Characterization and identification of an iron-oxidizing,Leptospirillum-like bacterium,present in the high sulfate leaching solution of a commercial bioleaching plant

Most copper bioleaching plants operate with a high concentration of sulfate salts, caused by the continuous addition of sulfuric acid and the recycling of the leaching solution. Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population present in the high-sulfate (150 gl(-1)) leaching solution, employed in a copper production plant, was investigated. The iron-oxidizing bacteria able to grow in the leaching solution were enriched by several batch cultivations and, after serial dilution, an abundant bacterial strain was isolated. This strain, called LA, exhibited a relatively constant rate of iron-oxidation in media containing sulfate ions at concentrations ranging from 10 to 150 gl(-1). Culture collection strains of Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans showed limited abilities to grow at sulfate ion concentrations higher than 70 gl(-1). In spite of its tolerance to high sulfate concentrations, strain LA was as sensitive to NaCl as A. ferrooxidans. Comparative sequence analysis of the 16S rRNA gene of strain LA indicated that it is phylogenetically related to strains described as Leptospirillum ferrooxidans. Bacterial community DNA restriction patterns of 16S rRNA genes suggested that strain LA was a minor component of the bacterial population present in leaching solution, but is abundant in ore leached with this solution.     

Differentiation of Acidithiobacillus ferrooxidans and A. thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli.     

Extracellular polymeric substances mediate bioleaching/biocorrosion via interfacial processes involving iron(III) ions and acidophilic bacteria

Extracellular polymeric substances seem to play a pivotal role in biocorrosion of metals and bioleaching, biocorrosion of metal sulfides for the winning of precious metals as well as acid rock drainage. For better control of both processes, the structure and function of extracellular polymeric substances of corrosion-causing or leaching bacteria are of crucial importance. Our research focused on the extremophilic bacteria Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, because of the "simplicity" and knowledge about the interactions of these bacteria with their substrate/substratum and their environment. For this purpose, the composition of the corresponding extracellular polymeric substances and their functions were analyzed. The extracellular polymeric substances of both species consist mainly of neutral sugars and lipids. The functions of the exopolymers seem to be: (i) to mediate attachment to a (metal) sulfide surface, and (ii) to concentrate iron(III) ions by complexation through uronic acids or other residues at the mineral surface, thus, allowing an oxidative attack on the sulfide. Consequently, dissolution of the metal sulfide is enhanced, which may result in an acceleration of 20- to 100-fold of the bioleaching process over chemical leaching. Experiments were performed to elucidate the importance of the iron(III) ions complexed by extracellular polymeric substances for strain-specific differences in oxidative activity for pyrite. Strains of A. ferrooxidans with a high amount of iron(III) ions in their extracellular polymeric substances possess greater oxidation activity than those with fewer iron(III) ions. These data provide insight into the function of and consequently the advantages that extracellular polymeric substances provide to bacteria. The role of extracellular polymeric substances for attachment under the conditions of a space station and resulting effects like biofouling, biocorrosion, malodorous gases, etc. will be discussed.     

Micro-Raman analysis and AFM imaging of Acidithiobacillus ferrooxidans biofilm grown on uranium ore

Acidithiobacillus ferrooxidans biofilm grown on uranium ore substrate was analyzed by a micro-Raman spectrometer and an atomic force microscope (AFM). The bacterium employed for this study, A. ferrooxidans BM1, was isolated from a uranium mine (Jaduguda, India). Micro-Raman analysis revealed the different constituents of molecular fragments present in microbial cells and in secreted extracellular polymeric substances (EPSs). AFM images clearly revealed bacterial cells surrounded by EPS. From Raman spectral data, the composition of EPS from A. ferrooxidans BM1 appeared to be similar to that of EPS secreted in a different Pseudomonas bacterium.     

Structure and function of the B and D genes of the Actinobacillus actinomycetemcomitans leukotoxin complex

The Actinobacillus actinomycetemcomitans leukotoxin gene complex, consisting of four genes, has been cloned and the sequence of the AaLtC and AaLtA genes reported. The present paper details the sequences of the AaLtB and AaLtD genes which, like AaLtC and AaLTA, are also homologues of genes found in other cytolytic toxin complexes of several other Gram-negative bacterial pathogens. When tested in a recombinant expression system, the AaLtB and/or AaLtD genes are required for the translocation and insertion of the A. actinomycetemcomitans leukotoxin (AaLtA) into the cell membrane of Escherichia coli.     

The interferon system of non-mammalian vertebrates

Genetic analysis in mice and other mammals showed that the interferon (IFN) system is important for resistance to viral and bacterial infections. IFN is thought to play a similar role in non-mammalian species, although knowledge is lagging behind. Molecular cloning revealed low but significant conservation of IFN genes in birds and fish. Whereas chickens and other birds have multiple genes for IFN-alpha and single genes for IFN-beta and IFN-gamma like most mammals, fish appear to have fewer IFN genes. Nonetheless, regulation of IFN-alpha and IFN-beta genes in response to viral and non-viral activators is well conserved in birds and fish, and similar sets of genes are induced in response to IFN in mammalian and non-mammalian vertebrates. IFN-like activities were also observed in reptiles and amphibians, but genes encoding these activities have not yet been cloned. Insights into the IFN system of non-mammalian species may lead to improved prophylactic and therapeutic approaches against virus infections in poultry and fish.     

Changes in gene expression profiles in developing B cells of murine bone marrow.

Gene expression profiles of five consecutive stages of mouse B cell development were generated with high-density oligonucleotide arrays from as few as 2 x 10(4) ex vivo isolated and flow-cytometrically purified cells. Between 2.8% and 6.8% of all genes change on differentiation from one cellular stage to the next by at least twofold. The entire pathway involves differential expression of 10.7% of all genes. Previously known expression patterns of 15 genes (like surrogate light chain, RAG-1/2, MHC class II, mel-14 antigen) are confirmed. The gene expression patterns of the proliferating pre-BI and large pre-BII cells on the one hand, and the resting immature and mature B cells on the other hand, are most similar to each other. Small pre-BII cells display a pattern that is transitional between these two groups. Most of the genes expressed in early precursors are involved in general processes, like protein folding or cell cycle regulation, whereas more mature precursors express genes involved in more specific molecular programs (cell surface receptors, secreted factors, and adhesion molecules, among others). Between 19 and 139 genes share a given expression pattern. Combining knowledge about gene function and expression pattern allows identification of novel candidate genes potentially involved in self-maintenance of pre-BI cells, allelic exclusion and pre-B cell receptor signaling in large pre BII cells, cell-cycle arrest of small pre-BII cells, propensity toward apoptosis or anergization in immature B cells, propensity toward cell division and activation in mature B cells, and stage-specific interactions with stromal cells in the bone marrow.     

Four twist genes in zebrafish, four expression patterns.

Twist genes code for regulatory bHLH proteins essential for embryonic development and conserved across the metazoa. There are four genes that constitute the zebrafish twist family: twist1a, twist1b, twist2--orthologs of the mammalian twist1 and twist2 genes; and twist3--a gene from a new clade that does not exist in mammals. Presented here are their embryonic mRNA expression profiles. The study extends the known conservation of twist developmental patterns in tetrapods to the fish, e.g., expression in cephalic neural crest, sclerotome and lateral plate mesoderm. Some other expression domains are unique, like hypochord and dorsal aorta; some, like the notochord, may be ancestral patterns retained from protochordates; and the expression in invaginating/migrating cells may have been retained from the jellyfish. Perhaps this is one of the more ancient functions of twist--conserved from diploblasts to humans--to facilitate cell movement.     

Amino-acid substitutions in the zinc finger of NIT2, the nitrogen regulatory protein of Neurospora crassa,alter promoter element recognition

NIT2, the major nitrogen regulatory protein of Neurospora crassa mediates nitrogen catabolite derepression of the structural genes which specify enzymes of nitrogen catabolism. The promoter of the structural gene for L-amino acid oxidase, a nitrogen-regulated enzyme, was found to contain two NIT2 binding sites, each with two copies of a GATA core consensus sequence. Site-directed mutagenesis was employed to create amino-acid substitutions within the single zinc-finger region of NIT2, which serves as the DNA-binding domain. The affect of those mutations upon NIT2 function in vivo in the activation of three separate structural genes was examined by transformation assays and relevant enzyme activities, and DNA-binding activity in vitro was determined by gel band mobility-shift assays. It was shown that specific amino-acid residues within the zinc-finger loop region of NIT2 are important for DNA-binding activity, whereas other residues influence the specificity of DNA binding. Mutant NIT2 proteins were obtained which retain DNA-binding activity and alter the specificity of DNA recognition, thus allowing a distinction between related DNA elements.     

The major histocompatibility complex in swine

Summary: In swine, the major histocompatibility complex ( Mhc ) or swine leukocyte antigen ( SLA ) is located on chromosome 7 and divided by the centromere. Thus, the telomeric class I and more centromeric class III regions are located on the p arm and the class II region is located on the q arm. The SLA region spans about 2 Mb, in which more than 70 genes have so far been characterized. Despite its division by the centromere, the spatial relationships between the genes in the class II and class III regions, and between the well-conserved non-class I genes of the class I region, are similar to those found in the human HLA complex. On the other hand, no orthologous relationships have been found between the Mhc class I genes in man and swine. In swine, the 12 SLA class I sequences constitute two distinct clusters. One chister comprises six classical class 1-related sequences, while the other comprises five class I-distantly related sequences including two swine homologous genes of the HLA Mhc class I chain-related gene ( MIC ) sequence family. The number of functional SLA classical class I genes, as defined by serology, probably varies from one to four, depending on the haplotype. Some of the SLA class I-distantly related sequences are clearly transcribed. As regards the SLA class II genes, some of them clearly code for at least one functional SLA-DR and one SLA-DQ heterodimer product, but none code for any DP product. The amino acid alignment of the variable domains of 33 SLA classical class I chains, and 62 DRβ and 20 DQβ chains confirmed the exceptionally polymorphic pattern of these polypeptides. Among the class II genes, the genes are either monomorphic, like the DRA gene, or oligomorphic, like the DQA genes. In contrast, the DRB and DQB genes display considerable polymorphism, which seems more marked in DRB than DQB genes.     

Insights into the pathways of iron- and sulfur-oxidation,and biofilm formation from the chemolithotrophic acidophile Acidithiobacillus ferrivorans CF27

The iron-oxidizing acidithiobacilli cluster into at least four groups, three of which (Acidithiobacillus ferrooxidans, Acidithiobacillus ferridurans and Acidithiobacillus ferrivorans) have been designated as separate species. While these have many physiological traits in common, they differ in some phenotypic characteristics including motility, and pH and temperature minima. In contrast to At. ferrooxidans and At. ferridurans, all At. ferrivorans strains analysed to date possess the iro gene (encoding an iron oxidase) and, with the exception of strain CF27, the rusB gene encoding an iso-rusticyanin whose exact function is uncertain. Strain CF27 differs from other acidithiobacilli by its marked propensity to form macroscopic biofilms in liquid media. To identify the genetic determinants responsible for the oxidation of ferrous iron and sulfur and for the formation of extracellular polymeric substances, the genome of At. ferrivorans CF27 strain was sequenced and comparative genomic studies carried out with other Acidithiobacillus spp.. Genetic disparities were detected that indicate possible differences in ferrous iron and reduced inorganic sulfur compounds oxidation pathways among iron-oxidizing acidithiobacilli. In addition, strain CF27 is the only sequenced Acidithiobacillus spp. to possess genes involved in the biosynthesis of fucose, a sugar known to confer high thickening and flocculating properties to extracellular polymeric substances.     

Genetic and evolutional mechanisms explain associated malformations--a 'G-E-M' concept

During the process of evolution, segmented structures like heart and kidneys appeared earlier than vertebral column. Single piece notochord was transformed into segmented vertebral column. Ribs followed the segmented vertebral column but preceded the fore limbs in evolution. Segmentation is the underlying principle of the body plan even in annelids and arthropods. In these animals apart from vertebral column; segmentation is obvious in other structures like kidneys and heart. Somewhere on the temporal axis of evolution--vertebral column, heart and kidneys have evolved together; and have shared the genetic control of embryological morphogenesis. Mutations or micro-evolution in homeotic--Hox--genes led to macro evolution and a sudden change in morphology, when six-legged insects diverged from crustacean-like arthropod ancestors with multiple limbs. The control of embryonic morphology has been highly conserved in evolution between vertebrates and invertebrates and Hox genes occupy a central role in the scheme of molecular control of early morphogenesis. Mutations affecting regulatory genes, including those containing homeobox sequences, have been important. Malformations and association like VACTERL can be rationally explained considering the genetic and evolutional mechanisms controlling morphogenesis.