Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutropbus pbbB and to Rbizobium meliloti nodG

Summary A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. mehloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the pbbB gene and involved in poly--hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli ⁷⁰ consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains.     

The gltF gene of Klebsiella pneumoniae: cloning and initial characterization

Summary From a gene bank of Klebsiella pneumoniae M5a1, a 1.7 kb gene fragment was isolated which was able to restore the Ntr⁺ phenotype and ammonium (methylammonium) transport, but not glutamate synthase in an Escherichia coli glt mutant (glutamate synthase deficiency). The fragment strongly hybridized with the gltF regulatory gene from E. coli. After subcloning the fragment into an overexpression vector, a protein with a molecular weight of 27000 dalton was identified as the gene product. The results indicate that the fragment cloned contains the gltF gene from K. pneumoniae.     

Cloning of histidine genes of Azospirillum brasilense: organization of the ABFH gene cluster and nucleotide sequence of the hisB gene

Summary A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxyterminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.Abbreviations IGP imidazole glycerolphosphate - HP histidinolphosphate     

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 by was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined –16 to –25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.     

Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.     

The Pik m gene, conferring stable resistance to isolates of Magnaporthe oryzae , was finely mapped in a crossover-cold region on rice chromosome 11

The Pik ^m gene in rice confers a high and stable resistance to many isolates of Magnaporthe oryzae collected from southern China. This gene locus was roughly mapped to the long arm of rice chromosome 11 with restriction fragment length polymorphic (RFLP) markers in the previous study. To effectively utilize the resistance, a linkage analysis was performed in a mapping population consisting of 659 highly susceptible plants collected from four F₂ populations using the publicly available simple sequence repeat (SSR) markers. The result showed that the locus was linked to the six SSR markers and defined by RM254 and RM144 with ≈13.4 and ≈1.2 cM, respectively. To fine map this locus, additional 10 PCR-based markers were developed in a region flanked by RM254 and RM144 through bioinformatics analysis (BIA) using the reference sequence of cv. Nipponbare. The linkage analysis with these 10 markers showed that the locus was further delimited to a 0.3-cM region flanked by K34 and K10, in which three markers, K27, K28, and K33, completely co-segregated with the locus. To physically map the locus, the Pik ^m -linked markers were anchored to bacterial artificial chromosome clones of the reference cv. Nipponbare by BIA. A physical map spanning ≈278 kb in length was constructed by alignment of sequences of the clones anchored by BIA, in which only six candidate genes having the R gene conserved structure, protein kinase, were further identified in an 84-kb segment.     

Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)^– RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)^– RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.     

Cloning and characterization of a secY homolog from Chlamydia trachomatis

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc- ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50 195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SceY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.     

Detection of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in seeds using a specific TaqMan probe

Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ Nucleotide Sequence Databanks and have been assigned the accession number X78413     

Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.     

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression

Summary Nearly isogenic lines (NILs) of rice (Oryza sativa) differing at a locus conferring resistance to the pathogen Xanthomonas oryzae pv. oryzae were surveyed with 123 DNA markers and 985 random primers using restriction fragment length plymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. One chromosome 11 marker (RG103) detected polymorphism between the NILs that cosegregated with Xa21. All other chromosome 11 DNA markers tested were monomorphic between the NILs, localizing the Xa21 introgressed region to an 8.3 cM interval on chromosome 11. Furthermore, we identified two polymerase chain reaction (PCR) products (RAPD2148 and RAPD818) that detected polymorphisms between the NILs. Genomic sequences hybridizing with RAPD818, RAPD248 and RG103 were duplicated specifically in the Xa21 NIL. All three markers cosegregated with the resistance locus, Xa21, in a F₂ population of 386 progeny. Based on the frequency with which we recovered polymorphic Xa21-linked markers, we estimated the physical size of the introgressed region to be approximately 800 kb. This estimation was supported by physical mapping (using pulsed field gel electrophoresis) of the sequences hybridizing with the three Xa21-linked DNA markers. The results showed that the three Xa21-linked markers are physically close to each other, with one copy of the RAPD818 sequences located within 60 kb of RAPD248 and the other copy within 270 kb of RG103. None of the enzymes tested generated a DNA fragment that hybridized with all three of the markers indicating that the introgressed region containing the resistance locus Xa21 is probably larger than 270 kb.     

Primary structure of the tms and prs genes of Bacillus subtilis

Summary The nucleotide sequence was determined of a 3211 nucleotide pair EcoRI-PvuII DNA fragment containing the tms and prs genes as well as a part of the ctc gene of Bacillus subtilis. The prs gene encodes phosphoribosylpyrophosphate (PRPP) synthetase, whereas the functioning of the tms and ctc gene products remains to be established. The prs gene contains an open reading frame of 317 codons resulting in a subunit M_r of 34828. An open reading frame comprising the tms gene contained 456 codons resulting in a putative translation product with an M_r of 49554. Comparison of the deduced B. subtilis PRPP synthetase amino acid sequence with PRPP synthetases from Escherichia coli and rat liver showed extensive similarity. The deduced Tms amino acid sequence was found to be 43% similar to the deduced amino acid sequence of ecourfl, a gene of E. coli with unknown function.     

Characterization of an Aspartic Proteinase of Mucor pusillus expressed in Aspergillus oryzae

The aspartic proteinase (MPP) gene from the zygomycete fungus Mucor pusillus was introduced into an ascomycete fungus, Aspergillus oryzae, by protoplast transformation using the nitrate reductase (niaD) gene as the selective marker. Southern blot analysis indicated that the MPP gene was integrated into the resident niaD locus at a copy number of 1–2. MPP secreted by the recombinant A. oryzae was correctly processed but was more highly glycosylated than that produced in the original M. pusillus strain. Treatment with endo--N-acetyl-glucosaminidase H and analysis of the carbohydrate composition of the secreted MPP revealed that the extra glycosylation of the MPP secreted by the recombinant A. oryzae was due to altered processing of mannose residues. The extra glycosylation of MPP affected its enzyme properties including its milk-clotting and proteolytic activities.     

Cloning of the trp genes from the archaebacterium Methanococcus voltae: nucleotide sequence of the trpBA genes

Summary A cosmid bank of Methanococcus voltae DNA was obtained in Escherichia coli after ligation of partially HindIII-digested M. voltae DNA in the HindIII site of the transferable cosmid pVK100. The bank was used to perform complementation experiments with E. coli auxotrophic mutants. Five cosmids complementing trpA shared three adjacent HindIII fragments of 2.1, 2.3 and 14 kb. Two of these cosmids also complemented trpD and carried an additional 4.2 kb HindIII fragment. The trpA- and trpD-complementing regions were more precisely localized using Tn5 mutagenesis. A 1.7 kb PstI fragment, cloned into pUC9 in both orientations, was responsible for the trpA complementation. This fragment was sequenced and an open reading frame (ORF) of 852 nucleotides (ORFtrpA) encoding a 284 amino acid polypeptide of mol. wt. 31938 was found. The amino acid sequence was compared with that of the subunit of tryptophan synthase (trpA gene product) from nine eubacterial species and to the N-terminal part of the tryptophan synthase of Saccharomyces cerevisiae (TRP5 gene product). Similarity varied from 24% (Brevibacterium lactofermentum) to 35% (S. cerevisiae). The nucleotide sequence of the region upstream from M. voltae ORFtrpA was determined and revealed the presence of an ORF of 1227 nucleotides (ORFtrpB) encoding a 409 amino acid polypeptide of mol. wt. 44634. The polypeptide sequence was similar to the subunit of tryptophan synthase (trpB gene product) from six eubacterial species and to the C-terminal part of the tryptophan synthase of S. cerevisiae. Similarity varied from 49% (S. cerevisiae, B. lactofermentum) to 58% (Pseudomonas aeruginosa). This high conservation supports the hypothesis of a common ancestor for the trpA and trpB genes of archaebacteria, eubacteria and eucaryotes. M. voltae ORFtrpA and ORFtrpB, which are transcribed in the same direction, are separated by a 37 bp AT-rich region. Immediately upstream from ORFtrpB, the 3 end of an ORF homologous to E. coli and Bacillus subtilis trpF was found. As the trpD-complementing region was located upstream from the trpFBA sequenced region, the organization of trp genes in the archaebacterium might thus be trpDFBA. Such an organization resembles that of enteric eubacteria, in which the trpEDCFBA genes are grouped in a single operon. However, M. voltae ORFtrpA and ORFtrpB do not overlap, in contrast with what is found in most eubacteria.     

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation

Summary A DNA fragment that codes for the 364 amino-terminal amino acid residues of a putative Bacillus subtilis SecA homologue has been cloned using the Escherichia coli SecA gene as a probe. The deduced amino acid sequence showed 58% identity to the aminoterminus of the E. coli SecA protein. A DNA fragment which codes for 275 amino-terminal amino acid residues of the B. subtilis SecA homologue was expressed in E. coli and the corresponding gene product was shown to be recognized by anti-E. coli SecA antibodies. This polypeptide, although only about 30% the size of the E. coli SecA protein, also restored growth of E. coli MM52 (secA ^ts) at the non-permissive temperature and the translocation defect of proOmpA in this mutant was relieved to a substantial extent.     

Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus

Summary The cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a M_r of 43 622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a M_r of 34532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10–12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus.