Histone-like protein H1 (H-NS), DNA supercoiling, and gene expression in bacteria

Changes in DNA supercoiling in response to environmental signals such as osmolarity, temperature, or anaerobicity appear to play an underlying role in the regulation of gene expression in bacteria. Extensive genetic analyses have implicated the osmZ gene in this regulatory process: osmZ mutations are highly pleiotropic and alter the topology of cellular DNA. We have shown that the product of the osmZ gene is the "histone-like" protein H1 (H-NS). Protein H1 is one of the most abundant components of bacterial chromatin and binds to DNA in a relatively nonspecific fashion. These data imply a regulatory role for one of the major components of bacterial chromatin and provide support for the notion that changes in DNA topology and/or chromatin structure play a role in regulating gene expression.     

Molecular cloning of a determinant coding for fimbrial antigen F165(1), a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli.

The genetic determinant coding for F165(1) fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K-:H51:F165). The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established. On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated. The cloned F165(1) fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa. Strains expressing F165(1) fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads. Antiserum against the cloned F165(1) fimbriae recognized a 18.5 kDa band in the parent strain 4787.     

Chromosomal mapping of genes encoding mannose-sensitive (type I) and mannose-resistant F8 (P) fimbriae of Escherichia coli O18:K5:H5

DNA hybridization experiments demonstrated that the gene clusters encoding the F8 fimbriae (fei) as well as the type I fimbriae (pil) exist in a single copy on the chromosome of E. coli O18:K5 strain 2980. In conjugation experiments with appropriate donors, the chromosomal site of these gene clusters was determined. The pil genes were mapped close to the gene clusters thr and leu controlling the biosynthesis of threonine and leucine, respectively. The fei genes were found to be located close to the galactose operon (gal) between the position 17 and 21 of the E. coli chromosomal linkage map.     

Functional relationship among the gene clusters encoding F7(1), F7(2), F9, and F11 fimbriae of human uropathogenic Escherichia coli.

The P fimbrial gene clusters encoding the serologically different F7(1), F7(2), F9, and F11 fimbriae were compared functionally. The results show that these gene clusters are closely related.     

Effect of DNA supercoiling and catabolite repression on the expression of the tetA genes in Escherichia coli

The effect of novobiocin, a gyrase inhibitor, and of catabolite repression on the expression of different classes of tetA genes in Escherichia coli was studied. The results obtained showed that the complete induction of the tetA genes corresponding to classes A, B, and D depends on DNA supercoiling, and that the expression of the tetA genes corresponding to classes A and C is not under catabolite repression.     

CS3抗原基因的表达及纤毛装配元件的研究

在肠毒素大肠杆菌(ETEC)的已知定居因子中,CS3是临床分离株中最常见的抗原之一。为了研究CS3纤毛装配的基本元件,绘制了CS3亚基结构基因和辅助蛋白编码区的限制酶谱。通过亚克隆的亚基基因和不同辅助蛋白基因之间的互补性表达结果,确定了CS3纤毛装配所需要的辅助蛋白的DNA功能片段。微细胞分析结果显示,CS3基因的有效表达和纤毛装配至少需要6条蛋白多肽,分子量分别为(×10~3)15、17、24、27、48和90。除了15×10~3/17×10~3的蛋白多肽为CS3亚基外,其余的蛋白多肽参与CS3亚基的转运及纤毛的装配。根据以上结果初步确定了上述相关基因的相对位置。     

Characterization of the second long polar (LP) fimbriae of Escherichia coli O157:H7 and distribution of LP fimbriae in other pathogenic E. coli strains

A second region containing five genes homologous to the long polar fimbrial operon of Salmonella enterica serovar Typhimurium is located in the chromosome of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A non-fimbriated E. coli K-12 strain carrying the cloned EHEC lpf (lpf2) genes expressed thin fibrillae-like structures on its surface and displayed reduced adherence to tissue culture cells. Neither mutation in the lpfA2 gene in either the parent or lpfA1 mutant strains showed an effect in adherence or in the formation of A/E lesions on HeLa cells. lpfA2 isogenic mutant strains adhere to Caco-2 cells almost as well as the wild-type at 5 h, but they were deficient in adherence at early time points. A collection of diarrheagenic E. coli strains were investigated for the presence of lpfA1 and lpfA2 and results showed that these genes are present in specific serogroups which are phylogenetically related. Our results suggest that LP fimbriae 2 may contribute to the early stages of EHEC adhesion and that genes encoding the major LP fimbrial subunits are present in a small group of EHEC and EPEC serotypes.     

The effect of supercoiling of DNA from colicinogenic plasmids on the expression of col, imm and lys genes

The expression of colicin genes is controlled by the SOS-system (Lex A repressor) and the adenylate-cyclase system (cAMP-CAP complex). The effect of plasmid DNA supercoiling on the expression of the operons of colicins E1, E2, and E3 has been studied by using E. coli minicells. It has been shown for the colicin E1 operon that it is the promoter that is influenced by supercoiling: an increase in negative supercoiling elevates the expression and, vice versa, DNA relaxation reduces the expression. The effect of supercoiling on gene activity of the colicin E1 immunity protein has not been observed, which may be due to the specific orientation of this gene. With the two other colicins supercoiling affects the expression of all genes which constitute the operon. The regulation of the colicin operon expression has been confirmed to occur at three levels: by the LexA protein, by the cAMP-CAP complex, and by the plasmid DNA supercoiling.     

Structure and expression of the genes coding for human alpha 1-acid glycoprotein.

alpha 1-acid glycoprotein (alpha AGP) is a well-characterized human plasma protein. Its structural properties have been studied for many years but little is known about its function. Amino acid sequence analysis of purified human alpha AGP from plasma pooled from several individuals showed considerable heterogeneity. We have cloned the genomic DNA segment encoding alpha AGP and we show that it contains three adjacent alpha AGP coding regions, AGP-A, B and B', identical in exon--intron organization but with slightly different coding potential. These results account for the heterogeneity observed by protein sequencing. Southern blot analysis indicates that the cloned cluster contains all the alpha AGP coding sequences present in the human genome. The larger majority of alpha AGP mRNA in human liver is transcribed from AGP-A, whose promoter and cap site have been determined while the level of AGP-B and B' mRNA in human liver is very low. Using Hep3B hepatoma cells as a model system for the in vitro study of the acute phase reaction, we show that only AGP-A is strongly induced by treatment with culture medium of LPS stimulated monocytes.     

Chromosomal assignment of four rat genes coding for the spermatid-specific proteins proacrosin (ACR), transition proteins 1 (TNP1) and 2 (TNP2), and protamine 1 (PRM1)

The genes for proacrosin, protamines, and transition proteins are exclusively expressed in haploid spermatogenic cells. From the analysis of mouse x rat cell hybrids which segregate rat chromosomes, the rat gene for proacrosin (ACR) was assigned to chromosome 7, that for transition protein 1 (TNP1) to chromosome 9, and the genes for transition protein 2 (TNP2) and protamine 1 (PRM1) to chromosome 10.     

F1651 fimbriae of the P fimbrial family inhibit the oxidative response of porcine neutrophils

The F165(1) fimbrial system has been associated with the resistance of Escherichia coli O115:K"V165" to phagocytic killing by porcine polymorphonuclear leukocytes (PMNLs). One mechanism of this resistance seemed to be inhibition of the oxidative response as observed following induction of PMNLs by phorbol myristate acetate (PMA) and treatment with bacteria possessing the F165(1) fimbriae. In order to confirm whether or not the F165(1) fimbriae are involved in this inhibition, we evaluated the effect of F165(1)-positive strains (a pathogenic wild-type strain 5131, and a recombinant strain HB101(pCJ7)) or an F165(1)-negative strain HB101 (used as negative control) on the oxidative response of porcine neutrophils (pNs) stimulated with PMA. Incubation of pNs with pathogenic E. coli strain 5131 resulted in significant inhibition of the oxidative response as compared to that observed for pNs incubated without bacteria, as assessed by hydrogen peroxide (H2O2) and superoxide anion (O2-) release from the phagocytes, and by the chemiluminescence assay. Similarly, incubation of pNs with the F165(1)-producing cloned strain HB101(pCJ7) resulted in significant inhibition of the pN oxidative response as compared to that observed for pNs incubated without bacteria or with strain HB101. In contrast, addition of purified F165(1) fimbriae to the pNs had no effect on the oxidative response.