The extremophile Acidithiobacillus ferrooxidans possesses a c-di-GMP signalling pathway that could play a significant role during bioleaching of minerals.

AIMS: The primary goal of this study was to characterize the existence of a functional c-di-GMP pathway in the bioleaching bacterium Acidithiobacillus ferrooxidans. METHODS AND RESULTS: A bioinformatic search revealed that the genome sequence of At. ferrooxidans ATCC 23270 codes for several proteins involved in the c-di-GMP pathway, including diguanylate cyclases (DGC), phosphodiesterases and PilZ effector proteins. Overexpression in Escherichia coli demonstrated that four At. ferrooxidans genes code for proteins containing GGDEF/EAL domains with functional DGC activity. MS/MS analysis allowed the identification of c-di-GMP in nucleotide preparations obtained from At. ferrooxidans cells. In addition, c-di-GMP levels in cells grown on the surface of solid energetic substrates such as sulfur prills or pyrite were higher than those measured in ferrous iron planktonic cells. CONCLUSIONS: At. ferrooxidans possesses a functional c-di-GMP pathway that could play a key role in At. ferrooxidans biofilm formation during bioleaching processes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first global study about the c-di-GMP pathway in an acidophilic bacterium of great interest for the biomining industry. It opens a new way to explore the regulation of biofilm formation by biomining micro-organisms during the bioleaching process.

High copper concentration reduces biofilm formation in Acidithiobacillus ferrooxidans by decreasing production of extracellular polymeric substances and its adherence to elemental sulfur.

Acidithiobacillus ferrooxidans is an acidophilic bacterium able to grow in environments with high concentrations of metals. It is a chemolithoautotroph able to form biofilms on the surface of solid minerals to obtain its energy. The response of both planktonic and sessile cells of A. ferrooxidans ATCC 23270 grown in elemental sulfur and adapted to high copper concentration was analyzed by quantitative proteomics. It was found that 137 proteins varied their abundance when comparing both lifestyles. Copper effllux proteins, some subunits of the ATP synthase complex, porins, and proteins involved in cell wall modification increased their abundance in copper-adapted sessile lifestyle cells. On the other hand, planktonic copper-adapted cells showed increased levels of proteins such as: cupreredoxins involved in copper cell sequestration, some proteins related to sulfur metabolism, those involved in biosynthesis and transport of lipopolysaccharides, and in assembly of type IV pili. During copper adaptation a decreased formation of biofilms was measured as determined by epifluorescence microscopy. This was apparently due not only to a diminished number of sessile cells but also to their exopolysaccharides production. This is the first study showing that copper, a prevalent metal in biomining environments causes dispersion of A. ferrooxidans biofilms. SIGNIFICANCE: Copper is a metal frequently found in high concentrations at mining environments inhabitated by acidophilic microorganisms. Copper resistance determinants of A. ferrooxidans have been previously studied in planktonic cells. Although biofilms are recurrent in these types of environments, the effect of copper on their formation has not been studied so far. The results obtained indicate that high concentrations of copper reduce the capacity of A. ferrooxidans ATCC 23270 to form biofilms on sulfur. These findings may be relevant to consider for a bacterium widely used in copper bioleaching processes.

In vitro methylation of the elongation factor EF-Tu from Escherichia coli.

The in vitro methylation of the elongation factor EF-Tu from Escherichia coli was investigated. The methylation of newly synthesized EF-Tu was obtained using lambda rifd 18 DNA as template and S-adenosyl [methyl-3H]methionine as methyl donor. About 3 mol methyl residues were incorporated for every 10 mol EF-Tu synthesized. Analysis of the nature of the methyl-containing residues by protein hydrolysis followed by paper chromatography showed that both mono- and dimethyllysine were present. The methylation of EF-Tu was also studied separately from its synthesis by using cell-free systems with artificially undermethylated components.

Phosphate-starvation induced changes in Thiobacillus ferrooxidans.

We have analysed the response of the acidophilic chemolithotroph Thiobacillus ferrooxidans to phosphate starvation. Cultivation of the bacteria in the absence of added phosphate induced a remarkable filamentation of the cells. Polyacrylamide gel electrophoresis revealed several proteins whose levels increased upon phosphate limitation, as well as some polypeptides that were exclusively synthesized under this growth limitation. One of the proteins whose level increased by the lack of phosphate was apparently an acid phosphatase with a pH optimum of about 3.8, and a molecular mass of 26 kDa, which was located in the periplasm. The N-terminal sequence of a 26 kDa protein derepressed by starvation, which may correspond to the T. ferrooxidans starvation, which may correspond to the T. ferrooxidans phosphatase, showed 30% and 35% identity with the known sequence of Lysobacter enzymogenes and Escherichia coli alkaline phosphatases, respectively.

Identification and characterization of GroEL and DnaK homologues in Thiobacillus ferrooxidans.

The major heat shock proteins from Thiobacillus ferrooxidans were identified as DnaK and GroEL equivalents by Western blotting and analysis of the N-terminal amino acid sequence of spots isolated from dried 2-D polyacrylamide electrophoresis gels. The T. ferrooxidans chaperonins showed 70% and 80% identity with the Escherichia coli GroEL and DnaK, respectively. By using electrophoresis with a transverse pore gradient of cross-linked polyacrylamide and nondenaturing conditions followed by Western blotting, we found that the GroEL proteins from both bacteria formed a 14-mer, whereas E. coli DnaK protein existed partially as a dimer and the T. ferrooxidans DnaK-equivalent showed only a monomeric nature under our experimental conditions.

In vivo and in vitro methylation of the elongation factor EF-Tu from Euglena gracilis chloroplast.

Based on amino acid sequence similarities between the methylated elongation factor EF-Tu from Escherichia coli and the EF-Tu from Euglena gracilis chloroplast, we predicted that the latter could also be methylated in the presence of an appropriate methyltransferase. We found that, as reported for the eubacterial homologous protein, the organellar factor could be methylated in vivo and in vitro to yield monomethyllysine.

Phosphorylation of GroEL, DnaK and other proteins from Thiobacillus ferrooxidans grown under different conditions.

The levels of phosphorylation of the chaperones DnaK and GroEL and other proteins varied when cells of Thiobacillus ferrooxidans were subjected to phosphate starvation. The phosphorylated amino acid of GroEL was found to be threonine. Our results show that not only heat shock, but also a nutrient starvation stress leads to phosphorylation of chaperones and, in addition, support the possible role of phosphorylation of these proteins in the sensing and regulation of stress responses in bacteria.

Phosphate starvation affects the synthesis of outer membrane proteins in Thiobacillus ferrooxidans.

The outer membrane protein (omp40) component from the chemolithoautotrophic acidophilic Thiobacillus ferrooxidans is apparently regulated by the external pH and the concentration of phosphorus. Its amino-terminal sequence showed little identity with the Escherichia coli OmpC, OmpF or PhoE porins, but was 38.5% identical to the outer membrane channel-forming protein NosA from Pseudomonas stutzeri, whose expression is also regulated environmentally. In addition, the partial amino acid sequence of T. ferrooxidans omp40 showed between 34 and 38% identity with the amino-terminal end of the small outer membrane proteins Rck and PagC from Salmonella typhimurium and OmpX from Enterobacter cloacae.

Chemotaxis of Leptospirillum ferrooxidans and other acidophilic chemolithotrophs: comparison with the Escherichia coli chemosensory system.

Ni2+, Fe2+ and Cu2+ were attractants and aspartate was an apparent repellent for Leptospirillum ferrooxidans, a behaviour opposite to that for Escherichia coli. Membranes from L. ferrooxidans contained proteins with a molecular mass in the range of 80 kDa which were methylated in vitro. Methylation was stimulated in the presence of a membrane-free extract from E. coli, showing the response pattern expected for L. ferrooxidans, increased methylation by Ni2+, and demethylation by aspartate. This suggests the existence of sensory transducers having a common methylation domain with the E. coli methyl-accepting chemotaxis proteins. Total chromosomal DNA digests from L. ferrooxidans, Thiobacillus ferrooxidans and T. thiooxidans hybridized with probes containing different domains of the tar gene from E. coli, implying the presence of tar type genes in the acidophilic bacteria studied.

Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.

Immunological methods for the quantitation of the industrially important bioleaching microorganisms.

To monitor the levels of Thiobacillus ferrooxidans in bioleaching operations, we have developed a specific and very sensitive dot-immunobinding assay. Polyclonal antisera against whole T. ferrooxidans cells was used, and the bacteria-antibody reaction was visualized by employing either 125I-labeled or peroxidase-conjugated protein A or 125I-labeled or peroxidase-conjugated goat anti-rabbit immunoglobulin G. A minimum of 10(3) cells per dot could be easily detected. Therefore, the method allows the sensitive, and specific simultaneous processing of numerous samples in a short time.

Methylation of ribosomal proteins in bacteria: evidence of conserved modification of the eubacterial 50S subunit.

Methylation of the 50S ribosomal proteins from Bacillus stearothermophilus, Bacillus subtilis, Alteromonas espejiana, and Halobacterium cutirubrum was measured after the cells were grown in the presence of [1-14C]methionine or [methyl-3H]methionine or both. Two-dimensional polyacrylamide gel electrophoretic analysis revealed, in general, similar relative electrophoretic mobilities of the methylated proteins from each eubacterium studied. Proteins known to be structurally and functionally homologous in several microorganisms were all methylated. Thus, the following group of proteins, which appear to be involved in peptidyltransferase or in polyphenylalanine-synthesizing activity in B. stearothermophilus (P.E. Auron and S. R. Fahnestock, J. Biol. Chem. 256:10105-10110, 1981), were methylated (possible Escherichia coli methylated homologs are indicated in parentheses): BTL5(EL5), BTL6(EL3), BTL8(EL10), BTL11(EL11), BTL13(EL7L12) and BTL20b(EL16). In addition, the pentameric ribosomal complex BTL13 X BTL8, analogous to the complex EL7L12 X EL10 of E. coli, contained methylated proteins. Analysis of the methylated amino acids in the most heavily methylated proteins, BSL11 from B. subtilis and BTL11 from B. stearothermophilus, showed the presence of epsilon-N-trimethyllysine as the major methylated amino acid in both proteins, in agreement with known data for E. coli. In addition, BSL11 appeared to contain trimethylalanine, a characteristic, modified amino acid previously described only in EL11 from E. coli. These results and those previously obtained from other bacteria indicate a high degree of conservation for ribosomal protein methylation and suggest an important, albeit unknown, role for the modification of these components in eubacterial ribosomes.

Methylation of ribosomal proteins in Bacillus subtilis.

We measured the methylation of ribosomal proteins from the 30S and 50S subunits of Bacillus subtilis after growing the cells in the presence of [1-14C]methionine and [methyl-3H]methionine. Two-dimensional polyacrylamide gel electrophoretic analysis revealed a preferential methylation of the 50S ribosomal proteins. Proteins L11 and L16, and possibly L9, L10, L18, and L20, were methylated. On the other hand, only two possibly methylated proteins were found on the 30S subunit. A comparison of these results with those for Escherichia coli suggests a common methylation pattern for the bacterial ribosomal proteins.

Partial Removal of Lipopolysaccharide from Thiobacillus ferrooxidans Affects Its Adhesion to Solids.

Conditions for the partial removal of lipopolysaccharide (LPS) from Thiobacillus ferrooxidans are described. Raising the pH of the solution containing the cells from pH 1.5 to pH 6.8 to 8.0 releases about 50% of the LPS without cell lysis. The release of LPS begins at pH 3.5, and it was not affected by EDTA concentration. Partial removal of LPS exposed higher amounts of a 40-kDa outer membrane protein in the bacteria, as detected by a dot immunoassay employing an antiserum against the T. ferrooxidans surface protein. This higher protein exposure and the reduced LPS content increased the hydrophobicity of the cell surface, as determined by an increased adhesion (50%) to hydrophobic sulfur prills and C-dodecanoic acid binding (2.5-fold) compared with control cells. In addition, adhesion of radioactively labeled microorganisms to a sulfide mineral was inhibited (40%) in the presence of previously added LPS. Our results suggest that not only LPS but also surface proteins probably play important roles in T. ferrooxidans adhesion to solid surfaces.

The glycogen-bound polyphosphate kinase from Sulfolobus acidocaldarius is actually a glycogen synthase.

Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya, Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162-5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.

Molecular cloning, sequencing, and expression of a chemoreceptor gene from Leptospirillum ferrooxidans.

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both sigma 28-like and sigma 70-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.

An immunological assay for detection and enumeration of thermophilic biomining microorganisms.

A specific, fast, and sensitive nonradioactive immunobinding assay for the detection and enumeration of the moderate thermophile Thiobacillus caldus and the thermophilic archaeon Sulfolobus acidocaldarius was developed. It employs enhanced chemiluminescence or peroxidase-conjugated immunoglobulins in a dot or slot blotting system and is very convenient for monitoring thermophilic bioleaching microorganisms in effluents from industrial bioleaching processes.

Effect of external pH perturbations on in vivo protein synthesis by the acidophilic bacterium Thiobacillus ferrooxidans.

The response of the obligate acidophilic bacterium Thiobacillus ferrooxidans to external pH changes is reported. When T. ferrooxidans cells grown at pH 1.5 were shifted to pH 3.5, there were several changes in the general protein synthesis pattern, including a large stimulation of the synthesis of a 36-kDa protein (p36). The apparent low isoelectric point of p36, its location in the membrane fraction, and its cross-reaction with anti-OmpC from Salmonella typhi suggested that it may be a porin whose expression is regulated by extracellular pH.

Methylation of proteins from the translational apparatus: an overview.

Several of the translational apparatus proteins are methylated in all kinds of organisms. Although most of the modified proteins play key roles during protein biosynthesis, the biological function of these chemical modifications still remains elusive. Our recent data indicate a highly conserved pattern of ribosomal protein methylation in eubacteria, with methylated proteins being both structurally and functionally homologous in several microorganisms. Chloroplast ribosomes also appear to have a rather eubacterial pattern of ribosomal protein methylation. On the other hand, there is an apparently ubiquitous methylation of some of the translational factors in several organisms. These findings suggest an important, albeit unknown role for the post-synthetic methylation of the translational machinery. The analysis of the sequences of known methylation target sites and the search of similar sites in other proteins of known sequence, allows to predict those ribosomal proteins or translational factors that may be subjected to post-translational modifications with one or more methyl groups. Although a definitive answer with respect to the biological role of these N-methylations is still missing, a direct correlation between the methylation of some proteins and their biological activity is just beginning to emerge.