Organization and nucleotide sequences of two lactococcal bacteriocin operons

Two distinct regions of the Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6, each of which specified bacteriocin production as well as immunity, have been sequenced and analyzed by deletion and frameshift mutation analyses. On a 1.8-kb ScaI-ClaI fragment specifying low antagonistic activity, three open reading frames (ORFs) were present, which were organized in an operon. The first two ORFs, containing 69 and 77 codons, respectively, were involved in bacteriocin activity, whereas the third ORF, containing 154 codons, was essential for immunity. Primer extension analysis indicated the presence of a promoter upstream of the ORFs. Two ORFs were present on a 1.3-kb ScaI-HindII fragment specifying high antagonistic activity. The first ORF, containing 75 codons, specified bacteriocin activity. The second ORF, containing 98 codons, specified immunity. The nucleotide sequences of both fragments upstream of the first ORFs as well as the first 20 bp of the first ORF of both bacteriocin operons appeared to be identical.     

Organization and nucleotide sequences of two lactococcal bacteriocin operons.

Two distinct regions of the Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6, each of which specified bacteriocin production as well as immunity, have been sequenced and analyzed by deletion and frameshift mutation analyses. On a 1.8-kb ScaI-ClaI fragment specifying low antagonistic activity, three open reading frames (ORFs) were present, which were organized in an operon. The first two ORFs, containing 69 and 77 codons, respectively, were involved in bacteriocin activity, whereas the third ORF, containing 154 codons, was essential for immunity. Primer extension analysis indicated the presence of a promoter upstream of the ORFs. Two ORFs were present on a 1.3-kb ScaI-HindII fragment specifying high antagonistic activity. The first ORF, containing 75 codons, specified bacteriocin activity. The second ORF, containing 98 codons, specified immunity. The nucleotide sequences of both fragments upstream of the first ORFs as well as the first 20 bp of the first ORF of both bacteriocin operons appeared to be identical.     

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.     

Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134.

Plasmid pJP4 of Alcaligenes eutrophus JMP134 encodes the degradation of 2,4-dichlorophenoxyacetic acid. A 1.2-kb BamHI-XhoI region of the restriction fragment BamHI-E has been proposed to contain the regulatory gene tfdR (A. R. Harker, R. H. Olsen, and R. J. Seidler, J. Bacteriol. 171:314-320, 1989; B. Kaphammer, J. J. Kukor, and R. H. Olsen, J. Bacteriol. 172:2280-2286, 1990). When sequenced and analyzed, the region is shown to contain two incomplete open reading frames (ORFs) positioned divergently. The complete DNA sequence for one of the two ORFs was obtained by sequencing the adjacent restriction fragment BamHI-F. The DNA sequence reveals 100% identify with the regulatory gene tfdS of pJP4. An XbaI-PstI fragment, containing the complete ORF, encodes a 32,000-Da protein which binds to the promoter regions upstream from tfdA and tfdDII. The deduced amino acid sequence of the complete ORF shows similarity with sequences of activator proteins TcbR, CatM, and CatR of the LysR family. The complete ORF represents the regulatory gene tfdR. The deduced amino acid sequence of the incomplete ORF, situated divergently from tfdR, indicates similarity to chloromuconate cycloisomerases produced by genes tfdD and tcbD of plasmids pJP4 and pP51, respectively. This ORF is identified as part of a putative isofunctional gene, tfdDII.     

Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.     

Identification of a lysA-like gene required for tabtoxin biosynthesis and pathogenicity in Pseudomonas syringae pv. tabaci strain PTBR2.024.

Pseudomonas syringae pv. tabaci strain PTBR2.024 produces tabtoxin and causes wildfire disease on tobacco and green bean. PTBR7.000, a Tn5 mutant of PTBR2.024, does not produce tabtoxin, is nonpathogenic on tobacco, and is prototrophic. A 3-kb fragment from a genomic library of the parent strain PTBR2.024 complemented both mutant phenotypes. This 3-kb fragment contains two open reading frames (ORFs), ORF1 and ORF2, and two truncated ORFs, ORF3 and ORF4. The Tn5 insert in PTBR7.000 was mapped to ORF2, and complementation studies showed that an intact ORF2 was sufficient to restore tabtoxin production and pathogenicity. The deduced amino acid sequences of ORF2 and truncated ORF3 contain significant homology to bacterial lysine biosynthetic enzymes, diaminopimelate decarboxylase, and delta 1-piperidine-2,6-dicarboxylate succinyl transferase, respectively. ORF2, however, is not required for lysine biosynthesis. We designated the sequence corresponding to ORF2 as gene tabA and propose that the product of tabA is an enzyme in the tabtoxin biosynthetic pathway that recognizes a substrate analogue of a compound in the lysine biosynthetic pathway.     

The ribulose-1,5-bisphosphate carboxylase/oxygenase gene cluster of Methylococcus capsulatus (Bath)

The genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Methylococcus capsulatus (Bath) were localised to an 8.3-kb EcoRI fragment of the genome. Genes encoding the large subunit ( cbbL), small subunit ( cbbS) and putative regulatory gene ( cbbQ) were shown to be located on one cluster. Surprisingly, cbbO, a second putative regulatory gene, was not located in the remaining 1.2-kb downstream (3') of cbbQ. However, probing of the M. capsulatus (Bath) genome with cbbO from Nitrosomonas europaea demonstrated that a cbbO homologue was contained within a separate 3.0-kb EcoRI fragment. Instead of a cbbR ORF being located upstream (5') of cbbL, there was a moxR-like ORF that was transcribed in the opposite direction to cbbL. There were three additional ORFs within the large 8.3-kb EcoRI fragment: a pyrE-like ORF, an rnr-like ORF and an incomplete ORF with no sequence similarity to any known protein. Phylogenetic analysis of cbbL from M. capsulatus (Bath) placed it within clade A of the green-type Form 1 Rubisco. cbbL was expressed in M. capsulatus (Bath) when grown with methane as a sole carbon and energy source under both copper-replete and copper-limited conditions. M. capsulatus (Bath) was capable of autotrophic growth on solid medium but not in liquid medium. Preliminarily investigations suggested that other methanotrophs may also be capable of autotrophic growth. Rubisco genes were also identified, by PCR, in Methylococcus-like strains and Methylocaldum species; however, no Rubisco genes were found in Methylomicrobium album BG8, Methylomonas methanica S1, Methylomonas rubra, Methylosinus trichosporium OB3b or Methylocystis parvus OBBP.     

阴沟肠杆菌B8拮抗活性基因‘admA’及上游调控序列的克隆与功能鉴定

为了阐明水稻白叶枯病拮抗菌阴沟肠杆菌B8的作用机理,文章采用转座子标签法和染色体步移技术克隆到突变株B8B中Tn5插入位点周边拮抗活性相关片段,并通过基因敲除验证了获得的拮抗相关片段admA’上游调控序列的功能。以转座子中Kan抗性基因为标签,克隆了B8B菌株中Tn5插入位点左侧2 608 bp序列,经两次染色体步移得到Tn5插入位点右侧的2 354 bp序列。序列拼接后获得B8菌株拮抗相关序列4 611 bp的Bcontig。生物信息学分析显示该序列含有7个ORF,分别对应于3-磷酸甘油醛脱氢酶(GADPH)基因的部分编码区、2个LysR家族转录调控因子、弧菌假设蛋白VSWAT3-20465及成团泛菌(Pantoea agglomerans)andrimid生物合成基因簇的admA、admB和部分admC基因序列。B8B菌株Tn5插入分别位于同源于弧菌假设蛋白的anrPORF及‘admA’基因上游200 bp和894 bp处。通过同源重组技术,借助敲除质粒pMB-BG,获得拮抗活性消失的突变株B-1和B-3。结果表明B8B突变株中Tn5的插入可能影响了anrP蛋白的转录和表达,进而调控拮抗物质编码基因簇的生物合成。B8菌株中拮抗物质相关基因是类似于andrimid生物合成基因簇的基因家族,其上游调控区对该抗生素的生物合成具有重要的作用。     

Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.     

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

Biosynthetic Pathway of Cephabacins in Lysobacter lactamgenus: Molecular and Biochemical Characterization of the Upstream Region of the Gene Clusters for Engineering of Novel Antibiotics

The cephabacins, one of the beta-lactam antibiotics, are produced by Lysobacter lactamgenus. The previous studies the cephabacin biosynthesis were limited to a gene cluster that encodes the gene products responsible for the biosynthesis of the cephem nucleus. The long-term goal of this research is to elucidate the metabolic diversity and biosynthetic pathway of cephabacins and to design and/or discover new pharmacologically active compounds by engineering the cephabacin biosynthetic pathway in L. lactamgenus. In this study, we have cloned and sequenced a 24-kb fragment of a DNA locus upstream of the previously reported but incomplete putative ORF9 of L. lactamgenus. This contains three putative ORFs (the complete ORF9, ORF10, and ORF11) transcribed in the same direction and one putative ORF (ORF12) in the opposite direction. The isolated DNA locus extends the previously cloned part of the DNA locus containing the genes responsible for biosynthesis of the cephem nucleus up to 45 kb. The 42-kb fragment of the 45-kb gene cluster is located between a potential TATA box just upstream of the ORF11 and a termination loop just downstream of the previously reported bla gene. The complete ORF9 contains three nonribosomal peptide synthetase (NRPS) modules and one polyketide synthase (PKS) module and the ORF11 contains one NRPS module. The complete ORF9 also contains a putative thioesterase domain at the C-terminal end. We predicted the amino acid specificity of the four NRPSs by generating specificity binding pockets and expressed one of the NRPSs to confirm the amino acid specificity. The adenylation domain of the NRPS1, which is the last module of the NRPSs, showed significant amino acid specificity for L-arginine. These findings are in perfect agreement with the composition that was expected for the structure of cephabacins which contain an acetate residue, an L-arginine, and one to three L-alanines at the C-3' position of the cephem nucleus of cephabacins. The ORF10, encoding a putative ABC transporter which might be involved in conferring resistance against cephabacins, was identified between the complete ORF9 and the ORF11. Therefore, the complete ORF9, ORF10, ORF11 reported here and the other genes previously reported constitute an operon for the biosynthesis of cephabacins in L. lactamgenus. Based on our results, the biosynthetic pathways of acetate and elongated peptide moieties and a mechanism by which cephabacins are assembled by connecting the peptide moiety synthesized by the gene products of the complete ORF9 and the ORF11 to the C-3' position of the cephem nucleus synthesized by the gene products of pcbAB, pcbC, cefE, cefF, and cefD have been elucidated.     

nifV is contiguous to nifHDK in Frankia strain FaC1

The organization of genes with the capacity to code for four proteins involved in nitrogen fixation in Frankia strain FaC1 was determined by restriction fragment mapping and nucleotide sequence analysis. Analysis of the 44-kb genomic cosmid clone pFAH 1. isolated from a cosmid library made from Frankia strain FaCl, resulted in the identification of a 7.2-kb Pst I fragment to which Klebsiella nif H, nif D and nif K probes hybridized. This nif -hybridizing fragment was subcloned and analyzed by restriction fragment mapping. Further subcloning of the 7.2-kb fragment and subsequent sequence analysis of approximately 6.8 kb revealed the presence of six open reading frames (ORFs). Four of these ORFs have the potential to code for nif V-, nif H-, nif D- and nif K-like gene products and the two others are unidentified ORFs. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes, but the positioning of the nif V-like gene relative to the nif HDK cluster differs, A consensus nif -promoter-like sequence, found 5'to nif H. was not detected upstream of the nif V-like gene. Nine copies of a 7-bp direct repeat were found 5'to ORFA.     

Cloning and characterization of a regulatory gene of the SARP family and its flanking region from Streptomyces ambofaciens

In a search for activators of secondary metabolism we isolated a 12.6-kb DNA fragment from a genomic library of Streptomyces ambofaciens NRRL 2240 (the spiramycin producer ). Sequencing of 6 kb of the cloned fragment revealed a cluster of four ORFs (ORF1–4) whose deduced products showed similarities to those of other genes involved in polyketide biosynthesis, including a pathway-specific regulatory gene of the SARP family. The results of insertional inactivation of some of the cloned genes clearly indicate that the isolated cluster does not code for spiramycin production, suggesting that some other polyketide compound might well be produced by this strain. Received: 14 May 1999 / Accepted: 28 July 1999