and characterized. Four of these suppressor mutations were found to be clustered in the single putative transmembrane helix of TolR (TolR-I) and one was located at the extreme C terminus of the protein. In addition, we isolated a spontaneous intragenic suppressor localized in the first transmembrane helix of TolQ (TolQ-I). These observations strongly suggest that TolR and TolQ interact via their transmembrane segments. Sequence analysis indicates that Ala177 lies on the alpha-helix face of TolQ-III that, according to its composition and evolutionary conservation, is the most likely to be involved in protein/protein interaction. Energy minimization of atomic models of the wild-type and mutated forms of TolQ-III and TolR-I suggests that the deleterious effect of the A177V substitution arises from a direct steric hindrance of this residue with neighboring transmembrane segments, and that suppressor mutations may alleviate this effect either directly or indirectly, e.g. by affecting the stability of conformational equilibrium of the transmembrane region of the complex.

Identification and preliminary characterization of temperature-sensitive mutations affecting HlyB, the translocator required for the secretion of haemolysin (HlyA) from Escherichia coli.

We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesis. From random mutagenesis, three mutants, temperature sensitive (Ts) for secretion, were isolated and the DNA sequenced: Gly10Arg close to the N-terminus, Gly408Asp in a highly conserved small periplasmic loop region PIV, and Pro624Leu in another highly conserved region, within the ATP-binding region. Despite the Ts character of the Gly10 substitution, a derivative of HlyB, in which the first 25 amino acids were replaced by 21 amino acids of the lambda Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispensable for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a conditional lethal mutation in HlyB. We have isolated 4 additional mutations in PIV by directed mutagenesis, giving a total of 5 out of 12 residues substituted in this region, with 4 mutations rendering HlyB defective in secretion. The Pro624 mutation, close to the Walker B-site for ATP binding in the cytoplasmic domain is identical to a mutation in HisP that leads to uncoupling of ATP hydrolysis from the transport of histidine. The expression of a fully functional haemolysin translocation system comprising HlyC,A,B and D increases the sensitivity of E. coli to vancomycin 2.5-fold, compared with cells expressing HlyB and HlyD alone. Thus, active translocation of HlyA renders the cells hyperpermeable to the drug. Mutations in hlyB affecting secretion could be assigned to two classes: those that restore the level of vancomycin resistance to that of E. coli not secreting HlyA and those that still confer hypersensitivity to the drug in the presence of HlyA. We propose that mutations that promote vancomycin resistance will include mutations affecting initial recognition of the secretion signal and therefore activation of a functional transport channel. Mutations that do not alter HlyA-dependent vancomycin sensitivity may, in contrast, affect later steps in the transport process.

Maturation and localization of the TolB protein required for colicin import.

The tolB gene has been shown previously to encode two proteins of 47.5 kDa (TolB) and 43 kDa (TolB*). To explain the presence of these two forms, two hypotheses have been proposed: TolB might be posttranslationally processed to TolB*, or an internal in-frame translation initiation resulting in TolB* may occur (S. K. Levengood and R. E. Webster, J. Bacteriol. 171:6600-6609, 1989). To address this question, TolB was tagged by inserting in its C-terminal region an epitope recognized by monoclonal antibody 1C11 without altering the function of TolB. It was then demonstrated that the functional protein corresponded to TolB*, the mature periplasmic protein, and that TolB was its precursor form, which was observed only when the protein was overexpressed. These two forms were purified by immunoprecipitation, and their N-terminal sequences were determined. An antibody directed against TolB was raised, which confirmed the results obtained with the tagged TolB.

Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.

TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic membrane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a large loop after the first transmembrane region are cytoplasmic. Amino-terminal sequence analysis of TolQ purified from the membrane revealed the presence of the initiating formyl methionine group, suggesting a rapid translocation of the amino-terminal region across the cytoplasmic membrane. Analysis of various tolQ mutant strains suggests that the third transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.

Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity.

TolR is a 142-amino-acid protein required for the import of colicins and bacteriophage and for maintenance of cell envelope integrity. The topology of TolR in the inner membrane was analyzed by two methods. First, bacteria expressing a series of TolR-beta-galactosidase, TolR-alkaline phosphatase, and TolR-beta-lactamase fusions were assayed for the appropriate enzymatic activity. Second, the accessibility of TolR to proteinase K was determined in permeabilized cells and everted vesicles with an antibody elicited against the carboxyl-terminal 70% of TolR. The results are consistent with TolR spanning the inner membrane once via residues 23 to 43 and with the carboxyl-terminal moiety being exposed to the periplasm. Quantitative studies with the anti-TolR antibody indicated the presence of 2 x 10(3) to 3 x 10(3) TolR molecules per cell.

The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein (PAL).

The excC mutants of Escherichia coli are hypersensitive to drugs such as cholic acid and release periplasmic proteins into the extracellular medium. A 1884 bp fragment carrying the excC gene was isolated and sequenced. It contains the 3' end of the tolB gene which maps at min 17 on the E. coli map and an open reading frame which encodes the 18,748 Da ExcC protein. The protein is composed of a hydrophobic region of 22 residues and displayed an overall hydrophilic configuration. It was shown that the ExcC protein is indeed the PAL (peptidoglycan-associated lipoprotein) described by Mizuno (1979). The pal gene had not yet been characterized on the E. coli linkage map since no obvious phenotype could be identified for mutations in this gene. A topologic analysis of the PAL protein using PAL-PhoA translational fusions showed that PAL is associated with the outer membrane only by its N-terminal moiety. The carboxy-terminal part of the protein is necessary for correct interaction of PAL with the peptidoglycan layer.

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.

Cloning of the excC and excD genes involved in the release of periplasmic proteins by Escherichia coli K12.

Strains of Escherichia coli K12 carrying a tolA, tolB, lky or exc mutation located at min 16.5 on the genetic map released periplasmic proteins into the extracellular medium. Wild-type genes defined by these mutations have been cloned from E. coli genomic bank made with plasmid pBR328. Subcloning experiments and complementation studies showed that lky and exc mutations were located either in the previously described tolA and tolB genes or in the newly characterized excC and excD genes. Using minicells, excC and excD gene products were identified as proteins with a molecular mass of 19 and 21 kDa, respectively.

tolA, tolB and excC, three cistrons involved in the control of pleiotropic release of periplasmic proteins by Escherichia coli K12.

Mutants of Escherichia coli K12 carrying exc mutations inducing the release of the plasmid pBR322-encoded beta-lactamase (EC 3.5.2.6) into the extracellular medium were analysed and compared with previously described excretory mutants carrying lky mutations associated with the release of alkaline phosphatase and to tolA and tolB mutants, originally described as tolerant towards various colicins. The exc, lky and tol mutations mapped near the gal operon at min 16.5 of the E. coli linkage map. A genetic analysis presented in this paper showed that some exc and lky mutations belonged to the tolA and tolB complementation groups. Furthermore, we identified a third cistron, excC, involved in the excretion of periplasmic enzymes but distinct from the two others.

Cloning of the lkyB (tolB) gene of Escherichia coli K12 and characterization of its product.

The lkyB gene of Escherichia coli K12 has been cloned from the Clarke and Carbon colony bank by selecting a ColE1 plasmid conferring cholic acid resistance to lkyB mutants. The lkyB gene was localized on hybrid plasmid pJC778 by analysis of mutated plasmids generated by Tn5 insertions. Restriction analysis and complementation studies indicated that plasmid pJC778 carried genes nadA, lkyB and sucA which mapped at min 16.5; the lkyB+ allele was dominant over the lkyB207 mutant allele. Analysis of cell envelope proteins from strains carrying plasmids pJC778 (lkyB+), pJC2578 or pJC2579 (lkyB::Tn5), as well as plasmid-coded proteins in a maxicell system, made it likely that the ikyB gene product was a membrane protein of molecular weight 42,000.