Functional metagenomics of oil-impacted mangrove sediments reveals high abundance of hydrolases of biotechnological interest.

Mangroves are located in coastal wetlands and are susceptible to the consequences of oil spills, what may threaten the diversity of microorganisms responsible for the nutrient cycling and the consequent ecosystem functioning. Previous reports show that high concentration of oil favors the incidence of epoxide hydrolases and haloalkane dehalogenases in mangroves. This finding has guided the goals of this study in an attempt to broaden the analysis to other hydrolases and thereby verify whether oil contamination interferes with the prevalence of particular hydrolases and their assigned microorganisms. For this, an in-depth survey of the taxonomic and functional microbial diversity recovered in a fosmid library (Library_Oil Mgv) constructed from oil-impacted Brazilian mangrove sediment was carried out. Fosmid DNA of the whole library was extracted and submitted to Illumina HiSeq sequencing. The resulting Library Oil_Mgv dataset was further compared with those obtained by direct sequencing of environmental DNA from Brazilian mangroves (from distinct regions and affected by distinct sources of contamination), focusing on hydrolases with potential use in biotechnological processes. The most abundant hydrolases found were proteases, esterases and amylases, with similar occurrence profile in all datasets. The main microbial groups harboring such hydrolase-encoding genes were distinct in each mangrove, and in the fosmid library these enzymes were mainly assigned to Chloroflexaceae (for amylases), Planctomycetaceae (for esterases) and Bradyrhizobiaceae (for proteases). Assembly and analysis of Library_Oil Mgv reads revealed three potentially novel enzymes, one epoxide hydrolase, one xylanase and one amylase, to be further investigated via heterologous expression assays.

Genomic and chemical insights into biosurfactant production by the mangrove-derived strain Bacillus safensis CCMA-560.

Many Bacillus species can produce biosurfactant, although most of the studies on lipopeptide production by this genus have been focused on Bacillus subtilis. Surfactants are broadly used in pharmaceutical, food and petroleum industry, and biological surfactant shows some advantages over the chemical surfactants, such as less toxicity, production from renewable, cheaper feedstocks and development of novel recombinant hyperproducer strains. This study is aimed to unveil the biosurfactant metabolic pathway and chemical composition in Bacillus safensis strain CCMA-560. The whole genome of the CCMA-560 strain was previously sequenced, and with the aid of bioinformatics tools, its biosurfactant metabolic pathway was compared to other pathways of closely related species. Fourier transform infrared (FTIR) and high-resolution TOF mass spectrometry (MS) were used to characterize the biosurfactant molecule. B. safensis CCMA-560 metabolic pathway is similar to other Bacillus species; however, some differences in amino acid incorporation were observed, and chemical analyses corroborated the genetic results. The strain CCMA-560 harbours two genes flanked by srfAC and srfAD not present in other Bacillus spp., which can be involved in the production of the analogue gramicidin. FTIR and MS showed that B. safensis CCMA-560 produces a mixture of at least four lipopeptides with seven amino acids incorporated and a fatty acid chain with 14 carbons, which makes this molecule similar to the biosurfactant of Bacillus pumilus, namely, pumilacidin. This is the first report on the biosurfactant production by B. safensis, encompassing the investigation of the metabolic pathway and chemical characterization of the biosurfactant molecule.

A Novel Multifunctional ß-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove.

The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a ß-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a ß-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-ß-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-ß-d-galactopyranoside, and 4-Nitrophenyl ß-d-glucopyranoside, presenting ß-N-acetylglucosaminidase, ß-glucosidase, and ß-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel ß-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product.

Bioremediation potential of microorganisms derived from petroleum reservoirs.

Bacterial strains and metagenomic clones, both obtained from petroleum reservoirs, were evaluated for petroleum degradation abilities either individually or in pools using seawater microcosms for 21 days. Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) analyses were carried out to evaluate crude oil degradation. The results showed that metagenomic clones 1A and 2B were able to biodegrade n-alkanes (C14 to C33) and isoprenoids (phytane and pristane), with rates ranging from 31% to 47%, respectively. The bacteria Dietzia maris CBMAI 705 and Micrococcus sp. CBMAI 636 showed higher rates reaching 99% after 21 days. The metagenomic clone pool biodegraded these compounds at rates ranging from 11% to 45%. Regarding aromatic compound biodegradation, metagenomic clones 2B and 10A were able to biodegrade up to 94% of phenanthrene and methylphenanthrenes (3-MP, 2-MP, 9-MP and 1-MP) with rates ranging from 55% to 70% after 21 days, while the bacteria Dietzia maris CBMAI 705 and Micrococcus sp. CBMAI 636 were able to biodegrade 63% and up to 99% of phenanthrene, respectively, and methylphenanthrenes (3-MP, 2-MP, 9-MP and 1-MP) with rates ranging from 23% to 99% after 21 days. In this work, isolated strains as well as metagenomic clones were capable of degrading several petroleum compounds, revealing an innovative strategy and a great potential for further biotechnological and bioremediation applications.

Prevalence of enteropathogens in normal feces from healthy children at an infant day care in Brazil.

INTRODUCTION: The diarrhea associated with gastroenteritis is a major cause of morbidity and mortality worldwide, affecting mainly infants. The characterization of both viral and bacterial agents associated with gastroenteritis can establish policies for surveillance, prevention and treatment of infections. Group A rotaviruses are the major infectious agent associated with dehydration in children, followed by pathotypes of Escherichia coli. There are three main types of clinical infections caused by E. coli strains that have acquired virulence genes: (i) enteric and diarrheal diseases, (ii) urinary tract infections, and (iii) sepsis and meningitis. METHODOLOGY: In this study, the objective was to identify the presence of rotavirus and diarrhogenic E. coli in the feces of children 4 to 14 months of age who displayed no gastroenteritis symptoms and stayed all day in a day-care center. We analyzed 188 samples using PAGE and PCR to identify rotaviruses and E. coli virulence genes, respectively. RESULTS: Thirty-six samples (19.1%) were positive for at least one pathotype of E. coli. Nineteen were identified to be of the EPEC group and fifteen of the EAEC group. Rotaviruses were not identified. CONCLUSIONS: As EPEC and EAEC are potential pathogens for children less than one year of age or immunocompromised individuals, our results show the importance of appropriate monitoring by public health agencies. In the situation that we have studied, children can be considered asymptomatic carriers of these pathogens and can transmit them to other susceptible children.