Tardiphaga alba sp. nov., a heavy-metal-tolerant bacterium isolated from garden soil.

A previously undescribed, heavy-metal-tolerant, motile, Gram-negative bacterium, designated strain SK50-23T, was characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SK50-23T was closely related to Tardiphaga robiniae LMG 26467T and the non-phototrophic 'Rhodopseudomonas boonkerdii' NS23T (98.1 and 97.3 % 16S rRNA gene sequence similarity, respectively). Strain SK50-23T possessed a circular genome of 5.86 Mb, with a DNA G+C content of 61.9 mol%. Digital DNA-DNA hybridization showed 20.8-21.6 % similarity between strain SK50-23T and related species. In addition, the whole-genome average nucleotide identity values between strain SK50-23T and related species ranged from 75.1 to 83.5 %. The major cellular fatty acid identified in strain SK50-23T was C18 : 1ω7c, and the main isoprenoid quinone present was ubiquinone Q-10. Strain SK50-23T could be assigned to the genus Tardiphaga with the species name Tardiphaga alba sp. nov. based on morphological, chemotaxonomic and genome-based taxonomic characteristics, and 16S rRNA gene-based phylogenetic characteristics. The type strain of the proposed novel species is SK50-23T (=NBRC 108825T=CGMCC No. 1.12037T).

Detection and isolation of a new member of Burkholderiaceae-related endofungal bacteria from Saksenaea boninensis sp. nov., a new thermotolerant fungus in Mucorales.

Thermotolerance in Mucorales (Mucoromycotina) is one of the factors to be opportunistic pathogens, causing mucormycosis. Among thermotolerant mucoralean fungi, Burkholderiaceae-related endobacteria (BRE) are rarely found and the known range of hosts is limited to Rhizopus spp. The phylogenetic divergence of BRE has recently expanded in other fungal groups such as Mortierellaceae spp. (Mortierellomycotina); however, it remains unexplored in Mucorales. Here, we found a thermotolerant mucoralean fungus obtained from a litter sample collected from Haha-jima Island in the Ogasawara (Bonin) Islands, Japan. The fungus was morphologically, phylogenetically, and physiologically characterized and proposed as a new species, Saksenaea boninensis sp. nov. Besides the fungal taxonomy, we also found the presence of BRE in isolates of this species by diagnostic PCR amplification of the 16S rRNA gene from mycelia, fluorescence microscopic observations, and isolation of the bacterium in pure culture. Phylogenetic analysis of the 16S rRNA gene of BRE revealed that it is distinct from all known BRE. The discovery of a culturable BRE lineage in the genus Saksenaea will add new insight into the evolutional origin of mucoralean fungus-BRE associations and emphasize the need to pay more attention to endofungal bacteria potentially associated with isolates of thermotolerant mucoralean fungi causing mucormycosis.

Enhanced supply of acetyl-CoA by exogenous pantothenate kinase promotes synthesis of poly(3-hydroxybutyrate).

BACKGROUND: Coenzyme A (CoA) is a carrier of acyl groups. This cofactor is synthesized from pantothenic acid in five steps. The phosphorylation of pantothenate is catalyzed by pantothenate kinase (CoaA), which is a key step in the CoA biosynthetic pathway. To determine whether the enhancement of the CoA biosynthetic pathway is effective for producing useful substances, the effect of elevated acetyl-CoA levels resulting from the introduction of the exogenous coaA gene on poly(3-hydroxybutyrate) [P(3HB)] synthesis was determined in Escherichia coli, which express the genes necessary for cyanobacterial polyhydroxyalkanoate synthesis (phaABEC). RESULTS: E. coli containing the coaA gene in addition to the pha genes accumulated more P(3HB) compared with the transformant containing the pha genes alone. P(3HB) production was enhanced by precursor addition, with P(3HB) content increasing from 18.4% (w/w) to 29.0% in the presence of 0.5 mM pantothenate and 16.3%-28.2% by adding 0.5 mM ß-alanine. Strains expressing the exogenous coaA in the presence of precursors contained acetyl-CoA in excess of 1 nmol/mg of dry cell wt, which promoted the reaction toward P(3HB) formation. The amount of acetate exported into the medium was three times lower in the cells carrying exogenous coaA and pha genes than in the cells carrying pha genes alone. This was attributed to significantly enlarging the intracellular pool size of CoA, which is the recipient of acetic acid and is advantageous for microbial production of value-added materials. CONCLUSIONS: Enhancing the CoA biosynthetic pathway with exogenous CoaA was effective at increasing P(3HB) production. Supplementing the medium with pantothenate facilitated the accumulation of P(3HB). ß-Alanine was able to replace the efficacy of adding pantothenate.

Complete genome sequence of Pseudoalteromonas sp. PS1M3, a psychrotrophic bacterium isolated from deep-sea sediment off the Boso Peninsula, Japan Trench.

Herein, we report the complete genome sequence of Pseudoalteromonas sp. PS1M3 (= NCBI 87791), which is a psychrotrophic bacterium that inhabits in seabed off the Boso Peninsula, Japan Trench. Analysis of the genomic sequence revealed that PS1M3 possesses 2 circular chromosomal DNAs and 2 circular plasmid DNAs. The genome of PS1M3 had a total size of 4,351,630 bp, an average GC content of 39.9%, and contained a total of 3811 predicted protein coding sequences, 28 rRNAs, and 100 tRNAs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was utilized to annotate the genes and KofamKOALA within KEGG assigned a gene cluster involved in glycogen biosynthesis and metabolic pathways with regard to heavy metal resistance (copper; cop and mercury; mer), indicating that PS1M3 can potentially use a stored glycogen as an energy source under oligotrophic environment and cope with multi-heavy metal contamination. To assess available genome relatedness indices, whole-genome average nucleotide identity analysis was examined using the complete genome sequences of Pseudoalteromonas spp., showing that 67.29-97.40% sequence similarity with PS1M3. This study may be useful in understanding the roles of a psychrotrophic Pseudoalteromonas in cold deep-sea sediment adaptation mechanisms.

A Nitrate-Transforming Bacterial Community Dominates in the Miscanthus Rhizosphere on Nitrogen-Deficient Volcanic Deposits of Miyake-jima.

The perennial gramineous grass Miscanthus condensatus functions as a major pioneer plant in colonizing acidic volcanic deposits on Miyake-jima, Japan, despite a lack of nitrogen nutrients. The nitrogen cycle in the rhizosphere is important for the vigorous growth of M. condensatus in this unfavorable environment. In the present study, we identified the nitrogen-cycling bacterial community in the M. condensatus rhizosphere on these volcanic deposits using a combination of metagenomics and culture-based analyses. Our results showed a large number of functional genes related to denitrification and dissimilatory nitrate reduction to ammonium (DNRA) in the rhizosphere, indicating that nitrate-transforming bacteria dominated the rhizosphere biome. Furthermore, nitrite reductase genes (i.e., nirK and nirS) related to the denitrification and those genes related to DNRA (i.e., nirB and nrfA) were mainly annotated to the classes Alpha-proteobacteria, Beta-proteobacteria, and Gamma-proteobacteria. A total of 304 nitrate-succinate-stimulated isolates were obtained from the M. condensatus rhizosphere and were classified into 34 operational taxonomic units according to amplified 16S rRNA gene restriction fragment pattern analysis. Additionally, two strains belonging to the genus Cupriavidus in the class Beta-proteobacteria showed a high in vitro denitrifying activity; however, metagenomic results indicated that the DNRA-related rhizobacteria appeared to take a major role in the nitrogen cycle of the M. condensatus rhizosphere in recent Miyake-jima volcanic deposits. This study elucidates the association between the Miscanthus rhizosphere and the nitrate-reducing bacterial community on newly placed volcanic deposits, which furthers our understanding of the transformation of nitrogen nutrition involved in the early development of vegetation.

Investigating Endobacteria that Thrive Within Mucoromycota.

Metagenomics approaches have revealed the importance of Mucoromycota in the evolution and functioning of plant microbiomes. Comprised of three subphyla (Glomeromycotina, Mortierellomycotina, and Mucoromycotina), this early diverging lineage of fungi encompasses species of mycorrhizal fungi, root endophytes, plant pathogens, and many decomposers of plant debris. Interestingly, several taxa of Mucoromycota share a common feature, that is, the presence of endobacteria within their mycelia and spores. The study of these endosymbiotic bacteria is still a challenging task. However, given recent improvements in the sensitivity of culture-free approaches, a deeper understanding of such microbial interactions is now possible and fuels an emerging research field. In this chapter, we report how Mucoromycota, in particular Mortierellomycotina, and their endobacteria can be investigated using a combination of diverse cellular biology, microscopy, and molecular techniques.

Mitigation of Paddy Field Soil Methane Emissions by Betaproteobacterium Azoarcus Inoculation of Rice Seeds.

Paddy fields are a major source of atmospheric methane, a greenhouse gas produced by methanogens and consumed by methanotrophs in flooded soil. The inoculation of rice seeds with the bacterium Azoarcus sp. KH32C alters the rice root-associated soil bacterial community composition. The present study investigated the effects of KH32C-inoculated rice cultivation on soil methanogens and methanotrophs involved in methane emissions from a rice paddy field. KH32C-inoculated and non-inoculated rice (cv. Nipponbare) were cultivated in a Japanese rice paddy with and without nitrogen fertilizer. Measurements of methane emissions and soil solution chemical properties revealed increases in methane flux over the waterlogged period with elevations in the concentrations of dissolved methane, dissolved organic carbon, and ferrous iron, which is an indicator of soil reduction levels. Reverse transcription quantitative PCR and amplicon sequencing were used to assess the transcription of the methyl-coenzyme M reductase gene (mcrA) from methanogens and the particulate methane monooxygenase gene (pmoA) from methanotrophs in paddy soil. The results obtained showed not only the transcript copy numbers, but also the compositions of mcrA and pmoA transcripts were related to methane flux. KH32C-inoculated rice cultivation recruited soil methanogens and methanotrophs that suppressed high methane synthesis, increased methane consumption, and decreased methane emissions by 23.5 and 17.2% under non-fertilized and nitrogen-fertilized conditions, respectively, while maintaining rice grain yield. The present study demonstrated the mitigation of paddy field methane emissions arising from the use of KH32C in rice cultivation due to its influence on the compositions of soil methanogen and methanotroph populations.

Complete Genome Sequence of a Psychrophilic Bacterium, Pseudoalteromonas sp. Strain APM04, Isolated from the Seafloor of the South Mariana Trough, Pacific Ocean.

The complete genome sequence of Pseudoalteromonas sp. strain APM04, which is a psychrophilic bacterium that inhabits the seabed of the South Mariana Trough, Pacific Ocean, was determined to characterize the genetic features associated with evolution in extremophilic and oligotrophic deep seawater.

Complete Genome Sequence of a Chemolithoautotrophic Iron-Oxidizing Bacterium, Acidithiobacillus ferrooxidans Strain NFP31, Isolated from Volcanic Ash Deposits on Miyake-Jima, Japan.

The genome sequence of Acidithiobacillus ferrooxidans strain NFP31, which is a chemolithoautotrophic iron-oxidizing bacterium that inhabits acidified volcanic deposits on Mount Oyama, Miyake Island (Miyake-jima), Japan, was determined to identify the genetic characteristics associated with pioneer microbes in newly placed pyroclastic deposits.

Whole-Genome Sequence of Entomortierella parvispora E1425, a Mucoromycotan Fungus Associated with Burkholderiaceae-Related Endosymbiotic Bacteria.

Some mucoromycotan fungi establish symbiotic associations with endohyphal bacteria. Here, the genome of Entomortierella parvispora E1425 (synonymously known as Mortierella parvispora E1425), which harbors a cultured Burkholderiaceae-related endobacterium (BRE) designated Mycoavidus sp. strain B2-EB, was sequenced. We provide genomic information to elucidate fungal-BRE symbiotic features.

Mycoavidus sp. Strain B2-EB: Comparative Genomics Reveals Minimal Genomic Features Required by a Cultivable Burkholderiaceae-Related Endofungal Bacterium.

Obligate bacterial endosymbionts are critical to the existence of many eukaryotes. Such endobacteria are usually characterized by reduced genomes and metabolic dependence on the host, which may cause difficulty in isolating them in pure cultures. Family Burkholderiaceae-related endofungal bacteria affiliated with the Mycoavidus-Glomeribacter clade can be associated with the fungal subphyla Mortierellomycotina and Glomeromycotina. In this study, a cultivable endosymbiotic bacterium, Mycoavidus sp. strain B2-EB, present in the fungal host Mortierella parvispora was obtained successfully. The B2-EB genome (1.88 Mb) represents the smallest genome among the endofungal bacterium Mycoavidus cysteinexigens (2.64-2.80 Mb) of Mortierella elongata and the uncultured endosymbiont "Candidatus Glomeribacter gigasporarum" (1.37 to 2.36 Mb) of arbuscular mycorrhizal fungi. Despite a reduction in genome size, strain B2-EB displays a high genome completeness, suggesting a nondegenerative reduction in the B2-EB genome. Compared with a large proportion of transposable elements (TEs) in other known Mycoavidus genomes (7.2 to 11.5% of the total genome length), TEs accounted for only 2.4% of the B2-EB genome. This pattern, together with a high proportion of single-copy genes in the B2-EB genome, suggests that the B2-EB genome reached a state of relative evolutionary stability. These results represent the most streamlined structure among the cultivable endofungal bacteria and suggest the minimal genome features required by both an endofungal lifestyle and artificial culture. This study allows us to understand the genome evolution of Burkholderiaceae-related endosymbionts and to elucidate microbiological interactions.IMPORTANCE This study attempted the isolation of a novel endobacterium, Mycoavidus sp. B2-EB (JCM 33615), harbored in the fungal host Mortierella parvispora E1425 (JCM 39028). We report the complete genome sequence of this strain, which possesses a reduced genome size with relatively high genome completeness and a streamlined genome structure. The information indicates the minimal genomic features required by both the endofungal lifestyle and artificial cultivation, which furthers our understanding of genome reduction in fungal endosymbionts and extends the culture resources for biotechnological development on engineering synthetic microbiomes.

Aposymbiosis of a Burkholderiaceae-Related Endobacterium Impacts on Sexual Reproduction of Its Fungal Host.

Bacterial endosymbionts inhabit diverse fungal lineages. Although the number of studies on bacteria is increasing, the mechanisms by which bacteria affect their fungal hosts remain unclear. We herein examined the homothallic isolate, Mortierella sugadairana YTM39, harboring a Burkholderiaceae-related endobacterium, which did not produce sexual spores. We successfully eliminated the bacterium from fungal isolates using ciprofloxacin treatment and asexual spore isolation for germinated asexual spores. Sexual spore formation by the fungus was restored by eliminating the bacterium from isolates. These results indicate that sexual reproduction by the fungus was inhibited by the bacterium. This is the first study on the sexual spore infertility of fungal hosts by endofungal bacteria.

Genome Sequence of Novoherbaspirillum sp. UKPF54, a Plant Growth-Promoting Rhizobacterial Strain with N2O-Mitigating Abilities, Isolated from Paddy Soil.

Novoherbaspirillum sp. strain UKPF54, a plant growth-promoting rhizobacterium with the ability to mitigate nitrous oxide emission from agriculture soils, has been successfully isolated from paddy soil in Kumamoto, Japan. Here, we report the whole-genome sequence of this strain.

Genome Sequence of Arthrobacter sp. UKPF54-2, a Plant Growth-Promoting Rhizobacterial Strain Isolated from Paddy Soil.

Arthrobacter sp. strain UKPF54-2, a plant growth-promoting rhizobacterium having the potential ability to control fungal and bacterial pathogens, was isolated from paddy soil in Kumamoto, Japan. We report here the whole-genome sequence of this strain.

Genome Sequences of Two Azospirillum sp. Strains, TSA2S and TSH100, Plant Growth-Promoting Rhizobacteria with N2O Mitigation Abilities.

Azospirillum sp. strains TSA2S and TSH100 are plant growth-promoting rhizobacteria with the capacity to mitigate N2O from agricultural soil. They were isolated from the rhizosphere of paddy soil in Tokyo, Japan. Here, we present the genome sequences of these two strains.

Comparative Characterization of Bacterial Communities in Moss-Covered and Unvegetated Volcanic Deposits of Mount Merapi, Indonesia.

Microbial colonization, followed by succession, on newly exposed volcanic substrates represents the beginning of the development of an early ecosystem. During early succession, colonization by mosses or plants significantly alters the pioneer microbial community composition through the photosynthetic carbon input. To provide further insights into this process, we investigated the three-year-old volcanic deposits of Mount Merapi, Indonesia. Samples were collected from unvegetated (BRD) and moss-covered (BRUD) sites. Forest site soil (FRS) near the volcanic deposit-covered area was also collected for reference. An analysis of BRD and BRUD revealed high culturable cell densities (1.7-8.5×105 CFU g-1) despite their low total C (<0.01%). FRS possessed high CFU (3×106 g-1); however, its relative value per unit of total C (2.6%) was lower than that of the deposit samples. Based on the tag pyrosequencing of 16S rRNA genes, the BRD bacterial community was characterized by a higher number of betaproteobacterial families (or genus), represented by chemolithotrophic Methylophilaceae, Leptothrix, and Sulfuricellaceae. In contrast, BRUD was predominated by different betaproteobacterial families, such as Oxalobacteraceae, Comamonadaceae, and Rhodocyclaceae. Some bacterial (Oxalobacteraceae) sequences were phylogenetically related to those of known moss-associated bacteria. Within the FRS community, Proteobacteria was the most abundant phylum, followed by Acidobacteria, whereas Burkholderiaceae was the most dominant bacterial family within FRS. These results suggest that an inter-family succession of Betaproteobacteria occurred in response to colonization by mosses, followed by plants.

Comparative Analysis of the Genetic Basis of Branched Nonylphenol Degradation by Sphingobium amiense DSM 16289T and Sphingobium cloacae JCM 10874T.

Branched nonylphenol (BNP), a degradation product of nonylphenol polyethoxylates, exerts estrogenic effects on various organisms. The genes underlying BNP degradation by Sphingobium amiense DSM 16289T were analyzed by complete genome sequencing and compared with those of the versatile BNP-degrading Sphingobium cloacae JCM 10874T. An opdA homolog (opdADSM16289) encoding BNP degradation activity was identified in DSM 16289T, in contrast with JCM 10874T, possessing both the opdA homolog and nmoA. The degradation profile of different BNP isomers was examined by Escherichia coli transformants harboring opdADSM16289, opdAJCM10874, and nmoAJCM10874 to characterize and compare the expression activities of these genes.

Prevalence and Intra-Family Phylogenetic Divergence of Burkholderiaceae-Related Endobacteria Associated with Species of Mortierella.

Endofungal bacteria are widespread within the phylum Mucoromycota, and these include Burkholderiaceae-related endobacteria (BRE). However, the prevalence of BRE in Mortierellomycotinan fungi and their phylogenetic divergence remain unclear. Therefore, we examined the prevalence of BRE in diverse species of Mortierella. We surveyed 238 isolates of Mortierella spp. mainly obtained in Japan that were phylogenetically classified into 59 species. BRE were found in 53 isolates consisting of 22 species of Mortierella. Among them, 20 species of Mortierella were newly reported as the fungal hosts of BRE. BRE in a Glomeribacter-Mycoavidus clade in the family Burkholderiaceae were separated phylogenetically into three groups. These groups consisted of a group containing Mycoavidus cysteinexigens, which is known to be associated with M. elongata, and two other newly distinguishable groups. Our results demonstrated that BRE were harbored by many species of Mortierella and those that associated with isolates of Mortierella spp. were more phylogenetically divergent than previously reported.

Complete Genome Sequence of a Microcystin-Degrading Bacterium, Sphingosinicella microcystinivorans Strain B-9.

Sphingosinicella microcystinivorans strain B-9 has the ability to degrade cyanobacterial hepatotoxic cyclic peptides, microcystins, and nodularins. This is the first report of the complete genome sequence of the microcystin-degrading bacterium.

Presence of Cu-Type (NirK) and cd1-Type (NirS) Nitrite Reductase Genes in the Denitrifying Bacterium Bradyrhizobium nitroreducens sp. nov.

Nitrite reductase is a key enzyme for denitrification. There are two types of nitrite reductases: copper-containing NirK and cytochrome cd1-containing NirS. Most denitrifiers possess either nirK or nirS, although a few strains been reported to possess both genes. We herein report the presence of nirK and nirS in the soil-denitrifying bacterium Bradyrhizobium sp. strain TSA1T. Both nirK and nirS were identified and actively transcribed under denitrification conditions. Based on physiological, chemotaxonomic, and genomic properties, strain TSA1T (=JCM 18858T=KCTC 62391T) represents a novel species within the genus Bradyrhizobium, for which we propose the name Bradyrhizobium nitroreducens sp. nov.