Molecular characterisation of Halobacillus strains isolated from different medieval wall paintings and building materials in Austria

We previously reported on the detection and isolation of an indigenous population of Halobacillus from salt-damaged medieval wall paintings and building materials of Herberstein castle in St. Johann bei Herberstein in Styria, Austria. Several moderately halophilic, Gram-positive, endospore-forming Halobacillus-like bacteria could be again isolated by conventional enrichment from salt efflorescences collected in the medieval St. Virgil's chapel in Vienna. Comparative 16S rDNA sequence analyses showed that the St. Virgil isolates are most closely related (>98.5% sequence similarity) to Halobacillus trueperi, Halobacillus litoralis, and to our previous halobacilli strains obtained from the castle Herberstein. Based on 16S rDNA sequence analysis, the strains could be clustered in three different groups. Group I: St. Virgil strains S3, S4, S21, and S22 (99.8–100% sequence similarity); group II: Herberstein strains K3-1, I7, and the St. Virgil strain S20 (99.3–99.7% sequence similarity); and group III: Herberstein strains I3, I3A, and I3R (100% sequence similarity). Molecular typing by denaturing gradient gel electrophoresis (DGGE), random amplified polymorphic DNA (RAPD-PCR), and internal transcribed spacer-homoduplex–heteroduplex polymorphism (ITS-HHP) fingerprinting showed that all isolates are typeable by each of the methods. RAPD was the most discriminatory method. With respect to their physiological characteristics—i.e., growth in the presence of 5–20% (w/v) NaCl, no growth in the absence of NaCl, optimum growth at 37 °C in media containing 5–10% (w/v) NaCl, and optimum pH around 7.5–8.0—the St. Virgil isolates resembled our previously isolated strains. However, the St. Virgil strains showed some differences in their biochemical properties. St. Virgil isolates hydrolysed Tween 80, two isolates reduced nitrate, and no isolate liquefied gelatine. The recurrent isolation of halobacilli from salt efflorescences on historic buildings and monuments at two different geographical locations may indicate that this group of bacteria is common in salt-affected ruins.     

Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium

In this work we analysed different chromosomal and symbiotic markers in rhizobial strains nodulating Lupinus albus (white lupin) in several continents. Collectively the analysis of their rrs and atpD genes, and 16S-23S intergenic spacers (ITS), showed that they belong to at least four chromosomal lineages within the genus Bradyrhizobium. Most isolates from the Canary Islands (near to the African continent) grouped with some strains isolated on mainland Spain and were identified as Bradyrhizobium canariense. These strains are divided into two ITS subgroups coincident with those previously described from isolates nodulating Ornithopus. The remaining strains isolated on mainland Spain grouped with most isolates from Chile (American continent) forming a new lineage related to Bradyrhizobium japonicum. The strains BLUT2 and ISLU207 isolated from the Canary Islands and Chile, respectively, formed two new lineages phylogenetically close to different species of Bradyrhizobium depending on the marker analyzed. The analysis of the nodC gene showed that all strains nodulating L. albus belong to the biovar genistearum; nevertheless they form four different nodC lineages of which lineage C is at present exclusively formed by L. albus endosymbionts isolated from different continents.     

A polyphasic taxonomic study of thermophilic bacilli from shallow, marine vents

Eighty-seven thermophilic, aerobic, spore-forming bacteria were isolated from shallow, marine, thermal vents of the Eolian Islands (Italy) and tested for a broad spectrum of phenotypic characteristics. A numerical taxonomy study was performed on these isolates and 8 thermophilic Bacillus and Geobacillus reference strains by 89 selected features. Results from cluster analysis showed the formation of nine clusters. Most of the isolates (83%) fell into several phenetically well distinguished clusters, loosely related to Geobacillus thermodenitrificans. The remaining isolates grouped together with different reference strains. Eighteen isolates, representative of the different clusters, were selected for subsequent genotypic characterisation, including partial 16S rDNA sequence analysis of 18 strains and almost complete 16S rDNA sequences of 9 strains. Subsequent DNA/DNA reassociation studies and determination of the base composition of DNA identified seven isolates as Geobacillus thermodenitrificans, two isolates as G. thermoleovorans and one isolate as Bacillus pallidus. Four isolates represented two novel species of Bacillus. The remaining four represented novel Geobacillus species, one of which has recently been described as Bacillus vulcani DSMZ 13174 T.     

Isolation and Identification of Actinobacteria from Surface-Sterilized Wheat Roots

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.     

Morphological and Biochemical Properties of a Sphaerotilus sp. Isolated From Paper Mill Slimes

Four strains of filamentous bacteria were isolated from slimes collected in different paper mill factories. Morphological and physiological characterization of the isolates indicated an affiliation with the genus Sphaerotilus. However, while the physiological properties of the isolates were almost identical, pronounced physiological differences between the isolates and Sphaerotilus natans DSM 6575^T, DSM 565, and DSM 566 with respect to their ability to metabolize complex polysaccharides, sugars, polyalcohols, or organic acids as carbon sources were detected. In contrast to the analyzed culture collection strains of S. natans, all paper mill isolates were able to grow at elevated temperatures of up to 40°C. Comparative sequence analysis of nearly complete 16S ribosomal DNA (rDNA) sequences from the four new isolates demonstrated that the retrieved sequences were highly similar to each other (99.6 to 99.8% similarity) and to previously published partial 16S rDNA sequences of S. natans DSM 6575^T and ATCC 15291. Polyphasic characterization of the isolated Sphaerotilus strains revealed interesting adaptations of the strains to the environmental paper mill conditions with regard to temperature tolerance and utilization of cellulose and starch.     

A cluster of atypical Yersinia strains with a distinctive 16S rRNA signature

Thirty-eight bacterial isolates from raw milk samples in Queensland, Australia were identified as members of the genus Yersinia on the basis of biochemical profile, ability to hybridize with a genus-specific DNA probe, comparative 16S rDNA sequence analysis, and the presence of characteristic 16S rDNA signature nucleotides which occur in all Yersinia spp. Twenty-five of these isolates reacted with typing sera (O:22 or O:58) of Y. enterocolitica; the remainder were non-typable. None of the isolates displayed any of the phenotypic or genetic virulence-associated characteristics of Y. enterocolitica. Comparative 16S rDNA sequence analysis revealed that members of this group appear to represent a new sub-line within the genus Yersinia, most closely related to Y. frederiksenii hybridization group 2 (unnamed genomospecies 2). This finding was confirmed by DNA hybridization studies which indicated that the strains belonged to the unnamed genomospecies, Yersinia frederiksenii genomospecies 2, which is biochemically indistinguishable from Y. frederiksenii (Y. frederiksenii genomospecies 1). A 23-nucleotide 16S rDNA signature stretch which characterised these strains was identified.     

Cytomorphological and Genetic Characterization of Troglobitic Leptolyngbya Strains Isolated from Roman Hypogea

Six Leptolyngbya strains, isolated from the archaeological surfaces of hypogean sites, were phenotypically and genetically characterized by light and electron microscopy and 16S rRNA gene and 16S-23S internally transcribed spacer (ITS) sequencing. Three phycoerythrin-rich (red) and three phycocyanin-rich (green) isolates were assigned to different operational taxonomic units (OTUs). Among the green isolates, one strain showed an OTU intraspecific variation due to differences in the ITS sequences and genomic polymorphism. Within the ITS sequence, variable regions, conserved domains and tRNA^Ile and tRNA^Ala genes showed high sequence identity among the phylotypes. Together, these data indicated a relatedness of the six strains to other Leptolyngbya from subaerophytic and geothermal environments and allowed the definition of novel Leptolyngbya OTUs.     

Genetic Diversity of Pierce's Disease Strains and Other Pathotypes of Xylella fastidiosa

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.     

Intraspecies diversity of Coxiella burnetii as revealed by com1 and mucZ sequence comparison

Coxiella burnetii is classified within the γ subgroup of the Proteobacteria. All strains tested to date have an identical 16S rRNA sequence but 20 different genotypes have been determined by pulsed field gel electrophoresis (PFGE). In this study, intraspecies genetic diversity was investigated by sequence comparison of 715 bp of the Com1 encoding gene (com1) and 774 bp of the MucZ encoding gene (mucZ) in 37 strains isolated from animals and humans with acute or chronic Q fever in Europe, North America and Africa. Five and four groups were established from sequence analysis of com1 and mucZ, respectively. Neither relation of the defined groups to geographical distribution of the isolates was noted nor relation to disease form (acute/chronic). The same isolates were grouped together regardless of the gene being investigated. Comparison of the five proposed groups to previous groups, yielded after digestion by NotI PFGE, allowed for an intermediate classification of C. burnetii isolates between those obtained by using 16S rDNA (one group) and PFGE (20 groups).     

Molecular characterisation of Halobacillus strains isolated from different medieval wall paintings and building materials in Austria

We previously reported on the detection and isolation of an indigenous population of Halobacillus from salt-damaged medieval wall paintings and building materials of Herberstein castle in St. Johann bei Herberstein in Styria, Austria. Several moderately halophilic, Gram-positive, endospore-forming Halobacillus-like bacteria could be again isolated by conventional enrichment from salt efflorescences collected in the medieval St. Virgil's chapel in Vienna. Comparative 16S rDNA sequence analyses showed that the St. Virgil isolates are most closely related (>98.5% sequence similarity) to Halobacillus trueperi, Halobacillus litoralis, and to our previous halobacilli strains obtained from the castle Herberstein. Based on 16S rDNA sequence analysis, the strains could be clustered in three different groups. Group I: St. Virgil strains S3, S4, S21, and S22 (99.8–100% sequence similarity); group II: Herberstein strains K3-1, I7, and the St. Virgil strain S20 (99.3–99.7% sequence similarity); and group III: Herberstein strains I3, I3A, and I3R (100% sequence similarity). Molecular typing by denaturing gradient gel electrophoresis (DGGE), random amplified polymorphic DNA (RAPD-PCR), and internal transcribed spacer-homoduplex–heteroduplex polymorphism (ITS-HHP) fingerprinting showed that all isolates are typeable by each of the methods. RAPD was the most discriminatory method. With respect to their physiological characteristics—i.e., growth in the presence of 5–20% (w/v) NaCl, no growth in the absence of NaCl, optimum growth at 37 °C in media containing 5–10% (w/v) NaCl, and optimum pH around 7.5–8.0—the St. Virgil isolates resembled our previously isolated strains. However, the St. Virgil strains showed some differences in their biochemical properties. St. Virgil isolates hydrolysed Tween 80, two isolates reduced nitrate, and no isolate liquefied gelatine. The recurrent isolation of halobacilli from salt efflorescences on historic buildings and monuments at two different geographical locations may indicate that this group of bacteria is common in salt-affected ruins.     

Molecular characterization of novel Bradyrhizobium strains nodulating Eriosema chinense and Flemingia vestita,important unexplored native legumes of the sub-Himalayan region (Meghalaya) of India

Root nodule bacterial strains were isolated from the little-studied legumes Eriosema chinense and Flemingia vestita (both in tribe Phaseoleae, Papilionoideae) growing in acidic soil of the sub-Himalayan region of the Indian state of Meghalaya (ME), and were identified as novel strains of Bradyrhizobium on the basis of their 16S rRNA sequences. Seven isolates selected on the basis of phenotypic characters and assessment of ARDRA and RAPD patterns were subjected to multilocus sequence analysis (MLSA) using four protein-coding housekeeping genes (glnII, recA, dnaK and gyrB). On the basis of 16S rRNA phylogeny as well as a concatenated MLSA five strains clustered in a single separate clade and two strains formed novel lineages within the genus Bradyrhizobium. The phylogenies of the symbiotic genes (nodA and nifH) were in agreement with the core gene phylogenies. It appears that genetically diverse Bradyrhizobium strains are the principal microsymbionts of these two important native legumes. The novel genotypes of Bradyrhizobium strains isolated in the present study efficiently nodulate the Phaseoloid crop species Glycine max, Vigna radiata and Vigna umbellata. These strains are genetically different from strains of Bradyrhizobium isolated earlier from a different agro-climatic region of India suggesting that the acidic nature of the soil, high precipitation and other local environmental conditions are responsible for the evolution of these newly-described Bradyrhizobium strains. In global terms, the sub-Himalayan region of India is geographically and climatically distinct and the Bradyrhizobium strains nodulating its legumes appear to be novel and potentially unique to the region.     

Biodiversity of Actinomycetes Associated with Caribbean Sponges and Their Potential for Natural Product Discovery

Marine actinomycetes provide a rich source of structurally unique and bioactive secondary metabolites. Numerous genera of marine actinomycetes have been isolated from marine sediments as well as several sponge species. In this study, 16 different species of Caribbean sponges were collected from four different locations in the coastal waters off Puerto Rico in order to examine diversity and bioactive metabolite production of marine actinomycetes in Caribbean sponges. Sediments were also collected from each location, in order to compare actinomycete communities between these two types of samples. A total of 180 actinomycetes were isolated and identified based on 16S rRNA gene analysis. Phylogenetic analysis revealed the presence of at least 14 new phylotypes belonging to the genera Micromonospora, Verruscosispora, Streptomyces, Salinospora, Solwaraspora, Microbacterium and Cellulosimicrobium. Seventy-eight of the isolates (19 from sediments and 59 from sponges) shared 100 % sequence identity with Micromonospora sp. R1. Despite having identical 16S rRNA sequences, the bioactivity of extracts and subsequent fractions generated from the fermentation of both sponge- and sediment-derived isolates identical to Micromonospora sp. R1 varied greatly, with a marked increase in antibiotic metabolite production in those isolates derived from sponges. These results indicate that the chemical profiles of isolates with high 16S rRNA sequence homology to known strains can be diverse and dependent on the source of isolation. In addition, seven previously reported dihydroquinones produced by five different Streptomyces strains have been purified and characterized from one Streptomyces sp. strain isolated in this study from the Caribbean sponge Agelas sceptrum.     

Identification of extracellular enzyme producing thermophilic bacilli from Balcova (Agamemnon) geothermal site by ITS rDNA RFLP

AIMS: Molecular characterization of extracellular enzyme producing thermophilic bacilli from Balcova geothermal site. METHODS AND RESULTS: Three types of geothermal samples were collected: mud, re-injection water, and samples from uncontrolled hydrothermal vents. Isolates grown at 55 degrees C in culture media prepared in sterilized re-injection water, were screened for extracellular enzyme activity by using eight different substrates: casein, carboxymethylcellulose, pectin, polygalacturonic acid (PGA), soluble starch, Tween 20 and 80, and xylan. In total, 109 thermoaerophilic isolates were selected. All of the isolates could hydrolyse Tween 20 (100%) but not Tween 80. Soluble starch was hydrolysed by 96%, casein by 55%, xylan and carboxymethylcellulose by 9%, and pectin and PGA by 2% of the isolates. The isolates were grouped into 14 different homology groups by the restriction pattern analysis of 16S-internal transcribed spacer (ITS) rDNA RFLP. Each of the RFLP groups was also studied by 16S rRNA gene partial sequence analysis. Plasmid DNA profiles revealed that 15 of the isolated strains contained small plasmid DNA molecules ranging in size from 12 000 to 35 000 bp. CONCLUSIONS: Combined analysis of 16S-ITS rDNA RFLP and 16S rRNA gene partial sequence results indicated the presence of novel or existing species of Anoxybacillus (nine species) and Geobacillus (three species). SIGNIFICANCE AND IMPACT OF THE STUDY: In this study 16S-ITS rDNA RFLP was applied for the first time to differentiate thermophilic bacilli. It was also the first study on thermophilic bacilli of Balcova geothermal site.     

Molecular characterization and estimation of cellulolytic potential of gut bacteria isolated from four white grub species native to Indian Himalayas

An investigation was carried out to isolate, identify and molecularly characterize the cellulose-degrading bacterial isolates from the guts of four white grub species (Anomala bengalensis, Brahmina coriacea, Holotrichia longipennis and Holotrichia setticollis) native to Uttarakhand, Himalayas through 16S rRNA sequencing. A total of 178 bacterial strains were isolated from different gut compartments of selected white grub species, of which 95 bacterial isolates showed cellulose metabolizing activities in the CMC assay. Maximum degraders i.e., 38 were isolated from A. bengalensis, of which 18 were isolated from the fermentation chamber. The value of cellulolytic index ranged between 0.05 and 16 showing a variable cellulolytic activity by degraders. A total of 25 potent strains of cellulose-degrading bacteria recording cellulolytic activity > 1 were isolated and sequenced for 16S rRNA gene. Bacillus stratosphericus strain CBG4MG1 (10.78 ± 4.18), Bacillus cereus strain CBG2FC1 (10.33 ± 3.53), Bacillus sp. strain CBG3MG2 (7.28 ± 0.16) and Paenibacillus ginsengagri strain CBG1FC2 (5.66 ± 2.67) were the most potent cellulose-degrading bacteria isolated from the gut of B. coriacea, H. longipennis, H. setticollis and A. bengalensis, respectively. Thus, the cellulolytic bacteria isolated from the gut of selected white grub species may be good sources for profiling novel isolates for industrial use besides identifying eco-friendly solutions for agro-waste management.     

Ecotypes of Planktonic Actinobacteria with Identical 16S rRNA Genes Adapted to Thermal Niches in Temperate, Subtropical, and Tropical Freshwater Habitats

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6°C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.     

Phylogeny of Bacteria Isolated from Rhabditis sp. (Nematoda) and Identification of Novel Entomopathogenic Serratia marcescens Strains

Twenty-five bacterial strains isolated from entomopathogenic nematodes were characterized to the genus level by 16S rRNA phylogeny and BLAST analyses. Bacteria strains isolated could be affiliated with seven genera. Microbacterium-like isolates phylogenetically affiliated with M. oxydans while those of Serratia were highly similar to S. marcescens. 16S rRNA sequences of Bacillus isolates matched those of both B. mycoides and B. weihenstephanesis. One isolate each matched Pseudomonas mosselii, Rheinheimera aquimaris, Achromobacter marplatensis, or Staphylococcus hominis. Serratia isolates were examined further for their pathogenicity to Galleria mellonella larvae. All the Serratia isolates exhibited potent pathogenicity toward G. mellonella larvae and possessed a metalloprotease gene encoding for a novel serralysin-like protein. The nucleotide sequence of the metalloprotease gene had 60 synonymous and 8 nonsynonymous substitutions when compared to the closest genBank entry, S. marcescens E-15, with an insertion of a new aspartic acid residue. Tajima’s test for equality of evolutionary rate was significant between the metalloprotease gene sequence of S. marcescens strain DOAB 216-82 (this study) and strain E-15. This new insecticidal metalloprotease gene and/or its product could have applications in agricultural biotechnology.     

Multi-locus sequence typing (MLST) of non-fermentative Gram-negative bacilli isolated from bloodstream infections in southern Poland

Non-fermentative Gram-negative bacilli are now one of the most important causes of severe infections in Polish hospitals. Acinetobacter species are serious concern because of the high prevalence of multi-drug resistance among strains. Resistance profiles for 53 Gram-negative non-fermentative blood isolates were done. MLST was carried out using 44 strains representing the most commonly isolated species: A. baumannii, P. aeruginosa, and S. maltophilia. MLST revealed that all 22 A. baumannii belonged to sequence type (ST) 2. The P. aeruginosa isolates belonged to 10 different STs. Four S. maltophilia isolates matched STs present in the database (ST4, ST15, ST116, ST142), seven isolates showing novel sequence types. Among P. aeruginosa and S. maltophilia PFGE confirmed the genetical variety of strains.     

rpoB-Based Microbial Community Analysis Avoids Limitations Inherent in 16S rRNA Gene Intraspecies Heterogeneity

Contemporary microbial community analysis frequently involves PCR-amplified sequences of the 16S rRNA gene (rDNA). However, this technology carries the inherent problem of heterogeneity between copies of the 16S rDNA in many species. As an alternative to 16S rDNA sequences in community analysis, we employed the gene for the RNA polymerase beta subunit (rpoB), which appears to exist in one copy only in bacteria. In the present study, the frequency of 16S rDNA heterogeneity in bacteria isolated from the marine environment was assessed using bacterial isolates from the red alga Delisea pulchra and from the surface of a marine rock. Ten strains commonly used in our laboratory were also assessed for the degree of heterogeneity between the copies of 16S rDNA and were used to illustrate the effect of this heterogeneity on microbial community pattern analysis. The rock isolates and the laboratory strains were also used to confirm nonheterogeneity of rpoB, as well as to investigate the versatility of the primers. In addition, a comparison between 16S rDNA and rpoB PCR-DGGE (denaturing gradient gel electrophoresis)-based community analyses was performed using a DNA mixture of nine isolates from D. pulchra. Eight out of 14 isolates from D. pulchra, all rock isolates, and 6 of 10 laboratory strains displayed multiple bands for 16S rDNA when analyzed by DGGE. There was no indication of heterogeneity for either the rock isolates or the laboratory strains when rpoB was used for PCR-DGGE analysis. Microbial community pattern analysis using 16S rDNA PCR-DGGE showed an overestimation of the number of laboratory strains in the sample, while some strains were not represented. Therefore, the 16S rDNA PCR-DGGE-based community analysis was proven to be severely limited by 16S rDNA heterogeneity. The mixture of isolates from D. pulchra proved to be more accurately described using rpoB, compared to the 16S rDNA-based PCR-DGGE.     

New bacilli from shallow hydrothermal vents of Panarea Island (Italy) and their biotechnological potential

Aims: To characterize bacilli isolated from shallow hydrothermal vents of Panarea Island (Italy) and evaluate their biotechnological potential. Methods and Results: Fifteen isolates were characterized by culture and molecular methods. Eleven isolates were thermophilic, six isolates were alkalophilic and four of them were haloalkalophilic. After 16S rRNA gene sequencing, four strains, exhibiting sequence similarity below 95% with deposited strains, may represent novel species of bacilli. One strain was strictly related to Geobacillus subterraneus, but shared phenotypic characteristics for which it could be considered a new strain of this species. Four strains were affiliated with different Bacillus spp. Most isolates produced gelatinase, lipases and amylase, and some were mercury tolerant. Exopolysaccharides (EPS) production was tested adding different sugars (glucose, sucrose, trehalose, fructose, ribose, xylose and mannose, 1% w/v) as a carbon source in a minimal medium. The highest EPS yield (185 mg l^?1) was reached by strain 1A70 utilizing ribose as a carbon source. Conclusions: Novel strains of Geobacillus and indigenous ribotypes of Bacillus with biotechnological potential inhabit shallow vents of Panarea Island. Significance and Impact of the Study: New strains of thermophilic bacilli from Panarea are producers of useful biomolecules for industrial purposes as well as environmental and biotechnological applications.     

Characterisation and identification of spoilage psychotrophic Gram-negative bacteria originating from Tunisian fresh fish

The contamination of fresh fish by spoilage bacteria is undesirable, particularly when Gram-negative bacteria, which produce thermo-resistant protease and lipase, can grow. The purpose of the present work was, therefore, to isolate and identify psychrotrophic Gram-negative (Psy G(-)) bacteria, isolated from 80 samples of 12 species of wild and aquacultured fresh seafood, by biochemical and molecular methods using 16S rRNA gene sequencing. Twenty-eight identified strains were studied to evaluate their catalase, nitrate reductase, lipolytic and proteolytic activities, as well as growth ability, at different temperatures, pH and NaCl concentrations. Among 150 Psy G(-) strains, the most dominant species found were: Pseudomonas fluorescens, Aeromonas hydrophila, Pseudomonas putida and Photobacterium damselae. All strains of Psy G(-) had catalase activity and were able to reduce nitrates to nitrites. Proteolytic activity on milk and on gelatin agar was demonstrated for the majority of the isolates. However, extracellular proteolytic activity as assessed by the azocasein method wasn’t very high in all the strains. Lipolytic activity, as assessed by the agar method, showed that 92.9 % of strains could hydrolyse egg yolk, against 82.1 % and 57 % that could hydrolyse Tween 20 and Tween 80, respectively.