Implantation of Bacillus pseudomycoides Chromate Transporter Increases Chromate Tolerance in Bacillus subtilis

Chromium of anthropogenic origin contaminates the environment worldwide. The toxicity of chromium, a group I human carcinogen, is greatest when it is in a hexavalent oxidation state, Cr(VI). Cr(VI) is actively transported into the cell, triggering oxidative damage intracellularly. Due to the abundance of unspecific intracellular reductants, any microbial species is capable of bio-transformation of toxic Cr(VI) to innocuous Cr(III), however, this process is often lethal. Only some bacterial species are capable of sustaining the vegetative growth in the presence of a high concentration of Cr(VI) and thus operate as self-sustainable bioremediation agents. One of the successful microbial Cr(VI) detoxification strategies is the activation of chromate efflux pumps. This work describes transplantation of the chromate efflux pump from the potentially pathogenic but highly Cr resistant Bacillus pseudomycoides environmental strain into non-pathogenic but only transiently Cr tolerant Bacillus subtilis strain. In our study, we compared the two Bacillus spp. strains harboring evolutionarily diverged chromate efflux proteins. We have found that individual cells of the Cr-resistant B. pseudomycoides environmental strain accumulate less Cr than the cells of B. subtilis strain. Further, we found that survival of the B. subtilis strain during the Cr stress can be increased by the introduction of the chromate transporter from the Cr resistant environmental strain into its genome. Additionally, the expression of B. pseudomycoides chromate transporter ChrA in B. subtilis seems to be activated by the presence of chromate, hinting at versatility of Cr-efflux proteins. This study outlines the future direction for increasing the Cr-tolerance of non-pathogenic species and safe bioremediation using soil bacteria.

The porous surface model, a novel experimental system for online quantitative observation of microbial processes under unsaturated conditions

Gamze Gülez, Dani Or

Water is arguably the most important constituent of microbial microhabitats due to its control of physical and physiological processes critical to microbial activity. In natural environments, bacteria often live on unsaturated surfaces, in thin (micrometric) liquid films. Nevertheless, no experimental systems are available that allow real-time observation of bacterial processes in liquid films of controlled thickness. We propose a novel, inexpensive, easily operated experimental platform, termed the porous surface model (PSM) that enables quantitative real-time microscopic observations of bacterial growth and activity under controlled unsaturated conditions. Bacteria are inoculated on a porous ceramic plate, wetted by a liquid medium. The thickness of the liquid film at the surface of the plate is set by imposing suction, corresponding to soil matric potential, to the liquid medium. The utility of the PSM was demonstrated using Pseudomonas putida KT2440 tagged with gfp as a model bacterium. Single cells were inoculated at the surface of the PSM, and the rate at which colonies expanded laterally was measured for three matric potentials (-0.5, -1.2, and -3.6 kPa). The matric potential exerted significant influence on colony expansion rates, with a faster rate of spreading at -0.5 than at -1.2 or -3.6 kPa (diameter increase rate, ca. 1,000, 200, and 17 mu m h(-1), respectively). These differences can be attributed to cell motility, strongly limited under the most negative matric potential. The PSM constitutes a tool uniquely adapted to study the influence of liquid film geometry on microbial processes. It should therefore contribute to uncovering mechanisms of microbial adaptation to unsaturated environments.

2008

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

The physiological state of competence for natural transformation allows bacteria to take up free DNA from the environment and recombine it into their genome. As one of three modes of horizontal gene transfer, natural transformation has hence contributed substantially to the enormous diversity and plasticity of bacterial genomes. With its potential to promote the spread of antibiotic resistances and virulence factors of a variety of pathogens, natural transformation also has an important impact on human health. Even though conserved components required to undergo natural transformation have been identified in many bacterial species, our knowledge on the underlying mechanism of DNA transport remains very limited. To better understand this aspect, we established a first model of DNA uptake in the naturally competent human pathogen Vibrio cholerae. We showed that at least twenty proteins are required for efficient uptake, protection and recombination of transforming DNA in a non-species-specific manner. 14 of these proteins are needed to produce a type IV pilus, which we were able to visualize for the first time. We showed that this competence pilus is not restricted to cell poles, as has been shown for other competent bacterial species (e.g. Bacillus subtilis). Furthermore, we compared the position of this pilus to the localization of other competence proteins and determined their role in the assembly of a surface exposed pilus fiber. While this type IV pilus is important for efficient DNA uptake, we also observed rare transformation events in its absence. In contrast, three pilus-unrelated proteins that are likely involved in the transport process were absolutely required for transformation. Two of them were implicated in DNA transport across the inner membrane. Deletion of the corresponding genes led to a strong accumulation of transforming DNA within the periplasm. However, one of them, ComEA, was essential for DNA uptake across the outer membrane, independently of competence pili. We further characterized the role of this protein by showing that it is localized to the periplasm of competent V. cholerae cells and that it binds to DNA in vivo. Based on our results, we proposed that ComEA directly contributes to DNA transport. Although we showed that DNA uptake across the outer membrane and translocation of DNA into the cytoplasm are functionally uncoupled, our results suggest that the two events coincide spatially. In summary, these findings allow us to present a first mechanistic model of the DNA uptake process in V. cholerae. Many aspects of this model likely apply to other naturally competent bacteria, and therefore represent an important contribution to the general understanding of this basic biological process.

EPFL2014

Metal reduction and sporulation in Desulfotomaculum reducens

Elena Cristina Dalla Vecchia

The genera Desulfotomaculum and Clostridium, belonging to the phylum Firmicutes, comprise Gram-positive, low G+C genomic content, anaerobic, spore- forming bacteria. Desulfotomaculum is a metabolically and environmentally versatile genus capable of growing fermentatively as well as by respiring sulfate. D. reducens is also capable of metal reduction and has been reported to conserve energy from this process. In this thesis, iron (Fe(III)) reduction by D. reducens in the presence of pyruvate or lactate as electron donors was investigated. When pyruvate is present, D. reducens grows fermentatively. If Fe(III) is present, it can act as an electron sink alternative to protons and consume excess reducing equivalents accumulated during pyruvate oxidation. Electron transfer to Fe(III) by D. reducens in the presence of pyruvate is mediated by a soluble electron carrier, likely riboflavin, released by cells during fermentation. This mechanism allows the microorganism to reduce extracellular, solid phase Fe(III) even if it is not directly accessible by physical contact. In the presence of lactate, D. reducens is unable to conserve significant energy from electron donor oxidation and Fe(III) reduction. In contrast to pyruvate, during lactate oxidation, no redox-active compound is released by the cells, and extracellular, solid phase Fe(III) can only be reduced by direct contact. This implies that a surface exposed reductase must be present in D. reducens cells. The investigation of the surface proteome of this organism led to the identification of three proteins, most likely localized to the cell surface, potentially involved in Fe(III) reduction. One is a heterodisulfide reductase subunit A, a putative intermediate in the Fe(III) reduction chain. The second is a membrane-associated hydrogenase. The third protein is annotated as alkyl hydroperoxide reductase although its actual function may be thiol-disulfide oxidoreduction; several lines of evidence suggest the involvement of the latter in metal reduction by D. reducens. Clostridium species are widespread in the environment and are capable of utilizing a great variety of organic substrates for fermentative growth, while they are unable to conserve energy through respiration. However, they can reduce terminal electron acceptors to eliminate excess reducing equivalents from fermentation. Among the electron acceptors used by Clostridium spp., are metals, such as iron and uranium (U(VI)). One species whose vegetative cells are capable of U(VI) reduction is C. acetobutylicum, although little information is available on the mechanism underlying this process. In this thesis, the potential involvement of the spores of C. acetobutylicum in U(VI) reduction is probed. It was found that the spores of this species can reduce the radionuclide, and that to do so they require the presence of an unidentified soluble compound released by growing cells and H2 as the electron donor. The involvement of spores in metal reduction is very interesting because generally spores are considered not to interact directly with the surrounding environment. In fact, spores are dormant, thus metabolically inactive, cells. Spore formation is a cell differentiation process activated in response to environmental stress and conditions that are hostile to vegetative growth. Sporulation has been extensively studied and characterized in Bacillus subtilis, for which a model has been developed. Some information is also available on sporulation in the clostridia. In this thesis, a genomic investigation of sporulation in the Desulfotomaculum genus was undertaken. The B. subtilis model was used as a reference and the information on Clostridium as a comparison. Significant similarities were found in the core regulatory process across genera, while some differences were identified in the initiation of sporulation. Substantial differences were highlighted in the spore structural proteins.

EPFL2014

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

EPFL2014

Metal reduction and sporulation in Desulfotomaculum reducens

Elena Cristina Dalla Vecchia

EPFL2014