Limited carbon sources prevent sulfate remediation in circumneutral abandoned mine drainage.

Passive remediation systems (PRS) use both biotic and abiotic processes to precipitate contaminants from abandoned mine drainage (AMD) so that the contaminants do not spread into local watersheds. PRS are efficient at removing heavy metals but sulfate remediation frequently does not occur. To understand the reasons for the lack of sulfate remediation, we studied four PRS that treat circumneutral AMD and one raw mine drainage discharge. Using 16S sequencing analysis, microbial community composition revealed a high relative abundance of bacterial families with sulfur cycling genera. Anaerobic abiotic studies showed that sulfide was quickly geochemically oxidized in the presence of iron hydroxides, leading to a buildup of sulfur intermediates. Supplementation of laboratory grown microbes from the PRS with lactate demonstrated the ability of actively growing microbes to overcome this abiotic sulfide oxidation by increasing the rate of sulfate reduction. Thus, the lack of carbon sources in the PRS contributes to the lack of sulfate remediation. Bacterial community analysis of 16S rRNA gene revealed that while the microbial communities in different parts of the PRS were phylogenetically distinct, the contaminated environments selected for communities that shared similar metabolic capabilities.

Microbial Communities Associated With Passive Acidic Abandoned Coal Mine Remediation.

Acid mine drainage (AMD) is an environmental issue that can be characterized by either acidic or circumneutral pH and high dissolved metal content in contaminated waters. It is estimated to affect roughly 3000 miles of waterways within the state of Pennsylvania, with half being acidic and half being circumneutral. To negate the harmful effects of AMD, ∼300 passive remediation systems have been constructed within the state of Pennsylvania. In this study, we evaluated the microbial community structure and functional capability associated with Middle Branch passive remediation system in central PA. Sediment and water samples were collected from each area within the passive remediation system and its receiving stream. Environmental parameters associated with the remediation system were found to explain a significant amount of variation in microbial community structure. This study revealed shifts in microbial community structure from acidophilic bacteria in raw AMD discharge to a more metabolically diverse set of taxa (i.e., Acidimicrobiales, Rhizobiales, Chthoniobacteraceae) toward the end of the system. Vertical flow ponds and the aerobic wetland showed strong metabolic capability for sulfur redox environments. These findings are integral to the understanding of designing effective passive remediation systems because it provides insight as to how certain bacteria [sulfate reducing bacteria (SRBs) and sulfur oxidizing bacteria (SOBs)] are potentially contributing to a microbially mediated AMD remediation process. This study further supports previous investigations that demonstrated the effectiveness of SRBs in the process of removing sulfate and heavy metals from contaminated water.

A seasonal study of a passive abandoned coalmine drainage remediation system reveals three distinct zones of contaminant levels and microbial communities.

A passive remediation system that treats coalmine drainage was sampled to determine the impact seasonal changes had on water quality and microbial diversity. Every quarter for 1 year, water-soil slurries were collected at the influent of the 5 settling ponds and the wetlands, and the effluent of the system. The concentration of 12 metals and sulfate, as well as sequences from the V4 region of the bacterial 16S rrn genes were determined. The water quality analysis revealed high levels of iron and sulfate, and measurable levels of Al, Ba, Cu, Pb, Mn, Sr, and Zn. Iron increased 25-fold in the summer and spikes in metal concentrations were observed during several seasons in pond 3 and the wetlands. These spikes cannot be explained by abiotic chemical reactions in the neutral pH found in the pond. Based on contaminant levels and microbial community composition, our results indicate that there were 3 unique environments in the system (ponds 1 and 2; pond 3; pond 4 through the end) and that changes in contaminant levels and bacterial composition in these environments correlated with seasonal variation. Iron and sulfate are the most prevalent contaminants in the system. An examination of sequences from known iron- and sulfur-cycling bacteria demonstrated that there were more iron-reducing (IRB) bacterial sequences than iron-oxidizing (IOB) (137,912 IRB vs. 98,138 IOB), the two groups of bacteria were found mainly in the fall and winter samples, and were prevalent in different ponds. There were more sulfur/sulfide-oxidizing (SOB) bacterial sequences than sulfur/sulfate-reducing (SRB) bacterial sequences (72,978 SOB vs 30,504 SRB), they were found mainly in the fall and winter samples, and the sequences were mixed in ponds 4, 5 and the wetlands effluent. Iron is remediated in this system but sulfate is not.

Mutations in the E. coli Rep helicase increase the amount of DNA per cell.

The mbrA4 mutation confers camphor resistance, severe growth defects and up to a two-fold increase in the amount of chromosomal DNA per cell. The extra DNA is replicated from oriC in a synchronous fashion. Cells containing mbrA4 are more resistant to X-rays, indicating that the extra DNA represents complete or nearly complete chromosomes. I report here that mbrA4 is an unusual allele of the leading strand DNA helicase, Rep. Eight independently isolated alleles of rep(mbrA) contain the same three changes in the rep gene: a G to A at position -44 from the start of the mRNA (+1); an opal stop at codon 142; and a glycine to serine at codon 414 (G414S). My data indicate that rep(mbrA4) is not a null mutation and that the third mutation, G414S, is necessary for camphor resistance, the phenotype associated with increased DNA content per cell. I also show that increase in DNA content does not lead to independently segregating chromosomes.

Expression of csp genes in E. coli K-12 in defined rich and defined minimal media during normal growth, and after cold-shock.

Cold-shock proteins (Csps) are a family of small nucleic acid-binding proteins found in 72% of sequenced bacterial genomes. Where it has been examined, at least one csp gene is required for cell viability. In Escherichia coli K-12, there are nine homologous csp genes named A-I. Regulation studies performed on individual members of this family have suggested that cspA, cspB, cspG, and cspI are cold-induced, cspC and cspE are constitutively expressed, cspD is stationary phase induced, and the induction patterns for cspF and cspH have yet to be determined. Aside from microarray studies, transcript levels from all nine csp genes have never been assayed using the same technique or in the same cells. The purpose of this study was to use quantitative RT-PCR to establish csp expression patterns for all nine csp genes at 37°C in defined rich and defined minimal media, and after a shift to 15°C for either 1h or 4h. We found that transcript levels for each of the csp genes changed throughout the growth curve. Transcripts for cspA, -B, and -E were more abundant than those detected for the other csp genes in defined rich medium. cspE mRNA levels in defined minimal medium were drastically higher than mRNA for the other csp genes. Of the nine csp genes, only cspI showed a significant increase in mRNA accumulation after cold-shock in defined rich medium. When mRNA accumulation was compared across the nine csp genes, there were more cspE transcripts in the cell than cspA, -B, -G, or -I transcripts after 1h cold-shock in either defined rich or defined minimal media. In defined minimal medium, transcription of cspA, -B, -G, and -I was induced after cold-shock.

GCAT-SEEKquence: genome consortium for active teaching of undergraduates through increased faculty access to next-generation sequencing data.

To transform undergraduate biology education, faculty need to provide opportunities for students to engage in the process of science. The rise of research approaches using next-generation (NextGen) sequencing has been impressive, but incorporation of such approaches into the undergraduate curriculum remains a major challenge. In this paper, we report proceedings of a National Science Foundation-funded workshop held July 11-14, 2011, at Juniata College. The purpose of the workshop was to develop a regional research coordination network for undergraduate biology education (RCN/UBE). The network is collaborating with a genome-sequencing core facility located at Pennsylvania State University (University Park) to enable undergraduate students and faculty at small colleges to access state-of-the-art sequencing technology. We aim to create a database of references, protocols, and raw data related to NextGen sequencing, and to find innovative ways to reduce costs related to sequencing and bioinformatics analysis. It was agreed that our regional network for NextGen sequencing could operate more effectively if it were partnered with the Genome Consortium for Active Teaching (GCAT) as a new arm of that consortium, entitled GCAT-SEEK(quence). This step would also permit the approach to be replicated elsewhere.

In vivo modulation of a DnaJ homolog, CbpA, by CbpM.

CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.

Specificity of DNA binding and dimerization by CspE from Escherichia coli.

The CspE protein from Escherichia coli K12 is a single-stranded nucleic acid-binding protein that plays a role in chromosome condensation in vivo. We report here that CspE binds to single-stranded DNA containing 6 or more contiguous dT residues with high affinity (K(D) < 30 nM). The interactions are predominantly through base-specific contacts. When an oligonucleotide contains fewer than 6 contiguous dT residues, the CspE interactions with single-stranded DNA are primarily electrostatic. The minimal length of single-stranded DNA to which CspE binds in a salt-resistant manner is eight nucleotides. We also show that CspE exists as a dimer in solution. We present a possible mechanism to explain the role of CspE in chromosome condensation in vivo by CspE binding to distant DNA regions in the chromosome and dimerizing, thereby condensing the intervening DNA.

Phenotypic characterization of overexpression or deletion of the Escherichia coli crcA, cspE and crcB genes.

The authors have previously shown that overexpression of the Escherichia coli K-12 crcA, cspE and crcB genes protects the chromosome from decondensation by camphor. In this study they examine the phenotypic consequences of deleting or overexpressing crcA, cspE and crcB. Overexpressing crcA, cspE and crcB increases supercoiling levels of plasmids in wild-type cells and in temperature-sensitive (Ts) gyrase mutants, suppresses the sensitivity of gyrase and topoisomerase IV (topo IV) Ts mutants to nalidixic acid, makes gyrase and topo IV Ts mutants more resistant to camphor and corrects the nucleoid morphology defects in topo IV Ts mutants. Overexpression of crcA, cspE and crcB results in a slight (2.2-fold) activation of the rcsA gene. Deleting crcA, cspE and crcB is not lethal to cells but results in an increase in sensitivity to camphor. Deletion of crcA, cspE and crcB exacerbates the nucleoid morphology defects of the topo IV Ts mutants. When the individual crcA, cspE or crcB genes were tested for their effects on camphor resistance and regulation of rcsA, cspE alone conferred 10-fold camphor resistance and 1.7-fold activation of rcsA. These activities were augmented when crcB was overexpressed with cspE (100-fold camphor resistance and 2.1-fold induction of rcsA).