Showing papers in "Environmental Microbiology in 2002"

A fluorimetric method for the estimation of G+C mol% content in microorganisms by thermal denaturation temperature.

Abstract: G+C mol% content in microorganisms is one of the recommended characteristics for the standard description of bacterial species. In this study we present a novel fluorimetric method to estimate the G+C mol% content in microorganisms. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence using a real-time PCR thermocycler. Unlike most previous determinations of G+C mol%, in this study only DNA from microorganisms with an available completely sequenced genome were used to prepare the calibration curves. Calibration curves showed a linear relationship between G+C mol% content and melting temperature and they were performed both in the absence and presence of 30% formamide. This protocol proves to be a rapid and inexpensive method to estimate DNA base ratios of novel microorganisms.

Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440

Abstract: Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns

Abstract: Technical developments in molecular biology have found extensive applications in the field of microbial ecology. Among these techniques, fingerprinting methods such as denaturing gel electrophoresis (DGE, including the three options: DGGE, TGGE and TTGE) has been applied to environmental samples over this last decade. Microbial ecologists took advantage of this technique, originally developed for the detection of single mutations, for the analysis of whole bacterial communities. However, until recently, the results of these high quality fingerprinting patterns were restricted to a visual interpretation, neglecting the analytical potential of the method in terms of statistical significance and ecological interpretation. A brief recall is presented here about the principles and limitations of DGE fingerprinting analysis, with an emphasis on the need of standardization of the whole analytical process. The main content focuses on statistical strategies for analysing the gel patterns, from single band examination to the analysis of whole fingerprinting profiles. Applying statistical method make the DGE fingerprinting technique a promising tool. Numerous samples can be analysed simultaneously, permitting the monitoring of microbial communities or simply bacterial groups for which occurrence and relative frequency are affected by any environmental parameter. As previously applied in the fields of plant and animal ecology, the use of statistics provides a significant advantage for the non-ambiguous interpretation of the spatial and temporal functioning of microbial communities.

Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern.

Abstract: Summary Microbial communities inhabiting a multipond solar saltern were analysed and compared using SSU rRNA polymerase chain reaction (PCR)-based fingerprintings carried out in parallel by four laboratories. A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied for Bacteria, Archaea and Eukarya, and laboratories applied their own techniques and protocols on the same set of samples. Members of all three domains were retrieved from all salt concentrations. Three fingerprinting techniques were used: denaturing gradient gel electrophoresis (DGGE), ribosomal internal spacer analysis (RISA), and terminal-restriction fragments length polymorphism (T-RFLP). In addition, each laboratory used its own biomass collection method and DNA extraction protocols. Prokaryotes were addressed using DGGE and RISA with different ‘domain-specific’ primers sets. Eukaryotes were analysed by one laboratory using DGGE and T-RFLP, but targeting the same 18S rDNA site. Fingerprints were compared through cluster analysis and non-metric multidimensional scaling plots. This exercise allowed fast comparison of microbial assemblages and determined to what extent the picture provided by each laboratory was similar to those of others. Formation of two main, salinity-based groups of samples in prokaryotes (4–15% and 22–37% salinity) was consistent for all the laboratories. When other clusters appeared, this was a result of the particular technique and the protocol used in each case, but more affected by the primers set used. Eukaryotic microorganisms changed more from pond to pond; 4–5% and 8–37% salinity were but the two main groups detected. Archaea showed the lowest number of bands whereas Eukarya showed the highest number of operational taxonomic units (OTUs) in the initial ponds. Artefacts appeared in the DGGE from ponds with extremely low microbial richness. On the other hand, different 16S rDNA fragments with the same restriction or internal transcribed spacer (ITS) length were the main limitations for T-RFLP and RISA analyses, respectively, in ponds with the highest OTUs richness. However, although the particular taxonomic composition could vary among protocols, the general structure of the microbial assemblages was maintained.

Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440

Abstract: Summary Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the β-ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two additional gene clusters that might be involved in the catabolism of N-heterocyclic aromatic compounds (nic cluster) and in a central meta-cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n-phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P. putida KT2440. The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.

In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area.

Abstract: Anaerobic oxidation of methane (AOM) and sulphate reduction were examined in sediment samples from a marine gas hydrate area (Hydrate Ridge, NE Pacific). The sediment contained high numbers of microbial consortia consisting of organisms that affiliate with methanogenic archaea and with sulphate-reducing bacteria. Sediment samples incubated under strictly anoxic conditions in defined mineral medium (salinity as in seawater) produced sulphide from sulphate if methane was added as the sole organic substrate. No sulphide production occurred in control experiments without methane. Methane-dependent sulphide production was fastest between 4 degree C and 16 degree C, the average rate with 0.1 MPa (approximately 1 atm) methane being 2.5 micro mol sulphide day(-1) and (g dry mass sediment)(-1). An increase of the methane pressure to 1.1 MPa (approximately 11 atm) resulted in a four to fivefold increase of the sulphide production rate. Quantitative measurements using a special anoxic incubation device without gas phase revealed continuous consumption of dissolved methane (from initially 3.2 to 0.7 mM) with simultaneous production of sulphide at a molar ratio of nearly 1:1. To test the response of the indigenous community to possible intermediates of AOM, molecular hydrogen, formate, acetate or methanol were added in the absence of methane; however, sulphide production from sulphate with any of these compounds was much slower than with methane. In the presence of methane, such additions neither stimulated nor inhibited sulphate reduction. Hence, the experiments did not provide evidence for one of these compounds acting as a free extracellular intermediate (intercellular shuttle) during AOM by the presently investigated consortia.

Cultivation of globally distributed soil bacteria from phylogenetic lineages previously only detected in cultivation-independent surveys

Abstract: The culturability of microorganisms in a 10 cm core of an Australian pasture soil was investigated using a minimal agar medium with xylan as the growth substrate. Culturability decreased with increasing depth, from a maximum of 19% of the total microscopically countable cells in the 0-2 cm section to 2.4% in the 8-10 cm section. Seventy-one isolates from the core were identified by comparative 16S rRNA gene sequence analysis. Many of these isolates belong to groups of globally distributed soil bacteria, including well-characterized families of the classes Alphaproteobacteria and Betaproteobacteria, and of the subclass Actinobacteridae. Other isolates belong to groups with few or no cultivated representatives: 10 isolates in two subdivisions of the phylum Acidobacteria, five isolates in a new order and nine isolates in a new family of the class Alphaproteobacteria, two isolates in a new order of the class Gammaproteobacteria, three isolates in two new families of the subclass Actinobacteridae, and two isolates in the subclass Rubrobacteridae. These new isolates represent the first laboratory cultures able to be assigned to some of these groups and greatly increase the number of cultivated strains known for others. This demonstrates that a minimal change in cultivation strategy (using a polymeric growth substrate and longer incubation times) can result in the isolation of globally distributed but previously uncultured phylogenetically novel soil bacteria.

Metabolic modelling of microbes: the flux-balance approach.

Abstract: One area of active research in this area has focused on bacterial metabolism (van Gulik and Heijnen, 1995; Liao et al., 1996; Lee et al., 1997; Sauer et al., 1998; Edwards and Palsson, 1999; Sauer and Bailey, 1999; Schilling et al., 1999; Edwards and Palsson, 2000a, b; Schilling et al., 2000; Edwards et al., 2001a, b). Genomic information, coupled with biochemical and strain-specific information, has been used to reconstruct whole-cell metabolic networks for sequenced organisms (Edwards and Palsson, 1999; Schilling and Palsson, 2000) (Fig. 1). However, this information is not sufficient to specify completely the metabolic phenotypes that will be expressed under given environmental conditions. Metabolic phenotypes can be defined in terms of flux distributions through a metabolic network. Interpreting and predicting metabolic flux distributions requires the application of mathematical modelling and computer simulation. There exists a long history of quantitative metabolic modelling (Bailey, 1998) that will not be detailed here. Currently, several well developed mathematical approaches exist for the dynamic analysis of cellular metabolism and its regulation (Shuler and Domach, 1983; Liao, 1993; Palsson and Lee, 1993; Fell, 1996; Barkai and Leibler, 1997; Bailey, 1998; Novak et al., 1999; Tomita et al., 1999; Varner and Ramkrishna, 1999; Vaseghi et al., 1999). Most of these methods require detailed kinetic and concentration information about enzymes and various cofactors. Even though biological information is growing rapidly, we still do not have enough information to describe cellular metabolism in mathematical detail for a single cell (Bailey, 2001). The human red blood cell remains the only exception (Holzhutter et al., 1985; Schuster et al., 1988; Joshi and Palsson, 1989; Rae et al., 1990; Lee and Palsson, 1991; Mulquiney and Kuchel, 1999)

Microbial diseases of corals and global warming.

Abstract: Coral bleaching and other diseases of corals have increased dramatically during the last few decades. As outbreaks of these diseases are highly correlated with increased sea-water temperature, one of the consequences of global warming will probably be mass destruction of coral reefs. The causative agent(s) of a few of these diseases have been reported: bleaching of Oculina patagonica by Vibrio shiloi; black band disease by a microbial consortium; sea-fan disease (aspergillosis) by Aspergillus sydowii; and coral white plague possibly by Sphingomonas sp. In addition, we have recently discovered that Vibrio coralyticus is the aetiological agent for bleaching the coral Pocillopora damicornis in the Red Sea. In the case of coral bleaching by V. shiloi, the major effect of increasing temperature is the expression of virulence genes by the pathogen. At high summer sea-water temperatures, V. shiloi produces an adhesin that allows it to adhere to a beta-galactoside-containing receptor in the coral mucus, penetrate into the coral epidermis, multiply intracellularly, differentiate into a viable-but-not-culturable (VBNC) state and produce toxins that inhibit photosynthesis and lyse the symbiotic zooxanthellae. In black band disease, sulphide is produced at the coral-microbial biofilm interface, which is probably responsible for tissue death. Reports of newly emerging coral diseases and the lack of epidemiological and biochemical information on the known diseases indicate that this will become a fertile area of research in the interface between microbial ecology and infectious disease.

The regulation of biofilm development by quorum sensing in Aeromonas hydrophila.

Abstract: Aeromonas hydrophila is an opportunistic Gram-negative pathogen that readily attaches to stainless steel to produce a thin biofilm with a complex 3D structure covering 40-50% of the available surface and producing large microcolonies. As A. hydrophila possesses an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system based on the ahyRI locus, the presence of the AhyI protein and C4-HSL within the biofilm phase was first established by Western blot and AHL biosensor analysis respectively. The ability of the A. hydrophila AH-1 N strain to form biofilms in a continuous-flow chamber was compared with isogenic ahyI and ahyR mutants. The ahyI mutant, which cannot produce C4-HSL, failed to form a mature biofilm. In addition, the viable count of biofilm, but not planktonic phase ahyI mutants, was significantly lower that the parent or ahyR mutant. This defect in the differentiation of the ahyI mutant biofilm could be partially restored by the addition of exogenous C4-HSL. A mutation in ahyR increased coverage of the available surface to around 80% with no obvious effect upon biofilm microcolony formation. These data support a role for AHL-dependent quorum sensing in A. hydrophila biofilm development. Exposure of the A. hydrophila AH-1N biofilm to N-(3-oxodecanoyl)homoserine lactone, which inhibits exoprotease production in planktonic cells, however, had no effect on biofilm formation or architecture within the continuous-flow chamber.

Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern

Abstract: Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined.

Multiple influences of nitrate on uranium solubility during bioremediation of uranium-contaminated subsurface sediments

Abstract: Microbiological reduction of soluble U(VI) to insoluble U(IV) has been proposed as a remediation strategy for uranium-contaminated groundwater. Nitrate is a common co-contaminant with uranium. Nitrate inhibited U(VI) reduction in acetate-amended aquifer sediments collected from a uranium-contaminated site in New Mexico. Once nitrate was depleted, both U(VI) and Fe(III) were reduced concurrently. When nitrate was added to sediments in which U(VI) had been reduced, U(VI) reappeared in solution. Parallel studies with the dissimilatory Fe(III)-, U(VI)- and nitrate-reducing microorganism, Geobacter metallireducens, demonstrated that nitrate inhibited reduction of Fe(III) and U(VI) in cell suspensions of cells that had been grown with nitrate as the electron acceptor, but not in Fe(III)-grown cells. Suspensions of nitrate-grown G. metallireducens oxidized Fe(II) and U(IV) with nitrate as the electron acceptor. U(IV) oxidation was accelerated when Fe(II) was also added, presumably due to the Fe(III) being formed abiotically oxidizing U(IV). These studies demonstrate that although the presence of nitrate is not likely to be an impediment to the bioremediation of uranium contamination with microbial U(VI) reduction, it is necessary to reduce nitrate before U(VI) can be reduced. These results also suggest that anaerobic oxidation of U(IV) to U(VI) with nitrate serving as the electron acceptor may provide a novel strategy for solubilizing and extracting microbial U(IV) precipitates from the subsurface.

Characterization of the bacterial consortium associated with black band disease in coral using molecular microbiological techniques.

Abstract: The bacterial community associated with black band disease (BBD) of the scleractinian corals Diploria strigosa, Montastrea annularis and Colpophyllia natans was examined using culture-independent techniques. Two complementary molecular screening techniques of 16S rDNA genes [amplified 16S ribosomal DNA restriction analysis (ARDRA) of clone libraries and denaturing gradient gel electrophoresis (DGGE)] were used to give a comprehensive characterization of the community. Findings support previous studies indicating low bacterial abundance and diversity associated with healthy corals. A single cyanobacterial ribotype was present in all the diseased samples, but this was not the same as that identified from Phormidium corallyticum culture isolated from BBD. The study confirms the presence of Desulfovibrio spp. and sulphate-reducing bacteria that have previously been associated with the BBD consortium. However, the species varied between diseased coral samples. We found no evidence of bacteria from terrestrial, freshwater or human sources in any of the samples. We report the presence of previously unrecognized potential pathogens [a Cytophaga sp. and an alpha-proteobacterium identified as the aetiological agent of juvenile oyster disease (JOD)] that were consistently present in all the diseased coral samples. The molecular biological approach described here gives an increasingly comprehensive and more precise picture of the bacterial population associated with BBD. To understand the pathogenesis of BBD, our attention should be focused on the pervasive ribotypes identified in this study (the Cyanobacterium sp., the Cytophaga sp. and the JOD pathogen).

Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines.

Abstract: Fluorescent pseudomonads are gamma-proteobacteria known for their capacity to colonize various ecological niches. This adaptability is reflected by their sophisticated and diverse iron uptake systems. The majority of fluorescent pseudomonads produce complex peptidic siderophores called pyoverdines or pseudobactins, which are very efficient iron scavengers. A tremendous variety of pyoverdines has been observed, each species producing a different pyoverdine. This variety can be used as an interesting tool to study the diversity and taxonomy of fluorescent pseudomonads. Other siderophores, including newly described ones, are also produced by pseudomonads, sometimes endowed with interesting properties in addition to iron scavenging, such as formation of complexes with other metals or antimicrobial activity. Factors other than iron limitation, and different regulatory proteins also seem to influence the production of siderophores in pseudomonads and are reviewed here as well. Another peculiarity of pseudomonads is their ability to use a large number of heterologous siderophores via different TonB-dependent receptors. A first genomic analysis of receptors in four different fluorescent pseudomonads suggests that their siderophore ligand repertoire is likely to overlap, and that not all receptors recognize siderophores as ligands.

Compatible solutes of organisms that live in hot saline environments

Abstract: Summary The accumulation of organic solutes is a prerequisite for osmotic adjustment of all microorganisms. Thermophilic and hyperthermophilic organisms generally accumulate very unusual compatible solutes namely, di-myo-inositol-phosphate, di-mannosyl-di-myo-­inositol-phosphate, di-glycerol-phosphate, mannosylglycerate and mannosylglyceramide, which have not been identified in bacteria or archaea that grow at low and moderate temperatures. There is also a growing awareness that some of these compatible solutes may have a role in the protection of cell components against thermal denaturation. Mannosylglycerate and di-glycerol-phosphate have been shown to protect enzymes and proteins from thermal denaturation in vitro as well, or better, than compatible solutes from mesophiles. The pathways leading to the synthesis of some of these compatible solutes from thermophiles and hyperthermophiles have been elucidated. However, large numbers of questions remain unanswered. Fundamental and applied interest in compatible ­solutes and osmotic adjustment in these organisms, drives research that, will, in the near future, allow us to understand the role of compatible solutes in osmotic protection and thermoprotection of some of the most fascinating organisms known on Earth.

Predominant growth of Alcanivorax strains in oil-contaminated and nutrient-supplemented sea water

Abstract: Summary We found that bacteria closely related to Alcanivorax became a dominant bacterial population in petroleum-contaminated sea water when nitrogen and phosphorus nutrients were supplied in adequate quantity The predominance of Alcanivorax bacteria was demonstrated under three experimental conditions: (i) in batch cultures of sea water containing heavy oil; (ii) in columns packed with oil-coated gravel undergoing a continuous sea water flow; and (iii) in a large-scale tidal flux reactor that mimics a beach undergoing tidal cycles with fresh sea water These results suggest that bacteria related to Alcanivorax are major players in the bioremediation of oil-contaminated marine environments

Reduction of Fe(III) oxide by methanogens in the presence and absence of extracellular quinones

Abstract: Summary Five methanogens (Methanosarcina barkeri MS, Methanosphaera cuniculi 1R7, Methanobacterium palustre F, Methanococcus voltaei A3 and Methanolobus vulcani PL-12/M) were investigated for their ability to reduce Fe(III) oxide and the soluble quinone anthraquinone-2,6-disulphonate (AQDS). Two species (M. barkeri and M. voltaei) reduced significant amounts of Fe(III) oxide using hydrogen as the electron donor, and 0.1 mM AQDS greatly accelerated Fe(III) reduction by these organisms. Although Fe(III) appeared to inhibit growth and methanogenesis of some strains, hydrogen partial pressures under donor-limited conditions were much lower (<0.5 Pa) in the presence of Fe(III) than in normal media (1‐10 Pa) for all species except for M. vulcani. These results demonstrate that electrons were transferred to Fe(III) by hydrogen-utilizing methanogens even when growth and methanogenesis were inhibited. All species except the obligate methylotroph M. vulcani were able to reduce AQDS when their growth substrates were present as electron donors, and rates were highest when organisms used hydrogen as the electron donor. Purified soil humic acids could also be reduced by the AQDS-reducing methanogens. The ability of methanogens to interact with extracellular quinones, humic acids and Fe(III) oxides raises the possibility that this functional group of organisms contributes to Fe(III) and humic acid reduction under certain conditions in the environment and provides an alternative explanation for the inhibition of methanogenesis in some Fe(III)-containing ecosystems.

Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida.

Abstract: Summary The TOL plasmid pWW0 (117 kb) is the best studied catabolic plasmid and the archetype of the IncP-9 plasmid incompatibility group from Pseudomonas. It carries the degradative (xyl) genes for toluenes and xylenes within catabolic transposons Tn4651 and Tn4653. Analysis of the complete pWW0 nucleotide sequence revealed 148 putative open reading frames. Of these, 77 showed similarity to published sequences in the available databases predicting functions for: plasmid replication, stable maintenance and transfer; phenotypic determinants; gene regulation and expression; and transposition. All identifiable transposition functions lay within the boundaries of the 70 kb transposon Tn4653, leaving a 46 kb sector containing all the IncP-9 core functions. The replicon and stable inheritance region was very similar to the mini-replicon from IncP-9 antibiotic resistance plasmid pM3, with their Rep proteins forming a novel group of initiation proteins. pWW0 transfer functions exist as two blocks encoding putative DNA processing and mating pair formation genes, with organizational and sequence similarity to IncW plasmids. In addition to the known Tn4651 and IS1246 elements, two additional transposable elements were identified as well as several putative transposition functions, which are probably genetic remnants from previous transposition events. Genes likely to be responsible for known resistance to ultraviolet light and free radicals were identified. Other putative phenotypic functions identified included resistance to mercury and other metal ions, as well as to quaternary ammonium compounds. The complexity and size of pWW0 is largely the result of the mosaic organization of the transposable elements that it carries, rather than the backbone functions of IncP-9 plasmids.

Persistence of Escherichia coli O157:H7 in soil and on plant roots.

Abstract: Soil microcosms were inoculated with Escherichia coli O157:H7 to test persistence in fallow soil, on roots of cover crops and in presence of manure. In fallow soils, E. coli O157:H7 persisted for 25-41 days, on rye roots for 47-96 days and on alfalfa roots, in a silt loam soil, for 92 days whereas on other legumes persistence ranged from 25-40 days, similar to fallow soil. Manure did not seem to affect the persistence of E. coli O157:H7 in these soils. Indigenous and manure-applied coliform populations often decreased faster when E. coli O157:H7 was applied, indicating possible competition between microflora. Coliform populations in microcosms not inoculated with E. coli O157:H7 decreased more slowly or increased. Microbial community analyses showed little effect for E. coli O157:H7 inoculation or addition of manure. Microbial community metabolic activity was enhanced from rye roots after 14 days and by 63 days from alfalfa roots. Microbial community lactose utilization increased over time on rye roots in all soils and on alfalfa roots in a silt loam soil when E. coli O157:H7 was inoculated. Lactose utilization also increased for uninoculated rye roots, soil around rye roots and in some fallow soils. Our data suggest that clay increases persistence and activity of E. coli O157:H7 and other coliforms. In frozen soil stored for over 500 days, E. coli O157:H7 was viable in 37% of tested samples. In summary, E. coli O157:H7 persisted longer and activity was enhanced with some cover crops in these soils due to plant roots, the presence of clay and freezing.

First insight into the genome of an uncultivated crenarchaeote from soil.

Abstract: Molecular phylogenetic surveys based on the characterization of 16S rRNA genes have revealed that soil is an environment particularly rich in microbial diversity. A clade of crenarchaeota (archaea) has frequently been detected among many other novel lineages of uncultivated bacteria. In this study we have initiated a genomic approach for the characterization of uncultivated microorganisms from soil. We have developed a procedure based on a two-phase electrophoresis technique that allows the fast and reliable purification of concentrated and clonable, high molecular weight DNA. From this DNA we have constructed complex large-insert genomic libraries. Using archaea-specific 16S rRNA probes we have isolated a 34 kbp fragment from a 900 Mbp fosmid library of soil DNA. The clone contained a complete 16S/23S rRNA operon and 17 genes encoding putative proteins. Phylogenetic analyses of the rRNA genes and of several protein encoding genes (e.g. DNA polymerase, FixAB, glycosyl transferase) confirmed the specific affiliation of the genomic fragment with the non-thermophilic clade of the crenarchaeota. Content and structure of the genomic fragment indicated that the archaea from soil differ significantly from their previously studied uncultivated marine relatives. The protein encoding genes gave the first insights into the physiological potential of these organisms and can serve as a basis for future genomic and functional genomic studies.

Diversity and distribution of a deeply branched novel proteobacterial group found in anaerobic–aerobic activated sludge processes

Abstract: A novel coccobacilli group found previously in enhanced biological phosphorus removal (EBPR) systems was further revealed to have a high degree of diversity and distribution in various activated sludge systems. Phylogenetic analysis based on 14 existing and 18 newly retrieved 16S rRNA sequences revealed that these sequences formed a novel cohesive cluster with seven subgroups in the gamma-Proteobacteria. Fluorescence in situ hybridization with a set of probes designed specifically targeting the novel group at different hierarchical levels showed that the novel group with a coccoid (2-4 micro m) to occasionally long-rod (up to 20 micro m) shape widely distributed and in some cases predominated in sludge samples taken from nine lab- and full-scale EBPR systems (10-50% of total cells) and four conventional activated sludge systems (1-10%). Variation of predominance was also observed among those subgroups in systems showing deteriorated or effective EBPR activity.

Reduction of humic substances by halorespiring, sulphate-reducing and methanogenic microorganisms

Abstract: Physiologically distinct anaerobic microorganisms were explored for their ability to oxidize different substrates with humic acids or the humic analogue, anthraquinone-2,6-disulphonate (AQDS), as a terminal electron acceptor. Most of the microorganisms evaluated including, for example, the halorespiring bacterium, Desulfitobacterium PCE1, the sulphate-reducing bacterium, Desulfovibrio G11 and the methanogenic archaeon, Methanospirillum hungatei JF1, could oxidize hydrogen linked to the reduction of humic acids or AQDS. Desulfitobacterium dehalogenans and Desulfitobacterium PCE1 could also convert lactate to acetate linked to the reduction of humic substances. Humus served as a terminal electron acceptor supporting growth of Desulfitobacterium species, which may explain the recovery of these microorganisms from organic rich environments in which the presence of chlorinated pollutants or sulphite is not expected. The results suggest that the ubiquity of humus reduction found in many different environments may be as a result of the increasing number of anaerobic microorganisms, which are known to be able to reduce humic substances.

Alkane hydroxylase homologues in Gram-positive strains.

Abstract: We isolated Gram-positive alkane-degraders from soil and a tricking-bed reactor, and show using polymerase chain reaction (PCR) with degenerate alkane hydroxylase primers and Southern blots that most Rhodococcus isolates contain three to five quite divergent homologues of the Pseudomonas putida GPo1 alkB gene. Two Mycobacterium isolates each contain one homologue, however there is no evidence for the presence of alkB homologues in the remaining strains.

Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata.

Abstract: Pseudoalteromonas tunicata is a marine bacterium with the ability to prevent biofouling by the production of at least four target-specific compounds. In addition to these antifouling compounds, P. tunicata produces at least two pigments. These include a yellow and a purple pigment which, when combined, give the bacterium a dark green appearance. Transposon mutagenesis was used in this study to investigate the correlation between pigment production and the expression of specific antifouling phenotypes in P. tunicata. Four different categories of pigmentation mutants were isolated including yellow, dark-purple, light-purple and white mutants. The mutants were tested for their ability to inhibit the settlement of invertebrate larvae, algal spore germination, fungal growth and bacterial growth. The results showed that the yellow-pigmented mutants retained full antifouling activity, whereas the purple and white mutant strains had lost some, or all, of their ability to inhibit target organisms. This demonstrates that the loss of antifouling capabilities correlates with the loss of yellow pigment and not purple pigment. Sequencing and analysis of the genes disrupted by the transposons in these mutants identified a number of potential biosynthetic enzymes and transport systems involved in the synthesis and regulation of pigmentation and fouling inhibitors in this organism.

Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries.

Abstract: A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated T(d) values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries.

Pseudomonas aeruginosa displays an epidemic population structure

Abstract: Bacteria can have population structures ranging from the fully sexual to the highly clonal. Despite numerous studies, the population structure of Pseudomonas aeruginosa is still somewhat contentious. We used a polyphasic approach in order to shed new light on this issue. A data set consisting of three outer membrane (lipo)protein gene sequences (oprI, oprL and oprD), a DNA-based fingerprint (amplified fragment length polymorphism), serotype and pyoverdine type of 73 P. aeruginosa clinical and environmental isolates, collected across the world, was analysed using biological data analysis software. We observed a clear mosaicism in the results, non-congruence between results of different typing methods and a microscale mosaic structure in the oprD gene. Hence, in this network, we also observed some clonal complexes characterized by an almost identical data set. The most recent clones exhibited serotypes O1, 6, 11 and 12. No obvious correlation was observed between these dominant clones and habitat or, with the exception of some recent clones, geographical origin. Our results are consistent with, and even clarify, some seemingly contradictory results in earlier epidemiological studies. Therefore, we suggest an epidemic population structure for P. aeruginosa, comparable with that of Neisseria meningitidis, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise.

Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates.

Abstract: Genomes are constantly evolving. Our report highlights the wide mutational diversity of clinical as well as environmental isolates, compared with the laboratory strain(s), through the systematic genetic analysis of a chromosomal porin gene (oprD) in relation to a specific antibiotic resistance. Mutational inactivation of the oprD gene is associated with carbapenem resistance in Pseudomonas aeruginosa. The sequence of the oprD gene of 55 Pseudomonas aeruginosa natural isolates obtained from across the world--from sources as diverse as patients and rhizospheres--was analysed. A microscale mosaic structure for this gene--resulting from multiple intra- and possibly interspecies recombinational events--is reported. An array of independent and seemingly fast-occurring defective oprD mutations were found, none of which had been described before. A burn wound isolate demonstrated unusually high overall sequence variability typical of mutator strains. We also present evidence for the existence of OprD homologues in other fluorescent pseudomonads.