Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism.

BACKGROUND: Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Experiments were performed to understand the effect of monosaccharides on gene expression during growth on the polysaccharide, WAX. RESULTS: Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolases were downregulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, showing a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. CONCLUSIONS: Glycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases were not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a broader substrate range than that represented by the glycoside hydrolase family in which the enzymes were categorized.

Concurrent metabolism of pentose and hexose sugars by the polyextremophile Alicyclobacillus acidocaldarius.

Alicyclobacillus acidocaldarius is a thermoacidophilic bacterium capable of growth on sugars from plant biomass. Carbon catabolite repression (CCR) allows bacteria to focus cellular resources on a sugar that provides efficient growth, but also allows sequential, rather than simultaneous use when more than one sugar is present. The A. acidocaldarius genome encodes all components of CCR, but transporters encoded are multifacilitator superfamily and ATP-binding cassette-type transporters, uncommon for CCR. Therefore, global transcriptome analysis of A. acidocaldarius grown on xylose or fructose was performed in chemostats, followed by attempted induction of CCR with glucose or arabinose. Alicyclobacillus acidocaldarius grew while simultaneously metabolizing xylose and glucose, xylose and arabinose, and fructose and glucose, indicating that CCR did not control carbon metabolism. Microarrays showed down-regulation of genes during growth on one sugar compared to two, and occurred primarily in genes encoding: (1) regulators; (2) enzymes for cell wall synthesis; and (3) sugar transporters.

Variation in Biological Soil Crust Bacterial Abundance and Diversity as a Function of Climate in Cold Steppe Ecosystems in the Intermountain West, USA.

Biological soil crust (biocrust) is a composite of mosses, lichens, and bacteria that performs many important soil system functions, including increasing soil stability, protecting against wind erosion, reducing nutrient loss, and mediating carbon and nitrogen fixation cycles. These cold desert and steppe ecosystems are expected to experience directional changes in both climate and disturbance. These include increased temperatures, precipitation phase changes, and increased disturbance from anthropogenic land use. In this study, we assessed how climate and grazing disturbance may affect the abundance and diversity of bacteria in biocrusts in cold steppe ecosystems located in southwestern Idaho, USA. To our knowledge, our study is the first to document how biocrust bacterial composition and diversity change along a cold steppe climatic gradient. Analyses based on 16S small subunit ribosomal RNA gene sequences identified the phylum Actinobacteria as the major bacterial component within study site biocrusts (relative abundance = 36-51%). The abundance of the phyla Actinobacteria and Firmicutes was higher at elevations experiencing cooler, wetter climates, while the abundance of Cyanobacteria, Proteobacteria, and Chloroflexi decreased. The abundance of the phyla Cyanobacteria and Proteobacteria showed no significant evidence of decline in grazed areas. Taken together, results from this study indicate that bacterial communities from rolling biocrusts found in cold steppe ecosystems are affected by climate regime and differ substantially from other cold desert ecosystems, resulting in potential differences in nutrient cycling and ecosystem dynamics.

Tyrosine phosphorylation of HSC70 and its interaction with RFC mediates methotrexate resistance in murine L1210 leukemia cells.

We previously identified and characterized a 66-68 kDa membrane-associated, tyrosine phosphorylated protein in murine leukemia L1210 cells as HSC70 which is a methotrexate (MTX)-binding protein. In order to further characterize the functional role of HSC70 in regulating MTX resistance in L1210 cells, we first showed that HSC70 colocalizes and interacts with reduced folate carrier (RFC) in L1210 cells by confocal laser scanning microscopy and Duolink in situ proximity ligation assay. The tyrosine phosphorylation status of HSC70 found in the membrane fraction was different from the parental L1210/0 and cisplatin (CDDP)-MTX cross resistant L1210/DDP cells. In MTX-binding assays, HSC70 from L1210/DDP cells showed less affinity for MTX-agarose beads than that of L1210/0 cells. In addition, genistein (a tyrosine phosphorylation inhibitor) significantly enhanced the resistance of L1210/0 cells to MTX. Moreover, site-directed mutation studies indicated the importance of tyrosine phosphorylation of HSC70 in regulating its binding to MTX. These findings suggest that tyrosine phosphorylation of HSC70 regulates the transportation of MTX into the cells via the HSC70-RFC system and contributes to MTX resistance in L1210 cells.

Inhibition of Batrachochytrium dendrobatidis Caused by Bacteria Isolated from the Skin of Boreal Toads, Anaxyrus (Bufo) boreas boreas, from Grand Teton National Park, Wyoming, USA.

The chytrid fungus Batrachochytrium dendrobatidis is a significant cause of the worldwide decline in amphibian populations; however, various amphibian species are capable of coexisting with B. dendrobatidis. Among them are boreal toads (Anaxyrus (Bufo) boreas boreas) located in Grand Teton National Park (GTNP) in Wyoming, USA. The purpose of this study was to identify cultivable bacterial isolates from the skin microbiota of boreal toads from GTNP and determine if they were capable of inhibiting B. dendrobatidis in vitro, and therefore might be a factor in the toad's coexistence with this pathogen. Isolates from 6 of 21 genera tested were found to inhibit the growth of B. dendrobatidis. These bacteria represent diverse lineages such as the Gammaproteobacteria, the Betaproteobacteria, and the Bacteroidetes/Chlorobium groups. We propose that these bacteria compete via microbial antagonism with B. dendrobatidis.

An anion-exchange method to concentrate dissolved DNA from aquifer water.

A rapid DNA isolation method was developed to concentrate dissolved DNA (dDNA) in aquifer water for molecular analysis. The aquifer dDNA from the Eastern Snake River Plain Aquifer (ESRPA) was extracted and concentrated using a new method with an anion-exchange Mustang® Q membrane. The concentration of aquifer dDNA in this study ranged from 60 to 264.5 ng l−1 in ESRPA aquifer wells. DNA stability in ESRPA aquifer water was also tested in this study. The dDNA extracted from aquifer water samples was used for PCR amplification of bacterial 16S rRNA genes for terminal restriction fragment length polymorphism (T-RFLP) analysis and construction of 16S rRNA gene clone libraries. The ureC gene, IncP, IncQ and IncW plasmid genes were also PCR amplified from dDNA samples. Based on the results, dDNA is relatively stable in aquifer water and can be concentrated by Q membrane method for molecular analysis. The quality of isolated dDNA was suitable as a PCR template.

Identification and characterization of a 66-68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells.

We previously observed an unidentified, tyrosine-phosphorylated, membrane-associated, 66-68-kDa protein which was present in the L1210 murine leukemia cells but not present, at least in the tyrosine-phosphorylated form, in cisplatin-methotrexate (CDDP-MTX) cross-resistant L1210/DDP cells. We purified and characterized this 66-68-kDa protein by affinity chromatography purification using its two identified properties, tyrosine phosphorylation and MTX-binding, and yielded a single band of 66-68 kDa. The purified protein was subjected to trypsin digestion and the isolated peptide fragments were sequenced and yielded two partial peptide sequences: VEIIANDQ and VTNAVVTVPAYFNDSQRQA. The two peptide sequences were used to search for the mouse genome at the national center for biotechnology information (NCBI) database for Open Reading Frame Sequence (ORFs) containing these peptides using the TBLASTN function. A single gene was identified containing both sequences, the HSPa8 gene, which codes for the heat shock family protein, HSC70. We further demonstrated that HSC70 is a MTX-binding protein using a binding assay with MTX-agarose beads followed by Western blotting. The HSC70 also existed in various cancer cell lines and showed binding to MTX. Additionally, the HSC70 protein, cloned from the L1210 murine leukemia cells, was expressed and purified from E. coli cells using a polyhistidine-tag purification system and it also showed the binding properties with MTX. DnaK, the HSC70 homologue in E. coli, also binds to MTX. By using the purified truncated HSC70 domains, we identified the adenosine triphosphatase (ATPase) domain of HSC70 that can bind to MTX. Thus, we have tentatively characterized a new, novel property of HSC70 as a MTX-binding protein.

Site-directed mutagenesis of a family 42 ß-galactosidase from an antarctic bacterium.

Site directed mutagenesis was used to modify the active site of a cold active beta-galactosidase taken from an Antarctic psychrotolerant Planococcus Bacterial isolate. The goal was to modify the active site such that there would be an increase in activity on certain substrates which showed little to no activity with the wild type enzyme. A total of 5 mutant enzymes were constructed with amino acid changes based on an analysis done via homology modeling. All 5 modified enzymes were assayed using 14 different nitrophenol substrates. In most cases there was a loss of activity on substrates that showed activity with the wild type enzymes. None of the expected activity was observed with any of the mutants, possibly in part due to a decrease in hydrogen bonding between the active site and the substrates. With the substrates p-nitrophenyl-ß-d-galacturonide and p-nitrophenyl-α-d-glucopyranoside we saw increased activity. With one of the mutants we measured a 320% increase in activity on p-nitrophenyl-ß-d-galacturonide. Two other mutants showed activity on p-nitrophenyl-α-d-glucopyranoside, which showed no activity at all with the wild type enzyme.

Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100-bp insertion in helix 6.

Two different versions of the 16S rRNA gene, one of which contained an unusual 100-bp insertion in helix 6, were detected in isolate UFO1 acquired from the Oak Ridge Integrated Field-Research Challenge (ORIFRC) site in Tennessee. rRNA was extracted from UFO1 and analyzed by reverse transcriptase-quantitative PCR with insert- and non-insert-specific primers; only the noninsert 16S rRNA gene sequence was detected. Similarly, PCR-based screening of a cDNA library (190 clones) constructed from reverse-transcribed rRNA from UFO1 did not detect any clones containing the 100-bp insert. Examination of cDNA with primers specific to the insert-bearing 16S rRNA gene, but downstream of the insert, suggests that the insert was excised from rRNA. Inspection of other 16S rRNA genes in the GenBank database revealed that a homologous insert sequence, also found in helix 6, has been reported in other environmental clones, including those acquired from ORIFRC enrichments. These findings demonstrate the existence of widely divergent copies of the 16S rRNA gene within the same organism, which may confound 16S rRNA gene-based methods of estimating microbial diversity in environmental samples.

Shiga toxins, and the genes encoding them, in fecal samples from native Idaho ungulates.

Cattle are a known reservoir of Shiga toxin-producing Escherichia coli. The prevalence and stability of Shiga toxin and/or Shiga toxin genes among native wild ungulates in Idaho were investigated. The frequency of both Shiga genes and toxin was similar to that reported for Idaho cattle ( approximately 19%).

Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis.

We assessed the diversity and phylogeny of Saprolegniaceae on amphibian eggs from the Pacific Northwest, with particular focus on Saprolegnia ferax, a species implicated in high egg mortality. We identified isolates from eggs of six amphibians with the internal transcribed spacer (ITS) and 5.8S gene regions and BLAST of the GenBank database. We identified 68 sequences as Saprolegniaceae and 43 sequences as true fungi from at least nine genera. Our phylogenetic analysis of the Saprolegniaceae included isolates within the genera Saprolegnia, Achlya and Leptolegnia. Our phylogeny grouped S. semihypogyna with Achlya rather than with the Saprolegnia reference sequences. We found only one isolate that grouped closely with S. ferax, and this came from a hatchery-raised salmon (Idaho) that we sampled opportunistically. We had representatives of 7-12 species and three genera of Saprolegniaceae on our amphibian eggs. Further work on the ecological roles of different species of Saprolegniaceae is needed to clarify their potential importance in amphibian egg mortality and potential links to population declines.

Phylogenetic analysis of Shiga toxin 1 and Shiga toxin 2 genes associated with disease outbreaks.

BACKGROUND: Shiga toxins 1 and 2 (Stx1 and Stx2) are bacteriophage-encoded proteins that have been associated with hemorrhagic colitis, hemolytic uremic syndrome and other severe disease conditions. Stx1 and Stx2 are genetically and immunologically distinct but share the same compound toxin structure, method of entry and enzymatic function. RESULTS: Phylogenetic analysis was performed using Stx1 and Stx2 amino acid and nucleotide sequences from 41 strains of Escherichia coli, along with known stx sequences available from GenBank. The analysis confirmed the Stx1 and Stx2 divergence, and showed that there is generally more sequence variation among stx2 genes than stx1. The phylograms showed generally flat topologies among our strains' stx1 and stx2 genes. In the stx2 gene, 39.5% of the amino acid sites display very low nonsynonymous to synonymous substitution ratios. CONCLUSION: The stx1 and stx2 genes used in this phylogenetic study show sequence conservation with no significant divergence with respect to place or time. These data could indicate that Shiga toxins are experiencing purifying selection.

PCR versus hybridization for detecting virulence genes of enterohemorrhagic Escherichia coli.

We compared PCR amplification of 9 enterohemorrhagic Escherichia coli virulence factors among 40 isolates (21 O/H antigenicity classes) with DNA hybridization. Both methods showed 100% of the chromosomal and phage genes: eae, stx, and stx2. PCR did not detect 4%-20% of hybridizable plasmid genes: hlyA, katP, espP, toxB, open reading frame (ORF) 1, and ORF2.

Low temperature reduction of hexavalent chromium by a microbial enrichment consortium and a novel strain of Arthrobacter aurescens.

BACKGROUND: Chromium is a transition metal most commonly found in the environment in its trivalent [Cr(III)] and hexavalent [Cr(VI)] forms. The EPA maximum total chromium contaminant level for drinking water is 0.1 mg/l (0.1 ppm). Many water sources, especially underground sources, are at low temperatures (less than or equal to 15 Centigrade) year round. It is important to evaluate the possibility of microbial remediation of Cr(VI) contamination using microorganisms adapted to these low temperatures (psychrophiles). RESULTS: Core samples obtained from a Cr(VI) contaminated aquifer at the Hanford facility in Washington were enriched in Vogel Bonner medium at 10 Centigrade with 0, 25, 50, 100, 200, 400 and 1000 mg/l Cr(VI). The extent of Cr(VI) reduction was evaluated using the diphenyl carbazide assay. Resistance to Cr(VI) up to and including 1000 mg/l Cr(VI) was observed in the consortium experiments. Reduction was slow or not observed at and above 100 mg/l Cr(VI) using the enrichment consortium. Average time to complete reduction of Cr(VI) in the 30 and 60 mg/l Cr(VI) cultures of the consortium was 8 and 17 days, respectively at 10 Centigrade. Lyophilized consortium cells did not demonstrate adsorption of Cr(VI) over a 24 hour period. Successful isolation of a Cr(VI) reducing organism (designated P4) from the consortium was confirmed by 16S rDNA amplification and sequencing. Average time to complete reduction of Cr(VI) at 10 Centigrade in the 25 and 50 mg/l Cr(VI) cultures of the isolate P4 was 3 and 5 days, respectively. The 16S rDNA sequence from isolate P4 identified this organism as a strain of Arthrobacter aurescens, a species that has not previously been shown to be capable of low temperature Cr(VI) reduction. CONCLUSION: A. aurescens, indigenous to the subsurface, has the potential to be a predominant metal reducer in enhanced, in situ subsurface bioremediation efforts involving Cr(VI) and possibly other heavy metals and radionuclides.

Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic arthrobacter isolate.

A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 beta-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18 degrees C and 50% activity remaining at 0 degrees C) and heat labile (inactivated within 10 min at 37 degrees C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both beta-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-beta-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20 degrees C than the comparable LacZ beta-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4 degrees C but dissociated into inactive monomers at 25 degrees C. Comparisons of family 2 beta-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.

Rhodoglobus vestalii gen. nov., sp. nov., a novel psychrophilic organism isolated from an Antarctic Dry Valley lake.

A novel, psychrophilic, gram-positive bacterium (designated strain LV3T) from a lake near the McMurdo Ice Shelf, Antarctica, has been isolated and characterized. This organism formed red-pigmented colonies, had an optimal growth temperature of 18 degrees C and grew on a variety of media between -2 and 21 degrees C. Scanning electron micrographs of strain LV3T that showed small rods with unusual bulbous protuberances during all phases of growth were of particular interest. The G + C content of the genomic DNA was approximately 62 mol%. The cell walls contained ornithine as the diamino acid. The major fatty acids were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. Cells grown at -2 degrees C contained significant amounts of anteiso-C15:1. The major menaquinones found in strain LV3T were MK-11 and MK-12. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain LV3T was a member of the family Microbacteriaceae and related to, but distinct from, organisms belonging to the genera Agreia, Leifsonia and Subtercola. In addition, alignments of 16S rRNA sequences showed that the sequence of strain LV3T contained a 13 bp insertion that was found in only a few related sequences. Based on the low growth temperature, unusual cell shape, distinct 16S rRNA gene sequence and structure and cell-wall amino acid and menaquinone compositions, Rhodoglobus vestalii gen. nov., sp. nov. is proposed, with the type strain LV3T (= ATCC BAA-534T = CIP 107482T).

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core.

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

Microbial communities from methane hydrate-bearing deep marine sediments in a forearc basin.

Microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35 degrees C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within the Euryarchaeota and Crenarchaeota domains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to the Bacteroidetes, Planctomycetes, Actinobacteria, Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments.