Barry M. Grail

Microbiology and Geochemistry of Mine Tailings Amended with Organic Carbon for Passive Treatment of Pore Water

A field-scale experiment was conducted to evaluate organic carbon amendment of mine tailings as a technique for pore water and drainage treatment. Six test cells were constructed by amending sulfide- and carbonate- rich tailings with varied mixtures of peat, spent-brewing grain and municipal biosolids. Samples were collected for microbial, geochemical and mineralogical analysis approximately three years after commencing this experiment. Test cells amended with spent-brewing grain promoted sulfate reduction and effective removal of sulfate and metal(loid)s. The addition of municipal biosolids did not sustain enhanced sulfidogenesis after three years, and peat was an ineffective source of organic carbon. Terminal-restriction fragment length polymorphism revealed that test cells which supported sulfidogenesis exhibited the greatest microbial diversity. Indigenous bacteria identified using molecular and cultivation analyses were found to be related to Cellulomonas, Thiobacillus, Bacteroides, Paludibacter and Desulfovibrio, which was the only sulfate-reducing bacterial (SRB) isolated. The results demonstrate that mixtures of solid organic materials which supported complex anaerobic microbial communities, including sulfate- reducing bacteria, were most effective in promoting pore-water treatment.

Conformational limitations of glycylsarcosine as a prototypic substrate for peptide transporters

Peptide transporters are present in all species to absorb the small peptides that occur ubiquitously as products of proteolysis. The broad substrate specificities of these systems allow them to be exploited therapeutically for delivery of peptidomimetic drugs in microbes and man. To this end, glycylsarcosine is currently used as a standard substrate for assaying peptidomimetic transport by peptide transporters. However, in this study we find it is unsuitable as a general substrate, based on assays of its transport by model bacterial peptide transporters and computer-based conformational analysis of its structure. Of the two generic transporters for di- and tripeptides, exemplified by Dpp and Tpp in Escherichia coli, only Dpp can transport glycylsarcosine. The explanation for this finding came from molecular modelling, which indicated that glycylsarcosine can adopt only a restricted range of conformers compared with typical dipeptides, and that of the conformers with a trans peptide bond, the majority have the specific psi and phi backbone torsion angles needed for molecular recognition and transport by Dpp but none possessed psi and phi torsions required for recognition by Tpp; moreover, 38% of its conformers have cis peptide bonds that are not substrates for any peptide transporter. Thus, using glycylsarcosine as substrate in competition assays with compounds that typically form conformers recognised by both types of peptide transporter will underestimate their transport. These findings have implications for assays of oral availability of peptidomimetic drugs such as beta-lactams, ACE inhibitors and anti-viral compounds, for which glycylsarcosine is routinely used.

Draft Genome Sequence of the Nominated Type Strain of "Ferrovum myxofaciens," an Acidophilic, Iron-Oxidizing Betaproteobacterium.

ABSTRACT “Ferrovum myxofaciens” is an iron-oxidizing betaproteobacterium with widespread distribution in acidic low-temperature environments, such as acid mine drainage streams. Here, we describe the genomic features of this novel acidophile and investigate the relevant metabolic pathways that enable its survival in these environments.

Predominant torsional forms adopted by dipeptide conformers in solution: parameters for molecular recognition.

In this paper, we describe the predominant conformational forms adopted by tripeptides and higher oligopeptides in aqueous solution. About 50 tripeptides and almost 20 higher oligopeptides (4–6 residues) were subjected to conformational analysis using SYBYL Random Search. As with dipeptides (Grail BM, Payne JW. J. Peptide Sci. 2000; 6: 186–199), both tripeptides and higher oligopeptides were found to occupy relatively few combinations of psi–phi space that were distinct from those associated with predominant protein secondary structures (e.g. helices and β‐sheets). Again, the preferred psi (ψ) values for the first residue (i−1) were in sectors encompassed by the ranges from +150° to ±180°, +60° to +90° and −60° to −90°, which were combined with preferred phi (ϕ) values for the second residue (i) in sectors with ranges from −150° to ±180°, −60° to −90° and +30° to +60°. It was notable that tripeptides and, to a greater extent, higher oligopeptides adopted an initial psi (ψ) (Tor2) from +150° to ±180°. For tripeptides, their N–C distances (distance between N‐terminal nitrogen and C‐terminal carbon atoms) distribute about 6.5 Å to give shorter, ‘folded’ conformers that are similar in length to dipeptides, and longer, ‘extended’ conformers that are distinct. Furthermore, for higher oligopeptides, their N–C distances did not increment in relation to their increasing number of residues and short, ‘folded’ conformers were still present. These findings have a bearing upon the recognition of these molecules as substrates for widely distributed peptidases and peptide transporters. Copyright

Draft Genome Sequence of "Acidibacillus ferrooxidans" ITV01, a Novel Acidophilic Firmicute Isolated from a Chalcopyrite Mine Drainage Site in Brazil.

ABSTRACT Here, we report the draft genome sequence of “Acidibacillus ferrooxidans” strain ITV01, a ferrous iron- and sulfide-mineral-oxidizing, obligate heterotrophic, and acidophilic bacterium affiliated with the phylum Firmicutes. Strain ITV01 was isolated from neutral drainage from a low-grade chalcopyrite from a mine in northern Brazil.

Column Bioleaching of a Saline, Calcareous Copper Sulfide Ore

“Deep in situ biomining”, widely considered to be a potentially environmentally-benign and cost effective biotechnology for extracting and recovering base metals from deep-buried base metal deposits, is being developed within the EU Horizon 2020 project “BioMOre”. Data are presented from non-aerated column experiments in which a saline, calcareous copper-rich ore (kupferschiefer) was subjected to a three-stage eaching protocol: (i) with water, to remove soluble salts; (ii) with sulfuric acid, to remove calcareous minerals and other acid-soluble salts; (iii) indirect bioleaching with a microbiologically-generated ferric iron lixiviant. Sequential leaching with water and acid removed ~85% of the chloride prior to bio-processing, while ~13% of the copper present in the ore was leached using sulfuric acid, and a further 39 - 59% by the lixiviant.

Identification of trehalose as a compatible solute in different species of acidophilic bacteria

The major industrial heap bioleaching processes are located in desert regions (mainly Chile and Australia) where fresh water is scarce and the use of resources with low water activity becomes an attractive alternative. However, in spite of the importance of the microbial populations involved in these processes, little is known about their response or adaptation to osmotic stress. In order to investigate the response to osmotic stress in these microorganisms, six species of acidophilic bacteria were grown at elevated osmotic strength in liquid media, and the compatible solutes synthesised were identified using ion chromatography and MALDI-TOF mass spectrometry. Trehalose was identified as one of, or the sole, compatible solute in all species and strains, apart from Acidithiobacillus thiooxidans where glucose and proline levels increased at elevated osmotic potentials. Several other potential compatible solutes were tentatively identified by MALDITOF analysis. The same compatible solutes were produced by these bacteria regardless of the salt used to produce the osmotic stress. The results correlate with data from sequenced genomes which confirm that many chemolithotrophic and heterotrophic acidophiles possess genes for trehalose synthesis. This is the first report to identify and quantify compatible solutes in acidophilic bacteria that have important roles in biomining technologies.

Salt Stress-Induced Loss of Iron Oxidoreduction Activities and Reacquisition of That Phenotype Depend on rus Operon Transcription in Acidithiobacillus ferridurans

ABSTRACT The type strain of the mineral-oxidizing acidophilic bacterium Acidithiobacillus ferridurans was grown in liquid medium containing elevated concentrations of sodium chloride with hydrogen as electron donor. While it became more tolerant to chloride, after about 1 year, the salt-stressed acidophile was found to have lost its ability to oxidize iron, though not sulfur or hydrogen. Detailed molecular examination revealed that this was due to an insertion sequence, ISAfd1, which belongs to the ISPepr1 subgroup of the IS4 family, having been inserted downstream of the two promoters PI and PII of the rus operon (which codes for the iron oxidation pathway in this acidophile), thereby preventing its transcription. The ability to oxidize iron was regained on protracted incubation of the culture inoculated onto salt-free solid medium containing ferrous iron and incubated under hydrogen. Two revertant strains were obtained. In one, the insertion sequence ISAfd1 had been excised, leaving an 11-bp signature, while in the other an ∼2,500-bp insertion sequence (belonging to the IS66 family) was detected in the downstream inverted repeat of ISAfd1. The transcriptional start site of the rus operon in the second revertant strain was downstream of the two ISs, due to the creation of a new “hybrid” promoter. The loss and subsequent regaining of the ability of A. ferriduransT to reduce ferric iron were concurrent with those observed for ferrous iron oxidation, suggesting that these two traits are closely linked in this acidophile. IMPORTANCE Iron-oxidizing acidophilic bacteria have primary roles in the oxidative dissolution of sulfide minerals, a process that underpins commercial mineral-processing biotechnologies (“biomining”). Most of these prokaryotes have relatively low tolerance to chloride, which limits their activities when only saline or brackish waters are available. The study showed that it was possible to adapt a typical iron-oxidizing acidophile to grow in the presence of salt concentrations similar to those in seawater, but in so doing they lost their ability to oxidize iron, though not sulfur or hydrogen. The bacterium regained its capacity for oxidizing iron when the salt stress was removed but simultaneously reverted to tolerating lower concentrations of salt. These results suggest that the bacteria that have the main roles in biomining operations could survive but become ineffective in cases where saline or brackish waters are used for irrigation.

Comparative Genome Analysis Provides Insights into Both the Lifestyle of Acidithiobacillus ferrivorans Strain CF27 and the Chimeric Nature of the Iron-Oxidizing Acidithiobacilli Genomes

The iron-oxidizing species Acidithiobacillus ferrivorans is one of few acidophiles able to oxidize ferrous iron and reduced inorganic sulfur compounds at low temperatures (<10°C). To complete the genome of At. ferrivorans strain CF27, new sequences were generated, and an update assembly and functional annotation were undertaken, followed by a comparative analysis with other Acidithiobacillus species whose genomes are publically available. The At. ferrivorans CF27 genome comprises a 3,409,655 bp chromosome and a 46,453 bp plasmid. At. ferrivorans CF27 possesses genes allowing its adaptation to cold, metal(loid)-rich environments, as well as others that enable it to sense environmental changes, allowing At. ferrivorans CF27 to escape hostile conditions and to move toward favorable locations. Interestingly, the genome of At. ferrivorans CF27 exhibits a large number of genomic islands (mostly containing genes of unknown function), suggesting that a large number of genes has been acquired by horizontal gene transfer over time. Furthermore, several genes specific to At. ferrivorans CF27 have been identified that could be responsible for the phenotypic differences of this strain compared to other Acidithiobacillus species. Most genes located inside At. ferrivorans CF27-specific gene clusters which have been analyzed were expressed by both ferrous iron-grown and sulfur-attached cells, indicating that they are not pseudogenes and may play a role in both situations. Analysis of the taxonomic composition of genomes of the Acidithiobacillia infers that they are chimeric in nature, supporting the premise that they belong to a particular taxonomic class, distinct to other proteobacterial subgroups.

Draft Genome Sequence of a Novel Acidophilic Iron-Oxidizing Firmicutes Species, “Acidibacillus ferrooxidans” (SLC66T)

ABSTRACT Here, we present the draft genome sequence of the type strain of “Acidibacillus ferrooxidans,” a mesophilic, heterotrophic, and acidophilic bacterium that was isolated from mine spoilage subjected to accelerated weathering in humidity cell tests carried out by the former U.S. Bureau of Mines in Salt Lake City, UT.