Linear plasmidlike DNA in the plant pathogenic fungus Fusarium oxysporum f. sp. conglutinans.

Double-stranded, 1.9-kilobase-pair (kbp) DNA molecules were found in 18 strains representing three pathogenic races of Fusarium oxysporum f. sp. conglutinans. The DNA element (pFOXC1) from a race 1 strain and the DNA element (pFOXC2) from a race 2 strain were shown by restriction endonuclease mapping to be linear. pFOXC2 was found in mitochondrial preparations and appears to have blocked 5' termini, as it was sensitive to 3'----5' exonuclease III but insensitive to 5'----3' lambda exonuclease. The major 1.8-kbp BglII restriction endonuclease fragment of pFOXC2 was cloned in plasmid pUC12. The recombinant plasmid (pCK1) was not homologous to the mitochondrial or nuclear genomes from F. oxysporum f. sp. conglutinans. This suggests that pFOXC2 is self-replicating. pCK1 was homologous to all 1.9-kbp DNA elements of race 2 but was not homologous to those of race 1 or race 5. All race 1 and 5 elements were also shown to share common DNA sequences.     

Molecular characterization of a cryptic plasmid from the psychrotrophic antarctic bacterium Pseudoalteromonas sp. 643A

We report the identification and nucleotide sequence analysis of pKW1, a plasmid of the psychrotrophic bacterium Pseudoalteromonas sp. 643A isolated from the stomach of Antarctic krill Euphasia superba. pKW1 consists of 4583 bp, has a G+C content of 43% and seven putative open reading frames (ORFs). The deduced amino acid sequence from ORF-1 shared significant similarity with the plasmid replicase protein of Psychrobacter cryohalolentis, strain K5. The DNA region immediately downstream of the ORF-1 showed some homology with the Rep-binding sequence of the theta-replicating ColE2-type plasmids. The ORF-3 amino acid sequence revealed amino acid sequence homology with the mobilization protein of Psychrobacter sp. PRwf-1 and Moraxella catarrhalis, with identities of 28% and 25%, respectively. The ORF-4 showed 46% amino acid sequence homology with the putative relaxase/mobilization nuclease MobA of Hafnia alvei and 44% homology with the putative mobilization protein A of Pasterulla multocida. The copy number of pKW1 in Pseudoalteromonas sp. 643A was estimated of 15 copies per chromosome.     

Identification of a replication initiation protein of the pVV8 plasmid from Thermus thermophilus HB8

Recently, the extremely thermophilic bacterium Thermus thermophilus HB8 has been demonstrated to harbor a circular plasmid designated by pVV8 in addition to two well-known plasmids, pTT8 and pTT27, and its entire sequence has been determined. The absence of any obvious replication initiation gene in the 81.2 kb plasmid prompted us to isolate its minimum replicon. By in vivo replication assays with fragments deleted in a stepwise manner, a minimum replicon containing a single ORF, TTHV001, was identified. A protein encoded by TTHV001 showed no amino acid sequence similarity to other function-known proteins. As the results of in vivo and in vitro experiments strongly suggested that the TTHV001 protein was involved in the replication initiation of pVV8, the protein and the gene were referred to as RepV and repV, respectively. The RepV protein binds to an inverted repeat sequence within its own repV gene and then triggers the unwinding of the DNA duplex in an A + T-rich region located just downstream from the inverted repeat. The in vivo replication assays with minimum replicon mutants in the RepV binding site or the unwinding region demonstrated that the unwinding in the region by the RepV binding was essential for pVV8 replication initiation.     

Cloning and sequencing of plasmid pLC494 isolated from human intestinal Lactobacillus casei: construction of an Escherichia coli-Lactobacillus shuttle vector

The complete nucleotide sequence of plasmid pLC494 isolated from Lactobacillus casei L-49 was determined. Plasmid pLC494 is an 8846-bp long circular molecule with a G+C content of 41.5%. Two putative open reading frames, ORF4 (282 amino acids) and ORF5 (169 amino acids), were identified as replication proteins A and B that revealed 100 and 99% similarity, respectively, with the replication proteins of plasmid pLA103 from Lactobacillus acidophilus TK8912. Upstream of ORF4 were the four repeat regions (three perfect 22-bp repeats and one imperfect motif), a putative ribosome binding site, a -10 region, and a -35 region. The shuttle vector pJLE4942 (5318 bp) was constructed using repA from pLC494, a multiple cloning site, ColE1 ori, the ori of gram-negative bacteria from vector pUC19, and the chloramphenicol resistance gene from pJIR418 as a selection marker. Transformation of several lactic acid bacteria with the vector pJLE4942 indicated that this vector might be useful as a genetic tool for the intestinal lactobacilli.     

Characterization of the mobilization region of a Bacteroides insertion element (NBU1) that is excised and transferred by Bacteroides conjugative transposons.

Many Bacteroides clinical isolates carry large conjugative transposons that, in addition to transferring themselves, excise, circularize, and transfer smaller, unlinked chromosomal DNA segments called NBUs (nonreplicating Bacteroides units). We report the localization and DNA sequence of a region of one of the NBUs, NBU1, that was necessary and sufficient for mobilization by Bacteroides conjugative transposons and by IncP plasmids. The fact that the mobilization region was internal to NBU1 indicates that the circular form of NBU1 is the form that is mobilized. The NBU1 mobilization region contained a single large (1.4-kbp) open reading frame (ORF1), which was designated mob. The oriT was located within a 220-bp region upstream of mob. The deduced amino acid sequence of the mob product had no significant similarity to those of mobilization proteins of well-characterized Escherichia coli group plasmids such as RK2 or of either of the two mobilization proteins of Bacteroides plasmid pBFTM10. There was, however, a high level of similarity between the deduced amino acid sequence of the mob product and that of the product of a Bacteroides vulgatus cryptic open reading frame closely linked to a cefoxitin resistance gene (cfxA).     

Nucleotide sequence of coat protein gene for GPV isolate of barley yellow dwarf virus and construction of expression plasmid for plant

GPV is a Chinese serotype isolate of barley yellow dwarf virus (BYDV) that has no reactionwith antiserum of MAV, PAV, SGV, RPV and RMV. The sequence of the coat protein (CP) of GPV isolate of BYDV was identified and its amino acid sequence was deduced. The coding region for the putative GPV CP is 603 bases nucleotides and encodes a Mr 22218 (22 ku) protein. The same as MAV, PAV and RPV, GPV contained a second ORF within the coat protein coding region. This protein of 17024 Mr (17 ku) is thought to correspond to the Virion protein genome linked (Vpg). Sequence comparisons of the CP coding region between the GPV isolate of BYDV and other isolates of BYDV have been done. The nucleotide and ammo acid sequence homology of GPV has a greater identity to the sequence of RPV than those of PAV and MAV. The GPV CP sequence shared 83.7% of nucleotide similarity and 77.5% of deduced amino add similarity, whereas that of the PAV and MAV shared 56.9%. 53.2% and 44.1%. 43.8% respectively. According to BYDV-GPV CP seque     

Molecular characterization and interstrain variability of pHPS1, a plasmid isolated from the Sydney strain (SS1) of Helicobacter pylori

The 5846-bp circular plasmid pHPS1 of Helicobacter pylori Sydney strain, SS1, was cloned, sequenced, and structurally characterized. The SS1 strain is widely used in animal studies of H. pylori infection. The sequence of pHPS1 revealed three open reading frames (ORFs), all of which are transcribed. Two ORFs encode putative plasmid replication proteins, RepA and RepB, similar to replicases resident on theta plasmids. In contrast, the function of ORF2 remains cryptic due to the absence of sequence similarity with any known protein in sequence databases. In addition, species specificity of these three coding regions was shown using DNA dot blot hybridization in 57 diverse clinical H. pylori isolates and 32 Helicobacter and Campylobacter strains. RepA appears to be the predominant plasmid replication protein of H. pylori and the deduced amino acid sequence was highly conserved (76-96%) in 8 H. pylori isolates, including SS1. RepB was detected in 3 H. pylori isolates examined in this study, 2 of which possess only the repB gene. Analysis of the protein sequences of these two replicases, together with previously characterized H. pylori plasmid replication proteins, supports the formation of a distinct class of H. pylori plasmid proteins. Moreover, comprehensive analysis of the whole genome sequence of H. pylori strain 26695, pHPS1, and other H. pylori plasmid sequences that are available revealed interesting insights as to the occurrence of plasmid-mediated recombination within H. pylori. Common regions between plasmids and chromosome sequences of H. pylori were identified in this study which could only have arisen by genetic recombination, thus providing the first line of evidence, albeit indirectly, of the contribution of H. pylori plasmids in generating an extensive genetic heterogeneity characteristic of this important gastroduodenal pathogen.     

禽流感病毒A/Chicken/Guangdong/SS/94(H9N2)HA基因的克隆及序列分析

禽流感是由A型流感病毒引起的禽的一种疾病综合症.1878年首次在意大利爆发流行,当时称该病为"鸡瘟".之后,许多国家和地区都有该病的报道,包括美国、英国、澳大利亚、爱尔兰、比利时、荷兰、法国、俄罗斯、加拿大、以色列、匈牙利、日本、中国(包括香港)等[1-3].     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans.

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing beta-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified alpha-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the alpha-amino acid ester hydrolase is a beta-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The alpha-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of beta-lactam antibiotic acylases.     

Cloning, sequencing, and sequence analysis of two novel plasmids from the thermophilic anaerobic bacterium Anaerocellum thermophilum

The nucleotide sequence of two novel plasmids isolated from the extreme thermophilic anaerobic bacterium Anaerocellum thermophilum DSM6725 (A. thermophilum), growing optimally at 70 degrees C, has been determined. pBAS2 was found to be a 3653 bp plasmid with a GC content of 43%, and the sequence revealed 10 open reading frames (ORFs). The two largest of these, namely Orf21 and Orf41, showed similarity to a Bacillus plasmid recombinase and a Pseudoalteromonas plasmid replication protein, respectively. A sequence with homology to double stranded replication origins from rolling circle plasmids was found, but no single stranded intermediates, characteristic of rolling circle replication, were found on Southern blots. The larger plasmid, pBAL, was found to be a 8294 bp plasmid with a GC content of 39%. It revealed 17 ORFs, of which three showed similarity at the amino acid (aa) level to known proteins. Orf22 showed the strongest similarity (33% aa) to replication proteins from large multiresistance Staphylococcal and Lactococcal plasmids, all of which are believed to replicate via a theta-like replication mechanism. Orf32 showed similarity to both DNA repair proteins and DNA polymerases with highest similarity to DNA repair protein from Campylobacter jejuni (25% aa). Orf34 showed similarity to sigma factors with highest similarity (28% aa) to the sporulation specific Sigma factor, Sigma 28(K) from Bacillus thuringiensis.     

Lack of homology between two haloacetate dehalogenase genes encoded on a plasmid from Moraxella sp. strain B.

Two genes encoding haloacetate dehalogenases, H-1 and H-2, are closely linked on a plasmid from Moraxella sp. strain B. H-1 predominantly acts on fluoroacetate, but H-2 does not. To elucidate the molecular relationship between the two enzymes, we compared their structural genes. Two restriction fragments of the plasmid DNA were subcloned on M13 phages and their nucleotide sequences were determined. The sequence of each fragment contained an open reading frame that was identified as the structural gene for each of the two dehalogenases on the basis of the following criteria; N-terminal amino acid sequence, amino acid composition, and molecular mass. The genes for H-1 and H-2, designated dehH1 and dehH2, respectively, had different sizes (885 bp and 675 bp) and G+C contents (58.3% and 53.4%). Sequence analysis revealed no homology between the two genes. We concluded that the dehalogenases H-1 and H-2 have no enzyme-evolutionary relationship. The deduced amino acid sequence of the dehH1 gene showed significant similarity to those of three hydrolases of Pseudomonas putida and a haloalkane dehalogenase of Xanthobacter autotrophicus. The dehH2 coding region was sandwiched between two repeated sequences about 1.8 kb long, which might play a part in the frequent spontaneous deletion of dehH2 from the plasmid.     

A cryptic plasmid from Pasteurella multocida has a predicted protein nearly identical to a transport protein from Actinobacillus actinomycetemcomitans

Several plasmids from Pasteurella multocida have been shown to carry antibiotic resistance genes but no other genes possibly related to the organism's pathogenesis. We report here that sequence from the plasmid pLEM from a fowl isolate of P. multocida, strain 1059, contained one open reading frame that had significant identity with a predicted protein from pVT745, a plasmid that was isolated from a human oral isolate of Actinobacillus actinomycetemcomitans. This predicted protein had significant homology at the amino acid level to cation transport proteins.     

Complete nucleotide sequence of an Amerindian human T-cell lymphotropic virus type II (HTLV-II) isolate: identification of a variant HTLV-II subtype b from a Guaymi Indian.

The complete nucleotide sequence of a human T-cell lymphotropic virus type II (HTLV-II) isolate from a Panamanian Guaymi Indian was determined and analyzed. When this new viral isolate (HTLV-IIG12) was compared with prototypic HTLV-IIMoT, the overall nucleotide sequence similarity was 95.4%, while the predicted amino acid sequence similarity was 97.5%. Although the overall percentage of nucleotide and amino acid identity with prototypic HTLV-IIMoT (subtype a) was high, HTLV-IIG12 displayed several distinctive features that defined it as an HTLV-II subtype b. However, there were several characteristics unique to this isolate, which included a cluster of nucleotide substitutions in the pre-gag region and changes in restriction enzyme sites within the pre-gag region and the gag, pol, env, and pX genes. In addition, two nucleotide changes in the C terminus of the Tax protein coding sequence inserted an Arg residue for a stop codon and appeared to result in a larger tax gene product in HTLV-IIG12. Although the HTLV-IIG12 isolate appears to be a variant of the prototypic HTLV-IIb, this information represents the first complete nucleotide sequence of any HTLV-II subtype b. These data will allow further studies on the evolutionary relationships between the HTLV-II subtypes and between HTLV-I and HTLV-II.     

Carbenicillin-hydrolysing penicillinase mediated by a plasmid of Proteus mirabilis and its relationship to the PSE-type enzymes of Pseudomonas aeruginosa

The nucleotide sequence of a carbenicillin-hydrolysing (carbenicillinase) gene occurring in an endogenous plasmid, pCS229, of Proteus mirabilis was determined. The amino acid sequence of the mature enzyme, comprising 271 amino acids with a molecular mass of 29 506 Da, was deduced. The pCS229 carbenicillinase showed only 46·4% similarity, in the overall amino acid sequence, to the chromosomal carbenicillinase of Pr. mirabilis GN79 ; however, the enzyme showed about 98% similarity to a Pseudomonas -specific plasmid-encoded carbenicillinase, PSE-4, that was isolated from Pseudomonas aeruginosa . Only five of 271 amino acids differed from those of PSE-4. This study proved the close relationship between the carbenicillinase genes distributed in Pr. mirabilis and Ps. aeruginosa .