Chemotaxonomic and phylogenetic analyses of Sphingomonas strains isolated from ears of plants in the family Gramineae and a proposal of Sphingomonas roseoflava sp. nov

Chemotaxonomic and phylogenetic characteristics of Sphingomonas strains isolated from plants of the family Gramineae were investigated. All strains contained the monosaccharide (glucuronic acid) type of glycosphingolipid (GSL-1). Most were found also to contain the oligosaccharide-type glycosphingolipids. Fatty acid and sphingosine profiles of the isolates were identical, although the ratio of the contents varies among the isolates. They all contained ubiquinone Q-10, and the G1C contents were from 66 to 68%. Phylogenetic analysis using 16S rRNA gene base sequences revealed that all the isolates were placed in the phylogenetic group of Sphingomonas paucimobilis in the alpha-4 subclass of Proteobacteria. By DNA-DNA hybridization experiments, the plant isolates were divided into five genotypic groups (groups 1 to 5). The strains of group 5 showed common physiological characteristics and formed pink-yellow colored colonies. Based on these results, Sphingomonas roseoflava sp. nov. was proposed for that homology group.     

一平浪盐矿古老岩盐沉积中可培养细菌的系统发育多样性研究

运用纯培养法和基于16S rRNA基因序列的系统发育分析对云南省一平浪盐矿古老岩盐沉积中可培养细菌的多样性进行了研究。用补充0.5~3.5mol/L NaCl的MBA和ISP2琼脂培养基从卤水、岩盐和盐土样品中分离到38株细菌,用细菌通用引物进行16S rRNA基因扩增和序列测定,用相关软件进行序列相似性搜索、比对和系统发育分析。结果表明,38个分离菌株可分为31个物种,属于4个大的系统发育类群(Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria)、17个科、24个属。多数菌株属于Proteobacteria门(18株,47.3%;Gamma-Proteobacteria,31.5%;Alpha-Proteobacteria,15.8%)和Firmicutes门(13株,34.2%)。这些分离菌株中,至少有3个菌株可能代表3个不同属的3个新物种:Y3、Y15和Y25分别代表Idiomarina属、Salinicoccus属和Saccharospirillum属的新物种;而菌株Y21有可能代表Staphylococcaceae科的一个新属。从以上结果可以看出,一平浪盐矿古老岩盐沉积中存在较为丰富的微生物物种多样性和系统发育多样性,并且潜藏着新的微生物资源。     

Proposal of Sphingomonas suberifaciens (van Bruggen, Jochimsen and Brown 1990) comb. nov., sphingomonas natatoria (Sly 1985) comb. nov., Sphingomonas ursincola (Yurkov et al. 1997) comb. nov., and emendation of the genus Sphingomonas.

Based on the results of a phylogenetic analysis of 16S rRNA and the presence of sphingoglycolipid in cellular lipids of the type strains, transfer of "Rhizomonas" suberifaciens, Blastomonas natatoria and Erythromonas ursincola to the genus Sphingomonas as Sphingomonas suberifaciens (van Bruggen et al 1990) comb. nov., Sphingomonas natatoria (Sly 1985) comb. nov., and Sphingomonas ursincola (Yurkov et al 1997) comb. nov. are herein proposed together with the emendation of genus Sphingomonas. The type strain of S. suberifaciens is van Bruggen Cal=ATCC 49382=NCPPB 3629=IFO 15211=JCM 8521, that of S. natatoria is ATCC 35951 =DSM 3183=NCIMB 12085=JCM10396, and that of S. ursincola is DSM 9006= KR-99.     

Occurrence and phylogenetic diversity of Sphingomonas strains in soils contaminated with polycyclic aromatic hydrocarbons

Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae.Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 10(4) CFU g of soil(-1). The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.     

Genetic characterization of microbial communities living at the surface of building stones

Aims:  The aim of the present study was to reveal the microbial genetic diversity of epilithic biofilms using a DNA-based procedure.
Methods and Results:  A DNA extraction protocol was first selected to obtain PCR-amplifiable metagenomic DNA from a limestone biofilm. Extracted DNA was used to amplify either 16S rRNA genes or ITS regions from prokaryotic and eukaryotic genomes, respectively. Amplified DNAs were subsequently cloned, amplified by colony PCR and screened by restriction analysis [restriction analyses of amplified ribosomal DNA (ARDRA)] for DNA sequencing. Phylogenetic analysis using 16S rDNA sequences showed that predominating bacteria were Alphaproteobacteria belonging to the genera Sphingomonas , Erythrobacter , Porphyrobacter , Rhodopila and Jannashia ; Cyanobacteria and Actinobacteria were also identified. Analysis of ITS rDNA sequences revealed the presence of algae of the Chlorophyceae family and fungi related either to Rhinocladiella or to a melanized ascomycete. Statistical analysis showed that the specific richness evidenced was representative of the original sampled biofilm.
Conclusions:  The molecular methodology developed here constitutes a valuable tool to investigate the genetic diversity of microbial biofilms from building stone.
Significance and Impact of the Study:  The easy-to-run molecular method described here has practical importance to establish microbiological diagnosis and to define strategies for protection and restoration of stone surfaces.     

Leifsonia gen. nov., a genus for 2,4-diaminobutyric acid-containing actinomycetes to accommodate "Corynebacterium aquaticum" Leifson 1962 and Clavibacter xyli subsp. cynodontis Davis et al. 1984

"Corynebacterium aquaticum" was first proposed by Leifson in 1962 but not included in the approved lists of bacterial names in 1980. This species has been left from reclassification of the genus Corynebacterium because of the unusual chemotaxonomic characteristics such as 2,4-diaminobutyric acid (DAB) in the peptidoglycan and menaquinones of MK-10 and MK-11. A close relationship of "C. aquaticum" to the genera Agromyces and Rathayibacter has been pointed out from the viewpoint of chemotaxonomic profiles and phylogeny based on the 16S rDNA sequences. An analysis of DAB isomers of the peptidoglycan distinguished "C. aquaticum" clearly from these genera by possessing both L-DAB and D-DAB. We also found that the type strain of Clavibacter xyli subsp. cynodontis and two strains of amine-decomposing bacteria showed the similar chemotaxonomic features and formed a cluster with "C. aquaticum" in the phylogenetic tree based on 16S rDNA sequences in the family Microbacteriaceae. Considering these results, we propose a new genus Leifsonia to accommodate the four strains. The four species, Leifsonia aquatica sp. nov., nom. rev., comb. nov. (type species, type strain=JCM 1368), Leifsonia shinshuensis sp. nov. (type strain=DB102=JCM 10591), Leifsonia naganoensis sp. nov. (type strain=DB103=JCM 10592), and Leifsonia cynodontis comb. nov. (type strain=JCM 9733=ICMP 8790), were proposed here for the strains.     

Development of PCR primers specific for the amplification and direct sequencing of gyrB genes from microbacteria, order Actinomycetales

PCR primer sets were developed for the specific amplification and sequence analyses encoding the gyrase subunit B (gyrB) of members of the family Microbacteriaceae, class Actinobacteria. The family contains species highly related by 16S rRNA gene sequence analyses. In order to test if the gene sequence analysis of gyrB is appropriate to discriminate between closely related species, we evaluate the 16S rRNA gene phylogeny of its members. As the published universal primer set for gyrB failed to amplify the responding gene of the majority of the 80 type strains of the family, three new primer sets were identified that generated fragments with a composite sequence length of about 900 nt. However, the amplification of all three fragments was successful only in 25% of the 80 type strains. In this study, the substitution frequencies in genes encoding gyrase and 16S rDNA were compared for 10 strains of nine genera. The frequency of gyrB nucleotide substitution is significantly higher than that of the 16S rDNA, and no linear correlation exists between the similarities of both molecules among members of the Microbacteriaceae. The phylogenetic analyses using the gyrB sequences provide higher resolution than using 16S rDNA sequences and seem able to discriminate between closely related species.     

Characterization of Porphyrobacter sanguineus sp. nov., an aerobic bacteriochlorophyll-containing bacterium capable of degrading biphenyl and dibenzofuran

Three strains of " Agrobacterium sanguineum", an aerobic marine bacterial species described previously, were re-characterized from phylogenetic and taxonomic viewpoints. 16S rDNA sequence comparisons showed that the " A. sanguineum" strains belong to the alpha-4 subgroup of alpha-Proteobacteria, with members of the genera Erythromicrobium and Porphyrobacter as their closest relatives. DNA-DNA hybridization studies indicated that the " A. sanguineum" strains were distinguishable from any previously known species of these genera. Bacteriochlorophyll a, monosaccharide-type glycosphingolipids, 2-OH fatty acids of C14:0, C15:0, C16:0, and C16:1, and ubiquinone-10 were detected in the " A. sanguineum" strains. The G+C of the DNA was 63.8-64.0 mol%. Two of the " A. sanguineum" strains, IAM 12620 (=ATCC 25659) and ATCC 25661, were able to grow with biphenyl and dibenzofuran as sole carbon source in the presence of 0.05% yeast extract. The medium in these cultures turned yellowish-orange at the exponential phase of growth due to the release of soluble chromogenic metabolites. The remaining " A. sanguineum" strain, ATCC 25660, and all test strains of Erythromicrobium and Porphyrobacter neither grew nor produced yellow-orange pigment with biphenyl or dibenzofuran. In PCR experiments, bphA1 gene, coding for the large subunit protein of biphenyl dioxygenase, was detected in " A. sanguineum" IAM 12620 and ATCC 25661. Based on these results, we propose classifying " A. sanguineum" IAM 12620 and ATCC 25661 as a new species of the genus Porphyrobacter with the name Porphyrobacter sanguineus sp. nov.     

Evolutionary relationships among Magnetospirillum strains inferred from phylogenetic analysis of 16S rDNA sequences.

We have investigated the evolutionary relationships between two facultatively anaerobic Magnetospirillum strains (AMB-1 and MGT-1) and fastidious, obligately microaerophilic species, such as Magnetospirillum magnetotacticum, using a molecular phylogenetic approach. Genomic DNA from strains MGT-1 and AMB-1 was used as a template for amplification of the genes coding for 16S rRNA (16S rDNA) by the polymerase chain reaction. Amplified DNA fragments were sequenced (1,424 bp) and compared with sequences for M. magnetotacticum MS-1 and Magnetospirillum gryphiswaldense MSR-1. Phylogenetic analysis of the aligned 16S rDNA sequences indicated that the two new magnetic spirilla, AMB-1 and MGT-1, lie within the alpha subdivision (alpha-1) of the eubacterial group Proteobacteria and are closely related to Rhodospirillum fulvum and to several endosymbiotic bacteria. Strains AMB-1, MGT-1, and MS-1 formed a cluster, termed group I, in which they were more closely related to each other than to group II, which contained M. gryphiswaldense MSR-1. Group I strains were also physiologically distinct from strain MSR-1. Sequence alignment studies allowed elucidation of genus-specific regions of the 16S rDNA, and oligonucleotide primers complementary to two of these regions were used to develop a specific polymerase chain reaction assay for detection of magnetic spirilla in natural samples.     

The phylogenetic relationships of cyanobacteria inferred from 16S rRNA,gyrB, rpoC1 and rpoD1 gene sequences

Phylogenetic analysis of cyanobacteria was carried out using the small subunit rRNA (16S rRNA), DNA gyrase subunit B (gyrB), DNA-dependent RNA polymerase gamma subunit (rpoC1) and a principal sigma factor of E. coli sigma(70) type for DNA-dependent RNA polymerase (rpoD1) gene sequences of 24 strains which contained 5 subgroups of cyanobacteria-3 strains of the Chroococcales, 5 strains of the Pluerocapsales, 7 strains of the Oscillatoriales, 7 strains of the Nostocales and 2 strains of the Stigonematales. Degenerated PCR primers of gyrB, rpoC1 and rpoD1 genes were designed using consensus amino acid sequences registered in GenBank. The phylogenetic positions of cyanobacteria were resolved through phylogenetic analysis based on 16S rDNA, gyrB, rpoC1 and rpoD1 gene sequences. Phylogenies of gyrB, rpoC1 and rpoD1 support 16S rRNA-based classification of cyanobacteria. Interestingly, phylogenies from amino acid sequences deduced from gyrB and combined amino acid sequences deduced from rpoC1 and rpoD1 genes strongly support that of 16S rRNA, but the branching pattens of the trees based on 16S rDNA, GyrB, rpoC1, rpoD1 and combined amino acid sequences deduced from rpoC1 and rpoD1 were not congruent. In this study, we showed the correlation among phylogenetic relationships of 16S rDNA, gyrB, rpoC1 and rpoD1 genes. The phylogenetic trees based on the sequences of 16S rDNA, GyrB, rpoC1, rpoD1 and the combined amino acid sequences deduced from rpoC1 and rpoD1 showed that the lateral gene transfer of rRNA might be suspected for Synechocystis sp. PCC 6803.     

Sphingomonas astaxanthinifaciens sp. nov., a novel astaxanthin-producing bacterium of the family Sphingomonadaceae isolated from Misasa, Tottori, Japan

A red-pigmented, Gram-negative, motile, strictly aerobic, mesophilic, oval- or short rod-shaped bacterium (TDMA-17(T)) was isolated from fresh water collected at Misasa, a radioactive site in Japan. TDMA-17(T) was slightly tolerant against gamma-ray irradiation, and effectively produced carotenoids (2.8 mg g(-1) dry cells) including, astaxanthin and astaxanthin isomers. Phylogenetic analysis based on 16S rRNA gene sequences placed TDMA-17(T) in a distinct lineage in the family Sphingomonadaceae, and the highest degree of sequence similarity determined were to Sphingomonas aerolata NW12(T) (94.5%), Sphingomonas aurantiaca MA101b(T) (94.0%), Sphingomonas melonis DAPP-PG 224(T) (94.0%), Sphingomonas asaccharolytica IFO 15499(T) (93.9%) and Sphingomonas abaci C42(T) (93.9%). The major fatty acids were C(17 : 1)omega6c (33.0%) and C(18 : 1)omega7c (20.8%). The DNA G+C content was 67.7 mol%. The presence of Q-10 as the main ubiquinone, the presence of Sphingomonadaceae-specific sphingoglycolipid in the polar lipid profiles, the presence of 2-hydroxy fatty acids and the absence of 3-hydroxy fatty acids supported the identification of this strain as a member of the genus Sphingomonas sensu stricto. Phylogenetic distinctiveness and unique phenotypic characteristics differentiated strain TDMA-17(T) from the closely related Sphingomonas species. The results of polyphasic taxonomic analyses suggested that TDMA-17(T) represents a novel Sphingomonas species, for which the name Sphingomonas astaxanthinifaciens sp. nov. is proposed. The type strain is TDMA-17(T) (=NBRC 102146=CCUG 53608).     

两个根瘤菌新群的系统发育学分析

在数值分类、ＳＤＳＰＡＧＥ 全细胞蛋白分析、ＤＮＡＤＮＡ 杂交、１６ＳｒＤＮＡＰＣＲＲＦＬＰ 的基础上,测定了两个分离自干旱地区苜蓿、草木樨根瘤菌新群１ 、２ 的中心株ＸＪ９６０６０ 、ＸＪ９６４０８ 的１６ＳｒＤＮＡ 全序列,并进一步将中心株和３１ 株已知菌、３ 株分自黄土高原的根瘤菌进行了系统发育学分析。结果表明,供试菌株在系统发育树中基本分成Ｓｉｎｏｒｈｉｚｏｂｉｕｍ 、Ｍｅｓｏｒｈｉｚｏｂｉｕｍ 、ＡｇｒｏｂａｃｔｅｒｉｕｍＲｈｉｚｏｂｉｕｍ 、Ｒｈｉｚｏｂｉｕ ｍ 、Ｂｒａｄｙｒｈｉｚｏｂｉｕｍ 、Ａｚｏｒｈｉｚｏｂｉｕｍ 六个分枝。群１ ,２ 落入Ｓｉｎｏｒｈｉｚｏｂｉｕ ｍ 分枝。     

Phylogenetic analysis of the fecal flora of the wild pygmy loris

The bacterial diversity in fecal samples from the wild pygmy loris was examined with a 16S rDNA clone library and restriction fragment length polymorphism analysis. The clones were classified as Firmicutes (43.1%), Proteobacteria (34.5%), Actinobacteria (5.2%), and Bacteroidetes (17.2%). The 58 different kinds of 16S rDNA sequences were classified into 16 genera and 20 uncultured bacteria. According to phylogenetic analysis, the major genera within the Proteobacteria was Pseudomonas, comprising 13.79% of the analyzed clone sequences. Many of the isolated rDNA sequences did not correspond to known microorganisms, but had high homology to uncultured clones found in human feces. Am. J. Primatol. 72:699–706, 2010. © 2010 Wiley‐Liss, Inc.     

16S rDNA phylogeny and distribution of lin genes in novel hexachlorocyclohexane-degrading Sphingomonas strains

Hexachlorocyclohexane (HCH) is a highly recalcitrant pesticide that persists in soils. Three novel HCH-degrading strains (DS2, DS2-2 and DS3-1) were isolated after enrichment from HCH-contaminated soil from Germany. These strains efficiently degraded the alpha-, gamma- and delta-isomers of HCH, while strain DS3-1 also degraded beta-HCH. Based on 16S rDNA analysis, strain DS3-1 was closely related to Sphingomonas taejonensis, while strains DS2 and DS2-2 were closely related to Sphingomonas flava and seven HCH-degrading strains recently isolated from HCH-contaminated Spanish soil. Hence, geographic origin of the strains was not reflected in their phylogenetic affiliation. Subsequently, lin genes involved in HCH degradation, virtually identical to those from Sphingomonas paucimobilis strains UT26 from Japan and B90A from India, were identified in strains DS3-1, DS2, DS2-2 and five of the strains from Spain. The conserved lin gene sequences and structural organization, as well as the close association with IS6100, suggest a shared lin gene origin and recent horizontal gene transfer among phylogenetically diverged Sphingomonas strains in remote geographic locations. The loss of the ability to degrade gamma-HCH was associated with the deletion of the linA gene, probably due to recombination involving IS6100 elements, of which several copies are located in the lin cluster region.     

Phylogenetic analysis of the genera Pseudonocardia and Actinobispora based on 16S ribosomal DNA sequences

The 16S rDNA sequences of 11 strains, nine type strains of validated Pseudonocardia species and Actinobispora yunnanensis, and two strains of unnamed Pseudonocardia species, were determined and compared with those of representatives of the family Pseudonocardiaceae. The phylogenetic analysis indicated that all of the validated species of the genera Pseudonocardia and Actinobispora consistently formed a monophyletic unit and separated well from the other genera of the family Pseudonocardiaceae. One unnamed Pseudonocardia strain was related to members of the genus Pseudonocardia, whereas the other unnamed Pseudonocardia strain formed a distinct clade within the radiation of the genus Amycolatopsis.     

Teichococcus ludipueritiae gen. nov. sp. nov., and Muricoccus roseus gen. nov. sp. nov. representing two new genera of the alpha-1 subclass of the Proteobacteria

Two bacterial isolates (170/96T and 173/96T) were recovered from the indoor building materials of a children's day care center. Phylogenetic analyses using the 16S rRNA gene sequences of both isolates indicated they both represent new lineages in the alpha-1-subclass of the Proteobacteria, with the highest sequence similarities of 93.7% and 93.6%, respectively to the type strain of Paracraurococcus ruber. When directly compared both isolates showed a 93.4% sequence similarity of their 16S rRNAs. The major respiratory quinone in both strains was a ubiquinone with 10 isoprenoid units and the major whole cell fatty acid of both strains was 18:1 omega7c. Both isolates also contained 18:1 2-OH and other fatty acids typical for members of the alpha-1 subclass of the Proteobacteria. Both strains were heterotrophic and strictly aerobic and formed slightly red-colored colonies on tryptone soy agar. Bacteriochlorophyll a could not be detected by direct spectrophotometric analyses of aerobically grown cells. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strains 170/96T and 173/96T represent two new genera and new species of the alpha-1 subclass of the Proteobacteria for which we propose the names Teichococcus ludipueritiae gen. nov. sp. nov., and Muricoccus roseus gen. nov. sp. nov., respectively.     

Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus.

The phylogenetic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy which does not rely on cultivation of the resident microorganisms. Small-subunit rRNA genes (16S rDNAs) were directly amplified from the mixed-population DNA of the termite gut by the PCR and were clonally isolated. Analysis of partial 16S rDNA sequences showed the existence of well-characterized genera as well as the presence of bacterial species for which no 16S rDNA sequence data are available. Of 55 clones sequenced, 45 were phylogenetically affiliated with four of the major groups of the domain Bacteria: the Proteobacteria, the spirochete group, the Bacteroides group, and the low-G+C-content gram-positive bacteria. Within the Proteobacteria, the 16S rDNA clones showed a close relationship to those of cultivated species of enteric bacteria and sulfate-reducing bacteria, while the 16S rDNA clones in the remaining three groups showed only distant relationships to those of known organisms in these groups. Of the remaining 10 clones, among which 8 clones formed a cluster, there was only very low sequence similarity to known 16S rRNA sequences. None of these clones were affiliated with any of the major groups within the domain Bacteria. The 16S rDNA gene sequence data show that the majority of the intestinal microflora of R. speratus consists of new, uncultured species previously unknown to microbiologists.     

秦岭地区野生细鳞鲑肠道微生物多样性及产酶活性分析

为探究秦岭地区野生细鳞鲑(Brachymystax lenok)肠道细菌组成多样性,筛选出产胞外酶菌株,利用传统分离培养并分子鉴定的方法和基于16S r RNA基因克隆的现代分子生物技术相结合测定秦岭野生细鳞鲑肠道细菌菌群多样性并构建系统发育树,利用淀粉酶、蛋白酶、纤维素酶及脂肪酶4种胞外酶筛选培养基筛选出产上述酶的细菌。细菌传统分离培养并分子鉴定法从细鳞鲑肠道获得18个属的细菌类群,分别归属于变形菌门、拟杆菌门和厚壁菌门,其中,气单胞菌属(Aeromonas)为优势菌群。基于16S r RNA基因克隆的现代分子方法获得22个属的细菌类群,分别归属于变形菌门、拟杆菌门、厚壁菌门和放线菌门,其中,鞘氨醇杆菌属(Sphingomonas)为优势菌群。4种胞外酶筛选获得53株细菌产胞外酶,其中21株可在低温(10℃)环境下产胞外酶。结果表明,传统分离培养法与基于16S r RNA基因克隆的现代分子生物技术相结合能够更有效全面地分析细鳞鲑鱼肠道微生物的多样性,并且细鳞鲑肠道微生物具有一定的产酶活性。