Genetic and Functional Diversity Among the Antagonistic Potential Fluorescent Pseudomonads Isolated from Tea Rhizosphere

Twenty-five fluorescent pseudomonads from rhizospheric soil of six tea gardens in four district of Upper Assam, India were isolated and screened for antagonistic activity against fungal pathogens such as Fusarium oxysporum f. sp. raphani (For), Fusarium oxysporum f. sp. ciceri (Foc), Fusarium semitectum (Fs), and Rhizoctonia solani (Rs); and bacterial pathogens—Staphylococcus aureus (Sa), Escherichia coli (Ec), and Klebsiella pneumoniae (Kp). Most of the isolates exhibited strong antagonistic activity against the fungal pathogens and gram-positive bacterium i.e. Staphylococcus aureus. Productions of siderophore, salicylic acid (SA), hydrogen cyanide (HCN), and cell wall-degrading enzyme (chitinase) were studied to observe the possible mechanisms of antagonistic activity of the isolates. Correlation between the antagonistic potentiality of some isolates and their levels of production of siderophore, salicylic acid, and hydrogen cyanide was observed. Out of the 25 isolates, antibiotic-coding genes, 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) were detected in the isolates, Pf12 and Pf373, respectively. Genetic diversity of these fluorescent pseudomonads were analyzed with reference to four strains of Pseudomonas fluorescens NICM 2099^T, P. aeruginosa MTCC 2582^T, P. aureofaciens NICM 2026^T, and P. syringae MTCC 673^T. 16S rDNA-RFLP analysis of these isolates using three tetra cutter restriction enzymes (HaeIII, AluI and MspI) revealed two distinct clusters. Cluster A comprised only two isolates Pf141 and 24-PfM3, and cluster B comprised 23 isolates along with four reference strains.     

<Emphasis Type="Italic">Flavobacterium koreense</Emphasis> sp. nov., <Emphasis Type="Italic">Flavobacterium chungnamense</Emphasis> sp. nov., and <Emphasis Type="Italic">Flavobacterium cheonanense</Emphasis> sp. nov., isolated from a freshwater reservoir

Taxonomic studies were performed on three strains isolated from Cheonho reservoir in Cheonan, Korea. The isolates were Gram-negative, aerobic, rod-shaped, non-motile, catalase-positive, and oxidase-positive. Colonies on solid media were cream-yellow, smooth, shiny, and circular. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belong to the genus Flavobacterium. The strains shared 98.6–99.4% sequence similarity with each other and showed less than 97% similarity with members of the genus Flavobacterium with validly published names. The DNA-DNA hybridization results confirmed the separate genomic status of strains ARSA-42^T, ARSA-103^T, and ARSA-108^T. The isolates contained menaqui-none-6 as the predominant menaquinone and iso-C_15:0, iso-C_15:0 3-OH, iso-Ci_15:1 G, and iso-C_16:0 3-OH as the major fatty acids. The genomic DNA G+C content of the isolates were 31.4–33.2 mol%. According to the phenotypic and genotypic data, these organisms are classified as representative of three novel species in the genus Flavobacterium, and the name Flavobacterium koreense sp. nov. (strain ARSA-42^T =KCTC 23182^T =JCM 17066^T =KACC 14969^T), Flavobacterium chungnamense sp. nov. (strain ARSA-103^T =KCTC 23183^T =JCM 17068^T =KACC 14971^T), and Flavobacterium cheonanense sp. nov. (strain ARSA-108^T =KCTC 23184^T =JCM 17069^T =KACC 14972) are proposed.     

Molecular characterization of Antarctic actinobacteria and screening for antimicrobial metabolite production

The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231^T, Staphylococcus aurues ATCC 51650^T, methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44^T and Pseudomonas aeruginosa ATCC 10145^T, respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus—polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.     

Arcobacter ellisii sp. nov., isolated from mussels

As part of a study carried out for detecting Arcobacter spp. in shellfish, three mussel isolates that were Gram-negative slightly curved rods, non-spore forming, showed a new 16S rDNA-RFLP pattern with a specific identification method for the species of this genus. Sequences of the 16S rRNA gene and those of the housekeeping genes rpoB, gyrB and hsp60 provided evidence that these mussel strains belonged to an unknown genetic lineage within the genus Arcobacter. The similarity between the 16S rRNA gene sequence of the representative strain (F79-6^T) and type strains of the other Arcobacter species ranged between 94.1% with A. halophilus and 99.1% with the recently proposed species A. defluvii (CECT 7697^T). DDH results between strain F79-6^T and the type strain of the latter species were below 70% (53 ± 3.0%). Phenotypic characteristics together with MALDITOF mass spectra differentiated the new mussel strains from all other Arcobacter species. All the results indicate that these strains represent a new species, for which the name Arcobacter ellisii sp. nov. with the type strain F79-6^T (=CECT 7837^T = LMG 26155^T) is proposed.     

Synthesis,molecular docking,binding free energy calculation and molecular dynamics simulation studies of benzothiazol-2-ylcarbamodithioates as Staphylococcus aureus MurD inhibitors

Abstract

A new series of benzothiazol-2-ylcarbamodithioate functional compounds 5a-f has been designed, synthesized and characterized by spectral data. These compounds were screened for their in vitro antibacterial activity against strains of Staphylococcus aureus (NCIM 5021, NCIM 5022 and methicillin-resistant isolate 43300), Bacillus subtilis (NCIM 2545), Escherichia coli (NCIM 2567), Klebsiella pneumoniae (NCIM 2706) and Psudomonas aeruginosa (NCIM 2036). Compounds 5a and 5d exhibited significant activity against all the tested bacterial strains. Specifically, compounds 5a and 5d showed potent activity against K. pneumoniae (NCIM 2706), while compound 5a also displayed potent activity against S. aureus (NCIM 5021). Compound 5d showed minimum IC₅₀ value of 13.37?μM against S. aureus MurD enzyme. Further, the binding interactions of compounds 5a-f in the catalytic pocket have been investigated using the extra-precision molecular docking and binding free energy calculation by MM-GBSA approach. A 30?ns molecular dynamics simulation of 5d/modeled S. aureus MurD enzyme was performed to determine the stability of the predicted binding conformation.     

Methylobacillus arboreus sp. nov., and Methylobacillus gramineus sp. nov., novel non-pigmented obligately methylotrophic bacteria associated with plants

Two newly isolated obligate methanol-utilizing bacteria (strains Iva^T and Lap^T) with the ribulose monophosphate pathway of C₁ assimilation are described. The isolates are strictly aerobic, Gram negative, asporogenous, motile rods multiplying by binary fission, mesophilic and neutrophilic, synthesize indole-3-acetate. The prevailing cellular fatty acids are straight-chain saturated C_16:0 and unsaturated C_16:1 acids. The major ubiquinone is Q-8. The predominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia is assimilated by glutamate dehydrogenase. The DNA G+C contents of strains Iva^T and Lap^T are 54.0 and 50.5 mol% (T_m), respectively.Based on 16S rRNA gene sequence analysis and DNA–DNA relatedness (38–45%) with type strains of the genus Methylobacillus, the novel isolates are classified as the new species of this genus and named Methylobacillus arboreus Iva^T (VKM B-2590^T, CCUG 59684^T, DSM 23628^T) and Methylobacillus gramineus Lap^T (VKM B-2591^T, CCUG 59687^T, DSM 23629^T).The GenBank accession numbers for the 16S rRNA gene and mxaF gene sequences of the strains Iva^T and Lap^T are GU937479, GU937478 and HM030736, HM030735, respectively.     

Bacillus locisalis sp. nov., a new haloalkaliphilic species from hypersaline and alkaline lakes of China, Kenya and Tanzania

A polyphasic taxonomic study was performed on seven Bacillus-like bacteria isolated from three hypersaline and alkaline lakes located in China, Kenya and Tanzania. All strains were moderately halophilic and alkaliphilic, Gram positive, motile rods. The DNA G+C content from the seven isolates ranged from 42.2 to 43.4 mol% and their major fatty acid was anteiso-C_15:0. Strain CG1^T, selected as representative strain of the isolates, possesses meso-diaminopimelic acid in the cell wall peptidoglycan, MK-7 as the predominant menaquinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as the major polar lipids. Comparative 16S rRNA gene sequence analysis indicated that the isolates belonged to the genus Bacillus. The seven isolates shared 97.7–99.9% 16S rRNA gene sequence similarity, and formed a branch that was distinct from the type strains of the recognized species of the genus Bacillus. They were most closely related to Bacillus agaradhaerens DSM 8721^T (92.6–93.8% 16S rRNA sequence similarity). DNA–DNA hybridization values between the seven isolates were 85–100%. According to the polyphasic characterization, the strains represent a novel species, for which the name Bacillus locisalis sp. nov. is proposed. The type strain is CG1^T (CCM 7370^T = CECT 7152^T = CGMCC 1.6286^T = DSM 18085^T).     

Branchiibius cervicis sp. nov., a novel species isolated from patients with atopic dermatitis

Novel actinobacterial strains, PAGU 1247^T, PAGU 1251 and PAGU 1252, were isolated from the skin of atopic dermatitis patients and were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that these isolates were located within the family Dermacoccaceae. The most closely related species of PAGU 1247^T in phylogenetic terms was Branchiibius hedensis Mer 29717^T, with 16S rRNA gene sequence similarity of 99.6%, although the DNA–DNA relatedness value was less than 43.9%. Some biochemical traits, such as lipase (C14) and α-galactosidase activity, could distinguish these isolates from B. hedensis. Strain PAGU 1247^T contained iso-C_16:0 and brC_18:0 as the major fatty acids. The quinone system consisted of menaquinone MK-8(H₆ and H₄). The G + C content of the genomic DNA was 67.6 mol%. On the basis of its phenotypic properties and genetic distinctiveness, strains PAGU 1247^T, PAGU 1251 and PAGU 1252 represents a novel species of the genus Branchiibius, for which the name Branchiibius cervicis sp. nov. is proposed. The type strain is PAGU 1247^T (=NBRC 106593^T = DSM 24166^T).     

Role of the Outer Membrane Protein OprD2 in Carbapenem-Resistance Mechanisms of Pseudomonas aeruginosa

We investigated the relationship between the outer membrane protein OprD₂ and carbapenem-resistance in 141 clinical isolates of Pseudomonas aeruginosa collected between January and December 2013 from the First Affiliated Hospital of Anhui Medical University in China. Agar dilution methods were employed to determine the minimum inhibitory concentration of meropenem (MEM) and imipenem (IMP) for P. aeruginosa. The gene encoding OprD₂ was amplified from141 P. aeruginosa isolates and analyzed by PCR and DNA sequencing. Differences between the effects of IMP^R and IMP^S groups on the resistance of the P. aeruginosa were observed by SDS-poly acrylamide gel electrophoresis (SDS-PAGE). Three resistance types were classified in the 141 carbapenem-resistant P. aeruginosa (CRPA) isolates tested, namely IMP^RMEM^R (66.7%), IMP^RMEM^S (32.6%), and IMP^RMEM^S (0.7%). DNA sequencing revealed significant diverse gene mutations in the OprD₂-encoding gene in these strains. Thirty-four strains had large fragment deletions in the OprD₂gene, in 6 strains the gene contained fragment inserts, and in 96 resistant strains, the gene featured small fragment deletions or multi-site mutations. Only 4 metallo-β-lactamase strains and 1 imipenem-sensitive (meropenem-resistant) strain showed a normal OprD₂ gene. Using SDS-PAGE to detect the outer membrane protein in 16 CRPA isolates, it was found that 10 IMP^RMEM^R strains and 5 IMP^RMEM^S strains had lost the OprD₂ protein, while the IMP^SMEM^R strain contained a normal 46-kDa protein. In conclusion, mutation or loss of the OprD₂-encoding gene caused the loss of OprD₂, which further led to carbapenem-resistance of P. aeruginosa. Our findings provide insights into the mechanism of carbapenem resistance in P. aeruginosa.     

Neokomagataea gen. nov., with Descriptions of Neokomagataea thailandica sp. nov. and Neokomagataea tanensis sp. nov., Osmotolerant Acetic Acid Bacteria of the α-Proteobacteria

Isolates AH11^T and AH13^T were isolated from flowers of lantana and candle bush respectively collected in Thailand. In phylogenetic trees based on 16S rRNA gene sequences, the two isolates formed an independent cluster, which was then connected to the type strain of Saccharibacter floricola. The calculated pair-wise 16S rRNA gene sequence similarities of isolate AH11^T were 95.7–92.3% to the type strains of the type species of the 12 genera of acetic acid bacteria. The DNA base composition was from 51.2 to 56.8 mol % G+C, with a range of 5.6 mol %. When isolate AH11^T was labeled, DNA-DNA similarities were 100, 12, 4, 5, and 4% respectively to isolates AH11^T and AH13^T and the type strains of Saccharibacter floricola, Gluconobacter oxydans, and Acetobacter aceti. The two isolates were non-motile and did not oxidize either acetate or lactate. No growth was found in the presence of 0.35% acetic acid w/v. The two isolates were not osmophilic but osmotolerant, produced 2,5-diketo-D-gluconate from D-glucose, and did not oxidize lactate, thus differing from strains of Saccharibacter floricola, which showed weak lactate oxidation. The two isolates contained unsaturated C_18:1ω7c fatty acid as the major fatty acid, and were unique in the presence of a considerable amount of straight-chain C_18:12OH fatty acid. Q-10 was present as the major isoprenoid quinone. Neokomagataea gen. nov. was proposed with the two species, Neokomagataea thailandica sp. nov. for isolate AH11^T (=BCC 25710 ^T =NBRC 106555^T), which has 56.8 mol % G+C, and Neokomagataea tanensis sp. nov. for isolate AH13^T (=BCC 25711^T=NBRC 106556^T), which has 51.2 mol % G+C.     

<Emphasis Type="Italic">Paenibacillus pini</Emphasis> sp. nov., a cellulolytic bacterium isolated from the rhizosphere of pine tree

Strain S22^T, a novel cellulolytic bacterium was isolated from the rhizosphere of pine trees. This isolate was Gram-reaction positive, motile and rods, and formed terminal or subterminal ellipsoidal spores. S22^T represented positive activity for catalase, oxidase, esterase (C4), esterase lipase (C8), β-galactosidase, leucine arylamidase, and hydrolysis of esculin. It contained meso-diaminopimelic acid as the diagnostic dia-mino acid in the cell-wall. The predominant isoprenoid quinone was menaquinone 7 (MK-7), and the major cellular fatty acids were anteiso-C_15:0 (52.9%), iso-Ci_16:0 (11.3%), and iso-C_15:0 (10.0%). The DNA G+C content was 43.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that this isolate belonged to the family Paenibacillaceae. S22^T exhibited less than 97.0% 16S rRNA gene similarity with all relative type strains in the genus Paenibacillus, and the most closely related strains were Paenibacillus anaericanus MH21^T and Paenibacillus ginsengisoli Gsoil 1638^T, with equal similarities of 95.8%. This polyphasic evidence suggested that strain S22^T should be considered a novel species in the genus Paenibacillus, for which the name, Paenibacillus pini sp. nov., is proposed. The type strain is S22^T (=KCTC 13694^T =KACC 14198^T =JCM 16418^T)     

Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa

Most clinical isolates identified as Pseudomonas aeruginosa grow on long-chain n-alkanes, while environmental P. aeruginosa isolates often grow on medium- as well as long-chain n-alkanes. Heterologous expression showed that the two alkane hydroxylase homologs of P. aeruginosa PAO1 (AlkB1 and AlkB2) oxidize C₁₂-C₁₆ n-alkanes, while two rubredoxin (RubA1 and RubA2) and a rubredoxin reductase (RubB) homologs can replace their P. putida GPo1 counterparts in n-octane oxidation. The two long-chain alkane hydroxylase genes are present in all environmental and clinical isolates of P. aeruginosa strains tested in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

Aeromonas piscicola sp. nov., isolated from diseased fish

Four Aeromonas strains (S1.2^T, EO-0505, TC1 and TI 1.1) isolated from moribund fish in Spain showed a restriction fragment length polymorphism (RFLP) pattern related to strains of Aeromonas salmonicida and Aeromonas bestiarum but their specific taxonomic position was unclear. Multilocus sequence analysis (MLSA) of housekeeping genes rpoD, gyrB, recA and dnaJ confirmed the allocation of these isolates to an unknown genetic lineage within the genus Aeromonas with A. salmonicida, A. bestiarum and Aeromonas popoffii as the phylogenetically nearest neighbours. Furthermore, a strain biochemically labelled as Aeromonas hydrophila (AH-3), showing a pattern of A. bestiarum based on 16S rDNA-RFLP, also clustered with the unknown genetic lineage. The genes rpoD and gyrB proved to be the best phylogenetic markers for differentiating these isolates from their neighbouring species. Useful phenotypic features for differentiating the novel species from other known Aeromonas species included their ability to hydrolyze elastin, produce acid from l-arabinose and salicin, and their inability to produce acid from lactose and use l-lactate as a sole carbon source. A polyphasic approach using phenotypic characterization, phylogenetic analysis of the 16S rRNA gene and of four housekeeping genes, as well as DNA–DNA hybridization studies and an analysis of the protein profiles by MALDI-TOF-MS, showed that these strains represented a novel species for which the name Aeromonas piscicola sp. nov. is proposed with isolate S1.2^T (=CECT 7443^T, =LMG 24783^T) as the type strain.     

Cohnella soli sp. nov. and Cohnella suwonensis sp. nov. Isolated from soil samples in Korea

Two bacterial isolates from soil samples taken in Korea, strains YM2-7^T and WD2-19^T, were characterized using a polyphasic approach. The cells were strictly aerobic, Gram-positive, motile with peritrichous flagella, and rod-shaped. Both strains formed ellipsoidal bulging positioned subterminal spores. Phylogenetic analysis of their 16S rRNA gene sequences revealed a clear affiliation with the Firmicutes. The 16S rRNA gene sequence similarity between YM2-7^T and WD2-19^T was 96.5%. Strains YM2-7^T and WD2-19^T showed 16S rRNA gene sequence similarities of 93.0–96.5% to type strains of recognized Cohnella species. The G+C contents of the DNA of strains YM2-7^T and WD2-19^T were 52.2 and 55.6 mol%, respectively. The major fatty acids of strains YM2-7^T and WD2-19^T were anteiso-C15:0 (44.4%), C16:0 (19.2%), and iso-C16:0 (16.8%) and anteiso-C15:0 (46.5%), iso-C16:0 (21.8%), and C16:0 (11.2%), respectively. Both strains contained menaquinone with seven isoprene units (MK-7) as the predominant quinone. Both strains had diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and lysophosphatidylglycerol as the major polar lipids. Comparative analysis of phenotypic and phylogenetic traits indicated that strains YM2-7^T and WD2-19^T represented two novel species of the genus Cohnella. The names Cohnella soli sp. nov. (type strain YM2-7^T =KACC 13346^T =NBRC 106486^T), and Cohnella suwonensis sp. nov. (type strain WD2-19T =KACC 13347^T =NBRC 106485^T) are proposed for these organisms.     

Spodiobacter cordis gen. nov. sp. nov., a member of the family Flavobacteriaceae isolated from patients with infective endocarditis

Two gram‐negative, catalase‐negative, oxidase‐positive strains (PAGU 1467^T and PAGU 1468) isolated from patients with infective endocarditis were investigated to determine their taxonomic status. 16S rRNA gene sequence analysis indicated that the two strains were members of the Bergeyella‐Chryseobacterium‐Riemerella branch of the family Flavobacteriaceae. Strains PAGU 1467^T and PAGU 1468 were highly related to each other (98.8% 16S rRNA gene sequence similarity). Phylogenetically closely‐related species to PAGU 1467^T comprised Bergeyella zoohelcum (95.0% 16S rRNA gene sequence similarity), Riemerella anatipestifer (94.3%) and Cloacibacterium normanense (94.3%). The major fatty acids of the two isolates were iso‐C_15:0, iso‐C_17:0 3‐OH and iso‐C_15:0 3‐OH. The presence of C_16:0 3‐OH and iso‐C_15:0 2‐OH allowed these isolates to be distinguished from B. zoohelcum. Menaquinone MK‐6 was the only respiratory quinone in these organisms; this is a consistent characteristic of the family Flavobacteriaceae. The guanine‐plus‐cytosine content of the genomic DNA was 42.0%, which is higher than that of other close phylogenetic relatives. On the basis of their phenotypic properties and genetic distinctiveness, isolates PAGU 1467^T and PAGU 1468 were classified within the novel genus Spodiobacter, as Spodiobacter cordis gen. nov., sp. nov., which is also the type species. The type strain of S. cordis is PAGU 1467^T ( = CCUG 65564^T = NBRC 109998^T).     

Pseudomonas sivasensis sp. nov. isolated from farm fisheries in Turkey

A study of 91 isolates from fish farms in Turkey showed that isolates P7^T, P11, P24b, P29, P72, P73 and P158 belonged to the genus Pseudomonas according to 16S rRNA nucleotide sequence analysis. The analysis of the sequences of the RNA polymerase sigma factor gene (rpoD) located these strains in the Pseudomonas fluorescens lineage of species within the P. fluorescens subgroup, close to the cluster composed of the species Pseudomonas grimontii, Pseudomonas marginalis and Pseudomonas panacis. Based on similarities in the 16S rRNA and rpoD gene sequences of three previously isolated strains from other origins (CCUG 57209, CCUG 62357 and W5.2-93) linked them to the same cluster. A polyphasic taxonomic approach including phenotypic characterization, fatty acid composition, and multilocus sequence analysis, together with whole-cell MALDI-TOF data, corroborated this assumption. The genome G+C mol% contents were 59.48 and 59.71, respectively. The average nucleotide indices based on BLAST analysis and the genome-to-genome distance calculation for the P7^T and CCUG 57209 strains with their closest relative, P. grimontii, were 88.16–88.29% and 38.10–38.20%, respectively. These data confirm that isolates P7^T, P11, P24b, P29, P72, P73, P158, CCUG 57209, CCUG 62357 and W5.2-93 represent a new species for which the name Pseudomonas sivasensis is proposed, with P7^T as a type strain (=CCUG 74260^T= and CECT30107^T).     

<Emphasis Type="Italic">Rhizobium sphaerophysae</Emphasis> sp. nov., a novel species isolated from root nodules of <Emphasis Type="Italic">Sphaerophysa salsula</Emphasis> in China

Four gram-negative, aerobic, motile, non-spore, forming rods with a wide pH and temperature range for growth (pH 7.0–11.0, optimum pH 8.0; 20–45°C, optimum 28°C) strains were isolated from root nodules of Sphaerophysa salsula and characterized by means of a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the four strains formed a new lineage related to the genus Rhizobium and the sequence similarities between the isolate and the most related type strain Rhizobium giardinii was 96.5%. These strains also formed a distinctive group from the reference strains for defined Rhizobium species based on housekeeping gene sequences (atpD and recA), BOX-PCR fingerprinting, phenotypic features and symbiotic properties. The representative strain CCNWGS0238^T has DNA-DNA relatedness of less than 33.4% with the most closely related species R. giardinii. It is therefore proposed as a new species, Rhizobium sphaerophysae sp. nov., with isolate CCNWGS0238^T (=ACCC17498^T = HAMBI3074^T) as the type strain.     

Impact of Heavy Metals on the Selective Phenotypical Markers of Pseudomonas aeruginosa

The aim of this study was to characterize the impact of heavy metals on phenotypical markers of Pseudomonas aeruginosa. Twenty-two isolates of P. aeruginosa, either clinical (20) or secondary treated wasterwater (2), were used to inoculate micro-ecosystems of sterile distilled water or secondary waste effluent in the presence of subminimal inhibitory concentrations of a variety of heavy metals commonly encountered in the aquatic naturally habitat (Ca²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, Ni²⁺, Zn²⁺). Micro-ecosystems were exposed to visible light at laboratory temperature and individual strains were reisolated after a 1-, 3-, or 6-month period. The re-isolates (129) were characterized using hierarchical classification analysis in order to define affinities among variants of P. aeruginosa. Subsequently, discriminant analysis was used to detect eventual relationships among the different phenotypical markers studied. Results of the hierarchical classification, based on qualitative or quantitative approaches, showed clearly that incubation of P. aeruginosa in the presence of heavy metals altered the studied phenotypical markers, namely serotype, phage type, MIC of metals, and pyocin type. Discriminant analysis showed that the studied phenotypical markers could be classified into four clusters: C1 (L1 and L2 phage types, Hg tolerance and/or resistance, S2 serotype), C2 (P2 pyocin type, Cd tolerance and/or resistance, S1 serotype), C3 (Co and Cr tolerance and/or resistance) and C4 (P1 pyocin type, Ni, Zn, and Cu tolerance and/or resistance).     

Proposal of three novel species of soil bacteria, <Emphasis Type="Italic">Variovorax ureilyticus,Variovorax rhizosphaerae</Emphasis>, and <Emphasis Type="Italic">Variovorax robiniae</Emphasis>, in the family <Emphasis Type="Italic">Comamonadaceae</Emphasis>

Three novel bacterial strains (UCM-2^T, UCM-G28^T, and UCM-G35^T) were obtained while isolating soil bacteria for the development of antibiotics. Cells of these strains were Gram-negative, non-spore forming, motile by means of a single flagellum, and rod shaped. In all strains, the predominant isoprenoid quinone was ubiquinone-8 (Q-8). Cells contained C_16:0, summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), summed feature 8 (C_18:1ω7c and/or C_18:1ω6c), and C_17:0 cyclo as the major fatty acids, and C_10:0 3-OH as the major hydroxy fatty acid. The polar lipid profiles of the three novel strains were dominated by diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The genomic DNA G + C contents of strains UCM-2^T, UCM-G28^T, and UCMG35^T were 67.5, 65.9, and 66.4 mol%, respectively. Phylogenetic analyses based on 16S rRNA sequences showed that strain UCM-2^T was most closely related to Variovorax soli NBRC 106424^T, whereas strains UCM-G28^T and UCM-G35^T were most similar to Variovorax ginsengisoli Gsoil 3165^T. Values indicating DNA-DNA hybridization between the novel isolates and closely related species in the genus Variovorax were lower than the 70% cut-off point. These phenotypic, chemotaxonomic, and phylogenetic data indicate that the three isolates should be classified as new members of the genus Variovorax, for which the names Variovorax ureilyticus sp. nov., Variovorax rhizosphaerae sp. nov., and Variovorax robiniae sp. nov. are proposed. The type strains are UCM-2^T (= KACC 18899^T = NBRC 112306^T), UCMG28^T (= KACC 18900^T = NBRC 112307^T), and UCM-G35^T (= KACC 18901^T = NBRC 112308^T), respectively.     

Alishewanella solinquinati sp. nov., Isolated from Soil Contaminated with Textile Dyes

A novel Gram-negative, motile, rod-shaped, facultative anaerobic bacterial strain, KMK6^T, was isolated from soil contaminated with textile dyes from an industrial estate located at Ichalkaranji, Maharashtra, India, and its taxonomical position was established by using a polyphasic approach. The major cellular fatty acids included C_17:1ω8c, summed feature 3 (C_16:1ω7c and/or iso-C_15:0 2-OH), C_17:0, C_16:0, and C_18:1ω7c. The DNA G+C content of strain KMK6^T was 48.8 mol %. 16S rRNA gene sequence analysis confirmed its placement in the genus Alishewanella, and exhibited sequence similarity levels of below 97 % to the type strains of validly published Alishewanella species. On the basis of genotypic and phenotypic evidence, strains KMK6^T is considered to be a novel species of the genus Alishewanella, for which we propose that strain KMK6^T (=NCIM 5295^T =BCRC 17848^T) is assigned to a novel species, Alishewanella solinquinati sp. nov.