Reciprocal Regulation between Fur and Two RyhB Homologs in / 生物医学与环境科学（英文）

Objective@#To investigate reciprocal regulation between Fur and two RyhB homologs in @*Methods@#Regulatory relationships were assessed by a combination of colony morphology assay, primer extension, electrophoretic mobility shift assay and DNase I footprinting.@*Results@#Fur bound to the promoter-proximal DNA regions of @*Conclusion@#Fur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in

IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage

Abstract Mycobacterium tuberculosis (MTB) adopts a special survival strategy to overcome the killing mechanism(s) of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3) of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ) and recombinant murine interleukin-10 (rmIL-10) on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50 ng/ml) of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50 ng/ml) significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.

Principales factores de virulencia de Listeria monocytogenesy su regulación / Main virulence factors of Listeria monocytogenesand its regulation

Listeria monocytogenesis a facultative intracellular pathogen, ubiquitous and aetiological agent of listeriosis. The main way of acquisition is the consumption of contaminated food and can cause serious medical conditions such as septicemia, meningitis and gastroenteritis, especially in children, immunocompromised individuals and seniors and abortions in pregnant women. An increase in cases of listeriosis worldwide has been reported and it is estimated that its prevalence in developed countries is in the range of 2 to 15 cases per one million population. This microorganism is characterized for the transition from the environment into the eukaryotic cell. Several virulence factors have been involved in the intracellular cycle that are regulated, pimarilly, by the PrfA protein, which in turn is regulated by different mechanisms operating at the transcriptional, translational and post-translational levels. Additionally, other regulatory mechanisms have been described as sigma factor, system VirR/S and antisense RNA, but PrfA is the most important control mechanism and is required for the expression of essential virulence factors for the intracellular cycle.

Listeria monocytogeneses un patógeno intracelular facultativo, ubicuo y agente etiológico de listeriosis. La principal vía de adquisición es el consumo de alimentos contaminados, pudiendo ocasionar cuadros clínicos muy graves como septicemia, meningitis y gastroenteritis, especialmente en niños, individuos inmunocomprometidos y de la tercera edad, y aborto en mujeres embarazadas. Se ha informado un aumento en los casos de listeriosis a escala mundial y se estima que su frecuencia en los países desarrollados está en un rango de 2 a 15 casos por millón de habitantes. Este microorganismo se caracteriza por realizar una transición desde el medio ambiente hacia la célula eucariota. Para este proceso se han descrito varios factores de virulencia, los cuales están involucrados en el ciclo intracelular y están regulados, principalmente, por la proteína PrfA, la cual a su vez está regulada por diferentes mecanismos que actúan a nivel transcripcional, traduccional y post-traduccional. Además, se han descrito otros mecanismos regulatorios como: factor Sigma, sistema VirR/S y ARN sin sentido. No obstante, PrfA es el mecanismo de control más importante y el cual es requerido para la expresión de los factores de virulencia esenciales para el ciclo intracelular.

Molecular characterization of Escherichia coli O157:H7 strains isolated from different sources and geographic regions

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.

Pathotyping avian pathogenic Escherichia coli strains in Korea

To examine the genetic background of avian pathogenic Escherichia coli (APEC) that affects virulence of this microorganism, we characterized the virulence genes of 101 APEC strains isolated from infected chickens between 1985~2005. Serotypes were determined with available anti-sera and median lethal doses were determined in subcutaneously inoculated chicks. The virulence genes we tested included ones encoding type 1 fimbriae (fimC), iron uptake-related (iroN, irp2, iucD, and fyuA), toxins (lt, st, stx1, stx2, and vat), and other factors (tsh, hlyF, ompT, and iss). Twenty-eight strains were found to be O1 (2.0%), O18 (3.0%), O20 (1.0%), O78 (19.8%), and O115 (2.0%) serotypes. The iroN (100%) gene was observed most frequently followed by ompT (94.1%), fimC (90.1%), hlyF (87.1%), iss (78.2%), iucD (73.3%), tsh (61.4%), fyuA (44.6%), and irp2 (43.6%). The strains were negative for all toxin genes except for vat (10.9%). All the strains were classified into 27 molecular pathotypes (MPs). The MP25, MP19, and MP10 pathotypes possessing iroN-fimC-ompT-hlyF-iucD-tsh-iss-irp2-fyuA (22.8%), iroN-fimC-ompT-hlyF-iucD-tsh-iss (21.8%), and iroN-fimC-ompT-hlyF-iss (11.9%) genotypes, respectively, were predominant. Redundancy of iron uptake-related genes was clearly observed and some strains were associated with higher mortality than others. Therefore, strains with the predominant genotypes can be used for diagnosis and vaccine.

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens

The histone-like protein H1 (H-NS) is an abundant structural component of the bacterial nucleoid and influences many cellular processes including recombination, transcription and transposition. Mutations in the hns gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes. We have studied the role of H-NS on the regulation of hemolysin gene expression in Serratia marcescens. The Escherichia coli hns mutant carrying S. marcescens hemolysin genes on a plasmid constructed by ligation of the 3.2-kb HindIII-SacI fragment of pR02 into pBluescriptIIKS, showed a high level of expression of this hemolytic factor. To determine the osmoregulation of wild-type and hns defective mutants the cells were grown to mid-logarithmic phase in LB medium with 0.06 or 0.3 M NaCl containing ampicillin and kanamycin, whereas to analyze the effect of pH on hemolysin expression, the cells were grown to late-logarithmic phase in LB medium buffered with 0.1 M Tris-HCl, pH 4.5 to 8.0. To assay growth phase-related hemolysin production, bacterial cells were grown in LB medium supplemented with ampicillin and kanamycin. The expression of S. marcescens hemolysin genes in wild-type E. coli and in an hns-defective derivative at different pH and during different growth phases indicated that, in the absence of H-NS, the expression of hemolysin did not vary with pH changes or growth phases. Furthermore, the data suggest that H-NS may play an important role in the regulation of hemolysin expression in S. marcescens and its effect may be due to changes in DNA topology influencing transcription and thus the amount of hemolysin expression. Implications for the mechanism by which H-NS influences gene expression are discussed.

Control of streptokinase gene expression in group A & C streptococci by two-component regulators.

BACKGROUND & OBJECTIVES: Group A streptococci (GAS) and human isolates of group C streptococci (GCS) have the stable capacity to produce the plasminogen activator streptokinase, albeit with varying efficiency. This property is subject to control by two two-component regulatory systems, FasCAX and CovRS, which act as activator and repressor, respectively. The present work aims at balancing these opposing activities in GAS and GCS, and at clarifying the phylogenetic position of the FasA response regulator, the less understood regulator of the two systems. METHODS: The GCS strain H46A and GAS strain NZ131 were used. Escherichia coli JM 109 was used as host for plasmid construction. Streptokinase activity of various wild type and mutant strains was measured. Phylogenetic trees of streptococcal FasA homologues were established. RESULTS: The streptokinase activities of the GAS strain NZ131 and the GCS strain H46A were attributable to more efficient CovR repressor action in NZ131 than in H46A. The FasA activator, on the other hand, functioned about equally efficient in the two strains. Phylogenetically, FasA homologues clustered distinctly in the proposed FasA-BlpR-ComE family of streptococcal response regulators and used the LytTR domain for DNA binding. INTERPRETATION & CONCLUSION: Assessing the apparent streptokinase activity of streptoccal strains require the dissection of the activities of the cov and fas systems. Although experimental evidence is still missing, FasA is closely related to a widely distributed family of streptococcal response regulators that is involved in behavioral processes, such as quorum sensing.

Gene expression in Chromobacterium violaceum

The repertoire of 4,431 open reading frames (ORFs), eight rRNA operons and 98 tRNA genes of Chromobacterium violaceum must be expressed in a regulated manner for successful adaptation to a wide variety of environmental conditions. To accomplish this feat, the organism relies on protein machineries involved in transcription, RNA processing and translation. Analysis of the C. violaceum genome showed that transcription initiation, elongation and termination are performed by the five well-known RNA polymerase subunits, five categories of sigma 70 factors, one sigma 54 factor, as well as six auxiliary elongation and termination factors. RNA processing is performed by a variety of endonucleases and exonucleases, such as ribonuclease H, ribonuclease E, ribonuclease P, and ribonuclease III, in addition to poly(A) polymerase and specific methyltransferases and pseudouridine synthases. ORFs for all ribosomal proteins, except S22, were found. Only 19 aminoacyl-tRNA synthetases were found, in addition to three aminoacyl-tRNA synthetase-related proteins. Asparaginyl-tRNA (Asn) is probably obtained by enzymatic modification of a mischarged aminoacyl-tRNA. The translation factors IF-1, IF-2, IF-3, EF-Ts, EF-Tu, EF-G, RF-1, RF-2 and RF-3 are all present in the C. violaceum genome, although the absence of selB suggests that C. violaceum does not synthesize selenoproteins. The components of trans-translation, tmRNA and associated proteins, are present in the C. violaceum genome. Finally, a large number of ORFs related to regulation of gene expression were also found, which was expected, considering the apparent adaptability of this bacterium