This study describes and validates a new method for metagenomic biomarker discovery by way of class comparison, tests of biological consistency and effect size estimation. This addresses the challenge of finding organisms, genes, or pathways that consistently explain the differences between two or more microbial communities, which is a central problem to the study of metagenomics. We extensively validate our method on several microbiomes and a convenient online interface for the method is provided at http://huttenhower.sph.harvard.edu/lefse/.

Metagenomic biomarker discovery and explanation

Short protocols in molecular biology

A major thrust of terrestrial microbial ecology is focused on understanding when and how the composition of the microbial community affects the functioning of biogeochemical processes at the ecosystem scale (meters-to-kilometers and days-to-years). While research has demonstrated these linkages for physiologically and phylogenetically “narrow” processes such as trace gas emissions and nitrification, there is less conclusive evidence that microbial community composition influences the “broad” processes of decomposition and organic matter turnover in soil. In this paper, we consider how soil microbial community structure influences C-cycling. We consider the phylogenetic level at which microbes form meaningful guilds, based on overall life history strategies, and suggest that these are associated with deep evolutionary divergences, while much of the species-level diversity probably reflects functional redundancy. We then consider under what conditions it is possible for differences among microbes to affect process dynamics, and argue that while microbial community structure may be important in the rate of OM breakdown in the rhizosphere and in detritus, it is likely not important in the mineral soil. In mineral soil, physical access to occluded or sorbed substrates is the rate-limiting process. Microbial community influences on OM turnover in mineral soils are based on how organisms allocate the C they take up—not only do the fates of the molecules differ, but they can affect the soil system differently as well. For example, extracellular enzymes and extracellular polysaccharides can be key controls on soil structure and function. How microbes allocate C may also be particularly important for understanding the long-term fate of C in soil—is it sequestered or not?

/pdf/microbial-control-over-carbon-cycling-in-soil-3rqyfpr4cd.pdf

Microbial control over carbon cycling in soil.

Manual for the Identification of Medical Bacteria.

It is one thing to show that herbivorous insects affect plant performance. It is an entirely different matter to demonstrate that insect herbivory affects plant population dynamics. There is a vast literature on insect pests of crop plants that shows how attack by defoliating, sucking, stem-mining, and gall-forming species can delay seed ripening, reduce seed production and individual seed weights, reduce the rates of shoot and root growth, increase the susceptibility of plants to disease, and reduce the competitive ability of plants relative to their un attacked neighbors. This literature tells us virtually nothing, however, about the importance of insects in natural communities, chiefly because we have so little information on the regulation of plant populations in the wild. For example, we do not know whether plant recruitment is seed limited , so we cannot predict whether there would be an increase in plant numbers if the simple experiment of sowing extra seeds were carried out. Information on the nature of population regulation is vital because if plant recruitment is not seed limited, then insects that reduce seed production will not have an important effect on plant population dynamics. This review concentrates on material published from 1 984 to 1987. Earlier material has been covered in other reviews (36, 64, 70, 94, 1 07, 12 1 , 1 82, 206) . The first part examines the impact of insect feeding on different aspects of plant performance. The second part examines two of the best sources of evidence on the impact of insect feeding on plant population dynamics: (a) the release of specialist insect herbivores against target weed species in classical

http://www.sysecol2.ethz.ch/Refs/EntClim/C/Cr080.pdf

Insect herbivores and plant population dynamics

An ultrasensitive bioassay system for the detection of N-acylhomoserine lactones (AHLs) was constructed in Agrobacterium tumefaciens by using the T7 expression system to overproduce the AHL receptor TraR. This strain detected many diverse AHLs, some at extremely low concentrations. We used this strain to detect for the first time AHLs made by Mesorhizobium huakuii, which symbiotically fixes nitrogen in association with the legume Astragalus sinicus, a source of green manure throughout eastern Asia.

Agrobacterium Bioassay Strain for Ultrasensitive Detection of N-Acylhomoserine Lactone-Type Quorum-Sensing Molecules: Detection of Autoinducers in Mesorhizobium huakuii

We have developed a fast and accurate method to engineer the Bacillus subtilis genome that involves fusing by PCR two flanking homology regions with an antibiotic resistance gene cassette bordered by two mutant lox sites (lox71 and lox66). The resulting PCR products were used directly to transform B. subtilis, and then transient Cre recombinase expression in the transformants was used to recombine lox71 and lox66 into a double-mutant lox72 site, thereby excising the marker gene. The mutation process could also be accomplished in 2 days by using a strain containing a cre isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression cassette in the chromosome as the recipient or using the lox site-flanked cassette containing both the cre IPTG-inducible expression cassette and resistance marker. The in vivo recombination efficiencies of different lox pairs were compared; the lox72 site that remains in the chromosome after Cre recombination had a low affinity for Cre and did not interfere with subsequent rounds of Cre/lox mutagenesis. We used this method to inactivate a specific gene, to delete a long fragment, to realize the in-frame deletion of a target gene, to introduce a gene of interest, and to carry out multiple manipulations in the same background. Furthermore, it should also be applicable to large genome rearrangement.

Cre/lox System and PCR-Based Genome Engineering in Bacillus subtilis

Isolation of a chlorpyrifos-degrading bacterium, Sphingomonas sp. strain Dsp-2, and cloning of the mpd gene.

We developed a self-formed adaptor PCR (termed SEFA PCR) which can be used for chromosome walking. Most of the amplified flanking sequences were longer than 2.0 kb, and some were as long as 6.0 kb. SEFA PCR is simple and efficient and should have broad applications in the isolation of unknown sequences in complex genomes.

Self-formed adaptor PCR: a simple and efficient method for chromosome walking.

A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many α/β-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.

Shunpeng Li

Papers

Agrobacterium Bioassay Strain for Ultrasensitive Detection of N-Acylhomoserine Lactone-Type Quorum-Sensing Molecules: Detection of Autoinducers in Mesorhizobium huakuii

Cre/lox System and PCR-Based Genome Engineering in Bacillus subtilis

Isolation of a chlorpyrifos-degrading bacterium, Sphingomonas sp. strain Dsp-2, and cloning of the mpd gene.

Self-formed adaptor PCR: a simple and efficient method for chromosome walking.

Cloning of a novel pyrethroid-hydrolyzing carboxylesterase gene from Sphingobium sp. strain JZ-1 and characterization of the gene product.