Genomic Plasticity in Ralstonia eutropha and Ralstonia pickettii: Evidence for Rapid Genomic Change and Adaptation

The proposed foci of our investigations were on Ralstonia eutropha and Rasltonia pickettii. We have 18 derived lineages of the former as well as their progenitor and eleven isolates of the latter. Our goal was to measure the level of plasticity in these strains and attempt to derive a mechanistic understanding of how genomic plasticity formed. Extensive attempts to reproducibly induce conformational changes in the genome of R. eutropha were unsuccessful. We thought that we had a reasonable lead on this inasmuch as we had shown that the ancestral strain along with many of the derivative lineages exhibited “temperature induced mutation and mortality akin to R. metallodurans. However we were unable to get subtractive hybridization working to the degree that it revealed differences between the lineages. During this time the R. pickettii analysis was proving quite fruitful and so we concentrated our efforts on our analyses of R. pickettii. These strains were isolated from a copper-contaminated lake sediment and were resistant to copper at 800 µg/ml (CuSO4). Our results in the investigation of R. pickettii permitted a view into the adaptation of a beta-proteobacteria to an extreme environment. Our worked revealed that within the same ecosystem two genomovars with structurally different genomes and genome sizes were present and apparently filling similar if not identical niches. The genomovars were detected with REP & BOX-PCR, pulse field gel electrophoresis, and DNA:DNA hybridizations. Moreover there were different metal resistance patterns associated with the different genomovars, one showing resistance to Zn and Cd while the other had resistance to Ni. Five of the isolates had a high-copy number extrachromosomal element that was identified as the replicative form of a filamentous phage. Mature virions were isolated from culture broth using PEG precipitation and CsCl density centrifugation. The DNA associated with the filamentous particles was single stranded and had a sequence identical to the intracellular replicative form. Using PCR targeting phage-specific genes we showed that seven out of the eleven isolates carried the phage. The seven isolates positive for phage were formed one of the genomovar groups, hence the presence of the phage may have generated the divergent lineage. One representative of each of these genomovars was sequence by JGI. While the genomes have not been closed completely, the results so far are provocative. Strain 12J, which carries the phage, revealed four integrated copies of the phage genome, each copy at a different level of divergence from the active replicative form. A comparative analysis of the common genes found in these integrated phage copies revealed that the gene complements were incongruent with one another within a phage copy, suggesting that the copies had become sites of recombination or that the cell had recruited genes for different functions. One of the integrated copies had the exact sequence as the replicative form and we assume this to be the most recent integration event. Evidence for a recent integration was revealed in a repeated sequence element found on each terminus of the phage genome. Finally, these isolates are of interest in bioremediation and reclamation of metals from waste streams. We have determined that each Ralstonia cell is capable of binding 6 x 107 Cu (II) ions and that 90% of the binding occurs within 12 hours of exposure to Cu(II). Our studies have revealed a species with a uniquely fluid genome. This Ralstonia population has been under extreme selective pressure over the past hundred years as it responded to the accumulation of copper in the lake sediment as a result of unregulated mining practices. In addition to the selective pressure of copper, the population was repeatedly infected with a filamentous phage that may have contributed to the divergence of the genomovars. This dynamic population could help reveal the selective forces and their consequences on genome structure in a proteobacterium that has potential for remediation

Monitoring Uranium Transformations Determined by the Evolution of Biogeochemical Processes

Our contribution to the larger project (ANL) was the phylogenetic analysis of evolved communities capable of reducing metals including uranium

Temporal Shifts in Microbial Communities in Nonpregnant African-American Women with and without Bacterial Vaginosis

Bacterial vaginosis (BV) has been described as an increase in the number of anaerobic and facultatively anaerobic bacteria relative to lactobacilli in the vaginal tract. Several undesirable consequences of this community shift can include irritation, white discharge, an elevated pH, and increased susceptibility to sexually transmitted infections. While the etiology of the condition remains ill defined, BV has been associated with adverse reproductive and pregnancy outcomes. In order to describe the structure of vaginal communities over time we determined the phylogenetic composition of vaginal communities from seven women sampled at multiple points using 16S rRNA gene sequencing. We found that women with no evidence of BV had communities dominated by lactobacilli that appeared stable over our sampling periods while those with BV had greater diversity and decreased stability overtime. In addition, only Lactobacillus iners was found in BV positive communities

Advancing Stage of Female Reproductive Life Associated with Bipolar Illness Exacerbation

Introduction: Perimenopause confers an increased risk of depression in the general population, yet bipolar disorder mood course remains unknown. Methods: Clinic visits in 519 premenopausal, 116 perimenopausal including 13 women transitioning from peri- to postmenopause, and 133 postmenopausal women with bipolar disorder who received naturalistic treatment in the multisite STEP-Bipolar Disorder study over 19.8 +/- 15.5 months were analyzed for mood state. Results: Advancing female reproductive stage was associated with significant decline in mood elevation; significant decline in euthymia; no significant difference in major depression; and symptomatic significant increase. Conclusions: Advancing stage of female reproductive life was associated with bipolar illness exacerbation. Women transitioning from peri- to postmenopause had significantly greater depression than other female reproductive groups

Defining the "core microbiome" of the microbial communities in the tonsils of healthy pigs

BACKGROUND: Porcine tonsils are the colonization site for many pathogenic as well as commensal microorganisms and are the primary lymphoid tissue encountered by organisms entering through the mouth or nares. The goal of this study was to provide an in-depth characterization of the composition and structure of the tonsillar microbial communities and to define the core microbiome in the tonsils of healthy pigs, using high throughput bar-coded 454-FLX pyrosequencing. RESULTS: Whole tonsils were collected at necropsy from 12 16-week-old finisher pigs from two healthy herds. Tonsil brushes were also used to collect samples from four of these animals. Bacterial DNA was isolated from each sample, amplified by PCR with universal primers specific for the bacterial 16S rRNA genes, and the PCR products sequenced using pyrosequencing. An average of 13,000 sequences were generated from each sample. Microbial community members were identified by sequence comparison to known bacterial 16S rRNA gene sequences. The microbiomes of these healthy herds showed very strong similarities in the major components as well as distinct differences in minor components. Pasteurellaceae dominated the tonsillar microbiome in all animals, comprising ~60% of the total, although the relative proportions of the genera Actinobacillus, Haemophilus, and Pasteurella varied between the herds. Also found in all animals were the genera Alkanindiges, Peptostreptococcus, Veillonella, Streptococcus and Fusobacterium, as well as Enterobacteriaceae and Neisseriaceae. Treponema and Chlamydia were unique to Herd 1, while Arcanobacterium was unique to Herd 2. Tonsil brushes yielded similar results to tissue specimens, although Enterobacteriaceae and obligate anaerobes were more frequently found in tissue than in brush samples, and Chlamydia, an obligately intracellular organism, was not found in brush specimens. CONCLUSIONS: We have extended and supported our previous studies with 16S clone libraries, using 16S rRNA gene pyrosequencing to describe the microbial communities in tonsils of healthy pigs. We have defined a core microbiome, dominated by Pasteurellaceae, in tonsil specimens, and have also demonstrated the presence of unique minor components of the tonsillar microbiome present in each herd. We have validated the use of noninvasive tonsil brushes, in comparison to tonsil tissue, which will facilitate future studies.Beth A. Lowe, Terence L. Marsh, Natasha Isaacs-Cosgrove, Roy N. Kirkwood, Matti Kiupel and Martha H. Mulk

Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction

<p>Abstract</p> <p>Background</p> <p>The genome of the Gram-positive, metal-reducing, dehalorespiring <it>Desulfitobacterium hafniense </it>DCB-2 was sequenced in order to gain insights into its metabolic capacities, adaptive physiology, and regulatory machineries, and to compare with that of <it>Desulfitobacterium hafniense </it>Y51, the phylogenetically closest strain among the species with a sequenced genome.</p> <p>Results</p> <p>The genome of <it>Desulfitobacterium hafniense </it>DCB-2 is composed of a 5,279,134-bp circular chromosome with 5,042 predicted genes. Genome content and parallel physiological studies support the cell's ability to fix N₂and CO₂, form spores and biofilms, reduce metals, and use a variety of electron acceptors in respiration, including halogenated organic compounds. The genome contained seven reductive dehalogenase genes and four nitrogenase gene homologs but lacked the Nar respiratory nitrate reductase system. The <it>D. hafniense </it>DCB-2 genome contained genes for 43 RNA polymerase sigma factors including 27 sigma-24 subunits, 59 two-component signal transduction systems, and about 730 transporter proteins. In addition, it contained genes for 53 molybdopterin-binding oxidoreductases, 19 flavoprotein paralogs of the fumarate reductase, and many other FAD/FMN-binding oxidoreductases, proving the cell's versatility in both adaptive and reductive capacities. Together with the ability to form spores, the presence of the CO₂-fixing Wood-Ljungdahl pathway and the genes associated with oxygen tolerance add flexibility to the cell's options for survival under stress.</p> <p>Conclusions</p> <p><it>D. hafniense </it>DCB-2's genome contains genes consistent with its abilities for dehalogenation, metal reduction, N₂and CO₂fixation, anaerobic respiration, oxygen tolerance, spore formation, and biofilm formation which make this organism a potential candidate for bioremediation at contaminated sites.</p

Effect of dietary prebiotics and probiotics on snakehead (Channa striata) health : Haematology and disease resistance parameters against Aeromonas hydrophila

This study examined the effect of dietary prebiotics and probiotics after 16 weeks, followed by 8 weeks of post feeding trial with the control unsupplemented diet on haematological and immune response against Aeromonas hydrophila infection in Channa striata fingerlings. Fish were raised on a 40% protein and 12% lipid feed containing three commercial prebiotics (β-glucan, GOS or galacto-oligosaccharide, MOS or mannan-oligosaccharide); and two probiotics- (Saccharomyces cerevisiae, Lactobacillus acidophilus), respectively and a control. Throughout the study, supplementation with dietary prebiotics and probiotics led to significant (P < 0.05) improvement in the red blood cells, white blood cells, packed cell volume, haemoglobin concentration and serum protein level and lysozyme activities; and these improvements were effective significantly (P < 0.05) when the fish were challenged with Aeromonas hydrophila at the dose of 2×106. The disease resistance against A. hydrophila was higher significantly (P < 0.05) in fish fed with probiotic feed supplements (L.acidophilus was highest) compared to prebiotics and control. The study is the first to report the absence of differences in sustaining the efficacies attained afterintakeofβ-glucan, GOSandMOSuponpost-feeding withanunsupplemented feed, over a prolonged period

Noncompetitive microbial diversity patterns in soils: their causes and implications for bioremediation

This funding provided support for over nine years of research on the structure and function of microbial communities in subsurface environments. The overarching goal during these years was to understand the impact of mixed contaminants, particularly heavy metals like uranium, on the structure and function of microbial communities. In addition we sought to identify microbial populations that were actively involved in the reduction of metals because these species of bacteria hold the potential for immobilizing soluble metals moving in subsurface water. Bacterial mediated biochemical reduction of metals like uranium, technetium and chromium, greatly reduces their mobility through complexation and precipitation. Hence, by taking advantage of natural metabolic capabilities of subsurface microbial populations it is possible to bioremediate contaminated subsurface environments with a cost-effective in situ approach. Towards this end we have i.) identified bacterial populations that have thrived under the adverse conditions at the contaminated FRC site, ii.) phylogenetically identified populations that respond to imposed remediation conditions at the FRC, iii.) used metagenomics to begin a reconstruction of the metabolic web in a contaminated subsurface zone, iv.) investigated the metal reducing attributes of a Gram-positive spore forming rod also capable of dechlorination

Characterizing microbial communities associated with northern root-knot nematode (Meloidogyne hapla) occurrence and soil health

The northern root-knot nematode (Meloidogyne hapla) causes extensive damage to agricultural crops globally. In addition, M. hapla populations with no known genetic or morphological differences exhibit parasitic variability (PV) or reproductive potential based on soil type. However, why M. hapla populations from mineral soil with degraded soil health conditions have a higher PV than populations from muck soil is unknown. To improve our understanding of soil bio-physicochemical conditions in the environment where M. hapla populations exhibited PV, this study characterized the soil microbial community and core- and indicator-species structure associated with M. hapla occurrence and soil health conditions in 15 Michigan mineral and muck vegetable production fields. Bacterial and fungal communities in soils from where nematodes were isolated were characterized with high throughput sequencing of 16S and internal transcribed spacer (ITS) rDNA. Our results showed that M. hapla-infested, as well as disturbed and degraded muck fields, had lower bacterial diversity (observed richness and Shannon) compared to corresponding mineral soil fields or non-infested mineral fields. Bacterial and fungal community abundance varied by soil group, soil health conditions, and/or M. hapla occurrence. A core microbial community was found to consist of 39 bacterial and 44 fungal sub-operational taxonomic units (OTUs) across all fields. In addition, 25 bacteria were resolved as indicator OTUs associated with M. hapla presence or absence, and 1,065 bacteria as indicator OTUs associated with soil health conditions. Out of the 1,065 bacterial OTUs, 73.9% indicated stable soil health, 8.4% disturbed, and 0.4% degraded condition; no indicators were common to the three categories. Collectively, these results provide a foundation for an in-depth understanding of the environment where M. hapla exists and conditions associated with parasitic variability

Development of the Tonsil Microbiome in Pigs and Effects of Stress on the Microbiome

Tonsils, lympho-epithelial tissues located at the junction of the oropharynx and nasopharynx, play a key role in surveillance, colonization, and persistence of inhaled and ingested pathogens. In pigs, the tonsils are a reservoir for numerous bacteria and viruses, including host-specific pathogens and potential zoonotic pathogens as well as commensal organisms. However, there are no in depth studies of the development of the tonsillar microbiome in pigs, or any mammal, over time. The goal of this study was to follow the development of the tonsil microbiome in healthy pigs from birth to market weight. Samples were collected using tonsil brushes from 16 piglets (4 each from 4 sows) at newborn, 1, 2, 3, and 4 weeks of age, and from 8 of those piglets at 6, 8, 10, 12, 16, and 19 weeks of age. Bacterial DNA was isolated from each sample and 16S rDNA genes were amplified and sequenced. Sequence analysis showed that members of the Streptococcaceae, Pasteurellaceae, and Moraxellaceae were present at all time points and represent the three most abundant families identified. Other community members appeared transiently or increased or decreased significantly with disruption events or stress. We observed four significant shifts in the tonsil community that coincided with well-defined disruption events: weaning plus addition of Carbadox plus movement to the nursery at week 3, removal of Carbadox and addition of Tylan at week 5, removal of Tylan and habitat change at week 9, and habitat change at week 16. Weaning triggered a bloom of Streptococcaeae and decrease of Moraxellaceae. The shift from Carbadox to Tylan led to reduction in Proteobacteria and Streptococcaceae but an increase in other Firmicutes, accompanied by a dramatic increase in community richness. Cessation of Tylan coincided with a return to a less rich community, and a bloom in Clostridiales. The final shift in habitat was accompanied by a decrease in Clostridiales and increase in Proteobacteria. The tonsillar microbiome of older pigs resembled the previously described mature core tonsillar microbiome. This study demonstrates a temporal succession in the development of the pig tonsillar microbiome, and significant community shifts that correlate with disruption events