DNA sequence analysis, gene product identification, and localization of flagellar motor components of Escherichia coli.

The Escherichia coli operon designated flaA contains seven flagellar genes; among them are two switch protein genes whose products are believed to interface with the motility and chemotaxis machinery of the cell. Complementation analysis using several plasmids carrying different portions of the flaA operon and analysis of expression of these plasmids in minicells allowed the identification of two flagellar gene products. The MotD (now called FliN) protein, a flagellar switch protein, was determined to have an apparent molecular weight of 16,000, and the FlaAI (FliL) protein, encoded by a previously unidentified gene, had an apparent molecular weight of 17,000. DNA sequence analysis of the motD gene revealed an open reading frame of 414 base pairs. There were two possible initiation codons (ATG) for motD translation, the first of which overlapped with the termination codon of the upstream gene, flaAII (fliN). The wild-type flaAI gene on the chromosome was replaced with a flaAI gene mutated in vitro. Loss of the flaAI gene product resulted in a nonmotile and nonflagellated phenotype. The subcellular location for both the MotD and FlaAI proteins was determined; the FlaAI protein partitioned exclusively in the insoluble fraction of a whole minicell sonic extract, whereas the MotD protein remained in both the soluble and insoluble fractions. In addition, we subcloned a 2.2-kilobase-pair DNA fragment capable of complementing the remaining four genes of the flaA operon (flbD [fliO], flaR [fliP], flaQ [fliQ], and flaP [fliR]).     

Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.     

HzAm1细胞rpL13基因的序列分析及病毒感染对其转录水平的影响

从美洲棉铃虫细胞(HzAM1)中克隆了核糖体大亚基蛋白L13(RibosomalproteinL13,RpL13)的cDNA及其基因组DNA序列,并进行了序列分析。其编码框为666bp,无内含子,预测编码大小约为25kDa的蛋白。通过与其它15种动物的RpL13编码框序列进行进化分析,发现聚类结果与传统物种分类一致。转录研究表明,棉铃虫核型多角体病毒(Helicoverpaarmigerasinglenucleocapsidnucleopolyhedrovirus,HaSNPV)感染HzAm1后使rpL13在细胞内的转录水平降低,在病毒感染后96h,rpL13mRNA的拷贝数降到对照健康细胞的8%。     

Role of the flaR gene in flagellar hook formation in Salmonella spp.

Flagellar filaments were reconstituted by polymerization with exogenously supplied flagellin monomers at the tips of normal hooks on Salmonella cells which were missing the filaments because of mutations in either the flaL or flaU gene or the flagellin genes H1 and H2. Reconstitution did not occur at the tips of polyhooks of the flaR mutant cells. Thus, the absence of flagellar filaments in the flaR mutant cells was probably caused by the inability of the polyhooks to work as polymerization nuclei for flagellin. A Phf+ mutant which produced polyhooks with flagellar filaments was isolated from a flaR polyhook mutant. Genetic analysis of the Phf+ mutant showed that it carried an intracistronic suppressor mutation of the original flaR mutation. This result indicated that the flaR gene regulates hook length and initiates flagellin formation.     

Nucleotide sequence and analysis of a gene encoding anthranilate synthase component I in Spirochaeta aurantia.

A Spirochaeta aurantia DNA fragment containing the trpE gene and flanking chromosomal DNA was cloned, and the sequence of the trpE structural gene plus 870 bp upstream and 1,257 bp downstream of trpE was determined. The S. aurantia trpE gene codes for a polypeptide of 482 amino acid residues with a predicted molecular weight of 53,629 that showed sequence similarity to TrpE proteins from other organisms. The S. aurantia TrpE polypeptide is not more closely related to the other published spirochete TrpE sequence (that of Leptospira biflexa) than to TrpE polypeptides of other bacteria. Two additional complete open reading frames and one partial open reading frame were identified in the sequenced DNA. One of the complete open reading frames and the partial open reading frame are upstream of trpE and are encoded on the DNA strand opposite that containing trpE. The other open reading frame is downstream of trpE and on the same DNA strand as trpE. On the basis of the results of a protein sequence data base search, it appears that trpE is the only tryptophan biosynthesis gene in the sequenced DNA. This is in contrast to L. biflexa, in which trpE is separated from trpG by only 64 bp.     

Identification of Listeria monocytogenes genes expressed in response to growth at low temperature

Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10 degrees C compared to those grown at 37 degrees C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses (flaA and flp), regulatory adaptive responses (rpoN, lhkA, yycJ, bglG, adaB, and psr), general microbial stress responses (groEL, clpP, clpB, flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alterations (fbp, psr, and flaA), and degradative metabolism (eutB, celD, and mleA). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10 degrees C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.