Bacterial whole cell protein profiles of the rRNA group II pseudomonads

Studies on bacterial whole cell protein profiles showed that members of the rRNA group II pseudomonads were distinct from other non-fluorescent and fluorescent pseudomonads, including Pseudomonas aeruginosa, the type species of the genus Pseudomonas. Strains of Ps. andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli, Ps. pickettii, Ps. pseudomallei and Ps. rubrisubalbicans showed uniform and distinct protein patterns, while strains of Ps. solanacearum and Ps. cepacia displayed differences within species. Numerical analysis of their protein profiles with GelManager and Taxan programs generated dendrograms comprising 16 clusters at 89% similarity. Each cluster included strains belonging to the same species with the exception of Ps. solanacearum, which fragmented into three clusters. Pseudomonas solanacearum showed different protein patterns correlating with different biovars and the two divisions of Cook et al. (1989), as well as the results of 16S rRNA gene sequencing. The whole cell protein profiles of a total of 83 strains belonging to 14 bacterial species were numerically analysed.     

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.     

Biocidal activity in plant pathogenic Acidovorax, Burkholderia, Herbaspirillum, Ralstonia and Xanthomonas spp.

Antibacterial and antifungal activity was investigated for strains of Acidovorax spp., Burkholderia spp., Herbaspirillum rubrisubalbicans and Ralstonia solanacearum ; strains representing 118 species and pathovars of Xanthomonas were also tested for phytotoxic capacity. Antibacterial activity was present in all Burkholderia spp. except B. andropogonis , in biovars II and III of R. solanacearum but not in biovars I and IV, and in two strains of Xanthomonas. Little antibacterial activity was recorded for Acidovorax spp. Antifungal activity was expressed by most strains of A. avenae ssp. avenae and A. avenae ssp. cattleyae. Weak or variable antifungal reactions were given by strains of A. avenae ssp. citrulli and no activity was expressed by A. konjaci. Most strains of B. caryophylli, B. cepacia, B. gladioli pv. agaricicola, B. gladioli pv. alliicola, B. gladioli pv. gladioli , B. glumae and B. plantari produced extensive inhibition zones against Rhodotorula mucilaginosa. Strains of H. rubrisubalbicans and R. solanacearum gave negative, weak or variable reactions. Strains of Xanthomonas spp. exhibited no antifungal activity. In all cases antifungal activity was caused by a low molecular weight toxin. Three Xanthomonas strains exhibited phytotoxic activity. The ecological implications of these data are discussed.     

Polynucleobacter necessarius, an obligate bacterial endosymbiont of the hypotrichous ciliate Euplotes aediculatus, is a member of the β-subclass of Proteobacteria

Abstract An almost full length 16S rRNA gene of the obligate bacterial endosymbiont Polynucleobacter necessarius was amplified using the polymerase chain reaction in combination with site-specific primers. The amplified DNA was directly sequenced and compared with other bacterial 16S rRNA sequences. P. necessarius belongs to the β-subclass of Proteobacteria and shows the closest relationship to Alcaligenes eutrophus, Burkholderia solanacearum , and B. pickettii . In situ hybridization with a specific oligonucleotide probe corroborated the assignment of the retrieved sequence to P. necessarius .     

Production and Use of Monoclonal Antibodies to Pseudomonas andropogonis

Pseudomonas andropogonis is an important pathogen of worldwide distribution in ornamental and other plant species from 15 families. This paper reports the production and characterization of monoclonal antibodies (MAbs) to P. andropogonis and evaluation of their use in the detection of the pathogen in carnation cuttings. Ten stable hybridoma cell lines were produced. Results of indirect ELISA and indirect immuno-fluorescence showed that MAb 6B3 was specific for P. andropogonis; MAb 3D5W1 reacted with both P. andropogonis and P. caryophylli; six other MAbs reacted with all strains of seven species of rRNA group II pseudomonads tested except P. solanacearum and P. pickettii. Eight of the ten MAbs failed to cross-react with other non-fluorescent or fluorescent pseudomonads, xanthomonads and other bacteria tested. P. andropogonis was similar in protein profile to other rRNA group II pseudomonads tested except P. solanacearum and P. pickettii. Epitopes were clearly located within the cell by immunogold labelling. Of four MAbs that were isotyped, two possessed IgGl and two the IgM heavy chain. P. andropogonis was readily detected by combining immunofluorescence and detached carnation leaf assay using an initial inoculum of 4 × 10° colony forming units (cfu) ml^?1 after enrichment at room temperature for 4 days.     

Identification of Pseudomonas tolaasi: the White Line in Agar and Mushroom Tissue Block Rapid Pitting Tests

A sharply defined white line of precipitate forms in Pseudomonas Agar F (Difco) between the opaque white colonies of Pseudomonas tolaasi and translucent colonies of certain unidentified pseudomonads. This visible interaction has been utilized in a specific and reliable method for the identification of Ps. tolaasi. The white line test was positive when 113 isolates of Ps. tolaasi from five different countries were examined, whereas 154 isolates of pseudomonads other than Ps. tolaasi , including Ps. corrugata, Ps. delphinii, Ps. fluorescens, Ps. lachrymans, Ps. marginalis, Ps. pastinaceae, Ps. phaseolicola, Ps. aeruginosa, Ps. putida, Ps. syringae, Ps. mors-prunorum, Ps. cichorii, Ps. antirrhini, Ps. viridiflava, Ps. caryophylli, Ps. cepacia, Ps. mendocina, Ps. stutzeri, Ps. acidivorus and Ps. lemoignei did not give the white line reaction with a reacting translucent colony pseudomonad. Browning of mushrooms in host tests does not help in the identification of Ps. tolaasi , but a conspicuous pitting produced in less than 10 min at the cut surface of mushroom tissue is as specific as the white line test in detecting Ps. tolaasi in suspension in distilled water.     

Construction of species-specific primers for Pseudomonas andropogonis based on 16S rDNA sequences

Approximately 94% of the total 16S rDNA of Pseudomonas andropogonis strain ACH 01053A was sequenced and compared with that of strain ATCC 23061 obtained from the GenBank database. The two sequences were highly homologous with 1·3% difference. Alignment of sequences with those from closely related bacteria revealed two possible regions for design of a specific primer suitable for detection of Ps. andropogonis . Using primers designed to these variable regions in a PCR test, an amplification product of approximately 410 bp was specifically produced by 40 strains of Ps. andropogonis . No other bacterial species showed an amplification product under optimized PCR conditions. As few as 1000 cells per reaction were detected.     

Identification of Pseudomonas spp. Isolated from Milk Produced in South Eastern Queensland

S ummary . Bacteria were isolated from raw and pasteurized milks produced throughout S. E. Queensland. Milk samples were plated initially on penicillin agar or on milk agar and incubated at 30° and 7°, respectively. On the basis of primary characterization, 167 of the 330 isolates obtained were identified as Pseudomonas spp. The pseudomonads were further characterized in accordance with the taxonomic studies of the genus by Stanier, Palleroni & Doudoroff (1966). Species designations were ascribed to the Pseudomonas isolates on the basis of distinctive species characteristics in conjunction with similarity coefficients between each isolate and the ideal species phenotype, as follows: Ps. fluorescens (121), Ps. aeruginosa (16), Ps. putida (12), Ps. maltophilia (9), Ps. pseudoalcaligenes (5), Ps. cepacia (3) and Ps. alcaligenes (1). A dendrogram obtained by cluster analysis of the Pseudomonas isolates is included.     

The specificity of an immune serum to the exocellular lipopolysaccharide of Pseudomonas wieringae

The serum obtained to exocellular lipopolysaccharide (ELPS) of Pseudomonas wieringae selectively agglutinated strains of pathovar of P. syringae and did not agglutinated strains of P. cichorii, P. solanacearum, P. gladioli pv. allicola, P. fluoroviolaceus, strains of nonphytopathogenic pseudomonads as well as bacteria of the genera Erwinia, Bacillus, Xanthomonas, Klebsiella. Consequently, the antigen determinant common with antigen of the species Pseudomonas syringae is present in the composition of ELPS.     

A new family of Alteromonadaceae fam. nov., including the marine proteobacteria species Alteromonas, Pseudoalteromonas, Idiomarina i Colwellia

The taxonomic position of the marine genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia within the gamma subclass of the class Proteobacteria were specified on the basis of their phenotypic, genotypic, and phylogenetic characteristics. Gram-negative aerobic bacteria of the genera Alteromonas, Pseudoalteromonas, and Idiomarina and facultatively anaerobic bacteria of the genus Colwellia were found to form a phylogenetic cluster with a 16S rRNA sequence homology of 90% or higher. The characteristics of these genera presented in this paper allow their reliable taxonomic identification. Based on the analysis of our experimental data and analyses available in the literature, we propose to combine the genera Alteromonas, Pseudoalteromonas, Idiomarina, and Colwellia into a new family, Alteromonadaceae fam. nov., with the type genus Alteromonas.     

Phylogenetic comparisons of a coastal bacterioplankton community with its counterparts in open ocean and freshwater systems

In order to extend previous comparisons between coastal marine bacterioplankton communities and their open ocean and freshwater counterparts, here we summarize and provide new data on a clone library of 105 SSU rRNA genes recovered from seawater collected over the western continental shelf of the USA in the Pacific Ocean. Comparisons to previously published data revealed that this coastal bacterioplankton clone library was dominated by SSU rRNA gene phylotypes originally described from surface waters of the open ocean, but also revealed unique SSU rRNA gene lineages of beta Proteobacteria related to those found in clone libraries from freshwater habitats. beta Proteobacteria lineages common to coastal and freshwater samples included members of a clade of obligately methylotrophic bacteria, SSU rRNA genes affiliated with Xylophilus ampelinus, and a clade related to the genus Duganella. In addition, SSU rRNA genes were recovered from such previously recognized marine bacterioplankton SSU rRNA gene clone clusters as the SAR86, SAR11, and SAR116 clusters within the class Proteobacteria, the Roseobacter clade of the alpha subclass of the Proteobacteria, the marine group A/SAR406 cluster, and the marine Actinobacteria clade. Overall, these results support and extend previous observations concerning the global distribution of several marine planktonic prokaryote SSU rRNA gene phylotypes, but also show that coastal bacterioplankton communities contain SSU rRNA gene lineages (and presumably bacterioplankton) shown previously to be prevalent in freshwater habitats.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

Rhodovarius lipocyclicus gen. nov. sp. nov., a new genus of the alpha-1 subclass of the Proteobacteria

A bacterial strain (CCUG 44693T) was recovered during an industrial hygiene control. Phylogenetic analyses using the 16S rRNA gene sequence of the isolate indicated that it represents a new lineage in the alpha-1 subclass of the Proteobacteria, with the highest sequence similarity of 93.3% to the type strain of Muricoccus roseus. In the polyamine pattern spermidine was the predominant compound. The polar lipid profile consisted of the major lipids phosphatidyl ethanolamine, phosphatidyl dimethylethanolamine, phosphatidyl glycerol, phosphatidyl cholin and an unknown amino lipid. The major respiratory quinone was a ubiquinone Q-10 and the major whole cell fatty acids were 19:0 cyclo omega8c and 18:1 omega7c. The isolate also contained 18:1 2-OH and other fatty acids typical for members of the alpha-1 subclass of the Proteobacteria in addition to 10:0 2-OH in low amounts, not detected in members of closely related genera. The strain grew heterotrophically and strictly aerobically and formed 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, chemotaxonomic and biochemical characteristics, we propose that strain CCUG 44693T (CIP 108310T) represents a new genus of the alpha-1 subclass of the Proteobacteria for which we propose the name Rhodovarius lipocyclicus gen. nov. sp. nov.     

Occurrence of Glycoside Hydrolases in Plant Pathogenic and Related Bacteria

One hundred and twenty-eight isolates representing 37 species and six genera of plant pathogenic and related bacteria were tested for the presence of /3–galactosidase, glucosidase. β-glucosidase and β-xylosidase; using nitrophenyl glycopyranosides as substrates. Agrobacterium tumefaciens, Corynebacterium flaccumfaciens, C. michiganense. Flavobacterium pectinovorum and Pseudomonas maltophilia showed activity on all of the four substrates. Xanthomonas albilineans and three nomenspecies of the X. campestris group had little or no o-glucosidase activity but all other tests with Xanthomonas spp . were positive. None of the fluorescen; pseudomonads examined possessed β-galactosidase but P. stizolobii, P. andropogonis and P. rubrisubalbicans , among the non-fluorescent pseudomonads showed activity.     

Diversity and differentiation among fluorescent pseudomonads in crop rhizospheres with whole-cell protein profiles

Fluorescent pseudomonads from rhizospheres of four different crops, grown under similar soil and climatic conditions were phenotypically characterized to differentiate them into biovars. In protein electrophenogram studies of relationships among fluorescent pseudomonads by UPGMA cluster analysis based on DICE similarity index, the isolates were mainly discerned into three major clusters representing four different biovars. The biovars generally matched the delineated phenotypic clusters with the exception of a strain belonging to biovar II. However, the isolates representing similar rhizospheres and geographic locations were generally distributed into different phenotypic clusters as influenced by factors yet to be determined. The studies reinstated the importance of whole-cell protein analyses in characterizing pseudomonads and assessing their diversity.     

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.     

Molecular characterization of bacterial populations in petroleum-contaminated groundwater discharged from underground crude oil storage cavities

Petroleum-contaminated groundwater discharged from underground crude oil storage cavities (cavity groundwater) harbored more than 10(6) microorganisms ml(-1), a density 100 times higher than the densities in groundwater around the cavities (control groundwater). To characterize bacterial populations growing in the cavity groundwater, 46 PCR-amplified almost full-length 16S ribosomal DNA (rDNA) fragments were cloned and sequenced, and 28 different sequences were obtained. All of the sequences were affiliated with the Proteobacteria; 25 sequences (43 clones) were affiliated with the epsilon subclass, 2 were affiliated with the beta subclass, and 1 was affiliated with the delta subclass. Two major clusters (designated clusters 1 and 2) were found for the epsilon subclass proteobacterial clones; cluster 1 (25 clones) was most closely related to Thiomicrospira denitrificans (88% identical in nucleotide sequence), while cluster 2 (11 clones) was closely related to Arcobacter spp. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rDNA fragments showed that one band was detected most strongly in cavity groundwater profiles independent of storage oil type and season. The sequence of this major band was identical to the sequences of most of the cluster 1 clones. Fluorescence in situ hybridization (FISH) indicated that the cluster 1 population accounted for 12 to 24% of the total bacterial population. This phylotype was not detected in the control groundwater by DGGE and FISH analyses. These results indicate that the novel members of the epsilon subclass of the Proteobacteria grow as major populations in the petroleum-contaminated cavity groundwater.     

Comparative sequence analysis and oligonucleotide probe design based on 23S rRNA genes of Alphaproteobacteria from North Sea bacterioplankton

Almost complete 23S rRNA gene sequences were obtained from 11 Alphaproteobacteria isolated from marine surface water of the German Bight. Five of the strains belong to the "marine alpha" group, a phylogenetic cluster which encompasses members of the genus Roseobacter and closely related bacteria. Phylogenetic sequence analysis based on 52 published as well as unpublished complete 23S rDNA sequences from Alphaproteobacteria including the newly obtained was in general consistent with the 16S rRNA gene sequence-derived phylogeny. 16S and 23S rRNA based phylogenies both showed a distinct cluster for strains associated with the "marine alpha" group. The suitability of both markers for the design of oligonucleotide probes targeting selected groups of Alphaproteobacteria was systematically evaluated and compared in silico. Six clusters of sequences covering different phylogenetic levels as well as two strains were selected in a case study. To compensate for the quantitative difference in the two data sets, the 16S rRNA dataset was truncated to sequences with an equivalent in the 23S rRNA data set. Our results show, that the overall number of phylogenetically redundant probes available could be more than doubled by extending probe design to the 23S rRNA. For small clusters of high sequence similarity and single strains, up to 8 times more discriminating binding sites were provided by the 23S rRNA.     

Analysis of bacterial diversity in the canine duodenum, jejunum, ileum, and colon by comparative 16S rRNA gene analysis

The study aim was to describe the diversity of the intraluminal intestinal microbial community in dogs by direct sequence analysis of the 16S rRNA gene. Intestinal content was collected from the duodenum, jejunum, ileum, and colon from six healthy dogs. Bacterial 16S rRNA gene was amplified with universal bacterial primers. Amplicons were ligated into cloning vectors and near-full-length 16S rRNA gene inserts were analyzed. From a total of 864 clones analyzed, 106 nonredundant 16S rRNA gene sequences were identified. Forty-two (40%) sequences showed<98% sequence similarity to 16S rRNA gene sequences reported previously. Operation taxonomic units were classified into four phyla: Firmicutes, Fusobacteria, Bacteroidetes, and Proteobacteria. Clostridiales predominated in the duodenum (40% of clones) and jejunum (39%), and were highly abundant in the ileum (25%) and colon (26%). Sequences affiliated with Clostridium cluster XI and Clostridium cluster XIVa dominated in the proximal small intestine and colon, respectively. Fusobacteriales and Bacteroidales were the most abundant bacterial order in the ileum (33%) and colon (30%). Enterobacteriales were more commonly observed in the small intestine than in the colon. Lactobacillales occurred commonly in all parts of the intestine.     

Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations.

The phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas were investigated by using small-subunit ribosomal DNA (rDNA) sequencing. Members of the genus Aeromonas formed a distinct line within the gamma subclass of the Proteobacteria. Plesiomonas shigelloides also clustered within the confines of the gamma subclass of the Proteobacteria but exhibited a closer association with members of the family Enterobacteriaceae than with members of the family Aeromonadaceae. Species of the genus Aeromonas exhibited very high levels of overall sequence similarity (ca. 98 to 100%) with each other. Several of the relationships derived from an analysis of the rDNA sequence data were in marked disagreement with the results of chromosomal DNA-DNA pairing experiments. Diagnostic rDNA signatures that have possible value for differentiating most Aeromonas species were discerned.