Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Summary The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 by in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 by in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84 — Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.     

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO

Summary The gene braB, encoding the Na^–-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na^–-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated M_r of 45279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with thebrnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.     

Suppression of gray leaf spot (blast) of perennial ryegrass turf by Pseudomonas aeruginosa from spent mushroom substrate

Bacteria isolated from spent mushroom substrate (SMS) were evaluated for the suppression of Pyricularia grisea, the causal agent of gray leaf spot of perennial ryegrass (Lolium perenne) turf. Thirty-two of 849 bacterial isolates (3.8%) from SMS significantly inhibited the mycelial growth of P. grisea in vitro. Six bacterial isolates that afforded maximum inhibition of P. grisea were also refractory to Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, and Fusarium culmorum. Each of the six isolates was identified as Pseudomonas aeruginosa by fatty acid profile analysis. The biocontrol activity of the bacterial isolates was not compromised by a prolonged exposure to UV radiation in vitro. In controlled-environment chamber experiments, all 32 bacterial isolates were tested for suppression of gray leaf spot on Pennfine perennial ryegrass when applied as seed treatment or foliar sprays. Foliar applications of the bacteria (10⁸ cfu/ml 0.1% carboxymethylcellulose), but not the seed treatment, significantly reduced disease severity and incidence. The three most efficient isolates from foliar application treatments, which were among the six bacterial isolates identified as P. aeruginosa, were further evaluated for suppression of gray leaf spot as a function of timing of application. The three isolates of P. aeruginosa suppressed gray leaf spot in perennial ryegrass in Cone-tainers when applied at 1, 3, and 7 days prior to inoculation with P. grisea both in controlled-environment chamber experiments, and in potted ryegrass plants maintained in the field. All application intervals, regardless of the bacterial isolate, provided significant reduction of gray leaf spot severity. Suppression of gray leaf spot by isolates of P. aeruginosa under controlled-environment chamber conditions was not different from that observed in potted ryegrass plants maintained in the field. In field experiments, an isolate of P. aeruginosa provided significant suppression of gray leaf spot when applied at 1, 7, and 14 days prior to inoculation with P. grisea. The bacterium proved effective against gray leaf spot of perennial ryegrass maintained as fairway and rough heights. These results indicate that P. aeruginosa may be a potential biocontrol agent for gray leaf spot of perennial ryegrass turf.     

Ornibactin production and transport properties in strains of Burkholderia vietnamiensis and Burkholderia cepacia (formerly Pseudomonas cepacia)

Several strains of Burkholderia vietnamiensis, isolated from the rhizosphere of rice plants, and four strains formerly known as Pseudomonas cepacia including two collection strains and two clinical isolates were compared for siderophore production and iron uptake. The B. vietnamiensis (TVV strains) as well as the B. cepacia strains (ATCC 25416 and ATCC 17759) and the clinical isolates K132 and LMG 6999 were all found to produce ornibactins under iron starvation. The two ATCC strains of B. cepacia additionally produced the previously described siderophores, pyochelin and cepabactin. Analysis of the ratio of isolated ornibactins (C4, C6 and C8) by HPLC revealed nearly identical profiles. Supplementation of the production medium with ornithine (20 mm) resulted in a 2.5-fold increase in ornibactin synthesis. Ornibactin-mediated iron uptake was independent of the length of the acyl side chain and was observed with all strains of B. vietnamiensis and B. cepacia, but was absent with strains of Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas stutzeri, known to produce pyoverdines or desferriferrioxamines as siderophores. These results suggest that ornibactin production is a common feature of all Burkholderia strains and that these strains develop an ornibactin-specific iron transport system which is distinct from the pyoverdine-specific transport in Pseudomonas strains.     

Combinative effects of a bacterial type-III effector and a biocontrol bacterium on rice growth and disease resistance

Expression of HpaG_Xoo, a bacterial type-III effector, in transgenic plants induces disease resistance. Resistance also can be elicited by biocontrol bacteria. In both cases, plant growth is often promoted. Here we address whether biocontrol bacteria and HpaG_Xoo can act together to provide better results in crop improvement. We studied effects ofPseudomonas cepacia on the rice variety R109 and the hpaG_Xoo-expressing rice line HER1. Compared to R109, HER1 showed increased growth, grain yield, and defense responses toward diseases and salinity stress. Colonization of roots byP. cepacia caused 20% and 13% increase, in contrast to controls, in root growth of R109 and HER1. Growth of leaves and stems also increased in R109 but that of HER 1 was inhibited. WhenP. cepacia colonization was subsequent to plant inoculation withRhizoctonia solani, a pathogen that causes sheath blight, the disease was less severe than controls in both R109 and HER1; HER1, nevertheless, was more resistant, suggesting thatP.cepacia and HpaG_Xoo cooperate in inducing disease resistance. Several genes that critically regulate growth and defense behaved differentially in HER1 and R109 while responding toP. cepacia. In R109 leaves, theOsARF1 gene, which regulates plant growth, was expressed in consistence with growth promotion byP. cepacia. Inversely,OsARF1 expression was coincident with inhibition in growth of HER1 leaves. In both plants, the expression ofOsEXP1, which encodes an expansin protein involved in plant growth, was concomitant with growth promotion in leaves instead of roots, in response toP. cepacia. We also studiedOsMAPK, a gene that encodes a mitogen-activated protein kinase and controls defense responses toward salinity and infection by pathogens in rice. In response toP. cepacia, an early expression ofOsMAPK was coincident with R109 resistance to the disease, while HER1 expressed the gene similarly whetherP. cepacia was present or not. Evidently,P. cepacia and G_Xoo-gene mediated resistance may act differently in rice growth and resistance. Whereas combinative effectsof P. cepacia and HpaG_Xoo in disease resistance have a great potential in agricultural use, it is interesting to study mechanisms that underlie interactions involving biocontrol bacteria, type-III effectors and pathogens. These authors contributed equally to this paper.     

Structural and functional analysis of the rcsA gene from Erwinia stewartii

Summary ResA is a positive regulator of genes for extracellular polysaccharide biosynthesis in the Enterobacteriaceae. The nucleotide sequence of the rcsA gene from Erwinia stewartii was determined and compared to rcsA sequences from E. amylovora, Escherichia coli, and Klebsiella pneumoniae. Three highly conserved regions of the gene were identified. The C-terminal end of the open reading frame (ORF) shared significant amino acid homology to the LuxR class of bacterial activator proteins. Insertion and deletion mutagenesis of the 5 non-coding region indicated that maximal expression of rcsA was dependent upon cis-acting regulatory sequences located more than 300 by upstream of the translational start site.     

Nucleotide sequence of genes hrdA, hrdC, and hrdD from Streptomyces coelicolor A3(2) having similarity to rpoD genes

Summary The complete nucleotide sequences were determined of hrdA, hrdC, and hrdD from Streptomyces coelicolor A3(2). They indicate the presence of a single open reading frame in each gene coding for polypeptides of 396 (43747 daltons), 339 (38173 daltons), and 332 amino acid residues (37190 daltons), respectively. These amino acid sequences revealed extensive similarities with the principal sigma factors of Bacillus subtilis, Escherichia coli, Mxyococcus xanthus, Pseudomonas aeruginosa, and also the katF gene product of E. coli. Besides the highly conserved amino acid residues in the rpoD box region, alignment of hrd gene products and the known principal sigma factors and sigma-related factors allowed us to postulate a common basic structure for the principal sigma type factors as distinct from the alternative sigma factors.     

-Amino acid contents of mitochondria and some purple bacteria

The endosymbiont most likely to have given rise to mitochondria is an aerobic bacterium belonging to the α subdivision of the so-called purple bacteria such as Rickettsia, Bradythizobium and Agrobacterium [1 and 2]. Contents of the -enantiomers of serine, alanine, proline, glutamate and aspartate in rat liver whole mitochondria, mitochondrial outer membranes, inner membranes and matrix, soluble proteins and free amino acids were detected. These values for -amino acid content were compared with those in soluble proteins and free amino acids from the purple bacteria Paracoccus denitrificans, Pseudomonas aeruginosa and Escherichia coli, members, respectively of the α, β, and γ subdivisions, to find any similarity between mitochondria and these purple bacteria. A similarity was observed in protein -amino acid contents which were low (<1.5%, D-type/D-type+L-type) both in the membrane and soluble protein fractions from mitochondria and in soluble protein from bacteria. Oddly, substantial amounts of free -serine and free -aspartate (around 2%) were found for the first time in mitochondria. The contents of -serine and -aspartate were higher than those of -alanine, -proline and -glutamate. In purple bacteria, the concentration of -serine (<2%) was the lowest of the five amino acids examined, and those of -alanine (27–32%) and -glutamate (7–26%) were high. Therefore, no similarity was shown in the free -amino acid content between mitochondria and any of the three purple bacteria.     

Comparative Genomics Reveals Long, Evolutionarily Conserved, Low-Complexity Islands in Yeast Proteins

Eukaryotic proteomes abound in low-complexity sequences, including tandem repeats and regions with significantly biased amino acid compositions. We assessed the functional importance of compositionally biased sequences in the yeast proteome using an evolutionary analysis of 2838 orthologous open reading frame (ORF) families from three Saccharomyces species (S. cerevisiae, S. bayanus, and S. paradoxus). Sequence conservation was measured by the amino acid sequence variability and by the ratio of nonsynonymous-to-synonymous nucleotide substitutions (K _a /K _s ) between pairs of orthologous ORFs. A total of 1033 ORF families contained one or more long (at least 45 residues), low-complexity islands as defined by a measure based on the Shannon information index. Low-complexity islands were generally less conserved than ORFs as a whole; on average they were 50% more variable in amino acid sequences and 50% higher in K _a /K _s ratios. Fast-evolving low-complexity sequences outnumbered conserved low-complexity sequences by a ratio of 10 to 1. Sequence differences between orthologous ORFs fit well to a selectively neutral Poisson model of sequence divergence. We therefore used the Poisson model to identify conserved low-complexity sequences. ORFs containing the 33 most conserved low-complexity sequences were overrepresented by those encoding nucleic acid binding proteins, cytoskeleton components, and intracellular transporters. While a few conserved low-complexity islands were known functional domains (e.g., DNA/RNA-binding domains), most were uncharacterized. We discuss how comparative genomics of closely related species can be employed further to distinguish functionally important, shorter, low-complexity sequences from the vast majority of such sequences likely maintained by neutral processes. [Reviewing Editor: Dr. Stuart Newfeld]     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

Flagellin (FliC) protein sequence diversity among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> does not correlate with H serotype diversity

In Escherichia coli, the fliC gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Here, the presence of the flagellin fliC gene was studied in 86 Bacillus thuringiensis strains encompassing 67 H serotypes. Nineteen strains from four additional species in the B. cereus sensu lato group, B. cereus, B. anthracis, B. mycoides, and B. weihenstephanensis, were added for comparison purposes. The fliC genes were amplified, cloned and their nucleotide sequences determined and translated into amino acid sequences. A bootstrapped neighbor-joining tree was generated from the alignment of the translated amino acid sequences of the amplicons. Although most B. thuringiensis H serotypes had different flagellin amino acid sequences, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. In addition, although serovars from the same H serotype were sometimes found clustered together, several serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. No correlations could be established between flagellin (FliC) protein sequence diversity among B. thuringiensis H serotypes and H serotype diversity. These suggest that the B. thuringiensis fliC gene does not code for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. In a previous study, the authors have shown that the B. thuringiensis hag gene codes for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. It is proposed that the B. thuringiensis fliC gene studied here be renamed and that the so-called hag gene studied before be renamed fliC, both in accordance with the E. coli nomenclature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequence analysis of the α-galactosidase <Emphasis Type="Italic">MEL</Emphasis> gene governing the efficient production of ethanol from raffinose-rich molasses in the yeast <Emphasis Type="Italic">Lachancea thermotolerans</Emphasis>

The yeast Lachancea thermotolerans, formerly Kluyveromyces thermotolerans, was tested for the ethanol fermentation of raffinose-rich molasses. Two melibiose-fermenting strains, NBRC 10066 and NBRC 10067, produced more ethanol than eight other strains. The concentration of ethanol synthesized by NBRC 10066 was slightly higher than that by NBRC 10067, probably on the basis of the expression of α-galactosidase. The regions corresponding to the α-galactosidase MEL1 gene of Saccharomyces cerevisiae were amplified. The nucleotide sequences of the two genes designated as MELth1 and MELth2 revealed single open reading frames of 1,416 bp encoding 472 amino acids but differed from each other in one base that converted the amino acid composition. The sequences of the 5′-upstream region from −1 to −515 of the two genes are identical except for one base.     

Streptomyces neyagawaensis as a control for the hazardous biomass of Microcystis aeruginosa (Cyanobacteria) in eutrophic freshwaters

An aquatic bacterium capable of eliminating the cyanobacterium Microcystis aeruginosa was isolated from the sediment of an eutrophic lake (Lake Juam, Korea). On the basis of 16S rDNA sequences and biochemical and morphological characteristics, the isolate was determined to be Streptomyces neyagawaensis. It grew optimally at 40 °C and pH 7. In the presence of this bacterium, the biomass of cyanobacterium M. aeruginosa NIES-298 was strongly suppressed, by up to 84.5% in abundance compared to the control. The antialgal activity of S. neyagawaensis depended on the growth phase of the cyanobacterium, but not of the antialgal bacterium. The antialgal activity of S. neyagawaensis was effective against a wide range of algae, including the green alga Chlorella sp., the diatoms Aulacoseira granulata and Stephanodiscus hantzschii, and four cyanobacteria, M. aeruginosa NIES-44, Anabaena cylindrica, Anabaena flos-aquae, and Oscillatoria sancta. S. neyagawaensis indirectly attacked M. aeruginosa by secretion of extracellular antialgal substances that were localized in the bacterial periplasm and had a specific activity of 7.7 U/μg. These results suggest that indigenous bacteria isolated from sediments may have potential application in controlling harmful cyanobacterial blooms in freshwaters.     

Nucleotide sequence ofatpB,rbcL,trnR,dedB andpsaI chloroplast genes from a fernAngiopteris lygodiifolia: a possible emergence of Spermatophyta lineage before the separation of Bryophyta and Pteridophyta

To elucidate the evolutionary relationship between the Spermatophyta, Pteridophyta and Bryophyta, we cloned a fragment of chloroplast DNA from the fernAngiopteris lygodiifolia (Pteridophyta) and determined its nucleotide sequence. The fragment contained theatpB,rbcL,trnR-CCG,dedB andpsaI genes. Comparisons of the deduced amino acid and nucleotide sequences of these genes from the three plant groups indicate thatAngiopteris sequences are more closely related to those of Bryophyta species (85% identity on average) than to those of seed plants (76% identity on average), supporting a hypothesis that the Bryophyta and Pteridophyta diverged more recently from one another than their common progenitor diverged from that of the Spermatophyta.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.     

Polymorphism of the bm86 Gene in South American Strains of the Cattle Tick Boophilus microplus

Thirty Boophilus microplus strains from various geographic regions of Brazil, Argentina, Uruguay, Venezuela and Colombia were analyzed for the bm86 and bm95 gene. A fragment of cDNA of 794 base pairs of the parasite larvae, included between nucleotides 278–1071s, was amplified and cloned on the pGEM-T vector. Two random clones were sequenced for each population and the nucleotides 278–1071 and predicted amino acid sequences compared with the bm86 and bm95 genes. Variations from 1.76 to 3.65% were detected in the nucleotides sequence when compared with the homologous sequence of the bm86 gene and a 3.4–6.08% in the homologous amino acid sequence of the Bm86 protein. When the sequences obtained were compared with the bm95 gene, variations from 0.50 to 3.15% were detected. Variations from 1.14 to 4.56% were detected for the Bm95 protein homologous sequences in the deduced amino acid sequence. Only five of the 30 strains analyzed presented two different types of alleles expressed and the two alleles of the Alegre population and allele 1 of the Betim population were the most divergent of all those analyzed.     

Cell hydrophobicity of <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. and <Emphasis Type="Italic">Bacillus</Emphasis> spp. bacteria and hydrocarbon biodegradation in the presence of Quillaya saponin

Biodegradation and hydrophobicity of Pseudomonas spp. and Bacillus spp. strains were tested at different concentrations of the biosurfactant Quillaya saponin. A model mixture of hydrocarbon (dodecane and hexadecane) was used for estimating the influence of surfactants on biodegradation. The bacterial adhesion to hydrocarbon method for determination of bacterial cell surface hydrophobicity was exploited. Among the tested bacterial strains the higher hydrophobicity was noticed for Pseudomonas aeruginosa TK. The hydrophobicity of this strain was 84%. The highest hydrocarbon biodegradation was observed for P. aeruginosa TK (49%) and Bacillus subtilis (35%) strains after 7 days of experiments. Generally the addition of Quillaya saponin increased hydrocarbon biodegradation remarkably. The optimal concentration proved to be 80 mg l⁻¹. The degree of hydrocarbon biodegradation was 75% for P. aeruginosa TK after the addition of saponin. However the most significant increase in biodegradation after addition of Quillaya saponin was in the case of P. aeruginosa 25 and Pseudomonas putida (the increase of biodegradation from 21 to 52% and from 31 to 66%, respectively). It is worth mentioning that decrease of hydrophobicity is correlated with the best biodegradation by P. aeruginosa strain. For the remaining strains, no significant hydrophobicity changes in relation to the system without surfactant were noticed.     

A Novel Alginate Lyase with High Activity on Acetylated Alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03

Summary To exploit alginate lyase which could degrade bacterial alginates, degenerate PCR and long range-inverse PCR (LR-IPCR) were used to isolate alginate lyase genes from soil bacteria. Gene algL, an alginate lyase-encoding gene from Pseudomonas sp. QD03 was cloned, and it was composed of a 1122 bp open reading frame (ORF) encoding 373 amino acid residues with the calculated molecular mass of 42.2 kDa. The deduced protein had a potential N-terminal signal peptide of 20 amino acid residues that was consistent with its proposed periplasmic location. Gene algL was expressed in pET24a (+)/E. coli BL21 (DE3) system. The recombinant AlgL was purified to electrophoretic homogeneity using affinity chromatography. The molecular weight of AlgL was estimated to be 42.8 kDa by SDS-PAGE. AlgL exhibited maximal activity at pH 7.5 and 37 °C. Na⁺, K⁺, Ca²⁺ and Ba²⁺ significantly enhanced the activity of AlgL. AlgL could degrade alginate and mannuronate blocks, but hardly degrade guluronate blocks. In particular, AlgL could degrade acetylated alginate of Pseudomonas aeruginosa FRD1 (approximately 0.54 mol of O-acetyl group per mol of alginate). It might be possible to use alginate lyase AlgL as an adjuvant therapeutic medicine for the treatment of disease associated with P. aeruginosa infection.     

Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences

Partial gyrA sequences were determined for twelve strains belonging to Bacillus amyloliquefaciens, B. atrophaeus, B. licheniformis, B. mojavensis,B. subtilis subsp. subtilis, B. subtilissubsp. spizizenii and B. vallismortis. The average nucleotide and translated amino acid similarities for the seven type strains were 83.7 and 95.1%, respectively, whereas the corresponding value for the 16S rRNA sequences was 99.1%. All of the type strains were sharply separated; the closest relationship was found between B. atrophaeus and B. mojavensis which shared a nucleotide similarity of 95.8%. Phylogenetic trees were inferred from gyrA nucleotide sequences using the neighbor-joining, Fitch–Margoliash and maximum parsimony algorithms. The test strains were divided into four groups, which generally reflected results previously reported in restriction digest and DNA-DNA hybridization studies. It is concluded from the comparative sequence analysis that the gyrA sequences provide a firm framework for the rapid and accurate classification and identification of Bacillus subtilis and related taxa.     

Identification of three Zwittermicin A biosynthesis-related genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> strain YBT-1520

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.