Looking for the mechanism of arsenate respiration of Fusibacter sp. strain 3D3, independent of ArrAB

The literature has reported the isolation of arsenate-dependent growing microorganisms which lack a canonical homolog for respiratory arsenate reductase, ArrAB. We recently isolated an arsenate-dependent growing bacterium from volcanic arsenic-bearing environments in Northern Chile, Fusibacter sp. strain 3D3 (Fas) and studied the arsenic metabolism in this Gram-positive isolate. Features of Fas deduced from genome analysis and comparative analysis with other arsenate-reducing microorganisms revealed the lack of ArrAB coding genes and the occurrence of two arsC genes encoding for putative cytoplasmic arsenate reductases named ArsC-1 and ArsC-2. Interestingly, ArsC-1 and ArsC-2 belong to the thioredoxin-coupled family (because of the redox-active disulfide protein used as reductant), but they conferred differential arsenate resistance to the E. coli WC3110 ΔarsC strain. PCR experiments confirmed the absence of arrAB genes and results obtained using uncouplers revealed that Fas growth is linked to the proton gradient. In addition, Fas harbors ferredoxin-NAD+ oxidoreductase (Rnf) and electron transfer flavoprotein (etf) coding genes. These are key molecular markers of a recently discovered flavin-based electron bifurcation mechanism involved in energy conservation, mainly in anaerobic metabolisms regulated by the cellular redox state and mostly associated with cytoplasmic enzyme complexes. At least three electron-bifurcating flavoenzyme complexes were evidenced in Fas, some of them shared in conserved genomic regions by other members of the Fusibacter genus. These physiological and genomic findings permit us to hypothesize the existence of an uncharacterized arsenate-dependent growth metabolism regulated by the cellular redox state in the Fusibacter genus.Fil: Acosta Grinok, Mauricio. Universidad Católica del Norte; ChileFil: Vázquez, Susana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología Industrial y Biotecnología; ArgentinaFil: Guiliani, Nicolás. Universidad de Chile; ChileFil: Marín, Sabrina. Universidad Católica del Norte; ChileFil: Demergasso, Cecilia. Universidad Católica del Norte; Chil

Nucleotide Second Messenger-Based Signaling in Extreme Acidophiles of the Acidithiobacillus Species Complex: Partition Between the Core and Variable Gene Complements

Cyclic and linear nucleotides are key elements of the signal transduction networks linking perception of the environment to specific cellular behavior of prokaryotes. These molecular mechanisms are particularly important in bacteria exposed to different, and frequently simultaneous, types of extreme conditions. This is the case in acidithiobacilli, a group of extremophilic bacteria thriving in highly acidic biotopes, that must also cope with significant variations in temperature, osmotic potentials and concentrations of various transition metals and metalloids. Environmental cues sensed by bacteria are transduced into differential levels of nucleotides acting as intracellular second messengers, promoting the activation or inhibition of target components and eliciting different output phenotypes. Cyclic (c) di-GMP, one of the most common bacterial second messengers, plays a key role in lifestyle changes in many bacteria, including acidithiobacilli. The presence of functional c-di-GMP-dependent signal transduction pathways in representative strains of the best-known linages of this species complex has been reported. However, a comprehensive panorama of the c-di-GMP modulated networks, the cognate input signals and output responses, are still missing for this group of extremophiles. Moreover, little fundamental understanding has been gathered for other nucleotides acting as second messengers. Taking advantage of the increasing number of sequenced genomes of the taxon, here we address the challenge of disentangling the nucleotide-driven signal transduction pathways in this group of polyextremophiles using comparative genomic tools and strategies. Results indicate that the acidithiobacilli possess all the genetic elements required to establish functional transduction pathways based in three different nucleotide-second messengers: (p)ppGpp, cyclic AMP (cAMP), and c-di-GMP. The elements related with the metabolism and transduction of (p)ppGpp and cAMP appear highly conserved, integrating signals related with nutrient starvation and polyphosphate metabolism, respectively. In contrast, c-di-GMP networks appear diverse and complex, differing both at the species and strain levels. Molecular elements of c-di-GMP metabolism and transduction were mostly found scattered along the flexible genome of the acidithiobacilli, allowing the identification of probable control modules that could be critical for substrate colonization, biofilm development and intercellular interactions. These may ultimately convey increased endurance to environmental stress and increased potential for gene sharing and adaptation to changing conditions

Proteomic and genomic analysis of the phosphate starvation response of Acidithiobacillus ferrooxidans

The recent availability of an incomplete genomic sequence from Acidithiobacillus ferrooxidans allowed us to continue and strengthen the demanding task of investigating the proteome and its functional implications in this extremophilic microorganism. The proteins of At. ferrooxidans were separated by two-dimensional polyacrylamide gel electrophoresis and their levels of synthesis and the microsequencing of their N-terminal end amino acids were determined. To link the 2D gel spots of interest with the genes that encodes them, we studied the global changes in gene expression of At. ferrooxidans when the bacterium was confronted with phosphate starvation. By comparing the amino acid sequences of the proteins whose synthesis was induced or repressed under these conditions, with the available At. ferrooxidans genomic database, we found several putative genes whose expression may be related to phosphate starvation. Analysis of the genome DNA sequences upstream and downstream of these genes showed us details of the structure of putative operons present in At. ferrooxidans, strongly suggesting the existence of a Pho regulon containing the putative genes phoB, phoR, pstS, pstC,pstA, pYtB, phoU, ppx and ppk. Some differences were seen in the organization of the genes in the possible Pho regulon of At. ferrooxidans when compared with the Pho operons from other microorganisms. This was specially evident in the organization of the genes involved in polyphosphate metabolism (ppk and ppx). Regulation of phosphate metabolism is of particular relevance when At, ferrooxidans grows in the presence of arsenopyrites, which release arsenate, a structural analog of phosphate. Structural comparison between the specific phosphate-binding protein PstS from Escherichia coli and the corresponding At. ferrooxidans homolog showed that both proteins are highly conserved, including the phosphate/arsenate binding site, which shares seven of the eight amino acid residues necessary for the hydrogen bonding to the four oxygens of phosphate

Biofilm formation by the acidophile bacterium acidithiobacillus thiooxidans involves c-di-GMP pathway and Pel exopolysaccharide

Acidophile bacteria belonging to the Acidithiobacillus genus are pivotal players for the bioleaching of metallic values such as copper. Cell adherence to ores and biofilm formation, mediated by the production of extracellular polymeric substances, strongly favors bioleaching activity. In recent years, the second messenger cyclic diguanylate (c-di-GMP) has emerged as a central regulator for biofilm formation in bacteria. C-di-GMP pathways have been reported in different Acidithiobacillus species; however, c-di-GMP effectors and signal transduction networks are still largely uncharacterized in these extremophile species. Here we investigated Pel exopolysaccharide and its role in biofilm formation by sulfur-oxidizing species Acidithiobacillusthiooxidans. We identified 39 open reading frames (ORFs) encoding proteins involved in c-di-GMP metabolism and signal transduction, including the c-di-GMP effector protein PelD, a structural component of the biosynthesis apparatus for Pel exopolysaccharide production. We found that intracellular c-di-GMP concentrations and transcription levels of pel genes were higher in At. thiooxidans biofilm cells compared to planktonic ones. By developing an At. thiooxidans pelD null-mutant strain we revealed that Pel exopolysaccharide is involved in biofilm structure and development. Further studies are still necessary to understand how Pel biosynthesis is regulated in Acidithiobacillus species, nevertheless these results represent the first characterization of a c-di-GMP effector protein involved in biofilm formation by acidophile species.CONICYT 21120064 FONDECYT 1160702 Universidad de Chile (UCH

Diguanylate Cyclase Null Mutant Reveals That C-Di-GMP Pathway Regulates the Motility and Adherence of the Extremophile Bacterium Acidithiobacillus caldus

Artículo de publicación ISIAn understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Delta c1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.FONDECYT 1080441 1120295 MECESUP UCH0407 UCH-0604 VID-Universidad de Chil

Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2) from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis

BACKGROUND: Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence FINDINGS: By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin CONCLUSIONS: Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties

Nucleotide second messenger-based signaling in extreme acidophiles of the Acidithiobacillus species complex: Partition between the core and variable gene complements

Cyclic and linear nucleotides are key elements of the signal transduction networks linking perception of the environment to specific cellular behavior of prokaryotes. These molecular mechanisms are particularly important in bacteria exposed to different, and frequently simultaneous, types of extreme conditions. This is the case in acidithiobacilli, a group of extremophilic bacteria thriving in highly acidic biotopes, that must also cope with significant variations in temperature, osmotic potentials and concentrations of various transition metals and metalloids. Environmental cues sensed by bacteria are transduced into differential levels of nucleotides acting as intracellular second messengers, promoting the activation or inhibition of target components and eliciting different output phenotypes. Cyclic (c) di-GMP, one of the most common bacterial second messengers, plays a key role in lifestyle changes in many bacteria, including acidithiobacilli. The presence of functional c-di-GMP-dependent signal transduction pathways in representative strains of the best-known linages of this species complex has been reported. However, a comprehensive panorama of the c-di-GMP modulated networks, the cognate input signals and output responses, are still missing for this group of extremophiles. Moreover, little fundamental understanding has been gathered for other nucleotides acting as second messengers. Taking advantage of the increasing number of sequenced genomes of the taxon, here we address the challenge of disentangling the nucleotide-driven signal transduction pathways in this group of polyextremophiles using comparative genomic tools and strategies. Results indicate that the acidithiobacilli possess all the genetic elements required to establish functional transduction pathways based in three different nucleotide-second messengers: (p)ppGpp, cyclic AMP (cAMP), and c-di-GMP. The elements related with the metabolism and transduction of (p)ppGpp and cAMP appear highly conserved, integrating signals related with nutrient starvation and polyphosphate metabolism, respectively. In contrast, c-di-GMP networks appear diverse and complex, differing both at the species and strain levels. Molecular elements of c-di-GMP metabolism and transduction were mostly found scattered along the flexible genome of the acidithiobacilli, allowing the identification of probable control modules that could be critical for substrate colonization, biofilm development a

Does bacterial elasticity affect adhesion to polymer fibers?

The factors governing bacterial adhesion to substrates with different topographies are still not fully identified. The present work seeks to elucidate for the first time and with quantitative data the roles of bacterial elasticity and shape and substrate topography in bacterial adhesion. With this aim, populations of three bacterial species, P. aeruginosa DSM 22644, B. subtilis DSM 10, and S. aureus DSM 20231 adhered on flat substrates covered with electrospun polycaprolactone fibers of different diameters ranging from 0.4 to 5.5 mu m are counted. Populations of bacterial cells are classified according to the preferred binding sites of the bacteria to the substrate. The colloidal probe technique was used to assess the stiffness of the bacteria and bacteria-polymer surface adhesion energy. A theoretical model is developed to interpret the observed populations in terms of a balance between stiffness and adhesion energy of the bacteria. The model, which also incorporates the radius of the fiber and the size and shape of the bacteria, predicts increased adhesion for a low level of stiffness and for a larger number of available bacteria-fiber contact points. Te adhesive propensity of bacteria depends in a nontrivial way on the radius of the fibers due to the random arrangement of fibers.Comisión Nacional de Investigación Cientifica y Tecnológica (CONICYT) CONICYT FONDECYT 11160230 1161010 1160702 1191467 1200853 Vicerrectoría de Investigación, Desarrollo e Innovación, Universidad de Santiago de Chile DICYT 041931HH Fondequip project EQM 130149 PAI-Conicyt 917001