Cloning of three Alnus sieboldiana type III polyketide synthases and formation of polyketides in recombinant Escherichia coli using cinnamic acid analogs as substrates.

Alnus sieboldiana is an actinorhizal plant that coexists with the nitrogen-fixing actinomycete Frankia via nodules. It produces a variety of polyketides, including flavonoids, stilbenoids, and diarylheptanoids. These compounds have beneficial biological activities. Plant polyketides are produced by type III polyketide synthases (PKSIII). In this study, three A. sieboldiana PKSIIIs (AsPKSIII1, AsPKSIII2, and AsPKSIII3) predicted from next-generation sequencing analysis of A. sieboldiana seedling RNA were amplified and cloned. Phylogenetic tree analysis classified AsPKSIII2 and AsPKSIII3 into the chalcone synthase (CHS) group, whereas AsPKSIII1 was not classified into this group. We attempted to produce polyketides by adding cinnamic acid analogs to the culture medium of Escherichia coli, in which the respective PKSIII gene and the acetyl-CoA carboxylase (ACC) and 4-coumarate: CoA ligase (4CL) genes were simultaneously recombined. AsPKSIII1 is an enzyme that condensed only one molecule of malonyl-CoA to cinnamoyl-CoAs. In contrast, AsPKSIII2 and AsPKSIII3 produced chalcones as shown in a phylogenetic tree analysis, but also produced triketide pyrone. The ratio of these products differed between the two enzymes. We determined the gene and amino acid sequences as well as the substrate specificities of the two enzymes involved in flavonoid production and one enzyme potentially involved in diarylheptanoid production in A. sieboldiana.

Complete genome sequence of Paraburkholderia sp. strain 22B1P capable of utilizing 3-chlorobenzoate as a carbon source.

Paraburkholderia sp. strain 22B1P utilizes 3-chlorobenzoate as a carbon source. Complete genome sequencing of strain 22B1P revealed two chromosomes and two plasmids. The genes involved in the conversion of 3-chlorobenzoate to 3-chlorocatechol and those involved in the conversion of 3-chlorocatechol to 3-oxoadipate were located on chromosomes 2 and 1, respectively.

Cloning and characterization of NADPH-dependent double-bond reductases from Alnus sieboldiana that recognize linear diarylheptanoids as substrates.

Diarylheptanoids are secondary metabolites of plants that comprise a C6-C7-C6 scaffold. They can be broadly classified into linear-type and cyclic-type diarylheptanoids based on their chemical structures. Actinorhizal trees, such as Casuarina, Alnus, and Myrica, which form nodule symbiosis with actinomycetes Frankia, produce cyclic diarylheptanoids (CDHs); in Alnus sieboldiana Matsum. in particular, we have reported that the addition of CDHs leads to an increase in the number of nodules. However, the information available on the biosynthesis of CDHs is scarce. A greater number of plants CDHs (including those isolated from actinorhizal trees) with a saturated heptane chain have been isolated compared with linear, non-cyclic diarylheptanoids. To identify the genes involved in the synthesis of these compounds, genes with significant sequence similarity to existing plant double-bond reductases were screened in A. sieboldiana. This report describes the isolation and characterization of two A. sieboldiana double-bond reductases (AsDBR1 and AsDBR2) that catalyze the NADPH-dependent reduction of bisdemethoxycurcumin and curcumin. The optimum pH for the two enzymes was 5.0. The apparent Km values for bisdemethoxycurcumin and NADPH were 4.24 and 3.53 µM in the case of AsDBR1, and 2.55 and 2.13 µM for AsDBR2. The kcat value was 9.4-fold higher for AsDBR1 vs. AsDBR2 when using the bisdemethoxycurcumin substrate. Interestingly, the two AsDBRs failed to reduce the phenylpropanoid monomer.

Isolation and Genomic Analysis of 3-Chlorobenzoate-Degrading Bacteria from Soil.

The compound 3-chlorobenzoate (3-CBA) is a hazardous industrial waste product that can harm human health and the environment. This study investigates the physiological and genetic potential for 3-chlorobenzoate (3-CBA) degradation. Six 3-CBA Gram-negative degraders with different degradation properties belonging to the genera Caballeronia, Paraburkholderia and Cupriavidus were isolated from the soil. The representative strains Caballeronia 19CS4-2 and Paraburkholderia 19CS9-1 showed higher maximum specific growth rates (µmax, h-1) than Cupriavidus 19C6 and degraded 5 mM 3-CBA within 20-28 h. Two degradation products, chloro-cis,cis-muconate and maleylacetate, were detected in all isolates using high-performance liquid chromatography and mass spectrometry. Genomic analyses revealed the presence of cbe and tfd gene clusters in strains 19CS4-2 and 19CS9-1, indicating that they probably metabolized the 3-CBA via the chlorocatechol ortho-cleavage pathway. Strain 19C6 possessed cbe genes, but not tfd genes, suggesting it might have a different chlorocatechol degradation pathway. Putative genes for the metabolism of 3-hydroxybenzoate via gentisate were found only in 19C6, which utilized the compound as a sole carbon source. 19C6 exhibited distinct characteristics from strains 19CS4-2 and 19CS9-1. The results confirm that bacteria can degrade 3-CBA and improve our understanding of how they contribute to environmental 3-CBA biodegradation.

Physiological and genomic analyses of cobalamin (vitamin B12)-auxotrophy of Lysobacter auxotrophicus sp. nov., a methionine-auxotrophic chitinolytic bacterium isolated from chitin-treated soil.

A novel bacterium, designated 5-21aT, isolated from chitin-treated upland soil, exhibits methionine (Met) auxotrophy and chitinolytic activity. A physiological experiment revealed the cobalamin (synonym, vitamin B12)(Cbl)-auxotrophic property of strain 5-21aT. The newly determined complete genomic sequence indicated that strain 5-21aT possesses only the putative gene for Cbl-dependent Met synthase (MetH) and lacks that for the Cbl-independent one (MetE), which implies the requirement of Cbl for Met-synthesis in strain 5-21aT. The set of genes for the upstream (corrin ring synthesis) pathway of Cbl synthesis is absent in the genome of strain 5-21aT, which explains the Cbl-auxotrophy of 5-21aT. This strain was characterized via a polyphasic approach to determine its taxonomic position. The nucleotide sequences of two copies of the 16S rRNA gene of strain 5-21aT indicated the highest similarities to Lysobacter soli DCY21T(99.8 and 99.9 %) and Lysobacter panacisoli CJ29T(98.7 and 98.8 %, respectively), whose Cbl-auxotrophic properties were revealed in this study. The principal respiratory quinone was Q-8. The predominant cellular fatty acids were iso-C15:0, iso-C16:0 and iso-C17:1 ω9c. The complete genome sequence of strain 5-21aT revealed that the genome size was 4 155 451 bp long and the G+C content was 67.87 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain 5-21aT and its most closely phylogenetic relative L. soli DCY21T were 88.8 and 36.5%, respectively. Based on genomic, chemotaxonomic, phenotypic and phylogenetic data, strain 5-21aT represents a novel species in the genus Lysobacter, for which the name Lyobacter auxotrophicus sp. nov. is proposed. The type strain is 5-21aT (=NBRC 115507T=LMG 32660T).

Complete Genome Sequence of Rhodococcus qingshengii N9T-4.

Rhodococcus qingshengii N9T-4 can grow on media without added carbon sources. Here, we report the complete nucleotide sequence of the N9T-4 genome, consisting of a chromosome (6,439,972 bp), a linear plasmid (pN9T4-1 [565,206 bp]), and two circular plasmids (pN9T-4-2 [99,662 bp] and pN9T-4-3 [30,419 bp]).

Hitherto-Unnoticed Self-Transmissible Plasmids Widely Distributed among Different Environments in Japan.

Various conjugative plasmids were obtained by exogenous plasmid capture, biparental mating, and/or triparental mating methods from different environmental samples in Japan. Based on phylogenetic analyses of their whole-nucleotide sequences, new IncP/P-1 plasmids that could be classified into novel subgroups were obtained. Mini-replicons of the plasmids were constructed, and each of them was incompatible with at least one of the IncP/P-1 plasmids, although they showed diverse iteron sequences in their oriV regions. There were two large clades of IncP/P-1 plasmids, clade I and II. Plasmids in clade I and II included antibiotic resistance genes. Notably, nucleotide compositions of newly found plasmids exhibited different tendencies compared with those of the previously well-studied IncP/P-1 plasmids. Indeed, the host range of plasmids of clade II was different from that of clade I. Although few PromA plasmids have been reported, the number of plasmids belonging to PromAß, and -γ subgroups detected in this study was close to that of IncP/P-1 plasmids. The host ranges of PromAγ and PromAδ plasmids were broad and transferred to different and distinct classes of Proteobacteria. Interestingly, PromA plasmids and many IncP/P-1 plasmids do not carry any accessory genes. These findings indicate the presence of "hitherto-unnoticed" conjugative plasmids, including IncP/P-1 or PromA derivative ones in nature. These plasmids would have important roles in the exchange of various genes, including antibiotic resistance genes, among different bacteria in nature. IMPORTANCE Plasmids are known to spread among different bacteria. However, which plasmids spread among environmental samples and in which environments they are present is still poorly understood. This study showed that unidentified conjugative plasmids were present in various environments. Different novel IncP/P-1 plasmids were found, whose host ranges were different from those of known plasmids, showing wide diversity of IncP/P-1 plasmids. PromA plasmids, exhibiting a broad host range, were diversified into several subgroups and widely distributed in varied environments. These findings are important for understanding how bacteria naturally exchange their genes, including antibiotic resistance genes, a growing threat to human health worldwide.

Draft Genome Sequence of Lactiplantibacillus plantarum NMZ-1139, Isolated from Whisky Mash.

Lactiplantibacillus plantarum NMZ-1139 was isolated from whisky mash and applied to sour beer production. Here, we report the draft genome sequence of L. plantarum NMZ-1139, which contains 3,117 protein-coding sequences, including genes associated with hop resistance, such as horA and hitA.

Imbalance in Carbon and Nitrogen Metabolism in Comamonas testosteroni R2 Is Caused by Negative Feedback and Rescued by L-arginine.

The collapse of Comamonas testosteroni R2 under chemostat conditions and the aerobic growth of strain R2 under batch conditions with phenol as the sole carbon source were investigated using physiological and transcriptomic techniques. Phenol-/catechol-degrading activities under chemostat conditions gradually decreased, suggesting that metabolites produced from strain R2 accumulated in the culture, which caused negative feedback. The competitive inhibition of phenol hydroxylase and catechol dioxygenase was observed in a crude extract of the supernatant collected from the collapsed culture. Transcriptomic analyses showed that genes related to nitrogen transport were up-regulated; the ammonium transporter amtB was up-regulated approximately 190-fold in the collapsed status, suggesting an increase in the concentration of ammonium in cells. The transcriptional levels of most of the genes related to gluconeogenesis, glycolysis, the pentose phosphate pathway, and the TCA and urea cycles decreased by ~0.7-fold in the stable status, whereas the activities of glutamate synthase and glutamine synthetase increased by ~2-fold. These results suggest that ammonium was assimilated into glutamate and glutamine via 2-oxoglutarate under the limited supply of carbon skeletons, whereas the synthesis of other amino acids and nucleotides was repressed by 0.6-fold. Furthermore, negative feedback appeared to cause an imbalance between carbon and nitrogen metabolism, resulting in collapse. The effects of amino acids on negative feedback were investigated. L-arginine allowed strain R2 to grow normally, even under growth-inhibiting conditions, suggesting that the imbalance was corrected by the stimulation of the urea cycle, resulting in the rescue of strain R2.

Complete Genome Sequence of Caballeronia sp. Strain NK8 (MAFF311271), a Chlorobenzoate-Degrading Bacterium.

Caballeronia sp. strain NK8 grows on 3-chlorobenzoate and shows chemotaxis toward 3-chlorobenzoate and its degradation products, such as chlorocatechols. Complete genome sequencing revealed a 9.2-Mb genome consisting of three chromosomes and four plasmids. The genes for degradation of 3-chlorobenzoate and chlorocatechols were located on plasmids pNK81 and pNK84, respectively.

Fluviispira sanaruensis sp., nov., Isolated from a Brackish Lake in Hamamatsu, Japan.

Strain RF1110005T, which was isolated from brackish lake water sampled at Lake Sanaru in Japan as a "filterable" bacterial strain, was characterized as a novel species in the genus Fluviispira, family Silvanigrellaceae, order Silvanigrellales, the class Oligoflexia and the phylum Bdellovibrionota. Cells of RF1110005T were aerobic, Gram stain negative, and show a pleomorphic morphology of spiral, filamentous and rod shapes. Catalase reaction was positive. Strain RF1110005T grew optimally at 30 °C, pH 7.0-8.0 and 0.5% NaCl (w/v). The major polar lipids in RF1110005T were phosphatidylethanolamine and phosphatidylglycerol. The predominant cellular fatty acids were iso-C15:0 and anteiso-C15:0. Phylogenetic analysis based on 16S rRNA gene sequences and concatenates of core gene sequence showed that the nearest neighbor of strain RF1110005T was Fluviispira multicolorata strain 33A1-SZDPT with 98.4% 16S rRNA gene sequence similarity. The genome size of strain RF1110005T was 3.5 Mbp with two plasmids (80 kb and 69 kb), and the G + C content was 33.7 mol%. Comparisons with genome-wide analyses and chemotaxonomic characters clearly showed that strain RF1110005T differed from F. multicolorata. Therefore, a novel species in Fluviispira sanaruensis, sp. nov., is proposed for strain RF1110005T (= JCM 31447 T = LMG 30360 T).

Complete Genome Sequence of Buttiauxella agrestis DSM 9389.

We report here the complete genome sequence of Buttiauxella agrestis DSM 9389, which harbors eight 16S rRNA genes classified into three types. The genome sequence of this strain showed a high average nucleotide identity (97.3%) with that of the highly membrane vesicle-producing strain B. agrestis ATCC 33320T.

Transcriptome differences between Cupriavidus necator NH9 grown with 3-chlorobenzoate and that grown with benzoate.

RNA-seq analysis of Cupriavidus necator NH9, a 3-chlorobenzoate degradative bacterium, cultured with 3-chlorobenzaote and benzoate, revealed strong induction of genes encoding enzymes in degradation pathways of the respective compound, including the genes to convert 3-chlorobenzaote and benzoate to chlorocatechol and catechol, respectively, and the genes of chlorocatechol ortho-cleavage pathway for conversion to central metabolites. The genes encoding transporters, components of the stress response, flagellar proteins, and chemotaxis proteins showed altered expression patterns between 3-chlorobenzoate and benzoate. Gene Ontology enrichment analysis revealed that chemotaxis-related terms were significantly upregulated by benzoate compared with 3-chlorobenzoate. Consistent with this, in semisolid agar plate assays, NH9 cells showed stronger chemotaxis to benzoate than to 3-chlorobenzoate. These results, combined with the absence of genes related to uptake/chemotaxis for 3-chlorobenzoate located closely to the degradation genes of 3-chlorobenzoate, suggested that NH9 has not fully adapted to the utilization of chlorinated benzoate, unlike benzoate, in nature.

16S rRNA Gene Amplicon Sequencing of Gut Microbiota in Three Species of Deep-Sea Fish in Suruga Bay, Japan.

We report here 16S rRNA gene amplicon sequence analysis of the gut microbiota in three species of deep-sea fish collected from Suruga Bay, Japan. Of the three species, two were dominated by the phylum Proteobacteria (genus Photobacterium), while one was dominated by the phyla Spirochaetes (genus Brevinema) and Tenericutes (unclassified Mycoplasmataceae).

Draft Genome Sequence of Phenol-Degrading Variovorax boronicumulans Strain c24.

We report the draft genome sequence of Variovorax boronicumulans strain c24, which was isolated from a soil-inoculated chemostat culture amended with phenol as a sole carbon and energy source. The genome data will provide insights into phenol and other xenobiotic compound degradation mechanisms for bioremediation applications.

Draft Genome Sequence of Phenol-Degrading Variovorax boronicumulans Strain HAB-30.

We report the draft genome sequence of Variovorax boronicumulans strain HAB-30, which was isolated from a phenol-degrading chemostat culture. This strain contains genes encoding a multicomponent type of phenol hydroxylase, with degradation pathways for catechol and other aromatic compounds. The genome sequence will be useful for understanding the metabolic pathways involved in phenol degradation.

Chryseotalea sanaruensis gen. nov., sp., nov., a Member of the Family Cytophagaceae, Isolated from a Brackish Lake in Hamamatsu Japan.

A strain-designated YsT was isolated as a filterable bacterial strain from Lake Sanaru, a brackish water lake in Hamamatsu Japan. YsT is aerobic, Gram-negative, and slender rod shaped. YsT grew optimally at 30 °C, pH 7.0-8.0 and without the addition of NaCl. MK-7 was the sole isoprenoid quinone. The main cellular polar lipids were phosphatidylethanolamine and unidentified amino- and polar-lipids. The predominant cellular fatty acids were C18:0, iso-C14:0 and iso-C15:0. Phylogenetic analysis of 16S rRNA gene sequence revealed the nearest neighbours of strain YsT to be members of the Ohtaekwangia and Chryseolinea genera with 91.2-92.1% sequence similarity. The percentages of conserved proteins (POCP) between the genomes of YsT and related strains were less than 50%. Phenotypic analyses suggested that YsT could not metabolize glucose and related sugars, which was discriminative from its phylogenetic relatives. We, therefore, propose a novel species in a new genus, Chryseotalea sanaruensis gen. nov., sp. nov. in the family Cytophagaceae (= JCM 30318T = LMG 30359T), based on cell size, the predominant cellular fatty acid composition, and the DNA GC content (38.9 mol%).

Draft Genome Sequence of Buttiauxella sp. Strain A111, Which Converts 2-Azahypoxanthine to 2-Aza-8-Oxohypoxanthine.

We report here the draft genome sequence of Buttiauxella sp. strain A111, isolated on the basis of bioconversion activity of the plant growth-regulating compound 2-azahypoxanthine to 2-aza-8-oxohypoxanthine. The genome contains 4,388 protein-coding sequences, including several genes possibly involved in the metabolism of the plant growth-regulating compound.

Isolation and characterization of a moderate thermophilic Paenibacillus naphthalenovorans strain 4B1 capable of degrading dibenzofuran from dioxin-contaminated soil in Vietnam.

A moderate thermophilic dibenzofuran (DF) degrader, strain 4B1, was isolated from dioxin-contaminated soil in Vietnam under thermophilic condition. A 16S rRNA gene sequence analysis assigned the strain to genus Paenibacillus. The optimum growth temperature of strain 4B1 was 45°C with a doubling time of 2.7 h in the presence of DF as a sole carbon and energy source. The rate of its growth and DF-degradation were approximately 3-fold higher than those of a reference Paenibacillus sp. strain. The 4B1 strain degraded 89% of 1000 mg L-1 DF within 48 h cultivation at the optimum temperature. TBLASTN analysis based on its draft genome sequence revealed that this strain possessed a dbf gene cluster. The open reading frames (dbfA1A2RBC) in the cluster shared 99-100% identity with those of Paenibacillus sp. YK5, indicating that DF was likely degraded by an angular dioxygenation pathway in strain 4B1. Four genes in the dbf gene cluster (dbfA1A2BC) were partially induced by DF, which was observed by semi-quantitative RT-PCR. Quantitative PCR analysis of dbfA1 transcripts, encoding the alpha subunit of DF dioxygenase, indicated that dbfA1 was expressed 4-times higher than that of strain YK5 at 45°C. These results suggest that the faster growth and degradation of DF in strain 4B1 could be due to differences in transcriptional regulation of dbf cluster genes.

Complete Genome Sequence of the Marine Bacterium Erythrobacter flavus Strain KJ5.

Erythrobacter flavus strain KJ5 (formerly called Erythrobacter sp. strain KJ5) is a yellowish marine bacterium that was isolated from a hard coral in the Karimunjawa Islands of Indonesia. Here, we report the complete genome sequence of the bacterium and provide a useful resource for studies of the biosynthetic pathways of its unique carotenoids.