Effect of inorganic ions, H(2)O(2) and pH on the photocatalytic inactivation of Escherichia coli with silver impregnated combustion synthesized TiO(2) catalyst

The photocatalytic antibacterial activity of Ag impregnated combustion synthesized TiO(2) (0.25 g/L) was studied against Escherichia coil in presence of UV irradiation. The effect of various parameters, such as anions, canons, hydrogen peroxide and pH, on the photocatalytic inactivation was investigated. The addition of inorganic ions showed a negative effect on inactivation. Among anions, the presence of chloride ions was observed to have a maximum negative effect and reduced the inactivation considerably. Among cations, the bacterial inactivation reduced significantly in the presence of Ca(2+) ions. Hydrogen peroxide addition in combination with Ag/TiO(2) photocatalysis, however, improved the inactivation. Photocatalysis with high concentration of H(2)O(2) yielded complete bacterial inactivation within few minutes. The photocatalytic inactivation of E. coil was not affected by variation in pH. (C) 2011 Elsevier B.V. All rights reserved.

The beta-Glucoside (bgl) Operon of Escherichia coli Is Involved in the Regulation of oppA, Encoding an Oligopeptide Transporter

We report that the bgl operon of Escherichia coli, encoding the functions necessary for the uptake and metabolism of aryl-beta-glucosides, is involved in the regulation of oligopeptide transport during stationary phase. Global analysis of intracellular proteins from Bgl-positive (Bgl(+)) and Bgl-negative (Bgl(-)) strains revealed that the operon exerts regulation on at least 12 downstream target genes. Of these, oppA, which encodes an oligopeptide transporter, was confirmed to be upregulated in the Bgl(+) strain. Loss of oppA function results in a partial loss of the growth advantage in stationary-phase (GASP) phenotype of Bgl(+) cells. The regulatory effect of the bgl operon on oppA expression is indirect and is mediated via gcvA, the activator of the glycine cleavage system, and gcvB, which regulates oppA at the posttranscriptional level. We show that BglG destabilizes the gcvA mRNA in vivo, leading to reduced expression of gcvA in the stationary phase. Deletion of gcvA results in the downregulation of gcvB and upregulation of oppA and can partially rescue the loss of the GASP phenotype seen in Delta bglG strains. A possible mechanism by which oppA confers a competitive advantage to Bgl(+) cells relative to Bgl(-) cells is discussed.

Chimeras of Escherichia coli and Mycobacterium tuberculosis Single-Stranded DNA Binding Proteins: Characterization and Function in Escherichia coli

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (m beta 1, m beta 1'beta 2, m beta 1-beta 5, m beta 1-beta 6 and m beta 4-beta 5) by transplanting beta 1, beta 1'beta 2, beta 1-beta 5, beta 1-beta 6 and beta 4-beta 5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, m beta 1'beta 2(ESWR) SSB was generated by mutating the MtuSSB specific `PRIY' sequence in the beta 2 strand of m beta 1'beta 2 SSB to EcoSSB specific `ESWR' sequence. Biochemical characterization revealed that except for m beta 1 SSB, all chimeras and a control construct lacking the C-terminal domain (Delta C SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, m beta 1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that m beta 1-beta 6, MtuSSB, m beta 1'beta 2 and m beta 1-beta 5 SSBs complemented E. coli Delta ssb in a dose dependent manner. Complementation by the m beta 1-beta 5 SSB was poor. In contrast, m beta 1'beta 2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

Crystal Structure of Escherichia coli Diaminopropionate Ammonia-lyase Reveals Mechanism of Enzyme Activation and Catalysis

Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of D-and L-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 angstrom resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with DL-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

Visible light photocatalytic inactivation of Escherichia coli with combustion synthesized TiO2

The photocatalytic inactivation of Escherichia coil was studied with combustion synthesized TiO2 photocatalysts in the presence of visible light. A series of 400W lamps irradiating in the visible region of the solar spectrum was used. The effect of various parameters, such as catalyst loading, light intensity, presence of inorganic ions, addition of hydrogen peroxide and pH, on the photocatalytic inactivation of E. coil was investigated. Photolysis alone had a small effect on inactivation while the dark experiment resulted in no inactivation and Ag/TiO2 showed the maximum inactivation. At a catalyst loading of 0.25 g/L, all the combustion synthesized catalysts showed better inactivation of E. coil compared to commercial Degussa P-25 (DP-25) TiO2 catalyst. An improved inactivation was observed with increasing lamp intensity and addition of H2O2. A negative effect on inactivation was observed by addition of inorganic ions such as HCO3-, SO42-, Cl-, NO3-, Na+, K+ and Ca2+. The photocatalytic inactivation of E. coli remained unaltered at different pH of the solution. The inactivation of E. coli was modeled with power law kinetics and was observed to follow first order kinetics. (C) 2012 Elsevier B.V. All rights reserved.

Unconventional initiator tRNAs sustain Escherichia coli

Of all tRNAs, initiator tRNA is unique in its ability to start protein synthesis by directly binding the ribosomal P-site. This ability is believed to derive from the almost universal presence of three consecutive G-C base (3G-C) pairs in the anticodon stem of initiator tRNA. Consistent with the hypothesis, a plasmid-borne initiator tRNA with one, two, or all 3G-C pairs mutated displays negligible initiation activity when tested in a WT Escherichia coli cell. Given this, the occurrence of unconventional initiator tRNAs lacking the 3G-C pairs, as in some species of Mycoplasma and Rhizobium, is puzzling. We resolve the puzzle by showing that the poor activity of unconventional initiator tRNAs in E. coli is because of competition from a large pool of the endogenous WT initiator tRNA (possessing the 3G-C pairs). We show that E. coli can be sustained on an initiator tRNA lacking the first and third G-C pairs; thereby reducing the 3G-C rule to a mere middle G-C requirement. Two general inferences following from our findings, that the activity of a mutant gene product may depend on its abundance in the cell relative to that of the WT, and that promiscuous initiation with elongator tRNAs has the potential to enhance phenotypic diversity without affecting genomic integrity, have been discussed.

Involvement of the Global Regulator H-NS in the Survival of Escherichia coli in Stationary Phase

Long-term batch cultures of Escherichia coli grown in nutrient-rich medium accumulate mutations that provide a growth advantage in the stationary phase (GASP). We have examined the survivors of prolonged stationary phase to identify loci involved in conferring a growth advantage and show that a mutation in the hns gene causing reduced activity of the global regulator H-NS confers a GASP phenotype under specific conditions. The hns-66 allele bears a point mutation within the termination codon of the H-NS open reading frame, resulting in a longer protein that is partially functional. Although isolated from a long-term stationary-phase culture of the parent carrying the rpoS819 allele that results in reduced RpoS activity, the hns-66 survivor showed a growth disadvantage in the early stationary phase (24 to 48 h) when competed against the parent. The hns-66 mutant is also unstable and reverts at a high frequency in the early stationary phase by accumulating second-site suppressor mutations within the ssrA gene involved in targeting aberrant proteins for proteolysis. The mutant was more stable and showed a moderate growth advantage in combination with the rpoS819 allele when competed against a 21-day-old parent. These studies show that H-NS is a target for mutations conferring fitness gain that depends on the genetic background as well as on the stage of the stationary phase.

A Comparative Kinetic and Thermodynamic Perspective of the sigma-Competition Model in Escherichia coli

Transcription is the most fundamental step in gene expression in any living organism. Various environmental cues help in the maturation of core RNA polymerase (RNAP; alpha(2)beta beta'omega) with different sigma-factors, leading to the directed recruitment of RNAP to different promoter DNA sequences. Thus it is essential to determine the sigma-factors that affect the preferential partitioning of core RNAP among various a-actors, and the role of sigma-switching in transcriptional gene regulation. Further, the macromolecular assembly of holo RNAP takes place in an extremely crowded environment within a cell, and thus far the kinetics and thermodynamics of this molecular recognition process have not been well addressed. In this study we used a site-directed bioaffinity immobilization method to evaluate the relative binding affinities of three different Escherichia coli sigma-factors to the same core RNAP with variations in temperature and ionic strength while emulating the crowded cellular milieu. Our data indicate that the interaction of core RNAP-sigma is susceptible to changes in external stimuli such as osmolytic and thermal stress, and the degree of susceptibility varies among different sigma-factors. This allows for a reversible sigma-switching from housekeeping factors to alternate sigma-factors when the organism senses a change in its physiological conditions.

The chbG Gene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

The chb operon of Escherichia coli is involved in the utilization of the beta-glucosides chitobiose and cellobiose. The function of chbG (ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show that chbG encodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of the chb promoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations in chbR conferring the ability to grow on both cellobiose and chitobiose are independent of chbG function for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-beta-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence of chbG, suggesting an additional role of chbG in the hydrolysis of chitooligosaccharides. The homologs of chbG in metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function of E. coli chbG has a broader significance.

Functional Analysis of the Genes Encoding Diaminopropionate Ammonia Lyase in Escherichia coli and Salmonella enterica Serovar Typhimurium

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on DL-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on DL-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on DL-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on DL-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of DL-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by DL-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by DL-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.

The Coil-to-Helix Transition in IlvN Regulates the Allosteric Control of Escherichia coli Acetohydroxyacid Synthase I

The solution structure of IlvN, the regulatory subunit of Escherichia coil acetohydroxyacid synthase I, in the valine-bound form has been determined using high-resolution multidimensional, multinuclear nuclear magnetic resonance (NMR) methods. IlvN in the presence or absence of the effector molecule is present as a 22.5 kDa dimeric molecule. The ensemble of 20 low-energy structures shows a backbone root-mean-square deviation of 0.73 +/- 0.13 angstrom and a root-mean-square deviation of 1.16 +/- 0.13 angstrom for all heavy atoms. Furthermore, more than 98% of the backbone phi and psi dihedral angles occupy the allowed and additionally allowed regions of the Ramachandran map, which is indicative of the fact that the structures are of high stereochemical quality. Each protomer exhibits a beta alpha beta beta alpha beta alpha topology that is a characteristic feature of the ACT domain seen in metabolic enzymes. In the valine-bound form, IlvN exists apparently as a single conformer. In the free form, IlvN exists as a mixture of conformational states that are in intermediate exchange on the NMR time scale. Thus, a large shift in the conformational equilibrium is observed upon going from the free form to the bound form. The structure of the valine-bound form of IlvN was found to be similar to that of the ACT domain of the unliganded form of IlvH. Comparisons of the structures of the unliganded forms of these proteins suggest significant differences. The structural and conformational properties of IlvN determined here have allowed a better understanding of the mechanism of regulation of branched chain amino acid biosynthesis.

Catalytic activity of Peptidase N is required for adaptation of Escherichia coli to nutritional downshift and high temperature stress

Peptidase N (PepN), the sole M1 family member in Escherichia coli, displays broad substrate specificity and modulates stress responses: it lowers resistance to sodium salicylate (NaSal)-induced stress but is required during nutritional downshift and high temperature (NDHT) stress. The expression of PepN does not significantly change during different growth phases in LB or NaSal-induced stress; however, PepN amounts are lower during NDHT stress. To gain mechanistic insights on the roles of catalytic activity of PepN in modulating these two stress responses, alanine mutants of PepN replacing E264 (GAMEN motif) and E298 (HEXXH motif) were generated. There are no major structural changes between purified wild type (WT) and mutant proteins, which are catalytically inactive. Importantly, growth profiles of Delta pepN upon expression of WT or mutant proteins demonstrated the importance of catalytic activity during NDHT but not NaSal-induced stress. Further fluorescamine reactivity studies demonstrated that the catalytic activity of PepN is required to generate higher intracellular amounts of free N-terminal amino acids; consequently, the lower growth of Delta pepN during NDHT stress increases with high amounts of casamino acids. Together, this study sheds insights on the expression and functional roles of the catalytic activity of PepN during adaptation to NDHT stress. (C) 2012 Elsevier GmbH. All rights reserved.

Non-neutral evolution in non-LEE-encoded type III effectors of attaching and effacing Escherichia coli

Attaching and effacing Escherichia coli (AEEC) employ type III secretion system (T3SS) to secrete effector proteins into host cells and regulate their function. Here we have investigated T3SS genes of AEEC for non-neutral evolution. Our analysis revealed non-neutral evolution in three genes (nleE1, nleB2 and nleD) which encode effector proteins. These genes are located outside the locus of enterocyte effacement (LEE). In general, non-LEE effector genes show greater deviation from neutral evolution than LEE effector genes. These results suggest that effector genes located outside LEE are under greater selection pressure than those present in LEE. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.