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-illycoavidus Glade 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 the Nonheterocystous Cyanobacterium Pseudanabaena sp. ABRG5-3

We report here the complete sequences of the main genome (4.8 Mb) and seven plasmids of the semifilamentous, nonheterocystous cyanobacterium Pseudanabaena sp. ABRG5-3, a strain isolated from a pond in Japan. These data are expected to enhance our understanding of the Pseudanabaena subclade near the root of cyanobacterial diversity

No Tillage Increases SOM in Labile Fraction but Not Stable Fraction of Andosols from a Long-Term Experiment in Japan

No tillage (NT) fosters carbon (C) sequestration, increases soil organic matter (SOM) stock, and improves soil health. However, its effect on SOM accumulation in Andosol, which has high OM stabilization characteristics due to its specific mineral properties, remains unclear. In this study, we evaluated the effect of NT on SOM content and its distribution by the physical fractionation method and assessed the quality of accumulated SOM in each fraction. We collected soil samples at 0–2.5, 2.5–7.5, and 7.5–15 cm depths from NT and conventional tillage (CT) plots in a long-term (19 years) field experiment of Andosols in Ibaraki, Japan. The soil samples were separated into light fraction (LF), coarse-POM (cPOM: 0.25–2 mm), fine-POM (fPOM: 0.053–0.25 mm), and silt + clay (mOM: <0.053 mm). The C, nitrogen (N), and organic phosphorus (Po) contents of each fraction were analyzed. The C content of cPOM and fPOM in NT at 0–7.5 cm was higher than in CT, while there was no clear difference in the mOM fraction or deeper layer (7.5–15 cm). NT increased the C, N, and Po contents in the labile POM fractions at the surface layers but did not increase the stable fraction or change the quality

Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium, Thermus thermophilus

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004)

Multiple sequence alignments: Detection and isolation of a new member of Burkholderiaceae‑related endofungal bacteria from Saksenaea boninensis sp. nov., a new thermotolerant fungus in Mucorales

&lt;p&gt;&lt;strong&gt;Methods:&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;Nucleotide sequences were aligned independently for each region using MAFFT v7.212 (Katoh and Standley, 2013). The obtained alignment blocks were subject to Gblocks 0.91b (Castresana, 2000) to remove poorly aligned positions with the relaxed selection setting described in Talavera &amp; Castresana (2007) using the following parameters (-t = d -b2 = 9 -b3 = 10 -b4 = 5 -b5 = h). After automatically removing gaps, the alignment blocks were viewed using MEGA 6.06 software (Tamura et al., 2013) and poorly aligned positions at either end of the alignments were removed manually. Pairwise distances of the nucleotide sequences (ITS2, ITS1-5.8S-ITS2, LSU, and tef1) of the ex-type strains of seven &lt;i&gt;Saksenaea&lt;/i&gt; spp. and the representative isolate &lt;i&gt;S. boninensis&lt;/i&gt; Sak4 were calculated by MEGA 6.06 software (Tamura et al. 2013). Multiple sequence alignment of 16S rRNA gene of the family &lt;i&gt;Burkholderiaceae&lt;/i&gt; was prepared for the phylogeny of a bacterial endosymbiont. Multiple sequence alignments of &nbsp;ITS, LSU, and tef1 genes of &lt;i&gt;Saksenaea&lt;/i&gt; spp. (Mucorales) were separately prepared for the phylogeny of a fungal host. Concatenated dataset of these genes were also prepared. All nucleotide sequences were retrieved from GenBank (See "Sequence_ID.csv" and taxon names of each alignment). &nbsp;&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Description of files:&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;A. Phylogeny of the family &lt;/strong&gt;&lt;i&gt;&lt;strong&gt;Burkholderiaceae&lt;/strong&gt;&lt;/i&gt;&lt;strong&gt; (Bacterial endosymbiont):&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;1. Burkholderiaceae_16S_RAW.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of 16S rRNA gene of the family &lt;i&gt;Burkholderiaceae&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;2. Burkholderiaceae_16S_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of 16S rRNA gene of the family &lt;i&gt;Burkholderiaceae&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;B. Phylogenies of &lt;/strong&gt;&lt;i&gt;&lt;strong&gt;Saksenaea&lt;/strong&gt;&lt;/i&gt;&lt;strong&gt; spp. (Fungal host):&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;1. Sequence_ID_v2.csv&lt;/p&gt;&lt;p&gt;Taxon names, accession numbers, and sequence ID for the concatenated multiple sequence alignment are listed.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;2. Saksenaea_ITS_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of ITS1-5.8S-ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. &nbsp;&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;3. Saksenaea_ITS_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS1-5.8S-ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. Only used for ITS1-5.8S-ITS2 phylogeny.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;4. Saksenaea_LSU_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of LSU gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. &nbsp;&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;5. Saksenaea_LSU_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of LSU gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. Only used for LSU phylogeny.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;6. Saksenaea_tef1_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of tef1 gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. &nbsp;&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;7. Saksenaea_tef1_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of tef1 gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. Only used for tef1 phylogeny.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;Concatenated dataset 1 (ITS2, LSU, tef1)&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;8. Saksenaea_ITS2_for_concatenated_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of a concatenated dataset 1.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;9. Saksenaea_ITS2_for_concatenated_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of a concatenated dataset 1.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;10. Saksenaea_LSU_for_concatenated_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of LSU gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of concatenated datasets 1 and 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;11. Saksenaea_LSU_for_concatenated_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of LSU gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of concatenated datasets 1 and 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;12. Saksenaea_tef1_for_concatenated_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of tef1 gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of concatenated datasets 1 and 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;13. Saksenaea_tef1_for_concatenated_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of tef1 gene region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of concatenated datasets 1 and 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;14. Saksenaea_ITS2_LSU_tef1_concatenated_dataset1.fasta&lt;/p&gt;&lt;p&gt;Concatenated dataset of three multiple sequence alignments (9, 11, and 13). This concatenated dataset was used for the main phylogeny of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;Concatenated dataset 2 (ITS1-5.8S-ITS2, LSU, tef1)&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;15. Saksenaea_ITS_for_concatenated_RAW_v2.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of ITS1-5.8S-ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of a concatenated dataset 2.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;16. Saksenaea_ITS_for_concatenated_aligned_v2.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS1-5.8S-ITS2 region of &lt;i&gt;Saksenaea&lt;/i&gt; spp. used for preparation of a concatenated dataset 2.&lt;/p&gt;&lt;p&gt;Blank sequences were inserted for five isolates of &lt;i&gt;Saksenaea longicolla&lt;/i&gt; after the alignment.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;17.Saksenaea_ITS_LSU_tef1_concatenated_dataset2.fasta&lt;/p&gt;&lt;p&gt;Concatenated dataset of three multiple sequence alignments (15, 11, and 13). This concatenated dataset was used for the main phylogeny of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;C. Pairwise distances of the ex-type strains of &lt;/strong&gt;&lt;i&gt;&lt;strong&gt;Saksenaea&lt;/strong&gt;&lt;/i&gt;&lt;strong&gt; spp.&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;1. Saksenaea_ITS_type_RAW.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of ITS1-5.8S-ITS2 region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;2. Saksenaea_ITS2_type_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS2 region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;3.Saksenaea_ITS_type_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of ITS1-5.8S-ITS2 region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp. without &lt;i&gt;Saksenaea longicolla&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;4. Saksenaea_LSU_type_RAW.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of LSU gene region of the ex-type strains of &lt;i&gt;Saksenaea &lt;/i&gt;spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;5. Saksenaea_LSU_type_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of LSU gene region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;6. Saksenaea_tef1_type_RAW.fasta&lt;/p&gt;&lt;p&gt;Non-aligned dataset of tef1 gene region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt;&lt;p&gt;&nbsp;&lt;/p&gt;&lt;p&gt;7. Saksenaea_tef1_type_aligned.fasta&lt;/p&gt;&lt;p&gt;Aligned dataset of tef1 gene region of the ex-type strains of &lt;i&gt;Saksenaea&lt;/i&gt; spp.&lt;/p&gt

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

Abstract 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)

Abstract 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 β-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). β-Alanine was able to replace the efficacy of adding pantothenate

A Nitrate-Transforming Bacterial Community Dominates in the <i>Miscanthus</i> 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