Characterization of the cellulase-secretome produced by the Antarctic bacterium Flavobacterium sp. AUG42.

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed.

Searching for novel photolyases in UVC-resistant Antarctic bacteria.

Ultraviolet (UV) light irradiation has serious consequences for cell survival, including DNA damage by formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6,4) pyrimidone photoproducts. In general, the Nucleotide Excision Repair pathway repairs these lesions; however, all living forms, except placental mammals and some marsupials, produce a flavoprotein known as photolyase that directly reverses these lesions. The aim of this work was the isolation and identification of Antarctic UVC-resistant bacteria, and the search for novel photolyases. Two Antarctic water samples were UVC-irradiated (254 nm; 50-200 J m- 2) and 12 UVC-resistant bacteria were isolated and identified by 16S rDNA amplification/analysis as members of the genera Pseudomonas, Janthinobacterium, Flavobacterium, Hymenobacter and Sphingomonas. The UVC 50% lethal dose and the photo-repair ability of isolates were analyzed. The occurrence of photolyase coding sequences in Pseudomonas, Hymenobacter and Sphingomonas isolates were searched by PCR or by searching in the draft DNA genome. Results suggest that Pseudomonas and Hymenobacter isolates produce CDP-photolyases, and Sphingomonas produces two CPD-photolyases and a 6,4-photolyase. Results suggest that the Antarctic environment is an important source of genetic material for the identification of novel photolyase genes with potential biotechnological applications.

The versatility of Delftia sp. isolates as tools for bioremediation and biofertilization technologies.

Two Pb(II)-resistant bacteria isolated from a soil containing 2,500 mg/kg of Pb were identified by 16S rRNA sequencing analysis as Delftia sp. and designated as 3C and 6C. Both isolates grew at a Pb(II) concentration of 62 mg/L and at the stationary phase showed a Pb(II)-sorption capability of 10 ± 1.5 (3C) and 5 ± 0.8 (6C) mg/g of biomass. Biochemical properties related to heavy metal resistance and plant growth promotion were analyzed and compared with the Cr(VI)-resistant plant growth-promoting Delftia sp. JD2, previously reported by our group. Both isolates and JD2 were resistant to Cr(VI), Pb(II) and many antibiotics, produced siderophores and the phytohormone indole-3-acetic, and showed clover growth-promoting activity in greenhouse conditions. Interestingly, the occurrence of integron class 1 was shown in all isolates. Our results add to previous reports and suggest that bacteria of the genus Delftia could be consider as good candidates for the design of technologies for cleaning up contaminated environments and/or the production of biofertilizers.

Delftia sp. JD2: a potential Cr(VI)-reducing agent with plant growth-promoting activity.

A chromium (Cr)-resistant bacterium isolated from soil containing 6,000 mg/kg of Cr was identified based on 16S rRNA gene sequence analysis as Delftia, and designated as JD2. Growth of JD2 was accompanied with reduction of Cr(VI) to Cr(III) in liquid medium initially containing 100 mg/L Cr(VI), the maximum concentration allowing growth. JD2 showed NADH/NADPH-dependent reductase activity associated with the soluble fraction of cells. The results suggest that JD2 might be a good candidate for the treatment of highly Cr(VI)-contaminated water and/or industrial effluents. The isolate produced indole-3-acetic acid in the presence and absence of Cr(VI) and showed free-living nitrogen-fixing activity possibly attributable to a V-nitrogenase. JD2 did not counteract the harmful effect of Cr(VI) during leguminous plant growth and nodulation by rhizobial strains but functioned as a "helper" bacterium to enhance the performance of rhizobial inoculant strains during inoculation of alfalfa and clover (used as model plants to study plant growth-promoting activity) in the absence of Cr(VI).