α-fur, an antisense RNA gene to fur in the extreme acidophile Acidithiobacillus ferrooxidans.

A large non-coding RNA, termed α-Fur, of ~1000 nt has been detected in the extreme acidophile Acidithiobacillus ferrooxidans encoded on the antisense strand to the iron-responsive master regulator fur (ferric uptake regulator) gene. A promoter for α-fur was predicted bioinformatically and validated using gene fusion experiments. The promoter is situated within the coding region and in the same sense as proB, potentially encoding a glutamate 5-kinase. The 3' termination site of the α-fur transcript was determined by 3' rapid amplification of cDNA ends to lie 7 nt downstream of the start of transcription of fur. Thus, α-fur is antisense to the complete coding region of fur, including its predicted ribosome-binding site. The genetic context of α-fur is conserved in several members of the genus Acidithiobacillus but not in all acidophiles, indicating that it is monophyletic but not niche specific. It is hypothesized that α-Fur regulates the cellular level of Fur. This is the fourth example of an antisense RNA to fur, although it is the first in an extreme acidophile, and underscores the growing importance of cis-encoded non-coding RNAs as potential regulators involved in the microbial iron-responsive stimulon.

The ferric iron uptake regulator (Fur) from the extreme acidophile Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a Gram-negative bacterium that lives at pH 2 in high concentrations of soluble ferrous and ferric iron, making it an interesting model for understanding the biological mechanisms of bacterial iron uptake and homeostasis in extremely acid conditions. A candidate fur(AF) (Ferric Uptake Regulator) gene was identified in the A. ferrooxidans ATCC 23270 genome. Fur(AF) has significant sequence similarity, including conservation of functional motifs, to known Fur orthologues and exhibits cross-reactivity to Escherichia coli Fur antiserum. The fur(AF) gene is able to complement fur deficiency in E. coli in an iron-responsive manner. Fur(AF) is also able to bind specifically to E. coli Fur regulatory regions (Fur boxes) and to a candidate Fur box from A. ferrooxidans, as judged by electrophoretic mobility shift assays. Fur(AF) represses gene expression from E. coli Fur-responsive promoters fiu and fhuF when expressed at high protein levels. However, it increases gene expression from these promoters at low concentrations and possibly from other Fur-regulated promoters involved in iron-responsive oxidative stress responses.

Apparent redundancy of electron transfer pathways via bc(1) complexes and terminal oxidases in the extremophilic chemolithoautotrophic Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can grow in the presence of either the weak reductant Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+). We have previously shown that the uphill electron transfer pathway between Fe(2+) and NAD(+) involved a bc(1) complex that functions only in the reverse direction [J. Bacteriol. 182, (2000) 3602]. In the present work, we demonstrate both the existence of a bc(1) complex functioning in the forward direction, expressed when the cells are grown on sulfur, and the presence of two terminal oxidases, a bd and a ba(3) type oxidase expressed more in sulfur than in iron-grown cells, besides the cytochrome aa(3) that was found to be expressed only in iron-grown cells. Sulfur-grown cells exhibit a branching point for electron flow at the level of the quinol pool leading on the one hand to a bd type oxidase, and on the other hand to a bc(1)-->ba(3) pathway. We have also demonstrated the presence in the genome of transcriptionally active genes potentially encoding the subunits of a bo(3) type oxidase. A scheme for the electron transfer chains has been established that shows the existence of multiple respiratory routes to a single electron acceptor O(2). Possible reasons for these apparently redundant pathways are discussed.

ISAfe1, an ISL3 family insertion sequence from Acidithiobacillus ferrooxidans ATCC 19859.

A 1.3-kb insertion sequence, termed ISAfe1 (U66426), from Acidithiobacillus ferrooxidans ATCC 19859 is described. ISAfe1 exhibits the features of a typical bacterial insertion sequence. It has 26-bp, imperfectly matched, terminal inverted repeats and an open reading frame (ORF) that potentially encodes a transposase (TPase) of 404 amino acids (AAB07489) with significant similarity to members of the ISL3 family of insertion sequences. A potential ribosome-binding site and potential -10 and -35 promoter sites for the TPase ORF were identified, and a +1 transcriptional start site was detected experimentally. A potential outwardly directed -35 site was identified in the right inverted repeat of ISAfe1. A second ORF (ORF B), of unknown function, was found on the complementary strand with significant similarity to ORF 2 of ISAe1 from Ralstonia eutropha. Southern blot analyses demonstrated that ISAfe1-like elements can be found in multiple copies in a variety of A. ferrooxidans strains and that they exhibit transposition. A codon adaptation index (CAI) analysis of the TPase of ISAfe1 indicates that is has a CAI of 0.726 and can be considered well adapted to its host, suggesting that ISAfe1 might be an ancient resident of A. ferrooxidans. Analysis of six of its target sites of insertion in the genome of A. ferrooxidans ATCC 19859 indicates a preference for 8-bp pseudopalindromic sequences, one of which resembles the termini of its inverted repeats. Evidence is presented here that is consistent with the possibility that ISAfe1 can promote both plasmid cointegrate formation and resolution in E. coli.

IST1 insertional inactivation of the resB gene: implications for phenotypic switching in Thiobacillus ferrooxidans.

Thiobacillus ferroxidans ATCC 19859 undergoes rapid phenotypic switching between a wild-type state characterized by the ability to oxidize ferrous iron (FeII) and reduced sulfur compounds and a mutant state where it has lost the capacity to oxidize FeII but retains the ability to oxidize sulfur. The mutant has also gained the capacity to swarm. It is proposed that loss of FeII oxidation is due to the reversible transposition of the insertion sequence IST1 into resB encoding a putative cytochrome c-type biogenesis protein. Downstream from resB and co-transcribed with it is resC, encoding another putative cytochrome biogenesis protein. IST1 insertional inactivation of resB could result in the loss of activity of its target c-type cytochrome(s). This putative target cytochrome(s) is proposed to be essential for FeII oxidation but not for sulfur oxidation. Curiously, resB and resC pertain to the proposed system II cytochrome biogenesis pathway whereas gamma Proteobacteria, of which T. ferrooxidans is a member, normally use system I. This could represent an example of lateral gene transfer.

Sequence and expression of the rusticyanin structural gene from Thiobacillus ferrooxidans ATCC33020 strain.

The periplasmic blue copper protein rusticyanin is thought to play an important role in iron oxidation by Thiobacillus ferrooxidans. We present the sequence of the gene, rus, encoding rusticyanin together with about 1.4 kb of upstream and 0.3 kb of downstream DNA. The rus gene is unique to T. ferrooxidans. Evidence is presented that it is the last gene of an operon and that it can be transcribed from its own promoter. In ATCC33020 strain, rusticyanin is synthesized in ferrous iron but also in sulfur growth conditions suggesting that it could play a role in both energetic metabolisms. The rus gene transcribed from a vector promoter in Escherichia coli leads to the production of a processed aporusticyanin in the periplasmic space, indicating that its signal sequence is correctly recognized by the secretion machinery and the signal peptidase of E. coli.

Bacterial populations in samples of bioleached copper ore as revealed by analysis of DNA obtained before and after cultivation.

The composition of bacterial populations in copper bioleaching systems was investigated by analysis of DNA obtained either directly from ores or leaching solutions or after laboratory cultures. This analysis consisted of the characterization of the spacer regions between the 16 and 23S genes in the bacterial rRNA genetic loci after PCR amplification. The sizes of the spacer regions, amplified from DNAs obtained from samples, were compared with the sizes of those obtained from cultures of the main bacterial species isolated from bioleaching systems. This allowed a preliminary assessment of the bacterial species present in the samples. Identification of the bacteria was achieved by partial sequencing of the 16S rRNA genes adjacent to the spacer regions. The spacer regions observed in DNA from columns leached at different iron concentrations indicated the presence of a mixture of different bacteria. The spacer region corresponding to Thiobacillus ferrooxidans was the main product observed at high ferrous iron concentration. At low ferrous iron concentration, spacer regions of different lengths, corresponding to Thiobacillus thiooxidans and "Leptospirillum ferrooxidans" were observed. However, T. ferrooxidans appeared to predominate after culture of these samples in medium containing ferrous iron as energy source. Although some of these strains contained singular spacer regions, they belonged within previously described groups of T. ferrooxidans according to the nucleotide sequence of the neighbor 16S rRNA. These results illustrate the bacterial diversity in bioleaching systems and the selective pressure generated by different growth conditions.

Thiobacillus ferrooxidans tyrosyl-tRNA synthetase functions in vivo in Escherichia coli.

The tyrosyl-tRNA synthetase gene (tyrZ) from Thiobacillus ferrooxidans, an acidophilic, autotrophic, gram-negative bacterium that participates in bioleaching of minerals, was cloned and sequenced. The encoded polypeptide (TyrRZ) is 407 amino acids in length (molecular mass; 38 kDa). The predicted protein sequence has an extensive overall identity (44%) to the sequence of the protein encoded by the Bacillus subtilus tyrZ gene, one of the two genes encoding tyrosyl-tRNA synthetases in this microorganism. Alignment with Escherichia coli TyrRS revealed limited overall identity (24%), except in the regions of the signature sequence for class I aminoacyl-tRNA synthetases. Complementation of an E. coli strain with a thermosensitive mutation in TyrRS showed that the protein encoded by the T. ferrooxidans tyrZ gene is functional and recognizes the E. coli tRNA(Tyr) as a substrate. TyrZ is a single-copy gene as revealed by Southern blot analysis. The gene was localized upstream from the putative promoters of the rrnT2 ribosomal RNA operon. Although no rho-independent transcription terminator was found between the two genes, a 1.3-kb RNA hybridized to a DNA probe derived from the tyrZ gene. The functional relationship between these two transcription units is discussed.

Transposition of IST2 in Thiobacillus ferrooxidans.

The genome of Thiobacillus ferrooxidans contains at least two different repetitive DNA elements. One of these elements, termed IST2 has been sequenced and shown to exhibit the characteristics of a typical prokaryotic insertion sequence. Furthermore, preliminary evidence has implicated IST2 in genomic rearrangements, although the mechanism of rearrangement, whether by transposition or recombination, has not been established. In this report we provide evidence from detailed restriction enzyme analyses and DNA sequencing data that support a model of transposition, consistent with the notion that IST2 is a mobile insertion sequence.

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase.

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.

Identification and structural analysis of a ribosomal RNA gene promoter from Thiobacillus ferrooxidans.

The 5'-terminus of a rRNA operon (rrnT2) from Thiobacillus ferrooxidans was characterized. The rRNA promoters from this microorganism were identified by means of a functional assay in Escherichia coli. DNA sequencing of the promoter region, upstream the 16 S rRNA gene, showed the presence of a consensus sequence for bacterial ribosomal promoters. Other features such as a 'discriminator' sequence, antiterminator elements and an upstream hexanucleotide common to several rRNA operons were also found. Two other putative transcription promoters were also identified.

Oxidation of Ferrous Iron and Elemental Sulfur by Thiobacillus ferrooxidans.

The oxidation of ferrous iron and elemental sulfur by Thiobacillus ferrooxidans that was absorbed and unabsorbed onto the surface of sulfur prills was studied. Unadsorbed sulfur-grown cells oxidized ferrous iron at a rate that was 3 to 7 times slower than that of ferrous iron-grown cells, but sulfur-grown cells were able to reach the oxidation rate of the ferrous iron-adapted cells after only 1.5 generations in a medium containing ferrous iron. Bacteria that were adsorbed to sulfur prills oxidized ferrous iron at a rate similar to that of unadsorbed sulfur-grown bacteria. They also showed the enhancement of ferrous iron oxidation activity in the presence of ferrous iron, even though sulfur continued to be available to the bacteria in this case. An increase in the level of rusticyanin together with the enhancement of the ferrous iron oxidation rate were observed in both sulfur-adsorbed and unadsorbed cells. On the other hand, sulfur oxidation by the adsorbed bacteria was not affected by the presence of ferrous iron in the medium. When bacteria that were adsorbed to sulfur prills were grown at a higher pH (ca. 2.5) in the presence of ferrous iron, they rapidly lost both ferrous iron and sulfur oxidation capacities and became inactive, apparently because of the deposition of a jarosite-like precipitate onto the surface to which they were attached.

A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries.

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts on PDE I activated by partial tryptic proteolysis, but the enzyme fully activated by trypsin is only slightly susceptible to inhibition by this protein. The inhibition of PDE I activation caused by this ovarian factor can be reversed by adding excess amounts of calmodulin or lysophosphatidylcholine. The presence of this inhibitor provides a possible explanation for the previously observed inactivity of PDE I in vivo.

Properties of a cyclic nucleotide phosphodiesterase of amphibian oocytes that is activated by calmodulin and calcium, by tryptic proteolysis, and by phospholipids.

A calmodulin-Ca2+-stimulated cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which hydrolyzed both cGMP and cAMP has been purified about 2000-fold from ovaries of the amphibian Xenopus laevis. Gel filtration through Sephadex G-200 indicated a molecular weight of 140,000. A single, major protein band of molecular weight 66,000 was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the stimulation by calmodulin-Ca2+, the enzyme was activated 5- to 10-fold by proteolysis and by certain phospholipids. Trypsin activation of the enzyme caused a reduction in the native molecular weight to 90,000 and a loss of the capacity to be stimulated by calmodulin-Ca2+ or by phospholipids. The phosphodiesterase was stimulated by low concentrations (0.1 microgram/ml) of lysophosphatidylcholine and lysophosphatidylethanolamine. This response did not require calcium ions. Phosphatidylinositol, fatty acids, progesterone, and phospholipase C had little or no effect on activity. Simultaneous addition of 1 mM 2-chloro-10-(3-aminopropyl)phenothiazine and lysophosphatidylcholine to the enzyme did not diminish the stimulatory effect of the phospholipid. The activation of the enzyme by all three agents resulted in an increase in the maximum velocity of the reaction without significant modification of the apparent Km values for cGMP (5 microM) or cAMP (30 microM). It was suggested that trypsin removed an inhibitory domain from the enzyme and that calmodulin and phospholipids interact with this same domain, eliminating its capacity to inhibit the active center of the enzyme.

Mu-1 directed inhibition of DNA breakdown in Escherichia coli, recA cells.

The spontaneous DNA breakdown exhibited by recA strains, is reduced after heat induction of a thermoinducible Mu-1 prophage. This inhibition is dependent upon RNA synthesis, suggesting that Mu-1 directs synthesis of a recBC nuclease inhibitor, analogous to the product of the lambda gam gene. The genetic evidence presented here shows that Mu-1 enables a lambda red gam phage to grow on a recA host. The in vitro assay for ATP-dependent exonuclease activity reveals a complete inhibition of this activity 30 min after induction of the Mu-1 prophage.

Partial purification and characterization of rat liver phenylalanine hydroxylase phosphatase.

A phosphatase which catalyzes the release of 32Pi from 32P-labeled phenylalanine hydroxylase has been purified about 50-fold from rat liver extracts. The phosphatase is able to catalyze the removal of only 70 to 80% of the 32Pi leaving about the same amount of Pi still bound to the hydroxylase as was originally present in the native enzyme. Dephosphorylation is accompanied by a corresponding loss in phenylalanine hydroxylase activity when the activity is measured with the natural cofactor, tetrahydrobiopterin, but not when measured with the synthetic cofactor, 6-methyltetrahydropterin. The partially purified phosphatase has very low activity toward p-nitrophenylphosphate, histone phosphate, and phosphorylase a. The activity toward these substrates has not been purified to the same extent as the phenylalanine hydroxylase phosphatase activity.