Evolution of temperature optimum in <Emphasis Type="Italic">Thermotogaceae</Emphasis> and the prediction of trait values of uncultured organisms

Quantitative characterization of the mode and rate of phenotypic evolution is rarely applied to prokaryotes. Here, we present an analysis of temperature optimum (T _opt) evolution in the thermophilic family Thermotogaceae, which has a large number of cultured representatives. We use log-rate-interval analysis to show that T _opt evolution in Thermotogaceae is consistent with a Brownian motion (BM) evolutionary model. The properties of the BM model are used to a establish confidence intervals on the unknown phenotypic trait value of an uncultured organism, given its distance to a close relative with known trait value. Cross-validation by bootstrapping indicates that the predictions are robust.     

<Emphasis Type="Italic">Aliihoeflea aestuarii</Emphasis> gen. nov., sp. nov., a novel bacterium isolated from tidal flat sediment

A novel Gram-negative and rod-shaped bacterium, designated N8^T, was isolated from tidal flat sediment. Phylogenetic analysis based on 16S rRNA gene sequences showed that N8^T strain is associated with the family Phyllobacteriaceae: two uncultured clones (98.4 and 99.8% 16S rRNA gene sequence similarity) and the genus Mesorhizobium (≤97.0%). The novel strain formed a separate clade with uncultured clones in the phylogenetic tree based on 16S rRNA gene sequences. Cellular fatty acid profiles predominately comprised C_18:1 ω7c and C_19:0 cyclo ω8c. The major isoprenoid quinone is ubiquinone-10 and genomic DNA G+C content is 53.4 mol%. The polyphasic taxonomic study indicates that the novel strain N8^T represents a novel species of the new genus in the family Phyllobacteriaceae, named Aliihoeflea aestuarii. The type strain is N8^T (= KCTC 22052^T= JCM 15118^T= DSM 19536^T).     

Detection of Putatively Thermophilic Anaerobic Methanotrophs in Diffuse Hydrothermal Vent Fluids

The anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivated Euryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (P_GC) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between the P_GC and optimal growth temperatures (T_opt) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.     

Exploration and isolation of novel thermophiles in frozen enrichment cultures derived from a terrestrial acidic hot spring

An isolation strategy, exploring novel microorganisms in frozen enrichment cultures (ENFE), which uses a combination of enrichment culture and 16S rRNA gene clone analysis, was evaluated for isolating uncultured thermophiles from a terrestrial acidic hot spring. The procedure comprised (a) multiple enrichment cultures under various conditions, (b) cryostorage of all enrichments, (c) microbial community analyses of the enrichments using 16S rRNA gene sequences, and (d) purification of microorganisms from enrichments containing previously uncultured microorganisms. The enrichments were performed under a total of 36 conditions, and 16 of these enrichments yielded positive microbial growth with the detection of three previously uncultured archaea. Two of the three previously uncultured archaea, strains HS-1 and HS-3, were successfully isolated. Strain HS-1 and HS-3 represented a novel lineage of the order Sulfolobales and novel species of the genus Sulfolobus, respectively. Although innovative isolation methods play strategic roles in isolating previously uncultured microorganisms, the ENFE strategy showed potential for characterizing and isolating such microorganisms using conventional media and techniques.     

Dimensionless analysis of the microbial growth rate dependence on sub-optimal temperatures

The influence of sub-optimal temperatures (T) on the microbial growth rate (μ) has been assessed by means of dimensionless variables: T_dim = [T−T_min]/[T_opt−T_min] and μ_dim = μ/μ_opt. T_min represents the temperature at which there is no growth, T_opt the optimum temperature at which the growth rate, μ_opt, is maximum. Data sets, growth rate vs temperature, have been taken from the literature for 12 organisms (psychrotrophs, mesophiles and thermophiles). In order to compare these organisms, the power law function has been used: [μ_dim] = [T_dim]^α. The parameters μ_opt and T_opt are determined from direct readings whereas T_min and αare estimated by means of a non-linear regression. An accurate estimation of T_min is obtained providing low growth rate data are available. A wide range of optimal temperatures where the growth rate almost equals μ_opt prevents one from obtaining a narrow confidence interval forα. On the basis of the analysis hereafter developed, thermophiles are characterized by values of the power α less than mesophiles and psychrotrophs. Almost all of these values are significantly different from two, previously determined for Staphylococcus xylosus and widely used for predicting the microbial growth in foods. Received 15 May 1998/ Accepted in revised form 25 September 1998     

Relationships Between Genomic G+C Content,RNA Secondary Structures,and Optimal Growth Temperature in Prokaryotes

G:C pairs are more stable than A:T pairs because they have an additional hydrogen bond. This has led to many studies on the correlation between the guanine+cytosine (G+C) content of nucleic acids and temperature over the last 20 years. We collected the optimal growth temperatures (T_opt) and the G+C contents of genomic DNA; 23S, 16S, and 5S ribosomal RNAs; and transfer RNAs for 764 prokaryotic species. No correlation was found between genomic G+C content and T_opt, but there were striking correlations between the G+C content of ribosomal and transfer RNA stems and T_opt. Two explanations have been proposed—neutral evolution and selection pressure—for the approximate equalities of G and C (respectively, A and T) contents within each strand of DNA molecules. Our results do not support the notion that selection pressure induces complementary oligonucleotides in close proximity and therefore numerous secondary structures in prokaryotic DNA, as the genomic G+C content does not behave in the same way as that of folded RNA with respect to optimal growth temperature. Received: 25 September 1996 / Accepted: 21 January 1997     

Biodiversity of Denitrifying and Dinitrogen-Fixing Bacteria in an Acid Forest Soil

Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd₁-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N₂-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N₂ fixation are not genetic traits of most of the uncultured bacteria.     

Phylogenetic Diversity of Microorganisms from the Sludge of a Biogas Reactor Processing Oil-Containing and Municipal Waste

High-throughput sequencing of the 16S rRNA gene fragments was used to determine the phylogenetic diversity of prokaryotes, including human pathogens, in the liquid phase of the sludge of a biogas reactor processing oil-containing and municipal waste. A unique microbial community was found to develop in the sludge, which comprised the microorganisms of municipal wastewater (bacteria of human feces) and specific groups of aerobic and anaerobic microorganisms, which possibly arrived with oil-containing water. In the 16S rRNA gene library, the sequences of representatives of Firmicutes prevailed (54.9%), which belonged to anaerobic bacteria of the genera Gelria (26.6%), Syntrophomonas (6.0%), Lutispora (2.0%), and uncultured Clostridia (group MBA03, 11.1%). The Proteobacteria sequences (20.7%) belonged mostly to the metabolically diverse members of the genus Pseudomonas (13.8%). The phylum Bacteroidetes (7%) was represented by uncultured bacteria (VadinBC27 wastewater-sludge group), while members of the phylum Cloacimonetes were mainly syntrophic bacteria Candidatus Cloacamonas (7.5%). The sequences of bacteria commonly occurring in oilfields (Clostridia, Anaerolinea, Bacteroidetes, sulfate-reducing Deltaproteobacteria, members of the family Syntrophaceae, and of the genera Thauera, Pseudomonas, Dechloromonas, and Petrimonas) were revealed. No sequences of bacteria known to be pathogenic to humans were found. The cultured microorganisms were aerobic organotrophic and anaerobic fermenting, denitrifying, and methanogenic prokaryotes. Fermenting and methanogenic enrichments grew on a broad range of organic substrates (sucrose, glycerol, starch), producing volatile fatty acids (acetate, n-butyrate, and propionate), gases (Н₂, СО₂, and CH₄), and decreasing pH of the medium from 7.0 to 4.5–5.0. The possible application of the biogas reactor sludge as a source of fermenting and methanogenic anaerobic prokaryotes, as well as of aerobic hydrocarbonoxidizing bacteria for oilfield introduction and for production of new preparations for enhanced oil recovery and for bioremediation of oil contamination is discussed.     

Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.     

Methanogen Diversity Evidenced by Molecular Characterization of Methyl Coenzyme M Reductase A (mcrA) Genes in Hydrothermal Sediments of the Guaymas Basin

The methanogenic community in hydrothermally active sediments of Guaymas Basin (Gulf of California, Mexico) was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Members of the Methanomicrobiales and Methanosarcinales dominated the mcrA and 16S rRNA clone libraries from the upper 15 cm of the sediments. Within the H₂/CO₂- and formate-utilizing family Methanomicrobiales, two mcrA and 16S rRNA lineages were closely affiliated with cultured species of the genera Methanoculleus and Methanocorpusculum. The most frequently recovered mcrA PCR amplicons within the Methanomicrobiales did not branch with any cultured genera. Within the nutritionally versatile family Methanosarcinales, one 16S rRNA amplicon and most of the mcrA PCR amplicons were affiliated with the obligately acetate utilizing species Methanosaeta concilii. The mcrA clone libraries also included phylotypes related to the methyl-disproportionating genus Methanococcoides. However, two mcrA and two 16S rRNA lineages within the Methanosarcinales were unrelated to any cultured genus. Overall, the clone libraries indicate a diversified methanogen community that uses H₂/CO₂, formate, acetate, and methylated substrates. Phylogenetic affiliations of mcrA and 16S rRNA clones with thermophilic and nonthermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas sediments.     

16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order “Rhodocyclales”

For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

Sabulilitoribacter multivorans gen. nov., sp. nov., a polysaccharide-degrading bacterium of the family Flavobacteriaceae isolated from seashore sand

A Gram-negative, aerobic, non-flagellated, non-gliding and rod-shaped bacterial strain, designated M-M16^T, was isolated from seashore sand around a seaweed farm on the South Sea, South Korea, and its taxonomic position was investigated by using a polyphasic study. Strain M-M16^T grew optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2 % (w/v) NaCl. Strain M-M16^T exhibited the highest 16S rRNA gene sequence similarity values to the type strains of Gaetbulibacter lutimaris (96.5 %) and Flaviramulus basaltis (95.8 %). Neighbour-joining and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain M-M16^T clustered with the type strains of Gaetbulibacter species and F. basaltis. Strain M-M16^T contained MK-6 as the predominant menaquinone and iso-C_15:1 G, iso-C_15:0 and iso-C_17:0 3-OH as the major fatty acids. The major polar lipids detected in strain M-M16^T were phosphatidylethanolamine and one unidentified lipid. The DNA G+C content of strain M-M16^T was 37.4 mol%. The phylogenetic and chemotaxonomic data and other phenotypic properties revealed that strain M-M16^T represents a novel genus and species within the family Flavobacteriaceae, for which the name Sabulilitoribacter multivorans gen. nov., sp. nov. is proposed. The type strain of S. multivorans is M-M16^T (= KCTC 32326^T = CCUG 63831^T).     

Paenibacillus pinihumi sp. nov., a cellulolytic bacterium isolated from the rhizosphere of Pinus densiflora

A novel cellulolytic bacterium, strain S23^T, was isolated from the rhizosphere of the pine trees in Daejeon, Republic of Korea. This isolate was Gram-positive, strictly aerobic, rod-shaped, catalase-negative, oxidase-positive, motile by means of peritrichous flagella, and tested positive for alkaline phosphatase, esterase lipase, leucine arylamidase, α-galactosidase, and β-galactosidase activities. The DNA G+C content was 49.5 mol%. The main cellular fatty acids were anteiso-C_15:0 (51.9%), iso-C_16:0 (14.7%), and iso-C_15:0 (13.2%). The major isoprenoid quinone was menaquinone 7 (MK-7). Diagnostic diamino acid in the cell-wall pepti-doglycan was meso-diaminopimelic acid. Comparative 16S rRNA gene sequence analysis showed that this strain clustered with Paenibacillus species. The 16S rRNA gene sequence similarity values between S23^T and other Paenibacillus species were between 89.9% and 95.9%, and S23^T was most closely related to Paenibacillus tarimensis SA-7-6^T. On the basis of phylogenetic and phenotypic properties of strain S23^T, the isolate is considered as a novel species belonging to the genus Paenibacillus. Therefore, the name, Paenibacillus pinihumi sp. nov., is proposed for the rhizosphere isolate; the type strain is S23^T (=KCTC 13695^T =KACC 14199^T =JCM 16419^T)     

Flavobacterium aquaticum sp. nov., a member of the Bacteroidetes isolated from a freshwater reservoir

A novel bacterial strain, designated ARSA-111^T, was isolated from a freshwater reservoir in Cheonan, Korea. Phylogenetic analysis based on 16S rRNA gene sequences suggested that the isolate belonged to the genus Flavobacterium of phylum Bacteroidetes. The 16S rRNA gene sequence of strain ARSA-111^T showed a high degree of sequence similarity to those of Flavobacteium cheonanense KACC 14972^T (97.3%), F. aquatile JCM 20475^T (97.1%), and other type strains of the genus Flavobacterium (< 97.0%). The phylogenetic tree and network analysis (i.e. median-joining) based on 16S rRNA gene sequences showed that strain ARSA-111^T is most closely related to F. aquatile JCM 20475^T. DNA-DNA hybridization experiment revealed 70% of genomic relatedness among strain ARSA-111^T, F. aquatile JCM 20475^T and F. cheonanense KACC 14972^T. The isolate had iso-C_15:1, iso-C_15:0, and iso-C_15:0 3-OH as predominant cellular fatty acids and MK-6 as a predominant menaquinone. The genomic DNA G+C content of the isolate was 35.6 mol%. On the basis of these data, strain ARSA-111^T is considered to be a novel species of the genus Flavobacterium, for which the name Flavobacterium aquaticum sp. nov. is proposed. The type strain is strain ARSA-111^T (=KACC 14973^T =KCTC 23185^T = JCM 17070^T).     

原核生物全基因组中16S rRNA基因的识别

【目的】识别原核生物全基因组中的16S rRNA基因。【方法】本文依据基因序列的GC碱基含量、碱基3-周期性和马尔可夫链3个方面的特性,构建了识别原核生物全基因组中16S rRNA基因的三层过滤模型。【结果】经检验,模型的特异性、敏感性和马修斯相关系数分别为99.58%、91.60%和91.49%。【结论】结果表明,本文所提出的方法可以高效、准确地识别出16S rRNA基因。     

Algivirga pacifica gen. nov., sp. nov., a Novel Agar-Degrading Marine Bacterium of the Family Flammeovirgaceae Isolated from Micronesia

An aerobic, Gram-negative, coccoid to short rod-shaped and non-flagellated marine bacterial strain S354^T was isolated from seawater of Micronesia. The strain was capable to degrade agar-forming slight depression into agar plate. Growth occurred at a temperature range of 12–44 °C, a pH range of 5–9, and a salinity range of 1–7 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences suggested that S354^T belongs to the family Flammeovirgaceae. The novel strain was most closely related to Limibacter armeniacum YM 11-185^T with similarity of 92.5 %. The DNA G+C content was 43.8 mol%. The major fatty acids (>10 %) were iso-C_15:0 and C_16:1 ω5c. The predominant isoprenoid quinone was determined to be MK-7. Polar lipid profile of S354^T consisted of phosphatidylethanolamine, unknown polar lipid, and unknown glycolipids. Based on the phenotypic, phylogenetic, biochemical, and physiological tests conducted in this study, S354^T is proposed to represent a type strain of a novel genus and species. The 16S rRNA gene sequence of S354^T is registered in GenBank under the accession number JQ639084. The type of strain Algivirga pacifica gen. nov., sp. nov. is S354^T (=KCCM 90107^T=JCM 18326^T).     

Pacificamonas flava gen. nov., sp. nov., a Novel Member of the Family Sphingomonadaceae Isolated from the Southeastern Pacific

Strain JLT2015^T was isolated from surface seawater of the Southeastern Pacific. The strain was Gram-negative, aerobic, motile by gliding, and rod shaped. The dominant fatty acids were C_18:1ω7c, C_16:0, and C_16:1ω7c. The major respiratory ubiquinone was Q-10, and the predominant polyamine pattern was spermidine. The components of the polar lipid profile were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and sphingoglycolipid. The DNA G+C content was 64.2 %. Phylogenetic analysis based on 16S rRNA gene sequence revealed strain JLT2015^T belonged to belong to the family Sphingomonadaceae, exhibiting 94.7 % 16S rRNA gene sequence similarity with Novosphingobium pentaromativorans. On the basis of the taxonomic data presented, together with phylogenetic and genetic characteristics, strain JLT2015^T is considered to represent a novel genus, for which the name Pacificamonas flava gen. nov., sp. nov. is proposed. The type strain of Pacificamonas flava is JLT2015^T (=LMG27364^T = CGMCC1.12401^T).     

Hymenobacter amundsenii sp. nov. resistant to ultraviolet radiation,isolated from regoliths in Antarctica

A group of thirteen bacterial strains was isolated from rock samples collected in a deglaciated northern part of James Ross Island, Antarctica. The cells were rod-shaped, Gram-stain-negative, non-motile, catalase positive, and produced moderately slimy, ultraviolet light (UVC)-irradiation-resistant and red–pink pigmented colonies on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, extensive biotyping, fatty acid profile, chemotaxonomy analyses, and whole genome sequencing were applied in order to clarify the taxonomic position of these isolates. Phylogenetic analysis based on the 16S rRNA gene indicated that all isolates constituted a coherent group belonging to the genus Hymenobacter. The closest relatives to the representative isolate P5136^T were Hymenobacter psychrophilus BZ33r^T and Hymenobacter rubripertinctus CCM 8852^T, exhibiting 97.53% and 97.47% 16S rRNA pairwise similarity, respectively. Average nucleotide identity calculated from the whole-genome sequencing data supported the finding that P5136^T represents a distinct Hymenobacter species. The major components in fatty acid profiles were Summed Feature 3 (C_16:1 ω7c/C_16:1 ω6c), C_16:1 ω5c, C_15:0 iso and C_15:0 anteiso. The cellular quinone content contained unanimously menaquinone MK-6 and MK-7 (ratio 1:5.1). The predominant polar lipid was phosphatidylethanolamine, and moderate to minor amounts of two unknown polar lipids, two unknown aminolipids, one unknown glycolipid and two unknown glycophospholipids were present. The G + C content of genomic DNAs is 60.31 mol%. Based on all the obtained results, we propose a novel species for which the name Hymenobacter amundsenii sp. nov. is suggested, with the type strain P5136^T (= CCM 8682^T = LMG 29687^T).     

Nonhongiella spirulinensis gen. nov., sp. nov., a bacterium isolated from a cultivation pond of Spirulina platensis in Sanya, China

A Gram-negative, aerobic, motile rod strain, designated Ma-20^T, was isolated from a pool of marine Spirulina platensis cultivation, Sanya, China, and was subjected to a polyphasic taxonomy study. Strain Ma-20^T can grow in the presence of 0.5–11 % (w/v) NaCl, 10–43 °C and pH 6–10, and grew optimally at 30 °C, pH 7.5–9.0 in natural seawater medium. The polar lipids were composed of phosphatidylethanolamine, three unidentified phospholipids and three unidentified polar lipids. The respiratory quinone was ubiquinone 8 (Q-8) and the major fatty acids were C_18:1ω6c/C_18:1ω7c (summed feature 8, 32.84 %), C_16:1ω6c/C_16:1ω7c (summed feature 3, 30.76 %), C_16:0 (13.54 %), C_12:03-OH (4.63 %), and C_12:0 (4.09 %). The DNA G+C content of strain Ma-20^T was 58 mol %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Ma-20^T belonging to Gammaproteobacteria, it shared 88.46–91.55 and 89.21–91.26 % 16S rRNA gene sequence similarity to the type strains in genus Hahella and Marinobacter, respectively. In addition to the large 16S rRNA gene sequence difference, Ma-20^T can also be distinguished from the reference type strains Hahella ganghwensis FR1050^T and Marinobacter hydrocarbonoclasticus sp. 17^T by several phenotypic characteristics and chemotaxonomic properties. On the basis of phenotypic, chemotaxonomic and phylogenetic properties, strain Ma-20^T is suggested to represent a novel species of a new genus in Gammaproteobacteria, for which the name Nonhongiella spirulinensis gen. nov., sp. nov. is proposed. The type strain is Ma-20^T (=KCTC 32221^T=LMG 27470^T).     

Paenibacillus brassicae sp. nov., isolated from cabbage rhizosphere in Beijing, China

A novel Gram-positive, rod-shaped, motile, spore-forming, nitrogen-fixing bacterium, designated strain 112^T, was isolated from cabbage rhizosphere in Beijing, China. The strain was found to grow at 10–40 °C and pH 4–11, with an optimum of 30 °C and pH 7.0, respectively. Phylogenetic analysis based on a fragment of the full-length 16S rRNA gene sequence revealed that strain 112^T is a member of the genus Paenibacillus. High levels of 16S rRNA gene similarities were found between strain 112^T, Paenibacillus sabinae DSM 17841^T (97.82 %) and Paenibacillus forsythiae DSM 17842^T (97.22 %). However, the DNA–DNA hybridization values between strain 112^T and the type strains of these two species were 10.36 and 6.28 %, respectively. The predominant menaquinone was found to be menaquinone 7 (MK-7). The major fatty acids were determined to be anteiso-C_15:0 and C_16:0. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unknown aminophospholipids. The cell wall peptidoglycan was found to contain meso-diaminopimelic acid. The DNA G+C content was determined to be 55.4 mol%. On the basis of its phenotypic characteristics, 16S rRNA gene sequence analysis and the value of DNA–DNA hybridization, strain 112^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus brassicae sp. nov. is proposed. The type strain is 112^T (= ACCC 01125^T = DSM 24983^T).