Balbina Nogales

Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

ABSTRACT The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia andPseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteriain the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of theProteobacteria, and to the genus Burkholderiain particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study.

Polychlorinated biphenyl-degrading microbial communities in soils and sediments

Recent advances in the degradation of polychlorinated biphenyls (PCBs) have focussed on the use of experimental enrichment cultures to obtain PCB-degrading communities, and the use of culture-independent approaches to characterize natural and experimental PCB-degrading communities and to identify the key members in this process. PCB-degrading communities can be surprisingly diverse. Novel types of composite bacteria-mineral biofilm communities have been described. Community metabolism of PCBs may lead to the formation of protoanemonin, a dead-end product in some instances but, in others, a seemingly productive intermediate. Analysis of isotope fractionation and preferred enantiomer degradation has provided new information on degradation of PCBs in anaerobic settings. The first defined community capable of dehalorespiration of PCBs has been described, and important community members identified. Here, we provide an overview of the current knowledge of aerobic and anaerobic degradation of PCBs in microbial consortia and in the environment, including novel approaches to determine in situ PCB degradation.

Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription-PCR amplification from mRNA.

ABSTRACT The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.

Anthropogenic perturbations in marine microbial communities

Human activities impact marine ecosystems at a global scale and all levels of complexity of life. Despite their importance as key players in ecosystem processes, the stress caused to microorganisms has been greatly neglected. This fact is aggravated by difficulties in the analysis of microbial communities and their high diversity, making the definition of patterns difficult. In this review, we discuss the effects of nutrient increase, pollution by organic chemicals and heavy metals and the introduction of antibiotics and pathogens into the environment. Microbial communities respond positively to nutrients and chemical pollution by increasing cell numbers. There are also significant changes in community composition, increases in diversity and high temporal variability. These changes, which evidence the modification of the environmental conditions due to anthropogenic stress, usually alter community functionality, although this aspect has not been explored in depth. Altered microbial communities in human-impacted marine environments can in turn have detrimental effects on human health (i.e. spread of pathogens and antibiotic resistance). New threats to marine ecosystems, i.e. related to climate change, could also have an impact on microbial communities. Therefore, an effort dedicated to analyse the microbial compartment in detail should be made when studying the impact of anthropogenic activities on marine ecosystems.

Comparative Proteogenomics of Twelve Roseobacter Exoproteomes Reveals Different Adaptive Strategies Among These Marine Bacteria

Roseobacters are generalist bacteria abundantly found in the oceans. Because little is known on how marine microorganisms interact in association or competition, we focused our attention on the microbial exoproteome, a key component in their interaction with extracellular milieu. Here we present a comparative analysis of the theoretically encoded exoproteome of twelve members of the Roseobacter group validated by extensive comparative proteogenomics. In silico analysis revealed that 30% of the encoded proteome of these microorganisms could be exported. The ratio of the different protein categories varied in accordance to the ecological distinctness of each strain, a trait reinforced by quantitative proteomics data. Despite the interspecies variations found, the most abundantly detected proteins by shotgun proteomics were from transporter, adhesion, motility, and toxin-like protein categories, defining four different plausible adaptive strategies within the Roseobacter group. In some strains the toxin-secretion strategy was over-represented with repeats-in-toxin-like proteins. Our results show that exoproteomes strongly depend on bacterial trophic strategy and can slightly change because of culture conditions. Simulated natural conditions and the effect of the indigenous microbial community on the exoproteome of Ruegeria pomeroyi DSS-3 were also assayed. Interestingly, we observed a significant depletion of the toxin-like proteins usually secreted by R. pomeroyi DSS-3 when grown in presence of a natural community sampled from a Mediterranean Sea port. The significance of this specific fraction of the exoproteome is discussed.

Halophilic archaea in the human intestinal mucosa.

The human gastrointestinal tract microbiota, despite its key roles in health and disease, remains a diverse, variable and poorly understood entity. Current surveys reveal a multitude of undefined bacterial taxa and a low diversity of methanogenic archaea. In an analysis of the microbiota in colonic mucosal biopsies from patients with inflammatory bowel disease we found 16S rDNA sequences representing a phylogenetically rich diversity of halophilic archaea from the Halobacteriaceae (haloarchaea), including novel phylotypes. As the human colon is not considered a salty environment and haloarchaea are described as extreme halophiles, we evaluated and further discarded the possibility that these sequences originated from pre-colonoscopy saline lavage solutions. Furthermore, aerobic enrichment cultures prepared from a patient biopsy at low salinity (2.5% NaCl) yielded haloarchaeal sequence types. Microscopic observation after fluorescence in situ hybridization provided evidence of the presence of viable archaeal cells in these cultures. These results prove the survival of haloarchaea in the digestive system and suggest that they may be members of the mucosal microbiota, even if present in low numbers in comparison with methanogenic archaea. Investigation of a potential physiological basis of this association may lead to new insights into gastrointestinal health and disease.

Bacterial Community Dynamics during Bioremediation of Diesel Oil-Contaminated Antarctic Soil

The effect of nutrient and inocula amendment in a bioremediation field trial using a nutrient-poor Antarctic soil chronically contaminated with hydrocarbons was tested. The analysis of the effects that the treatments caused in bacterial numbers and hydrocarbon removal was combined with the elucidation of the changes occurring on the bacterial community, by 16S rDNA-based terminal restriction fragment length polymorphism (T-RFLP) typing, and the detection of some of the genes involved in the catabolism of hydrocarbons. All treatments caused a significant increase in the number of bacteria able to grow on hydrocarbons and a significant decrease in the soil hydrocarbon content, as compared to the control. However, there were no significant differences between treatments. Comparison of the soil T-RFLP profiles indicated that there were changes in the structure and composition of bacterial communities during the bioremediation trial, although the communities in treated plots were highly similar irrespective of the treatment applied, and they had a similar temporal dynamics. These results showed that nutrient addition was the main factor contributing to the outcome of the bioremediation experiment. This was supported by the lack of evidence of the establishment of inoculated consortia in soils, since their characteristic electrophoretic peaks were only detectable in soil profiles at the beginning of the experiment. Genetic potential for naphthalene degradation, evidenced by detection of nahAc gene, was observed in all soil plots including the control. In treated plots, an increase in the detection of catechol degradation genes (nahH and catA) and in a key gene of denitrification (nosZ) was observed as well. These results indicate that treatments favored the degradation of aromatic hydrocarbons and probably stimulated denitrification, at least transiently. This mesocosm study shows that recovery of chronically contaminated Antarctic soils can be successfully accelerated using biostimulation with nutrients, and that this causes a change in the indigenous bacterial communities and in the genetic potential for hydrocarbon degradation.

Proteomic insights into the lifestyle of an environmentally relevant marine bacterium

In terms of lifestyle, free-living bacteria are classified as either oligotrophic/specialist or opportunist/generalist. Heterogeneous marine environments such as coastal waters favour the establishment of marine generalist bacteria, which code for a large pool of functions. This is basically foreseen to cope with the heterogeneity of organic matter supplied to these systems. Nevertheless, it is not known what fraction of a generalist proteome is needed for house-keeping functions or what fraction is modified to cope with environmental changes. Here, we used high-throughput proteomics to define the proteome of Ruegeria pomeroyi DSS-3, a model marine generalist bacterium of the Roseobacter clade. We evaluated its genome expression under several natural environmental conditions, revealing the versatility of the bacterium to adapt to anthropogenic influence, poor nutrient concentrations or the presence of the natural microbial community. We also assayed 30 different laboratory incubations to increase proteome coverage and to dig further into the functional genomics of the bacterium. We established its core proteome and the proteome devoted to adaptation to general cellular physiological variations (almost 50%). We suggest that the other half of its theoretical proteome is the opportunist genetic pool devoted exclusively to very specific environmental conditions.

Pseudomonas Diversity in Crude-Oil-Contaminated Intertidal Sand Samples Obtained after the Prestige Oil Spill

ABSTRACT The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifying Pseudomonas strains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index for Pseudomonas species was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the species P. stutzeri, P. putida, P. anguilliseptica, and P. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novel Pseudomonas species. One isolate was considered representative of a novel P. stutzeri genomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by the rpoD PCR method was a useful tool for evaluating Pseudomonas communities and also for microdiversity studies of Pseudomonas populations.

Bacterial diversity in dry modern freshwater stromatolites from Ruidera Pools Natural Park, Spain.

Ruidera Pools Natural Park, Spain, constitutes one of the most representative systems of carbonate precipitation in Europe. The prokaryotic community of a dry modern stromatolite recovered from the park has been analyzed by molecular techniques that included denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, together with microscopic observations from the sample and cultures. Ribosomal RNA was directly extracted to study the putatively active part of the microbial community present in the sample. A total of 295 16S rRNA gene sequences were analyzed. Libraries were dominated by sequences related to Cyanobacteria, most frequently to the genus Leptolyngbya. A diverse and abundant assemblage of non-cyanobacterial sequences was also found, including members of Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Acidobacteria,Planctomycetes and Chloroflexi groups. No amplification was obtained when using archaeal primers. The results showed that at the time of sampling, when the pool was dry, the bacterial community of the stromatolites was dominated by groups of highly related Cyanobacteria, including new groups that had not been previously reported, although a high diversity outside this phylogenetic group was also found. The results indicated that part of the Cyanobacteria assemblage was metabolically active and could thus play a role in the mineralization processes inside the stromatolites.