Fur in Magnetospirillum gryphiswaldense Influences Magnetosomes Formation and Directly Regulates the Genes Involved in Iron and Oxygen Metabolism

Magnetospirillum gryphiswaldense strain MSR-1 has the unique capability of taking up large amounts of iron and synthesizing magnetosomes (intracellular magnetic particles composed of Fe3O4). The unusual high iron content of MSR-1 makes it a useful model for studying biological mechanisms of iron uptake and homeostasis. The ferric uptake regulator (Fur) protein plays a key role in maintaining iron homeostasis in many bacteria. We identified and characterized a fur-homologous gene (MGR_1314) in MSR-1. MGR_1314 was able to complement a fur mutant of E. coli in iron-responsive manner in vivo. We constructed a fur mutant strain of MSR-1. In comparison to wild-type MSR-1, the mutant strain had lower magnetosome formation, and was more sensitive to hydrogen peroxide and streptonigrin, indicating higher intracellular free iron content. Quantitative real-time RT-PCR and chromatin immunoprecipitation analyses indicated that Fur protein directly regulates expression of several key genes involved in iron transport and oxygen metabolism, in addition it also functions in magnetosome formation in M. gryphiswaldense

Proteome Analysis Identifies the Dpr Protein of Streptococcus mutans as an Important Factor in the Presence of Early Streptococcal Colonizers of Tooth Surfaces

Oral streptococci are primary colonizers of tooth surfaces and Streptococcus mutans is the principal causative agent of dental caries in humans. A number of proteins are involved in the formation of monospecies biofilms by S. mutans. This study analyzed the protein expression profiles of S. mutans biofilms formed in the presence or absence of S. gordonii, a pioneer colonizer of the tooth surface, by two-dimensional gel electrophoresis (2-DE). After identifying S. mutans proteins by Mass spectrometric analysis, their expression in the presence of S. gordonii was analyzed. S. mutans was inoculated with or without S. gordonii DL1. The two species were compartmentalized using 0.2-μl Anopore membranes. The biofilms on polystyrene plates were harvested, and the solubilized proteins were separated by 2-DE. When S. mutans biofilms were formed in the presence of S. gordonii, the peroxide resistance protein Dpr of the former showed 4.3-fold increased expression compared to biofilms that developed in the absence of the pioneer colonizer. In addition, we performed a competition assay using S. mutans antioxidant protein mutants together with S. gordonii and other initial colonizers. Growth of the dpr-knockout S. mutans mutant was significantly inhibited by S. gordonii, as well as by S. sanguinis. Furthermore, a cell viability assay revealed that the viability of the dpr-defective mutant was significantly attenuated compared to the wild-type strain when co-cultured with S. gordonii. Therefore, these results suggest that Dpr might be one of the essential proteins for S. mutans survival on teeth in the presence of early colonizing oral streptococci

Oxidative Inactivation of Mitochondrial Aconitase Results in Iron and H2O2-Mediated Neurotoxicity in Rat Primary Mesencephalic Cultures

BACKGROUND:Mitochondrial oxidative stress is a contributing factor in the etiology of numerous neuronal disorders. However, the precise mechanism(s) by which mitochondrial reactive oxygen species (ROS) modify cellular targets to induce the death of neurons remains unknown. The goal of this study was to determine if oxidative inactivation of mitochondrial aconitase (m-aconitase) resulted in the release of redox-active iron (Fe2+) and hydrogen peroxide (H2O2) and whether this contributes to cell death. METHODOLOGY/PRINCIPAL FINDINGS:Incubation of rat primary mesencephalic cultures with the redox cycling herbicide paraquat (PQ2+) resulted in increased production of H2O2 and Fe2+ at times preceding cell death. To confirm the role of m-aconitase as a source of Fenton reagents and death, we overexpressed m-aconitase using an adenoviral construct thereby increasing the target available for inactivation by ROS. Co-labeling studies identified astrocytes as the predominant cell type expressing transduced m-aconitase although neurons were identified as the primary cell type dying. Oxidative inactivation of m-aconitase overexpressing cultures resulted in exacerbation of H2O2 production, Fe2+ accumulation and increased neuronal death. Increased cell death in m-aconitase overexpressing cultures was attenuated by addition of catalase and/or a cell permeable iron chelator suggesting that neuronal death occurred in part via astrocyte-derived H2O2. CONCLUSIONS:These results suggest a role of ROS-sensitive m-aconitase as a source of Fe2+ and H2O2 and as a contributing factor to neurotoxicity

Ribonucleotide reductases of Salmonella Typhimurium : transcriptional regulation and differential role in pathogenesis

Ribonucleotide reductases (RNRs) are essential enzymes that carry out the de novo synthesis of deoxyribonucleotides by reducing ribonucleotides. There are three different classes of RNRs (I, II and III), all having different oxygen dependency and biochemical characteristics. Salmonella enterica serovar Typhimurium (S. Typhimurium) harbors class Ia, class Ib and class III RNRs in its genome. We have studied the transcriptional regulation of these three RNR classes in S. Typhimurium as well as their differential function during infection of macrophage and epithelial cells. Deletion of both NrdR and Fur, two main transcriptional regulators, indicates that Fur specifically represses the class Ib enzyme and that NrdR acts as a global repressor of all three classes. A Fur recognition sequence within the nrdHIEF promoter has also been described and confirmed by electrophoretic mobility shift assays (EMSA). In order to elucidate the role of each RNR class during infection, S. Typhimurium single and double RNR mutants (as well as Fur and NrdR mutants) were used in infection assays with macrophage and epithelial cell lines. Our results indicate class Ia to be mainly responsible for deoxyribonucleotide production during invasion and proliferation inside macrophages and epithelial cells. Neither class Ib nor class III seem to be essential for growth under these conditions. However, class Ib is able to maintain certain growth in an nrdAB mutant during the first hours of macrophage infection. Our results suggest that, during the early stages of macrophage infection, class Ib may contribute to deoxyribonucleotide synthesis by means of both an NrdR and a Fur-dependent derepression of nrdHIEF due to hydrogen peroxide production and DNA damage associated with the oxidative burst, thus helping to overcome the host defenses

Mitochondrial Changes in Ageing Caenorhabditis elegans – What Do We Learn from Superoxide Dismutase Knockouts?

One of the most popular damage accumulation theories of ageing is the mitochondrial free radical theory of ageing (mFRTA). The mFRTA proposes that ageing is due to the accumulation of unrepaired oxidative damage, in particular damage to mitochondrial DNA (mtDNA). Within the mFRTA, the “vicious cycle” theory further proposes that reactive oxygen species (ROS) promote mtDNA mutations, which then lead to a further increase in ROS production. Recently, data have been published on Caenorhabditis elegans mutants deficient in one or both forms of mitochondrial superoxide dismutase (SOD). Surprisingly, even double mutants, lacking both mitochondrial forms of SOD, show no reduction in lifespan. This has been interpreted as evidence against the mFRTA because it is assumed that these mutants suffer from significantly elevated oxidative damage to their mitochondria. Here, using a novel mtDNA damage assay in conjunction with related, well established damage and metabolic markers, we first investigate the age-dependent mitochondrial decline in a cohort of ageing wild-type nematodes, in particular testing the plausibility of the “vicious cycle” theory. We then apply the methods and insights gained from this investigation to a mutant strain for C. elegans that lacks both forms of mitochondrial SOD. While we show a clear age-dependent, linear increase in oxidative damage in WT nematodes, we find no evidence for autocatalytic damage amplification as proposed by the “vicious cycle” theory. Comparing the SOD mutants with wild-type animals, we further show that oxidative damage levels in the mtDNA of SOD mutants are not significantly different from those in wild-type animals, i.e. even the total loss of mitochondrial SOD did not significantly increase oxidative damage to mtDNA. Possible reasons for this unexpected result and some implications for the mFRTA are discussed

A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence

<p>Abstract</p> <p>Background</p> <p>The genome of <it>Pseudomonas aeruginosa </it>contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, <it>ppkA</it>, has been implicated in <it>P. aeruginosa </it>virulence. Together with the adjacent <it>pppA </it>phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a <it>pppA-ppkA </it>double mutant and characterised its phenotype and transcriptomic profiles.</p> <p>Results</p> <p>Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that <it>pppA-ppkA </it>deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the <it>pppA-ppkA </it>mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the <it>pppA </it>and <it>ppkA </it>genes were expressed ectopically.</p> <p>Conclusions</p> <p>Our results suggest that in addition to its crucial role in controlling the activity of <it>P. aeruginosa </it>H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions.</p

Proteomic and Physiological Responses of Kineococcus radiotolerans to Copper

Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0–1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R2 = 0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration

Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved

The spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a >10-fold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens, and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. +/- NO), and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO