Phototrophic microbial mats are ideal model systems for ecological and evolutionary analysis of highly diverse microbial communities. Microbial mats are small-scale, nearly closed, and self-sustaining benthic ecosystems that comprise the major element cycles, trophic levels, and food webs. The steep and fluctuating physicochemical microgradients, that are the result of the ever changing environmental conditions and of the microorganisms' own activities, give rise to a plethora of potential niches resulting in the formation of one of the most diverse microbial ecosystems known to date. For several decades, microbial mats have been studied extensively and more recently molecular biological techniques have been introduced that allowed assessing and investigating the diversity and functioning of these systems. These investigations also involved metagenomics analyses using high-throughput DNA and RNA sequencing. Here, we summarize some of the latest developments in metagenomic analysis of three representative phototrophic microbial mat types (coastal, hot spring, and hypersaline). We also present a comparison of the available metagenomic data sets from mats emphasizing the major differences between them as well as elucidating the overlap in overall community composition.

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product.

A long-term experiment on the effect of chitin addition to soil on the suppression of soilborne pathogens was set up and monitored for 8 years in an experimental field, Vredepeel, The Netherlands. Chitinous matter obtained from shrimps was added to soil top layers on two different occasions, and the suppressiveness of soil toward Verticillium dahliae, as well as plant-pathogenic nematodes, was assessed, in addition to analyses of the abundances and community structures of members of the soil microbiota. The data revealed that chitin amendment had raised the suppressiveness of soil, in particular toward Verticillium dahliae, 9 months after the (second) treatment, extending to 2 years following treatment. Moreover, major effects of the added chitin on the soil microbial communities were detected. First, shifts in both the abundances and structures of the chitin-treated soil microbial communities, both of total soil bacteria and fungi, were found. In addition, the abundances and structures of soil actinobacteria and the Oxalobacteraceae were affected by chitin. At the functional gene level, the abundance of specific (family-18 glycoside hydrolase) chitinase genes carried by the soil bacteria also revealed upshifts as a result of the added chitin. The effects of chitin noted for the Oxalobacteraceae were specifically related to significant upshifts in the abundances of the species Duganella violaceinigra and Massilia plicata. These effects of chitin persisted over the time of the experiment.

SummaryEstrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 ␤ -estradiol (E 2 ) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E 2 also directly induced osteoclast apoptosis in a dose-and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E 2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptormediated mechanism.

Lake Meyghan is one of the largest and commercially most important salt lakes in Iran. Despite its inland location and high altitude, Lake Meyghan has a thalassohaline salt composition suggesting a marine origin. Inputs of fresh water by rivers and rainfall formed various basins characterized by different salinities. We analyzed the microbial community composition of three basins by isolation and culturing of microorganisms and by analysis of the metagenome. The basins that were investigated comprised a green ~50 g kg−1 salinity brine, a red ~180 g kg−1 salinity brine and a white ~300 g kg−1 salinity brine. Using different growth media, 57 strains of Bacteria and 48 strains of Archaea were isolated. Two bacterial isolates represent potential novel species with less than 96% 16S rRNA gene sequence identity to known species. Abundant isolates were also well represented in the metagenome. Bacteria dominated the low salinity brine, with Alteromonadales (Gammaproteobacteria) as a particularly important taxon, whereas the high salinity brines were dominated by haloarchaea. Although the brines of Lake Meyghan differ in geochemical composition, their ecosystem function appears largely conserved amongst each other while being driven by different microbial communities.

Coastal microbial mats form a nearly closed micro-scale ecosystem harboring a complex microbial community. Previous DNA based analysis did not necessarily provide information about the active fraction of the microbial community because it includes dormant, inactive cells as well as a potential stable pool of extracellular DNA. Here we focused on the active microbial community by comparing 16S rRNA sequences obtained from the ribosomal RNA pool with gene sequences obtained from the DNA fraction. In addition, we aimed to establish an optimal and feasible sampling protocol that takes potential spatial and temporal heterogeneity into account. The coastal microbial mat investigated here was sampled randomly and at regular time points during one 24-h period. DNA and RNA was extracted and after conversion of the RNA fraction to cDNA, the V1-V3 and the V3-V4 regions of the 16S rRNA gene were targeted for high-throughput amplicon sequencing. We show that the community composition varies little in time and space whereas two amplified 16S regions gave significant different results. The largest differences were found when comparing the “resident community” (DNA) with the “active community” (cDNA/RNA); in the latter, Cyanobacteria dominated for almost 95% while they represented 60% of the resident fraction.

Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of sizes of 21 to 40 kb, yielding a total of approximately 5.8 GB of cloned soil DNA. Using genetic screenings by repeated PCR cycles aimed to detect gene sequences of the bacterial chitinase A-class (hereby named chi A genes), we identified and characterized five fosmids carrying candidate genes for chitinolytic enzymes. The analysis thus allowed access to the genomic (fosmid-borne) context of these genes. Using the chiA-targeted PCR, which is based on degenerate primers, the five fosmids all produced amplicons, of which the sequences were related to predicted chitinolytic enzyme-encoding genes of four different host organisms, including Stenotrophomonas maltophilia. Sequencing and de novo annotation of the fosmid inserts confirmed that each one of these carried one or more open reading frames that were predicted to encode enzymes active on chitin, including one for a chitin deacetylase. Moreover, the genetic contexts in which the putative chitinolytic enzyme-encoding genes were located were unique per fosmid. Specifically, inserts from organisms related to Burkholderia sp., Acidobacterium sp., Aeromonas veronii, and the chloroflexi Nitrolancetus hollandicus and/or Ktedonobacter racemifer were obtained. Remarkably, the S. maltophilia chiA-like gene was found to occur in two different genetic contexts (related to N. hollandicus/K. racemifer), indicating the historical occurrence of genetic reshufflings in this part of the soil microbiota. One fosmid containing the insert composed of DNA from the N. hollandicus-like organism (denoted 53D1) was selected for further work. Using subcloning procedures, its putative gene for a chitinolytic enzyme was successfully brought to expression in an E. coli host. On the basis of purified protein preparations, the produced protein was characterized as a chitobiosidase of 43.6 kDa, with a pI of 4.83. Given its activity spectrum, it can be typified as a halotolerant chitobiosidase.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6639-5) contains supplementary material, which is available to authorized users.

Macroalgae belonging to the genus Padina are known to produce antibacterial compounds that may inhibit growth of human- and animal pathogens. Hitherto, it was unclear whether this antibacterial activity is produced by the macroalga itself or by secondary metabolite producing epiphytic bacteria. Here we report antibacterial activities of epiphytic bacteria isolated from Padina pavonica (Peacocks tail) located on northern coast of Tunisia. Eighteen isolates were obtained in pure culture and tested for antimicrobial activities. Based on the 16S rRNA gene sequences the isolates were closely related to Proteobacteria (12 isolates; 2 Alpha- and 10 Gammaproteobacteria), Firmicutes (4 isolates) and Actinobacteria (2 isolates). The antimicrobial activity was assessed as inhibition of growth of 12 species of pathogenic bacteria (Aeromonas salmonicida, A. hydrophila, Enterobacter xiangfangensis, Enterococcus faecium, Escherichia coli, Micrococcus sp., Salmonella typhimurium, Staphylococcus aureus, Streptococcus sp., Vibrio alginoliticus, V. proteolyticus, V. vulnificus) and one pathogenic yeast (Candida albicans). Among the Firmicutes, isolate P8, which is closely related to Bacillus pumilus, displayed the largest spectrum of growth inhibition of the pathogenic bacteria tested. The results emphasize the potential use of P. pavonica associated antagonistic bacteria as producers of novel antibacterial compounds.