Application of the profiles of amino acid utilization as the sole carbon and nitrogen sources for pseudomonad taxonomy

Profiles of utilization of 20 amino acids were determined for 218 strains of 10 Pseudomonas species (P. aeruginosa, P. putida, P. fluorescens, P. stutzeri, P. alcaligenes, P. pseudoalcaligenes, P. luteola, P. oryzihabitans, P. mendocina, and P. chlororaphis), including the type strains of these species. Amino acid utilization was determined on minimal salt agar with an amino acid as the sole source of nitrogen and carbon. All the investigated pseudomonad species had species-specific profiles of amino acid utilization. For the type strains of all species, Jaccard’s coefficients of community were different (Sj = 0.31–0.82), in accordance with the interspecies differences. The similarity between the intraspecies variants of the profiles and that of the type strain was high; for 98% of P. aeruginosa strains, Sj = 0.85−1.0; for 100% of P. putida, P. stutzeri, and P. alcaligenes strains, Sj was 0.87–1.0, 0.90, and 0.86–1.0, respectively. Only for P. fluorescens and P. pseudoalcaligenes were low Sj of the intraspecies profiles revealed, in accordance with the known phenotypic heterogeneity of these species. These results agree with the known pseudomonad classification, and the method is therefore valid for identification of known species and for determination of the new members of the genus Pseudomonas.     

Lines of Evidence for Horizontal Gene Transfer of a Phenazine Producing Operon into Multiple Bacterial Species

Phenazines are secondary metabolites with broad-spectrum antibiotic activity against bacteria, fungi, and eukaryotes. In pseudomonad species, a conserved seven-gene phenazine operon (phzABCDEFG) is required for the conversion of chorismic acid to the broad-spectrum antibiotic phenazine-1-carboxylate. Previous analyses of genes involved in phenazine production from nonpseudomonad species uncovered a high degree of sequence similarity to pseudomonad homologues. The analyses undertaken in this study wished to eluciadate the evolutionary history of genes involved in the production of phenazines. Furthermore, I wanted to determine if the phenazine operon has been transferred through horizontal gene transfer. Analyses of GC content, codon usage patterns, frequency of 3:1 dinucleotides, sequence similarities, and phylogenetic reconstructions were undertaken to map the evolutionary history of phenazine genes from multiple bacterial species. Patchy phyletic distribution, high sequence similarities, and phylogenetic evidence infer that pseudomonad, Streptomyces cinnamonensis, Pantoea agglomerans, Burkholderia cepacia, Pectobacterium atrosepticum, Brevibacterium linens, and Mycobacterium abscessus species all contain a phenazine operon which has most likely been transferred among these species through horizontal gene transfer. The acquisition of an antibiotic-associated operon is significant, as it may increase the relative fitness of the recipient species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pseudomonas syringae pv. helianthi (Kawamura 1934) Dye, Wilkie et Young 1978, a Pathogen of Sunflower (Helianthus annuus L.)

Among the bacterial strains isolated from diseased sunflower leaves, eight were studied in some detail. A fluorescent pseudomonad isolated from necrotic tissues and its reisolates belong to group Ia of phytopathogenic pseudomonads which includes Pseudomonas syringae bacterium. A study of host range indicated that the pathogen infects only sunflower but not the other plant species. Based on the pathogenicity study and biochemical and physiological tests, it was concluded that the pathogen belongs to the bacterium Pseudomonas syringae pv. helianthi.     

Pseudomonas syringae pv. helianthi (Kawamura 1934) Dye,Wilkie et Young 1978, a Pathogen of Sunflower (Helianthus annuus L.)

Among the bacterial strains isolated from diseased sunflower leaves, eight were studied in some detail. A fluorescent pseudomonad isolated from necrotic tissues and its reisolates belong to group Ia of phytopathogenic pseudomonads which includes Pseudomonas syringae bacterium. A study of host range indicated that the pathogen infects only sunflower but not the other plant species. Based on the pathogenicity study and biochemical and physiological tests, it was concluded that the pathogen belongs to the bacterium Pseudomonas syringae pv. helianthi.     

Molecular and phenotypic characterization of potential plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> from rice and maize rhizospheres

In this study, Pseudomonas species were isolated from the rhizospheres of two plant hosts: rice (Oryza sativa cultivar Pathum Thani 1) and maize (Zea mays cultivar DK888). The genotypic diversity of isolates was determined on basis of amplified rDNA restriction analysis (ARDRA). This analysis showed that both plant varieties selected for two distinct populations of Pseudomonas. The actual biocontrol and plant promotion abilities of these strains was confirmed by bioassays on fungal (Verticillum sp., Rhizoctonia solani and Fusarium sp.) and bacterial (Ralstonia solanacearum and Bacillus subtilis) plant pathogens, as well as indole-3-acetic acid (IAA) production and carbon source utilization. There was a significant difference between isolates from rice and maize rhizosphere in terms of biological control against R.  solanacearum and B.  subtilis. Interestingly, none of the pseudomonads isolated from maize rhizosphere showed antagonistic activity against R.  solanacearum. This study indicated that the percentage of pseudomonad isolates obtained from rice rhizosphere which showed the ability to produce fluorescent pigments was almost threefold higher than pseudomonad isolates obtained from maize rhizosphere. Furthermore, the biocontrol assay results indicated that pseudomonad isolated from rice showed a higher ability to control bacterial and fungal root pathogens than pseudomonad isolates obtained from maize. This work clearly identified a number of isolates with potential for use as plant growth-promoting and biocontrol agents on rice and maize.     

Immunofluorescence assay for effects on field abundance of a naturally occurring pseudomonad during passage through the gut of a marine deposit feeder,Abarenicola pacifica

In a seasonal study we used immunofluorescence to follow a specific bacterial population, as well as total numbers, through the fore-, mid-, and hindgut of a deposit feeder, Abarenicola pacifica. We chose a pseudomonad because of its high ambient abundance. On five dates, we collected A. pacifica gut contents, with concurrent measurements of sedimentary food quality (chlorophyll a, protein, bacterial abundance), animal egestion rates (inversely proportional to gut residence time), and temperature. Increasing bacterial numbers from ingested sediment to foregut contents, and decreases from foregut to midgut indicate significant selection and digestion, respectively, of both the pseudomonad and the total bacterial community. Inverse correlations between egestion rate and digestive removal of bacteria offer some support for the prediction that digestion of bacteria is proportional to time spent exposed to digestive enzymes, although the significance of the associated statistical tests is marginal. No hindgut growth of the pseudomonad was observed, likely due to the short gut residence time of A. pacifica. The pseudomonad showed variation of less than a factor of 3 in its ambient sedimentary abundance over the year. Off print requests to: C. Plante.     

Bacterial diversity of soil in the vicinity of Pindari glacier,Himalayan mountain ranges,India, using culturable bacteria and soil 16S rRNA gene clones

Three 16S rRNA gene clone libraries (P1L, P4L and P8L) were constructed using three soil samples (P1S, P4S and P8S) collected near Pindari glacier, Himalayas. The three libraries yielded a total of 703 clones. Actinobacteria, Firmicutes and Proteobacteria were common to the three libraries. In addition to the above P1L and P8L shared the phyla Acidobacteria, Bacteroidetes, Gemmatimonadetes and Planctomycetes. Phyla Chlamydiae, Chlorobi, Chloroflexi, Dictyoglomi, Fibrobacteres, Nitrospirae, Verrucomicrobia, candidate division SPAM and candidate TM7s TM7a phylum were present only in P1L. Rarefaction analysis indicated that the bacterial diversity in P4S and P8S soil samples was representative of the sample. Principal component analysis (PCA) revealed that P1S and P8S were different from P4S soil sample. PCA also indicated that arsenic content, pH, Cr and altitude influence the observed differences in the percentage of specific OTUs in the three 16S rRNA gene clone libraries. The observed bacterial diversity was similar to that observed for other Himalayan and non-polar cold habitats. A total of 40 strains of bacteria were isolated from the above three soil samples and based on the morphology 20 bacterial strains were selected for further characterization. The 20 bacteria belonged to 12 different genera. All the isolates were psychro-, halo- and alkalitolerant. Amylase and urease activities were detected in majority of the strains but lipase and protease activities were not detected. Long chain, saturated, unsaturated and branched fatty acids were predominant in the psychrotolerant bacteria.     

Molecular genetic delineation of Phaeocystis species (Prymnesiophyceae) using coding and non-coding regions of nuclear and plastid genomes

Sequence variation among 22 isolates representing a global distribution of the prymnesiophyte genus Phaeocystis has been compared using nuclear-encoded 18S rRNA genes and two non-coding regions: the ribosomal DNA internal transcribed spacer 1 (ITS1) separating the 18S rRNA and 5.8S rRNA genes and the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) spacer flanked by short stretches of the adjacent large and small subunits (rbcL and rbcS). 18S rRNA can only resolve major species complexes. The analysis suggests that an undescribed unicellular Phaeocystis sp. (isolate PLY 559) is a sister taxon to the Mediterranean unicellular Phaeocystis jahnii; this clade branched prior to the divergence of all other Phaeocystis species, including the colonial ones. Little divergence was seen among the multiple isolates sequenced from each colonial species complex. RUBISCO spacer regions are even more highly conserved among closely related colonial Phaeocystis species and are identical in Phaeocystis antarctica, Phaeocystis pouchetii and two warm-temperate strains of Phaeocystis globosa, with a single base substitution in two cold-temperate strains of P. globosa. The RUBISCO spacer sequences from two predominantly unicellular Phaeocystis isolates from the Mediterranean Sea and PLY 559 were clearly different from other Phaeocystis strains. In contrast, ITS1 exhibited substantial inter- and intraspecific sequence divergence and showed more resolution among the taxa. Distinctly different copies of the ITS1 region were found in P. globosa, even among cloned DNA from a single strain, suggesting that it is a species complex and making this region unsuitable for phylogenetic analysis in this species. However, among nine P. antarctica strains, four ITS1 haplotypes could be separated. Using the branching order in the ITS1 tree we have attempted to trace the biogeographic history of the dispersal of strains in Antarctic coastal waters.     

The phylogeny of fluorescent pseudomonads in an unflooded rice paddy soil

The purpose of this research was to determine the diversity and distribution of fluorescent pseudomonads in an unflooded rice paddy soil. A region of the 16S ribosomal RNA gene from isolates was amplified using PCR and subsequently analysed by sequence analysis for bacterial identification and phylogenetic classification. A total of 117 fluorescent pseudomonads, representing between 10 and 21 species, were isolated from two sampling sites within the same paddy (designated as soils C and S). The isolates were found to be ≥96% homologous with known sequences, and were most closely related to the followingPseudomonas species:P. antarctica, P. costantini, P. extremorientalis, P. frederiksbergensis, P. kilonensis, P. koreensis, P. lini, P. mandelii, P. poae, P. rhodesiae, andP. veronii. Of these matches, the bulk of the isolates (49%) were affiliated withP. mandelii. In soils C and S, phylogenetic analysis revealed that 35 and 82 isolates co-clustered with 39 and 59% of 66 fluorescent pseudomonad type strains, respectively.     

Isolation of Bacteria Able to Metabolize High Concentrations of Formaldehyde

Summary Nineteen bacterial strains able to degrade and metabolize formaldehyde as a sole carbon source were isolated from soil and wastewater of a formaldehyde production factory. The samples were cultured in complex and mineral salts media containing 370 mg formaldehyde/l. The bacterial strains were identified to be Pseudomonas pseudoalcaligenes, P. aeruginosa, P. testosteroni, P. putida, and Methylobacterium extorquens. After adaptation of these microorganisms to high concentrations of formaldehyde; two isolated strains of M. extorquens (strains ESS and PSS) and four strains of P. pseudoalcaligenes (strains LSW, SSW, NSW and OSS) degraded 1850 mg formaldehyde/l, where as P. pseudoalcaligenes strain OSS completely consumed 3700 mg of formaldehyde/l after 24 h and degraded 70% of 5920 mg of formaldehyde/l after 72 h.     

Genetic Diversity in Pseudomonads Associated with Cereal Cultures Infected with Basal Bacteriosis

The genetic properties of 45 pseudomonad strains isolated from cereal cultures exhibiting symptoms of basal bacteriosis have been investigated. Considerable genetic diversity has been demonstrated using DNA fingerprints obtained by amplification with REP, ERIC, and BOX primers. Restriction analysis of the 16S–23S internal transcribed spacer (ITS1) allowed the strains to be subdivided into two major groups. In a phylogenetic tree, the ITS1s of these groups fell into two clusters, which also included the ITS1 of Pseudomonas syringae (“Syringae” cluster) and the ITS1 of P. fluorescens, P. tolaasii, P. reactans, P. gingeri, and P. agarici (“Fluorescens” cluster) from the GenBank database. Comparison of the ITS1 divergence levels within the “Fluorescens” cluster suggests expediency of treating P. tolaasii, P. reactans, various P. fluorescens groups, and, possibly, P. gingeri and P. agarici as subspecies of one genospecies. The intragenomic heterogeneity of ITS1s was observed in some of the pseudomonad strains studied. The results of amplification with specific primers and subsequent sequencing of the amplificate suggest the possibility of the presence of a functionally active syrB gene involved in syringomycin biosynthesis in the strains studied.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 537–544.Original Russian Text Copyright © 2005 by Bobrova, Milyutina, Troitskii.     

Application of fluorescence internal transcribed spacer-PCR (f-ITS) for the cluster analysis of bacteria isolated from air and deteriorated fresco surfaces

The aim of this work was the evaluation of fluorescence ITS-PCR (f-ITS) as a molecular tool to analyze the microbial community involved in the biodeterioration of cultural heritage surfaces. As a case study we analyzed by f-ITS ninety-two bacterial strains isolated from a medieval fresco and the surrounding air environment. The internal transcribed spacer between the 16S and 23S rRNA genes was amplified, and then the fluorescently labeled PCR products were separated by capillary electrophoresis. Bacterial strains were identified by 16S rDNA sequencing. The f-ITS electropherograms showed different profiles coherent with the affiliation of the strains at the genus and species levels. Among the isolates obtained from the fresco surface, those belonging to the genus Bacillus were the most prevailing exhibiting 8 different f-ITS profiles. The airborne bacilli exhibited only 2 of these 8 profiles. Staphylococcus were mostly isolated from air and produced 4 different profiles. Pseudomonas isolates presented 3 different profiles, and one of them was typical of Pseudomonas putida. Members of the other genera produced their distinctive profiles. Our results show that f-ITS is a promising molecular tool for the rapid selection and clustering of strains isolated from different sources.     

Alteromonas flava sp. nov. and Alteromonas facilis sp. nov., two novel copper tolerating bacteria isolated from a sea cucumber culture pond in China

Two bacterial strains, P0211^T and P0213^T, were isolated from a sea cucumber culture pond in China. The strains were able to resist high copper levels. These two strains were characterized at the phenotypic, chemotaxonomic, and genomic level. They were completely different colors, but the 16S rRNA genes showed 99.30% similarity. Phylogenetic analysis based on the sequences of the 16S rRNA gene and five housekeeping genes (dnaK, sucC, rpoB, gyrB, and rpoD) supported the inclusion of these strains within the genus Alteromonas, and the two isolated strains formed a group separated from the closest species Alteromonas aestuariivivens KCTC 52655^T. Genomic analyses, including average nucleotide identity (ANIb and ANIm), DNA–DNA hybridization (DDH), and the percentage of conserved proteins (POCP), clearly separated strains P0211^T and P0213^T from the other species within the genus Alteromonas with values below the thresholds for species delineation. The chemotaxonomic features (including fatty acid and polar lipid analysis) of strains P0211^T and P0213^T also confirmed their differentiation from the related taxa.The results demonstrated that strains P0211^T and P0213^T represented two novel species in the genus Alteromonas, for which we propose the names Alteromonas flava sp. nov., type strain P0211^T (= KCTC 62078^T = MCCC 1H00242^T), and Alteromonas facilis sp. nov., type strain P0213^T (= KCTC 62079^T = MCCC 1H00243^T).     

Metabolic Activity and Phylogenetic Diversity of Reed (<Emphasis Type="Italic">Phragmites australis</Emphasis>) Periphyton Bacterial Communities in a Hungarian Shallow Soda Lake

In the present study, the species composition and potential metabolic activities of bacterial communities of reed Phragmites australis (Cav.) (Trin. ex Steudel) periphyton from Lake Velencei were studied by cultivation-based and metabolic fingerprinting methods. Serially diluted spring biofilm samples were used to test the community-level physiological profiling (CLPP) using BIOLOG microplates, and for plating onto different media. On the basis of their morphological, biochemical, and physiological test results, 173 strains were clustered by numerical analysis. Representatives of amplified ribosomal DNA restriction analysis (ARDRA) groups were identified by their 16S rDNA sequence comparison. Based on the results of the CLPP investigations, regional differences were detected among the utilized substrate numbers and types, parallel with the increase in incubation time. The phenotypic test results of the strains showed considerable variability with respect to the sampling sites and the media used for cultivation. The most frequently isolated strains were identified as members of genera Agrobacterium, Pseudomonas (P. anguilliseptica, P. marginalis, P. alcaligenes, P. fragi) with aerobic or facultative anaerobic respiratory metabolism, and the species Aeromonas sobria and A. veronii with strong facultative fermentative metabolism. Other strains were identified as Gram-positive Arthrobacter, Bacillus, and Kocuria species. The rarely isolated strains were members of β-Proteobacteria (Acidovorax, Delftia, Hydrogenophaga, and Rhodoferax), γ-Proteobacteria (Psychrobacter and Shewanella), low G + C Gram-positives (Brevibacillus, Paenibacillus, and Exiguobacterium) and high G + C Gram-positives (Aureobacterium and Microbacterium).     

Growth promotion and inhibition by antibiotic-producing fluorescent pseudomonads on citrus roots

Summary Forty-three strains of feeder root colonizing fluorescent pseudomonads from rough lemon (Citrus jambhiri Lush.) roots were examined for effects on rough lemon and sweet orange (Citrus sinensis Osbeck) seedlings. Plants inoculated with a single bacterial soil-drench had, after 10 months, a range of stimulatory (to 116%) and inhibitory effects (to 52%). Stimulatory bacteria particularly increased growth of root systems. Cultivar-specific inhibition and stimulation was evident in inoculations of rough lemon and sweet orange seedlings. Populations of fluorescent rhizobacteria on inoculated and noninoculated, as well as on stimulated and nonstimulated seedlings, did not differ significantly (10.8×10⁶ to 30.3×10⁶ CFU/g root). Population of fluorescent rhizobacteria on seedlings were higher than populations on feeder roots from grove trees (2.8 to 5.7×10⁶ CFU/g). Ninety-four and 81% of 251 fluorescent strains produced antibiotics against the fungusGeotrichum candidum and the bacteriumErwinia stewartii, respectively. Antibiotic activities of 90% of the antibiotic producing strains were repressed by Fe³⁺, indicating siderophore production. In comparison, only 9.6 and 15% of 94 randomly selected nonfluorescentPseudomonas strains were antibiotic producers. Differences between stimulatory and inhibitory or neutral bacteria were not apparent from antibiosis tests. On the basis of physiological tests,Pseudomonas putida was the most abundant (>62%) pseudomonad species on rough lemon roots. Growth stimulating strains appeared to be in bothP. putida andP. fluorescens groups. FewP. aeruginosa strains were identified on citrus roots.Florida Agricultural Experiment Stations Journal Series No.     

Phylogenetic identification of epibiotic bacteria possessing antimicrobial activities isolated from red algal species of Japan

We investigated the diversity of epibiotic bacteria possessing antimicrobial activity isolated from nine species of red algae, and identified their phylogenetic position. For the isolation of epibiotic bacteria, nine species of red algae, Pachymeniopsis lauceolata, Plocamium telfairiae, Gelidium amansii, Chondrus oncellatus, Grateloupia filicina, Ceramium kondoi, Lomentaria catenata, Schizymenia dubyi and Porphyra yezoensis, were collected from the intertidal zone of Awaji Island, Japan. In total 92 bacteria were collected from the above red algal species. Primary screening results using disc diffusion assay revealed that 33% of bacteria possess antibacterial activity. Ten bacteria that showed high antibacterial activity were further studied for their ability to inhibit a set of fouling bacteria, some luminescent Vibrio and Photobacterium species and a panel of pathogenic bacteria. In general, the inhibitory activities were high against fouling and luminescent bacteria, while low against various pathogenic bacteria tested. These results suggest that some epibiotic bacteria have adapted to defend their position in their surface environment through the production of antibacterial metabolites giving defense against a broad spectrum of bacterial competitors. The phylogenetic analysis using 16 S rRNA sequences identified 7 of the 10 strains as belonging to the genus Bacillus, and other strains each 1 belonging to genus Microbacterium, Psychrobacter, and Vibrio species.     

毛泡桐内生真菌的分离、拮抗细菌菌株的筛选和鉴定

为寻找新的能产生抗生素的植物内生真菌,采用3种培养基对毛泡桐进行内生真菌的分离,以大肠埃希菌、枯草芽胞杆菌、金黄色葡萄球菌、青枯假单胞菌为指示菌,利用琼脂块法和滤纸片扩散法筛选能抑制细菌的菌株;通过形态特征和ITS序列分析鉴定高活性菌株。结果从毛泡桐的根、茎、叶中共分离得到46株内生真菌,至少能抑制一种指示菌的有9株,其中菌株KLBMP-Pt630、KLBMP-Pt675和KLBMP-Pt686活性较强,鉴定结果显示:KLBMP-Pt630为三线镰刀菌、KLBMP-Pt675为棒曲霉、KLBMP-Pt686属于肉座菌目真菌。     

Paenibacillus tylopili sp.nov., a chitinolytic bacterium isolated from the mycorhizosphere of Tylopilus felleus

Two chitinolytic bacterial strains (designated MK2^T and V7) were isolated from the mycorhizosphere of the fungus Tylopilus felleus. The strains were facultatively anaerobic G⁺ endospore formers. Physiological analysis and 16S rRNA gene PCR-RFLP assays revealed nearly identical profiles for both strains, demonstrating their relationship at the species level. Sequences specific for the genus Paenibacillus were found within the 16S rRNA gene sequence of the strain MK2^T. The 16S rRNA gene sequence showed the highest similarity to the sequences of Paenibacillus amylolyticus, P. pabuli and P. xylanilyticus. DNA-DNA relatedness of the strain with the type strain of P. amylolyticus was 4.95 %, of P. pabuli 38.0 %, and of P. xylanilyticus 46.3 %, indicating no relatedness between MK2^T and any of them at the species level. The most abundant fatty acids in strains MK2^T and V7 were anteiso-C_15:0, iso-C_16:0, iso-C_15:0 and n-C_16:0. DNA-DNA relatedness, morphological, physiological and chemotaxonomic analyses, and phylogenetic data based on 16S rRNA gene sequencing made it possible to describe both strains as the novel species of the genus Paenibacillus, for which the name Paenibacillus tylopili is proposed, the type strain being MK2^T (DSM 18927^T, LMG 23975^T).     

Marine Bacillus spp. Associated With the Egg Capsule of Concholepas concholepas (Common Name “Loco”) Have an Inhibitory Activity Toward the Pathogen Vibrio parahaemolyticus

The pandemic bacterium Vibrio parahaemolyticus, isolated from seawater, sediment, and marine organisms, is responsible for gastroenteric illnesses in humans and also cause diseases in aquaculture industry in Chile and other countries around the world. In this study, bacterial flora with inhibitory activity against pathogenic V. parahaemolyticus were collected from egg capsules of Concholepas concholepas and evaluated. The 16S rRNA fragment was sequenced from each isolated strain to determine its identity using the GenBank database. A phylogenetic analysis was made, and tests for the productions of antibacterial substance were performed using the double-layer method. Forty-five morphotypes of bacterial colonies were isolated, 8 of which presented an inhibitory effect on the growth of V. parahaemolyticus. 16S rRNA sequence and phylogenetic analysis show that these strains constitute taxa that are phylogenetically related to the Bacillus genus and are probably sister species or strains of the species Bacillus pumilus, Bacillus licheniform, or Bacillus sp. It is important to determine the nature of the antibacterial substance to evaluate their potential for use against the pathogen species V. parahaemolyticus.