Repression of btuB gene transcription in Escherichia coli by the GadX protein

<p>Abstract</p> <p>Background</p> <p>BtuB (B twelve uptake) is an outer membrane protein of <it>Escherichia coli</it>, it serves as a receptor for cobalamines uptake or bactericidal toxin entry. A decrease in the production of the BtuB protein would cause <it>E. coli </it>to become resistant to colicins. The production of BtuB has been shown to be regulated at the post-transcriptional level. The secondary structure switch of 5' untranslated region of <it>butB </it>and the intracellular concentration of adenosylcobalamin (Ado-Cbl) would affect the translation efficiency and RNA stability of <it>btuB</it>. The transcriptional regulation of <it>btuB </it>expression is still unclear.</p> <p>Results</p> <p>To determine whether the <it>btuB </it>gene is also transcriptionally controlled by trans-acting factors, a genomic library was screened for clones that enable <it>E. coli </it>to grow in the presence of colicin E7, and a plasmid carrying <it>gadX </it>and <it>gadY </it>genes was isolated. The <it>lacZ </it>reporter gene assay revealed that these two genes decreased the <it>btuB </it>promoter activity by approximately 50%, and the production of the BtuB protein was reduced by approximately 90% in the presence of a plasmid carrying both <it>gadX </it>and <it>gadY </it>genes in <it>E. coli </it>as determined by Western blotting. Results of electrophoretic mobility assay and DNase I footprinting indicated that the GadX protein binds to the 5' untranslated region of the <it>btuB </it>gene. Since <it>gadX </it>and <it>gadY </it>genes are more highly expressed under acidic conditions, the transcriptional level of <it>btuB </it>in cells cultured in pH 7.4 or pH 5.5 medium was examined by quantitative real-time PCR to investigate the effect of GadX. The results showed the transcription of <it>gadX </it>with 1.4-fold increase but the level of <it>btuB </it>was reduced to 57%.</p> <p>Conclusions</p> <p>Through biological and biochemical analysis, we have demonstrated the GadX can directly interact with <it>btuB </it>promoter and affect the expression of <it>btuB</it>. In conclusion, this study provides the first evidence that the expression of <it>btuB </it>gene is transcriptionally repressed by the acid responsive genes <it>gadX </it>and <it>gadY</it>.</p

A putative lytic transglycosylase tightly regulated and critical for the EHEC type three secretion

Open reading frame l0045 in the pathogenic island of enterohemorrhagic Escherichia coli O157:H7 has been predicted to encode a lytic transglycosylase that is homologous to two different gene products encoded by the same bacteria at loci away from the island. To deduce the necessity of the presence in the island, we created an l0045-deleted strain of EHEC and observed that both the level of cytosolic EspA and that of the other type III secreted proteins in the media were affected. In a complementation assay, a low level-expressing L0045 appeared to recover efficiently the type III secretion (TTS). On the other hand, when l0045 was driven to express robustly, the intracellular levels of representative TTS proteins were severely suppressed. This suppression is apparently caused by the protein of L0045 per se since introducing an early translational termination codon abolished the suppression. Intriguingly, the authentic L0045 was hardly detected in all lysates of EHEC differently prepared while the same construct was expectedly expressed in the K-12 strain. A unique network must exist in EHEC to tightly regulate the presence of L0045, and we found that a LEE regulator (GrlA) is critically involved in this regulation

Factors Affecting Daughter Cells' Arrangement during the Early Bacterial Divisions

On agar plates, daughter cells of Escherichia coli mutually slide and align side-by-side in parallel during the first round of binary fission. This phenomenon has been previously attributed to an elastic material that restricts apparently separated bacteria from being in string. We hypothesize that the interaction between bacteria and the underneath substratum may affect the arrangement of the daughter bacteria. To test this hypothesis, bacterial division on hyaluronic acid (HA) gel, as an alternative substratum, was examined. Consistent with our proposition, the HA gel differs from agar by suppressing the typical side-by-side alignments to a rare population. Examination of bacterial surface molecules that may contribute to the daughter cells' arrangement yielded an observation that, with disrupted lpp, the E. coli daughter cells increasingly formed non-typical patterns, i.e. neither sliding side-by-side in parallel nor forming elongated strings. Therefore, our results suggest strongly that the early cell patterning is affected by multiple interaction factors. With oscillatory optical tweezers, we further demonstrated that the interaction force decreased in bacteria without Lpp, a result substantiating our notion that the side-by-side sliding phenomenon directly reflects the strength of in-situ interaction between bacteria and substratum

A role of ygfZ in the Escherichia coli response to plumbagin challenge

Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. Escherichia coli strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin treatment triggered up-regulation of many genes in E. coli including ahpC, mdaB, nfnB, nfo, sodA, yggX and ygfZ. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, ygfZ and sodA were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide stress but not restore the resistance of bacteria when encountering plumbagin at the absence of ygfZ. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance when sodA was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for the resistance of E. coli to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished apparently from the culture of YgfZ-expressing E. coli. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture supernatant of the wild type E. coli but not in the ΔygfZ mutant. Our results showed that the presence of ygfZ is not only critical for the E coli resistance to plumbagin but also facilitates the plumbagin degradation

Search for a Very Light CP-Odd Higgs Boson in Top Quark Decays from p(p)over-bar Collisions at root s=1.96 TeV

We present the results of a search for a very light CP-odd Higgs boson a(1)(0) originating from top quark decays t -> H(+/-)b -> W-+/-(*) a(1)(0)b, and subsequently decaying into tau(+)tau(-). Using a data sample corresponding to an integrated luminosity of 2.7 fb(-1) collected by the CDF II detector in p (p) over bar collisions at 1.96 TeV, we perform a search for events containing a lepton, three or more jets, and an additional isolated track with transverse momentum in the range 3 to 20 GeV/c. Observed events are consistent with background sources, and 95% C.L. limits are set on the branching ratio of t -> H(+/-)b for various masses of H-+/- and a(1)(0)

Varied Immunity Generated in Mice by DNA Vaccines with Large and Small Hepatitis Delta Antigens

Whether the hepatitis delta virus (HDV) DNA vaccine can induce anti-HDV antibodies has been debatable. The role of the isoprenylated motif of hepatitis delta antigens (HDAg) in the generation of immune responses following DNA-based immunization has never been studied. Plasmids p2577L, encoding large HDAg (L-HDAg), p2577S, expressing small HDAg (S-HDAg), and p25L-211S, encoding a mutant form of L-HDAg with a cysteine-to-serine mutation at codon 211, were constructed in this study. Mice were intramuscularly injected with the plasmids. The anti-HDV antibody titers, T-cell proliferation responses, T-helper responses, and HDV-specific, gamma interferon (IFN-γ)-producing CD8(+) T cells were analyzed. Animals immunized with p2577S showed a strong anti-HDV antibody response. Conversely, only a low titer of anti-HDV antibodies was detected in mice immunized with p2577L. Epitope mapping revealed that the anti-HDV antibodies generated by p2577L vaccination hardly reacted with epitope amino acids 174 to 194, located at the C terminus of S-HDAg. All of the HDAg-encoding plasmids could induce significant T-cell proliferation responses and generate Th1 responses and HDV-specific, IFN-γ-producing CD8(+) T cells. In conclusion, HDAg-specific antibodies definitely exist following DNA vaccination. The magnitudes of the humoral immune responses generated by L-HDAg- and S-HDAg-encoding DNA vaccines are different. The isoprenylated motif can mask epitope amino acids 174 to 195 of HDAg but does not interfere with cellular immunity following DNA-based immunization. These findings are important for the choice of a candidate HDV DNA vaccine in the future

Interaction and Replication Activation of Genotype I and II Hepatitis Delta Antigens

The nucleotide sequences of hepatitis D viruses (HDV) vary 5 to 14% among isolates of the same genotype and 23 to 34% among different genotypes. The only viral-genome-encoded antigen, hepatitis delta antigen (HDAg), has two forms that differ in size. The small HDAg (HDAg-S) trans-activates viral replication, while the large form (HDAg-L) is essential for viral assembly. Previously, it has been shown that the packaging efficiency of HDAg-L is higher for genotype I than for genotype II. In this study, the question of whether other functional properties of the HDAgs are affected by genotype differences is addressed. By coexpression of the two antigens in HuH-7 cells followed by specific antibody precipitation, it was found that HDAgs of different origins interacted without genotypic discrimination. Moreover, in the presence of hepatitis B virus surface antigen, HDAg-S was incorporated into virion-like particles through interaction with HDAg-L without genotype restriction. As to the differences in replication activation of genotype I HDV RNA, all HDAg-S clones tested had some trans-activation activity, and this activity varied greatly among isolates. As to the support of HDV genotype II replication, only clones of HDAg-S from genotype II showed trans-activation activity, and this activity also varied among isolates. In conclusion, genotype has no effect on HDAg interaction and genotype per se only partly predicts how much the HDAg-S of an HDV isolate affects the replication of a second HDV isolate

Expression Analysis of Up-Regulated Genes Responding to Plumbagin in Escherichia coli

Plumbagin is found in many medicinal plants and has been reported to have antimicrobial activities. We examined the molecular responses of Escherichia coli to plumbagin by using a proteomic approach to search for bacterial genes up-regulated by the drug. The protein profile obtained was compared with that of E. coli without the plumbagin treatment. Subsequent analyses of the induced proteins by mass spectroscopy identified several up-regulated genes, including ygfZ, whose function has not been defined. Analyses of the 5′-flanking sequences indicate that most of these genes contain a marbox-like stretch, and several of them are categorized as members of the mar/sox regulon. Representatives of these genes were cloned into plasmids, and the marbox-like sequences were modified by site-directed mutagenesis. It was proven that mutations in these regions substantially repressed the level of proteins encoded by the downstream genes. Furthermore, plumbagin's early effect was demonstrated to robustly induce SoxS rather than MarA, an observation distinctly different from that seen with sodium salicylate