Genome Sequence of the Polymyxin-Producing Plant-Probiotic Rhizobacterium Paenibacillus polymyxa E681

Paenibacillus polymyxa E681, a spore-forming, low-G+C, Gram-positive bacterium isolated from the rhizosphere of winter barley grown in South Korea, has great potential for agricultural applications due to its ability to promote plant growth and suppress plant diseases. Here we present the complete genome sequence of P. polymyxa E681. Its 5.4-Mb genome encodes functions specialized to the plant-associated lifestyle and characteristics that are beneficial to plants, such as the production of a plant growth hormone, antibiotics, and hydrolytic enzymes.Among the plant-associated microbes, some are beneficial to plants, as they antagonize various plant pathogens, induce immunity, or even promote growth (2, 21, 29). These “plant-probiotic” bacteria (15, 16, 19, 22, 23, 28) have been isolated and commercially developed for use in the biological control of plant diseases or biofertilization (7, 10). Spore-forming bacteria, in particular, members of the phylum Firmicutes and streptomycetes, are considered advantageous in product formulation and stable maintenance in soil (9).The genus Paenibacillus (1) has grown to encompass more than 110 species (http://www.bacterio.cict.fr/p/paenibacillus.html), but its genome information is severely underrepresented. Paenibacillus spp. are important members of soil- or plant-associated ecosystems (3, 8, 20), with Paenibacillus polymyxa as one of the most industrially significant bacteria (13, 17, 25, 31). P. polymyxa E681, an endospore former isolated from the rhizosphere of winter barley in South Korea (14, 27), suppresses plant diseases, produces antibiotics and a plant hormone, secretes a variety of hydrolytic enzymes, and has good root-colonizing ability (4, 26).We determined the genome sequence of a rifampin-resistant clone of E681. About 62,000 chromatograms (∼6.7-fold genome coverage) were produced from plasmid/fosmid/bacterial artificial chromosome libraries with an AB 3700/377 DNA analyzer. Base calling, fragment assembly, contig/scaffold editing, and finishing were performed with Phred/Phrap/Consed. Gaps were closed by primer walking. To improve the sequence quality, 2.4 Gb of 76-bp single-ended sequences were obtained from Illumina Genome Analyzer IIx. Errors were identified using Maq/MapView and rectified by confirmatory sequencing. Yacop-predicted coding sequences were translated and subjected to transitive annotation by searches against UniProt, COG, KEGG Genes, and TIGRFAMs.The genome is composed of one circular chromosome of 5,394,884 bp (45.8% G+C). It has as many as 12 rRNA operons. No plasmid was found. Three-quarters of the 4,805 genes were assigned putative functions. Protein-coding genes are distributed preferentially on the leading strand. Apparently to cope with an ever-changing environment in the rhizosphere, the genome hosts at least 13 extracytoplasmic function sigma factors (12). There are 19 complete/disrupted insertion sequence elements but few phage-related genes.Some antibiotic-biosynthetic genes have been characterized. Polymyxin, produced and transported by PmxA to -E (5), is a potent antimicrobial that recently attracted attention for the treatment of multidrug-resistant Gram-negative bacteria (11, 18, 30). Fusaricidin, an antifungal antibiotic consisting of six amino acids, is synthesized by a single-chain nonribosomal peptide synthetase (6). E681 may also synthesize a polyketide, a tridecaptin-like nonribosomal peptide, and a hybrid of polyketide and nonribosomal peptide. A gene cluster is responsible for the production of a novel lantibiotic.Based on sequence investigation and biochemical analysis, auxin biosynthesis via the indole-3-pyruvic acid pathway was proposed as the only possible mechanism (24). The bacterium also produces volatile compounds that may promote growth and induce resistance of plants and one or more N-acyl-l-homoserine lactonases. Genome analysis revealed a rich set of secreted enzymes that degrade various plant-derived polysaccharides. They include xylanases, pectic enzymes, cellulases, and amylases. Genes involved in nitrogen fixation were not identified.     

Thiosulfate oxidation and mixotrophic growth of Methylobacterium oryzae

Thiosulfate oxidation and mixotrophic growth with succinate or methanol plus thiosulfate was examined in nutrient-limited mixotrophic condition for Methylobacterium oryzae CBMB20, which was recently characterized and reported as a novel species isolated from rice. Methylobacterium oryzae was able to utilize thiosulfate in the presence of sulfate. Thiosulfate oxidation increased the protein yield by 25% in mixotrophic medium containing 18.5 mmol.L-1 of sodium succinate and 20 mmol.L-1 of sodium thiosulfate on day 5. The respirometric study revealed that thiosulfate was the most preferable reduced inorganic sulfur source, followed by sulfur and sulfite. Thiosulfate was predominantly oxidized to sulfate and intermediate products of thiosulfate oxidation, such as tetrathionate, trithionate, polythionate, and sulfur, were not detected in spent medium. It indicated that bacterium use the non-S4 intermediate sulfur oxidation pathway for thiosulfate oxidation. Thiosulfate oxidation enzymes, such as rhodanese and sulfite oxidase activities appeared to be constitutively expressed, but activity increased during growth on thiosulfate. No thiosulfate oxidase (tetrathionate synthase) activity was detected.     

Competitiveness of Diverse Methylobacterium Strains in the Phyllosphere of Arabidopsis thaliana and Identification of Representative Models, Including M. extorquens PA1

Facultative methylotrophic bacteria of the genus Methylobacterium are consistently found in association with plants, particularly in the phyllosphere. To gain a better understanding of the mechanisms underlying the dispersal and occurrence of Methylobacterium on plants, diverse strains were isolated, identified, and studied with regard to their competitiveness on the model plant Arabidopsis thaliana. As a basis for this study a comprehensive collection of Methylobacterium isolates was established. Isolates were obtained from five different naturally grown A. thaliana populations and diverse other plant genera at these and further sites. They were classified using automated ribosomal internal spacer analysis (ARISA) and a representative subset was identified based on 16S rRNA gene sequence analysis. A comparison of their ARISA patterns with those generated based on a cultivation-independent approach from the same sampling material confirmed that the isolates were abundant colonizers of the studied plants. In competition experiments, colonization efficiency of the strains was found to be linked to phylogeny, rather than to the geographical origin or plant genus from which they were isolated. The most competitive colonizers were related to the species Methylobacterium tardum and Methylobacterium extorquens. Higher cell numbers were observed in the phyllosphere of A. thaliana when a mixture of different strains was applied relative to inoculation with only one strain, suggesting partial niche heterogeneity. Based on the results of the competition experiments, representative strains with different colonization efficiencies were selected, which will serve as models in future studies aiming at a better understanding of plant colonization by this bacterial genus. Among them is the meanwhile genome-sequenced strain M. extorquens PA1, which represents a competitive species of plant colonizers with a broad dispersal. This strain was characterized in more detail including physiological, morphological, and chemotaxonomical properties.     

Draft Genome Sequence of Aspergillus oryzae Strain 3.042

Aspergillus oryzae is the most important fungus for the traditional fermentation in China and is particularly important in soy sauce fermentation. We report the 36,547,279-bp draft genome sequence of A. oryzae 3.042 and compared it to the published genome sequence of A. oryzae RIB40.     

Genome Sequence of Methylobacterium sp. Strain GXF4, a Xylem-Associated Bacterium Isolated from Vitis vinifera L. Grapevine

Methylobacterium sp. strain GXF4 is an isolate from grapevine. Here we present the sequence, assembly, and annotation of its genome, which may shed light on its role as a grapevine xylem inhabitant. To our knowledge, this is the first genome announcement of a plant xylem-associated strain of the genus Methylobacterium.     

Reclassification of Methylobacterium chloromethanicum and Methylobacterium dichloromethanicum as later subjective synonyms of Methylobacterium extorquens and of Methylobacterium lusitanum as a later subjective synonym of Methylobacterium rhodesianum

Phylogenetic analysis based on 16S rDNA sequences was performed on all type strains of the 14 validly described Methylobacterium species to ascertain the genealogic relationships among these species. The results showed that type strains of Methylobacterium were divided into two monophyletic groups whose members were distinct species with sequence similarity values greater than 97.0% between any two of the members in the same group. Only M. organophilum JCM 2833(T) and ATCC 27886(T) were not divided into those two groups. In particular, strains of M. dichloromethanicum and M. chloromethanicum exhibited extremely high similarity values (99.9 and 100%, respectively) with the type strain of M. extorquens. To clarify the relationships among Methylobacterium species in more detail, phylogenetic analysis based on the 5' end hyper-variable region of 16S rDNA (HV region), ribotyping analysis, fatty acid analysis, G+C content analysis and DNA-DNA hybridization experiments was performed on 58 strains of Methylobacterium species. Results of the ribotyping analysis and the phylogenetic analysis based on HV region sequences indicated that many Methylobacterium strains, including M. 'organophilum' DSM 760(T), have been erroneously identified. The DNA G+C content of Methylobacterium strains were between 68.1 and 71.3%. Results of whole-cell fatty-acid profiles showed that all strains contained 18 : 1omega7c as the primary fatty acid component (82.8-90.1%), with 16 : 0 and 18 : 0 as minor components. M. dichloromethanicum DSM 6343(T), M. chloromethanicum NCIMB 13688(T), and M. extorquens IAM 12631(T) exhibited high DNA-DNA relatedness values between each other (69-80%). M. lusitanum NCIMB 13779(T) also showed a close relationship with M. rhodesianum DSM 5687(T) at DNA-DNA relatedness levels of 89-92%. According to these results, many Methylobacterium strains should be reclassified, with M. dichloromethanicum and M. chloromethanicum regarded as a synonym of M. extorquens, and M. lusitanum a synonym for M. rhodesianum.     

Distribution of Methanotrophs in the Phyllosphere

A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.     

Soluble Cytochromes c of a KCN-resistant Mutant of an Obligate Methylotroph,Methylomonas sp.

A KCN-resistant mutant, Methylomonas sp. YK 56, contained three kinds of soluble cytochromes c (cytochromes c-I, c-II, and c-III) though the wild type strain contained two kinds (cytochromes c-I and c-III). The proportion of the three cytochromes c of the mutant were 2.4, 71.5, and 26.1%, and that of the two cytochromes c of the wild type strain were 2.1 and 97.9%, respectively.

Cytochromes c-II and c-III of the mutant were purified by a procedure involving ammonium sulfate fractionation and DEAE-, CM-cellulose, and Sephadex G-150 column chromatography. Cytochrome c-II was obtained as crystals with ammonium sulfate. Both absorption peaks of the α-band of the two cytochromes c were at 551.5 nm at room temperature and the β-band of cytochrome c-II had a shoulder at 530 nm. Molecular weights Of the two cytochromes c were 16,000 and 20,000, respectively and their isoelectric points were 4.1 and 3.5, respectively.     

Genome re-sequencing analysis uncovers pathogenecity-related genes undergoing positive selection in Magnaporthe oryzae

Rice blast caused by Magnaporthe oryzae(M.oryzae) is one of the most destructive diseases,which causes significant rice yield losses and affects global food security.To better understand genetic variations among different isolates of M.oryzae in nature,we re-sequenced the genomes of two field isolates,CH43 and Zhong-10-8-14,which showed distinct pathogenecity on most of the rice cultivars.Genome-wide genetic variation analysis reveals that ZHONG-10-8-14 exhibits higher sequence variations than CH43.Structural variations(SVs) detection shows that the sequence variations primarily occur in exons and intergenic regions.Bioinformatics analysis for gene variations reveals that many pathogenecity-related pathways are enriched.In addition,193 candidate effectors with various DNA polymorphisms were identified,including two known effectors AVR-Pik and AVR-Pital.Comparative polymorphism analysis of thirteen randomly selected effectors suggests that the genetic variations of effectors are under positive selection.The expression pattern analysis of several pathogenecity-related variant genes indicates that these genes are differentially regulated in two isolates,with much higher expression levels in Zhong-10-8-14 than CH43.Our data demonstrate that the genetic variations of effectors and pathogenecity-related genes are under positive selection,resulting in the distinct pathogenicities of CH43 and Zhong-10-8-14 on rice.     

Nitrogen Fixation in the Phyllosphere of the Douglas Fir, Pseudotsuga douglasii

Nitrogen-fixing bacteria have been isolated from the leaf surfacesof the Douglas Fir. ¹⁵N tracer studies have shown that thesebacteria fix nitrogen in culture and in the field on the trees.A considerable proportion of the annual requirement for nitrogenby Douglas Fir may be provided by nitrogen-fixing bacteria onthe leaves and in the soil.     

Characterization of Rhodococcus wratislaviensis,a New Gram-Positive Facultative Methylotroph,and Properties of Its C1 Metabolism

Microbiology - A facultative methylotroph, strain 2AzMo (VKM Ac-2782), was isolated from the coastal zone of the Sea of Azov. The cells of the isolate are aerobic gram-positive nonmotile rods....     

Production of a polysaccharide, methylan, in Methylobacterium organophilum by controlling ammonium ion

Summary Cell growth increased proportionally to the initial concentration of ammonium ion, however, methylan production was significantly inhibited at the high concentration of ammonium ion. The control of ammonium ion within the desired level(usually 0.45 g/l) was needed to reduce the inhibition. Methylan production was increased to 12.5 g/l by maintaining ammonium ion below 0.15 g/l.     

Genome of Xanthomonas oryzae bacteriophage Xp10: an odd T-odd phage

Xp10 is a lytic bacteriophage of the phytopathogenic bacterium Xanthomonas oryzae. Though morphologically Xp10 belongs to the Syphoviridae family, it encodes its own single-subunit RNA polymerase characteristic of T7-like phages of the Podoviridae family. Here, we report the determination and analysis of the 44,373 bp sequence of the Xp10 genome. The genome is a linear, double-stranded DNA molecule with 3' cohesive overhangs and no terminal repeats or redundancies. Half of the Xp10 genome contains genes coding for structural proteins and host lysis functions in an arrangement typical for temperate dairy phages that are related to the Escherichia coli lambda phage. The other half of the Xp10 genome contains genes coding for factors of host gene expression shut-off, enzymes of viral genome replication and expression. The two groups of genes are transcribed divergently and separated by a regulatory region, which contains divergent promoters recognized by the host RNA polymerase. Xp10 has apparently arisen through a recombination between genomes of widely different phages. Further evidence of extensive gene flux in the evolution of Xp10 includes a high fraction (10%) of genes derived from an HNH-family endonuclease, and a DNA-dependent DNA polymerase that is closer to a homolog from Leishmania than to DNA polymerases from other phages or bacteria.     

Crystal structure of XoLAP, a leucine aminopeptidase, from Xanthomonas oryzae pv. oryzae

Aminopeptidases are metalloproteinases that degrade N-terminal residues from protein and play important roles in cell growth and development by controlling cell homeostasis and protein maturation. We determined the crystal structure of XoLAP, a leucyl aminopeptidase, at 2.6 Å resolution from Xanthomonas oryzae pv. oryzae, causing the destructive rice disease of bacterial blight. It is the first crystal structure of aminopeptidase from phytopathogens as a drug target. XoLAP existed as a hexamer and the monomer structure consisted of an N-terminal cap domain and a C-terminal peptidase domain with two divalent zinc ions. XoLAP structure was compared with BlLAP and EcLAP (EcPepA) structures. Based on the structural comparison, the molecular model of XoLAP in complex with the natural aminopeptidase inhibitor of microginin FR1 was proposed. The model structure will be useful to develop a novel antibacterial drug against Xoo.     

Transformation of a Methylotrophic Bacterium, Methylobacterium extorquens, with a Broad-Host-Range Plasmid by Electroporation

An efficient method for the transformation of the methylotrophic bacterium Methylobacterium extorquens NR-2 with a broad-host-range plasmid, pLA2917, by electroporation was examined. Transformants of M. extorquens NR-2 expressing resistance to kanamycin were obtained after electric pulse. These transformants were found to harbor a single plasmid which was electrophoretically identical and homologous to pLA2917 obtained from Escherichia coli. Several factors which determined the transformation efficiency were optimized, resulting in a transformation efficiency of up to 8 × 10³ transformants per μg of plasmid DNA by 10 pulses at a field strength of 10 kV/cm and a pulse duration of 300 μs.