Complete nucleotide sequence of a cryptic plasmid from the ruminal bacterium Selenomonas ruminantium HD4 and identification of two predicted open reading frames.

A cryptic plasmid (pSR1) isolated from Selenomonas ruminantium HD4 was previously cloned into the HindIII site of pBR322 and a restriction map was constructed using HindIII, ClaI, BamHI, and PvuII (S. A. Martin and R. G. Dean, Appl. Environ. Microbiol. 55(12), 3035-3038, 1989). Analysis of the nucleotide sequence of pSR1 revealed two major open reading frames (ORFs) located in the minus strand at different frames. Analysis of ORF-1 revealed that it has 325 amino acids with a predicted MW of 36,588, and ORF-2 has 379 amino acids with a predicted MW of 42,651. The ORF-1 amino acids showed 30 to 32% sequence homology to the hypothetical protein YtqA in Bacillus subtilis and another hypothetical protein in the thermophilic bacterium Aquifex aeolicus. ORF-2 showed limited homology (23%) to the hypothetical protein ICFG in the photosynthetic cyanobacteria Synechocystis PCC6803.     

溶藻弧菌质粒pVAE259全序列测定与分子生物学特征分析

【目的】获得溶藻弧菌环状质粒pVAE259全序列,分析其分子生物学特征并探索该质粒可能具备的功能。【方法】使用酶切、克隆测序的方法获得pVAE259的全序列,利用软件分析DNA序列和可能的编码蛋白,推测质粒的生物学信息。【结果】pVAE259为闭合环状质粒,全长6,075 bp,GC含量为42.16%。在NCBI中比对发现pVAE259与Vibriosp.41隐蔽性质粒pPS41具有较高的相似性。我们在序列中找到一个oriT位点,另外全序列的4118-5494 bp推测为质粒复制区域。pVAE259中存在7个氨基酸序列长度大于100的开放式阅读框(ORF):ORF1-ORF7。其中ORF1编码蛋白属于释放酶超级家族(Relaxase Super-family)蛋白,在NCBI数据库中它与大肠杆菌(Escherichia coli)的MobA-like蛋白最相似;ORF2编码蛋白属于复制酶超级家族(Replicase Super-family),它与嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)的复制蛋白RepA最相似;ORF5与伸长盐单胞菌(Halomonas elongata)质粒pHE1的转移蛋白MobC相似。【结论】根据上述结果及相关文献分析,pVAE259可能是具有转移能力的质粒,该质粒是否影响宿主菌的表型性状还不清楚。     

Complete Sequence of Plasmid pMP1 from the Marine Environmental Vibrio vulnificus and Location of Its Replication Origin

A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine–cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.     

Nucleotide sequence of the gene for an alkaline endoglucanase from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis.

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

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.     

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.     

Identification of an enzyme system for daidzein-to-equol conversion in Slackia sp. strain NATTS

An Escherichia coli library comprising 8,424 strains incorporating gene fragments of the equol-producing bacterium Slackia sp. strain NATTS was constructed and screened for E. coli strains having daidzein- and dihydrodaidzein (DHD)- metabolizing activity. We obtained 3 clones that functioned to convert daidzein to DHD and 2 clones that converted DHD to equol. We then sequenced the gene fragments inserted into plasmids contained by these 5 clones. All of the gene fragments were contiguous, encoding three open reading frames (ORF-1, -2, and -3). Analysis of E. coli strains containing an expression vector incorporating one of the orf-1, -2, or -3 genes revealed that (i) the protein encoded by orf-1 was involved in the conversion of cis/trans-tetrahydrodaidzein (cis/trans-THD) to equol, (ii) the protein encoded by orf-2 was involved in the conversion of DHD to cis/trans-THD, and (iii) the protein encoded by orf-3 was involved in the conversion of daidzein to DHD. ORF-1 had a primary amino acid structure similar to that of succinate dehydrogenase. ORF-2 was presumed to be an enzyme belonging to the short-chain dehydrogenase/reductase superfamily. ORF-3 was predicted to have 42% identity to the daidzein reductase of Lactococcus strain 20-92 and belonged to the NADH:flavin oxidoreductase family. These findings showed that the daidzein-to-equol conversion reaction in the Slackia sp. NATTS strain proceeds by the action of these three enzymes.     

Genetic Transformation System for a Psychrotrophic Deep-sea Bacterium: Isolation and Characterization of a Psychrotrophic Plasmid

A versatile system that permits genetic manipulation of a psychrotrophic deep-sea bacterium, Pseudoalteromonas sp. PS1M3, has been developed. A cryptic indigenous plasmid, pPS1M3, of 3.1 kb from the above strain was isolated and characterized. The nucleotide sequence analysis of plasmid pPS1M3 revealed the presence of one open reading frame, and its deduced amino acid sequence was identified as the essential protein for plasmid maintenance. Transformation with the pPS1M3 harboring antibiotic resistance genes by electroporation was fully successful using the pPS1M3-cured strain as a host. This plasmid was quite stable under nonselective culture conditions for about 100 generations at 4°C. The copy number of this plasmid in the cell was about 5 copies per chromosome. Received May 30, 2000; accepted October 11, 2000.     

Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M_r of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M_r of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGC-GGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

Studies on the Biosynthesis of Fosfomycin. Conversion of 2-Hydroxyethylphosphonic Acid and 2-Aminoethylphosphonic Acid to Fosfomycin

Two types of 130 kDa insecticidal protein genes isolated from large plasmid DNAs of Bacillus thuringiensis var. israelensis HDS22 were cloned in an Escherichia coli plasmid vector. Analysis of the nucleotide sequences revealed that the two genes encoded a 127,500-dalton protein (ISRH3) consisting of 1,135 amino acids and a 134,400-dalton protein (ISRH4) consisting of 1,180 amino acids. The two insecticidal proteins were identical in a region of the C-terminal 467 amino acids.     

Cloning, Characterization, and Possible Origin of the Prevotella loescheii dnaK Homolog

Heat shock proteins play an important role in bacterial survival and response to environmental stress. We cloned the Prevotella loescheii HSP70 homolog (dnaK) and characterized the coding sequence, regulatory regions, and evolutionary relationships to other bacteria. Predicted proteins encoded by the P. loescheii dnaK homolog (open reading frame ORF-1) and two downstream coding regions, ORF-2 and ORF-3, are highly homologous to the proteins encoded by ORF-4 (dnaK), ORF-5, and ORF-6 from the dnaK region of Porphyromonas gingivalis. The dnaK promoter resembles other HSP (heat shock protein) promoters. Alignment of the predicted protein encoded by ORF-2 showed significant homology to the Bacteroides fragilis tnpA gene from the transposon Tn4555, whereas the ORF-3 protein showed homology to B. fragilis transposase (Tn5220) and integrase (Tn4555) proteins. This suggests a transposition-like event may be responsible for transfer of these genes between Porphyromonas and Prevotella. Received: 8 June 2000 / Accepted: 11 August 2000     

Respiratory syncytial virus envelope glycoprotein (G) has a novel structure.

Amino acid sequence of human respiratory syncytial virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino acid microsequencing of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated in vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane insertion at 41 residues from the N-terminus. There was no N-terminal signal sequence and the hydrophilic N-terminal 20 residues probably represent the cytoplasmic tail of the protein. The N-terminally oriented membrane insertion was somewhat analogous to paramyxovirus hemagglutinin-neuraminidase (HN) and influenza neuraminidase (NA). The protein was moderately hydrophilic and rich in hydroxy-amino acids. It was both N- and O-glycosylated with the latter contributing significantly to the net molecular mass 90K.     

Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.     

Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24

A gene encoding chitinases from Aeromonas sp. No. 10S-24 was cloned into Escherichia coli DH5α using pUC19, and its nucleotides were sequenced. The chitinase gene was clustered in ORFs (open reading frame) 1 to 4, in a 8-kb fragment of DNA. ORF-1 consisted of 1608 bp encoding 535 amino acid residues, and ORF-2 consisted of 1425 bp encoding 474 amino acid residues. ORF-3 was 1617 bp long and encodes a protein consisting of 538 amino acids. ORF-4 encodes 287 amino acids of the N-terminal region. The amino acid sequences of ORF-1 and ORF-3 share sequence homology with chitinase D from Bacillus circulans, and chitinase A and B from Streptomyces lividans. The amino acid sequence of ORF-2 shared sequence homology with chitinase II from Aeromonas sp. No. 10S-24, and chitinase from Saccharopolyspora erythraea. A region of the sequence starting from Ala-28 of the amino acid sequence of ORF-3 coincided with the N-terminal amino acid sequence of chitinase III from Aeromonas sp. No. 10S-24.     

Sequence analysis of a cryptic plasmid pCJ419 from Campylobacter jejuni and construction of an Escherichia coli-Campylobacter shuttle vector

A small cryptic plasmid, pCJ419, was identified in a human clinical isolate of Campylobacter jejuni, cloned and sequenced. pCJ419 is a circular molecule of 4013 bp with a G+C content of 27.1%. The products of four open reading frames (ORFs) share significant sequence similarity with putative proteins from known C. jejuni and Campylobacter coli plasmids. ORF-1 encodes a putative mobilisation protein (Mob). ORF-2 and ORF-3 encode proteins that have high identity to putative RepA and RepB proteins, respectively, of known C. jejuni and C. coli plasmids. ORF-4 encodes a protein that has high identity to a hypothetical protein of unknown function, Cjp32, previously described in a pVir plasmid of C. jejuni. Tandem repeating 22-bp sequences typical of a plasmid replication origin (ori) were identified upstream of the DNA sequences encoding putative replication initiation proteins. An Escherichia coli-Campylobacter shuttle cloning vector, pGU0202, was constructed using plasmid pMW2 that harbours a Campylobacter-derived kanamycin resistance gene [aph(3')-III]. The sequences encoding pCJ419 mob, RepA and RepB proteins were inserted upstream of aph(3')-III resulting in a stable construct of 6174 bp that was used to transform both E. coli and Campylobacter.     

Identification of an insertion-sequence-like genetic element in the newly recognized human pathogen Mycoplasma incognitus

Cloned 2.2-kb DNA (plasmid psb-2.2) of Mycoplasma incognitus, a pathogen in AIDS and non-AIDS patients [Lo et al., Am. J. Trop. Med. Hyg. 41 (1989) 364-376; 601-616], contains a 1405-bp genetic element closely resembling bacterial insertion sequence (IS) elements. This IS-like element has 29-bp terminal inverted repeats with seven mismatches, is immediately flanked by 3-bp direct repeats, and has typical stem-and-loop structures at or near both the termini. Two potential open reading frames (ORF-1 and ORF-2) encode 143 amino acids (aa) and 103 aa, respectively, in this IS-like element. Part (57 aa) of the deduced aa sequence of ORF-2 has a significant homology (43%) with the putative transposase of Escherichia coli IS3. In this study, a series of synthetic oligodeoxyribonucleotides each containing a specific sequence of a selected segment in psb-2.2, have been used as probes which reveal that the IS-like element occurs more than ten times in the genome of M. incognitus. This potentially transposable element has many characteristic features in common with bacterial IS elements.     

Characterization of the traT gene and mutants that increase outer membrane permeability from the Salmonella typhimurium virulence plasmid

The nucleotide sequence of the traT gene present in the virulence-associated plasmid of Salmonella typhimurium was determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino-terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previously localized to the traT gene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6-5: the introduction, by site-directed mutagenesis, of either positively or negatively charged amino acids or the helix-disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer membrane.