A novel sequence framework (bla(TEM-1G)) encoding the parental TEM-1 beta-lactamase

A novel parental bla(TEM) gene (bla(TEM-1G)), encoding a TEM-1 beta-lactamase (pI of 5.4) produced by the uropathogenic Escherichia coli strain FMV194 was isolated from a dog. We report PCR-restriction fragment length polymorphism analysis and nucleotide sequencing of this gene. The bla(TEM-1G) sequence was identical to the bla(TEM-1C) gene framework in the coding and promoter (P3) regions, except for a silent G(604)-->T mutation in the coding region. Molecular phylogenetic analysis of parental bla(TEM) genes indicated two distinct groups, one comprising bla(TEM-1F) and bla(TEM-2). The other group comprises bla(TEM-1C) which is the probable ancestor of bla(TEM-1A), bla(TEM-1D) and bla(TEM-1G). The bla(TEM-1G) gene has the same framework as a gene encoding an inhibitor-resistant TEM beta-lactamase produced by an E. coli strain of human origin. Thus, parental bla(TEM) genes encoding beta-lactamases in E. coli strains isolated from different host species, in this case human and canine, may be phylogenetically very close.     

Structural and functional characterization of the 5'-flanking region of the rat and human cytochrome P450 2E1 genes: identification of a polymorphic repeat in the human gene.

Cytochrome P450 2E1 (CYP2E1) is a toxicologically very important enzyme with a high extent of interindividual variability in expression. We sequenced and characterized the 5'-flanking region of the human and rat CYP2E1 genes. The identity between the human and rat sequences (-3.8 kb to +1 kb) was generally between 35 and 60%, and the most similar regions were found in the proximal part of the sequence. Two more distant regions at -1.6 to -2.0 kb and -2.5 to -2. 8 kb in the human sequence were also found to have high identity to the rat sequence. A polymorphic repeat sequence in the human gene was found between -2178 to -1945 bp. The common allele (CYP2E1*1C) contained 6 repeats (each 42-60 bp long) and the rare allele (CYP2E1*1D) had 8 repeats with an allele frequency of 1% among Caucasians and 23% among Chinese. The CYP2E1 5'-flanking regions of the human (-3712 bp to +10 bp) and rat (-3685 bp to +28 bp) genes were ligated in front of a luciferase reporter gene and transfected into rat hepatoma Fao and human hepatoma B16A2 cells. Important species specificity was noted in the control of gene expression and regions of negative and positive cis-acting elements were localized. No difference was seen in the constitutive expression between the two polymorphic forms. The importance of this repeat polymorphism for high and low inducible CYP2E1 phenotypes is discussed.     

Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop.

We have compared the recF genes from Escherichia coli K-12, Salmonella typhimurium, Pseudomonas putida, and Bacillus subtilis at the DNA and amino acid sequence levels. To do this we determined the complete nucleotide sequence of the recF gene from Salmonella typhimurium and we completed the nucleotide sequence of recF gene from Pseudomonas putida begun by Fujita et al. (1). We found that the RecF proteins encoded by these two genes contain respectively 92% and 38% amino acid identity with the E. coli RecF protein. Additionally, we have found that the S. typhimurium and P. putida recF genes will complement an E. coli recF mutant, but the recF gene from Bacillus subtilis [showing about 20% identity with E. coli (2)] will not. Amino acid sequence alignment of the four proteins identified four highly conserved regions. Two of these regions are part of a putative phosphate binding loop. In one region (position 36), we changed the lysine codon (which is essential for ATPase, GTPase and kinase activity in other proteins having this phosphate binding loop) to an arginine codon. We then tested this mutation (recF4101) on a multicopy plasmid for its ability to complement a recF chromosomal mutation and on the E. coli chromosome for its effect on sensitivity to UV irradiation. The strain with recF4101 on its chromosome is as sensitive as a null recF mutant strain. The strain with the plasmid-borne mutant allele is however more UV resistant than the null mutant strain. We conclude that lysine-36 and possibly a phosphate binding loop is essential for full recF activity. Lastly we made two chimeric recF genes by exchanging the amino terminal 48 amino acids of the S. typhimurium and E. coli recF genes. Both chimeras could complement E. coli chromosomal recF mutations.     

系统发生分析发现牛病毒性腹泻病病毒新基因亚型

本研究对我国首次分离获得的牛源牛病毒性腹泻病毒(BVDV)毒株Changchun 184(CC-184)和猪源牛病毒 性腹泻病毒ZM-95进行了遗传衍化关系研究。选择主要抗原E2基因为研究对象,首先应用RT-PCR及套式PCR 克隆得到CC-184和ZM-95的E2片段,通过序列测定发现CC-184和ZM-95 E2基因长度分别为1,122bp和1, 125bp,各自编码374和375个氨基酸残基。核酸序列同源性比较和系统发生分析表明2株病毒均属于BVDV-1, CC-184与Osloss亲缘关系最近,都属于已有的b基因亚型,其E2基因同源性达91．8％。而ZM-95的E2基因有 一个特征性的变异区,包含一个密码子序列插入,这一变异区编码了一段有别于其他瘟病毒的五肽氨基酸序列 HYKKK。结果还表明ZM-95与BVDV-1现有的5个基因亚型的亲缘关系均较远,E2基因同源性最高(与Oregon c24v)只有72．4％。而BVDV-1亚型内毒株间的同源性大于85％,亚型间的同源性在69％～75％之间,充分说明 ZM-95是BVDV-1中一个新发现的基因亚型。通常认为猪源BVDV来源于牛,应该与牛源BVDV有十分近的遗 传关系,但是本研究发现ZM-95与其他已知牛源BVDV较低的基因同源性说明猪源BVDV还具有独立的遗传衍 化与传播来源的可能性。     

Role for E2F in Control of Both DNA Replication and Mitotic Functions as Revealed from DNA Microarray Analysis

We have used high-density DNA microarrays to provide an analysis of gene regulation during the mammalian cell cycle and the role of E2F in this process. Cell cycle analysis was facilitated by a combined examination of gene control in serum-stimulated fibroblasts and cells synchronized at G(1)/S by hydroxyurea block that were then released to proceed through the cell cycle. The latter approach (G(1)/S synchronization) is critical for rigorously maintaining cell synchrony for unambiguous analysis of gene regulation in later stages of the cell cycle. Analysis of these samples identified seven distinct clusters of genes that exhibit unique patterns of expression. Genes tend to cluster within these groups based on common function and the time during the cell cycle that the activity is required. Placed in this context, the analysis of genes induced by E2F proteins identified genes or expressed sequence tags not previously described as regulated by E2F proteins; surprisingly, many of these encode proteins known to function during mitosis. A comparison of the E2F-induced genes with the patterns of cell growth-regulated gene expression revealed that virtually all of the E2F-induced genes are found in only two of the cell cycle clusters; one group was regulated at G(1)/S, and the second group, which included the mitotic activities, was regulated at G(2). The activation of the G(2) genes suggests a broader role for E2F in the control of both DNA replication and mitotic activities.     

A novel phenanthrene dioxygenase from Nocardioides sp. Strain KP7: expression in Escherichia coli

Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the alpha and beta subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8. 3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the alpha and beta subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.     

The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro

The UspA1 and UspA2 proteins of Moraxella catarrhalis are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of uspA1 and uspA2 mutants of M. catarrhalis strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113-3119, 1998). In the present study, isogenic uspA1, uspA2, and uspA1 uspA2 mutations were constructed in three additional M. catarrhalis strains: 012E, TTA37, and 046E. The uspA1 mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the uspA1 gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the uspA2 gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the uspA2 genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this "hybrid" protein was designated uspA2H. PCR-based analysis revealed that approximately 20% of M. catarrhalis strains apparently possess a uspA2H gene instead of a uspA2 gene. The M. catarrhalis uspA1, uspA2, and uspA2H genes were cloned and expressed in Haemophilus influenzae cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.     

Characterization of a unique chromosomal copper resistance gene cluster from Xanthomonas campestris pv. vesicatoria

We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.     

Identification of the monooxygenase gene clusters responsible for the regioselective oxidation of phenol to hydroquinone in mycobacteria

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.     

Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx2) in the outbreak strain or with the loss of this gene. The two stx2 alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx2 genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.     

DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca.

The DNA sequences of the Thermomonospora fusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined. Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon. There were no significant homologies between the coding regions of the three genes. The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes. E2 belongs to a family of cellulases that includes celA from M. bispora, cenA from Cellulomonas fimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei. E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family. There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C. fimi and Pseudomonas fluorescens cellulose binding domains.     

A newly discovered Bacteroides conjugative transposon,CTnGERM1, contains genes also found in gram-positive bacteria

Results of a recent study of antibiotic resistance genes in human colonic Bacteroides strains suggested that gene transfer events between members of this genus are fairly common. The identification of Bacteroides isolates that carried an erythromycin resistance gene, ermG, whose DNA sequence was 99% identical to that of an ermG gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into Bacteroides species from other genera. Six of seven ermG-containing Bacteroides strains tested were able to transfer ermG by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying ermG in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in Bacteroides isolates-CTns of the CTnERL/CTnDOT family-which sometimes carry another type of erm gene, ermF. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the ermG-containing strains but also to DNA from strains that did not carry ermG. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired ermG. The percentage of G+C content of the ermG region was significantly lower than that of the chromosome of Bacteroides species-an indication that CTnGERM1 may have entered Bacteroides strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered Bacteroides species relatively recently. One of the genes located upstream of ermG encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from Streptococcus pyogenes. Our having found >90% sequence identity of two upstream genes, including mefA, and the remnants of two transposon-carried genes downstream of ermG with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.     

Evolution in the laboratory: the genome of Halobacterium salinarum strain R1 compared to that of strain NRC-1

We report the sequence of the Halobacterium salinarum strain R1 chromosome and its four megaplasmids. Our set of protein-coding genes is supported by extensive proteomic and sequence homology data. The structures of the plasmids, which show three large-scale duplications (adding up to 100 kb), were unequivocally confirmed by cosmid analysis. The chromosome of strain R1 is completely colinear and virtually identical to that of strain NRC-1. Correlation of the plasmid sequences revealed 210 kb of sequence that occurs only in strain R1. The remaining 350 kb shows virtual sequence identity in the two strains. Nevertheless, the number and overall structure of the plasmids are largely incompatible. Also, 20% of the protein sequences differ despite the near identity at the DNA sequence level. Finally, we report genome-wide mobility data for insertion sequences from which we conclude that strains R1 and NRC-1 originate from the same natural isolate. This exemplifies evolution in the laboratory.