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The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V . parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella . Our results showed that two subpopulations (P1 and P2) of V . parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:Δ lldD ) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:Δ lldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

Background Selenite (SeO 3 2− ) oxyanion shows severe toxicity to biota. Different bacterial strains exist that are capable of reducing SeO 3 2− to non-toxic elemental selenium (Se 0 ), with the formation of Se nanoparticles (SeNPs). These SeNPs might be exploited for technological applications due to their physico-chemical and biological characteristics. The present paper discusses the reduction of selenite to SeNPs by a strain of Bacillus sp., SeITE01, isolated from the rhizosphere of the Se-hyperaccumulator legume Astragalus bisulcatus . Results Use of 16S rRNA and GyrB gene sequence analysis positioned SeITE01 phylogenetically close to B. mycoides . On agarized medium, this strain showed rhizoid growth whilst, in liquid cultures, it was capable of reducing 0.5 and 2.0 mM SeO 3 2− within 12 and 24 hours, respectively. The resultant Se 0 aggregated to form nanoparticles and the amount of Se 0 measured was equivalent to the amount of selenium originally added as selenite to the growth medium. A delay of more than 24 hours was observed between the depletion of SeO 3 2 and the detection of SeNPs. Nearly spherical-shaped SeNPs were mostly found in the extracellular environment whilst rarely in the cytoplasmic compartment. Size of SeNPs ranged from 50 to 400 nm in diameter, with dimensions greatly influenced by the incubation times. Different SeITE01 protein fractions were assayed for SeO 3 2− reductase capability, revealing that enzymatic activity was mainly associated with the membrane fraction. Reduction of SeO 3 2− was also detected in the supernatant of bacterial cultures upon NADH addition. Conclusions The selenite reducing bacterial strain SeITE01 was attributed to the species Bacillus mycoides on the basis of phenotypic and molecular traits. Under aerobic conditions, the formation of SeNPs were observed both extracellularly or intracellullarly. Possible mechanisms of Se 0 precipitation and SeNPs assembly are suggested. SeO 3 2− is proposed to be enzimatically reduced to Se 0 through redox reactions by proteins released from bacterial cells. Sulfhydryl groups on peptides excreted outside the cells may also react directly with selenite. Furthermore, membrane reductases and the intracellular synthesis of low molecular weight thiols such as bacillithiols may also play a role in SeO 3 2− reduction. Formation of SeNPs seems to be the result of an Ostwald ripening mechanism.

During selenate respiration by Thauera selenatis, the reduction of selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approximately 150 nm in diameter. We report that the Se nanospheres are associated with a protein of approximately 95 kDa. Subsequent experiments to investigate the expression and secretion profile of this protein have demonstrated that it is up-regulated and secreted in response to increasing selenite concentrations. The protein was purified from Se nanospheres, and peptide fragments from a tryptic digest were used to identify the gene in the draft T. selenatis genome. A matched open reading frame was located, encoding a protein with a calculated mass of 94.5 kDa. N-terminal sequence analysis of the mature protein revealed no cleavable signal peptide, suggesting that the protein is exported directly from the cytoplasm. The protein has been called Se factor A (SefA), and homologues of known function have not been reported previously. The sefA gene was cloned and expressed in Escherichia coli, and the recombinant His-tagged SefA purified. In vivo experiments demonstrate that SefA forms larger (approximately 300 nm) Se nanospheres in E. coli when treated with selenite, and these are retained within the cell. In vitro assays demonstrate that the formation of Se nanospheres upon the reduction of selenite by glutathione are stabilized by the presence of SefA. The role of SefA in selenium nanosphere assembly has potential for exploitation in bionanomaterial fabrication.

The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that 11168H forms persister cells at a frequency of 10 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.

Many species of Bacteria and Archaea respire nitrate using a molybdenum-dependent membrane-bound respiratory system called Nar. Classically, the 'Bacterial' Nar system is oriented such that nitrate reduction takes place on the inside of this membrane. However, the active site subunit of the 'Archaeal' Nar systems has a twin arginine ('RR') motif, which is a suggestion of translocation to the outside of the cytoplasmic membrane. These 'Archaeal' type of nitrate reductases are part of a group of molybdoenzymes with an 'RR' motif that are predicted to have an aspartate ligand to the molybdenum ion. This group includes selenate reductases and possible sequence signatures are described that serve to distinguish the Nar nitrate reductases from the selenate reductases. The 'RR' sequences of nitrate reductases of Archaea and some that have recently emerged in Bacteria are also considered and it is concluded that there is good evidence for there being both Archaeal and Bacterial examples of Nar-type nitrate reductases with an active site on the outside of the cytoplasmic membrane. Finally, the bioenergetic consequences of nitrate reduction on the outside of the cytoplasmic membrane have been explored.

For the study of the dinuclear center of heme-copper oxidases cytochrome bo3 from Escherichia coli offers several advantages over the extensively characterized bovine cytochrome c oxidase. The availability of strains with enhanced levels of expression allows purification of the significant amounts of enzyme required for detailed spectroscopic studies. Cytochrome bo3 is readily prepared as the fast form, with a homogeneous dinuclear center which gives rise to characteristic broad EPR signals not seen in CcO. The absence of CuA and the incorporation of protohemes allows for a detailed interpretation of the MCD spectra arising from the dinuclear center heme o3. Careful analysis allows us to distinguish between small molecules that bind to heme o3, those which are ligands of CuB, and those which react to yield higher oxidation states of heme o3. Here we review results from our studies of the reactions of fast cytochrome bo3 with formate, fluoride, chloride, azide, cyanide, NO, and H2O2.

Enterobacter cloacae SLD1a-1 is capable of the complete reduction of selenate to selenium and the initial reaction is catalysed by a membrane-bound selenate reductase. In the present study, continuous culture experiments were employed to investigate the possibility that selenate reduction, via the selenate reductase, might provide sufficient energy to maintain cell viability when deprived of the preferred anaerobic terminal electron acceptor nitrate. The evidence presented indicates that the selenate reductase supports slow growth that retards the wash-out of the culture when switching to nitrate-depleted selenate-rich medium, and provides a proton motive force for sustained cell maintenance. In contrast, a strain of E. cloacae (sub sp. cloacae) that does not readily reduce selenate, cannot sustain cell maintenance when switching to a selenate-rich medium. This work demonstrates for the first time that respiratory linked selenate reduction gives E. cloacae SLD1a-1 a selective advantage when inhabiting selenate-contaminated environments and highlights the suitability of utilising E. cloacae SLD1a-1 when developing selenium remediation strategies.

Enterobacter cloacae SLD1a-1 is capable of reducing selenium oxyanions to elemental selenium under both aerobic and anaerobic conditions. In this study the enzyme that catalyses the initial reduction of selenate (SeO 4 2−) to selenite (SeO 3 2−) has been localised to isolated cytoplasmic membrane fractions. Experiments with intact cells have shown that the putative selenate reductase can accept electrons more readily from membrane-impermeable methyl viologen than membrane-permeable benzyl viologen, suggesting that the location of the catalytic site is towards the periplasmic side of the cytoplasmic membrane. Enzyme activity was enhanced by growing cells in the presence of 1 mM sodium molybdate and significantly reduced in cells grown in the presence of 1 mM sodium tungstate. Non-denaturing polyacrylamide gel electrophoresis (PAGE) gels stained for selenate and nitrate reductase activity have revealed that two distinct membrane-bound enzymes catalyse the reduction of selenate and nitrate. The role of this membrane-bound molybdenum-dependent reductase in relation to selenate detoxification and energy conservation is discussed.

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 μM) reduced nitrate at a rate of V max=3.41±0.16 μmol [NO 3 −] min −1 mg −1 with an affinity for nitrate of K m=0.24±0.05 mM and was heat-stable up to 50°C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 μM) reduced nitrate at a much slower rate, with much lower affinity ( V max=0.05±0.002 μmol [NO 3 −] min −1 mg −1 and K m=3.91±0.45 mM) and was labile during prolonged incubation at >20°C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g′ = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The “slow” form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a 3 and the single unpaired electron of Cu B. The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo 3 , has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232–4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (| J| ≈ 1 cm −1), with the axial zero-field splitting ( D ≈ 5 cm −1) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.