Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

Aim:  To investigate the applicability of rpoB gene, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP‐ and BOX‐polymerase chain reaction) were also used. Methods and Results:  rpoB DNA (458 bp) were amplified from 21 Geobacillus‐ and Bacillus type strains, producing different BOX‐ and REP‐PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. Conclusion:  BOX‐ and REP‐PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. Significance and Impact of the Study:  The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.     

Biochemical and phylogenetic characterization of two novel deep-sea Thermococcus isolates with potentially biotechnological applications

The partial 16S rDNA gene sequences of two thermophilic archaeal strains, TY and TYS, previously isolated from the Guaymas Basin hydrothermal vent site were determined. Lipid analyses and a comparative analysis performed with 16S rDNA sequences of similar thermophilic species showed that the strains isolated from deep-sea vents were not identical to the other species belonging to the genus Thermococcus. On the basis of the results of the phylogenetic analyses, lipid analyses, and previously reported physiological data, we believe that strains TY and TYS are significantly different from the previously described Thermococcus species. According to specific physiological and molecular features, we propose the use of these isolates as potential tools for the development of biotechnological applications in the field of starch processing and DNA technology. Received: 19 August 1996 / Accepted: 6 November 1996     

Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.     

Chemical Characterization of Exopolysaccharides from Antarctic Marine Bacteria

Exopolysaccharides (EPS) may have an important role in the Antarctic marine environment, possibly acting as ligands for trace metal nutrients such as iron or providing cryoprotection for growth at low temperature and high salinity. Ten bacterial strains, isolated from Southern Ocean particulate material or from sea ice, were characterized. Whole cell fatty acid profiles and 16S rRNA gene sequences showed that the isolates included representatives of the genera Pseudoalteromonas, Shewanella, Polaribacter, and Flavobacterium as well as one strain, which constituted a new bacterial genus in the family Flavobacteriaceae. The isolates are, therefore, members of the “Gammaproteobacteria” and Cytophaga-Flexibacter-Bacteroides, the taxonomic groups that have been shown to dominate polar sea ice and seawater microbial communities. Exopolysaccharides produced by Antarctic isolates were characterized. Chemical composition and molecular weight data revealed that these EPS were very diverse, even among six closely related Pseudoalteromonas isolates. Most of the EPS contained charged uronic acid residues; several also contained sulfate groups. Some strain produced unusually large polymers (molecular weight up to 5.7 MDa) including one strain in which EPS synthesis is stimulated by low temperature. This study represents a first step in the understanding of the role of bacterial EPS in the Antarctic marine environment.     

Isolation and characterization of Bacillus thuringiensis strains from olive-related habitats in Turkey

Aims: To isolate Bacillus thuringiensis strains from different olive‐related habitats (olive groves and olive oil factories) in Turkey and to characterize these strains by molecular methods. Methods and Results: A total of 150 samples, consisting of olive grove soil, green olive leaves, olive leaf residues, animal faeces, olive pomace and dust, were examined for the presence of B. thuringiensis. One hundred B. thuringiensis strains were isolated from 54 environmental samples (36%) and characterized in terms of crystal morphology, cry and cyt gene content by polymerase chain reaction, plasmid profiles and 16S‐internal transcribed spacer ribosomal DNA restriction fragment length polymorphism (16S‐ITS rDNA RFLP). The highest percentage of samples containing B. thuringiensis was found in 38 out of 54 total soil samples (70%). Of the 100 B. thuringiensis isolates, the most frequent crystal shapes were irregularly shaped (24%), spherical‐irregular pointed (19%), cuboidal (17%) and spherical (16%). The cry1 plus cry4 genotype was the most abundant genotype in our collection (21%). RFLP analysis of the amplified 16S‐ITS rDNA revealed 11 distinct patterns for the isolates and 10 reference strains. Conclusions: Bacillus thuringiensis isolates showed a great genetic diversity and crystal shape heterogeneity. Significance and Impact of the Study: This is the first study on the isolation and characterization of B. thuringiensis from olive‐related habitats in Turkey. No correlation was observed between the cry genotypes and insecticidal crystal shapes of the isolates. Restriction profiles of 23% of the isolates were found to be different from those of the 10 reference strains used.     

Phylogenetic diversity based on <Emphasis Type="Italic">rrs,atpD, recA</Emphasis> genes and 16S–23S intergenic sequence analyses of rhizobial strains isolated from <Emphasis Type="Italic">Vicia faba</Emphasis> and <Emphasis Type="Italic">Pisum sativum</Emphasis> in Peru

In this study 17 isolates from effective nodules of Vicia faba and Pisum sativum var. macrocarpum growing in different soils from Peru were isolated and characterized. The isolates, presenting 11 different RAPD profiles, were distributed in three groups on the basis of their 16S-RFLP patterns. The 16S rRNA gene sequences of strains from 16S-RFLP groups I, II and III were closely related (identities higher than 99.5%) to Rhizobium leguminosarum bv. trifolii DSM 30141 (=ATCC 14480), R. leguminosarum bv. viciae DSM 30132^T and Rhizobium etli CFN42^T (=USDA 9032^T), respectively. The analysis of the 16S–23S intergenic spacer (ITS) and two housekeeping genes, atpD and recA, confirmed the identification of strains from group I, however those from groups II and III were phylogenetically divergent to strains DSM 30132^T and CFN42^T. These results support the fact that the 16S rRNA gene is not adequate for identification at species level within genus Rhizobium and suggest the existence of putative new species within the phylogenetic group of R. leguminosarum. They also confirm the need of a taxonomic revision of R. leguminosarum since the reference strains of the three biovars included in this study are phylogenetically divergent according to their ITS, atpD and recA gene sequences.     

Escherichia coli isolated from seafood: toxicity and plasmid profiles

Thirty-two Escherichia coli strains were isolated from red snapper (Lutjanus purpureus) and from seabob shrimp (Xiphopenaeus kroyeri). The strains were numbered S1–S16, and F1–F16, which corresponds to the isolation origin from shrimp (S) and fish (F). The isolates were biologically and antigenically characterized by agglutination tests with enteropathogenic E. coli (EPEC)-, enteroinvasive E. coli (EIEC)- and enterohemorrhagic E. coli (EHEC) O157-specific antisera. The ETEC enterotoxins were characterized by GMI-ELISA for enterotoxin LT-1 (thermolabile) and by inoculation of supernatants prepared from newly born mice for enterotoxin Sta. A total of 14 strains produced exotoxins, of which seven were thermolabile (LT) and seven were thermostable (ST). Antimicrobial susceptibility profiles were determined by disc diffusion in agar using ampicillin, cephalothin, cefoxitin, ceftriaxone, imipenem, nalidixic acid, ciprofloxacin, chloramphenicol, gentamicin, nitrofurantoin, sulfamethoxazole–trimethoprim, and tetracycline. Four isolates showed lower susceptibility to some antibiotics, two strains were resistant to ampicillin, tetracycline, and sulfamethoxazole-trimethoprim, and two were resistant to tetracycline and nitrofurantoin. Plasmids were extracted in the four resistant isolates; two of them contained plasmids whose molecular weight varied from low to high. The characterization of LT- and ST-toxin-producing E. coli strains displaying multiresistance and containing plasmids suggests the need for tightening current control measures for the use of antimicrobials. Electronic Publication     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.     

Characterization of freshwater benthic biofilm‐forming Hydrocoryne (Cyanobacteria) isolates from Antarctica

The aims of this work were to study cyanobacterial isolates resembling the genus Hydrocoryne using a combination of morphology and phylogeny of 16S rRNA and nifH sequences and to investigate genes involved in cyanotoxin and protease inhibitor production. Four new cyanobacterial strains, isolated from biofilm samples collected from King George Island, Antarctica, were studied. In terms of morphology, these new strains share traits similar to true Anabaena morphotypes (benthic ones), whereas phylogenetic analysis of their 16S rRNA gene sequences grouped them with the sequence of the type species Hydrocoryne spongiosa (H. Schwabe ex Bornet and Flahault 1886–1888), but not with sequences of the type species from the genus Anabaena. This cluster is the sister group of Anabaena morphotypes isolated only from the Gulf of Finland. In addition, this cluster is related to two other clusters formed by sequences of Anabaena isolated from different sites. Partial nifH genes were sequenced from two strains and the phylogenetic tree revealed that the Antarctic nifH sequences clustered with sequences from Anabaena. Furthermore, two strains were tested, using PCR with specific primers, for the presence of genes involved in cyanotoxins (microcystin and saxitoxin) and protease inhibitor (aeruginosin, and cyanopeptolin). Only cyanopeptolin was amplified using PCR. These four Hydrocoryne strains are the first to be isolated and sequenced from Antarctica, which improves our knowledge on this poorly defined cyanobacterial genus.     

Genotypic Diversity of Oscillatoriacean Strains Belonging to the Genera <Emphasis Type="Italic">Geitlerinema</Emphasis> and <Emphasis Type="Italic">Spirulina</Emphasis> Determined by 16S rDNA Restriction Analysis

Genotypic diversity of several cyanobacterial strains mostly isolated from marine or brackish waters, belonging to the genera Geitlerinema and Spirulina, was investigated by amplified 16S ribosomal DNA restriction analysis and compared with morphological features and response to salinity. Cluster analysis was performed on amplified 16S rDNA restriction profiles of these strains along with profiles obtained from sequence data of five Spirulina-like strains, including three representatives of the new genus Halospirulina. Our strains with tightly coiled trichomes from hypersaline waters could be assigned to the Halospirulina genus. Among the uncoiled strains, the two strains of hypersaline origin clustered together and were found to be distant from their counterparts of marine and freshwater habitat. Moreover, another cluster, formed by alkali-tolerant strains with tightly coiled trichomes, was well delineated. Received: 14 May 2002 / Accepted: 22 July 2002     

Comparison of 16S rRNA gene sequences of genus <Emphasis Type="Italic">Methanobrevibacter</Emphasis>

Background  

The phylogeny of the genus Methanobrevibacter was established almost 25 years ago on the basis of the similarities of the 16S rRNA oligonucleotide catalogs. Since then, many 16S rRNA gene sequences of newly isolated strains or clones representing the genus Methanobrevibacter have been deposited. We tried to reorganize the 16S rRNA gene sequences of this genus and revise the taxonomic affiliation of the isolates and clones representing the genus Methanobrevibacter.     

Analysis and comparison of the microflora isolated from fresco surface and from surrounding air environment through molecular and biodegradative assays

The aim of this study was to find a correlation among the environmental isolated microflora and the fresco colonizators through the investigation of their biodegradative abilities and DNA characteristics. A molecular technique named RAMP (Random Amplified Microsatellite Polymorphisms) was utilized in order to analyze the DNA diversity of bacterial and fungal species isolated from fresco as well as from air samples. The RAMP-PCR results were combined with the screening of some biodegradative properties obtained through the use of specific agar plate assays detecting the proteolytic, solubilization and biomineralization abilities of the isolated microflora. This comparative analysis showed that only in few cases a direct link among the fresco and airborne isolates of specific microbial group existed. The investigation clearly evidenced that colonization of surface of Ladislav’s fresco occurred in different time and by different strains than those observed at the moment of sampling campaign. Furthermore, the microflora investigation permitted the identification of taxonomically interesting bacteria with particular biodegradative properties, which had been less studied until now.     

Phylogenetically Diverse New Sulfur Chemolithotrophs of α-Proteobacteria Isolated from Indian Soils

Five facultative sulfur chemolithotrophs were isolated from soils to study the diversity of sulfur lithotrophy. Phenotypic characteristics, including sulfur lithotrophic properties and chemotaxonomic features of the isolates, were similar to those of the members of the colorless sulfur bacteria. 16S rDNA sequence analyses rendered placing the isolates to three distinct phylogenetic clusters of -proteobacteria. Three isolates, AS001, AS002, and KCT002, were identified as members of the genus Paracoccus. The strains AS001 and AS002, having identical 16S-rDNA sequence, showed significant 16S rDNA sequence similarity (99.1%) to Paracoccus versutus. The strain KCT002 showed highest (98%) 16S rDNA sequence similarity to P. alcaliphilus and 96% similarity to the pair AS001 and AS002. Isolate KCT001 appeared to be closely related to Pseudaminobacter salicylatoxidans, although sulfur lithotrophy of P. salicylotoxidans is not known. The other isolate, TCK, showed almost identical 16S rDNA (99.9%) sequence with two recently described unclassified chemolithoautotrophic arsenite oxidizing strains. Physiological and chemotaxonomic characteristics and phylogenetic analyses of the five new strains emphasize the need of polyphasic bacterial taxonomy of sulfur lithotrophs.     

Identification of cultured and uncultured picocyanobacteria from a mesotrophic freshwater lake based on the partial sequences of 16S rDNA

Eight cultured strains (OK01, OK02, OK03, OK05, OK07, OK08, OK09, and OK10) of picocyanobacteria were isolated from Lake Okutama. Five cyanobacterial DNA fragments (DGGE bands; B4, B5, B6, B7, and B8) were obtained from the lake water samples by denaturing gradient gel electrophoresis (DGGE) after polymerase chain reaction (PCR) amplification of 16S ribosomal genes. To classify the picocyanobacterial strains and the DGGE bands, a partial sequence of 16S rDNA was used. Among seven strains, OK01, OK07, and OK09 were identified as the genus Synechococcus and OK02 and OK05 as the genus Phormidium. OK03 was identified as the genus Oscillatoria and was closely related to B4 (100% homology). B5, B6, B7, and B8 were related to the genus Synechococcus. These results revealed that the picocyanobacteria in the lake are phylogenetically diverse. PCR-DGGE analysis is a useful tool to determine picocyanobacterial community structure in freshwater environments. Received: February 25, 2001 / Accepted: July 27, 2001     

Genome profiling implies high genetic diversity in microsporidia isolated from the common cutworm, <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Lepidoptera: Noctuidae), in Vietnam

In order to evaluate the potential application of microsporidia as a microbial control agent against lepidopteran insect pests, microsporidian infection in a field population of the common cutworm, Spodoptera litura (Fabricius), was surveyed in vegetable crop fields in Can Tho City, Vietnam, in March 2007. The infection rate of microsporidia was 46.7% (99/212 individuals) in adult S. litura, and 16 samples of infected adults were used to characterize the microsporidia at the molecular level. Analysis of the small subunit ribosomal RNA (SSU rRNA) sequences indicated that microsporidian strains isolated from S. litura were closely related to Nosema bombycis from the silkworm, Bombyx mori (Linnaeus); however, phylogenetic analysis based on genome profiling produced a different result from the SSU rRNA sequences. Temperature gradient gel electrophoresis profiles of 12 microsporidian strains from S. litura were closely related to N. bombycis strains, while the profiles of three microsporidian strains formed a different cluster. The Vietnamese strains did not form a single group, but were classified into at least three groups. These results suggested that the microsporidia isolated from S. litura in the Mekong Delta, Vietnam, are genetically diverse.     

Characterization of rhizospheric bacteria isolated from Deschampsia antarctica Desv.

Deschampsia antarctica Desv. is the only gramineae capable of colonizing the Antarctic due to the region’s extreme climate and soil environment. In the present research, bacteria colonizing the rhizospheric soil of D. antarctica were isolated and characterized. The soil studies showed that D. antarctica possesses a wide spectrum of psychrotolerant bacteria with extensive and varied antibiotic resistance, as well as heavy metal tolerance. The bacterial strains isolated from the rhizosphere of D. antarctica also produced a diverse pattern of enzymes. Based on the strain identification with partial characterization of the 16S rRNA gene, the majority of the isolates correspond to different Pseudomonas species, and species of the genus Flavobacterium sp. and Arthrobacter sp. The isolated strains collected from this research constitute a unique collection for future, more detailed taxonomic analysis and physiological characterization, contributing to the search for potential biotechnological uses. These findings and others have great potential for developing new biotechnological products from Antarctic microorganisms.     

Distribution and identification of red yeasts in deep-sea environments around the northwest Pacific Ocean

We isolated 99 yeast strains, including 40 red yeasts, from benthic animals and sediments collected from the deep-sea floor in various areas in the northwest Pacific Ocean. Comparing the yeast isolates from animals and sediments collected from shallow locations, the proportion of red yeasts differed considerably, comprising 81.5% and 10.6% of the isolates from animals and sediments, respectively. All of the red yeast isolates belonged to the genera Rhodotorula and Sporobolomyces. On the basis of morphological and physiological characteristics, the isolates were identified as R. aurantiaca, R. glutinis, R. minuta and R. mucilaginosa of the genus Rhodotorula, and S. salmonicolor and S. shibatanus of the genus Sporobolomyces. Only R. glutinis and R. mucilaginosa were isolated from sediments. All of the others were isolated from animal sources. Phylogenetic analyses based on internal transcribed spacer (ITS) regions and 5.8S rRNA gene sequences allowed us to establish the precise taxonomic placement of each of the isolates and thereby investigate the intraspecific relationships among the isolates. Twenty-two strains identified as members of R. glutinis, which showed a wide distribution in the deep-sea, and five isolates identified as R. minuta, which were isolated only from benthic animals, showed substantial heterogeneity within the species. The isolates phenotypically identified as Sporobolomyces species and R. mucilaginosa phylogenetically occupied the placements corresponding to these species. Some strains assigned to known species on the basis of phenotypic features should be regarded as new species as suggested by the results of molecular analysis.     

Haloarcula spp able to biosynthesize exo- and endopolymers

Three halobacteria strains (T5, T6, T7), growing optimally at a sodium chloride concentration of 3.5 M, were isolated from samples collected in the Tunisian marine saltern located close to Monastir. Although they were different in colony and cell morphology, the isolates were similar in most respects and all produced a homopolyester identified spectroscopically as polyhydroxybutyrate when grown on carbohydrates. Under the same conditions, the isolates formed acidic exopolysaccharides. Acid methanolysis of their complex lipids released archaeol (2,3-di-O-phytanyl-sn-glycerol). All the isolates had polar lipid patterns characteristic of representatives of the genus Haloarcula. Partial sequence analysis of 16S rRNA genes from strain T5 confirmed its assignment to the genus Haloarcula. Although strain T5 differed phenotypically from the species Haloarcula japonica in several respects, DNA–DNA hybridization indicated that the T5 isolate is a new strain of H. japonica. Received 10 June 1998/ Accepted in revised form 29 July 1999     

Identification and characterization of chitinolytic bacteria isolated from a freshwater lake

To develop a novel type of biocontrol agent, we focus on bacteria that are characterized by both chitinase activity and biofilm development. Chitinolytic bacteria were isolated from sediments and chitin flakes immersed in the water of a sand dune lake, Sakata, in Niigata, Japan. Thirty-one isolates from more than 5100 isolated strains were examined chitinase activity and biofilm formation. Phylogenetic analysis of these isolates based on the 16S rRNA gene sequences revealed that most isolates belonged to the family Aeromonadaceae, followed by Paenibacillaceae, Enterobacteriaceae, and Neisseriaceae. The specific activity of chitinase of four selected strains was higher than that of a reference strain. The molecular size of one chitinase produced by Andreprevotia was greater than that of typical bacterial chitinases. The dialyzed culture supernatant containing chitinases of the four strains suppressed hyphal growth of Trichoderma reesei. These results indicate that these four strains are good candidates for biocontrol agents.     

Bias Caused by Using Different Isolation Media for Assessing the Genetic Diversity of a Natural Microbial Population

Abstract The influence of isolation medium on the biodiversity of Burkholderia cepacia strains recovered from the rhizosphere of Zea mays was evaluated by comparing the genetic diversity of isolates obtained by plating serial dilutions of root macerates on the two selective media TB-T and PCAT. From each medium, 50 randomly chosen colonies were isolated. On the basis of the restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA), all strains isolated from TB-T medium were assigned to the B. cepacia species, whereas among PCAT isolates only 74% were assigned to the B. cepacia species. Genetic diversity among the PCAT and TB-T isolates was evaluated by the random amplified polymorphic DNA (RAPD) technique. The analysis of molecular variance (AMOVA) method was applied to determine the variance component for RAPD patterns. Most of the genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001). Mean genetic distances among PCAT isolates (10.39) and TB-T isolates (9.36) were significantly different (P < 0.0001). The results indicate that the two different isolation media select for B. cepacia populations with a different degree of genetic diversity. Moreover, a higher degree of genetic diversity was observed among strains isolated from PCAT medium than among those isolated from TB-T medium. Received: 29 April 1999; Accepted: 27 January 2000; Online Publication: 28 August 2000