Integrated Analysis of Protein Complexes and Regulatory Networks Involved in Anaerobic Energy Metabolism of Shewanella Oneidensis MR-1

Anaerobic Nitrate Reduction. Nitrate is an extensive co-contaminant at some DOE sites making metal and radionuclide reduction problematic. Hence, we sought to better understand the nitrate reduction pathway and its control in S. oneidensis MR-1. It is not known whether the nitrate reduction is by denitrification or dissimilatory nitrate reduction into ammonium (DNRA). By both physiological and genetic evidence, we proved that DNRA is the nitrate reduction pathway in this organism. Using the complete genome sequence of S. oneidensis MR-1, we identified a gene encoding a periplasmic nitrate reductase based on its 72% sequence identity with the napA gene in E. coli. Anaerobic growth of MR-1 on nitrate was abolished in a site directed napA mutant, indicating that NapA is the only nitrate reductase present. The anaerobic expression of napA and nrfA, a homolog of the cytochrome b552 nitrite reductase in E. coli, increased with increasing nitrate concentration until a plateau was reached at 3 mM KNO3. This indicates that these genes are not repressed by increasing concentrations of nitrate. The reduction of nitrate can generate intermediates that can be toxic to the microorganism. To determine the genetic response of MR-1 to high concentrations of nitrate, DNA microarrays were used to obtain a complete gene expression profile of MR-1 at low (1 mM) versus high (40 mM) nitrate concentrations. Genes encoding transporters and efflux pumps were up-regulated, perhaps as a mechanism to export toxic compounds. In addition, the gene expression profile of MR-1, grown anaerobically with nitrate as the only electron acceptor, suggested that this dissimilatory pathway contributes to N assimilation. Hence the nitrate reduction pathway could serve a dual purpose. The role of EtrA, a homolog of Fnr (global anaerobic regulator in E. coli) was examined using an etrA deletion mutant we constructed, S. oneidensis EtrA7-1

A general framework for quantitatively assessing ecological stochasticity.

Understanding the community assembly mechanisms controlling biodiversity patterns is a central issue in ecology. Although it is generally accepted that both deterministic and stochastic processes play important roles in community assembly, quantifying their relative importance is challenging. Here we propose a general mathematical framework to quantify ecological stochasticity under different situations in which deterministic factors drive the communities more similar or dissimilar than null expectation. An index, normalized stochasticity ratio (NST), was developed with 50% as the boundary point between more deterministic (<50%) and more stochastic (>50%) assembly. NST was tested with simulated communities by considering abiotic filtering, competition, environmental noise, and spatial scales. All tested approaches showed limited performance at large spatial scales or under very high environmental noise. However, in all of the other simulated scenarios, NST showed high accuracy (0.90 to 1.00) and precision (0.91 to 0.99), with averages of 0.37 higher accuracy (0.1 to 0.7) and 0.33 higher precision (0.0 to 1.8) than previous approaches. NST was also applied to estimate stochasticity in the succession of a groundwater microbial community in response to organic carbon (vegetable oil) injection. Our results showed that community assembly was shifted from more deterministic (NST = 21%) to more stochastic (NST = 70%) right after organic carbon input. As the vegetable oil was consumed, the community gradually returned to be more deterministic (NST = 27%). In addition, our results demonstrated that null model algorithms and community similarity metrics had strong effects on quantifying ecological stochasticity

Use of Gene Probes to Aid in Recovery and Identification of Functionally Dominant 2,4-Dichlorophenoxyacetic Acid-Degrading Populations in Soil

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to soils in microcosms, and degradation was monitored after each of five repeated additions. Total DNAs were isolated from soil bacterial communities after each 2,4-D treatment. The DNA samples were analyzed on slot blots and Southern blots by using a tfdA gene probe subcloned from plasmid pJP4 and a Spa probe derived from a different 2,4-D-degrading isolate, a Sphingomonas paucimobilis strain. 2,4-D applied to soil was quickly degraded by indigenous microbial populations. As determined by slot blot analyses of DNA from a Michigan soil, the increase in hybridization signal in response to 2,4-D treatments was greater with the Spa probe than with the tfdA probe. In contrast, the DNA from a Saskatchewan soil exhibited an increase in hybridization signal with the tfdA probe. This indicated that a population with 2,4-D-degradative gene sequences different from the tfdA gene sequence was dominant in the Michigan site, but not in the Saskatchewan site. A Southern blot analysis of DNA from Michigan soil showed that the dominant 2,4-D-degrading population was S. paucimobilis 1443. A less dominant 2,4-D-degrading population was detected with the tfdA probe; further analysis revealed that this population was a Pseudomonas pickettii 712. These gene probe analyses revealed that an important population carrying out 2,4-D degradation was not detected when the canonical tfdA gene probe was used. After a series of new strains were isolated, we identified a probe to detect and identify the dominant members of this new group

Analysis of Competition in Soil among 2,4-Dichlorophenoxyacetic Acid-Degrading Bacteria

Competition among indigenous and inoculated 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was studied in a native Kansas prairie soil following 2,4-D additions. The soil was inoculated with four different 2,4-D-degrading strains at densities of 10(3) cells per g of soil; the organisms used were Pseudomonas cepacia DBO1(pJP4) and three Michigan soil isolates, strain 745, Sphingomonas paucimobilis 1443, and Pseudomonas pickettii 712. Following 2,4-D additions, total soil DNA was extracted and analyzed on Southern blots by using a tfdA gene probe which detected three of the strains and another probe that detected the fourth strain, S. paucimobilis 1443, which belongs to a different class of 2,4-D degraders. P. cepacia DBO1(pJP4), a constructed strain, outcompeted the other added strains and the indigenous 2,4-D-degrading populations. The S. paucimobilis population was the secondary dominant population, and strain 745 and P. pickettii were not detected. Relative fitness coefficients determined in axenic broth cultures predicted the outcome of competition in soil for some but not all strains. Lag time was shown to be a principal determinant of competitiveness among the strains, but the lag times were significantly reduced in mixed broth cultures, which changed the competitive outcome. Plasmids containing the genes for the 2,4-D pathway were important determinants of competitiveness since plasmid pKA4 in P. cepacia DBO1 resulted in the slower growth characteristic of its original host, P. pickettii, rather than the rapid growth observed when this strain harbors pJP4

Genetic and Phenotypic Diversity of 2,4-Dichlorophenoxyacetic Acid (2,4-D)-Degrading Bacteria Isolated from 2,4-D-Treated Field Soils

Forty-seven numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated at different times from 1989 through 1992 from eight agricultural plots (3.6 by 9.1 m) which were either not treated with 2,4-D or treated with 2,4-D at three different concentrations. Isolates were obtained from the most dilute positive most-probable-number tubes inoculated with soil samples from the different plots on seven sampling dates over the 3-year period. The isolates were compared by using fatty acid methyl ester (FAME) profiles, chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences, and hybridization patterns obtained with probes for the tfd genes of plasmid pJP4 and a probe (Spa probe) that detects a distinctly different 2,4-D-degrading isolate, Sphingomonas paucimobilis (formerly Pseudomonas paucimobilis). A total of 57% of the isolates were identified to the species level by the FAME analysis, and these isolates were strains of Sphingomonas, Pseudomonas, or Alcaligenes species. Hybridization analysis revealed four groups. Group I strains, which exhibited sequence homology with tfdA, -B, -C, and -D genes, were rather diverse, as determined by both the FAME analysis and the REP-PCR analysis. Group II, which exhibited homology only with the tfdA4 gene, was a small group and was probably a subset of group I. All group I and II strains had plasmids. Hybridization analysis revealed that the tfd genes were located on plasmids in 75% of these strains and on the chromosome or a large plasmid in the other 25% of the strains. One strain exhibited tfdA and -B hybridization associated with a plasmid band, while tfdC and -D hybridized with the chromosomal band area. The group III strains exhibited no detectable homology to tfd genes but hybridized to the Spa probe. The members of this group were tightly clustered as determined by both the FAME analysis and the REP-PCR analysis, were distinctly different from group I strains as determined by the FAME analysis, and had very few plasmids; this group contained more of the 47 isolates than any other group. The group III strains were identified as S. paucimobilis. The group IV strains, which hybridized to neither the tfd probe nor the Spa probe, were as diverse as the group I strains as determined by the FAME and REP-PCR analyses. Most of group IV strains could not be identified by the FAME analysis. Strains belonging to groups I and III were more frequently recovered from soils that had greater field exposure to 2,4-D, suggesting that they were the best competitors for 2,4-D under field conditions. The selection regimen which we used led to two successful but dissimilar groups; the members of one group were similar at the plasmid level but not at the organism level, and the members of the other group were similar at the organism level. Since the members of the latter group are ecologically successful and degradative genes unlike tfd genes, they deserve more attention

Assembling large, complex environmental metagenomes

The large volumes of sequencing data required to sample complex environments deeply pose new challenges to sequence analysis approaches. De novo metagenomic assembly effectively reduces the total amount of data to be analyzed but requires significant computational resources. We apply two pre-assembly filtering approaches, digital normalization and partitioning, to make large metagenome assemblies more comput\ ationaly tractable. Using a human gut mock community dataset, we demonstrate that these methods result in assemblies nearly identical to assemblies from unprocessed data. We then assemble two large soil metagenomes from matched Iowa corn and native prairie soils. The predicted functional content and phylogenetic origin of the assembled contigs indicate significant taxonomic differences despite similar function. The assembly strategies presented are generic and can be extended to any metagenome; full source code is freely available under a BSD license.Comment: Includes supporting informatio

Diazotrophs Show Signs of Restoration in Amazon Rain Forest Soils with Ecosystem Rehabilitation.

Biological nitrogen fixation can be an important source of nitrogen in tropical forests that serve as a major CO2 sink. Extensive deforestation of the Amazon is known to influence microbial communities and the biogeochemical cycles they mediate. However, it is unknown how diazotrophs (nitrogen-fixing microorganisms) respond to deforestation and subsequent ecosystem conversion to agriculture, as well as whether they can recover in secondary forests that are established after agriculture is abandoned. To address these knowledge gaps, we combined a spatially explicit sampling approach with high-throughput sequencing of nifH genes. The main objectives were to assess the functional distance decay relationship of the diazotrophic bacterial community in a tropical forest ecosystem and to quantify the roles of various factors that drive the observed changes in the diazotrophic community structure. We observed an increase in local diazotrophic diversity (α-diversity) with a decrease in community turnover (β-diversity), associated with a shift in diazotrophic community structure as a result of the forest-to-pasture conversion. Both diazotrophic community turnover and structure showed signs of recovery in secondary forests. Changes in the diazotrophic community were primarily driven by the change in land use rather than differences in geochemical characteristics or geographic distances. The diazotroph communities in secondary forests resembled those in primary forests, suggesting that at least partial recovery of diazotrophs is possible following agricultural abandonment.IMPORTANCE The Amazon region is a major tropical forest region that is being deforested at an alarming rate to create space for cattle ranching and agriculture. Diazotrophs (nitrogen-fixing microorganisms) play an important role in supplying soil N for plant growth in tropical forests. It is unknown how diazotrophs respond to deforestation and whether they can recover in secondary forests that establish after agriculture is abandoned. Using high-throughput sequencing of nifH genes, we characterized the response of diazotrophs' β-diversity and identified major drivers of changes in diazotrophs from forest-to-pasture and pasture-to-secondary-forest conversions. Studying the impact of land use change on diazotrophs is important for a better understanding of the impact of deforestation on tropical forest ecosystem functioning, and our results on the potential recovery of diazotrophs in secondary forests imply the possible restoration of ecosystem functions in secondary forests

Detection in Soil of a Deletion in an Engineered DNA Sequence by Using DNA Probes

Two Pseudomonas strains were engineered to contain the nptII gene and plasmid vector sequences in their chromosomes. After incubation of these strains in nonsterile soil, total bacterial DNA was isolated and analyzed by Southern blot hybridization with the nptII gene and the plasmid vector as probes. In addition to the expected bands of hybridization, a new band corresponding to the loss of vector sequences from the chromosome while retaining the nptII gene was observed for one of the strains. The more stressful conditions encountered in soil appeared to increase the frequency of loss of the vector sequences from this strain

Integrated genome-based studies of Shewanella Ecophysiology

The aim of the work reported is to study Shewanella population genomics, and to understand the evolution, ecophysiology, and speciation of Shewanella. The tasks supporting this aim are: to study genetic and ecophysiological bases defining the core and diversification of Shewanella species; to determine gene content patterns along redox gradients; and to Investigate the evolutionary processes, patterns and mechanisms of Shewanella