Proteomic and transcriptomic analysis of selenium utilization in Methanococcus maripaludis.

Methanococcus maripaludis utilizes selenocysteine- (Sec-) containing proteins (selenoproteins), mostly active in the organism's primary energy metabolism, methanogenesis. During selenium depletion, M. maripaludis employs a set of enzymes containing cysteine (Cys) instead of Sec. The genes coding for these Sec-/Cys-containing isoforms were the only genes known of which expression is influenced by the selenium status of the cell. Using proteomics and transcriptomics, approx. 7% and 12%, respectively, of all genes/proteins were found differentially expressed/synthesized in response to the selenium supply. Some of the genes identified involve methanogenesis, nitrogenase functions, and putative transporters. An increase of transcript abundance for putative transporters under selenium depletion indicated the organism's effort to tap into alternative sources of selenium. M. maripaludis is known to utilize selenite and dimethylselenide as selenium sources. To expand this list, a selenium-responsive reporter strain was assessed with nine other, environmentally relevant selenium species. While the effect of some was very similar to that of selenite, others were effectively utilized at lower concentrations. Conversely, selenate and seleno-amino acids were only utilized at unphysiologically high concentrations and two compounds were not utilized at all. To address the role of the selenium-regulated putative transporters, M. maripaludis mutant strains lacking one or two of the putative transporters were tested for the capability to utilize the different selenium species. Of the five putative transporters analyzed by loss-of-function mutagenesis, none appeared to be absolutely required for utilizing any of the selenium species tested, indicating they have redundant and/or overlapping specificities or are not dedicated selenium transporters. IMPORTANCE: While selenium metabolism in microorganisms has been studied intensively in the past, global gene expression approaches have not been employed so far. Furthermore, the use of different selenium sources, widely environmentally interconvertible via biotic and abiotic processes, was also not extensively studied before. Methanococcus maripaludis JJ is ideally suited for such analyses, thanks to its known selenium usage and available genetic tools. Thus, an overall view on the selenium regulon of M. maripaludis was obtained via transcriptomic and proteomic analyses, which inspired further experimentation. This led to demonstrating the use of selenium sources M. maripaludis was previously not known to employ. Also, an attempt-although so far unsuccessful-was made to pinpoint potential selenium transporter genes, in order to deepen our understanding of trace element utilization in this important model organism.

Functional Metagenomics Reveals a New Catalytic Domain, the Metallo-ß-Lactamase Superfamily Domain, Associated with Phytase Activity.

Inositol-6-phosphate, also known as phytic acid, is a phosphorus source that plays several important roles in the phosphorus cycle and in cell metabolism. The known characterized enzymes responsible for its degradation, the phytases, are mostly derived from cultured individual microorganisms. The catalytic signatures of phytases are restricted to the molecular domains of four protein superfamilies: histidine phosphatases, protein tyrosine phosphatases, the purple acid phosphatases and the ß-propeller phosphatases. During function-based screening of previously generated forest soil metagenomic libraries for Escherichia coli clones conferring phytase activity, two positive clones harboring the plasmids pLP05 and pLP12 were detected. Analysis of the insert sequences revealed the absence of classic phosphatase/phytase signatures of the proteins deduced from the putative genes, but the genes mblp01 (pLP05) and mblp02 (pLP12) encoded putative metallo-ß-lactamases (MBLs). Several MBL representatives are promiscuous proteins with phosphoesterase activity, but phytase activity was previously not reported. Both mblp01 and mblp02 were subcloned, expressed, and analyzed. Mblp01 and Mblp02 are members of the lactamase B2 family. Protein modeling showed that the closest structural homologue of both proteins was ZipD of E. coli Mblp01 and Mblp02 showed activity toward the majority of the tested phosphorylated substrates, including phytate. The maximal enzyme activities were recorded for Mblp01 at 50°C under acidic conditions and for Mblp02 at 35°C and a neutral pH. In the presence of Cu2+ or SDS, the activities of Mblp01 and Mblp02 were strongly inhibited. Analyses of the minimal inhibitory concentrations of several ß-lactam antibiotics revealed that recombinant E. coli cells carrying mblp01 or mblp02 showed reduced sensitivity toward ß-lactam antibiotics.IMPORTANCE Phytic acid is a phosphorus storage molecule in many plant tissues, a source of phosphorus alternative to phosphate rocks, but it can also be a problematic antinutrient. In comparison to other phosphorus sources, phytic acid exhibits reduced bioavailability. Additionally, it influences functions of secondary messengers and acts as antioxidant in tumor growth prevention. The enzymatic capability to process phytate has been reported for a limited number of protein families. This might be due to the almost exclusive use of proteins derived from individual microorganisms to analyze phytase activity. With such a restriction, the study of the complexity and diversity of the phytases remains incomplete. By using metagenome-derived samples, this study demonstrates the existence of phytase activity in one of the most promiscuous superfamilies, the metallo-ß-lactamases. Our results increase the general knowledge on phytase diversity in environmental samples and could provide new avenues for the study and engineering of new biocatalysts.

Characteristics of the First Protein Tyrosine Phosphatase with Phytase Activity from a Soil Metagenome.

Protein tyrosine phosphatases (PTPs) fulfil multiple key regulatory functions. Within the group of PTPs, the atypical lipid phosphatases (ALPs) are known for their role as virulence factors associated with human pathogens. Another group of PTPs, which is capable of using inositol-hexakisphosphate (InsP6) as substrate, are known as phytases. Phytases play major roles in the environmental phosphorus cycle, biotechnology, and pathogenesis. So far, all functionally characterized PTPs, including ALPs and PTP-phytases, have been derived exclusively from isolated microorganisms. In this study, screening of a soil-derived metagenomic library resulted in identification of a gene (pho16B), encoding a PTP, which shares structural characteristics with the ALPs. In addition, the characterization of the gene product (Pho16B) revealed the capability of the protein to use InsP6 as substrate, and the potential of soil as a source of phytases with so far unknown characteristics. Thus, Pho16B represents the first functional environmentally derived PTP-phytase. The enzyme has a molecular mass of 38 kDa. The enzyme is promiscuous, showing highest activity and affinity toward naphthyl phosphate (Km 0.966 mM). Pho16B contains the HCXXGKDR[TA]G submotif of PTP-ALPs, and it is structurally related to PtpB of Mycobacterium tuberculosis. This study demonstrates the presence and functionality of an environmental gene codifying a PTP-phytase homologous to enzymes closely associated to bacterial pathogenicity.

First Insights into the Genome Sequence of the Cellulolytic Bacterium Clostridium hungatei DSM 14427.

Clostridium hungatei is an obligate anaerobic and spore-forming bacterium, which was isolated from soil. It ferments carbohydrates, such as cellulose or d-glucose. C. hungatei is able to fix nitrogen. The draft genome consists of 1 chromosome (4.902 Mb) with 4,246 predicted protein-coding genes.