Craig Baker-Austin

Characterization of Ferroplasma isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments.

ABSTRACT Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum YT by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate “Ferroplasma acidarmanus” Fer1T required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42°C and the pH optima were 1.0 to 1.7, and “F. acidarmanus” Fer1T was capable of growing at pH 0. The optimum yeast extract concentration for “F. acidarmanus” Fer1T was higher than that for the other three isolates. Phenotypic results suggested that isolate “F. acidarmanus” Fer1T is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and YT group as a single species. “F. acidarmanus” Fer1T groups separately, and we propose the new species “F. acidarmanus” Fer1T sp. nov.

Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities

The acquisition of new genetic material via horizontal gene transfer allows bacteria to rapidly evolve. One key to estimating the contribution of horizontal gene transfer to bacterial evolution is to quantify the abundance of mobile genetic elements (MGEs) in bacterial communities under varying degrees of selective pressure. We quantified class 1 integrase (intI1) gene abundance in total community DNA extracted from contaminated and reference riverine and estuarine microhabitats, and in metal- or antibiotic-amended freshwater microcosms. The intI1 gene was more abundant in all contaminant-exposed communities indicating that relative gene transfer potential is higher in these communities. A second key to assessing the contributions of MGEs to bacterial evolution is to examine the structure and function of the MGE-associated gene pool. We determined that the gene cassette pool is a novel and diverse resource available for bacterial acquisition, but that contamination has no discernible effect on cassette richness. Gene cassette profiles were more similar within sites than among sites, yet bacterial community profiles were not, suggesting that selective pressures can shape the structure of the gene cassette pool. Of the 46 sequenced gene cassette products, 37 were novel sequences, while the 9 gene cassettes with similarity to database sequences were primarily to hypothetical proteins. That class 1 integrons are ubiquitous and abundant in environmental bacterial communities indicates that this group of MGEs can play a substantial role in the acquisition of a diverse array of gene cassettes beyond their demonstrated impact in mediating multidrug resistance in clinical bacteria.

Extreme arsenic resistance by the acidophilic archaeon ‘Ferroplasma acidarmanus’ Fer1

Abstract‘Ferroplasma acidarmanus’ Fer1 is an arsenic-hypertolerant acidophilic archaeon isolated from the Iron Mountain mine, California; a site characterized by heavy metals contamination. The presence of up to 10xa0g arsenate per litre [As(V); 133xa0mM] did not significantly reduce growth yields, whereas between 5 and 10xa0g arsenite per litre [As(III); 67–133xa0mM] significantly reduced the yield. Previous bioinformatic analysis indicates that ‘F. acidarmanus’ Fer1 has only two predicted genes involved in arsenic resistance and lacks a recognizable gene for an arsenate reductase. Biochemical analysis suggests that ‘F. acidarmanus’ Fer1 does not reduce arsenate indicating that ‘F. acidarmanus’ Fer1 has an alternative resistance mechanism to arsenate other than reduction to arsenite and efflux. Primer extension analysis of the putative ars transcriptional regulator (arsR) and efflux pump (arsB) demonstrated that these genes are co-transcribed, and expressed in response to arsenite, but not arsenate. Two-dimensional polyacrylamide gel electrophoresis analysis of ‘F. acidarmanus’ Fer1 cells exposed to arsenite revealed enhanced expression of proteins associated with protein refolding, including the thermosome Group II HSP60 family chaperonin and HSP70 DnaK type heat shock proteins. This report represents the first molecular and proteomic study of arsenic resistance in an acidophilic archaeon.

Antibiotic Resistance in the Shellfish Pathogen Vibrio parahaemolyticus Isolated from the Coastal Water and Sediment of Georgia and South Carolina, USA

Vibrio parahaemolyticus is a gram-negative pathogen commonly encountered in estuarine and marine environments, and a common cause of seafood-related gastrointestinal infections. We isolated 350 V. parahaemolyticus strains from water and sediment at three locations along the Atlantic coast of Georgia and South Carolina during various seasons. These isolates were tested for susceptibility to 24 antibiotics. Isolate virulence was determined through PCR of tdh and trh genes. The breadth of resistance to antibiotics was unexpectedly high, with 24% isolates demonstrating resistance to 10 or more agents. A significant fraction of isolates were resistant to diverse beta-lactams, aminoglycosides, and other classes of antibiotics. Fifteen of the 350 strains possessed virulence genes, with no apparent correlation between virulence and site, sample type, or season of isolation. Antibiotic resistance was slightly reduced among the virulent strains. This study represents one of the largest surveys to date of the virulence and antibiotic resistance in environmental V. parahaemolyticus strains. The observed antibiotic susceptibility patterns suggest that current guidelines for the antibiotic treatment of non-cholerae Vibrio should be reevaluated and extended.

Biofilm development in the extremely acidophilic archaeon ‘Ferroplasma acidarmanus’ Fer1

Abstract‘Ferroplasma acidarmanus’ Fer1 is an iron-oxidizing extreme acidophile isolated from the Iron Mountain mine, California, USA. This archaeon is predominantly found in biofilm-associated structures in the environment, and produces two distinct biofilm morphologies. Bioinformatic analysis of the ‘F. acidarmanus’ Fer1 genome identified genes annotated as involved in attachment and biofilm formation. No putative quorum sensing signaling genes were identified and no N-acyl homoserine lactone-like compounds were found in ‘F. acidarmanus’ Fer1 biofilm supernatant. Scanning confocal microscopy analysis of biofilm development on the surface of pyrite demonstrated the temporal and spatial development of biofilm growth. Furthermore, two-dimensional polyacrylamide gel electrophoresis was used to examine differential protein expression patterns between biofilm and planktonic populations. Ten up-regulated proteins were identified that included six enzymes associated with anaerobic growth, suggesting that the dominating phenotype in the mature biofilm was associated with anaerobic modes of growth. This report increases our knowledge of the genetic and proteomic basis of biofilm formation in an extreme acidophilic archaeon.

Towards determining details of anaerobic growth coupled to ferric iron reduction by the acidophilic archaeon 'Ferroplasma acidarmanus' Fer1.

Elucidation of the different growth states of Ferroplasma species is crucial in understanding the cycling of iron in acid leaching sites. Therefore, a proteomic and biochemical study of anaerobic growth in ‘Ferroplasmaacidarmanus’ Fer1 has been carried out. Anaerobic growth in Ferroplasma spp. occurred by coupling oxidation of organic carbon with the reduction of Fe3+; but sulfate, nitrate, sulfite, thiosulfate, and arsenate were not utilized as electron acceptors. Rates of Fe3+ reduction were similar to other acidophilic chemoorganotrophs. Analysis of the ‘F.acidarmanus’ Fer1 proteome by 2-dimensional polyacrylamide gel electrophoresis revealed ten key proteins linked with central metabolic pathwaysxa0≥4 fold up-regulated during anaerobic growth. These included proteins putatively identified as associated with the reductive tricarboxylic acid pathway used for anaerobic energy production, and others including a putative flavoprotein involved in electron transport. Inhibition of anaerobic growth and Fe3+ reduction by inhibitors suggests the involvement of electron transport in Fe3+ reduction. This study has increased the knowledge of anaerobic growth in this biotechnologically and environmentally important acidophilic archaeon.