Gene Sequence Phylogenies of the Family <Emphasis Type="Italic">Microbacteriaceae</Emphasis>

The type strains of 32 species of 13 genera of the family Microbacteriaceae were analysed with respect to gene-coding phylogeny for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA), and polyphosphate kinase (ppk). The resulting gene trees were compared with the 16S rRNA gene phylogeny of the same strains. The topology of neighbour-joining and maximum parsimony phylogenetic trees, based on nucleic-acid sequences and protein sequences of housekeeping genes, differed from one another, and no gene tree was identical to that of the 16S rRNA gene tree. Most genera analysed containing >1 strain formed phylogenetically coherent taxa. The three pathovars of Curtobacterium flaccumfaciens clustered together to the exclusion of the type strains of other Curtobacterium species in all DNA - and protein-based analyses. In no tree did the distribution of a major taxonomic marker, i.e., diaminobutyric acid versus lysine and/or ornithine in the peptidoglycan, or acyl type of peptidoglycan, correlate with the phylogenetic position of the organisms. The changing phylogenetic position of Agrococcus jenensis was unexpected: This strain defined individual lineages in the trees based on 16S rRNA and gyrB and showed identity with Microbacterium saperdae in the other three gene trees.     

The gyrB gene is a useful phylogenetic marker for exploring the diversity of Flavobacterium strains isolated from terrestrial and aquatic habitats in Antarctica

Within the phylum Bacteroidetes, the gyrB gene, encoding for the B subunit of the DNA gyrase, has been used as a phylogenetic marker for several genera closely related to Flavobacterium. The phylogenies of the complete 16S rRNA gene and the gyrB gene were compared for 33 Antarctic Flavobacterium isolates and 23 type strains from closely related Flavobacterium species. gyrB gene sequences provided a higher discriminatory power to distinguish between different Flavobacterium groups than 16S rRNA gene sequences. The gyrB gene is therefore a promising molecular marker for elucidating the phylogenetic relationships among Flavobacterium species and should be evaluated for all the other type strains of described Flavobacterium species. Combining the phylogeny of both genes, the new Antarctic Flavobacterium strains constitute 15 Flavobacterium groups, including at least 13 potentially new species together with one group of isolates probably belonging to the species Flavobacterium micromati and one group close to Flavobacterium gelidilacus.     

Evaluation of arabinofuranosidase and xylanase activities of Geobacillus spp. isolated from some hot springs in Turkey

Some hot springs located in the west of Turkey were investigated with respect to the presence of thermophilic microorganisms. Based on phenotyping characteristics and 16S rRNA gene sequence analysis, 16 of the isolates belonged to the genus Geobacillus and grew optimally at about 60 degrees C on nutrient agar. 16S rRNA gene sequence analysis showed that these isolates resembled Geobacillus species by > or = 97%, but SDS-PAGE profiles of these 16 isolates differ from some of the other species of the genus Geobacillus. However, it is also known that analysis of 16S rRNA gene sequences may be insufficient to distinguish between some species. It is proposed that recN sequence comparisons could accurately measure genome similarities for the Geobacillus genus. Based on recN sequence analysis, isolates 11, IT3, and 12 are strains of G stearothermophilus; isolate 14.3 is a strain of G thermodenitrificans; isolates 9.1, IT4.1, and 4.5 are uncertain and it is required to make further analysis. The presence of xylanase and arabinofuranosidase activities, and their optimum temperature and pH were also investigated. These results showed that 7 of the strains have both xylanase and arabinofuranosidase activities, 4 of them has only xylanase, and the remaning 5 strains have neither of these activities. The isolates 9.1, 7.1, and 3.3 have the highest temperature optima (80 degrees C), and 7.2, 9.1, AO4, 9.2, and AO17 have the highest pH optima (pH 8) of xylanase. Isolates 7.2, AO4, AC15, and 12 have optimum arabinofuranosidase activities at 75 degrees C, and only isolate AC 15 has the lowest pH of 5.5.     

Genetic markers for analysing symbiotic relationships and lateral gene transfer in Neotropical bradyrhizobia

Assays with seven sets of lineage-specific polymerase chain reaction (PCR) primers in the ribosomal RNA region were performed on 96 isolates of the Bradyrhizobium sp. nodule bacteria from Barro Colorado Island, Panama. The isolates were derived from 10 legume host species in six genera (Centrosema, Desmodium, Dioclea, Inga, Machaerium and Vigna). The PCR assays differentiated 13 composite genotypes, and sequencing of a 5' 23S rRNA region indicated that all but one had a unique sequence. The most common genotype (seen in 44% of the isolates) was associated with all six legume host genera, and had a marker profile and 5' 23S rRNA sequence identical to a Bradyrhizobium lineage associated with several other legume genera in Panama and Costa Rica. Another 46% of the isolates had genotypes found to be associated with two to three legume genera. Bradyrhizobium strains with low host specificity thus appear to be prevalent in this tropical forest. Based on 16S rRNA and 5' 23S rRNA markers, most of the isolates had clear affinities to either B. japonicum or B. elkanii. However, one strain (Cp5-3) with a B. elkanii-type 16S rRNA marker had a 5' 23S rRNA region resembling B. japonicum. A partition homogeneity test indicated that relationships of strain Cp5-3 were significantly discordant for 16S rRNA vs. 23S rRNA sequences, and a runs test detected significant mosaic structure across the rRNA region. Lateral gene transfer events have therefore played a role in the evolution of symbiotic bacteria in this environment.     

Diversity of culturable actinobacteria isolated from marine sponge <Emphasis Type="Italic">Haliclona</Emphasis> sp.

This study describes actinobacteria isolated from the marine sponge Haliclona sp. collected in shallow water of the South China Sea. A total of 54 actinobacteria were isolated using media selective for actinobacteria. Species diversity and natural product diversity of isolates from marine sponge Haliclona sp. were analysed. Twenty-four isolates were selected on the basis of their morphology on different media and assigned to the phylum Actinobacteria by a combination of 16S rRNA gene based restriction enzymes digestion and 16S rRNA gene sequence analysis. The 16S rRNA genes of 24 isolates were digested by restriction enzymes TaqI and MspI and assigned to different groups according to their restriction enzyme pattern. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Streptomyces, Nocardiopsis, Micromonospora and Verrucosispora; one other isolate was recovered that does not belong to known genera based on its unique 16S rRNA gene sequence. To our knowledge, this is the first report of a bacterium classified as Verrucosispora sp. that has been isolated from a marine sponge. The majority of the strains tested belong to the genus Streptomyces and three isolates may be new species. All of the 24 isolates were screened for genes encoding polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). PKS and NRPS sequences were detected in more than half of the isolates and the different "PKS-I-PKS-II-NRPS" combinations in different isolates belonging to the same species are indicators of their potential natural product diversity and divergent genetic evolution.     

基于16S rRNA和HSP60基因序列分析粘细菌孢囊杆菌亚目形态分类的种属之间的亲缘关系

[目的]利用16S rRNA和HSP60基因分子标记分析鉴定形态分类特征不稳定的粘细菌种属.[方法]利用粘细菌的传统分离纯化方法从土壤中分离粘细菌,根据菌株的形态特征进行分类,PCR方法扩增菌株的16S rRNA和HSP60基因序列并进行系统发育关系分析.[结果]根据形态特征,分离得到的15株粘细菌菌株归入孢囊杆菌亚目(Cystobacterineae)的2个科3个属.其中11株粘细菌具有典型的所在种属的子实体结构,而菌株0085-4、0121-3、NM03和Myx9736的子实体结构发生了不同程度退化.15株粘细菌的16S rRNA基因序列的相似性在95.4%到99.5%之间.而HSP60基因序列差异较大.[结论]在属水平上,粘细菌形态分类特征和16S rRNA基因系统进化关系具有很好的一致性;在揭示粘细菌种间系统发育关系中,HSP60基因序列更为适用.     

Phylogenetic diversity of Flavobacteria isolated from the North Sea on solid media

Flavobacteria are abundant in the North Sea, an epeiric sea on the continental shelf of Europe. However, this abundance has so far not been reflected by the number of strains in culture collections. In this study, Flavobacteria were isolated from pelagic and benthic samples, such as seawater, phytoplankton, sediment and its porewater, and from surfaces of animals and seaweeds on agar plates with a variety of carbon sources. Dilution cultivation with a new medium, incubation at low temperatures and with long incubation times, and colony screening by a Flavobacteria-Cytophagia-specific PCR detecting 16S rRNA gene sequences led to a collection of phylogenetically diverse strains. Two strains affiliated with Flammeovirgaceae and seven strains affiliated with Cyclobacteriaceae, whereas within the Flavobacteriaceae 20 isolated strains presumably represented seven novel candidate genera and 355 strains affiliated with 26 of 80 validly described marine Flavobacteriaceae genera, based on a genus boundary of 95.0% 16S rRNA gene sequence identity. The majority of strains (276) affiliated with 37 known species in 16 genera (based on a boundary of 98.7% 16S rRNA gene sequence identity), whereas 79 strains likely represented 42 novel species in 22 established Flavobacteriaceae genera. Pigmentation, iridescence, gliding motility, agar lysis, and flexirubin as a chemical marker supported the taxonomy at the species level. This study demonstrated the culturability on solid medium of phylogenetically diverse Flavobacteria originating from the North Sea.     

How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity.

16S rRNA (genes coding for rRNA) sequence comparisons were conducted with the following three psychrophilic strains: Bacillus globisporus W25T (T = type strain) and Bacillus psychrophilus W16AT, and W5. These strains exhibited more than 99.5% sequence identity and within experimental uncertainty could be regarded as identical. Their close taxonomic relationship was further documented by phenotypic similarities. In contrast, previously published DNA-DNA hybridization results have convincingly established that these strains do not belong to the same species if current standards are used. These results emphasize the important point that effective identity of 16S rRNA sequences is not necessarily a sufficient criterion to guarantee species identity. Thus, although 16S rRNA sequences can be used routinely to distinguish and establish relationships between genera and well-resolved species, very recently diverged species may not be recognizable.     

The phylogenetic significance of peptidoglycan types: Molecular analysis of the genera Microbacterium and Aureobacterium based upon sequence comparison of gyrB, rpoB, recA and ppk and 16SrRNA genes

The type strains of 27 species of the genus Microbacterium, family Microbacteriaceae, were analyzed with respect to the phylogeny of the housekeeping genes coding for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA) and polyphosphate kinase (ppk). The resulting gene trees were compared to the 16S rRNA gene phylogeny of the same species. The topology of neighbour-joining and maximum parsimony phylogenetic trees based upon nucleic acid sequences and protein sequences of housekeeping genes differed among each other and no gene tree was identical to that of the 16S rRNA gene tree. Only some species showed consistent clustering by all genes analyzed, but the majority of species branched with different neighbours in most gene trees. The failure to phylogenetically cluster type strains into two groups based upon differences in the amino acid composition of peptidoglycan on the basis of 16S rRNA gene sequence similarity, once leading to the union of the genera Microbacterium and Aureobacterium, was also seen in the analysis of recA, rpoB and gyrB gene and protein phylogenies. Analysis of the pkk gene and protein as well as of a concatenate tree, combining sequences of all five genes (total of 3.700 nucleotides), sees members of the former genus Aureobacterium and other type strains with lysine as diagnostic diamino acid to form a coherent cluster that branches within the radiation of Microbacterium species with ornithine in the peptidoglycan.     

Outer membrane efflux protein (OMEP) is a suitable molecular marker for resolving the phylogeny and taxonomic status of closely related cyanobacteria

Taxonomy of Cyanobacteria, the oldest phototrophic prokaryotes, is problematic for many years due to their simple morphology, high variability and adaptability to diverse ecological niches. After introduction of the polyphasic approach, which is based on the combination of several criteria (molecular sequencing, morphological and ecological), the whole classification system of these organisms is subject to reorganization. The aim of this study was to evaluate whether the outer membrane efflux protein (OMEP) sequences can be used as a molecular marker for resolving the phylogeny and taxonomic status of closely related cyanobacteria. We have performed phylogenetic analyses based on the amino acid sequences of the OMEP and the DNA sequences of the 16S rRNA gene from 86 cyanobacterial species/strains with completely sequenced genomes. Phylogenetic trees based on the OMEP showed that most of the cyanobacterial species/strains belonging to different genera are clustered in separate clades supported by high bootstrap values. Comparing the OMEP trees with the 16S rDNA tree clearly showed that the OMEP is more suitable marker in resolving phylogenetic relationships within Cyanobacteria at generic and species level.     

Neoscardovia arbecensis gen. nov., sp. nov., isolated from porcine slurries

Three Gram-positive, anaerobic, pleomorphic strains (PG10(T), PG18 and PG22), were selected among five strains isolated from pig slurries while searching for host specific bifidobacteria to track the source of fecal pollution in water. Analysis of the 16S rRNA gene sequence showed a maximum identity of 94% to various species of the family Bifidobacteriaceae. However, phylogenetic analyses of 16S rRNA and HSP60 gene sequences revealed a closer relationship of these strains to members of the recently described Aeriscardovia, Parascardovia and Scardovia genera, than to other Bifidobacterium species. The names Neoscardovia gen. nov. and Neoscardovia arbecensis sp. nov. are proposed for a new genus and for the first species belonging to this genus, respectively, and for which PG10(T) (CECT 8111(T), DSM 25737(T)) was designated as the type strain. This new species should be placed in the Bifidobacteriaceae family within the class Actinobacteria, with Aeriscardovia aeriphila being the closest relative. The prevailing cellular fatty acids were C(16:0) and C(18:1)ω9c, and the major polar lipids consisted of a variety of glycolipids, diphosphatidyl glycerol, two unidentified phospholipids, and phosphatidyl glycerol. The peptidoglycan structure was A1γmeso-Dpm-direct. The GenBank accession numbers for the 16S rRNA gene and HSP60 gene sequences of strains PG10(T), PG18 and PG22 are JF519691, JF519693, JQ767128 and JQ767130, JQ767131, JQ767133, respectively.     

南海深海沉积物放线菌多样性分析

【目的】免培养和纯培养相结合分析南海深海沉积物放线菌多样性。【方法】免培养方法通过提取沉积物宏基因组DNA,利用放线菌门特异性引物扩增放线菌16S r RNA基因序列,构建放线菌16S r RNA基因克隆文库,文库经RFLP(Restriction fragment length polymorphism)分析后挑选代表序列测序并进行多样性指数分析和系统发育分析。可培养方法利用8种培养基进行菌株分离,对排重后的菌株进行16S r RNA基因序列多样性分析。【结果】构建的两个深海位点的16S r RNA基因克隆文库在放线菌门的放线菌纲(Actinobacteria)、酸微菌纲(Acidimicrobiia)、腈基降解菌纲(Nitriliruptoria)和嗜热油菌纲(Thermoleophilia)4个纲中均有分布;两个位点中的种群结构有差异,N40-4位点的优势种群是放线菌纲的链霉菌目(Streptomycetales);N63-4位点的优势种群是腈基降解菌纲的腈基降解菌目(Nitriliruptorales)。8种培养基共分离出41株放线菌,根据形态特征排重后得到的19株菌分布于10个不同的属,12个不同的种,其中稀有放线菌属比例较高,菌株OAct400为潜在的微杆菌属(Microbacterium)新种。【结论】南海深海沉积物蕴含着丰富的放线菌物种资源及大量未知种群,具有进一步研究的价值。     

干酪乳杆菌的近缘种及亚种部分看家基因的系统发育分析

【背景】16S rRNA基因序列分析已广泛应用于细菌的分类鉴定,但是存在一定局限性,而使用看家基因作为分子标记在近缘种及亚种间的系统发育分析中具有其独特的优势。【目的】研究16S rRNA、uvr C (核酸外切酶ABC,C亚基)和mur E (UDP-N-乙酰胞壁酰三肽合酶)基因序列对干酪乳杆菌的近缘种及亚种的区分能力。【方法】采用分离自传统发酵乳中的6株干酪乳杆菌为研究对象,选取uvr C和mur E基因片段,通过PCR扩增、测序,结合已公布的干酪乳杆菌的近缘种或亚种的相应序列计算遗传距离、构建系统发育树,并与16S rRNA基因序列分析技术进行比较。【结果】研究发现Lactobacilluscasei及相近种间的uvr C、mur E和联合基因(uvr C-mur E)构建的系统发育树拓扑结构与16S rRNA基因结果基本一致,区别在于相似性的不同,其分别为79.00%-99.16%、89.08%-99.20%、76.56%-99.69%和99.58%-100%。基于16S rRNA基因不能区分干酪乳杆菌的近缘种及亚种,而看家基因uvr C和mur E基因序列能够很好地区分干酪乳杆菌的近缘种及亚种,并且将uvr C和mur E基因串联使用后,试验菌株与参考菌株的分类关系更加清晰。【结论】联合基因(uvr C-mur E)可作为16SrRNA基因的辅助工具用于干酪乳杆菌的近缘种及亚种的快速准确鉴定。     

cpcHID操纵子序列用于钝顶节旋藻品系分类与鉴定的研究

克隆并测定7株钝顶节旋藻品系的cpcHID操纵子序列,以及16SrRNA和16S-23SrRNA转录单元内间隔区(ITS)序列,进一步通过生物信息学和分子系统学等研究发现:(1)7株品系的cpcHID序列,以及16SrRNA和ITS序列具有很高的相似性。(2)基于7株品系cpcHID序列的GC含量绝对偏差平均值、碱基变异率和遗传距离系数普遍比基于16SrRNA和ITS序列的大。(3)基于cpcHID序列的分类结果与基于16SrRNA和ITS序列的十分相近。因此,cpcHID可作为节旋藻等蓝细菌分类与鉴定的一种新的分子标记,特别是以其丰富的信息量而在品系水平的分类鉴定中占有优势。     

青海可可西里嗜碱芽胞杆菌资源调查

【目的】了解可可西里嗜碱芽胞杆菌资源多样性及产酶多样性,为芽胞杆菌功能资源挖掘和菌剂开发提供基础。【方法】采用Horikoshi I培养基,通过可培养法分离青海可可西里土壤中的嗜碱芽胞杆菌,利用16S r RNA基因序列初步鉴定分离获得的芽胞杆菌。采用透明圈法分析分离菌株的产蛋白酶、纤维素酶及木聚糖酶活性。【结果】从青海可可西里土壤中共分离获得66株嗜碱芽胞杆菌,根据16S r RNA基因序列相似性分析,发现它们隶属于6个属22个种,分别为芽胞杆菌属(Bacillus)、纤细芽胞杆菌属(Gracilibacillus)、喜盐芽胞杆菌属(Halobacillus)、咸海鲜芽胞杆菌属(Jeotgalibacillus)、类芽胞杆菌属(Paenibacillus)和嗜冷芽胞杆菌属(Psychrobacillus),其中以芽胞杆菌属(Bacillus)为优势属。2株嗜碱芽胞杆菌与它们最近匹配模式菌株的16S r RNA基因序列相似性为97.00%和98.65%,为潜在新种。三种酶活检测结果表明产酶菌株约占总分离菌株的95.00%,其中55株具有产蛋白酶活性,27株具有产纤维素酶活性,8株能够产木聚糖酶。【结论】青海可可西里蕴藏着较丰富的嗜碱芽胞杆菌资源及丰富的产酶资源,为后续嗜碱芽胞杆菌的挖掘提供理论基础。     

An early origin of plastids within the cyanobacterial divergence is suggested by evolutionary trees based on complete 16S rRNA sequences

It is generally accepted that the plastids arose from a cyanobacterial ancestor, but the exact phylogenetic relationships between cyanobacteria and plastids are still controversial. Most studies based on partial 16S rRNA sequences suggested a relatively late origin of plastids within the cyanobacterial divergence. In order to clarify the exact relationship and divergence order of cyanobacteria and plastids, we studied their phylogeny on the basis of nearly complete 16S rRNA gene sequences. The data set comprised 15 strains of cyanobacteria from different morphological groups, 1 prochlorophyte, and plastids belonging to 8 species of plants and 12 species of diverse algae. This set included three cyanobacterial sequences determined in this study. This is the most comprehensive set of complete cyanobacterial and plastidial 16S rRNA sequences used so far. Phylogenetic trees were constructed using neighbor joining and maximum parsimony, and the reliability of the tree topologies was tested by different methods. Our results suggest an early origin of plastids within the cyanobacterial divergence, preceded only by the divergence of two cyanobacterial genera, Gloeobacter and Pseudanabaena.      

台湾海峡沉积物放线菌多样性及抗菌活性研究

探究台湾海峡海洋沉积物中放线菌的多样性并进行抗菌活性筛选,为发现新的药物先导化合物提供新菌源。采用7种选择性培养基分离10份来自台湾海峡沉积物样品中的海洋放线菌,并对分离菌株的16S rRNA基因序列进行系统进化分析。采用滤纸片法对部分代表放线菌菌株发酵液进行生物活性筛选,共分离到532株放线菌,挑选其中170株代表菌株进行鉴定,分属于8个目10个科14个属。有2株菌的16S rRNA基因序列显示属于潜在的新种,170株菌中有68.8%的菌株显示出至少对一种指示菌有抑菌活性。结果表明台湾海峡海洋沉积物中蕴藏着丰富的放线菌资源,是发现新药的有效途径。     

A broad-host-range, generalized transducing phage (SN-T) acquires 16S rRNA genes from different genera of bacteria

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 x 10(-9) transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.     

大连湾原油降解菌的分离和多样性分析

[目的]研究大连湾原油污染海域可培养原油降解菌的多样性,并获得新的原油降解菌.[方法]通过大连湾海水、海泥和海绵样品采集,以原油作为唯一碳源,培养、富集、分离筛选原油降解菌,根据16S rRNA基因序列确定其系统进化地位.[结果]通过形态观察和16S rRNA基因分析,共获得22个属的50株菌.其中,有6株菌的16S rRNA序列与最相近的菌株序列一致性仅为95％-97％,可能是潜在的新菌.单菌实验表明,45株菌具有石油降解能力.[结论]揭示了大连湾可培养原油降解菌的多样性,并获得了新的原油降解菌,为海洋石油污染的生物治理提供新资源.