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

Complexity of the Mycoplasma fermentans M64 Genome and Metabolic Essentiality and Diversity among Mycoplasmas

Recently, the genomes of two Mycoplasma fermentans strains, namely M64 and JER, have been completely sequenced. Gross comparison indicated that the genome of M64 is significantly bigger than the other strain and the difference is mainly contributed by the repetitive sequences including seven families of simple and complex transposable elements ranging from 973 to 23,778 bps. Analysis of these repeats resulted in the identification of a new distinct family of Integrative Conjugal Elements of M. fermentans, designated as ICEF-III. Using the concept of “reaction connectivity”, the metabolic capabilities in M. fermentans manifested by the complete and partial connected biomodules were revealed. A comparison of the reported M. pulmonis, M. arthritidis, M. genitalium, B. subtilis, and E. coli essential genes and the genes predicted from the M64 genome indicated that more than 73% of the Mycoplasmas essential genes are preserved in M. fermentans. Further examination of the highly and partly connected reactions by a novel combinatorial phylogenetic tree, metabolic network, and essential gene analysis indicated that some of the pathways (e.g. purine and pyrimidine metabolisms) with partial connected reactions may be important for the conversions of intermediate metabolites. Taken together, in light of systems and network analyses, the diversity among the Mycoplasma species was manifested on the variations of their limited metabolic abilities during evolution

Proteomics Characterization of Cytoplasmic and Lipid-Associated Membrane Proteins of Human Pathogen Mycoplasma fermentans M64

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen

Characterization of stm3030 and stm3031 genes of Salmonella enterica serovar Typhimurium in relation to cephalosporin resistance

Background/purpose: The outer membrane protein STM3031 had been shown to confer Salmonella enterica serovar Typhimurium resistance to ceftriaxone. In this study, the STM3030 was increased in strain R200 and decreased in strain R200(Δstm3031). How stm3030 and stm3031 contributing to antibiotic resistance was investigated. Methods: The level of STM3030 protein in R200(Δstm3031) were compared between 01–4, R200, and R200(Δstm3031) by 2-DE analysis. The stm3030 gene deleted strain, R200(Δstm3030), was generated by the one-step inactivation chromosome gene method. The various antibiotic susceptibility of strains 01–4, R200, R200(Δstm3031) and R200(Δstm3030) were determined by agar dilutions assays and E-test. The co-transcription of stm3031 and stm3030 were determined by RT-PCR. The promoter activities of these two genes fused with LacZ were determined. The binding of the regulatory protein BaeR on the promoter of both genes was detected by EMSA. The interaction between STM3030 and STM3031 proteins was determined by GST pull-down assay. Results: Strain R200(Δstm3030) displayed a 32- to 64-fold reduction in resistance to cephalosporin drugs. Transcription analyses revealed that stm3030 and stm3031 are independent genes and that the promoter of stm3030 is stronger than that of stm3031. The regulator BaeR binds to the promoter region of stm3031 but not that of stm3030. The STM3031 decreased in R200(Δstm3030) compared to R200 by western blot analysis. The pull-down assay revealed that STM3030 and STM3031 bind to each other. Conclusion: Our data indicate that STM3030 has a chaperone-like activity and may modulate or stabilize STM3031, leading to resistance of S. enterica serovar Typhimurium to cephalosporin drugs. Keywords: Cephalosporin resistance, Salmonella Typhimurium, stm3030, stm303

Correlation between Ceftriaxone Resistance of Salmonella enterica Serovar Typhimurium and Expression of Outer Membrane Proteins OmpW and Ail/OmpX-Like Protein, Which Are Regulated by BaeR of a Two-Component System

Mutant 7F2 of Salmonella enterica serovar Typhimurium has a transposon inserted in the regulator gene baeR of a two-component system and showed a more-than-fourfold reduction in resistance to ceftriaxone. Complementation analysis suggested an association among the outer membrane proteins OmpW and STM3031, ceftriaxone resistance, and baeR

The Expression Levels of Outer Membrane Proteins STM1530 and OmpD, Which Are Influenced by the CpxAR and BaeSR Two-Component Systems, Play Important Roles in the Ceftriaxone Resistance of Salmonella enterica Serovar Typhimurium▿

Significant increases in STM3031, STM1530, and AcrD protein levels and significant decreases in OmpC and OmpD protein levels are present when the ceftriaxone-resistant Salmonella enterica serovar Typhimurium R200 strain is compared with the ceftriaxone-susceptible strain 01-4. AcrD is known to be involved in drug export, and STM3031 seems to play a key role in ceftriaxone resistance. Here, we examine the roles of STM1530, OmpC, and OmpD in ceftriaxone resistance. An ompD gene deletion mutant showed 4-fold higher ceftriaxone resistance than 01-4. An ompC gene deletion mutant showed 4-fold higher cephalothin and erythromycin resistance than 01-4, but there was no effect on ceftriaxone resistance. However, a stm1530 deletion mutant did show >64-fold lower ceftriaxone resistance than R200. Moreover, the STM3031 protein was significantly decreased in R200(Δstm1530) compared to R200. STM3031 expression has been shown to be influenced by the two-component system regulator gene baeR. CpxR seems to modulate BaeR. A cpxA-cpxR gene deletion mutant showed >2,048-fold lower ceftriaxone resistance than R200. The outer membrane protein profile of R200(ΔcpxAR) showed significant decreases in STM3031 and STM1530 compared to R200, while OmpD had returned to the level found in 01-4. Furthermore, the stm3031, stm1530, and ompD mRNA levels were correlated with their protein expression levels in these strains, while decreases in the mRNA levels of the efflux pump acrB, acrD, and acrF genes were found in R200(ΔcpxAR). Findings similar to those for R200(ΔcpxAR) were found for R200(ΔbaeSR). These results, together with those for STM3031 and the fact that STM1530 is an outer membrane protein, suggest that STM1530 and OmpD are influenced by the CpxAR and BaeSR two-component systems and that this contributes to S. enterica serovar Typhimurium ceftriaxone resistance

An OXA-66/OXA-51-Like Carbapenemase and Possibly an Efflux Pump Are Associated with Resistance to Imipenem in Acinetobacter baumannii▿ †

We investigated the mechanisms involved in imipenem resistance in 23 clinical strains of Acinetobacter baumannii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of a 30-kDa protein in imipenem-intermediate A. baumannii (IIAB) and imipenem-resistant A. baumannii (IRAB) strains; this protein was almost undetectable in imipenem-susceptible A. baumannii (ISAB) strains. The 30-kDa protein was identified as an OXA-51-like carbapenemase using two-dimensional gel electrophoresis and mass spectrometry. Similar to other recent findings, blaOXA-51-like genes were found to exist in all 23 clinical strains; however, the transcript levels of blaOXA-51-like in the IIAB and IRAB were higher than in the ISAB strains using reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays. This change was due to the presence of an insertion sequence, ISAba1, upstream of blaOXA-51-like in the IIAB and IRAB strains that was not present in the ISAB strains. The introduction of blaOXA-66 (a blaOXA-51-like gene), identified in ISAB ab1254 and IRAB ab1266, into Escherichia coli TOP10 cells resulted in 3.95-fold and 7.90-fold elevations in resistance to imipenem, respectively. Furthermore, when ISAB ab8 and ISAB ab1254 and their in vitro-selected imipenem-resistant mutants ISAB ab8(r) and ISAB ab1254(r) were compared, the results showed no change in the blaOXA-66/blaOXA-51-like gene sequences, in expression of the gene, and in the outer membrane protein profiles. However, there was a four- to eightfold reduction in imipenem resistance upon adding carbonyl cyanide m-chlorophenylhydrazone. Taken together, these results suggest that the OXA-66/OXA-51-like carbapenemase contributes to intrinsic resistance to imipenem; however, drug export by an efflux pump may be more important and/or occur more frequently in imipenem-resistant A. baumannii. Furthermore, this is the first report of a Taiwanese strain of an OXA-66/OXA-51-like carbapenemase that confers imipenem resistance in A. baumannii