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Many Escherichia coli phages have been sequenced, but in most cases their sequences alone do not suffice to predict their host specificity. Analysis of phage resistant E. coli K-12 mutants have uncovered a certain set of outer membrane proteins and polysaccharides as receptors. In this review, a compilation of E. coli K12 phage receptors is provided and their functional characterization, often driven by studies on phage resistant mutants, is discussed in the historical context. While great progress has been made in this field thus far, several proteins in the outer membrane still await characterization as phage receptors.

Pyruvate, the key regulator in connection of a variety of metabolic pathways, influences transcription of the Escherichia coli genome through controlling the activity of two pyruvate-sensing two-component systems (TCSs), BtsSR and PyrSR. Previously, we identified the whole set of regulatory targets of PyrSR with low-affinity to pyruvate. Using gSELEX screening system, we found here that BtsSR with high-affinity to pyruvate regulates more than 100 genes including as many as 13 transcription factors genes including the csgD gene encoding the master regulator of biofilm formation. CsgD regulates more than 20 target genes including the csg operons encoding the Curli fimbriae. In addition, we identified the csgBAC as one of the regulatory targets of BtsR, thus indicating the involvement of two pyruvate-dependent regulatory pathways of the curli formation: indirect regulation by CsgD; and direct regulation by BtsR. Based on the findings of the whole set of regulatory targets by two pyruvate-sensing BtsR and PyrR, we further propose an innovative concept that the pyruvate level-dependent regulation of different gene sets takes place through two pyruvate-sensing TCS systems, high-affinity BtsSR and low-affinity PyrSR to pyruvate.

Soluble forms of tellurium are environmental contaminants that are toxic to microorganisms. While tellurite [Te(IV)] is a well-characterized antimicrobial agent, little is known about the interactions of tellurate [Te(VI)] with bacterial cells. In this study, we investigated the role of sulfate transporters in the uptake of tellurate in Escherichia coli K-12. Mutant strains carrying a deletion of the cysW gene in the CysPUWA sulfate transporter system accumulated less cellular tellurium and exhibited higher resistance to tellurate compared with the wild-type strain. Complementation of the mutation restored tellurate sensitivity and uptake. These results indicate that tellurate enters E. coli cells to cause toxic effects via the CysPUWA sulfate transporter.

The genome of Escherichia coli K-12 contains ten cryptic phages, altogether constituting about 3.6% of the genome in sequence. Among more than 200 predicted genes in these cryptic phages, 14 putative transcription factor (TF) genes exist, but their regulatory functions remain unidentified. As an initial attempt to make a breakthrough for understanding the regulatory roles of cryptic phage-encoded TFs, we tried to identify the regulatory function of CP4-6 cryptic prophage-encoded YagI with unknown function. After SELEX screening, YagI was found to bind mainly at a single site within the spacer of bidirectional transcription units, yagA (encoding another uncharacterized TF) and yagEF (encoding 2-keto-3-deoxy gluconate aldolase, and dehydratase, respectively) within this prophage region. YagEF enzymes are involved in the catabolism of xylose downstream from xylonate. We then designated YagI as XynR (regulator of xylonate catabolism), one of the rare single-target TFs. In agreement with this predicted regulatory function, the activity of XynR was suggested to be controlled by xylonate. Even though low-affinity binding sites of XynR were identified in the E. coli K-12 genome, they all were inside open reading frames, implying that the regulation network of XynR is still fixed within the CR4-6 prophage without significant influence over the host E. coli K-12.

The OmpW family consists of a ubiquitous group of small outer membrane (OM) β-barrel proteins of Gram-negative bacteria with proposed roles in environmental adaptation but poorly understood mechanisms of expression. We report here that Escherichia coli K-12 OmpW contents are drastically modified by temperature changes compatible with the leap from the environment to warm-blooded hosts and/or vice versa. Thus, while OmpW is present in the OM of bacteria grown at 37 °C, it sharply disappears at 23 °C with the concomitant acquisition of colicin S4 resistance by the cells. ompW::lacZY fusions indicated that temperature regulation operates at the level of transcription, being ompW expression almost abolished at 23 °C as compared to 37 °C. Moreover, E. coli Δhns mutants lacking H-NS showed reductions in ompW transcription and OmpW contents at 37 °C, indicating positive modulatory roles for this nucleoid-structuring protein in ompW expression. Also, ΔhnsΔstpA double mutants simultaneously lacking H-NS and its paralog StpA showed more severe reductions in ompW expression at 37 °C, resulting in the complete loss of OmpW. The overall results indicate that OmpW contents in E. coli are regulated by both temperature and H-NS and reinforce OmpW functions in bacterial adaptation to warm-blooded hosts Fil: Brambilla, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Moran Barrio, Jorgelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Viale, Alejandro Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Next-generation sequencing has significantly reduced the cost of genome-sequencing projects, resulting in an expressive increase in the availability of genomic data in public databases. The cheaper and easier is to sequence new genomes, the more accurate the annotation steps have to be to avoid both the loss of information and the accumulation of erroneous features that may affect the accuracy of further analysis. In the case of bacteria genomes, a range of web annotation software has been developed; however, many applications have yet to incorporate the steps required to improve their result, including the removal of false-positive/spurious and a more complete identification of non-coding features. We present Genix, a new web-based bacterial genome annotation pipeline. A comparison of the results generated by Genix for four reference genomes against those generated by other annotation tools indicated that our pipeline is able to provide results that are closer to the reference genome annotation, with a smaller amount of false-positive proteins and missing functional annotated proteins. Additionally, the metrics obtained by Genix were slightly better than those obtained by Prokka, a state-of-art standalone annotation system. Our results indicate that Genix is a useful tool that is able to provide a more refined result, and may be a user-friendly way to obtain high-quality results.

It has been widely reported that pH mediates cadmium toxicity to bacteria. We used a tripartite approach to investigate mechanisms by which pH affects cadmium toxicity that included analyses of: (1) growth kinetics, (2) global gene expression, and (3) cadmium speciation. Cadmium extended the lag phase at pH 7, but not at pH 5. DNA microarray analysis revealed that stress response genes including hdeA, otsA , and yjbJ were more highly expressed at pH 5 than at pH 7 after only 5 min of exposure to cadmium, suggesting that acidic pH more rapidly induced genes that confer cadmium resistance. In addition, genes involved in transport and many hypothetical genes were more highly expressed at pH 5 than at pH 7 in the presence of cadmium. Concentrations of two cadmium species, including one previously implicated in the mechanism by which pH mediates cadmium toxicity (CdOH+), increased with pH. Our data demonstrate that transcriptional responses of Escherichia coli to cadmium are substantially affected by pH and suggest that several stress response, transport, and hypothetical genes play roles in the mechanism by which pH mediates cadmium toxicity.

The Rcs phosphorelay is an important signalling pathway that is conserved throughout the Enterobacteriaceae. The Rcs phosphorelay is composed of the RcsC sensor kinase, a membrane-localized HPt-containing protein RcsD and the cytoplasmic response regulator RcsB. In this study we were interested in studying the degree of functional conservation between the different enteric RcsC homologues. Therefore, we tested for the ability of RcsC homologues from pathogenic Escherichia coli, Salmonella enterica and Yersinia pestis to functionally complement an rcsC mutation in E. coli K-12. Complementation was measured as (1) an increase in cpsB-lacZ expression in response to DjlA overproduction, a well-established signal for RcsC activation and (2) the ability to restore biofilm formation. All of the homologues increased cpsB-lacZ expression in response to DjlA overproduction confirming that the different RcsC homologues are able to sense this stimulus and to transduce the signal to the downstream components of the Rcs pathway in E. coli. This suggests that the core signalling domains of RcsC are functionally conserved throughout the Enterobacteriaceae. However, we also show that RcsC from Y. pestis was unable to restore normal biofilm formation in the E. coli K-12 rcsC mutant and we argue that this is due to the increased net kinase activity observed with this homologue.

Transport of three synthesized endothiopeptides: AlaPsi[CSNH]Ala, AlaPsi[CSNH]Leu, and AlaPsi[CSNH]Phe, into Escherichia coli K12 mutant strains and enzymatic degradation studies were carried out. These compounds, well transported by permeases, but significantly more resistant to enzymatic cleavage than the corresponding natural peptides, seem to be useful as enzyme inhibitor carriers.

The metabolic pathway involving dihydroxyacetone is poorly characterized although novel enzymes associated with this metabolite have recently been demonstrated. The role of GldA in dihydroxyacetone and methylglyoxal metabolism was investigated by purifying the enzyme and characterizing its catalytic ability using nuclear magnetic resonance (NMR) spectroscopy. At neutral pH, the enzyme exhibits much higher affinities towards dihydroxyacetone, methylglyoxal, and glycolaldehyde than glycerol with K(m) values of 0.30, 0.50, 0.85, and 56 mM, respectively. This is consistent with NMR data with crude extracts, showing that the conversion from dihydroxyacetone to glycerol by GldA is far more efficient than the reverse reaction. Dihydroxyacetone was found to be lethal at higher concentration with an LC(50) value of 28 mM compared with 0.4 mM of methylglyoxal, while lactaldehyde was found to exhibit significant growth inhibition in Escherichia coli cells. The toxicity of dihydroxyacetone appears to be due to its intracellular conversion to an aldehyde compound, presumably methylglyoxal, since the glyoxalase mutant becomes sensitive to dihydroxyacetone. Based on information that gldA is preceded in an operon by the ptsA homolog and talC gene encoding fructose 6-phosphate aldolase, this study proposes that the primary role of gldA is to remove toxic dihydroxyacetone by converting it into glycerol.

Escherichia coli K-12 contains nine paralogs of CspA, namely CspA-CspI. In spite of considerable sequence similarity among these genes, the individual members of this family show significant differences in their expression regulation. Among these nine members, cspA, B, G and I have been reported to be cold-induced. The unusually long 5'-untranslated region (5'-UTR) of these four and other cold-induced genes has often been associated with their inducibility. Sequence analysis of the cspE upstream region revealed two promoter-like motifs having high scores. We identified the promoter site and established that cspE has a much shorter 5'-UTR compared to other cold-induced genes. Our results showed that cspE is induced to about threefold at both the transcript and the protein level in response to cold-shock. Its transcript half-life increases significantly upon cold-shock. Furthermore, we demonstrated that RNase E, a key endonuclease responsible for mRNA degradation in E. coli, regulates cspE transcript stability, possibly through the assembly of a degradosome. In silico analysis of the cspE 5'-UTR revealed alternative secondary structures at 37 and 15 degrees C. A point mutation that was predicted to relax the secondary structure of the 5'-UTR at 15 degrees C showed considerable reduction in transcript stability, indicating that alternative transcript secondary structures might be the cause of the differential stability.

Antibiotic-resistant bacteria are a great threat to human health and food safety and there is an urgent need to understand the mechanisms of resistance for combating these bacteria. In the current study, comparative proteomic methodologies were applied to identify Escherichia coli K-12 outer membrane (OM) proteins related to kanamycin resistance. Mass spectrometry and western blotting results revealed that OM proteins TolC, Tsx and OstA were up-regulated, whereas MipA, OmpA, FadL and OmpW were down-regulated in kanamycin-resistant E. coli K-12 strain. Genetic deletion of tolC (ΔtolC-Km) led to a 2-fold decrease in the minimum inhibitory concentration (MIC) of kanamycin and deletion of mipA (ΔmipA-Km) resulted in a 4-fold increase in the MIC of kanamycin. Changes in the MICs for genetically modified strains could be completely recovered by gene complementation. Compared with the wild-type strain, the survival capability of ΔompA-Km was significantly increased and that of Δtsx-Km was significantly decreased. We further evaluated the role and expression of MipA in response to four other antibiotics including nalidixic acid, streptomycin, chloramphenicol and aureomycin, which suggested that MipA was a novel OM protein related to antibiotic resistance.

There are many physiological differences between Escherichia coli B and K-12 strains, owing to their different origins. Deeper insight into the metabolic and regulative mechanisms of these strains will inform improved usage of these industrial workhorses. In the present study, we observed that BL21 fermentation broth gradually turned yellow during cultivation. By spectral analysis and liquid chromatography-mass spectrometry identification, we confirmed for the first time that the yellow substance accumulated in the fermentation broth is riboflavin. Comparing the enzyme sequences involved in riboflavin metabolism between BL21 and MG1655, we identified a site mutation on the 115 residue of bifunctional riboflavin kinase/FMN adenylyltransferase (RibF) in BL21. This His115Leu mutation was found to reduce enzyme activity to 55% of that of MG1655, which is probably one reason for riboflavin accumulation in BL21. Quantitative PCR analysis showed that genes of the entire branch of the riboflavin and FAD biosynthesis pathways in BL21 were up-regulated. Several physiological and metabolic characteristics of BL21 and MG1655 were found to be different, and may also be related to the riboflavin accumulation.

The mobile insertion element IS5 is a relatively small but genetically compact DNA sequence of 1195 bp found in variable copy number in the genome of Escherichia coli strains. This study presents a detailed transcript analysis of the population of IS5 elements present in E. coli strains MC4100 and MG1655. The findings indicate that the ins5A gene comprising 978 bp is transcribed from its own promoter, which is located close to the right-hand end of the element. The two divergently transcribed genes ins5C and in5B form an operon, and this transcript is fully contained within the borders of the ins5A transcript. Although transcription out of IS5 from element-internal promoters was negligible, in the case of MG1655 a major transcript was found to extend into the insertion element. This suggests that IS5-specific transcription can be influenced by the specific location of the element in the chromosome, the orientation it adopts and the gene it interrupts.

Subclonal analysis of spontaneous zygogenesis (Z-mating) products of Escherichia coli K12 was undertaken to grasp the extent of vertical and horizontal evolution in unstable strains expressing one parental or recombinant genome. Isolates were obtained following serial cultures or serial intercrosses. A high diversity of strains was obtained, among which some resumed the phenotype of the partners of the initial or subsequent Z-matings. When non-complementing diploids are infected with a mixture of distinct temperate bacteriophages, lysogenisation occurs at the expenses of the active chromosome only. This event is associated with an alternate expression of prophage and chromosomal genes. Present work provides further evidence for the existence of non-complementing diploidy and opens a novel route for virus research in general.

Glyoxal (GO) belongs to the reactive electrophilic species generated in vivo in all organisms. In order to identify targets of GO and their response mechanisms, we attempted to screen for GO-sensitive mutants by random insertions of Tn phoA -132. The genes responsible for GO susceptibility were functionally classified as the following: (i) tRNA modification; trmE , gidA and truA , (ii) DNA repair; recA and recC , (iii) toxin–antitoxin; mqsA and (iv) redox metabolism; yqhD and caiC . In addition, an insertion in the crp gene, encoding the cAMP responsive transcription factor, exhibits a GO-resistant phenotype, which is consistent with the phenotype of adenylate cyclase ( cya ) mutant showing GO resistance. This suggests that global regulation involving cAMP is operated in a stress response to GO. To further characterize the CRP-regulated genes directly associated with GO resistance, we created double mutants deficient in both crp and one of the candidate genes including yqhD , gloA and sodB . The results indicate that these genes are negatively regulated by CRP as confirmed by real-time RT-PCR. We propose that tRNA as well as DNA are the targets of GO and that toxin/antitoxin, antioxidant and cAMP are involved in cellular response to GO.

The mercury-resistance transposon Tn5053 inhibits restriction activity of the type I restriction-modification endonuclease EcoKI in Escherichia coli K12 cells. This is the first report of antirestriction activity of a non-conjugative transposon. The gene (ardD) coding for the antirestriction protein has been cloned. The ardD gene is located within the tniA gene, coding for transposase, on the complementary strand. The direction of transcription is opposite to transcription of the tniA gene.

Bacteria are present extensively in the environment. Investigation of their antioxidant properties will be useful for further study on atrazine stress tolerance of bacteria and the defense mechanism of antioxidant enzymes against atrazine or other triazine herbicides. Superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and total antioxidant capacity (T-AOC) from one Gram-negative representative strain Escherichia coli K12 and one Gram-positive representative strain Bacillus subtilis B19, respectively, were tested for response to atrazine stress. The results indicated that SOD, CAT, GST and T-AOC were induced upon exposure to atrazine. The growth of two bacteria was better in the absence than in the presence of atrazine, indicating that atrazine can decrease bacterial growth. The changes of enzyme activities indicate the presence of oxidative stress. Oxidative stress induced by atrazine may be due to imbalance of redox potential in bacterial cells, which leads to bacterial metabolic disorder.

The synthesis of total cellular proteins in Escherichia coli K12 was studied in batch culture following exposure of cells to low concentrations of monochlorophenol, pentachlorophenol and cadmium chloride. Changes in protein patterns were identified after pulse-chase labelling of proteins with [35S]methionine and subsequent two-dimensional gel electrophoresis (2D-PAGE). We demonstrated that besides the induction of some stress proteins, also a transient decrease in the rate of synthesis of other proteins occurred. Two of these proteins were identified as OmpF and aspartate transcarbamoylase (ATCase). Their transient repression appeared to be a general response to stress elicited by different pollutants and may therefore be used as a general and sensitive early warning system for pollutant stress.

Escherichia coli has been used widely in laboratory and the biotech industry. However, the genetic and metabolic characteristics remain inadequately studied, particularly for those strains with extensive genetic manipulations that might have resulted in unknown mutations. Here, we demonstrate a comparative proteomics and genetics approach to identify unknown mutations in E. coli K-12 derivatives. The comparative proteomic and genetic analyses revealed an IS5 disruption of the kdgR gene in two commonly used derivative strains of E. coli K-12, XL1-Blue and DH5α, compared with K-12 wild-type strain W3110. In addition, a controversial deoR mutation was clarified as a wild type in E. coli DH5α using the same approach. This approach should be useful in characterizing the unknown mutations in various mutant strains developed. At the same time, comparative proteomic analysis also revealed the distinct metabolic characteristic of the two derivatives: higher biosynthetic flux to purine nucleotides. This is potentially beneficial for the synthesis of plasmid DNA.

TraJ is an activator of the transfer (tra) operon in the F plasmid that counteracts H-NS silencing at the main transfer promoter (P(Y)). TraJ contains 226 aa (26 670 kDa), not 229 aa as reported previously, and forms homodimers. TraJ binds DNA containing P(Y)in vivo as demonstrated using a chromatin-immunoprecipitation assay. Mutations within a predicted helix-turn-helix DNA-binding motif reduced binding and decreased mating efficiency. The deletion of four or more residues from the C-terminus of TraJ blocked its activity, but did not interfere with DNA binding. This feature, as well as homology to the C-terminal region of RovA and SlyA within the MarR/SlyA family, suggests that TraJ might counteract H-NS repression via a mechanism similar to these desilencing proteins.

Random mutagenesis has been used to identify the target DNA sites for the MalI repressor at the divergent Escherichia coli K-12 malX-malI promoters. The malX promoter is repressed by MalI binding to a DNA site located from position -24 to position -9, upstream of the malX promoter transcript start. The malI promoter is repressed by MalI binding from position +3 to position +18, downstream of the malI transcript start. MalI binding at the malI promoter target is not required for repression of the malX promoter. Similarly, MalI binding at the malX promoter target is not required for repression of the malI. Although the malX and malI promoters are regulated by a single DNA site for cyclic AMP receptor protein, they function independently and each is repressed by MalI binding to a different independent operator site.

In a previous work, it was observed that the swarming of polyamine-deficient Proteus mirabilis (speB::sm) was severely inhibited on Luria-Bertani (LB) swarming plates (LBSw) (LB, 0.5% glucose, 0.5% agar), and it was clarified that extracellular putrescine was important as a signaling molecule for the induction of swarming in P. mirabilis. However, a polyamine-deficient strain (delta-speAB delta-speC) of Escherichia coli swarmed as well as the parental strain on LBSw plates. We report that the swarming phenotype of a polyamine-deficient E. coli strain is dependent on spermidine and PotABCD, a spermidine importer.

Little is known about the dynamics of cellular growth, death, and evolution within bacterial biofilms. Here we show evidence of evolution within single-species biofilms in real time. Escherichia coli harvested from 22-day-old biofilms express a competitive advantage over cells incubated in biofilms for shorter periods of time. This advantage is manifested as the ability of aged cells to outcompete younger cells in the presence of a pre-existing biofilm, even though cells from older biofilms do not express an increased ability to form initial biofilms on a fresh, unoccupied surface. This phenomenon is similar to the growth advantage in stationary phase, or GASP, phenotype exhibited by planktonically grown cells when incubated under competitive conditions. The ability of bacteria in biofilms to show rapid heritable change has implications for our understanding of the adaptive abilities of biofilms in a wide variety of natural and man-made environments.

The Escherichia coli K-12 malI-malX intergenic region contains two divergent promoters, which have been investigated by both mutational and biochemical analysis. The malX promoter drives transcription initiation from a location that is 43 bp upstream from the malX translation start codon. Expression from the malX promoter is dependent on binding of the cyclic AMP receptor protein (CRP) to a DNA site centred 41.5 bp upstream of the transcript start. The malI promoter drives transcription initiation from a location 85 bp upstream from the malX transcript start and it is active without the CRP. Expression from the malI promoter can be stimulated by the CRP. Mutational analysis suggests that the malI promoter has an unusual organization.

Peroxiredoxins are a class of peroxide-scavenging enzymes having a conserved cysteine residue(s) in their active centers. Thiol peroxidase (Tpx) is one of the peroxiredoxins identified in Escherichia coli. Despite the absence of the N-terminal signal sequence for transport across the membrane, it has been characterized as a periplasmic protein. Reanalysis of Tpx localization, using active site cysteine mutants of thioredoxin 1 (Trx1), demonstrated that Tpx forms a mixed-disulfide complex with cytoplasmic Trx1, indicating that Tpx localizes in the cytoplasm.

The Tol system is an interactive set of envelope proteins that includes both outer and cytoplasmic membrane proteins. Central to this system is TolA, which spans the periplasmic space to communicate with residents of each membrane. To identify motifs involved in the protein/protein interactions through which TolA acts, the ability of a phylogenetically distinct TolA protein from Yersinia enterocolitica to function in the Tol system of Escherichia coli was examined. Although at least 59 codons shorter and only c. 67% identical to its E. coli homologue, the Y. enterocolitica gene encoded a protein that supported the physiological function of the Tol system in E. coli, and conferred sensitivity to the TolA-dependent colicins A, K, and E1, but interestingly, not to colicin N. The correlation of conferred phenotype with sequence similarities and differences provides a first step in defining essential structural motifs and their specific contributions to function.

Siderocalin is part of the innate immune system and is secreted by epithelial cells in the early stages of inflammation. This protein binds catecholate siderophores such as enterobactin with high affinity. As a consequence, the iron supply and growth of the siderophore-producing bacteria is disturbed. Recombinant siderocalin isolated from strains of Escherichia coli K-12 contained bound enterobactin. Very low amounts of siderocalin were isolated from another K-12 strain unable to produce enterobactin, which indicated that enterobactin might stabilize the recombinant protein. Siderocalin isolated from E. coli strain Nissle 1917, which produces the glucosylated enterobactin salmochelin, contained a mixture of bound salmochelin (55%) and enterobactin (45%). The growth of an enterobactin-producing E. coli K-12 strain but not of the same strain carrying a plasmid encoding iroBCDEN and therefore able to produce salmochelin was suppressed when siderocalin was added to the medium, which indicated that salmochelin is bound by siderocalin before siderocalin folds into its final conformation in the cell, and that binding of salmochelin to matured siderocalin is not possible. Salmochelin is mainly produced by pathogenic enterobacteria. Glucosylation of enterobactin may be a mechanism by which these bacteria evade trapping of the enterobactin by siderocalin.

Recent genomic studies with Escherichia coli K-12 have suggested scores of previously unexplored targets for the cyclic AMP receptor protein (CRP) global transcription regulator. Eleven of these loci were cloned and CRP binding was demonstrated at eight of these targets. It is shown that CRP can activate transcription at five of these targets and the functional DNA sites for CRP are identified. It is reported that CRP functions as a Class I activator at the aer promoter and as a Class II activator at the gatY, sdaC, ychH and malX promoters.

Previous reports have suggested that Escherichia coli K12 mutants defective in the expression of oligogopeptide permease protein A (OppA) exhibit reduced sensitivity to aminoglycosides due to altered permeability of the cell envelope. In this work, the role of the OppA protein, and the oligogopeptide permease (Opp) transport system has been evaluated, in the resistance to aminoglycosides using derivatives of the E. coli K12 SS320 strain selected for triornithine resistance or with a deletion of the complete opp operon. All tested mutants were defective in the uptake of tri- and tetra-peptides but did not expressed resistance to aminoglycosides. Additionally, complementation tests carried out with a plasmid encoding the OppA protein did not affect the sensitivity of the strains to these antibiotics. Taken together, these evidences indicate that the Opp uptake system, as well as the OppA protein, does not play a direct role in the sensitivity to aminoglycosides in E. coli K12.

We describe a genetic β-galactoside reporter system using a disk diffusion assay on MacConkey Lactose agar petri plates to monitor maintenance of the bacteriophage λ prophage state and viral induction in Escherichia coli K-12. Evidence is presented that the phage λ major lytic promoters, pL and pR , are activated when cells containing the reporters are exposed to the energy poison carbonyl cyanide m -chlorophenyl hydrazine, CCCP. This uncoupler of oxidative phosphorylation inhibits ATP synthesis by collapsing the proton motive force. Expression of the λ lytic promoters in response to CCCP requires host RecA function and an auto-cleavable CI repressor, as does SOS induction of the λ prophage that occurs by a DNA damage-dependent pathway. λ Cro function is required for CCCP-mediated activation of the λ lytic promoters. CCCP does not induce an sfi-lacZ SOS reporter.

Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.6 kb. Initial attempts to localize the gene(s) responsible for antimicrobial activity by subcloning into pUC-based vectors failed. A new plasmid for toxic gene expression (pTGEX) was designed enabling localization of the antibacterial activity to a 4.7-kb HindIII fragment. Transposon mutagenesis localized the gene to an open reading frame of 483 bp designated antibacterial protein1 (abp1). Abp1 was 94% identical to a hypothetical protein of Neisseria subflava (accession number WP_004519448.1). An Escherichia coli clone expressing Abp1 exhibited antibacterial activity against Bacillus subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and B4268, and S. aureus NCTC 12493,ATCC 35696 and NCTC 11561. However, no antibacterial activity was observed against Pseudomonas aeruginosa ATCC 9027, N. subflava ATCC A1078, E. coli K12 JM109 and BL21(DE3) Fusobacterium nucleatum ATCC 25586 and NCTC 11326, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 or Lactobacillus casei NCTC 6375.

Bacterial surface components have a major role in the development of biofilms. In the present study, the effect of Escherichia coli O8-antigen on biofilms was investigated using two E. coli K-12 derived strains that differed only in the O8-antigen biosynthesis. In the presence of O8-antigen both bacterial adhesion and biofilm formation slightly decreased under static conditions whereas a substantial increase in adhesion and biofilm formation was observed under agitated conditions. It was noted that, irrespective of the O8-antigen status, the hydrophobic interactions played an important role in bacterial adhesion under both static and agitated conditions. However, under agitated conditions, the extent of bacterial adhesion in the O8-antigen bearing strain was predominantly determined by the electrostatic interactions. Results showed that the presence of O8-antigen decreases the surface hydrophobicity and surface charge. Moreover, O8-antigen facilitates adhesion on hydrophilic and hydrophobic surfaces as revealed through tests with modified substrata. Our results indicate that O8-antigen, which appears dispensable for biofilm formation under static conditions, actually enhances E. coli biofilm formation under agitated conditions.

gamma-Glutamyl-gamma-aminobutyrate hydrolase (PuuD) was purified and the properties of the enzyme were characterized. The active center of PuuD was identified as Cys-114 by site-directed mutagenesis. The expression of PuuD was induced by putrescine and O2 (substrates of the Puu pathway), while the addition of succinate or NH4Cl (products of the Puu pathway) to the medium reduced the expression of PuuD. The findings that the puuD-deficient strain accumulated gamma-glutamyl-gamma-aminobutyrate (gamma-Glu-GABA) and could not grow on putrescine as a sole nitrogen source indicate that PuuD is physiologically important as a gamma-Glu-GABA hydrolase.