DNA-Repair Potential of <Emphasis Type="Italic">Halomonas</Emphasis> spp. from the Salt Plains Microbial Observatory of Oklahoma

The Great Salt Plains (GSP), an unvegetated, barren salt flat that is part of the Salt Plains National Wildlife Refuge near Cherokee, Oklahoma, is the site of the Salt Plains Microbial Observatory. At the GSP the briny remains of an ancient sea rise to the surface, evaporate under dry conditions, and leave crusts of white salt. Adaptation to this environment requires development of coping mechanisms providing tolerance to desiccating conditions due to the high salinity, extreme temperatures, alkaline pH, unrelenting exposure to solar UV radiation, and prevailing winds. Several lines of evidence suggest that the same DNA repair mechanisms that are usually associated with UV light or chemically induced DNA damage are also important in protecting microbes from desiccation. Because little is known about the DNA repair capacity of microorganisms from hypersaline terrestrial environments, we explored the DNA repair capacity of microbial isolates from the GSP. We used survival following exposure to UV light as a convenient tool to assess DNA repair capacity. Two species of Halomonas (H. salina and H. venusta) that have been isolated repeatedly from the GSP were chosen for analysis. The survival profiles were compared to those of Escherichia coli, Pseudomonas aeruginosa, and Halomonas spp. from aquatic saline environments. Survival of GSP organisms exceeded that of the freshwater organism P. aeruginosa, although they survived no better than E. coli. The GSP isolates were much more resistance to killing by UV than were the aquatic species of Halomonas reported in the literature [Martin et al. (2000) Can J Microbiol 46:180–187]. Unlike E. coli, the GSP isolates did not appear to have an inducible, error-prone repair mechanism. However, they demonstrated high levels of spontaneous mutation.     

HYPERSALINE SOIL SUPPORTS A DIVERSE COMMUNITY OF DUNALIELLA (CHLOROPHYCEAE)1

Numerous isolates of the green halophile Dunaliella were studied as part of a survey of microbial diversity at the Great Salt Plains (GSP) in Oklahoma, USA. The GSP is a large (～65 km²) salt flat with extreme temporal and spatial fluctuations in salinity and temperature. Although the flagellate halophile Dunaliella is common worldwide, nearly all cultured isolates are from saline habitats that are primarily aquatic rather than primarily terrestrial. The diverse GSP Dunaliella strains exhibit three morphotypes: a predominantly motile form, a motile form with a prominent palmelloid phase (nonmotile, mucilage rich), and a palmelloid form with a weakly motile phase. All had broad salinity optima well below typical in situ salinities at the GSP, and two of the palmelloid isolates grew as well in freshwater as in highly saline media. Molecular phylogenetic and evolutionary analyses revealed that Dunaliella from the GSP (and two similar habitats in the Great Basin, USA) are allied with D. viridis Teodor. but possess phylogenetic diversity in excess of existing global isolates from aquatic habitats. In addition, isolates from primarily terrestrial habitats exhibit statistically higher rates of nucleotide substitution than the phylogenetically homogeneous set of primarily aquatic Dunaliella taxa. We hypothesize that dynamically extreme saline soil habitats may select for different and more diverse Dunaliella lineages than more stable saline aquatic habitats. We also propose Dunaliella as a tractable microbial model for in situ testing of evolutionary and phylogeographic hypotheses.     

Cyanobacterial Diversity and Halotolerance in a Variable Hypersaline Environment

The Great Salt Plains (GSP) in north-central Oklahoma, USA is an expansive salt flat (∼65 km²) that is part of the federally protected Salt Plains National Wildlife Refuge. The GSP serves as an ideal environment to study the microbial diversity of a terrestrial, hypersaline system that experiences wide fluctuations in freshwater influx and diel temperature. Our study assessed cyanobacterial diversity at the GSP by focusing on the taxonomic and physiological diversity of GSP isolates, and the 16S rRNA phylogenetic diversity of isolates and environmental clones from three sites (north, central, and south). Taxonomic diversity of isolates was limited to a few genera (mostly Phormidium and Geitlerinema), but physiological diversity based on halotolerance ranges was strikingly more diverse, even between strains of the same phylotype. The phylogenetic tree revealed diversity that spanned a number of cyanobacterial lineages, although diversity at each site was dominated by only a few phylotypes. Unlike other hypersaline systems, a number of environmental clones from the GSP were members of the heterocystous lineage. Although a number of cyanobacterial isolates were close matches with prevalent environmental clones, it is not certain if these clones reflect the same halotolerance ranges of their matching isolates. This caveat is based on the notable disparities we found between strains of the same phylotype and their inherent halotolerance. Our findings support the hypothesis that variable or poikilotrophic environments promote diversification, and in particular, select for variation in ecotype more than phylotype.     

Archaeal Diversity at the Great Salt Plains of Oklahoma Described by Cultivation and Molecular Analyses

The Great Salt Plains of Oklahoma is a natural inland terrestrial hypersaline environment that forms evaporite crusts of mainly NaCl. Previous work described the bacterial community through the characterization of 105 isolates from 46 phylotypes. The current report describes the archaeal community through both microbial isolation and culture-independent techniques. Nineteen distinct archaea were isolated, and ten were characterized phenetically. Included were isolates phylogenetically related to Haloarcula, Haloferax, Halorubrum, Haloterrigena, and Natrinema. The isolates were aerobic, non-motile, Gram-negative organisms and exhibited little capacity for fermentation. All of the isolates were halophilic, with most requiring at least 15% salinity for growth, and all grew at 30% salinity. The isolates were mainly mesothermic and could grow at alkaline pH (8.5). A 16S rRNA gene library was generated by polymerase chain reaction amplification of direct soil DNA extracts, and 200 clones were sequenced and analyzed. At 99% and 94% sequence identity, 36 and 19 operational taxonomic units (OTUs) were detected, respectively, while 53 and 22 OTUs were estimated by Chao1, respectively. Coverage was relatively high (100% and 59% at 89% and 99% sequence identity, respectively), and the Shannon Index was 3.01 at 99% sequence identity, comparable to or somewhat lower than hypersaline habitats previously studied. Only sequences from Euryarchaeota in the Halobacteriales were detected, and the strength of matches to known sequences was generally low, most near 90% sequence identity. Large clusters were observed that are related to Haloarcula and Halorubrum. More than two-thirds of the sequences were in clusters that did not have close relatives reported in public databases.     

CaCO3 and MgCO3 Dissolving Halophilic Bacteria

In the current study, fifteen halophilic and halotolerant bacteria were isolated from salt-affected soil of ?anl?urfa, Turkey. The isolates were characterized by conventional and molecular techniques (16S rDNA sequence analyses) as belonging to seven different genus including Bacillus (5 isolates), Halobacillus (1 isolate), Oceanobacillus (2 isolates), Halomonas (3 isolate), Nesterenkonia (1 isolate), Chromohalobacter (2 isolates) and Jeotgalibacillus (2 isolates). According to the results obtained, the investigated bacterial strains have high salt tolerance and significant enzyme activities which can improve soil nutrient cycling and fertility. Furthermore, these bacterial strains have been investigated for their ability to dissolve common salts available in salt-affected soils. Salt dissolving experiments showed that two Chromohalobacter isolates were able to dissolve CaCO₃ and one of the Halomonas isolate was able to dissolve both CaCO₃ and MgCO₃. As these bacterial isolates can dissolve CaCO₃ and MgCO₃, the availability of Ca²⁺ and Mg²⁺ ions may increase which can enhance the removal of the excess Na⁺ in soil profile.     

ALGAL COMMUNITY DYNAMICS AND HALOTOLERANCE IN A TERRESTRIAL,HYPERSALINE ENVIRONMENT1

We studied the algal community of the Great Salt Plains (GSP), an expansive (65 km²) salt flat situated in north‐central Oklahoma, USA that has been designated as the Salt Plains Microbial Observatory (SPMO) by the National Science Foundation. The GSP offered a unique opportunity to study a terrestrial, hypersaline algal community that experiences wide‐ranging environmental conditions. We were able to show that ammonium‐N, rather than salinity, was the most important predictor of total algal biomass. However, salinity was found to be a significant controlling variable in diatom distribution at the GSP, where diatom abundance was negatively correlated with porewater salinity concentrations. Overall, chlorophytes (likely dominated by Dunaliella spp.) were the most abundant algal group at the consistently hypersaline (>300 ppt) south site. Diatom and cyanobacterial biomass were on average highest at the central site, which experienced greater fluctuations in salinity. While taxonomic diversity was limited to three algal groups (chlorophytes, diatoms, and cyanobacteria), the salinity preferences and halotolerance ranges of isolated strains were quite variable. Although porewater salinities at the GSP are commonly near saturation (>300 ppt), the large majority of isolates had halotolerance ranges below 150 ppt. This suggests that algae at the GSP rarely achieve maximum growth rates, and could only do so when intermittent rain events reduce salinity to optimal levels. Because the vast majority of our strains were isolated from salt‐saturated soil samples, maintaining viability (rather than growth efficiency) appears to be the most successful adaptation to the extreme conditions at the GSP.     

Phylogenetic and metabolic bacterial diversity of <Emphasis Type="Italic">Phragmites australis</Emphasis> periphyton communities in two Hungarian soda ponds

Bacterial diversity of reed (Phragmites australis) periphyton communities of Kelemen-szék and Nagy-Vadas (two Hungarian soda ponds) was investigated using molecular cloning and cultivation-based techniques. The majority of the 80 Kelemen-szék and 72 Nagy-Vadas bacterial isolates proved to be moderately halophilic and alkaliphilic. A great proportion of the isolates showed phosphatase and urease activity, utilized aesculin, citrate and certain biopolymers (e.g., gelatine and tween 80). Partial 16S rDNA sequence analysis of 33 Kelemen-szék and 20 Nagy-Vadas ARDRA group representatives showed Gram-positive (Nesterenkonia, Cellulomonas, Dietzia, Bacillus and Planococcus) dominance at both sampling sites. Species of the genera Acidovorax, Hydrogenophaga (β-Proteobacteria) and Flavobacterium, Sphingobacterium (Bacteroidetes) were represented only from Kelemen-szék. Altogether 16 isolates showed low sequence similarity with yet described bacteria and may represent novel taxa. Screening of the 16S rRNA gene libraries of 129 Kelemen-szék and 158 Nagy-Vadas clones resulted in 30 and 28 different ARDRA groups, respectively. Sequence analysis revealed a Gram-negative (Rheinheimera, Aquimonas, Cellvibrio, Flavobacterium and Sphingobacterium) dominated phylogenetic diversity. A high number of the clones were affiliated with uncultured bacterial clones described from diverse environmental samples.     

Biocontrol Potential of Soybean Bacterial Endophytes Against Charcoal Rot Fungus, <Emphasis Type="Italic">Rhizoctonia bataticola</Emphasis>

A total of 137 bacterial isolates from surface sterilized root, stem, and nodule tissues of soybean were screened for their antifungal activity against major phytopathogens like Rhizoctonia bataticola, Macrophomina phaseolina, Fusarium udam, and Sclerotium rolfsii. Nine bacterial endophytes suppressed the pathogens under in vitro plate assay. These were characterized biochemically and identified at the genus level based on their partial sequence analysis of 16S rDNA. Eight of the isolates belonged to Bacillus and one to Paenibacillus. The phylogenetic relationship among the selected isolates was studied and phylogenetic trees were generated. The selected isolates were screened for biocontrol traits like production of hydrogen cyanide (HCN), siderophore, hydrolytic enzymes, antibiotics, and plant growth promoting traits like indole 3-acetic acid production, phosphate solubilization, and nitrogen fixation. A modified assessment scheme was used to select the most efficient biocontrol isolates Paenibacillus sp. HKA-15 (HKA-15) and Bacillus sp. HKA-121 (HKA-121) as potential candidates for charcoal rot biocontrol as well as soybean plant growth promotion.     

The isolation of heavy-metal resistant culturable bacteria and resistance determinants from a heavy-metal-contaminated site

In this study we performed a phylogenetic analysis of a culturable bacterial community isolated from heavymetal-contaminated soil from southwest Slovakia using 16S rRNA (16S rDNA) and heavy-metal resistance genes. The soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and a trace amount of cadmium (<0.25 mg/kg). A total of 100 isolates were grown on rich (Nutrient agar No. 2) or minimal (soil-extract agar medium) medium. The isolates were identified by phylogenetic analysis using partial sequences of their 16S rRNA (16S rDNA) genes. Representatives of two broad taxonomic groups, Firmicutes and Proteobacteria, were found on rich medium, whereas four taxonomic groups, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, were represented on minimal medium. Forty-two isolates grown on rich medium were assigned to 20 bacterial species, while 58 bacteria grown on minimal medium belonged to 49 species. Twenty-three isolates carried czcA- and/or nccA-like heavy-metal-resistance determinants. The heavy-metalresistance genes of nine isolates were identified by phylogenetic analysis of their protein sequences.     

Cultivable Bacterial Diversity of Alkaline Lonar Lake, India

Aerobic, alkaliphilic bacteria were isolated and characterized from water and sediment samples collected in the winter season, January 2002 from alkaline Lonar lake, India, having pH 10.5. The total number of microorganisms in the sediment and water samples was found to be 10²–10⁶ cfu g⁻¹ and 10²–10⁴ cfu ml⁻¹, respectively. One hundred and ninety-six strains were isolated using different enrichment media. To study the bacterial diversity of Lonar lake and to select the bacterial strains for further characterization, screening was done on the basis of pH and salt tolerance of the isolates. Sixty-four isolates were subjected to phenotypic, biochemical characterization and 16S rRNA sequencing. Out of 64, 31 bacterial isolates were selected on the basis of their enzyme profile and further subjected to phylogenetic analysis. Phylogenetic analysis indicated that most of the Lonar lake isolates were related to the phylum Firmicutes, containing Low G+C, Gram-positive bacteria, with different genera: Bacillus, Paenibacillus, Alkalibacillus, Exiguobacterium, Planococcus, Enterococcus and Vagococcus. Seven strains constituted a Gram-negative bacterial group, with different genera: Halomonas, Stenotrophomonas and Providencia affiliated to γ-Proteobacteria, Alcaligenes to β-Proteobacteria and Paracoccus to α-Proteobacteria. Only five isolates were High G+C, Gram-positive bacteria associated with phylum Actinobacteria, with various genera: Cellulosimicrobium, Dietzia, Arthrobacter and Micrococcus. Despite the alkaline pH of the Lonar lake, most of the strains were alkalitolerant and only two strains were obligate alkaliphilic. Most of the isolates produced biotechnologically important enzymes at alkaline pH, while only two isolates (ARI 351 and ARI 341) showed the presence of polyhydroxyalkcanoate (PHA) and exopolysaccharide (EPS), respectively.     

Culture-Dependent and Culture-Independent Characterization of Microbial Assemblages Associated with High-Temperature Petroleum Reservoirs

Recent investigations of oil reservoirs in a variety of locales have indicated that these habitats may harbor active thermophilic prokaryotic assemblages. In this study, we used both molecular and culture-based methods to characterize prokaryotic consortia associated with high-temperature, sulfur-rich oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at temperatures ranging from 60 to 90°C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling Thermoanaerobacter, and Thermococcus-like isolates. The predominant autotrophic microorganisms recovered from inorganic enrichments using H₂, acetate, and CO₂ as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus species. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universal oligonucleotide primer sets. Sequence analysis of the universal library indicated that a large percentage of clones were highly similar to known bacterial and archaeal isolates recovered from similar habitats. Represented genera in rDNA clone libraries included Thermoanaerobacter, Thermococcus, Desulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Acinetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The archaeal library was dominated by methanogen-like rDNAs, with a lower percentage of clones belonging to the Thermococcales. Our results strongly support the hypothesis that sulfur-utilizing and methane-producing thermophilic microorganisms have a widespread distribution in oil reservoirs and the potential to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.     

Culturable Bacteria Present in the Fluid of the Hooded-Pitcher Plant <Emphasis Type="Italic">Sarracenia Minor</Emphasis> Based on 16S rDNA Gene Sequence Data

The culturable microbial community within the pitcher fluid of 93 Sarracenia minor carnivorous plants was examined over a 2-year study. Many aspects of the plant/bacterial/insect interaction within the pitcher fluid are minimally understood because the bacterial taxa present in these pitchers have not been identified. Thirteen isolates were characterized by 16S rDNA sequencing and subsequent phylogenetic analysis. The Proteobacteria were the most abundant taxa and included representatives from Serratia, Achromobacter, and Pantoea. The Actinobacteria Micrococcus was also abundant while Bacillus, Lactococcus, Chryseobacterium, and Rhodococcus were infrequently encountered. Several isolates conformed to species identifiers (>98% rDNA gene sequence similarity) including Serratia marcescens (isolates found in 27.5% of pitchers), Achromobacter xylosoxidans (37.6%), Micrococcus luteus (40.9%), Bacillus cereus (isolates found in 10.2%), Bacillus thuringiensis (5.4%), Lactococcus lactis (17.2%), and Rhodococcus equi (2.2%). Species–area curves suggest that sampling efforts were sufficient to recover a representative culturable bacterial community. The bacteria present represent a diverse community probably as a result of introduction by insect vectors, but the ecological significance remains under explored.     

Carbon substrate utilization, antibiotic sensitivity, and numerical taxonomy of bacterial isolates from the Great Salt Plains of Oklahoma

The current work extends the phenotypic characterization of a bacterial culture collection from the Great Salt Plains of Oklahoma. This barren expanse of mud flats is typically crusted with thalassohaline salt evaporites. The initial account of the aerobic heterotrophic bacteria from the Great Salt Plains described 105 halotolerant isolates that represented 47 phylotypes. Extensive phenotypic analyses were performed on 76 isolates representing 37 unique phylotypes. The current report extends these observations for 60 of the isolates by measuring a wider set of phenotypic characteristics. Utilization patterns for 45 carbon substrates were used to assign the isolates into seven coherent phenons, along with several singletons and a group of isolates that did not grow on single carbon substrates. Most of the isolates were able to utilize nearly all of the nitrogen sources tested, with nitrate being the least utilized. Little antibiotic resistance was seen in the collection as a whole; however, certain phenons were enriched for antibiotic-resistant organisms. A total of 81 phenotypic characteristics were used to generate dendrograms. The numerical taxonomy trees essentially agreed with those generated using 16S rRNA gene sequences. The pattern of carbon substrate utilization showed substantial changes at different salinities that may have relevance to the variable salinities microbes experience at the Salt Plains over time.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Isolation and Characterization of Salt-Tolerant Bacterial Strains in Salt-Affected Soils of East Anatolian Region

In the current study, 18 salt-tolerant bacteria were isolated from salt-affected soil in the east Anatolian region. The obtained isolates were identified and characterized by conventional (morphology, physiology, and biochemical tests) and molecular techniques 16 rDNA. Among 18 sequenced isolates, 6 Bacillus, 1 Halomonas, 2 Halobacillus, 2 Zhihengliuella, 2 Oceanobacillus, 1 Virgibacillus, 1 Staphylococcus, 1 Thalassobacillus, 1 Exiguobacterium were identified with high similarity to previously identified strains in the literature. According to the results obtained, investigated bacterial strains have high salt tolerance and significant enzyme activities that can improve soil nutrient cycling and fertility. To the best of our knowledge, the current article is the first study in evaluation and diversity of potential halophlic/halotolerant bacterial strains in salt-affected soils of the east Anatolian region.     

Phylogenetic analysis of culturable marine bacteria in sediments from South Korean Yellow Sea

Biogeochemical and microbiological characterization of marine sediments taken from the Yellow Sea of South Korea was carried out. One hundred and thirty six bacterial strains were isolated, characterized and phylogenetic relationship was evaluated. The gene sequences of 16S rDNA regions were examined to study the phylogenetic analysis of bacterial community in the marine sediments. Among 136 isolates, 5 bacterial isolates were identified as novel members, remaining 131 isolates were fall into 5 major linkages of bacterial phyla represented as follows: Firmicutes,, -@Proteobacteria, High G + C and Bacteroidetes. Bacterial community in sediments mainly dominated by Firmicute (58.77%) and followed by @-Proteobacteria (38.16%). @-Proteobacteria domain highly diverged and mainly consists of the genera Vibrio, Marinobacterium, Photobacterium, Pseudoalteromonas, Oceanisphaera, Halomonas, Alteromonas, Stenotrophomas and Pseudomonas. Total N and Organic matter content in Yellow Sea of South Korea were relatively high. The Total-N content in the sediments was varied from 177.31 to 1974.96 (mg/kg) and organic matter ranged from 0.82 to 4.23 (g/100 g⁻¹). The current research work provides clear explanation obtained for the phylogenetic affiliation of the culturable bacterial community in sediments of South Korean Yellow Sea and revealed the relationship with biogeochemical characteristics of the sediments.     

Isolation of sulfate‐reducing bacteria from deep sediment layers of the pacific ocean

Bacterial populations exist at great depths in marine sediments, but little is known about the type and characteristics of organisms in this unique bacterial environment. Cascadia Margin sediments from the Pacific Ocean have deep bacterial activity and bacterial populations, which are stimulated around a gas hydrate zone (215–225 m below sea floor [mbsf]). Bacterial sulfate reduction is the dominant anaerobic process within these sediments, and the depth distribution of sulfate‐reducing activity corresponds with distributions of viable sulfate‐reducing bacteria (SRB). Anaerobically stored sediments from this site were used to isolate sulfate‐reducing bacteria using a temperature‐gradient system, elevated pressure and temperatures, different media, and a range of growth substrates. A variety of enrichments on lactate were obtained from 0.5 and 222 mbsf, with surprisingly more rapid growth from the deeper sediments. The temperature range of enrichments producing strong growth from 222 mbsf was markedly wider than those from the near surface sediment (15–45°C and 9–19°C, respectively). This presumably reflects a temperature increase in deeper sediments. Only a few of these enrichments were successfully isolated due to very slow or no growth on subculture, despite the use of a wide range of different media and growth conditions. Psychrophilic and mesophilic sulfate‐reducing isolates were obtained from 0.5 m depth. As the minimum growth temperature of the mesophile (probably a Desulfotomaculum sp.) was above the in situ temperature of 3°C, it must have been present in the sediment as spores. A larger number of isolates (23) was obtained from 222 mbsf, and these barophilic SRB were closely related (based on 16S rRNA gene analysis), but not identical to, Desulfovibrio profundus, recently isolated from deep sediments from the Japan Sea. Bacteria related to D. profundus may be widespread in deep marine sediments.     

Phylogenetic and physiological characterization of mesophilic and thermophilic bacteria from a sewage sludge composting process in Sapporo,Japan

The phylogenetic and physiological characteristics of mesophilic and thermophilic bacteria isolated from a field-scale sewage sludge composter were determined by 16S rDNA and phenotype analyses. Of the 34 mesophilic isolates, 5 (15%), 16 (47%), and 3 (9%) displayed amylase, protease, and lipase activities, respectively. Among these isolates, the following species were identified based on their 16S rRNA gene sequences: Aneurinibacillus aneurinilyticus, Bacillus fortis, Bacillus subtilis, Brachybacterium paraconglomeratum, Brevibacterium otitidis, Dietzia maris, Pseudomonas xiamenensis, Staphylococcus lentus, Thermobifida fusca, Ureibacillus thermosphaericus, and Vagococcus lutrae. However, 15 isolates could not be identified as known taxa, thus indicating new bacterial taxa. Of these new taxa, it is likely that NoID A plays an important role in organic matter decomposition during composting based on its physiological characteristics. Sapporo sewage sludge compost contains a microbial ecosystem with novel bacterial biodiversity, comprising a high percentage of previously unrecognized species. This study improves our knowledge of the unique bacteria in sewage sludge compost, providing a future resource for bacterial genetic information and bacterial species of agricultural benefit.     

Isolation and Biodiversity of Hitherto Undescribed Soil Bacteria Related to <Emphasis Type="Italic">Bacillus niacini</Emphasis>

The hitherto largely not described phylogenetic neighborhood of Bacillus niacini has been explored by a comprehensive cultivation experiment and genomic variety studies. Previous culture-independent studies demonstrated that ~15% of all Bacillus 16S rDNA directly extracted from soils worldwide was affiliated to B. niacini. Seven different media were inoculated with soil suspensions in serial dilutions and incubated at different temperatures. Then, bacterial colonies were picked and analyzed by sequencing. A mineral medium with acetate as carbon source yielded a B. niacini rate of >3% of all picked colonies. Other media were less efficient but also successful. Applying this culturing approach, we succeeded in obtaining 64 isolates from different Dutch soils. The isolates turned out to be diverse, although closely related to B. niacini as revealed by 16S rDNA sequencing. Close matches with environmental clones were also found, thus demonstrating much more diversity beyond previously known 16S rDNA sequences. The rep-PCR fingerprinting method revealed a high genomic variety, redundancy could not be observed among our isolates. Hence, the hitherto neglected B. niacini lineage, apparently among the most abundant soil Bacillus, was accessible to our cultivation approach.     

Characterization of halophilic bacteria from environmental samples from the brackish water of Pulicat Lake, India

Culture dependent phenotypic characterization and 16S rDNA based phylogenetic analyses were applied to study the aerobic halophilic bacterial population present in the Pulicat brackish-water Lake of India. Five different media were employed for isolation of bacteria. A total of 198 morphotypes were recovered, purified and screened for salt tolerance in nutrient agar medium amended with 5–25% NaCl. Based on 16S rDNA restriction fragment length polymorphism analysis with three restriction endonucleases, 51 isolates tolerant to 5% or more NaCl were grouped into 29 clusters. Phylogenetic analysis using 16S rRNA gene sequences revealed that 29 strains could further be allocated into two clades: 19 to Firmicutes and 10 to γ-Proteobacteria. Firmicutes included low G+C Gram-positive bacteria related to family Bacillaceae, which included five genera Bacillus, Virgibacillus, Rummelibacillus, Alkalibacillus and Halobacillus. Another genera included in Firmicutes was Salimicrobium halophilum. In the γ-Proteobacteria group, all the isolates belonged to one genus Halomonas, represented by six different species Halomonas salina, H. shengliensis, H. salifodinae, H. pacifica, H. aquamarina and H. halophila. Most of the isolates exhibited cellulase, xylanase, amylase and protease activities.     

Phylogeny of Sphingomonas species that degrade pentachlorophenol

Four pentachlorophenol (PCP)-degrading bacteria isolated from geographically diverse areas have been examined in detail as regards their physiology and phylogeny. According to traditional biochemical methods, these strains had been classified as members of the genera Arthrobacter, Flavobacterium, Pseudomonas, and Sphingomonas. The PCP degradation pathway has been studied extensively in Sphingomonas (Flavobacterium) sp strain ATCC 39723 and the first three degradation steps catalyzed by a PCP-4-monooxygenase (PcpB) and a reductive dehalogenase (PcpC) that functions twice are well established. A fourth step appears to involve ring-fission of the aromatic nucleus (PcpA). Molecular analyses revealed that the PCP degradation pathway in these four strains was rather conserved, leading to a phylogenetic analysis using 16S rDNA. The results revealed a much closer phylogenetic relationship between these organisms than traditional classification indicated, placing them into the more recently established genus Sphingomonas where they may even represent a single species. With 16S rDNA analysis, many bacterial isolates involved in degradation of xenobiotic compounds that were previously classified into diverse genera have been reclassified into the genus Sphingomonas. Received 14 April 1999/ Accepted in revised form 20 July 1999