The bacterial metallome: composition and stability with specific reference to the anaerobic bacterium Desulfovibrio desulfuricans.

In bacteria, the intracellular metal content or metallome reflects the metabolic requirements of the cell. When comparing the composition of metals in phytoplankton and bacteria that make up the macronutrients and the trace elements, we have determined that the content of trace elements in both of these microorganisms is markedly similar. The trace metals consisting of transition metals plus zinc are present in a stoichometric molar formula that we have calculated to be as follows: Fe(1)Mn(0.3)Zn(0.26)Cu(0.03)Co(0.03)Mo(0.03). Under conditions of routine cultivation, trace metal homeostasis may be maintained by a series of transporter systems that are energized by the cell. In specific environments where heavy metals are present at toxic levels, some bacteria have developed a detoxification strategy where the metallic ion is reduced outside of the cell. The result of this extracellular metabolism is that the bacterial metallome specific for trace metals is not disrupted. One of the microorganisms that reduces toxic metals outside of the cell is the sulfate-reducing bacterium Desulfovibrio desulfuricans. While D. desulfuricans reduces metals by enzymatic processes involving polyhemic cytochromes c3 and hydrogenases, which are all present inside the cell; we report the presence of chain B cytochrome c nitrite reductase, NrfA, in the outer membrane fraction of D. desulfuricans ATCC 27774 and discuss its activity as a metal reductase.

Arsenate detoxification in a Pseudomonad hypertolerant to arsenic.

Pseudomonas sp. strain As-1, obtained from an electroplating industrial effluent, was capable of growing aerobically in growth medium supplemented with up to 65 mM arsenate (As (V)), significantly higher concentrations than those tolerated by other reference arsenic resistant bacteria. The majority of the arsenic was detected in culture supernatants as arsenite (As (III)) and X-ray absorbance spectroscopy suggested that 30% of this cell-bound arsenic was As (V), 65% As (III) and 5% of arsenic was associated with sulphur. PCR analysis using primers designed against arsenic resistance genes of other Gram-negative bacteria confirmed the presence of an arsenic resistance operon comprising of three genes, arsR, arsB and arsC in order of predicted transcription, and consistent with a role in intracellular reduction of As (V) and efflux of As (III). In addition to this classical arsenic resistance mechanism, other biochemical responses to arsenic were implicated. Novel arsenic-binding proteins were purified from cellular fractions, while proteomic analysis of arsenic-induced cultures identified the upregulation of additional proteins not normally associated with the metabolism of arsenic. Cross-talk with a network of proteins involved in phosphate metabolism was suggested by these studies, consistent with the similarity between the phosphate and arsenate anions.

Cr(VI) detoxification by Desulfovibrio vulgaris strain Hildenborough: microbe-metal interactions studies.

Toxic heavy metals constitute a worldwide environmental pollution problem. Bioremediation technologies represent efficient alternatives to the classic cleaning-up of contaminated soil and ground water. Most toxic heavy metals such as chromium are less soluble and toxic when reduced than when oxidized. Sulfate-reducing bacteria (SRB) are able to reduce heavy metals by a chemical reduction via the production of H2S and by a direct enzymatic process involving hydrogenases and c3 cytochromes. We have previously reported the effects of chromate [Cr(VI)] on SRB bioenergetic metabolism and the molecular mechanism of the metal reduction by polyhemic cytochromes. In the current work, we pinpoint the bacteria-metal interactions using Desulfovibrio vulgaris strain Hildenborough as a model. The bacteria were grown in the presence of high Cr(VI) concentration, where they accumulated precipitates of a reduced form of chromium, trivalent chromium [Cr(III)], on their cell surfaces. Moreover, the inner and outer membranes exhibited precipitates that shared the spectroscopic signature of trivalent chromium. This subcellular localization is consistent with enzymatic metal reduction by cytochromes and hydrogenases. Regarding environmental significance, our findings point out the Cr(VI) immobilization mechanisms of SRB; suggesting that SRB are highly important in metal biogeochemistry.

Functional refolding of the Campylobacter jejuni MOMP (major outer membrane protein) porin by GroEL from the same species.

Functional and structural studies of outer membrane proteins from Gram-negative bacteria are frequently carried out using refolded proteins. Recombinant proteins are produced in Escherichia coli as inclusion bodies and then tediously refolded by dilution in buffered detergent solutions. In the present work, we obtained the refolding of MOMP (major outer membrane protein) from Campylobacter assisted by the molecular chaperone GroEL. Refolded MOMP recovered its native pore-forming activity when reconstituted in planar lipid bilayers. Both proteins were purified from the Campylobacter jejuni strain 85H. The purity of GroEL was assessed by silver staining and MS. Its native ultrastructure was observed by the use of transmission electron microscopy. Denaturation of MOMP was performed in urea at 65 degrees C followed by dialysis against 100 mM acetic acid, and was assessed by CD analysis. MOMP refolding reached a maximum efficiency in the presence of GroEL (at a MOMP/GroEL molar ratio of 9:1) and ATP. Under these conditions, 95% of denatured MOMP was refolded after a 15 min incubation. This approach represents an alternative method in studies of membrane protein refolding.

Oral microbial heat-shock proteins and their potential contributions to infections.

The oral cavity is a complex ecosystem in which several hundred microbial species normally cohabit harmoniously. However, under certain special conditions, the growth of some micro-organisms with a pathogenic potential is promoted, leading to infections such as dental caries, periodontal disease, and stomatitis. The physiology and pathogenic properties of micro-organisms are influenced by modifications in environmental conditions that lead to the synthesis of specific proteins known as the heat-shock proteins (HSPs). HSPs are families of highly conserved proteins whose main role is to allow micro-organisms to survive under stress conditions. HSPs act as molecular chaperones in the assembly and folding of proteins, and as proteases when damaged or toxic proteins have to be degraded. Several pathological functions have been associated with these proteins. Many HSPs of oral micro-organisms, particularly periodontopathogens, have been identified, and some of their properties-including location, cytotoxicity, and amino acid sequence homology with other HSPs-have been reported. Since these proteins are immunodominant antigens in many human pathogens, studies have recently focused on the potential contributions of HSPs to oral diseases. The cytotoxicity of some bacterial HSPs may contribute to tissue destruction, whereas the presence of common epitopes in host proteins and microbial HSPs may lead to autoimmune responses. Here, we review the current knowledge regarding HSPs produced by oral micro-organisms and discuss their possible contributions to the pathogenesis of oral infections.

Stress response in Actinobacillus actinomycetemcomitans: induction of general and specific stress proteins.

Stress proteins are highly conserved proteins that are essential for cell survival. In this study, the induction of general and specific stress proteins in Actinobacillus actinomycetemcomitans cells subjected to different stress conditions was evaluated by two-dimensional SDS-PAGE analysis. Twenty-eight (up- or down)regulated proteins, including DnaK and GroEL proteins, were identified as general stress proteins. In addition, eighteen regulated proteins were classified as pH stress-specific proteins, ten as acid stress-specific proteins, five as alkaline stress-specific proteins, three as heat stress-specific proteins, and ten as acid/heat stress-specific proteins. Further proteomic studies are required to determine the exact nature of the proteins regulated during the stress response of A. actinomycetemcomitans.