16S rRNA Sequences and Differences in Bacteria Isolated from the Muztag Ata Glacier at Increasing Depths

Small subunit 16S rRNA sequences, growth temperatures, and phylogenetic relationships have been established for 129 bacterial isolates recovered under aerobic growth conditions from different regions of a 22-m ice core from the Muztag Ata Mountain glacier on the Pamirs Plateau (China). Only 11% were psychrophiles (grew at 2°C or −2°C up to ~20°C), although the majority (82%) were psychrotolerant (grew at 2°C or −2°C up to 37°C). The majority of the isolates had 16S rRNA sequences similar to previously determined sequences, ranging from 85% to 100% identical to database sequences. Based on their 16S rRNA sequences, 42.6% of the isolates were high-G+C (HGC) gram-positive bacteria, 23.3% were γ-Proteobacteria, 14.7% were α-Proteobacteria, 14.7% were Flavobacteria, and 4.7% were low-G+C (LGC) gram-positive bacteria. There were clear differences in the depth distribution, with Proteobacteria, HGC/Cytophaga-Flavobacterium-Bacteroides (CFB), Proteobacteria, LGC/CFB/HGC, Cryobacterium psychrophilum, HGC/CFB, Proteobacteria/HGC/CFB, and HGC/CFB being the predominant isolates from ice that originated from 2.7 to 3.8, 6.2, 7.5, 8.3, 9.0, 9.7, 12.5, and 15.3 m below the surface, respectively. This layered distribution of bacterial isolates presumably reflects both differences in bacteria inhabiting the glacier's surface, differences in bacteria deposited serendipitously on the glacier's surface by wind and snowfall, and nutrient availability within the ice.     

Detection and Isolation of Ultrasmall Microorganisms from a 120,000-Year-Old Greenland Glacier Ice Core

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 μm³) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5°C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-μm, 0.2-μm, and even 0.1-μm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-μm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria; Cytophaga-Flavobacteria-Bacteroides; high-G+C gram-positive; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.     

Depth Distribution of Microbial Diversity in Mono Lake, a Meromictic Soda Lake in California

We analyzed the variation with depth in the composition of members of the domain Bacteria in samples from alkaline, hypersaline, and currently meromictic Mono Lake in California. DNA samples were collected from the mixolimnion (2 m), the base of the oxycline (17.5 m), the upper chemocline (23 m), and the monimolimnion (35 m). Composition was assessed by sequencing randomly selected cloned fragments of 16S rRNA genes retrieved from the DNA samples. Most of the 212 sequences retrieved from the samples fell into five major lineages of the domain Bacteria: α- and γ-Proteobacteria (6 and 10%, respectively), Cytophaga-Flexibacter-Bacteroides (19%), high-G+C-content gram-positive organisms (Actinobacteria; 25%), and low-G+C-content gram-positive organisms (Bacillus and Clostridium; 19%). Twelve percent were identified as chloroplasts. The remaining 9% represented β- and δ-Proteobacteria, Verrucomicrobiales, and candidate divisions. Mixolimnion and oxycline samples had low microbial diversity, with only 9 and 12 distinct phylotypes, respectively, whereas chemocline and monimolimnion samples were more diverse, containing 27 and 25 phylotypes, respectively. The compositions of microbial assemblages from the mixolimnion and oxycline were not significantly different from each other (P = 0.314 and 0.877), but they were significantly different from those of chemocline and monimolimnion assemblages (P < 0.001), and the compositions of chemocline and monimolimnion assemblages were not significantly different from each other (P = 0.006 and 0.124). The populations of sequences retrieved from the mixolimnion and oxycline samples were dominated by sequences related to high-G+C-content gram-positive bacteria (49 and 63%, respectively) distributed in only three distinct phylotypes, while the population of sequences retrieved from the monimolimnion sample was dominated (52%) by sequences related to low-G+C-content gram-positive bacteria distributed in 12 distinct phylotypes. Twelve and 28% of the sequences retrieved from the chemocline sample were also found in the mixolimnion and monimolimnion samples, respectively. None of the sequences retrieved from the monimolimnion sample were found in the mixolimnion or oxycline samples. Elevated diversity in anoxic bottom water samples relative to oxic surface water samples suggests a greater opportunity for niche differentiation in bottom versus surface waters of this lake.     

Comparative Genomics of DNA Fragments from Six Antarctic Marine Planktonic Bacteria

Six environmental fosmid clones from Antarctic coastal water bacterioplankton were completely sequenced. The genome fragments harbored small-subunit rRNA genes that were between 85 and 91% similar to those of their nearest cultivated relatives. The six fragments span four phyla, including the Gemmatimonadetes, Proteobacteria (α and γ), Bacteroidetes, and high-G+C gram-positive bacteria. Gene-finding and annotation analyses identified 244 total open reading frames. Amino acid comparisons of 123 and 113 Antarctic bacterial amino acid sequences to mesophilic homologs from G+C-specific and SwissProt/UniProt databases, respectively, revealed widespread adaptation to the cold. The most significant changes in these Antarctic bacterial protein sequences included a reduction in salt-bridge-forming residues such as arginine, glutamic acid, and aspartic acid, reduced proline contents, and a reduction in stabilizing hydrophobic clusters. Stretches of disordered amino acids were significantly longer in the Antarctic sequences than in the mesophilic sequences. These characteristics were not specific to any one phylum, COG role category, or G+C content and imply that underlying genotypic and biochemical adaptations to the cold are inherent to life in the permanently subzero Antarctic waters.     

Characterization of the Dominant and Rare Members of a Young Hawaiian Soil Bacterial Community with Small-Subunit Ribosomal DNA Amplified from DNA Fractionated on the Basis of Its Guanine and Cytosine Composition

The small-subunit ribosomal DNA (rDNA) diversity was found to be very high in a Hawaiian soil community that might be expected to have lower diversity than the communities in continental soils because the Hawaiian soil is geographically isolated and only 200 years old, is subjected to a constant climate, and harbors low plant diversity. Since an underlying community structure could not be revealed by analyzing the total eubacterial rDNA, we first fractionated the DNA on the basis of guanine-plus-cytosine (G+C) content by using bis-benzimidazole and equilibrium centrifugation and then analyzed the bacterial rDNA amplified from a fraction with a high biomass (63% G+C fraction) and a fraction with a low biomass (35% G+C fraction). The rDNA clone libraries were screened by amplified rDNA restriction analysis to determine phylotype distribution. The dominant biomass reflected by the 63% G+C fraction contained several dominant phylotypes, while the community members that were less successful (35% G+C fraction) did not show dominance but there was a very high diversity of phylotypes. Nucleotide sequence analysis revealed taxa belonging to the groups expected for the G+C contents used. The dominant phylotypes in the 63% G+C fraction were members of the Pseudomonas, Rhizobium-Agrobacterium, and Rhodospirillum assemblages, while all of the clones sequenced from the 35% G+C fraction were affiliated with several Clostridium assemblages. The two-step rDNA analysis used here uncovered more diversity than can be detected by direct rDNA analysis of total community DNA. The G+C separation step is also a way to detect some of the less dominant organisms in a community.     

Geomicrobiology of High-Level Nuclear Waste-Contaminated Vadose Sediments at the Hanford Site, Washington State

Sediments from a high-level nuclear waste plume were collected as part of investigations to evaluate the potential fate and migration of contaminants in the subsurface. The plume originated from a leak that occurred in 1962 from a waste tank consisting of high concentrations of alkali, nitrate, aluminate, Cr(VI), ¹³⁷Cs, and ⁹⁹Tc. Investigations were initiated to determine the distribution of viable microorganisms in the vadose sediment samples, probe the phylogeny of cultivated and uncultivated members, and evaluate the ability of the cultivated organisms to survive acute doses of ionizing radiation. The populations of viable aerobic heterotrophic bacteria were generally low, from below detection to ~10⁴ CFU g⁻¹, but viable microorganisms were recovered from 11 of 16 samples, including several of the most radioactive ones (e.g., >10 μCi of ¹³⁷Cs/g). The isolates from the contaminated sediments and clone libraries from sediment DNA extracts were dominated by members related to known gram-positive bacteria. Gram-positive bacteria most closely related to Arthrobacter species were the most common isolates among all samples, but other phyla high in G+C content were also represented, including Rhodococcus and Nocardia. Two isolates from the second-most radioactive sample (>20 μCi of ¹³⁷Cs g⁻¹) were closely related to Deinococcus radiodurans and were able to survive acute doses of ionizing radiation approaching 20 kGy. Many of the gram-positive isolates were resistant to lower levels of gamma radiation. These results demonstrate that gram-positive bacteria, predominantly from phyla high in G+C content, are indigenous to Hanford vadose sediments and that some are effective at surviving the extreme physical and chemical stress associated with radioactive waste.     

(Per)chlorate Reduction by the Thermophilic Bacterium Moorella perchloratireducens sp. nov., Isolated from Underground Gas Storage

A thermophilic bacterium, strain An10, was isolated from underground gas storage with methanol as a substrate and perchlorate as an electron acceptor. Cells were gram-positive straight rods, 0.4 to 0.6 μm in diameter and 2 to 8 μm in length, growing as single cells or in pairs. Spores were terminal with a bulged sporangium. The temperature range for growth was 40 to 70°C, with an optimum at 55 to 60°C. The pH optimum was around 7. The salinity range for growth was between 0 and 40 g NaCl liter⁻¹ with an optimum at 10 g liter⁻¹. Strain An10 was able to grow on CO, methanol, pyruvate, glucose, fructose, cellobiose, mannose, xylose, and pectin. The isolate was able to respire with (per)chlorate, nitrate, thiosulfate, neutralized Fe(III) complexes, and anthraquinone-2,6-disulfonate. The G+C content of the DNA was 57.6 mol%. On the basis of 16S rRNA analysis, strain An10 was most closely related to Moorella thermoacetica and Moorella thermoautotrophica. The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell extracts. Strain An10 is the first thermophilic and gram-positive bacterium with the ability to use (per)chlorate as a terminal electron acceptor.     

Phylogenetic Analysis of Bacterial Communities in Mesophilic and Thermophilic Bioreactors Treating Pharmaceutical Wastewater

The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied. These reactors were operated at both mesophilic (28 to 32°C) and thermophilic (50 to 58°C) temperatures. Community fingerprint analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domain Bacteria revealed that these seven reactors supported three distinct microbial communities. A band-counting analysis of the PCR-DGGE results suggested that elevated reactor temperatures corresponded with reduced species richness. Cloning of nearly complete 16S rRNA genes also suggested a reduced species richness in the thermophilic reactors by comparing the number of clones with different nucleotide inserts versus the total number of clones screened. While these results imply that elevated temperature can reduce species richness, other factors also could have impacted the number of populations that were detected. Nearly complete 16S rDNA sequence analysis showed that the thermophilic reactors were dominated by members from the β subdivision of the division Proteobacteria (β-proteobacteria) in addition to anaerobic phylotypes from the low-G+C gram-positive and Synergistes divisions. The mesophilic reactors, however, included at least six bacterial divisions, including Cytophaga-Flavobacterium-Bacteroides, Synergistes, Planctomycetes, low-G+C gram-positives, Holophaga-Acidobacterium, and Proteobacteria (α-proteobacteria, β-proteobacteria, γ-proteobacteria and δ-proteobacteria subdivisions). The two PCR-based techniques detected the presence of similar bacterial populations but failed to coincide on the relative distribution of these phylotypes. This suggested that at least one of these methods is insufficiently quantitative to determine total community biodiversity—a function of both the total number of species present (richness) and their relative distribution (evenness).     

Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by Enterococcus faecalis BFE 1071

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.     

Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Shewanella spp. Genomic Evolution for a Cold Marine Lifestyle and In-Situ Explosive Biodegradation

Shewanella halifaxensis and Shewanella sediminis were among a few aquatic γ-proteobacteria that were psychrophiles and the first anaerobic bacteria that degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although many mesophilic or psychrophilic strains of Shewanella and γ-proteobacteria were sequenced for their genomes, the genomic evolution pathways for temperature adaptation were poorly understood. On the other hand, the genes responsible for anaerobic RDX mineralization pathways remain unknown. To determine the unique genomic properties of bacteria responsible for both cold-adaptation and RDX degradation, the genomes of S. halifaxensis and S. sediminis were sequenced and compared with 108 other γ-proteobacteria including Shewanella that differ in temperature and Na⁺ requirements, as well as RDX degradation capability. Results showed that for coping with marine environments their genomes had extensively exchanged with deep sea bacterial genomes. Many genes for Na⁺-dependent nutrient transporters were recruited to use the high Na⁺ content as an energy source. For coping with low temperatures, these two strains as well as other psychrophilic strains of Shewanella and γ-proteobacteria were found to decrease their genome G+C content and proteome alanine, proline and arginine content (p-value <0.01) to increase protein structural flexibility. Compared to poorer RDX-degrading strains, S. halifaxensis and S. sediminis have more number of genes for cytochromes and other enzymes related to RDX metabolic pathways. Experimentally, one cytochrome was found induced in S. halifaxensis by RDX when the chemical was the sole terminal electron acceptor. The isolated protein degraded RDX by mono-denitration and was identified as a multiheme 52 kDa cytochrome using a proteomic approach. The present analyses provided the first insight into divergent genomic evolution of bacterial strains for adaptation to the specific cold marine conditions and to the degradation of the pollutant RDX. The present study also provided the first evidence for the involvement of a specific c-type cytochrome in anaerobic RDX metabolism.     

A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.     

Ferrous Iron-Dependent Volatilization of Mercury by the Plasma Membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe²⁺ medium (pH 2.5) supplemented with 6 μM Hg²⁺. In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 μM Hg²⁺. When incubated for 3 h in a salt solution (pH 2.5) with 0.7 μM Hg²⁺, resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe²⁺ was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30°C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe²⁺-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 μM Hg²⁺ and 1 mM Fe²⁺, plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe²⁺-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe²⁺-dependent mercury volatilization activity of the plasma membrane.     

Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a Thermococcus sp.

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 μm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly α-cyclodextrin with small amounts of β- and γ-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.     

Rearrangement of a 1,3-trans-[Pt(NH3)2[(GXG)-N7G,N7G]] intrastrand cross-link into interstrand cross-links within RNA duplexes

The cross-linking reaction described previously in the DNA and 2′-O-methyl RNA series is extended to RNA duplexes. A 17mer single-stranded RNA containing the 1,3-trans-{Pt(NH₃)₂[(GAG)-N7G,N7G]} intrastrand chelate, named G*AG* (* indicating a platinated base) gives, upon pairing with the complementary RNA strand, the G*AG/CUC* interstrand cross-link. The rate of the reaction in 200 mM NaClO₄ is similar to that observed for DNA–RNA duplexes. It depends on the added Na⁺ or Mg²⁺ cation and on its concentration. RNA duplexes containing GA/GA or AG/AG tandem mismatches in the rearrangement triplet core were also studied. The major interstrand cross-links, G*AG/CGA* and G*AG/AGC*, are accompanied by a minor one involving the central G of the CGA or AGC complementary sequence G*AG/CG*A and G*AG/AG*C. In 200 mM NaClO₄, the G*A/GA tandem mismatch does not modify the rate of the cross-linking rearrangement whereas the AG*/AG mismatch slows it down by a factor of four. Our results reflect the predominance of the local structure of the rearrangement core over the nucleophility of the cross-linking base. They also show that the reaction could be used to trap tertiary structures of naturally occurring RNAs, including those with the commonly encountered GA/GA mismatch.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

A Novel β-N-Acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: Gene Cloning,Expression, and Assignment to Family 3 of the Glycosyl Hydrolases

A β-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an M_r of 66,329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned β-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical β-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable β-glucosidase activity, revealed homology with microbial β-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of β-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60°C and pH 5.0, and the apparent K_m and V_max values for p-nitrophenyl-β-N-acetylglucosamine were 425.7 μM and 24.8 μmol min⁻¹ mg of protein⁻¹, respectively.Streptomycetes are gram-positive, mycelial soil bacteria with a high G+C content. In addition to having the ability to synthesize a wide variety of antibiotics and chemotherapeutic agents, they produce extracellular hydrolytic enzymes to obtain nutrients and energy by solubilizing polymeric compounds in soil. These enzymes include proteases, nucleases, lipases, and a variety of enzymes that hydrolyze different types of polysaccharides such as cellulose, chitin, and xylan (13). This last class of enzymes has received considerable attention not only from the standpoint of the utilization of renewable resources but also from that of basic research. Among actinomycetes, Streptomyces spp. make up one group regarded as particularly efficient in the breakdown of chitin (10). Following cellulose, chitin is the second most abundant polymer (β-1,4-linked polymer of N-acetylglucosamine) in nature. Efficient degradation of chitin by microorganisms is achieved by the concerted action of chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.30) (1, 19, 20).We have been studying the chitinolytic system of Streptomyces thermoviolaceus OPC-520 to clarify the roles of individual enzymes involved in chitin degradation, the relationship between structure and function, and the regulation of gene expression. When S. thermoviolaceus OPC-520 is cultivated in the presence of chitin, this strain secretes three different chitinases and only one β-N-acetylglucosaminidase and the production is repressed by glucose (unpublished data). Previously, we purified and characterized a major chitinase (Chi40) produced by the strain, which shows a high optimum temperature (70 to 80°C), high optimum pH (pH 8.0 to 10.0), and heat stability (22), and recently reported the cloning and expression of the Chi40 gene (23).While a number of chitinase genes have been isolated from a wide variety of organisms, including bacteria, fungi, insects, plants, and animals, examples of cloning of the β-N-acetylglucosaminidase gene involved in a chitinolytic system are few. To understand the role of β-N-acetylglucosaminidase in chitin degradation by strain OPC-520, its relationship to similar proteins isolated from other sources, and the regulatory system involved in the induction of the enzyme, we have isolated and expressed the gene encoding β-N-acetylglucosaminidase. Here we report the molecular cloning and biochemical characterization of a β-N-acetylglucosaminidase, designated NagA, from S. thermoviolaceus OPC-520. This novel enzyme, which is clearly different from the N-acetylglucosaminidases so far reported, is assigned to family 3 of the glycosyl hydrolases on the basis of sequence comparison. This is the first report of a β-N-acetylglucosaminidase gene isolated from the genus Streptomyces.     

Thermal Inactivation of Susceptible and Multiantimicrobial-Resistant Salmonella Strains Grown in the Absence or Presence of Glucose

The heat resistance of susceptible and multiantimicrobial-resistant Salmonella strains grown to stationary phase in glucose-free tryptic soy broth supplemented with 0.6% yeast extract (TSBYE−G; nonadapted), in regular (0.25% glucose) TSBYE, or in TSBYE−G with 1.00% added glucose (TSBYE+G; acid adapted) was determined at 55, 57, 59, and 61°C. Cultures were heated in sterile 0.1% buffered peptone water (50 μl) in heat-sealed capillary tubes immersed in a thermostatically controlled circulating-water bath. Decimal reduction times (D values) were calculated from survival curves having r² values of >0.90 as a means of comparing thermal tolerance among variables. D_59°C values increased (P < 0.05) from 0.50 to 0.58 to 0.66 min for TSBYE−G, TSBYE, and TSBYE+G cultures, respectively. D_61°C values of antimicrobial-susceptible Salmonella strains increased (P < 0.05) from 0.14 to 0.19 as the glucose concentration increased from 0.00 to 1.00%, respectively, while D_61°C values of multiantimicrobial-resistant Salmonella strains did not differ (P > 0.05) between TSBYE−G and TSBYE+G cultures. When averaged across glucose levels and temperatures, there were no differences (P > 0.05) between the D values of susceptible and multiantimicrobial-resistant inocula. Collectively, D values ranged from 4.23 to 5.39, 1.47 to 1.81, 0.50 to 0.66, and 0.16 to 0.20 min for Salmonella strains inactivated at 55, 57, 59, and 61°C, respectively. z_D values were 1.20, 1.48, and 1.49°C for Salmonella strains grown in TSBYE+G, TSBYE, and TSBYE−G, respectively, while the corresponding activation energies of inactivation were 497, 493, and 494 kJ/mol. Study results suggested a cross-protective effect of acid adaptation on thermal inactivation but no association between antimicrobial susceptibility and the ability of salmonellae to survive heat stress.     

Development of a Gene Cloning and Inactivation System for Halorespiring Desulfitobacterium dehalogenans

Efficient host-vector systems have been developed for the versatile, strictly anaerobic, halo- and fumarate-respiring gram-positive bacterium Desulfitobacterium dehalogenans. An electroporation-based transformation procedure resulting in approximately 10³ to 10⁴ transformants per μg of the cloning vector pIL253 was developed and validated. The broad-host-range vector pG⁺host9 was shown to replicate at a permissive temperature of 30°C, whereas the replicon was not functional at 40°C. The D. dehalogenans frdCAB operon, predicted to encode a fumarate reductase, was cloned, characterized, and targeted for insertional inactivation by pG⁺host9 carrying a 0.6-kb internal frdA fragment. Single-crossover integration at the frdA locus occurred at a frequency of 3.3 × 10⁻⁴ per cell and resulted in partially impaired fumarate reductase activity. The gene cloning and inactivation systems described here provide a solid basis for the further elucidation of the halorespiratory network in D. dehalogenans and allow for its further exploitation as a dedicated degrader.