Marcela A. Ferrero

Thermostable alkaline proteases of Bacillus licheniformis MIR 29: isolation, production and characterization

Abstract Bacillus licheniformis MIR 29 has been isolated and produces extracellular proteases. It is able to grow at temperatures up to 60 °C and at pH values up to 9.0. Casein was the best carbon source for production of a thermostable protease activity which, in some conditions, is 90% extracellular. The synthesis of alkaline protease is not constitutive; different levels of production were found with different carbon and nitrogen sources. Casein was thought to be an inducer of enzyme synthesis. The optimal pH and temperature of the enzyme activity were 12 °C and 60 °C, respectively. The enzyme was stable up to 60 °C in the absence of stabilizers. The protease activity was inhibited with phenylmethylsulphonyl fluoride, indicating a serine-protease activity. The proteolytic activity was lowered by molecules present in the culture supernatant, which include amino acids and peptides, indicating end-product inhibition. Electrophoresis assay on denaturating gels showed two bands with alkaline protease activity, in the 25 to 40-kDa molecular mass range.

Diverse UV-B resistance of culturable bacterial community from high-altitude wetland water.

Isolation of most ultraviolet B (UV-B)-resistant culturable bacteria that occur in the habitat of Laguna Azul, a high-altitude wetland [4554 m above sea level (asl)] from the Northwestern Argentinean Andes, was carried out by culture-based methods. Water from this environment was exposed to UV-B radiation under laboratory conditions during 36 h, at an irradiance of 4.94 W/m2. It was found that the total number of bacteria in water samples decreased; however, most of the community survived long-term irradiation (312 nm) (53.3 kJ/m2). The percentage of bacteria belonging to dominant species did not vary significantly, depending on the number of UV irradiation doses. The most resistant microbes in the culturable community were Gram-positive pigmented species (Bacillus megaterium [endospores and/or vegetative cells], Staphylococcus saprophyticus, and Nocardia sp.). Only one Gram-negative bacterium could be cultivated (Acinetobacter johnsonii). Nocardia sp. that survived doses of 3201 kJ/m2 were the most resistant bacteria to UV-B treatment. This study is the first report on UV-B resistance of a microbial community isolated from high-altitude extreme environments, and proposes a method for direct isolation of UV-B-resistant bacteria from extreme irradiated environments.

Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia

BackgroundPolycyclic aromatic hydrocarbons (PAHs), widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination.ResultsGenes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs) were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (r = 0.834, p < 0.05). Overall, eight different ARHD gene types were detected in the sediments. In five of them, their deduced amino acid sequences formed deeply rooted branches with previously described ARHD peptide sequences, exhibiting less than 70% identity to them. They contain consensus sequences of the Rieske type [2Fe-2S] cluster binding site, suggesting that these gene fragments encode for ARHDs. On the other hand, three gene types were closely related to previously described ARHDs: archetypical nahAc-like genes, phnAc-like genes as identified in Alcaligenes faecalis AFK2, and phnA1-like genes from marine PAH-degraders from the genus Cycloclasticus.ConclusionThese results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information can be used to study the geographical distribution and ecological significance of bacterial populations carrying these genes, and to design molecular assays to monitor the progress and effectiveness of remediation technologies.

Enrichment of arsenic transforming and resistant heterotrophic bacteria from sediments of two salt lakes in Northern Chile

Microbial populations are involved in the arsenic biogeochemical cycle in catalyzing arsenic transformations and playing indirect roles. To investigate which ecotypes among the diverse microbial communities could have a role in cycling arsenic in salt lakes in Northern Chile and to obtain clues to facilitate their isolation in pure culture, sediment samples from Salar de Ascotán and Salar de Atacama were cultured in diluted LB medium amended with NaCl and arsenic, at different incubation conditions. The samples and the cultures were analyzed by nucleic acid extraction, fingerprinting analysis, and sequencing. Microbial reduction of As was evidenced in all the enrichments carried out in anaerobiosis. The results revealed that the incubation factors were more important for determining the microbial community structure than arsenic species and concentrations. The predominant microorganisms in enrichments from both sediments belonged to the Firmicutes and Proteobacteria phyla, but most of the bacterial ecotypes were confined to only one system. The occurrence of an active arsenic biogeochemical cycle was suggested in the system with the highest arsenic content that included populations compatible with microorganisms able to transform arsenic for energy conservation, accumulate arsenic, produce H2, H2S and acetic acid (potential sources of electrons for arsenic reduction) and tolerate high arsenic levels.

Indigo production by Pseudomonas sp. J26, a marine naphthalene-degrading strain

A technique developed to determine naphthalene dioxygenase (NDO) activity was optimized and used to study the biotransformation of indole to indigo by Pseudomonas sp. J26 whole cells. The maximum production of indigo was achieved at 25 °C using 2.5 mM indole when J26 was grown in the complex medium JPP, while indole concentrations higher than 4 mM proved toxic for cells. The maximum rate of indigo production was 0.56 nmol min–1 mg dry biomass–1, obtaining 75.5 μM of indigo after 8 h of incubation, while a maximal concentration (138.1 μM) of indigo was obtained after 20 h. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Simul taneous production of alpha and beta amylases byBacillus subtilis MIR-5 in batch and continuous culture

SummarySimultaneous production of α- and β-amylase was studied in batch and continuous culture using starch as substrate inB. subtilis MIR-5. By mainipulating the cultural condition, both enzymes could then be produced by the same strain.

Isolation of Sphaerotilus–Leptothrix strains from iron bacteria communities in Tierra del Fuego wetlands

Sheath-forming iron- and manganese-depositing bacteria belonging to the Sphaerotilus-Leptothrix group (SLG) are widespread in natural and artificial water systems. Known requirements for their growth include the presence of organic substrates and molecular oxygen. High concentrations of reduced iron or manganese, although not necessary for most species, make their growth a noticeable phenomenon. Such microbial communities have been studied mostly in the Northern Hemisphere. Here, we present descriptions of diverse ochre-depositing microbial communities in Tierra del Fuego, Argentina, using a combined approach of microscopical examination, clone library construction and cultivation focused on SLG bacteria. To date, only few SLG type strains are available. The present work increases the number and diversity of cultivated SLG bacteria by obtaining isolates from biofilms and sediment samples of wetlands in Tierra del Fuego. Thirty isolates were selected based on morphological features such as sheath formation and iron/manganese deposition. Five operational taxonomic units (OTUs) were deduced. Sequencing of 16S rRNA genes showed that one OTU is identical to the Leptothrix mobilis Feox-1(T) -sequence while the four remaining OTUs show similarity values related to previously described type strains. Similarity values ranged from 96.5% to 98.8%, indicating possible new species and subspecies.

Draft Genome Sequence of Brevibacterium linens AE038-8, an Extremely Arsenic-Resistant Bacterium

ABSTRACT To understand the arsenic biogeocycles in the groundwaters at Tucumán, Argentina, we isolated Brevibacterium linens sp. strain AE38-8, obtained from arsenic-contaminated well water. This strain is extremely resistant to arsenicals and has arsenic resistance (ars) genes in its genome. Here, we report the draft genome sequence of B. linens AE38-8.

Enhanced polyaromatic hydrocarbon degradation by adapted cultures of actinomycete strains.

Fifteen actinomycete strains were evaluated for their potential use in removal of polycyclic aromatic hydrocarbons (PAH). Their capability to degrade of naphthalene, phenanthrene, and pyrene was tested in minimal medium (MM) and MM with glucose as another substrate. Degradation of naphthalene in MM was observed in all isolates at different rates, reaching maximum values near to 76% in some strains of Streptomyces, Rhodococcus sp. 016 and Amycolatopsis tucumanensis DSM 45259. Maximum values of degradation of phenanthrene in MM occurred in cultures of A. tucumanensis DSM 45259 (36.2%) and Streptomyces sp. A12 (20%), while the degradation of pyrene in MM was poor and only significant with Streptomyces sp. A12 (4.3%). Because of the poor performance when growing on phenanthrene and pyrene alone, Rhodococcus sp. 20, Rhodococcus sp. 016, A. tucumanensis DSM 45259, Streptomyces sp. A2, and Streptomyces sp. A12 were challenged to an adaptation schedule of successive cultures on a fresh solid medium supplemented with PAHs, decreasing concentration of glucose in each step. As a result, an enhanced degradation of PAHs by adapted strains was observed in the presence of glucose as co‐substrate, without degradation of phenanthrene and pyrene in MM while an increase to up to 50% of degradation was seen with these strains in glucose amended media. An internal fragment of the catA gene, which codes for catechol 1,2‐dioxygenase, was amplified from both Rhodococcus strains, showing the potential for degradation of aromatic compounds via salycilate. These results allow us to propose the usefulness of these actinomycete strains for PAH bioremediation in the environment.

Selection of an extracellular esterase-producing microorganism

SummaryThe conventional esterase plate-screening technique has been modified in order to obtain a better differentiation of high producing strains. A short exposure to Lugols iodine solution after colony growth enhanced the contrast between the precipitation halo and the background. Batch cultures of a selected strain characterized as belonging to theBacillus subtilis group showed a high level of extracellular activity at pH 6.6 to 8.0 and 35°C. In crude extracts the optimum enzymatic activity was obtained at 35°C and pH 7.0.