TAIL-PCR方法快速分离Xcc致病相关基因序列

以mini-Tn5 gfp-km转座子中nptⅡ片段作为探针,对已获得的五株野油菜黄单胞菌野油菜黑腐病致病型（Xcc)非致病突变体进行了Southern blot分析,结果表明,这五株突变体确由mini-Tn5 gfp-km转座子插入致病相关基因所致,且为单拷贝不同位点的插入。提取这五株突变体总DNA作为模板,采用改进的热不对称交错PCR（TAIL－PCR）方法从其中克隆到了各自转座子插入区侧翼序列,对这些侧翼序列进行了序列测定并将分析结果与GenBank database及Xcc全基因组序列做了比较,结果表明,五个侧翼序列所在的基因确与Xcc致病性有关。这种改进后的TAIL－PCR方法为突变体特别是转座子插入突变体中目的基因的克隆提供了一种简要高效的新方法。     

A Xanthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity.

A DNA fragment from Xanthomonas campestris pv. campestris that partially restored the carbohydrate fermentation pattern of a cya crp Escherichia coli strain was cloned and expressed in E. coli. The nucleotide sequence of this fragment revealed the presence of a 700-base-pair open reading frame that coded for a protein highly similar to the catabolite activation factor (CAP) of E. coli (accordingly named CLP for CAP-like protein). An X. campestris pv. campestris clp mutant was constructed by reverse genetics. This strain was not affected in the utilization of various carbon sources but had strongly reduced pathogenicity. Production of xanthan gum, pigment, and extracellular enzymes was either increased or decreased, suggesting that CLP plays a role in the regulation of phytopathogenicity.     

The sequence of the ribosomal 16S RNA from Proteus vulgaris. Sequence comparison with E. coli 16S RNA and its use in secondary model building.

The complete nucleotide sequence of the 16S RNA from Proteus vulgaris has been determined. The molecule (1544 nucleotides) shows 93% homology with the sequence of E. coli 16S RNA. Six methylated nucleotides have been localized in positions homologous to those observed in the E. coli RNA molecule. Both E. coli and P. vulgaris 16S RNA chains can be folded up into a common secondary structure scheme. Comparative sequence analysis of the two molecules has provided a valuable contribution to 16S RNA secondary structure model building.     

oriC region and replication termination site,dif, of the Xanthomonas campestris pv. campestris 17 chromosome

A 13-kb DNA fragment containing oriC and the flanking genes thdF, orf900, yidC, rnpA, rpmH, oriC, dnaA, dnaN, recF, and gyrB was cloned from the gram-negative plant pathogen Xanthomonas campestris pv. campestris 17. These genes are conserved in order with other eubacterial oriC genes and code for proteins that share high degrees of identity with their homologues, except for orf900, which has a homologue only in Xylella fastidiosa. The dnaA/dnaN intergenic region (273 bp) identified to be the minimal oriC region responsible for autonomous replication has 10 pure AT clusters of four to seven bases and only three consensus DnaA boxes. These findings are in disagreement with the notion that typical oriCs contain four or more DnaA boxes located upstream of the dnaA gene. The X. campestris pv. campestris 17 attB site required for site-specific integration of cloned fragments from filamentous phage phiLf replicative form DNA was identified to be a dif site on the basis of similarities in nucleotide sequence and function with the Escherichia coli dif site required for chromosome dimer resolution and whose deletion causes filamentation of the cells. The oriC and dif sites were located at 12:00 and 6:00, respectively, on the circular X. campestris pv. campestris 17 chromosome map, similar to the locations found for E. coli sites. Computer searches revealed the presence of both the dif site and XerC/XerD recombinase homologues in 16 of the 42 fully sequenced eubacterial genomes, but eight of the dif sites are located far away from the 6:00 point instead of being placed opposite the cognate oriC. The differences in the relative position suggest that mechanisms different from that of E. coli may participate in the control of chromosome replication.     

Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.     

Comparative analysis of the 5'-terminal nucleotide sequence and central domains of 16S rRNA of Yersinia pestis and Escherichia coli]

Partial nucleotide sequence of 16S rRNA (16-989 nn.) of plague agent (Yersinia pestis) was determined after sequencing of cloned cDNA fragments. The comparison of Y. pestis 16S rRNA sequence with that of E. coli shows a number of point sequence variation due to base changes. The base changes are found in 16S rRNA secondary structure regions that are localized on the surface of 30S ribosome subunit (hairpins 6 and 18) as well as in the regions that bind the proteins S8, S15, S16 and S20. These proteins of Y. pestis differ from the same proteins of E. coli by electrophoretic mobility, when analyzed by two-dimensional co-electrophoresis in polyacrylamide gel. The correlation between the structure of the four proteins and the structure of their binding sites in 16S rRNA are discussed.     

Molecular cloning and nucleotide sequence of the gene for the ribosomal protein S11 from the archaebacterium Halobacterium marismortui

The gene encoding ribosomal protein S11 (Escherichia coli S15 homologue) from Halobacterium marismortui was cloned employing two synthetic oligonucleotide mixtures, 23 and 32 bases in length, as hybridization probes. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (1300 base pairs) were then determined by the dideoxy chain termination method. Comparison of the nucleotide sequence of the H. marismortui S11 gene with that of the E. coli S15 gene (rpsO) showed that the 3'-end of the S11 gene can be aligned with the entire E. coli S15 gene, sharing 44% identical nucleotides. It has been found that the S11 gene has a higher G + C content (G + C = 65%) than that of the E. coli S15 gene (G + C = 53%). This increase in G + C content specifically shows up as a preference for G + C in the 3rd position of the codon. Upstream of the S11 gene, an archaebacterial promoter sequence (GGACTTTCA) and a putative ribosomal binding site (GCGGT) have been found, 88 and 15 (or 24) base pairs from the initiation codon of the gene. In addition, an open reading frame could be identified immediately after the stop codon for the S11 gene. Northern blotting analysis using the S11 coding region as probe has shown that the S11 gene is located on a 2.4-kilobase mRNA, suggesting that it is cotranscribed with other downstream gene(s).     

Organization and codon usage of the streptomycin operon in Micrococcus luteus, a bacterium with a high genomic G + C content.

The DNA sequence of the Micrococcus luteus str operon, which includes genes for ribosomal proteins S12 (str or rpsL) and S7 (rpsG) and elongation factors (EF) G (fus) and Tu (tuf), has been determined and compared with the corresponding sequence of Escherichia coli to estimate the effect of high genomic G + C content (74%) of M. luteus on the codon usage pattern. The gene organization in this operon and the deduced amino acid sequence of each corresponding protein are well conserved between the two species. The mean G + C content of the M. luteus str operon is 67%, which is much higher than that of E. coli (51%). The codon usage pattern of M. luteus is very different from that of E. coli and extremely biased to the use of G and C in silent positions. About 95% (1,309 of 1,382) of codons have G or C at the third position. Codon GUG is used for initiation of S12, EF-G, and EF-Tu, and AUG is used only in S7, whereas GUG initiates only one of the EF-Tu's in E. coli. UGA is the predominant termination codon in M. luteus, in contrast to UAA in E. coli.     

Amino-Terminal Sequences of Some Escherichia coli 30S Ribosomal Proteins and Functionally Corresponding Bacillus stearothermophilus Ribosomal Proteins

Amino-terminal sequences of five purified Escherichia coli 30S ribosomal proteins (S4, S9, S10, S16, and S20) were compared with those of their functionally corresponding Bacillus stearothermophilus ribosomal proteins identified previously by the reconstitution technique. An automatic Edman degradation method was used for sequence determinations. The sequence of the first 30 residues is presented, except that only the first 25 residues are shown for the S20 pair. Substantial (40 to 70%) sequence homologies have been observed in every case. The results show that the pairs of functionally equivalent proteins, previously identified by the reconstitution technique, are also chemically related. Thus, the present chemical studies give further support for the previous conclusion that two ribosomes with different properties, 30S subunits from E. coli and B. stearothermophilus, have the same fundamental structural organization.     

Computer analysis of the sequence relationships among 4.5S RNA molecular species from various sources

Nucleotide sequence homology among 4.5S RNAs from various organisms was examined by computer analysis to evaluate their sequence relationships. Chloroplast 4.5S rRNAs of wheat and tobacco were not significantly related to Escherichia coli 4.5S RNA, but were closely related to the 3'-terminus of bacterial 23S rRNA. Significant sequence homology was found between rat Novikoff hepatoma 4.5S RNAI and mouse and hamster 4.5S RNAs, suggesting that these RNAs are products of a family of genes with diverged sequences. E. coli 4.5S RNA had no significant sequence homology with any rodent 4.5S RNAs as a whole sequence. The E. coli, mouse and hamster 4.5S RNAs, however, were found to share a homologous 14-nucleotide sequence at the center of the molecules, which is known to exist as a conserved sequence in both Alu and Alu-equivalent sequences of mammalian DNAs.     

Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae.

A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYI1) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine gamma-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine gamma-lyase (41%), Escherichia coli cystathionine gamma-synthase (36%), and cystathionine beta-lyase (25%). The N-terminal half of it was homologous (39%) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYI1) gene marginally complemented the E. coli metB mutation (cystathionine gamma-synthase deficiency) and conferred cystathionine gamma-synthase activity as well as cystathionine gamma-lyase activity to E. coli; cystathionine gamma-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine gamma-lyase and E. coli cystathionine gamma-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.     

Development of specific and rapid detection of bacterial pathogens in dairy products by PCR

A simple and specific method for direct detection of bovine mastitis pathogens (Streptococcus agalactiae (GBS), Staphylococcus aureus and Escherichia coli) in milk products, bacterial samples from milk and isolated bacterial DNA was developed. The method is based on polymerase chain reaction (PCR) using sequence-specific primers only for GBS and species-specific primers derived from 16S and 23S rRNA for all chosen species. The presence of the gene of surface immunogenic protein (Sip) in bovine GBS isolates, described previously only in human GBS isolates was confirmed. The GBS detection was performed with the sequence coding for surface immunogenic protein from GBS human isolates designated as Sip specific sequence (SSS); this sequence was selected for specific primer design. The sequence is unique for GBS and was designed from a consensus of all known sip genes. The specific identification was shown on a collection of 75 GBS bovine isolates from different localities in Slovakia. All isolates were positive to SSS, 16S and 23S rRNA sequence. The 16S and 23S rRNA PCR detection was also performed with S. aureus and E. coli isolates and specific PCR products were also detected. The detection limit of this assay for milk products was 6 CFU/microL (i.e. 6000 CFU/mL) for GBS and E. coli, and 16 CFU/microL for S. aureus. This rapid, sensitive and specific diagnostic method can be performed within hours and represents an innovative diagnostic tool for the detection of milk pathogens in dairy products.     

DNA sequence of the 16S rRNA/23S rRNA intercistronic spacer of two rDNA operons of the archaebacterium Methanococcus vannielii

The DNA sequence of the spacer (plus flanking) regions separating the 16S rRNA and 23S rRNA genes of two presumptive rDNA operons of the archaebacterium Methanococcus vannielii was determined. The spacers are 156 and 242 base pairs in size and they share a sequence homology of 49 base pairs following the 3' terminus of the 16S rRNA gene and of about 60 base pairs preceding the 5' end of the 23S rRNA gene. The 242 base pair spacer, in addition contains a sequence which can be transcribed into tRNAAla, whereas no tRNA-like secondary structure can be delineated from the 156 base pair spacer region. Almost complete sequence homology was detected between the end of the 16S rRNA gene and the 3' termini of either Escherichia coli or Halobacterium halobium 16S rRNA, whereas the putative 5' terminal 23S rRNA sequence shared partial homology with E. coli 23S rRNA and eukaryotic 5.8S rRNA.     

Phylogeny of the 5S ribosomal RNA from Synechococcus lividus II: the cyanobacterial/chloroplast 5S RNAs form a common structural class

The complete nucleotide sequence of the 5S ribosomal RNA from the cyanobacterium Synechococcus lividus II has been determined. The sequence is (sequence in text) This 5S RNA has the cyanobacterial- and chloroplast-specific nucleotide insertion between positions 30 and 31 (using the numbering system of the generalized eubacterial 5S RNA) and the chloroplast-specific nucleotide-deletion signature between positions 34 and 39. The 5S RNA of S. lividus II has 27 base differences compared with the 5S RNA of the related strain S. lividus III. This large difference may reflect an ancient divergence between these two organisms. The electrophoretic mobilities on nondenaturing polyacrylamide gels of renatured 5S RNAs from S. lividus II, S. lividus III, and spinach chloroplasts are identical, but differ considerably from that of Escherichia coli 5S RNA. This most likely reflects differences in higher-order structure between the 5S RNA of E. coli and these cyanobacterial and chloroplast 5S RNAs.     

Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35 degrees C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

Deoxyribonucleic Acid Adenine and Cytosine Methylation in Salmonella typhimurium and Salmonella typhi

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.     

Homology of the 3'' terminal sequences of the 18S rRNA of Bombyx mori and the 16S rRNA of Escherchia coli.

The terminal 220 base pairs (bp) of the gene for 18S rRNA and 18 bp of the adjoining spacer rDNA of the silkworm Bombyx mori have been sequenced. Comparison with the sequence of the 16S rRNA gene of Escherichia coli has shown that a region including 45 bp of the B. mori sequence at the 3' end is remarkably homologous with the 3' terminal E. coli sequence. Other homologies occur in the terminal regions of the 18S and 16S rRNAs, including a perfectly conserved stretch of 13 bp within a longer homology located 150--200 bp from the 3' termini. These homologies are the most extensive so far reported between prokaryotic and eukaryotic genomic DNA.     

Staphylococcus aureus helicase but not Escherichia coli helicase stimulates S. aureus primase activity and maintains initiation specificity

Bacterial primases are essential for DNA replication due to their role in polymerizing the formation of short RNA primers repeatedly on the lagging-strand template and at least once on the leading-strand template. The ability of recombinant Staphylococcus aureus DnaG primase to utilize different single-stranded DNA templates was tested using oligonucleotides of the sequence 5'-CAGA (CA)5 XYZ (CA)3-3', where XYZ represented the variable trinucleotide. These experiments demonstrated that S. aureus primase synthesized RNA primers predominately on templates containing 5'-d(CTA)-3' or TTA and to a much lesser degree on GTA-containing templates, in contrast to results seen with the Escherichia coli DnaG primase recognition sequence 5'-d(CTG)-3'. Primer synthesis was initiated complementarily to the middle nucleotide of the recognition sequence, while the third nucleotide, an adenosine, was required to support primer synthesis but was not copied into the RNA primer. The replicative helicases from both S. aureus and E. coli were tested for their ability to stimulate either S. aureus or E. coli primase. Results showed that each bacterial helicase could only stimulate the cognate bacterial primase. In addition, S. aureus helicase stimulated the production of full-length primers, whereas E. coli helicase increased the synthesis of only short RNA polymers. These studies identified important differences between E. coli and S. aureus related to DNA replication and suggest that each bacterial primase and helicase may have adapted unique properties optimized for replication.