Analysis of a phenol-adapted microbial community: degradation capacity, taxonomy and metabolic description.

AIMS: In this study, the ability of the consortium MR-01 to degrade phenol was determined. The effects of this chemical on the taxonomy and the metabolic behaviour were analysed through metagenomics. METHODS AND RESULTS: Consortium MR-01 was acclimated in a sublethal concentration of phenol. After this process, the capacity to degrade this molecule was analysed. Results showed that degradation increased with the increment of the initial phenol concentration. Metagenomic analysis indicates that the consortium metabolized phenol under aerobic conditions using phenol 2-monooxygenase and the meta-cleavage pathway. Sequence of the enzymes involved in the phenol degradation was ascribed to the Actinomycetales and Chloroflexales orders, with relative abundances <1%. The most abundant genera were part of the Sphingomonadales order; however, the role of these species in the consortium is not clear. CONCLUSIONS: Consortium MR-01 degrades efficiently high concentrations of phenol. The participation of extremophiles in the degradation process and the emergence of beneficial metabolic dependencies between the community members are some of the strategies used by the consortium to survive and develop under harsh environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the few studies describing the taxonomy and metabolic profile of a phenol degrading consortium.

Simple and inexpensive DNA extraction protocol for studying the bacterial composition of sludges used in microbial fuel cells.

Bacteria oxidize organic matter and nutrients to produce electric energy in microbial fuel cells (MFC) - a technology of increasing importance because of its sustainability. To improve the performance of MFCs, it is necessary not only to gain a better understanding of MFC engineering designs, but also to improve the understanding of the composition of the microbial communities in MFCs. Fast and efficient DNA extraction protocols that are suitable for extracting diverse bacterial genomes are necessary to identify the bacterial diversity present in MFCs and to further monitor the dynamic changes of microbial communities. This study focused on testing different direct cell lysis protocols to extract DNA from a microbial sludge harvested from an MFC. The protocol that achieved the best results was based on a previous study, but was modified by eliminating a chaotropic salt and the special columns used for nucleic acid purification. The efficiency of this less expensive and more straightforward protocol was confirmed by PCR amplification of the 16S rRNA gene and denaturing gradient gel electrophoresis analysis, which confirmed the extraction of multiple genomes. The sequences of 10 clones revealed the presence of phyla, Proteobacteria, Firmicutes and Actinobacteria, comprising both Gram-negative and Gram-positive bacteria. Some of these bacteria were identified at the genus level, e.g., Clostridium, Pseudoxanthomonas, Tistrella, and Enterobacter; these genera have been described in active sludges from wastewater treatment, supporting the congruency of our results. Therefore, this protocol is a useful tool for analysis of the bacteria responsible for energy production in MFCs.

A method for isolating RNA from metabolically active bacterial flora associated with octopus.

AIM: Development of a method for the isolation and purification of metagenomic RNA (mgRNA) from the ectopic bacterial flora of octopus. METHODS AND RESULTS: Modifications were made to the methods of Valenzuela-Avendaño et al. (Plant Mol Biol Rep, 2005, 23, 199a) and Chomczynski and Sacchi (Anal Biochem, 1987, 162, 156) to develop a protocol based on chemical lysis with Trizol. This proposed protocol effectively isolated mgRNA. The resulting bacterial RNA transcripts were amplified with universal primers directed to the hypervariable regions of the 16S rRNA gene by complementary DNA synthesis. Protocol efficacy in the study of metabolically active bacterial flora was proven using DGGE, which produced a banding pattern that recovered sequences mainly related to the Vibrionaceae family. CONCLUSION: The analysed samples were clearly complex, and the proposed protocol was proven to effectively isolate mgRNA from the metabolically active bacterial flora associated with octopus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first protocol proposed for the isolation of bacterial mgRNA that allows identification and study of metabolically active bacterial flora associated with octopus. This is an important step forward in understanding and controlling the microbial community of this economically important fishery resource, aimed at detecting its potentially pathogenic bacteria.

A species-specific polymerase chain reaction assay for rapid and sensitive detection of Colletotrichum capsici.

Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.

Culture-independent analysis of lactic acid bacteria diversity associated with mezcal fermentation.

Mezcal is an alcoholic beverage obtained from the distillation of fermented juices of cooked Agave spp. plant stalks (agave must), and each region in Mexico with denomination of origin uses defined Agave species to prepare mezcal with unique organoleptic characteristics. During fermentation to produce mezcal in the state of Tamaulipas, not only alcohol-producing yeasts are involved, but also a lactic acid bacterial community that has not been characterized yet. In order to address this lack of knowledge on this traditional Mexican beverage, we performed a DGGE-16S rRNA analysis of the lactic acid bacterial diversity and metabolite accumulation during the fermentation of a typical agave must that is rustically produced in San Carlos County (Tamaulipas, Mexico). The analysis of metabolite production indicated a short but important malolactic fermentation stage not previously described for mezcal. The denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes showed a distinctive lactic acid bacterial community composed mainly of Pediococcus parvulus, Lactobacillus brevis, Lactobacillus composti, Lactobacillus parabuchneri, and Lactobacillus plantarum. Some atypical genera such as Weissella and Bacillus were also found in the residual must. Our results suggest that the lactic acid bacteria could strongly be implicated in the organoleptic attributes of this traditional Mexican distilled beverage.

A simple silica-based method for metagenomic DNA extraction from soil and sediments.

A new method is described for extraction of metagenomic DNA from soil and sediments which is based on DNA adsorption to silica without the use of phenol, ethanol precipitation or a cesium chloride gradient. High-quality DNA was obtained, and PCR inhibition was overcome by adding bovine serum albumin and adjusting magnesium concentration. By using PCR-DGGE with Firmicutes and lactic acid bacteria-specific primers the extracted metagenomic DNA was shown to contain a mixture of bacterial genomes. This method can be used for screening bacterial diversity in soil and sediment samples.

Isolation and identification of lactic acid bacteria from sediments of a coastal marsh using a differential selective medium.

AIMS: To identify lactic acid bacteria (LAB) colonies isolated from sediments of a coastal marsh by the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) in MRS medium. METHODS AND RESULTS: Single colonies isolated from sediments of a coastal marsh by enrichment in MRS broth were selected from MRS-TTC plates and classified according to colony phenotype based on TTC reduction. A total of 37 colonies grouped in seven different phenotypes were identified by analysis of its 16S ribosomal gene sequence. Most isolates belonged to the Firmicutes phylum, mainly to orders Bacillales and Lactobacillales. LAB were represented by 20 isolates, 15 of which belong to the genus Weissella. CONCLUSIONS: Enrichment in MRS was highly selective for the isolation of bacteria belonging to phylum Firmicutes. Several different phenotypes were developed by LAB and must be considered during LAB isolation based on TTC reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study aimed at determining a relationship between colony phenotype from TTC reduction and a partial identification of isolates based on 16S ribosomal gene sequence similarities. Besides, this is the first report of isolation of W. cibaria from environmental samples.