Showing papers in "Applied and Environmental Microbiology in 1997"

The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations.

Abstract: The naturally occurring genetic heterogeneity of autotrophic ammonia-oxidizing populations belonging to the beta subclass of the Proteobacteria was studied by using a newly developed PCR-based assay targeting a partial stretch of the gene which encodes the active-site polypeptide of ammonia monooxygenase (amoA). The PCR yielded a specific 491-bp fragment with all of the nitrifiers tested, but not with the homologous stretch of the particulate methane monooxygenase, a key enzyme of methane-oxidizing bacteria. The assay also specifically detected amoA in DNA extracted from various aquatic and terrestrial environments. The resulting PCR products retrieved from rice roots, activated sludge, a freshwater sample, and an enrichment culture were used for the generation of amoA gene libraries. No false positives were detected in a set of 47 randomly selected clone sequences that were analyzed further. The majority of the environmental sequences retrieved from rice roots and activated sludge grouped within the phylogenetic radiation defined by cultured strains of the genera Nitrosomonas and Nitrosospira. The comparative analysis identified members of both of these genera in activated sludge; however, only Nitrosospira-like sequences with very similar amino acid patterns were found on rice roots. Further differentiation of these molecular isolates was clearly possible on the nucleic acid level due to the accumulation of synonymous mutations, suggesting that several closely related but distinct Nitrosospira-like populations are the main colonizers of the rhizosphere of rice. Each of the amoA gene libraries obtained from the freshwater sample and the enrichment culture was dominated by a novel lineage that shared a branch with the Nitrosospira cluster but could not be assigned to any of the known pure cultures. Our data suggest that amoA represents a very powerful molecular tool for analyzing indigenous ammonia-oxidizing communities due to (i) its specificity, (ii) its fine-scale resolution of closely related populations, and (iii) the fact that a functional trait rather than a phylogenetic trait is detected.

Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.

Abstract: A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.

Inhibition and facilitation of nucleic acid amplification.

Abstract: Factors that inhibit the amplification of nucleic acids by PCR are present with target DNAs from many sources. The inhibitors generally act at one or more of three essential points in the reaction in the following ways: they interfere with the cell lysis necessary for extraction of DNA, they interfere by nucleic acid degradation or capture, and they inhibit polymerase activity for amplification of target DNA. Although a wide range of inhibitors is reported, the identities and modes of action of many remain unclear. These effects may have important implications for clinical and public health investigations, especially if the investigations involve food and environmental screening. Common inhibitors include various components of body fluids and reagents encountered in clinical and forensic science (e.g., hemoglobin, urea, and heparin), food constituents (e.g., organic and phenolic compounds, glycogen, fats, and Ca 21 ), and environmental compounds (e.g., phenolic compounds, humic acids, and heavy metals). Other, more widespread inhibitors include constituents of bacterial cells, nontarget DNA and contaminants, and laboratory items such as pollen, glove powder, laboratory plasticware, and cellulose. This review discusses the findings of many studies related to clinical, food, and environmental microbiology, including approaches that have been used to overcome inhibition and facilitate amplification for detection and typing. Few areas of biological science remain untouched by the invention of PCR (34, 81, 99). Other methods for amplifying nucleic acids (72, 123), such as Qb replicase (18), ligase chain reaction (13, 128), single-stranded sequence replication (17, 47), strand displacement amplification (126, 127), and nucleic acid sequence-based amplification (23, 122), have been described, but these methods have received less attention. Problems sometimes occur with PCR, however (124). Despite early indications of great sensitivity, the sensitivity of PCR may be a negative aspect of the procedure, since the most commonly reported problem is false-positive results due to cross-contamination (98, 124). This problem can be overcome by UV irradiation (100), with sodium hypochlorite (92), and by photochemical or enzymic methods (25, 36, 40, 78). One problem that is less discussed is reaction inhibition. This may be total or partial and can manifest itself as complete reaction failure or as reduced sensitivity of detection. In some cases, inhibition may be the cause of false-negative reactions, since few workers incorporate internal controls in each reaction tube. Early evidence of exquisite sensitivity with mammalian cells (53) involving detection of a single molecule of DNA from a hair was not reproduced when PCR was applied to many microbial (and some mammalian) situations, where poor sensitivity, specificity, and reproducibility have been reported (16, 82, 86, 129, 132, 134). There may also be potentially important effects in PCR typing reactions (121), and difficulties can occur in post-PCR manipulation (61). Although systematic study of inhibition has seldom been the focus of published investigations, many workers have reported these effects in the course of other studies (12, 19, 21, 124, 129, 132, 133). Considering the prevalence of this problem, it is surprising that few systematic and mechanistic studies of PCR inhibition have been reported. Rossen et al. (97) contributed the most comprehensive study of PCR inhibition, identifying inhibitory factors in foods, bacterial culture media, and various chemical compounds. These inhibitory factors included organic and inorganic chemicals, detergents, antibiotics, buffers, enzymes, polysaccharides, fats, and proteins. This review lists and discusses inhibitors and methods that can overcome the attenuation of amplification in clinical, food, and environmental microbiology. It is beyond the scope of this paper to discuss in detail the various physical, enzymic, and chemical methods used in the extraction, purification, and quantitation of nucleic acids. Those methods are presented and discussed in commercial literature and elsewhere (14, 95, 96, 106, 134).

Analysis of Actinomycete Communities by Specific Amplification of Genes Encoding 16S rRNA and Gel-Electrophoretic Separation in Denaturing Gradients

Abstract: A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.

PCR primers to amplify 16S rRNA genes from cyanobacteria.

Abstract: We developed and tested a set of oligonucleotide primers for the specific amplification of 16S rRNA gene segments from cyanobacteria and plastids by PCR. PCR products were recovered from all cultures of cyanobacteria and diatoms that were checked but not from other bacteria and archaea. Gene segments selectively retrieved from cyanobacteria and diatoms in unialgal but nonaxenic cultures and from cyanobionts in lichens could be directly sequenced. In the context of growing sequence databases, this procedure allows rapid and phylogenetically meaningful identification without pure cultures or molecular cloning. We demonstrate the use of this specific PCR in combination with denaturing gradient gel electrophoresis to probe the diversity of oxygenic phototrophic microorganisms in cultures, lichens, and complex microbial communities.

Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

Abstract: The community structure of bacterioplankton in meromictic Lake Saelenvannet was examined by PCR amplification of the V3 region of 16S rRNA from microbial communities recovered from various depths in the water column. Two different primer sets were used, one for amplification of DNA from the domain Bacteria and another specific for DNA from the domain Archaea. Amplified DNA fragments were resolved by denaturing gradient gel electrophoresis (DGGE), and the resulting profiles were reproducible and specific for the communities from different depths. Bacterial diversity estimated from the number and intensity of specific fragments in DGGE profiles decreased with depth. The reverse was true for the Archaea, with the diversity increasing with depth. Hybridization of DGGE profiles with oligonucleotide probes specific for phylogenetic groups of microorganisms showed the presence of both sulfate-reducing bacteria and methanogens throughout the water column, but they appeared to be most abundant below the chemocline. Several dominant fragments in the DGGE profiles were excised and sequenced. Among the dominant populations were representatives related to Chlorobium phaeovibrioides, chloroplasts from eukaryotic algae, and unidentified Archaea.

Enumeration and Cell Cycle Analysis of Natural Populations of Marine Picoplankton by Flow Cytometry Using the Nucleic Acid Stain SYBR Green I.

Abstract: The novel dye SYBR Green I binds specifically to nucleic acids and can be excited by blue light (488-nm wavelength). Cell concentrations of prokaryotes measured in marine samples with this dye on a low-cost compact flow cytometer are comparable to those obtained with the UV-excited stain Hoechst 33342 (bis-benzimide) on an expensive flow cytometer with a water-cooled laser. In contrast to TOTO-1 and TO-PRO-1, SYBR Green I has the advantage of clearly discriminating both heterotrophic bacteria and autotrophic Prochlorococcus cells, even in oligotrophic waters. As with TOTO-1 and TO-PRO-1, two groups of heterotrophic bacteria (B-I and B-II-like types) can be distinguished. Moreover, the resolution of DNA distribution obtained with SYBR Green I is similar to that obtained with Hoechst 33342 and permits the analysis of the cell cycle of photosynthetic prokaryotes over the whole water column.

Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments.

Abstract: Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations. DGGE was evaluated for the identification of ammonia oxidizers of the beta subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes. Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 59 GC clamp, were designed to amplify a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains. The primers were tested against a representative selection of clones and cultures encompassing the currently recognized beta-subdivision ammonia oxidizer 16S rDNA sequence diversity. Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used. The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations of differing in pH and distance from the beach. The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs. Ammonia oxidizer-like sequences from different phylogenetic groupings isolated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples. Bands from the DGGE gels of environmental samples were excised, reamplified, and directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones. Six of the seven sequenced clusters of beta-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure between some sample sites were demonstrated. The most seaward dune site contained sequences showing affinity with sequence clusters previously isolated only from marine environments and was the only site where sequences relate to Nitrosomonas genes could be detected. Nitrosospira-like sequences were present in all sites, and there was some evidence of differences between Nitrosospira populations in acid and alkaline dune soils. Such differences in community structure may affect physiological differences within beta-subdivision ammonia oxidizers, with consequent effects on nitrification rates in response to key environmental factors.

Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

Abstract: The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.

Phylogenetic analysis and in situ identification of bacteria in activated sludge.

Abstract: The bacterial community structure of activated sludge of a large municipal wastewater treatment plant was investigated by use of the rRNA approach. Almost-full-length genes coding for the small-subunit rRNA (rDNA) were amplified by PCR and subsequently cloned into the pGEM-T vector. Clones were screened by dot blot hybridization with group-specific oligonucleotide probes. The phylogenetic affiliations of clones were compared with the results obtained with the original sample by in situ hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes and found to be in general agreement. Twenty-five 16S rDNA clones were fully sequenced, 11 were almost fully (> 80%) sequenced, and 27 were partially sequenced. By comparative sequence analyses, the majority of the examined clones (35 of 67) could be affiliated with the beta subclass of the class Proteobacteria. The gamma and alpha subclasses of Proteobacteria were represented by 13 and 4 clones, respectively. Eight clones grouped with the epsilon group of Proteobacteria, and five clones grouped with gram-positive bacteria with a low DNA G+C content. The 16S rDNA of two clones showed similarity with 16S rDNA genes of members of the phyla Chlamydiae and Planctomyces. 16S rRNA-targeted oligonucleotide probes were designed and used for the enumeration of the respective bacteria. Interestingly, potentially pathogenic representatives of the genus Arcobacter were present in significant numbers (4%) in the activated sludge sample examined. Pairs of probes targeted to the 5' and 3' regions were used for detection of chimeric sequences by in situ hybridization. Two clones could be identified as chimera by applying such a pair of probes.

Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation.

Abstract: Although the Amazon Basin is well known for its diversity of flora and fauna, this report represents the first description of the microbial diversity in Amazonian soils involving a culture-independent approach. Among the 100 sequences of genes coding for small-subunit rRNA obtained by PCR amplification with universal small-subunit rRNA primers, 98 were bacterial and 2 were archaeal. No duplicate sequences were found, and none of the sequences had been previously described. Eighteen percent of the bacterial sequences could not be classified in any known bacterial kingdom. Two sequences may represent a unique branch between the vast majority of bacteria and the deeply branching, predominantly thermophilic bacteria. Five sequences formed a clade that may represent a novel group within the class Proteobacteria. In addition, rRNA intergenic spacer analysis was used to show significant microbial population differences between a mature forest soil and an adjacent pasture soil.

Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel.

Abstract: Newly described phylogenetic lineages within the domain Archaea have recently been found to be significant components of marine picoplankton assemblages. To better understand the ecology of these microorganisms, we investigated the relative abundance, distribution, and phylogenetic composition of Archaea in the Santa Barbara Channel. Significant amounts of archaeal rRNA and rDNA (genes coding for rRNA) were detected in all samples analyzed. The relative abundance of archaeal rRNA as measured by quantitative oligonucleotide hybridization experiments was low in surface waters but reached higher values (20 to 30% of prokaryotic rRNA) at depths below 100 m. Probes were developed for the two major groups of marine Archaea detected. rRNA originating from the euryarchaeal group (group II) was most abundant in surface waters, whereas rRNA from the crenarchaeal group (group I) dominated at depth. Clone libraries of PCR-amplified archaeal rRNA genes were constructed with samples from 0 and 200 m deep. Screening of libraries by hybridization with specific oligonucleotide probes, as well as subsequent sequencing of the cloned genes, indicated that virtually all archaeal rDNA clones recovered belonged to one of the two groups. The recovery of cloned rDNA sequence types in depth profiles exhibited the same trends as were observed in quantitative rRNA hybridization experiments. One representative of each of 18 distinct restriction fragment length polymorphism types was partially sequenced. Recovered sequences spanned most of the previously reported phylogenetic diversity detected in planktonic crenarchaeal and euryarchaeal groups. Several rDNA sequences appeared to be harbored in archaeal types which are widely distributed in marine coastal waters. In total, data suggest that marine planktonic crenarchaea and euryarchaea of temperate coastal habitats thrive in different zones of the water column. The relative rRNA abundance of the crenarchaeal group suggests that its members constitute a significant fraction of the prokaryotic biomass in subsurface coastal waters.

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

Abstract: A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.

Isolation and Characterization of Novel Iron-Oxidizing Bacteria That Grow at Circumneutral pH

Abstract: A gel-stabilized gradient method that employed opposing gradients of Fe2+ and O2 was used to isolate and characterize two new Fe-oxidizing bacteria from a neutral pH, Fe(2+)-containing groundwater in Michigan. Two separate enrichment cultures were obtained, and in each the cells grew in a distinct, rust-colored band in the gel at the oxic-anoxic interface. The cells were tightly associated with the ferric hydroxides. Repeated serial dilutions of both enrichments resulted in the isolation of two axenic strains, ES-1 and ES-2. The cultures were judged pure based on (i) growth from single colonies in tubes at dilutions of 10(-7) (ES-2) (ES-2) and 10(-8) (ES-1); (ii) uniform cell morphologies, i.e., ES-1 was a motile long thin, bent, or S-shaped rod and ES-2 was a shorter curved rod; and (iii) no growth on a heterotrophic medium. Strain ES-1 grew to a density of 10(8) cells/ml on FeS with a doubling time of 8 h. Strain ES-2 grew to a density of 5 x 10(7) cells/ml with a doubling time of 12.5 h. Both strains also grew on FeCO3. Neither strain grew without Fe2+, nor did they grow with glucose, pyruvate, acetate, Mn, or H2S as an electron donor. Studies with an oxygen microelectrode revealed that both strains grew at the oxic-anoxic interface of the gradients and tracked the O2 minima when subjected to higher O2 concentrations, suggesting they are microaerobes. Phylogenetically the two strains formed a novel lineage within the gamma Proteobacteria. They were very closely related to each other and were equally closely related to PVB OTU 1, a phylotype obtained from an iron-rich hydrothermal vent system at the Loihi Seamount in the Pacific Ocean, and SPB OTU 1, a phylotype obtained from permafrost soil in Siberia. Their closest cultivated relative was Stenotrophomonas maltophilia. In total, this evidence suggests ES-1 and ES-2 are members of a previously untapped group of putatively lithotrophic, unicellular iron-oxidizing bacteria.

Diversity and association of psychrophilic bacteria in Antarctic sea ice.

Abstract: The bacterial populations associated with sea ice sampled from Antarctic coastal areas were investigated by use of a phenotypic approach and a phylogenetic approach based on genes encoding 16S rRNA (16S rDNA). The diversity of bacteria associated with sea ice was also compared with the bacterial diversity of seawater underlying sea ice. Psychrophilic (optimal growth temperature, 20 degrees C; growth can occur at approximately 4 degrees C) isolated from sea ice cores and under-ice seawater were quite similar. Overall, bacterial isolates from Antarctic sea ice were found to belong to four phylogenetic groups, the alpha and gamma subdivisions of the Proteobacteria, the gram-positive branch, and the Flexibacter-Bacteroides-Cytophaga phylum. Most of the sea ice strains examined appeared to be novel taxa based on phylogenetic comparisons, with 45% of the strains being psychrophilic. 16S rDNA sequence analysis revealed that psychrophilic strains belonged to the genera Colwellia, Shewanella, Marinobacter, Planococcus, and novel phylogenetic lineages adjacent to Colwellia and Alteromonas and within the Flexibacter-Bacteroides-Cytophaga phylum. Psychrotrophic strains were found to be members of the genera Pseudoalteromonas, Psychrobacter, Halomonas, Pseudomonas, Hyphomonas, Sphingomonas, Arthrobacter, Planococcus, and Halobacillus. From this survey, it is proposed that ice diatom assemblages provide niches conducive to the proliferation of a diverse array of psychrophilic bacterial species.

Frequency of Antibiotic-Producing Pseudomonas spp. in Natural Environments.

Abstract: The antibiotics phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) are major determinants of biological control of soilborne plant pathogens by various strains of fluorescent Pseudomonas spp. In this study, we described primers and probes that enable specific and efficient detection of a wide variety of fluorescent Pseudomonas strains that produce various phenazine antibiotics or Phl. PCR analysis and Southern hybridization demonstrated that specific genes within the biosynthetic loci for Phl and PCA are conserved among various Pseudomonas strains of worldwide origin. The frequency of Phl- and PCA-producing fluorescent pseudomonads was determined on roots of wheat grown in three soils suppressive to take-all disease of wheat and four soils conducive to take-all by colony hybridization followed by PCR. Phenazine-producing strains were not detected on roots from any of the soils. However, Phl-producing fluorescent pseudomonads were isolated from all three take-all-suppressive soils at densities ranging from approximately 5 x 10(sup5) to 2 x 10(sup6) CFU per g of root. In the complementary conducive soils, Phl-producing pseudomonads were not detected or were detected at densities at least 40-fold lower than those in the suppressive soils. We speculate that fluorescent Pseudomonas spp. that produce Phl play an important role in the natural suppressiveness of these soils to take-all disease of wheat.

Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei.

Abstract: Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression.

Reactivities of various mediators and laccases with kraft pulp and lignin model compounds.

Abstract: Laccase-catalyzed oxygen delignification of kraft pulp offers some potential as a replacement for conventional chemical bleaching and has the advantage of requiring much lower pressure and temperature. However, chemical mediators are required for effective delignification by laccase, and their price is currently too high at the dosages required. To date, most studies have employed laccase from Trametes versicolor. We have found significant differences in reactivity between laccases from different fungi when they are tested for pulp delignification in the presence of the mediators 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). A more detailed study of T. versicolor laccase with ABTS and HBT showed that HBT gave the most extensive delignification over 2 h but deactivated the enzyme, and therefore a higher enzyme dosage was required. Other mediators, including 1-nitroso-2-naphthol-3,6-disulfonic acid, 4-hydroxy-3-nitroso-1-naphthalenesulfonic acid, promazine, chlorpromazine, and Remazol brilliant blue, were also tested for their ability to delignify kraft pulp. Studies with dimeric model compounds indicated that the mechanisms of oxidation by ABTS and HBT are different. In addition, oxygen uptake by laccase is much slower with HBT than with ABTS. It is proposed that the dication of ABTS and the 1-oxide radical of HBT, with redox potentials in the 0.8- to 0.9-V range, are required for pulp delignification.

Effects of aerobic and microaerobic conditions on anaerobic ammonium-oxidizing (Anammox) sludge

Abstract: The anaerobic ammonium oxidation (Anammox) process is a promising novel option for removing nitrogen from wastewater. In this study it was shown that the Anammox process was inhibited reversibly by the presence of oxygen. Furthermore, aerobic nitrifiers were shown not to play an important role in the Anammox process.

Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum.

Abstract: Enterocin P is a new bacteriocin produced by Enterococcus faecium P13 isolated from a Spanish dry-fermented sausage. Enterocin P inhibited most of tested spoilage and food-borne gram-positive pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum. Enterocin P is produced during growth in MRS broth from 16 to 45 degrees C; it is heat resistant (60 min at 100 degrees C; 15 min at 121 degrees C) and can withstand exposure to pH between 2.0 and 11.0, freeze-thawing, lyophilization, and long-term storage at 4 and -20 degrees C. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation, gel filtration, cation-exchange, hydrophobic-interaction, and reverse-phase liquid chromatography. The sequence of 43 amino acids of the N terminus was obtained by Edman degradation. DNA sequencing analysis of a 755-bp region revealed the presence of two consecutive open reading frames (ORFs). The first ORF encodes a 71-amino-acid protein containing a hydrophobic N-terminal sec-dependent leader sequence of 27 amino acids followed by the amino acid sequence corresponding to the purified and sequenced enterocin P. The bacteriocin is apparently synthesized as a prepeptide that is cleaved immediately after the Val-Asp-Ala residues (positions -3 to -1), resulting in the mature bacteriocin consisting of 44 amino acids, and with a theoretical molecular weight of 4,493. A second ORF, encoding a putative immunity protein composed of 88 amino acids with a calculated molecular weight of 9,886, was found immediately downstream of the enterocin P structural gene. Enterocin P shows a strong antilisterial activity and has the consensus sequence found in the pediocin-like bacteriocins; however, enterocin P is processed and secreted by the sec-dependent pathway.

Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments.

Abstract: Although polycyclic aromatic hydrocarbons (PAHs) have usually been found to persist under strict anaerobic conditions, in a previous study an unusual site was found in San Diego Bay in which two PAHs, naphthalene and phenanthrene, were oxidized to carbon dioxide under sulfate-reducing conditions. Further investigations with these sediments revealed that methylnaphthalene, fluorene, and fluoranthene were also anaerobically oxidized to carbon dioxide in these sediments, while pyrene and benzo[a]pyrene were not. Studies with naphthalene indicated that PAH oxidation was sulfate dependent. Incubating the sediments with additional naphthalene for 1 month resulted in a significant increase in the oxidation of [14C]naphthalene. In sediments from a less heavily contaminated site in San diego Bay where PAHs were not readily degraded, naphthalene degradation could be stimulated through inoculation with active PAH-degrading sediments from the most heavily contaminated site. Sediments from the less heavily contaminated site that had been adapted for rapid anaerobic degradation of high concentrations of benzene did not oxidize naphthalene, suggesting that the benzene- and naphthalene-degrading populations were different. When fuels containing complex mixtures of alkanes were added to sediments from the two sites, there was significant degradation in the alkanes. [14C]hexadecane was also anaerobically oxidized to 14CO2 in these sediments. Molybdate, a specific inhibitor of sulfate reduction, inhibited hexadecane oxidation. These results demonstrate that a wide variety of hydrocarbon contaminants can be degraded under sulfate-reducing conditions in hydrocarbon-contaminated sediments, and they suggest that it may be possible to use sulfate reduction rather than aerobic respiration as a treatment strategy for hydrocarbon-contaminated dredged sediments.

Regulation of laccase gene transcription in Trametes versicolor

Abstract: The expression of laccase in the white rot fungus Trametes versicolor is regulated at the level of gene transcription by copper and nitrogen. We used reverse transcription-PCR to demonstrate that as the concentration of copper or nitrogen in fungal cultures was increased, an increase in laccase activity, corresponding to increased laccase gene transcription levels, was observed. In addition, we demonstrated that the amounts of laccase mRNA and laccase activity in 10-day-old cultures were a direct function of the concentration of either 1-hydroxybenzotriazole, a previously described laccase substrate, or 2,5-xylidine, a well-known laccase inducer, in the medium. No induction was observed after the addition of two aromatic acids, ferulic acid and veratric acid, which have been shown to induce laccase production in other white rot fungi. When either copper, 2,5-xylidine, or both compounds were added to cultures grown in the absence of copper, increased laccase transcript levels were detected within 15 min. Corresponding increases in laccase activity were observed after 24-h incubation only when copper was present.

Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene.

Abstract: A molecular phylogenetic analysis of 25 isolates of fish nodaviruses, the causative agents of viral nervous necrosis of marine fish, was performed based on the nucleotide sequences (427 bases) of the coat protein gene. These fish nodaviruses were classified into four clusters: tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, berfin flounder nervous necrosis virus, and red-spotted grouper nervous necrosis virus.

Numerical dominance of a group of marine bacteria in the alpha-subclass of the class Proteobacteria in coastal seawater

Abstract: A cluster of marine bacteria within the alpha-3 subclass of the class Proteobacteria accounted for up to 28% of the 16S ribosomal DNA (rDNA) sequences in seawater samples from the coast of the southeastern United States. Two independent oligonucleotide probes targeting 16S rDNA of this "marine alpha" cluster indicate that the group dominates bacterioplankton communities in estuarine and nearshore regions of the southeastern U.S. coast. Marine alpha bacteria decline predictably in abundance with decreasing salinity along estuarine transsects and are not detectable in low-salinity (5%) or freshwater samples. Sequences of 16S rDNA obtained from seawater by PCR with one group-specific oligonucleotide as a primer confirm that the oligonucleotide targets only members of this phylogenetic cluster. Likewise, sequences of 16S rDNA obtained from seawater by PCR with several different pairs of nonspecific primers show an unusually high abundance of marine alpha sequences (52 to 84%) among the clones, which possibly indicates a PCR bias toward the group. Members of the marine alpha group were readily cultured from coastal seawater, accounting for 40% of the colonies isolated on low-nutrient marine agar, based on hybridizations with the group-specific 16S rDNA probe and on sequence analysis. This is the first description of a numerically dominant cluster of coastal bacteria, identified by molecular techniques, that can be readily cultured and studied in the laboratory.

Adaptation to sublethal environmental stresses protects Listeria monocytogenes against lethal preservation factors.

Abstract: A sublethal dose of ethanol (5%, vol/vol), acid (HCl, pH 4.5 to 5.0), H2O2 (500 ppm), or NaCl (7%, wt/vol) was added to a Listeria monocytogenes culture at the exponential phase, and the cells were allowed to grow for 1 h. Exponential-phase cells also were heat shocked at 45 degrees C for 1 h. The stress-adapted cells were then subjected to the following factors at the indicated lethal levels--NaCl (25%, wt/vol), ethanol (17.5%, vol/vol), hydrogen peroxide (0.1%, wt/vol), acid (pH 3.5), and starvation on 0.1 M phosphate buffer at pH 7.0 (up to 300 h). Viable counts of the pathogen, after the treatment, were determined on Trypticase soy agar-yeast extract, and survivor plots were constructed. The area (h.log10 CFU/ml) between the control and treatment curves was calculated to represent the protective effect resulting from adaptation to the sublethal stress factor. Adaptation to pH 4.5 to 5.0 or 5% ethanol significantly (P < 0.05) increased the resistance of L. monocytogenes to lethal doses of acid, ethanol, and H2O2. Adaptation to ethanol significantly (P < 0.05) increased the resistance to 25% NaCl. When L. monocytogenes was adapted to 500 ppm of H2O2, 7% NaCl, or heat, resistance of the pathogen to 1% hydrogen peroxide increased significantly (P < 0.05). Heat shock significantly (P < 0.05) increased the resistance to ethanol and NaCl. Therefore, the occurrence of stress protection after adaptation of L. monocytogenes to environmental stresses depends on the type of stress encountered and the lethal factor applied. This "stress hardening" should be considered when current food processing technologies are modified or new ones are developed.

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.

Abstract: Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires’ disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a nonreplicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10 4 cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.

Diverse uncultivated bacterial groups from soils of the arid southwestern United States that are present in many geographic regions.

Abstract: We have performed a phylogenetic survey of microbial species present in two soils from northern Arizona. Microbial DNA was purified directly from soil samples and subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences (rDNAs). Clone libraries from the two soils were constructed, and 60 clone inserts were partially sequenced. Phylogenetic analysis of these sequences revealed extensive diversity. Most of the analyzed sequences (64%) fell into five novel clusters having no known cultured members. Extensive analysis of 10 nearly full-length rDNAs from clones representative of the novel groups indicated that four of the five groups probably cluster into a large "supergroup" which is as distinct from currently recognized bacterial divisions as the latter are from each other. From this we postulate the existence of a major bacterial lineage, previously known only from a single cultured representative, whose diversity and ecology we are only beginning to explore. Analysis of our data and that from other rDNA sequence-based studies of soils from different geographic regions shows considerable overlap of sequence types. Taken together, these groups encompass most of the novel rDNA sequences recovered in each comparable analysis reported to date, despite large differences in soil types and geographic sources. Our results indicate that members of these new groups comprise a phylogenetically diverse, geographically widespread, and perhaps numerically important component of the soil microbiota.

Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology.

Abstract: The utility of parallel hybridization of environmental nucleic acids to many oligonucleotides immobilized in a matrix of polyacrylamide gel pads on a glass slide (oligonucleotide microchip) was evaluated. Oligonucleotides complementary to small-subunit rRNA sequences of selected microbial groups, encompassing key genera of nitrifying bacteria, were shown to selectively retain labeled target nucleic acid derived from either DNA or RNA forms of the target sequences. The utility of varying the probe concentration to normalize hybridization signals and the use of multicolor detection for simultaneous quantitation of multiple probe-target populations were demonstrated.

Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria.

Abstract: Several propane-oxidizing bacteria were tested for their ability to degrade gasoline oxygenates, including methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). Both a laboratory strain and natural isolates were able to degrade each compound after growth on propane. When propane-grown strain ENV425 was incubated with 20 mg of uniformly labeled [14C]MTBE per liter, the strain converted > 60% of the added MTBE to 14CO2 in < 30 h. The initial oxidation of MTBE and ETBE resulted in the production of nearly stoichiometric amounts of tert-butyl alcohol (TBA), while the initial oxidation of TAME resulted in the production of tert-amyl alcohol. The methoxy methyl group of MTBE was oxidized to formaldehyde and ultimately to CO2. TBA was further oxidized to 2-methyl-2-hydroxy-1-propanol and then 2-hydroxy isobutyric acid; however, neither of these degradation products was an effective growth substrate for the propane oxidizers. Analysis of cell extracts of ENV425 and experiments with enzyme inhibitors implicated a soluble P-450 enzyme in the oxidation of both MTBE and TBA. MTBE was oxidized to TBA by camphor-grown Pseudomonas putida CAM, which produces the well-characterized P-450cam, but not by Rhodococcus rhodochrous 116, which produces two P-450 enzymes. Rates of MTBE degradation by propane-oxidizing strains ranged from 3.9 to 9.2 nmol/min/mg of cell protein at 28 degrees C, whereas TBA was oxidized at a rate of only 1.8 to 2.4 nmol/min/mg of cell protein at the same temperature.

Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample

Abstract: Numerous investigations applying the cloning and sequencing of rRNA genes (rDNAs) to the study of marine bacterioplankton diversity have shown that the sequences of genes cloned directly from environmental DNA do not correspond to the genes of cultured marine taxa. These results have been interpreted as support for the hypothesis that the most abundant heterotrophic marine bacterioplankton species are not readily culturable by commonly used methods. However, an alternative explanation is that marine bacterioplankton can be easily cultured but are not well represented in sequence databases. To further examine this question, we compared the small-subunit (SSU) rDNAs of 127 cellular clones isolated from a water sample collected off the Oregon coast to 58 bacterial SSU rDNAs cloned from environmental DNAs from the same water sample. The results revealed little overlap between partial SSU rDNA sequences from the cellular clones and the environmental clone library. An exception was the SSU rDNA sequence recovered from a cellular clone belonging to the Pseudomonas subgroup of the gamma subclass of the class Proteobacteria, which was related to a single gene cloned directly from the same water sample (OCS181) (similarity, 94.6%). In addition, partial SSU rDNA sequences from three of the cultured strains matched a novel rDNA clone related to the gamma subclass of the Proteobacteria found previously in an environmental clone library from marine aggregates (AGG53) (similarity, 94.3 to 99.6%). Our results support the hypothesis that many of the most abundant bacterioplankton species are not readily culturable by standard methods but also show that heterotrophic bacterioplankton that are culturable on media with high organic contents include many strains for which SSU rDNA sequences are not available in sequence databases.