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.

Oxygen concentration affects frequency and range of transconjugants for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1.

The frequency of transconjugants were compared for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1 under aerobic and anaerobic conditions. Filter mating assays were performed with one donor strain and one recipient strain using different donors of Pseudomonas and recipient strains, including Pseudomonas, Pantoea, and Buttiauxella. Under anaerobic condition, frequencies of transconjugants for both plasmids were 101-103-fold lower than those under aerobic condition regardless of whether aerobically or anaerobically grown donors and recipients were used. To compare the transconjugant ranges under aerobic and anaerobic conditions, conjugation was performed between the donor of pBP136 and recipient bacteria extracted from environmental samples. Several transconjugants were uniquely obtained from each aerobic or anaerobic condition. Our findings indicate that a plasmid can differently spread among bacteria depending on the oxygen concentrations of the environment.

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).

Identification of a transcriptional regulator for oligotrophy-responsive promoter in Rhodococcus erythropolis N9T-4.

Two genes, aldA, and mnoA, encoding an NAD-dependent aliphatic dehydrogenase and N,N'-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase, respectively, are strongly expressed when Rhodococcus erythropolis N9T-4 is grown under oligotrophic conditions. In this study, we found a transcriptional regulator required for the transcription of both aldA and mnoA. The transcriptional regulator was also found to be essential for the oligotrophic growth of N9T-4.

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%).

Algoriphagus sanaruensis sp. nov., a member of the family Cyclobacteriaceae, isolated from a brackish lake in Hamamatsu, Japan.

Strain M8-2T, which was isolated from brackish lake water (Lake Sanaru) in Japan, was characterized for representation of a novel species in the genus Algoriphagus. Cells of strain M8-2T were aerobic, Gram-stain-negative and curved-rod-shaped (0.2-0.5 µm wide and 0.7-1.9 µm long). Strain M8-2T grew optimally at 30 °C, pH 6.5-7.5 and in the presence of 0.5-1.0 % (w/v) NaCl. MK-7 was the sole isoprenoid quinone. The major polar lipids were phosphatidylethanolamine, an unidentified phospholipid and an unidentified polar lipid. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0. Phylogenetic analysis based on its 16S rRNA gene sequence showed that strain M8-2T belonged to the genus Algoriphagus and was closely related to Algoriphagus aquatilis A8-7T, Algoriphagus boseongensis BS-R1T, Algoriphagus aquaeductus T4T, Algoriphagus olei CC-Hsuan-617T, Algoriphagusshivajiensis NIO-S3T and Algoriphagus mannitolivorans DSM 15301T with sequence similarities of 96.6-97.4 %. Results of average nucleotide identity (<75 %) and digital DNA-DNA hybridization (<19 %) studies showed that M8-2T was distinct from its phylogenetic relatives. Based on the results of tests for acid production, the predominant cellular fatty acid composition, the DNA G+C content and phylogenetic position, a novel species in the genus Algoriphagus, with the name Algoriphagussanaruensis sp. nov., is proposed for strain M8-2T (=JCM 31446T=LMG 29969T).

Simultaneous enzymatic saccharification and comminution for the valorization of lignocellulosic biomass toward natural products.

BACKGROUND: Large-scale processing of lignocellulosics for glucose production generally relies on high temperature and acidic or alkaline conditions. However, extreme conditions produce chemical contaminants that complicate downstream processing. A method that mainly rely on mechanical and enzymatic reaction completely averts such problem and generates unmodified lignin. Products from this process could find novel applications in the chemicals, feed and food industry. But a large-scale system suitable for this purpose is yet to be developed. In this study we applied simultaneous enzymatic saccharification and communition (SESC) for the pre-treatment of a representative lignocellulosic biomass, cedar softwood, under both laboratory and large-scale conditions. RESULTS: Laboratory-scale comminution achieved a maximum saccharification efficiency of 80% at the optimum pH of 6. It was possible to recycle the supernatant to concentrate the glucose without affecting the efficiency. During the direct alcohol fermentation of SESC slurry, a high yield of ethanol was attained. The mild reaction conditions prevented the generation of undesired chemical inhibitors. Large-scale SESC treatment using a commercial beads mill system achieved a saccharification efficiency of 60% at an energy consumption of 50 MJ/kg biomass. CONCLUSION: SESC is very promising for the mild and clean processing of lignocellulose to generate glucose and unmodified lignin in a large scale. Economic feasibility is highly dependent on its potential to generate high value natural products for energy, specialty chemicals, feed and food application.

Proteobacteria and Bacteroidetes are major phyla of filterable bacteria passing through 0.22 µm pore size membrane filter, in Lake Sanaru, Hamamatsu, Japan.

141 filterable bacteria that passed through a 0.22 µm pore size filter were isolated from Lake Sanaru in Hamamatsu, Japan. These belonged to Proteobacteria, Bacteroidetes, Firmicutes, or Actinobacteria among which the first two phyla comprised the majority of the isolates. 48 isolates (12 taxa) are candidates assignable to new bacterial species or genera of Proteobacteria or Bacteroidetes.

Effects of phosphate addition on methane fermentation in the batch and upflow anaerobic sludge blanket (UASB) reactors.

Ammonia inhibition of methane fermentation is one of the leading causes of failure of anaerobic digestion reactors. In a batch anaerobic digestion reactor with 429 mM NH3-N/L of ammonia, the addition of 25 mM phosphate resulted in an increase in methane production rate. Similar results were obtained with the addition of disodium phosphate in continuous anaerobic digestion using an upflow anaerobic sludge blanket (UASB) reactor. While methane content and production rate decreased in the presence of more than 143 mM NH3-N/L of ammonium chloride in UASB, the addition of 5 mM disodium phosphate suppressed ammonia inhibition at 214 mM NH3-N/L of ammonium chloride. The addition prevented acetate/propionate accumulation, which might be one of the effects of the phosphate on the ammonia inhibition. The effects on the microbial community in the UASB reactor was also assessed, which was composed of Bacteria involved in hydrolysis, acidogenesis, acetogenesis, and dehydrogenation, as well as Archaea carrying out methanogenesis. The change in the microbial community was observed by ammonia inhibition and the addition of phosphate. The change indicates that the suppression of ammonia inhibition by disodium phosphate addition could stimulate the activity of methanogens, reduce shift in bacterial community, and enhance hydrogen-producing bacteria. The addition of phosphate will be an important treatment for future studies of methane fermentation.

Single-cell analyses revealed transfer ranges of IncP-1, IncP-7, and IncP-9 plasmids in a soil bacterial community.

The conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods. Pseudomonas putida, a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylum Proteobacteria was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phyla Actinobacteria, Bacteroidetes, and Firmicutes were detected only by the culture-independent method. Members of the genus Pseudomonas (class Gammaproteobacteria) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereas Delftia species (class Betaproteobacteria) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containing Delftia strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivable Delftia strains as recipients, fluorescence in situ hybridization detected pCAR1-containing Delftia cells, suggesting that Delftia was a "transient" host of pCAR1.

Extensive reduction of cell viability and enhanced matrix production in Pseudomonas aeruginosa PAO1 flow biofilms treated with a D-amino acid mixture.

Treatment of Pseudomonas aeruginosa PAO1 flow biofilms with a D-amino acid mixture caused significant reductions in cell biomass by 75% and cell viability by 71%. No biofilm disassembly occurred, and matrix production increased by 30%, thereby providing a thick protective cover for remaining viable or persister cells.

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.

Transconjugant range of PromA plasmids in microbial communities is predicted by sequence similarity with the bacterial host chromosome.

Nucleotide sequence similarity, including k-mer plasmid composition, has been used for prediction of plasmid evolutionary host range, representing the hosts in which a plasmid has replicated at some point during its evolutionary history. However, the relationships between the bacterial taxa of experimentally identified transconjugants and the predicted evolutionary host ranges are poorly understood. Here, four different PromA group plasmids showing different k-mer compositions were used as model plasmids. Filter mating assays were performed with a donor harbouring plasmids and recipients of bacterial communities extracted from environmental samples. A broad range of transconjugants was obtained with different bacterial taxa. A calculation of the dissimilarities in k-mer compositions as Mahalanobis distance between the plasmid and its sequenced transconjugant chromosomes revealed that each plasmid and transconjugant were significantly more similar than the plasmid and other non-transconjugant chromosomes. These results indicate that plasmids with different k-mer compositions clearly have different host ranges to which the plasmid will be transferred and replicated. The similarity of the nucleotide compositions could be used for predicting not only the plasmid evolutionary host range but also future host ranges.

Identification of genes involved in enhanced membrane vesicle formation in Pseudomonas aeruginosa biofilms: surface sensing facilitates vesiculation.

Membrane vesicles (MVs) are small spherical structures (20-400 nm) produced by most bacteria and have important biological functions including toxin delivery, signal transfer, biofilm formation, and immunomodulation of the host. Although MV formation is enhanced in biofilms of a wide range of bacterial species, the underlying mechanisms are not fully understood. An opportunistic pathogen, Pseudomonas aeruginosa, causes chronic infections that can be difficult to treat due to biofilm formation. Since MVs are abundant in biofilms, can transport virulence factors to the host, and have inflammation-inducing functions, the mechanisms of enhanced MV formation in biofilms needs to be elucidated to effectively treat infections. In this study, we evaluated the characteristics of MVs in P. aeruginosa PAO1 biofilms, and identified factors that contribute to enhanced MV formation. Vesiculation was significantly enhanced in the static culture; MVs were connected to filamentous substances in the biofilm, and separation between the outer and inner membranes and curvature of the membrane were observed in biofilm cells. By screening a transposon mutant library (8,023 mutants) for alterations in MV formation in biofilms, 66 mutants were identified as low-vesiculation strains (2/3 decrease relative to wild type), whereas no mutant was obtained that produced more MVs (twofold increase). Some transposons were inserted into genes related to biofilm formation, including flagellar motility (flg, fli, and mot) and extracellular polysaccharide synthesis (psl). ΔpelAΔpslA, which does not synthesize the extracellular polysaccharides Pel and Psl, showed reduced MV production in biofilms but not in planktonic conditions, suggesting that enhanced vesiculation is closely related to the synthesis of biofilm matrices in P. aeruginosa. Additionally, we found that blebbing occurred during bacterial attachment. Our findings indicate that biofilm-related factors are closely involved in enhanced MV formation in biofilms and that surface sensing facilitates vesiculation. Furthermore, this work expands the understanding of the infection strategy in P. aeruginosa biofilms.

Methylobacterium oxalidis sp. nov., isolated from leaves of Oxalis corniculata.

A pink-pigmented, facultatively methylotrophic bacterium, strain 35a(T), was isolated from the leaves of Oxalis corniculata. Cells of strain 35a(T) were Gram-reaction-negative, motile, non-spore-forming rods. The highest 16S rRNA gene pairwise sequence similarities for strain 35a(T) were found with the strains of Methylobacterium iners 5317S-33(T) (96.7%), 'Methylobacterium soli' YIM 48816 (96.6%) and Methylobacterium jeotgali S2R03-9(T) (96.3%). 16S rRNA gene sequence similarities with the type strains of all other recognized species of the genus Methylobacterium were below 96%. Major cellular fatty acids were C(18:1)ω7c, C(18:0) and C(16:0). The results of DNA-DNA hybridization experiments, analysis of cpn60 gene sequences, fatty acid profiles, whole-cell MALDI-TOF/MS spectral pattern analysis, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 35a(T) from its nearest phylogenetic neighbours. Strain 35a(T) is therefore considered to represent a novel species within the genus Methylobacterium, for which the name Methylobacterium oxalidis sp. nov. is proposed. The type strain is 35a(T) (=DSM 24028(T)=NBRC 107715(T)).

Methylobacterium gnaphalii sp. nov., isolated from leaves of Gnaphalium spicatum.

A pink-pigmented, facultatively methylotrophic bacterium, strain 23e(T), was isolated from the leaves of Gnaphalium spicatum (cudweed). The cells of strain 23e(T) were Gram-reaction negative, motile and non-spore-forming rods. On the basis of 16S rRNA gene sequence similarities, strain 23e(T) was related to Methylobacterium organophilum ATCC 27886(T) (97.1%) and Methylobacterium marchantiae JT1(T) (97%), and the phylogenetic similarities to all other Methylobacterium species with validly published names were less than 97%. Major cellular fatty acids were C(18:1)ω7c, C(16:00) and C(18:0). The results of DNA-DNA hybridization, phylogenetic analyses based on 16S rRNA and cpn60 gene sequences, fatty acid profiles, whole-cell matrix-assisted laser desorption/ionization time of flight/MS analysis, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 23e(T) from the phylogenetically closest relatives. We propose that strain 23e(T) represents a novel species within the genus Methylobacterium, for which the name Methylobacterium gnaphalii sp. nov. is proposed. The type strain is 23e(T) (=DSM 24027(T)=NBRC 107716(T)).

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]).

Pandoraea oxalativorans sp. nov., Pandoraea faecigallinarum sp. nov. and Pandoraea vervacti sp. nov., isolated from oxalate-enriched culture.

Five isolates, designated TA2, TA4, TA25(T), KOx(T) and NS15(T) were isolated in previous studies by enrichment in mineral medium with potassium oxalate as the sole carbon source and were characterized using a polyphasic approach. The isolates were Gram-reaction-negative, aerobic, non-spore-forming rods. Phylogenetic analyses based on 16S rRNA and DNA gyrase B subunit (gyrB) gene sequences confirmed that the isolates belonged to the genus Pandoraea and were most closely related to Pandoraea sputorum and Pandoraea pnomenusa (97.2-99.7 % 16S rRNA gene sequence similarity). The isolates could be differentiated from their closest relatives on the basis of several phenotypic characteristics. The major cellular fatty acid profiles of the isolates comprised C16:0, C18:1ω7c, C17:0 cyclo and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). On the basis of DNA-DNA hybridization studies and phylogenetic analyses, the isolates represent three novel species within the genus Pandoraea, for which the names Pandoraea oxalativorans sp. nov. (TA25(T)  = NBRC 106091(T)  = CCM 7677(T)  = DSM 23570(T)), Pandoraea faecigallinarum sp. nov. (KOx(T)  = NBRC 106092(T)  = CCM 2766(T)  = DSM 23572(T)) and Pandoraea vervacti sp. nov. (NS15(T)  = NBRC 106088(T)  = CCM 7667(T)  = DSM 23571(T)) are proposed.

Characterization of a cryptic plasmid, pSM103mini, from polyethylene-glycol degrading Sphingopyxis macrogoltabida strain 103.

A cryptic plasmid, pSM103mini, was found in polyethylene-glycol degrading bacterium Sphingopyxis macrogoltabida, strain 103. The plasmid was 4,328-bp long and its GC content was 57.5%. It contained four open reading frames, but only two of them showed significant similarity to reported proteins. ORF3 and ORF4 were assumed to encode resolvase and replication protein (RepA) respectively. Downstream of ORF4 we found complex repeat sequences. These together with ORF3 and 4 were necessary and sufficient for plasmid maintenance in strain 103, as analyzed by constructing deletion plasmids. The pHSG398-fused plasmid (pHSG-SM103mini) functioned as a shuttle vector between strain 103 and Escherichia coli. The plasmid constructed was maintained in strain 103 and its close relative, S. macrogoltabida strain 203, but not efficiently in PEG-degrading S. terrae.

Crystallization and preliminary crystallographic analysis of manganese(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from Bacillus sp. JF8.

A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized. The initial screening for crystallization was performed by the sitting-drop vapour-diffusion method using a crystallization robot, resulting in the growth of two crystal forms. The first crystal belonged to space group P1, with unit-cell parameters a = 62.7, b = 71.4, c = 93.6 A, alpha = 71.2, beta = 81.0, gamma = 64.0 degrees, and diffracted to 1.3 A resolution. The second crystal belonged to space group I222, with unit-cell parameters a = 74.2, b = 90.8, c = 104.3 A, and diffracted to 1.3 A resolution. Molecular-replacement trials using homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (28% amino-acid sequence identity) as a search model provided a satisfactory solution for both crystal forms.