The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin.

The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present. We previously showed that FlgM is also necessary for the virulence of S. typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence. In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S. typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S. typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S. typhimurium mutant resulted in a virulent phenotype. Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S. typhimurium. Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S. typhimurium.     

Nucleotide sequence and organization of Bacillus subtilis RNA polymerase major sigma (sigma 43) operon.

The gene coding for Bacillus subtilis RNA polymerase major sigma 43, rpoD, was cloned together with its neighboring genes in a 7 kb EcoRI fragment. The complete nucleotide sequence of a 5 kb fragment including the entire rpoD gene revealed the presence of two other genes preceding rpoD in the order P23-dnaE-rpoD. The dnaE codes for DNA primase while the function of P23 remains unknown. The three genes reside in an operon that is similar in organization to the E. coli RNA polymerase major sigma 70 operon, which is composed of genes encoding small ribosome protein S21 (rpsU), DNA primase (dnaG), and RNA polymerase sigma 70 (rpoD). There is a relatively high degree of base and amino acid homology between the DNA primase and sigma genes. The most significant differences between the two operons are observed in the molecular size of the first genes (P23 and rpsU), the complete lack of amino acid homology between P23 and S21, the molecular weights of the two rpoD genes, the size of the intercistronic region between the first two genes, and the regulatory elements of the operon.     

Cloning and analysis of the gene (rpoDA) for the principal sigma factor of Pseudomonas aeruginosa

A gene (rpoDA) of Pseudomonas aeruginosa whose gene product has a homologous function and structure with the principal sigma factor of Escherichia coli was cloned and sequenced. The DNA region corresponding to one of the two hybridization signals found in P. aeruginosa DNA with a synthetic oligonucleotide probe (rpoD probe) was shown to be able to complement a temperature sensitive mutation of Escherichia coli rpoD gene. The amino acid sequence deduced from the nucleotide sequence of rpoDA showed an extensive homology with that of the principal sigma factor of E. coli throughout the entire region, which indicates that the two gene products have an essentially identical domain structure. A common basic structure observed among principal sigma factors of different eubacterial strains was proposed. RpoDA protein was identified in the extract of the cell carrying a plasmid clone with the rpoDA gene insert by Western blot analysis.     

鼠伤寒沙门氏菌Ⅰ相鞭毛蛋白基因fliC~I的克隆和鉴定

以提纯的鼠伤寒沙门氏菌8705染色体DNA为材料,经EcoR Ⅰ消化,过SepharcylS-400柱,得到大于400bp的酶切片段;然后随机克隆到质粒pGEM-3Zf(—)中,转化大肠杆菌LC2a(hag~-,recA~-);在氨苄青霉素平板上共得到6013个转化子,从中筛选出1个有动力的克隆,小量制备质粒DNA,经酶切电泳鉴定,该克隆的外源片段大小为15.3kb,将其命名为pGI4015。动力、动力抑制试验和Southern blot分子杂交试验证明pGI4015中载有鼠伤寒沙门氏菌Ⅰ相鞭毛蛋白基因fliC~I;利用其BamH Ⅰ和Sal Ⅰ位点删除与鞭毛蛋白表达无关的序列,构建亚克隆质粒pGI4015BS,使得fliC~I定位于更小的区域——3.8kb的BamH Ⅰ/Sal Ⅰ插入片段上。     

Nucleotide sequence analysis of purH and purD genes from Salmonella typhimurium

The purH and purD genes coding for the 5'-phosphoribosyl 5-amino-imidazole-4-carboxamide (AICAR) transformylase and 5'-phosphoribosyl-glycinamide (GAR) synthetase, respectively, were identified on a 4.8 kb Eco RI fragment of chromosomal DNA from Salmonella typhimurium. Nucleotide sequence analysis of the cloned fragment revealed the presence of two large open reading frames (O.R.F.), which were separated by 11 base pairs (bp). Substantial DNA and amino acid sequence homology was noted between the purH and purD genes of S. typhimurium and Escherichia coli. Expression of the Salmonella purD gene in a T7 polymerase/promoter system revealed the presence of a 49 kDa protein band by SDS-PAGE and subsequent autoradiography. The purH gene of Salmonella was not expressed since the 5' end of this gene was not cloned.     

Genetic and molecular characterization of the polar flagellum of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.     

Genes encoding the beta-subunit of bacterial RNA-polymerases. I. Primary structure of the EcoRI-C fragment of the Salmonella typhimurium gene rpoB

BglII fragment of S. typhimurium DNA, containing rpoB gene coding for the RNA polymerase beta-subunit, was cloned. The nucleotide sequence of the rpoB gene EcoRI-C fragment (2873 b.p.) was determined.     

DNA hybridization probe for the Pseudomonas fluorescens group.

Plasmid pHF360 was constructed from cloned rRNA genes (rDNA) of Pseudomonas aeruginosa and used as hybridization probe for the Pseudomonas fluorescens group. The probe was tested by dot and in situ colony hybridizations to chromosomal DNAs from a wide variety of organisms. pHF360 DNA hybridized exclusively to chromosomal DNAs from bacteria representing the P. fluorescens group and separated them clearly from all other bacteria tested in the present study. Determination of the nucleotide sequence of the cloned DNA showed that it is a fragment from a 23S rRNA gene of P. aeruginosa. It was compared with the published 23S RNA sequence from Escherichia coli.     

Adenylate kinase isoenzymes in Aspergillus nidulans

Multiple HindIII-restriction fragments of Salmonella typhimurium and Salmonella typhi chromosomal DNA exhibited homology with the heat-labile enterotoxin (LT1) gene of Escherichia coli as determined by Southern blot analysis. A 9.4 kb HindIII restriction fragment identified in S. typhimurium and S. typhi chromosomal DNA reacted with both eltA and eltB gene probes. However, the homology of the 9.4 kb DNA fragment from these Salmonella species was greater with eltB than eltA. In addition, a synthetic oligonucleotide probe, made to a portion of the putative GM1-ganglioside binding region of cholera toxin (CT) and LT1, hybridized with the 9.4 kb DNA fragment of S. typhimurium but not with the 9.4 kb fragment found in S. typhi isolates. The hybridization of multiple restriction fragments of Salmonella DNA with eltA and eltB gene sequences further suggests duplication of the stx operon on the chromosome of these bacteria.     

DNA hybridization probe for the Pseudomonas fluorescens group

Plasmid pHF360 was constructed from cloned rRNA genes (rDNA) of Pseudomonas aeruginosa and used as hybridization probe for the Pseudomonas fluorescens group. The probe was tested by dot and in situ colony hybridizations to chromosomal DNAs from a wide variety of organisms. pHF360 DNA hybridized exclusively to chromosomal DNAs from bacteria representing the P. fluorescens group and separated them clearly from all other bacteria tested in the present study. Determination of the nucleotide sequence of the cloned DNA showed that it is a fragment from a 23S rRNA gene of P. aeruginosa. It was compared with the published 23S RNA sequence from Escherichia coli.