MiR-34a inhibits lymphatic metastasis potential of mouse hepatoma cells

Genetic adaptation is one of the key features of Escherichia coli (E. coli) that ensure its survival in different hostile environments. E. coli seems to initiate biofilm development in response to specific environmental cues. A number of properties inherent within bacterial biofilms indicate that their gene expression is different from that of planktonic bacteria. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to a varied number of stresses, as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stress environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. The purpose of this study was to understand and analyse the responses of rpoS and bolA gene to sudden change in the environment. In this study, E. coli K-12 MG1655, rpoS, and bolA mutant strains were used and gene expression was studied. Results show that both genes contribute to the ability to respond and adapt in response to various types of stresses. RpoS response to various stress environments was somehow constant in both the planktonic and biofilm phases, whereas bolA responded well under various stress conditions, in both planktonic and biofilm mode, up to 5-6-fold change in the expression was noticed in the case of pH variation and hydrogen peroxide stress (H(2)O(2)) as compared with rpoS.     

Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene.

The bolA gene, which is involved in the morphogenetic pathways of Escherichia coli, was sequenced and two potential promoters were identified. Expression from promoter P1, proximal to the bolA structural gene is specifically induced during the transition to the stationary phase of growth. This promoter contains an unusual--10 region (CGGCTAGTA), which defines a new class of E. coli promoters necessary for the dramatic increase in the rate of synthesis of a large set of proteins during the cessation of logarithmic growth. This conclusion was confirmed by identifying two additional E. coli promoters and one plasmid promoter, which also were induced during the transition to the stationary phase of growth. Analysis of proteins produced during the exponential and stationary phases of growth in a bolA null mutant suggest a possible role for the BolA protein in the induction of the expression of penicillin-binding protein 6 (PBP6) in the transition to the stationary phase. Supporting this hypothesis is the presence of a putative DNA-binding domain within the bolA coding sequence.     

Isolation and characterization of sporulation-specific promoters in the yeast Saccharomyces cerevisiae

A library of random yeast genomic DNA:lacZ fusions has been constructed using an episomal yeast-Escherichia coli shuttle vector (pCS1). Plasmid pCS1 requires insertion of a promoter and an in frame ATG codon upstream of its resident truncated lacZ gene to regulate expression in yeast. Yeast genomic DNA fragments of 4-6 kb were generated by partial digestion with Sau3A and ligated into the unique BamHI site of plasmid pCS1 to generate a library of 5 x 10(4) individual E. coli transformants. This library was screened to identify promoter-lacZ fusions that were expressed uniquely during sporulation. Of 342 yeast transformants that exhibited beta-galactosidase activity, two were found to express the lacZ gene in a sporulation-specific manner. This paper presents the characterization of two genomic yeast DNA fragments containing promoters that control lacZ expression during the sporulation process. Expression from the promoter present in plasmid pJC18 occurred from 11-21 hours into the sporulation process, while the promoter in plasmid pJC217 was active from 4-14 hours. Staining of nuclear DNA to correlate nuclear morphology with timing of gene expression showed when each of these promoters was active in terms of the morphological stages of sporulation.     

Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.     

Degradation of p22phox and inhibition of superoxide generation by Ehrlichia chaffeensis in human monocytes

Ehrlichia chaffeensis is an obligate intracellular bacterium which replicates in monocytes or macrophages, the primary producers of reactive oxygen species (ROS). However, effects of ROS on E. chaffeensis infection and whether E. chaffeensis modulates ROS generation in host monocytes are unknown. Here, E. chaffeensis was shown to lose infectivity upon exposure to O(2)(-) or hydrogen peroxide. Upon incubation with human monocytes, E. chaffeensis neither induced O(2)(-) generation by human monocytes, nor colocalized with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components. Instead, it actively blocked O(2)(-) generation by monocytes stimulated with phorbol myristate acetate and caused the rapid degradation of p22(phox), a component of NADPH oxidase. These effects were not seen in neutrophil, which is another potent ROS generator, but a cell type that E. chaffeensis does not infect. Trypsin pretreatment of monocytes prevented the inhibition of O(2)(-) generation by E. chaffeensis. The degradation of p22(phox) by E. chaffeensis was specific to subsets of monocytes with bound and/or intracellular bacteria, and the degradation could be reduced by heat treatment of the bacterium, lipopolysaccharide pretreatment of monocytes, or the incubation with haemin. The degradation of p22(phox) by E. chaffeensis and its prevention by haemin or protease inhibitors also occurred in isolated monocyte membrane fractions, indicating that host cytoplasmic signalling is not required for these processes. The amount of gp91(phox) was stable under all conditions examined in this study. These findings point to a unique survival mechanism of ROS-sensitive obligate intraleucocytic bacteria that involves the destabilization of p22(phox) following the binding of bacteria to host cell surface proteins.     

Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola.

The gene (tutA) encoding tyrosine phenol-lyase from Erwinia herbicola was cloned into Escherichia coli, and fusions to the lac and tac promoters were constructed. The enzyme was expressed at high levels in E. coli in the presence of isopropyl-beta-D-thiogalactopyranoside or lactose as an inducer. L-Dihydroxyphenylalanine was synthesized in high yield from catechol, pyruvate, and ammonia by induced cells.     

Intra-leukocyte expression of two-component systems in Ehrlichia chaffeensis and Anaplasma phagocytophilum and effects of the histidine kinase inhibitor closantel

The two-component system (TCS) composed of a pair of a sensor histidine kinase and a response regulator, allows bacteria to sense signals and respond to changes in their environment through specific gene activation or repression. The present study examined TCS in the obligatory intracellular bacteria Ehrlichia chaffeensis and Anaplasma phagocytophilum, that cause human monocytic ehrlichiosis (HME) and human granulocytic anaplasmosis (HGA) respectively. The genomes of E. chaffeensis and A. phagocytophilum were each predicted to encode three pairs of TCSs. All six genes encoding three histidine kinases and three response regulators were expressed in both E. chaffeensis and A. phagocytophilum cultured in human leukocytes. Pretreatment of host cell-free E. chaffeensis or A. phagocytophilum with closantel, an inhibitor of histidine kinases, completely blocked the infection of host cells. Treatment of infected cells 1 day post infection with closantel cleared infection in dose-dependent manner. All six genes in E. chaffeensis were cloned, recombinant proteins were expressed, and polyclonal antibodies were produced. Double immunofluorescence labelling and Western blot analysis revealed that all six proteins were expressed in cell culture. Autokinase activities of the three recombinant histidine kinases from E. chaffeensis were inhibited by closantel in vitro. A number of E. chaffeensis genes, including the six TCS genes, were downregulated within 5-60 min post closantel treatment. These results suggest that these TCSs play an essential role in infection and survival of E. chaffeensis and A. phagocytophilum in human leukocytes.     

Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor sigma S.

The novel sigma factor (sigma S) encoded by rpoS (katF) is required for induction of many growth phase-regulated genes and expression of a variety of stationary-phase phenotypes in Escherichia coli. Here we demonstrate that wild-type cells exhibit spherical morphology in stationary phase, whereas rpoS mutant cells remain rod shaped and are generally larger. Size reduction of E. coli cells along the growth curve is a continuous and at least biphasic process, the second phase of which is absent in rpoS-deficient cells and correlates with induction of the morphogene bolA in wild-type cells. Stationary-phase induction of bolA is dependent on sigma S. The "gearbox" a characteristic sequence motif present in the sigma S-dependent growth phase- and growth rate-regulated bolAp1 promoter, is not recognized by sigma S, since stationary-phase induction of the mcbA promoter, which also contains a gearbox, does not require sigma S, and other sigma S-controlled promoters do not contain gearboxes. However, good homology to the potential -35 and -10 consensus sequences for sigma S regulation is found in the bolAp1 promoter.     

Regulation of Aspergillus nidulans penicillin biosynthesis and penicillin biosynthesis genes acvA and ipnA by glucose.

Expression of the Aspergillus nidulans penicillin biosynthesis genes acvA and ipnA, encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, respectively, was analyzed. The intergenic region carrying the divergently oriented promoters was fused in frame in both orientations to Escherichia coli lacZ and E. coli uidA reporter genes. Each construct permits simultaneous expression studies of both genes. Transformants of A. nidulans carrying a single copy of either plasmid integrated at the chromosomal argB locus were selected for further investigations. Expression of both genes was directed by the 872-bp intergenic region. ipnA- and acvA-derived gene fusions were expressed from this region at different levels. ipnA had significantly higher expression than did acvA. Glucose specifically reduced the production of penicillin and significantly repressed the expression of ipnA but not of acvA gene fusions. The specific activities of isopenicillin N synthetase, the gene product of ipnA, and acyl coenzyme A:6-aminopenicillanic acid acyltransferase were also reduced in glucose-grown cultures.