Naoko Okibe

Enumeration and characterization of acidophilic microorganisms isolated from a pilot plant stirred-tank bioleaching operation

ABSTRACT Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45°C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.

Ferrimicrobium acidiphilum gen. nov., sp. nov. and Ferrithrix thermotolerans gen. nov., sp. nov.: heterotrophic, iron-oxidizing, extremely acidophilic actinobacteria

Two novel extremely acidophilic, iron-oxidizing actinobacteria were isolated, one from a mine site in North Wales, UK (isolate T23(T)), and the other from a geothermal site in Yellowstone National Park, Wyoming, USA (Y005(T)). These new actinobacteria belong to the subclass Acidimicrobidae, and in contrast to the only other classified member of the subclass (Acidimicrobium ferrooxidans), both isolates were obligate heterotrophs. The mine site isolate was mesophilic and grew as small rods, while the Yellowstone isolate was a moderate thermophile and grew as long filaments, forming macroscopic flocs in liquid media. Both isolates accelerated the oxidative dissolution of pyrite in yeast extract-amended cultures, but neither was able to oxidize reduced forms of sulfur. Ferrous iron oxidation enhanced growth yields of the novel mesophilic actinobacterium T23(T), though this was not confirmed for the Yellowstone isolate. Both isolates catalysed the dissimilatory reduction of ferric iron, using glycerol as electron donor, in oxygen-free medium. Based on comparative analyses of base compositions of their chromosomal DNA and of their 16S rRNA gene sequences, the isolates are both distinct from each other and from Acidimicrobium ferrooxidans, and are representatives of two novel genera. The names Ferrimicrobium acidiphilum gen. nov., sp. nov. and Ferrithrix thermotolerans gen. nov., sp. nov. are proposed for the mesophilic and moderately thermophilic isolates, respectively, with the respective type strains T23(T) (=DSM 19497(T)=ATCC BAA-1647(T)) and Y005(T) (=DSM 19514(T)=ATCC BAA-1645(T)).

Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences

Systematic screening of secretion proteins using an approach based on the completely sequenced genome of Corynebacterium glutamicum R revealed 405 candidate signal peptides, 108 of which were able to heterologously secrete an active-form alpha-amylase derived from Geobacillus stearothermophilus. These comprised 90 general secretory (Sec)-type, 10 twin-arginine translocator (Tat)-type and eight Sec-type with presumptive lipobox peptides. Only Sec- and Tat-type signals directed high-efficiency secretion. In two assays, 11 of these signals resulted in 50- to 150-fold increased amounts of secreted alpha-amylase compared with the well-known corynebacterial secretory protein PS2. While the presence of an AXA motif at the cleavage sites was readily apparent, it was the presence of a glutamine residue adjacent to the cleavage site that may affect secretion efficiency.

Toxicity of flotation reagents to moderately thermophilic bioleaching microorganisms

The toxicity of 15 flotation reagents (including xanthates, carbamates, thiophosphates, a mercaptobenzthiazole and a frothing reagent) used for concentrating sulfide minerals to five species of mineral-oxidising, moderately thermophilic and acidophilic microorganisms was assessed. The acidophiles tested included four bacteria (a Leptospirillum isolate, Acidimicrobium ferrooxidans, Acidithiobacillus caldus and a Sulfobacillusisolate) and one archaeon (a Ferroplasma isolate). There was wide variation both in terms of the relative toxicities of the different flotation reagents and the sensitivities of the microorganisms tested. In general, the dithiophosphates and the mercaptobenzothiol were the most toxic, while the Leptospirillum and Ferroplasma isolates were the most sensitive of the acidophilic microorganisms. The significance of these findings, in view of the expanding application of ore concentrates bioprocessing, is discussed.

Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid

Random chemical mutation of a Corynebacterium glutamicum-Escherichia coli shuttle vector derived from plasmid pCGR2 was done using hydroxylamine. It brought about amino acid substitutions G109D and E180K within the replicase superfamily domain of the plasmids RepA protein and rendered the plasmid highly unstable, especially at higher incubation temperatures. Colony formation of C. glutamicum was consequently completely inhibited at 37°C but not at 25°C. G109 is a semi-conserved residue mutation which resulted in major temperature sensitivity. E180 on the other hand is not conserved even among RepA proteins of closely related C. glutamicum pCG1 family plasmids and its independent mutation caused relatively moderate plasmid instability. Nonetheless, simultaneous mutation of both residues was required to achieve temperature-sensitive colony formation. This new pCGR2-derived temperature-sensitive plasmid enabled highly efficient chromosomal integration in a variety of C. glutamicum wild-type strains, proving its usefulness in gene disruption studies. Based on this, an efficient markerless gene replacement system was demonstrated using a selection system incorporating the temperature-sensitive replicon and Bacillus subtilis sacB selection marker, a system hitherto not used in this bacterium. Single-crossover integrants were accurately selected by temperature-dependent manner and 93% of the colonies obtained by the subsequent sucrose selection were successful double-crossover recombinants.

Isolation, evaluation and use of two strong, carbon source-inducible promoters from Corynebacterium glutamicum.

Aims:  To obtain strong, carbon source‐inducible promoters useful for industrial applications of Corynebacterium glutamicum.

“Bioshrouding”—a novel approach for securing reactive mineral tailings

A novel technique (“bioshrouding”) for safeguarding highly reactive sulfidic mineral tailings deposits is proposed. In this, freshly milled wastes are colonised with ferric iron-reducing heterotrophic acidophilic bacteria that form biofilms on reactive mineral surfaces, thereby preventing or minimising colonisation by iron sulfide-oxidising chemolithotrophs such as Acidithiobacillus ferrooxidans and Leptospirillum spp. Data from initial experiments showed that dissolution of pyrite could be reduced by between 57 and 75% by “bioshrouding” the mineral with three different species of heterotrophic acidophiles (Acidiphilium, Acidocella and Acidobacterium spp.), under conditions that were conducive to microbial oxidative dissolution of the iron sulfide.

Gene cloning and characterization of aldehyde dehydrogenase from a petroleum-degrading bacterium, strain HD-1.

The hd-ald gene encoding aldehyde dehydrogenase (hd-ALDH) from an mixotrophic petroleum-degrading bacterium, strain HD-1 was cloned and sequenced. hd-ALDH (506 amino acids) is a member of the NAD+-dependent aldehyde dehydrogenase group. The hd-ald gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically and enzymatically. The molecular weight of the enzyme was estimated to be 55,000 by SDS-PAGE, and 224,000 by gel filtration chromatography, suggesting that it acts as a tetramer. The CD spectrum suggests that the helical content of the enzyme is 10%. hd-ALDH was active on various aliphatic aldehyde substrates. The K(m) values of the enzyme were 6.4 microM for acetaldehyde, 4.2 microM for hexanal, 2.8 microM for octanal, and 0.84 microM for decanal, whereas the kcat values for these substrates were nearly equal (51-64 min(-1)). These results indicate that hd-ALDH acts preferentially on long-chain aliphatic aldehydes.

Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum

pCGR2 and pCG1 belong to different subfamilies of the pCG1 family of Corynebacterium glutamicum plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, crrI and cgrI, respectively. The genes in turn share identical positions complementary to the leader region of their respective repA (encoding plasmid replication initiator) genes. Determination of their precise transcriptional start- and end-points revealed the presence of short antisense RNA molecules (72 bp, CrrI; and 73 bp, CgrI). These short RNAs and their target mRNAs were predicted to form highly structured molecules comprising stem-loops with known U-turn motifs. Abolishing synthesis of CrrI and CgrI by promoter mutagenesis resulted in about sevenfold increase in plasmid copy number on top of an 11-fold (CrrI) and 32-fold (CgrI) increase in repA mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by parB, a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of repA. Simultaneous deactivation of CrrI and parB led to a drastic 87-fold increase in copy number of a pCGR2-derived shuttle vector. Moreover, the fact that changes in the structure of the terminal loops of CrrI and CgrI affected plasmid copy number buttressed the important role of the loop structure in formation of the initial interaction complexes between antisense RNAs and their target mRNAs. Similar antisense RNA control systems are likely to exist not only in the two C. glutamicum pCG1 subfamilies but also in related plasmids across Corynebacterium species.

Concentrate Mineralogy Dictates the Composition of Bioleaching Microbial Consortia

D. Barrie Johnson, Liu Yajie, Naoko Okibe, Kris Coupland and Kevin B. Hallberg School of Biological Sciences, University of Wales, Bangor, U.K. East China Institute of Technology, Fuzhou City, Jiangxi Province, P.R. China Research Institute for Innovative Technology for the Earth, (RITE), Kyoto, Japan d.b.johnson@bangor.ac.uk, lyjwonderful@yahoo.com.cn, okibe@rite.or.jp, mmmgirth@hotmail.com, k.hallberg@bangor.ac.uk