Characterization and Phylogenetic Diversity of Carboxymethyl Cellulase Producing <Emphasis Type="Italic">Bacillus</Emphasis> Species from a Landfill Ecosystem

Total population of cellulose degrading bacteria was studied in a landfill ecosystem as a part of microbial diversity study. Samples were obtained from 3 and 5 feet depth of a local landfill being operated for past 10 years. Among many isolates, 22 bacterial strains were selected based on their capability to decompose carboxymethyl cellulose (CMC). These isolates were cultivated on agar medium with CMC as the carbon source. All isolates were Gram positive, endospore forming and alkalophilic bacteria with optimum growth pH 9–10. They were grouped based on the phenotypic and chemotaxonomic characters and representative strains of different groups along with high carboxymethyl cellulase (CMCase) producing strains were included for further characterization. Analysis of 16S rRNA gene indicated that these strains belong to different species of the genus Bacillus. Maximum CMCase activity of 4.8 U/ml at 50°C was obtained by strain LFC15. Results in the present study indicated the potential of waste land ecosystems such as landfill are potential source for isolation of industrially important microorganisms.     

Following phylogenetic tracks of <Emphasis Type="Italic">Astragalus cicer</Emphasis> microsymbionts

A multilocus phylogenetic approach was applied to elucidate the phylogeny of Astragalus cicer rhizobia derived from Poland, Ukraine, and Canada. The strains selected for the studies represented three main geographically different phenons of these bacteria. Phylogenetic analyses were performed with three chromosomal housekeeping loci (16S rRNA, atpD, glnII) and three symbiotic genes located on a plasmid (nodA, nodC, nifH). The “core” and “auxiliary” gene trees revealed that A. cicer nodule isolates were intermingled with the strains of Mesorhizobium species, which implies that they are descendents of the same ancestor as mesorhizobia and fall into the Mesorhizobium genus. The noted congruence of the housekeeping and symbiotic gene phylogenies of A. cicer microsymbionts indicates that sym loci are transferred to these bacteria through vertical transmission without a significant participation of intergeneric horizontal gene spread. All the three sym gene sequences of the Polish and Ukrainian A. cicer nodule isolates were more closely related to one another than to the corresponding sequences of the Canadian isolates. The phylogeographic patterns of the sym genes of intercontinental strains point to their relatively long, separate, evolutionary history.     

Isolation and analyses of uranium tolerant <Emphasis Type="Italic">Serratia marcescens</Emphasis> strains and their utilization for aerobic uranium U(VI) bioadsorption

Enrichment-based methods targeted at uranium-tolerant populations among the culturable, aerobic, chemo-heterotrophic bacteria from the subsurface soils of Domiasiat (India’s largest sandstone-type uranium deposits, containing an average ore grade of 0.1 % U₃O₈), indicated a wide occurrence of Serratia marcescens. Five representative S. marcescens isolates were characterized by a polyphasic taxonomic approach. The phylogenetic analyses of 16S rRNA gene sequences showed their relatedness to S. marcescens ATCC 13880 (≥99.4% similarity). Biochemical characteristics and random amplified polymorphic DNA profiles revealed significant differences among the representative isolates and the type strain as well. The minimum inhibitory concentration for uranium U(VI) exhibited by these natural isolates was found to range from 3.5–4.0 mM. On evaluation for their uranyl adsorption properties, it was found that all these isolates were able to remove nearly 90–92% (21–22 mg/L) and 60–70% (285–335 mg/L) of U(VI) on being challenged with 100 μM (23.8 mg/L) and 2 mM (476 mg/L) uranyl nitrate solutions, respectively, at pH 3.5 within 10 min of exposure. his capacity was retained by the isolates even after 24 h of incubation. Viability tests confirmed the tolerance of these isolates to toxic concentrations of soluble uranium U(VI) at pH 3.5. This is among the first studies to report uranium-tolerant aerobic chemoheterotrophs obtained from the pristine uranium ore-bearing site of Domiasiat.     

Selecting Bacterial Strains for Use in the Biocontrol of Diseases Caused by <Emphasis Type="Italic">Phytophthora capsici</Emphasis> and <Emphasis Type="Italic">Alternaria alternata</Emphasis> in Sweet Pepper Plants

More than 500 isolates of bacteria were obtained from the aerial part and rhizosphere of sweet pepper (Capsicum annuum L.) plants harvested from different places in the Region of Murcia (Spain). The isolates were purified and assayed in vitro against Phytophthora capsici and Alternaria alternata. Sixty isolates (12 %) produced an inhibition zone against at least one of the pathogens, while ten had a strongly inhibitory effect on both pathogens assayed. Microscopic observation of interactions zone showed cell vacuolisation, hyphae lysis and spilling of cytoplasm content of the pathogens in the culture media. These ten isolates were then chosen for biocontrol of Phytophthora root rot and Alternaria leaf spots of pepper plants in vivo. Four of them denominated HS93, LS234, LS523 and LS674 reduced P. capsici root rot by 80, 51, 49 and 54 %, respectively, and A. alternata leaf spots by 54, 74, 62 and 53 %. HS93 belongs to the genus Bacillus and probably the species subtilis, while LS234, LS523 and LS674 belong to the genus Bacillus and probably the species licheniformis. Dry mass of plants treated with these bacteria was significantly higher than that of non-treated and inoculated plants.     

Phenotypic and genotypic characterization of tetracycline and minocycline resistance in Clostridium perfringens

The aim of this study was to determine the incidence of tetracycline resistance and the prevalence of tetracycline-resistance genes in strains of Clostridium perfringens isolated from different sources between 1994 and 2005. Susceptibility to tetracycline and minocycline in strains from humans (35 isolates), chickens (15 isolates), food (21 isolates), soil (16 isolates) and veterinary sources (6 isolates) was determined, and tetracycline-resistance genes were detected. Resistance was most common in strains isolated from chickens, followed by those from soils, clinical samples and foods. The most highly resistant strains were found among clinical and food isolates. tetA(P) was the most common resistance gene, and along with tetB(P) was found in all resistant strains and some sensitive strains. One tetracycline-resistant food isolate had an intact tet(M) gene. However, PCR fragments of 0.4 or 0.8 kb with high degrees of identity to parts of the tet(M) sequences of other bacteria were found, mainly in clinical isolates, and often in isolates with tetB(P). No correlation between level of sensitivity to tetracycline or minocycline and the presence of tetA(P), tetB(P) or part of tet(M) was found. The presence of part of tet(M) in some strains of C. perfringens containing tetB(P) may have occurred by recent gene transfer.     

Culturable bacterial diversity associated with cysts of <Emphasis Type="Italic">Eurhizococcus brasiliensis</Emphasis> (Hempel) (Hemiptera: Margarodidae)

The ground-pearl Eurhizococcus brasiliensis is an important insect pest of grapes. Nowadays, its biology is still barely known and studies related to its secondary symbionts are virtually non-existent. Our main goal was to evaluate the bacterial diversity associated with cysts of E. brasiliensis using a culture-dependent approach. Six different isolation media were used and shown to be suitable for culturing bacteria associated with E. brasiliensis. A total of 39 bacteria strains were isolated and classified into 10 different morphotypes. The ISP-4 medium was the most suitable for bacteria isolation from cysts of the ground-pearl, providing the highest number of morphotypes. 16S rDNA gene analysis demonstrated a high diversity of bacteria associated with cysts, with six Pseudomonas chlororaphis isolates (the most predominant morphotype) and three different morphotypes of Bacillus spp. as the most representative genera. The phylogenetic analysis showed close affinity between the isolated morphotypes and bacterial strains usually isolated from plant and soil samples. Sphingopyxis and Stenotrophomonas were isolated for the first time from arthropods. Although it was not possible to determine the primary source of infection by these bacteria, our data suggests that these microorganisms may be transitory and acquired from the environment.     

Isolation of bacteria of the genus <Emphasis Type="Italic">Variovorax</Emphasis> from the <Emphasis Type="Italic">Thioploca</Emphasis> mats of Lake Baikal

Three strains of gram-negative bacteria were isolated from the mats of colorless sulfur bacteria Thioploca (Lake Baikal). The cells of new strains are motile with peritrichous flagella. Bacteria are aerobic, obligate chemoorganoheterotrophs growing within the pH range of 3.0–8.8 with the optimum at 8.3 and within the temperature range of 5–42°C with the optimum at 28°C. The cells contained menaquinones MK-8 H2 as the major component, as well as MK-7 H2 (less than 15%), while the content of ubiquinone Q8 was at least an order of magnitude lower. The G+C content of DNA in the new strains varied from 67.4 to 69.9 mol %. The level of DNA-DNA hybridization between the strains ranged from 80 to 94%, indicating that all the isolates belonged to one species. Analysis of the 16S rRNA gene nucleotide sequences of the type strain (Gen-Bank HQ400611) revealed close homologues among the known species of the genus Variovorax: 98% resemblance with the type strains of the species V. paradoxus, V. soli, V. ginsengisoli, and V. boronicumulans and 96% similarity with the type strain of V. dokdonensis. However, since the isolates differed significantly in the composition of fatty acids and isoprenoid quinones from the nearest neighbors in the phylogenetic tree, they cannot be related implicitly to the known species.     

Genetic diversity of elite rhizobial strains of subtropical and tropical legumes based on the 16S rRNA and <Emphasis Type="Italic">glnII</Emphasis> genes

Biodiversity of diazotrophic symbiotic bacteria in the tropics is a valuable but still poorly studied resource. The objective of this study was to determine if a second housekeeping gene, glnII, in addition to the 16S rRNA, can be employed to improve the knowledge about taxonomy and phylogeny of rhizobia. Twenty-three elite rhizobial strains, very effective in fixing nitrogen with twenty-one herbal and woody legumes (including species from fourteen tribes in the three subfamilies of the family Leguminosae) were selected for this study; all strains are used as commercial inoculants in Brazil. Complete sequences of the 16S rRNA and partial sequences (480 bp) of the glnII gene were obtained. The same primers and amplification conditions were successful for sequencing the glnII genes of bacteria belonging to five different rhizobial genera—Bradyrhizobium, Mesorhizobium, Methylobacterium, Rhizobium, Sinorhizobium)—positioned in distantly related branches. The analysis of the concatenated genes (16S rRNA + glnII) considerably improved information about phylogeny and taxonomy of rhizobia in comparison to the single analysis of the 16S rRNA. Nine strains might belong to new species. The complementary analysis of the glnII gene was successful with all strains and improved the phylogenetic clustering and clarified the taxonomic position of several strains. The strategy of including the analysis of glnII, in addition to the 16S rRNA, is cost- and time- effective for the characterization of large rhizobial culture collections or in surveys of many isolates.     

Isolation and characterization of new poly(3HB)‐accumulating star‐shaped cell‐aggregates‐forming thermophilic bacteria

Aims: This study aimed at isolating thermophilic bacteria that utilize cheap carbon substrates for the economically feasible production of poly(3‐hydroxybutyrate), poly(3HB), at elevated temperatures. Methods and Results: Thermophilic bacteria were enriched from an aerobic organic waste treatment plant in Germany, and from hot springs in Egypt. Using the viable colony staining method for hydrophobic cellular inclusions with Nile red in mineral salts medium (MSM) containing different carbon sources, six Gram‐negative bacteria were isolated. Under the cultivation conditions used in this study, strains MW9, MW11, MW12, MW13 and MW14 formed stable star‐shaped cell‐aggregates (SSCAs) during growth; only strain MW10 consisted of free‐living rod‐shaped cells. The phylogenetic relationships of the strains as derived from 16S rRNA gene sequence comparisons revealed them as members of the Alphaproteobacteria. The 16S rRNA gene sequences of the isolates were very similar (>99% similarity) and exhibited similarities ranging from 93 to 99% with the most closely related species that were Chelatococcus daeguensis, Chelatococcus sambhunathii , Chelatococcus asaccharovorans, Bosea minatitlanensis, Bosea thiooxidans and Methylobacterium lusitanum. Strains MW9, MW10, MW13 and MW14 grew optimally in MSM with glucose, whereas strains MW11 and MW12 preferred glycerol as sole carbon source for growth and poly(3HB) accumulation. The highest cell density and highest poly(3HB) content attained were 4·8 g l^?l (cell dry weight) and 73% (w/w), respectively. Cells of all strains grew at temperatures between 37 and 55°C with the optimum growth at 50°C. Conclusions: New PHA‐accumulating thermophilic bacterial strains were isolated and characterized to produce poly(3HB) from glucose or glycerol in MSM at 50°C. SSCAs formation was reported during growth. Significance and Impact of the Study: To the best of our knowledge, this is the first report on the formation of SSCAs by PHA‐accumulating bacteria and also by thermophilic bacteria. PHA‐producing thermophiles can significantly reduce the costs of fermentative PHA production.     

Diversity of Arsenate Reductase Genes (<Emphasis Type="Italic">arsC</Emphasis> Genes) from Arsenic-Resistant Environmental Isolates of <Emphasis Type="Italic">E. coli</Emphasis>

A polymerase chain reaction (PCR) approach was used to assess the occurrence and diversity of arsenate reductase gene (arsC gene) in arsenic-resistant environmental E. coli strains. For this purpose, two different sets of primers were designed for the specific amplification of approximately 370-bp fragments from the arsC gene. These primers were used to screen a collection of 25 environmental arsenic-resistant strains isolated from different geographical regions of India, as well as Bangladesh. The PCR results showed that 17 out of the 25 environmental isolates (68%) contained a gene related to the arsC family. Phylogenetic analysis of the protein sequences deduced from the amplicons indicated a prevalence of arsC genes in the isolated strains. A significant divergence in the DNA sequence was found in the arsC genes among As-resistant environmental E. coli strains from this study, and arsenic resistance, a genetic character, arose from a common ancestral background.     

Isolation and characterization of sulphate-reducing bacteria <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> from Vajreshwari thermal springs in Maharashtra,India

Sulphate-reducing bacteria (SRB) in the thermal springs of Vajreshwari were investigated with combined microbiological and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 25 to 55 °C and could use pyruvate, lactate and ethanol as electron donors. Desulfoviridin was detected in all the isolates. The partial 16S rRNA and dissimilatory sulphite reductase (DSR) gene sequences of five representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris.     

Antimicrobial resistance and presence of <Emphasis Type="Italic">ileS-2</Emphasis> gene encoding mupirocin resistance in clinical isolates of methicillin-resistant <Emphasis Type="Italic">Staphylococcus</Emphasis> sp.

Seventy-eight staphylococcal strains were isolated from surgical-site, blood-stream and other hospital-acquired infections. Eighteen isolates were determined as methicillin (MET)-resistant S. aureus (MRSA), while the remaining were MET-resistant coagulase-negative staphylococci (CoNS). Fifty percent of CoNS strains were multiresistant, while 10 % of isolates were resistant only to β-lactams. Clinical isolates of CoNS were generally more resistant to antimicrobial agents than S. aureus strains. Thirty-nine % of S. aureus strains were resistant only to β-lactams. None of the MRSA strains carried ileS-2 gene; this gene was found in two strains of S. epidermidis.     

Diversity of Predominant Lactic Acid Bacteria Associated with Cocoa Fermentation in Nigeria

The fermentation of cocoa relies on a complex succession of bacteria and filamentous fungi, all of which can have an impact on cocoa flavor. So far, few investigations have focused on the diversity of lactic acid bacteria involved in cocoa fermentation, and many earlier investigations did not rely on polyphasic taxonomical approaches, which take both phenotypic and genotypic characterization techniques into account. In our study, we characterized predominant lactic acid bacteria from cocoa fermentations in Nigeria, using a combination of phenotypic tests, repetitive extragenic palindromic PCR, and sequencing of the 16S rRNA gene of representative strains for accurate species identification. Thus, of a total of 193 lactic acid bacteria (LAB) strains isolated from common media used to cultivate LAB, 40 (20.7%) were heterofermentative and consisted of either L. brevis or L. fermentum strains. The majority of the isolates were homofermentative rods (110 strains; 57% of isolates) which were characterized as L. plantarum strains. The homofermentative cocci consisted predominantly of 35 (18.1% of isolates) Pediococcus acidilactici strains. Thus, the LAB populations derived from these media in this study were accurately described. This can contribute to the further assessment of the effect of common LAB strains on the flavor characteristics of fermenting cocoa in further studies.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.     

Intra- and Interbacterial Genetic Exchange of Lyme Disease Spirochete <Emphasis Type="Italic">erp</Emphasis> Genes Generates Sequence Identity Amidst Diversity

All isolates of the spirochete Borrelia burgdorferi contain multiple, different plasmids of the cp32 family, each of which contains a locus encoding Erp surface proteins. Many of these proteins are known to bind host complement regulatory factor H, enabling the bacteria to avoid killing by the alternative complement pathway during vertebrate infection. In the present study, we characterized the erp loci and cp32 plasmids of strains N40, Sh-2-82, and 297 and compared them to the previously determined cp32 sequences of type strain B31. Bacteria of strain N40 contain 6 different cp32s, those of Sh-2-82 contain 10, and 297 bacteria contain 9 cp32s. Significant conservation between all strains was noted for the cp32 loci responsible for plasmid maintenance, indicating close relationships that appear to correspond with incompatibility groups. In contrast, considerable diversity was found between erp gene sequences, both within individual bacteria and between different strains. However, examples of identities among erp loci were found, with strains Sh-2-82, 297, and B31 each containing three identical loci that likely arose through intrabacterial genetic rearrangements. These studies also found the first evidence of large-scale genetic exchanges between Lyme disease spirochetes in nature, including the apparent transfer of an entire cp32 plasmid between two different bacteria.     

Gastrointestinal Colonization Rates for Human Clinical Isolates of <Emphasis Type="Italic">Aeromonas Veronii</Emphasis> Using a Mouse Model

A variety of environment-associated gastrointestinal infections have been associated with the Aeromonas group of bacteria which contain both non-virulent strains as well as virulent strains within a particular species. This study monitors the colonization rates of colon tissue in a mouse-streptomycin dose/response model involving isolates of Aeromonas veronii biovar sobria obtained from human clinical specimens. The ability to successfully colonize mouse colon tissues by the human clinical isolates was then compared with the rates achieved in a previous study of Aeromonas isolates obtained from environmental drinking water samples. Results suggest that strains of Aeromonas isolated from drinking water environmental samples contain pathogenic and virulence capabilities similar to those seen in Aeromonas veronii clinical isolates from human infections.     

DNA-DNA hybridization of some root-nodule bacteria indigenous in the salt-affected soils of egypt

The DNA-DNA hybridization was used to characterize thirty isolates of root-nodule bacteria indigenous to the salt-affected soils of Egypt. Total DNA from different bacterial isolates lacked homology with total DNA probes of the effective strains ofRhizobium leguminosarum andR. meliloti. It is suggested that the genomic structure of the root-nodule bacteria may be modified by salt stress and/or that the effective strains of these bacteria are to be eliminated from the salt-affected soil.     

Nodule Symbiosis of Invasive <Emphasis Type="Italic">Mimosa pigra</Emphasis> in Australia and in Ancestral Habitats: A Comparative Analysis

Ninety isolates of root nodule bacteria from an invasive Mimosa pigra population in Australia were characterized by PCR assays and by sequencing of ribosomal genes. All isolates belonged to the same bacterial genus (Burkholderia) that predominates on M. pigra in its native geographic range in tropical America. However, the Australian Burkholderia strains represented several divergent lineages, none of which had a close relationship to currently known Burkholderia strains in American M. pigra populations. Inoculation of M. pigra with Australian strains resulted in equal or higher plant growth and nodule nitrogenase activity (measured by acetylene reduction assays) relative to outcomes with bacteria from M. pigra’s native geographic region. The main difference in symbiotic phenotype for bacteria from the two regions involved responses to an alternate Mimosa host species: Central American strains failed to fix nitrogen in association with Mimosa pudica, while most Australian Burkholderia isolates tested had high nodule nitrogenase activity in association with both Mimosa species. Invasive M. pigra populations in Australia have therefore acquired a diverse assemblage of nodule bacteria that are effective nitrogen-fixing symbionts, despite having a broader host range and a distant genetic relationship to bacterial strains found in the plant’s ancestral region.     

The Genetic Diversity of Culturable Nitrogen-Fixing Bacteria in the Rhizosphere of Wheat

A total of 17 culturable nitrogen-fixing bacterial strains associated with the roots of wheat growing in different regions of Greece were isolated and characterized for plant-growth-promoting traits such as auxin production and phosphate solubilization. The phylogenetic position of the isolates was first assessed by the analysis of the PCR-amplified 16S rRNA gene. The comparative sequence analysis and phylogenetic analysis based on 16S rRNA gene sequences show that the isolates recovered in this study are grouped with Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. The diazotrophic nature of all isolates was confirmed by amplification of partial nifH gene sequences. The phylogenetic tree based on nifH gene sequences is consistent with 16S rRNA gene phylogeny. The isolates belonging to Azospirillum species were further characterized by examining the partial dnaK gene phylogenetic tree. Furthermore, it was demonstrated that the ipdC gene was present in all Azospirillum isolates, suggesting that auxin is mainly synthesized via the indole-3-pyruvate pathway. Although members of P. stutzeri and A. zeae are known diazotrophic bacteria, to the best of our knowledge, this is the first report of isolation and characterization of strains belonging to these bacterial genera associated with wheat.     

Phenotypical Assays and Partial Sequencing of the <Emphasis Type="Italic">hsp60</Emphasis> Gene for Identification of <Emphasis Type="Italic">Streptococcus equi</Emphasis>

Strangles is an acute and contagious disease characterized by inflammation of the upper respiratory tract of horses. The etiological agent of strangles is the bacteria S. equi subsp. equi, which belongs to the Lancefield group C. Opportunistic agents from the same group are frequently isolated from horses with strangles and may induce mistaken diagnoses. Among the subspecies of S. equi, the phenotypic features are almost undistinguishable; however, the pathogenic potential is widely differentiated. The aim of this study was to characterize S. equi isolates obtained from clinical samples of strangles by phenotypic tests and to analyze the partial sequences obtained from fragments of the hsp60 gene. In this work, 26 strains of Streptococcus spp. isolated from horse clinical samples were analyzed. By phenotypical assays, 18 were characterized as S. equi subsp. equi, five as S. equi subsp. zooepidemicus, two as S. dysgalactiae subsp. equisimilis, and one as Streptococcus sp. However 21 isolates were identified as S. equi subsp. equi and five as S. equi subsp. zooepidemicus by DNA sequencing. The sequencing of the partial hsp60 gene was demonstrated to be an alternative method to analyze and differentiate strains of Streptococcus spp. In addition, this method can be useful as a discriminatory tool for characterization of atypical isolates.