Taxonomy of the Clostridia: ribosomal ribonucleic acid homologies among the species.

rRNA homologies have been determined on reference strains representing 56 species of Clostridium. Competition experiments using tritium-labelled 23S rRNA were employed. The majority of the species had DNA with 27 to 28% guanine plus cytosine (%GC). These fell into rRNA homology groups I and II, which were well defined, and a third group which consisted of species which did not belong in groups I and II. Species whose DNA was 41 to 45% GC comprised a fourth group. Thirty species were placed into rRNA homology group I on the basis of having 50% or greater homology with Clostridium butyricum, C. perfringens, C. carnis, C. sporogenes, C. novyi or C. pasteurianum. Ten subgroups were delineated in homology group I. Species in each subgroup either had high homology with a particular reference species or a similar pattern of homologies to all of the reference organisms. The eleven species in rRNA homology group II had 69% or greater homology to C. lituseburense. Species in groups I and II had intergroup homologies of 20 to 40%. The six species in group II had very low homologies with groups I and II. Negligible homology also resulted when five of the species were tested against the sixth, C. ramosum. The five species having DNA with 41 to 45% GC were C. innocuum, C. sphenoides, C. indolis, C. barkeri and C. orotic um. Little rRNA homology was apparent between C. innocuum and the other high % GC species or with several Bacillus species having similar %GC DNA. Correlations between homology results and phenotypic characteristics are discussed.     

乳酸杆菌代谢产物对引起阴道炎常见细菌的抑制作用的初步探讨

目的探讨乳酸杆菌代谢产物对临床常见引起阴道炎的大肠埃希菌、铜绿假单胞菌、金黄色葡萄球菌、白色念珠菌、伤寒杆菌和肠球菌的抑菌作用。方法采用营养琼脂平板培养基定量涂菌,国际标准药敏杯给药的药敏试验法,检测乳酸杆菌代谢产物对大肠埃希菌、铜绿假单胞菌、金黄色葡萄球菌、白色念珠菌、伤寒杆菌和肠球菌的抑菌环的大小。结果乳酸杆菌代谢产物对大肠埃希菌、铜绿假单胞菌、金黄色葡萄球菌和伤寒杆菌有明显的抑菌作用,对肠球菌、白色念珠菌无抑菌作用。结论在临床上可应用乳酸杆菌及其制剂调节阴道微生态平衡,治疗细菌性阴道炎。     

Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov.

Based on the 16S rRNA sequences, DNA-DNA homology values, cellular lipid and fatty acid composition, and phenotypic characteristics, a new genus Burkholderia is proposed for the RNA homology group II of genus Pseudomonas. Seven species in this group were transferred to the new genus. Thus seven new combinations, Burkholderia cepacia (Palleroni and Holmes 1981), Burkholderia mallei (Zopf 1885), Burkholderia pseudomallei (Whitmore 1913), Burkholderia caryophylli (Burkholder 1942), Burkholderia gladioli (Severini 1913), Burkholderia pickettii (Ralston et al 1973) and Burkholderia solanacearum (Smith 1896) were proposed.     

Sequence and relatedness in other bacteria of the Pseudomonas aeruginosa oprP gene coding for the phosphate-specific porin P

The oprP gene encoding the Pseudomonas aeruginosa phosphate-specific outer membrane porin protein OprP was sequenced. Comparison of the derived amino acid sequence with the known sequences of other bacterial porins demonstrated that OprP could be no better aligned to these porin sequences than it could to the periplasmic phosphate-binding protein PhoS of Escherichia coli. Southern hybridization and restriction mapping of the oprP gene in 37 clinical isolates and the 17 serotype strains of P. aeruginosa revealed that restriction sites in the vicinity of the oprP gene were highly conserved. Several species from the Pseudomonas fluorescens rRNA homology group contained DNA that hybridized to an oprP gene probe.     

The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri.

The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.     

Outer membrane proteins of Pseudomonas

In this review, we describe the outer membrane proteins of Pseudomonas aeruginosa and related strains from the Pseudomonas fluorescens rRNA homology group of the Pseudomonadaceae, with emphasis on the physiological function and biochemical characteristics of these proteins. The use of opr (for outer membrane protein) is proposed as the genetic designation for the P. aeruginosa outer membrane proteins and letters are assigned, in conjunction with this designation, to known outer membrane proteins. Proteins whose primary functions involve pore formation, transport of specific substrates, cell structure determination and membrane stabilization are discussed. The conservation of selected proteins in the above Pseudomonas species is also examined.     

Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers

We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.     

Fluorescent Pseudomonas species categorized by using polymerase chain reaction (PCR)/restriction fragment analysis of 16S rDNA

A rapid procedure for the identification of fluorescent pseudomonads, based on the polymerase chain reaction (PCR) and restriction fragment analysis of 16S rDNA genes is described. Thirty-one strains belonging to 10 different Pseudomonas species of the Pseudomonas fluorescens rRNA branch were characterized. Amplified rDNA was digested with 13 different restriction endonucleases. The combined data from restriction analysis enabled the definition of 17 different 16S rDNA genotypes. All type strains belonging to different species were differentiated. The good correlation between grouping obtained using restriction analysis with other molecular classification criteria demonstrates the value of the described method to characterize rapidly fluorescent Pseudomonas strains at the species level.     

Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial degradation. Microbial communities present in MWFs lead to biofilm formation in the MWF systems, which act as a continuous source of contamination. Species of rRNA group I Pseudomonas dominate in contaminated MWFs. However, their actual distribution is typically underestimated when using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar. To overcome this, fluorescent in situ hybridization (FISH) was used to study their abundance along with biofilm formation by two species recovered from MWFs, Pseudomonas fluorescens MWF-1 and the newly described Pseudomonas oleovorans subsp. lubricantis. Based on 16S rRNA sequences, a unique fluorescent molecular probe (Pseudo120) was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas. The specificity of the probe was evaluated using hybridization experiments with whole cells of different Pseudomonas species. The probe's sensitivity was determined to be 10(3) cells/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (± 1.1) × 10(6) and 5.0 (± 2.3) × 10(6) cells/ml in four different industrial MWF samples collected from three different locations. Biofilm formation was visualized under stagnant conditions using high and low concentrations of cells for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120. On the basis of these observations, this molecular probe can be successfully be used in the management of MWF systems to monitor the levels and biofilm formation of rRNA group I pseudomonads.     

Isolation and Characterization of the algD gene of Pseudomonas syringae pv. phaseolicola and its distribution among other Pseudomonads and related Organisms

The gene coding for GDP-mannose dehydrogenase ( algD ) was isolated from a Pseudomonas syringae pv. phaseolicola genomic library using a polymerase chain reaction-generated heterologous DNA-probe from Pseudomonas aeruginosa . A total of 2123 base pairs were sequenced (accession number AF001555) and analysed for homologies to the alginate gene cluster of P. aeruginosa . Downstream from algD an alg8 homologue was found suggesting a similar arrangement of the alginate gene cluster in P. syringae pv. phaseolicola to that in P. aeruginosa . Also, the deduced amino acid sequence of algD shows high similarity to that of P. aeruginosa (0.9) and Azotobacter vinelandii (0.88). Southern hybridization experiments revealed that algD is widely distributed among members of the Pseudomonas rRNA homology group I. Among others, sequences homologous to algD were detected in the P. syringae pathovars lachrymans , mori , morsprunorum, pisi , savastanoi, tabaci and tomato as well as in Pseudomonas amygdali . For most of the algD positive organisms synthesis of alginate has been reported by other studies. However, algD homologues were also detected for the species Pseudomonas corrugata , Pseudomonas marginalis and Pseudomonas avenae ( Acidovorax avenae ), for which alginate biosynthesis has not yet been reported.     

Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35 degrees C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads

The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system.     

Complete denitration of nitroglycerin by bacteria isolated from a washwater soakaway

Four axenic bacterial species capable of biodegrading nitroglycerin (glycerol trinitrate [GTN]) were isolated from soil samples taken from a washwater soakaway at a disused GTN manufacturing plant. The isolates were identified by 16S rRNA gene sequence homology as Pseudomonas putida, an Arthrobacter species, a Klebsiella species, and a Rhodococcus species. Each of the isolates utilized GTN as its sole nitrogen source and removed nitro groups sequentially from GTN to produce glycerol dinitrates and mononitrates (GMN), with the exception of the Arthrobacter strain, which achieved removal of only the first nitro group within the time course of the experiment. The Klebsiella strain exhibited a distinct preference for removal of the central nitro group from GTN, while the other five strains exhibited no such regioselectivity. All strains which removed a second nitro group from glycerol 1,2-dinitrate showed regiospecific removal of the end nitro group, thereby producing glycerol 2-mononitrate. Most significant was the finding that the Rhodococcus species was capable of removing the final nitro group from GMN and thus achieved complete biodegradation of GTN. Such complete denitration of GTN has previously been shown only in mixed bacterial populations and in cultures of Penicillium corylophilum Dierckx supplemented with an additional carbon and nitrogen source. Hence, to the best of our knowledge, this is the first report of a microorganism that can achieve complete denitration of GTN.     

Accumulation of poly(3-hydroxybutyrate) from octanoate in different pseudomonas belonging to the rRNA homology group I

It is admitted that one of the characteristics of pseudomonads is their inability to accumulate poly(3-hydroxybutyrate). In this paper, we show that poly(3-hydroxyoctanoate) synthesis is restricted to Pseudomonas rRNA homology group I, which includes both fluorescent and nonfluorescent species. However, within the genus Pseudomonas, the P. aeruginosa complex can be subdivided into two groups: the "P. aeruginosa group", which includes P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, produce poly(3-hydroxyoctanoate) from octanoate and the "P. oleovorans group" which includes the type strain of P. oleovorans, P. pseudoalcaligenes and two Pseudomonas sp., produce poly(3-hydroxybutyrate) during cultivation on octanoate. Strain GPo1 (ATCC 29347) formely identified as P. oleovorans and known to produce various medium-side-chain PHAs such as poly(3-hydroxyoctanoate) has been reclassified in the P. putida complex.     

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.     

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.     

Streptococcus fryi sp. nov., a novel species with Lancefield group M antigens

The taxonomic characteristics of β-hemolytic streptococcal strains that reacted with Lancefield group M antisera were investigated. Group M streptococci have not been proposed as a species to date. Four strains of the group M streptococci isolated from dog were located within the pyogenic group of the genus Streptococcus on 16S rRNA gene-based phylogenetic analysis; the group M strains were located a short distance away from all other members of the group. The homology values of 16S rRNA gene sequences between group M strains and all other streptococci were<95.6%. Group M strains exhibited low levels of DNA-DNA homology to other streptococcal species. Some biochemical traits, such as β-galactosidase activity and acid production from glycogen, could distinguish these group M strains from other closely related species. Thus, these strains are proposed to constitute a new species -Streptococcus fryi sp. nov. The type strain is PAGU 653(T) (=NCTC 10235(T)=JCM 16387(T)).     

Phylogenetic studies of the rRNA group II pseudomonads based on 16S rRNA gene sequences

Nearly complete sequences of 16S rRNA genes were determined for eight bacterial strains representing five species of the rRNA homology group II pseudomonads that are members of the beta subclass of the class Proteobacteria. Comparative analysis with published sequence data indicated that Pseudomonas andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli and Ps. cepacia aggregate in one coherent cluster at 94·2% sequence similarity; Ps. solanacearum and Ps. pickettii shared 95·3% and 92·8% similarity with Alcaligenes eutrophus in another cluster. Both clusters joined at 87·8% similarity, which is similar to that for genera in this subclass of Proteobacteria. Based on this study and on comparison with other works we suggest that these species are separated from authentic pseudomonads and constitute a new genus or possibly two related genera accommodating Ps. andropogonis, Ps. caryophylli, Ps. gladioli, Ps. cepacia, and Ps. solanacearum, Ps. pickettii and A. eutrophus, respectively. Four strains of Ps. solanacearum representing Biovars 1, 2, 3 and 4 were subdivided into two clusters at 99·1% sequence similarity, in agreement with other published phenotypic and genotypic studies. The two clusters may be potentially regarded as subspecies.     

Acidovorax, a new genus for Pseudomonas facilis, Pseudomonas delafieldii, E. Falsen (EF) group 13, EF group 16, and several clinical isolates, with the species Acidovorax facilis comb. nov., Acidovorax delafieldii comb. nov., and Acidovorax temperans sp. nov

Pseudomonas facilis and Pseudomonas delafieldii are inappropriately assigned to the genus Pseudomonas. They belong to the acidovorans rRNA complex in rRNA superfamily III (i.e., the beta subclass of the Proteobacteria). The taxonomic relationships of both of these species, two groups of clinical isolates (E. Falsen [EF] group 13 and EF group 16), and several unidentified or presently misnamed strains were examined by using DNA:rRNA hybridization, numerical analyses of biochemical and auxanographic features and of fatty acid patterns, polyacrylamide gel electrophoresis of cellular proteins, and DNA:DNA hybridization. These organisms form a separate group within the acidovorans rRNA complex, and we propose to transfer them to a new genus, Acidovorax. We describe the following three species in this genus: the type species, Acidovorax facilis (formerly Pseudomonas facilis), with type strain LMG 2193 (= CCUG 2113 = ATCC 11228); Acidovorax delafieldii (for the former Pseudomonas delafieldii and most of the EF group 13 strains), with type strain LMG 5943 (= CCUG 1779 = ATCC 17505); and Acidovorax temperans (for several former Pseudomonas and Alcaligenes strains and most of the EF group 16 strains), with type strain CCUG 11779 (= LMG 7169).     

Evaluation of a novel 16S rRNA/tRNA mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea

In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA)^Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA^Val genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA^Val marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA^Val gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA^Val region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA^Val marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.