Widespread occurrence of bacterial human virulence determinants in soil and freshwater environments.

The occurrence of 22 bacterial human virulence genes (encoding toxins, adhesins, secretion systems, regulators of virulence, inflammatory mediators, and bacterial resistance) in beech wood soil, roadside soil, organic agricultural soil, and freshwater biofilm was investigated by nested PCR. The presence of clinically relevant bacterial groups known to possess virulence genes was tested by PCR of 16S and 23S rRNA genes. For each of the virulence genes detected in the environments, sequencing and NCBI BLAST analysis confirmed the identity of the PCR products. The virulence genes showed widespread environmental occurrence, as 17 different genes were observed. Sixteen genes were detected in beech wood soil, and 14 were detected in roadside and organic agricultural soils, while 11 were detected in the freshwater biofilm. All types of virulence traits were represented in all environments; however, the frequency at which they were detected was variable. A principal-component analysis suggested that several factors influenced the presence of the virulence genes; however, their distribution was most likely related to the level of contamination by polycyclic aromatic hydrocarbons and pH. The occurrence of the virulence genes in the environments generally did not appear to be the result of the presence of clinically relevant bacteria, indicating an environmental origin of the virulence genes. The widespread occurrence of the virulence traits and the high degree of sequence conservation between the environmental and clinical sequences suggest that soil and freshwater environments may constitute reservoirs of virulence determinants normally associated with human disease.

Studying plasmid horizontal transfer in situ: a critical review.

This review deals with the prospective, experimental documentation of horizontal gene transfer (HGT) and its role in real-time, local adaptation. We have focused on plasmids and their function as an accessory and/or adaptive gene pool. Studies of the extent of HGT in natural environments have identified certain hot spots, and many of these involve biofilms. Biofilms are uniquely suited for HGT, as they sustain high bacterial density and metabolic activity, even in the harshest environments. Single-cell detection of donor, recipient and transconjugant bacteria in various natural environments, combined with individual-based mathematical models, has provided a new platform for HGT studies.

Acclimation of subsurface microbial communities to mercury.

We studied the acclimation to mercury of bacterial communities of different depths from contaminated and noncontaminated floodplain soils. The level of mercury tolerance of the bacterial communities from the contaminated site was higher than those of the reference site. Furthermore, the level of mercury tolerance and functional versatility of bacterial communities in contaminated soils initially were higher for surface soil, compared with the deeper soils. However, following new mercury exposure, no differences between bacterial communities were observed, which indicates a high adaptive potential of the subsurface communities, possibly due to differences in the availability of mercury. IncP-1 trfA genes were detected in extracted community DNA from all soil depths of the contaminated site, and this finding was correlated to the isolation of four different mercury-resistance plasmids, all belonging to the IncP-1beta group. The abundance of merA and IncP-1 plasmid carrying populations increased, after new mercury exposure, which could be the result of selection as well as horizontal gene exchange. The data in this study suggest a role for IncP-1 plasmids in the acclimation to mercury of surface as well as subsurface soil microbial communities.

Root growth and exudate production define the frequency of horizontal plasmid transfer in the Rhizosphere.

To identify the main drivers of plasmid transfer in the rhizosphere, conjugal transfer was studied in the rhizospheres of pea and barley. The donor Pseudomonas putida KT2442, containing plasmid pKJK5::gfp, was coated onto the seeds, while the recipient P. putida LM24, having a chromosomal insertion of dsRed, was inoculated into the growth medium. Mean transconjugant-to-donor ratios in vermiculite were 4.0+/-0.8 x 10(-2) in the pea and 5.9+/-1.4 x 10(-3) in the barley rhizospheres. In soil, transfer ratios were about 10 times lower. As a result of a 2-times higher root exudation rate in pea, donor densities in pea (1 x 10(6)-2 x 10(9) CFU g(-1) root) were about 10 times higher than in barley. No difference in recipient densities was observed. In situ visualization of single cells on the rhizoplane and macroscopic visualization of the colonization pattern showed that donors and transconjugants were ubiquitously distributed in the pea rhizosphere, while they were only located on the upper parts of the barley roots. Because the barley root elongated about 10 times faster than the pea root, donors were probably outgrown by the elongating barley root. Thus by affecting the cell density and distribution, exudation and root growth appear to be key parameters controlling plasmid transfer in the rhizosphere.

Effects of stress and other environmental factors on horizontal plasmid transfer assessed by direct quantification of discrete transfer events.

Selection pressure may affect the horizontal transfer of plasmids. The inability to distinguish between gene transfer and the growth of transconjugants complicates testing. We have developed a method that enables the quantification of discrete transfer events. It uses large numbers of replicate matings (192 or 384) in microtiter wells and the counting of transfer-positive and transfer-negative wells. We applied the method to study the transfer of the IncP1 plasmid pRO103 between Escherichia coli and Pseudomonas putida strains. pRO103 encodes resistance to mercury and tetracycline and partial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). The results showed positive correlation between transfer and donor metabolic activity, and an optimal temperature for transfer of 29 degrees C. On stimulation of donor activity, the optimal temperature was decreased to 24.5 degrees C. HgCl(2) above 1.0 microg L(-1) negatively affected transfer, whereas 2,4-D up to 0.3 mM had no effect. The negative effect of mercury was shown to be a result of stressing of the recipient. No effects of mercury on transfer could be detected by traditional filter mating. Thus, the method is superior to filter mating and, as the experimental design allows the manipulation of individual parameters, it is ideal for the assessment and comparison of effects of environmental factors on plasmid transfer.

Bacterial Human Virulence Genes across Diverse Habitats As Assessed by In silico Analysis of Environmental Metagenomes.

The occurrence and distribution of clinically relevant bacterial virulence genes across natural (non-human) environments is not well understood. We aimed to investigate the occurrence of homologs to bacterial human virulence genes in a variety of ecological niches to better understand the role of natural environments in the evolution of bacterial virulence. Twenty four bacterial virulence genes were analyzed in 46 diverse environmental metagenomic datasets, representing various soils, seawater, freshwater, marine sediments, hot springs, the deep-sea, hypersaline mats, microbialites, gutless worms and glacial ice. Homologs to 16 bacterial human virulence genes, involved in urinary tract infections, gastrointestinal diseases, skin diseases, and wound and systemic infections, showed global ubiquity. A principal component analysis did not demonstrate clear trends across the metagenomes with respect to occurrence and frequency of observed gene homologs. Full-length (>95%) homologs of several virulence genes were identified, and translated sequences of the environmental and clinical genes were up to 50-100% identical. Furthermore, phylogenetic analyses indicated deep branching positions of some of the environmental gene homologs, suggesting that they represent ancient lineages in the phylogeny of the clinical genes. Fifteen virulence gene homologs were detected in metatranscriptomes, providing evidence of environmental expression. The ubiquitous presence and transcription of the virulence gene homologs in non-human environments point to an important ecological role of the genes for the activity and survival of environmental bacteria. Furthermore, the high degree of sequence conservation between several of the environmental and clinical genes suggests common ancestral origins.

Occurrence and expression of bacterial human virulence gene homologues in natural soil bacteria.

The presence and in vitro expression of homologues to 22 bacterial human virulence determinants amongst culturable soil bacteria were investigated. About 25% of the bacterial isolates contained virulence gene homologues representing toxin (hblA, cytK2), adhesin (fimH), regulator (phoQ) and resistance (yfbI) determinants in pathogenic bacteria. The homologues of the toxin genes were found in Actinobacteria and Firmicutes (hblA), and in Firmicutes and Alpha- and Gammaproteobacteria (cytK2). The homologues to the type 1 fimbrial adhesin gene, fimH, and the L-Ara4N transferase gene, yfbI, were observed in Actinobacteria, Firmicutes and Gammaproteobacteria. The regulator gene, phoQ, was only found in Gammaproteobacteria. The presence of cytK2 in Alpha- and Gammaproteobacteria, fimH in Actinobacteria and Firmicutes, and hblA in Actinobacteria has not previously been described. A close sequence similarity (84-100%) was observed between the genes of environmental and clinical isolates, and expression assays suggested that the genes in some cases were expressed in vitro. The presence of functional virulence gene homologues underpins their importance for the survival of environmental bacteria. Furthermore, the high degree of sequence conservation to clinical sequences indicates that natural environments may be 'evolutionary cribs' of emerging pathogens.

Mercuric reductase genes (merA) and mercury resistance plasmids in High Arctic snow, freshwater and sea-ice brine.

Bacterial reduction in Hg(2+) to Hg(0) , mediated by the mercuric reductase (MerA), is important in the biogeochemical cycling of Hg in temperate environments. Little is known about the occurrence and diversity of merA in the Arctic. Seven merA determinants were identified among bacterial isolates from High Arctic snow, freshwater and sea-ice brine. Three determinants in Bacteriodetes, Firmicutes and Actinobacteria showed < 92% (amino acid) sequence similarity to known merA, while one merA homologue in Alphaproteobacteria and 3 homologues from Betaproteobacteria and Gammaproteobacteria were > 99% similar to known merA's. Phylogenetic analysis showed the Bacteroidetes merA to be part of an early lineage in the mer phylogeny, whereas the Betaproteobacteria and Gammaproteobacteria merA appeared to have evolved recently. Several isolates, in which merA was not detected, were able to reduce Hg(2+) , suggesting presence of unidentified merA genes. About 25% of the isolates contained plasmids, two of which encoded mer operons. One plasmid was a broad host-range IncP-α plasmid. No known incompatibility group could be assigned to the others. The presence of conjugative plasmids, and an incongruent distribution of merA within the taxonomic groups, suggests horizontal transfer of merA as a likely mechanism for High Arctic microbial communities to adapt to changing mercury concentration.

Diversity and characterization of mercury-resistant bacteria in snow, freshwater and sea-ice brine from the High Arctic.

It is well-established that atmospheric deposition transports mercury from lower latitudes to the Arctic. The role of bacteria in the dynamics of the deposited mercury, however, is unknown. We characterized mercury-resistant bacteria from High Arctic snow, freshwater and sea-ice brine. Bacterial densities were 9.4 × 10(5), 5 × 10(5) and 0.9-3.1 × 10(3) cells mL(-1) in freshwater, brine and snow, respectively. Highest cultivability was observed in snow (11.9%), followed by freshwater (0.3%) and brine (0.03%). In snow, the mercury-resistant bacteria accounted for up to 31% of the culturable bacteria, but <2% in freshwater and brine. The resistant bacteria belonged to the Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Actinobacteria, and Bacteriodetes. Resistance levels of most isolates were not temperature dependent. Of the resistant isolates, 25% reduced Hg(II) to Hg(0). No relation between resistance level, ability to reduce Hg(II) and phylogenetic group was observed. An estimation of the potential bacterial reduction of Hg(II) in snow suggested that it was important in the deeper snow layers where light attenuation inhibited photoreduction. Thus, by reducing Hg(II) to Hg(0), mercury-resistant bacteria may limit the supply of substrate for methylation processes and, hence, contribute to lowering the risk that methylmercury is being incorporated into the Arctic food chains.

Effect of availability of nitrogen compounds on community structure of aquatic bacteria in model systems.

To test if the quality and concentration of dissolved nitrogen (N) species could be a selective force in shaping bacterioplankton community structure, competition for various N compounds among five heterotrophic marine bacteria (Pseudomonas strains B, B25, and AX; Bacillus strain A6; Erythrobacter strain F19) was examined. Two of the five strains (AX and B25) were capable of utilizing urea for growth. The five strains were inoculated into dilute (1/1,000 strength) ZoBell medium enriched with various N sources (free amino acids, casein, ammonium, nitrate, or urea). Regardless of the added N source, the communities were either dominated by strain B (at 50 microM N) or strain AX (at 250 microM N). Without any addition of N, strain F19 dominated. If F19 was not included in the community, strain B25 dominated. Despite these differences in community structure, consumption of the added N compounds was surprisingly similar and no advantages of urea for the urea-utilizing bacterium B25 were obvious. To examine if urea could be of selective advantage to the urea-degrading strains B25 and AX, communities with and without B25 were amended with urea N. As expected, strain B25 became dominant when present, but without this strain the non-urea-utilizing strain B outcompeted the urea-utilizing strain AX. Possibly, strain B benefited from N released during catabolism of urea by strain AX. Changes in community composition did not result in major changes in the nitrogen dynamics. The results indicate that dissolved N species can be a selective force in shaping microbial communities. Relative to nutrient generalists, nutrient specialists may either have competitive advantages or stimulate growth of other species by synergetic interactions. Results from the model communities suggest that there may be a large degree of unpredictability in the making of microbial communities, whereas major ecosystem functions such as N cycling appear relatively stable.

Cultivation-independent examination of horizontal transfer and host range of an IncP-1 plasmid among gram-positive and gram-negative bacteria indigenous to the barley rhizosphere.

The host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of the Proteobacteria, but also Arthrobacter sp., a gram-positive member of the Actinobacteria. The transfer frequency (transconjugants per donor) from the Pseudomonas putida donor to the indigenous bacteria was 7.03 x 10(-2) +/- 3.84 x 10(-2). This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.

Luteibacter rhizovicinus gen. nov., sp. nov., a yellow-pigmented gammaproteobacterium isolated from the rhizosphere of barley (Hordeum vulgare L.).

Three strains of Gram-negative, aerobic, yellow-pigmented, chemo-organotrophic bacteria, motile by a polar flagellum, were isolated from the rhizosphere of spring barley (Hordeum vulgare L.) at a research field near Copenhagen, Denmark. The three strains, LJ79, LJ96T and LJ99, formed visible colonies on one-tenth-strength tryptic soy broth supplemented with agar (1/10 TSBA) after incubation for 6 days at 15 degrees C. The strains hydrolysed starch, casein (skimmed milk), gelatin and various pentoses and hexoses and grew on MacConkey agar and full-strength TSBA. Growth on 1/10 TSBA occurred at 4-30 degrees C, pH 6-9 and 0-3 % (w/v) NaCl. The strains had identical 16S rRNA gene sequences and ERIC (enterobacterial repetitive intergenic consensus sequence) fingerprint profiles, but could be differentiated by their RAPD (random amplified polymorphic DNA) fingerprint patterns. Strain LJ96(T) had a DNA G+C content of 64.3 mol% and the major fatty acids were 15 : 0 iso (23.4 %), 17 : 1 iso omega9c (25.5 %) and 17 : 0 iso (18.1 %). Phylogenetic analysis of the 16S rRNA gene sequences of the three strains showed 96 % sequence similarity to Rhodanobacter lindaniclasticus LMG 18385T, 95 % to Frateuria aurantia DSM 6220T and 96 % to Fulvimonas soli LMG 19981T. Using LJ96T DNA as probe, DNA-DNA hybridizations documented the relationship of the three strains to a single species (87.4-98.7 % relatedness) and showed less than 30 % relatedness to Frateuria aurantia DSM 6220T and Fulvimonas soli DSM 14263T. Rhodanobacter lindaniclasticus LMG 18385T is not extant and the strain not available from any public strain collections, thus DNA-DNA hybridization could not include this strain. On the basis of genotypic and phenotypic characteristics, the three yellow-pigmented strains could also be differentiated from Frateuria aurantia, Fulvimonas soli and Rhodanobacter lindaniclasticus. The name Luteibacter rhizovicinus gen. nov., sp. nov. is proposed, with the type strain LJ96T (=DSM 16549T=ATCC BAA-1015T).

Transcriptional regulation of pWW0 transfer genes in Pseudomonas putida KT2440.

The conjugative IncP-9 plasmid pWW0 (TOL) carries transfer genes, many of whose functions can be predicted from sequence similarities to the well-studied IncW and IncP-1 plasmids, and that are clustered with the replication and maintenance genes of the plasmid core. In this study we show that the IncP-9 transfer genes are transcribed from at least three promoter regions. The promoters for traA and traD act divergently from the region found to encode the origin of transfer, oriT. These promoters regulate expression of traA, B, and perhaps traC in one direction and traD in the other, all of whose gene products are predicted to be involved in relaxasome formation and DNA processing during transfer, and they are repressed by TraA. The third promoter region, upstream of mpfR, is responsible for transcription of mpfR and mpfA to mpfJ, encoding proteins involved in mating pair formation. Transcription from this region is negatively autoregulated by MpfR. Thus the pWW0 transfer genes, like those of the IncP-1 plasmids, are expressed at all times, but kept in control by a negative feed back loop to limit the metabolic burden on the host. Although many of the related mating pair formation systems are, as in pWW0, transcribed divergently from an operon for replication and/or stable inheritance functions, MpfR is not related to the known regulatory proteins of these other transfer systems outside those of the IncP-9 family and indeed the regulators tend to be specific for each plasmid family. This suggests that the general pattern of genetic organisation exhibited by these systems has arisen a number of times independently and must therefore be highly favourable to plasmid survival and spread.

Tenacibaculum skagerrakense sp. nov., a marine bacterium isolated from the pelagic zone in Skagerrak, Denmark.

A number of bacteria were isolated from sea water in Skagerrak, Denmark, at 30 m depth. Two of the isolates, strains D28 and D30(T), belonged to the Flavobacteriaceae within the Cytophaga-Flavobacterium-Bacteroides group. Sequencing of 16S rRNA genes of the two strains indicated strongly that they belonged to the genus Tenacibaculum and that they showed greatest similarity to the species Tenacibaculum amylolyticum and Tenacibaculum mesophilum. DNA-DNA hybridization values, DNA base composition and phenotypic characteristics separated the Skagerrak strains from the other species within TENACIBACULUM: Thus, it is concluded that the strains belong to a novel species within the genus Tenacibaculum, for which the name Tenacibaculum skagerrakense sp. nov. is proposed, with strain D30(T) (=ATCC BAA-458(T)=DSM 14836(T)) as the type strain.

Functional characteristics of culturable bacterioplankton from marine and estuarine environments.

Information on the structure of bacterioplankton communities is continuously increasing, while knowledge of their metabolic capabilities remains limited. In this study, the metabolic capacity of bacterioplankton was investigated, as such information is necessary to fully understand carbon cycling and other biogeochemical processes. The diversity of dominant culturable chemoorganotrophic bacteria from one estuarine and three marine environments was analyzed by random isolation of colony-forming units on solid media, taxonomical identification by partial 16S rRNA gene sequence analysis, and functional characterization of the isolates. A total of 76 16S rRNA gene sequences, representing 19 different genotypes, were obtained from the four sampling localities, including Bacillus, Pseudomonas, Pseudoalteromonas, Vibrio, and Erythrobacter as the most frequently isolated genera. The range of metabolic functions possessed by the cultured bacterial assemblages differed significantly between sites. Similarly, the percentage at each sampling station of bacteria capable of performing a specific function was significantly different for 18 of the 25 investigated metabolic functions. At two localities, the bacterial assemblages were dominated by a single genus (Pseudoalteromonas or Erythrobacter) and appeared to be functionally specialized. More than 95% of the isolates were capable of utilizing dissolved free amino acids and protein as their sole nitrogen sources, and all isolates of the specialized assemblages expressed beta-glucosidase. Furthermore, only some of the isolates were able to utilize NH4+, while up to two thirds of the isolates of the two marine sites were able to grow on NO3-.

Plasmid transfer from Pseudomonas putida to the indigenous bacteria on alfalfa sprouts: characterization, direct quantification, and in situ location of transconjugant cells.

The transfer of the plasmids pJKJ5 and TOL (pWWO) from Pseudomonas putida to the indigenous bacterial community on alfalfa sprouts was studied. Tagging with fluorescent protein markers allowed direct quantification of the introduced donor bacteria and of indigenous bacteria that had received the plasmids. The sprouts were observed for 9 days; during this time alfalfa seeds, inoculated with donor bacteria, developed to edible and subsequently decaying sprouts. The first transconjugants were detected on day 6 after donor inoculation and occurred at frequencies of 3.4 x 10(-4) and 2.0 x 10(-6) transconjugant cells per donor cell for pKJK5::gfp and TOL::gfp, respectively. Confocal laser scanning microscopy revealed that the sprouts were heavily colonized with donors and that most transconjugants were located around the hypocotyl and root areas. Randomly selected members of the indigenous bacterial community from both inoculated and uninoculated sprouts, as well as a representative part of the community that had received the plasmids, were characterized by polymorphisms of PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by partial 16S rDNA sequencing. This showed that the initially dominating genera Erwinia and Paenibacillus were gradually replaced by Pseudomonas on the fully developed sprouts. Transconjugants carrying either of the investigated plasmids mainly belonged to the genera Pseudomonas and ERWINIA: The numbers of transconjugant cells did not reach detectable levels until 6 days after the onset of germination, at which point these species constituted the majority of the indigenous bacteria. In conclusion, the alfalfa sprouts provided an environment that allowed noteworthy frequencies of plasmid transfer from P. putida in the absence of selective pressure that could favor the presence of the investigated plasmids.

Functional characteristics of culturable bacterioplankton from marine and estuarine environments / Características funcionales del bacterioplancton cultivable de ambientes marinos y de estuario

Information on the structure of bacterioplankton communities is continuously increasing, while knowledge of their metabolic capabilities remains limited. In this study, the metabolic capacity of bacterioplankton was investigated, as such information is necessary to fully understand carbon cycling and other biogeochemical processes. The diversity of dominant culturable chemoorganotrophic bacteria from one estuarine and three marine environments was analyzed by random isolation of colony-forming units on solid media, taxonomical identification by partial 16S rRNA gene sequence analysis, and functional characterization of the isolates. A total of 76 16S rRNA gene sequences, representing 19 different genotypes, were obtained from the four sampling localities, including Bacillus, Pseudomonas, Pseudoalteromonas, Vibrio, and Erythrobacter as the most frequently isolated genera. The range of metabolic functions possessed by the cultured bacterial assemblages differed significantly between sites. Similarly, the percentage at each sampling station of bacteria capable of performing a specific function was significantly different for 18 of the 25 investigated metabolic functions. At two localities, the bacterial assemblages were dominated by a single genus (Pseudoalteromonas or Erythrobacter) and appeared to be functionally specialized. More than 95% of the isolates were capable of utilizing dissolved free amino acids and protein as their sole nitrogen sources, and all isolates of the specialized assemblages expressed beta-glucosidase. Furthermore, only some of the isolates were able to utilize NH4+, while up to two thirds of the isolates of the two marine sites were able to grow on NO3- (AU)

La información sobre la estructura del bacterioplancton aumenta continuamente, mientras que el conocimiento de sus capacidades metabólicas sigue siendo limitado. En este estudio se investigó la capacidad metabólica del bacterioplancton, dado que dicha información es necesaria para comprender completamente el ciclo del carbono y otros procesos biogeoquímicos. La diversidad de las bacterias quimioorganotrofas cultivables que predominaban en un ambiente de estuario y en tres ambientes marinos se estudió aislando al azar unidades formadoras de colonias en medios sólidos, realizando la identificación taxonómica por medio del análisis de secuencias de genes del 16S rRNA, y mediante la caracterización funcional de los aislados. A partir de las cuatro localidades de muestreo se obtuvieron 76 secuencias de genes del 16S rRNA, que representaban 19 genotipos diferentes. Los géneros aislados con mayor frecuencia fueron Bacillus, Pseudomonas, Pseudoalteromonas, Vibrio y Erythrobacter. El margen de las funciones metabólicas que tenían los conjuntos (assemblages) de bacterias cultivadas difería notablemente entre las distintas localidades de muestreo. De manera similar, en cada estación de muestreo el porcentaje de bacterias que podían realizar alguna función específica era muy diferente para 18 de las 25 funciones metabólicas investigadas. En dos localidades predominaba un sólo género (Pseudoalteromonas o Erythrobacter) y parecían desempeñar funciones especializadas. Más del 95% de los aislados podían utilizar como única fuente de nitrógeno aminoácidos libres y proteínas disueltos, y todos los aislados de los conjuntos especializados expresaban β-glucosidasa. Además, sólo algunos de los aislados podían usar NH4+, mientras que hasta un tercio de los aislados en las dos localidades marinas podían crecer con NO3- (AU)