Supplementation Of Growth Medium Research Articles

The Vibrio cholerae fatty acid regulatory protein, FadR, represses transcription of plsB, the gene encoding the first enzyme of membrane phospholipid biosynthesis

Glycerol-3-phosphate (sn-glycerol-3-P, G3P) acyltransferase catalyses the first committed step in the biosynthesis of membrane phospholipids, the acylation of G3P to form 1-acyl G3P (lysophosphatidic acid). The paradigm G3P acyltransferase is the Escherichia coli plsB gene product which acylates position-1 of G3P using fatty acids in thioester linkage to either acyl carrier protein (ACP) or CoA as acyl donors. Although the E. coli plsB gene was discovered about 30 years ago, no evidence for transcriptional control of its expression has been reported. However A.E. Kazakov and co-workers (J Bacteriol 2009; 191: 52-64) reported the presence of a putative FadR binding site upstream of the candidate plsB genes of Vibrio cholerae and three other Vibrio species suggesting that plsB might be regulated by FadR, a GntR family transcription factor thus far known only to regulate fatty acid synthesis and degradation. We report that the V. cholerae plsB homologue restored growth of E. coli strain BB26-36 which is a G3P auxotroph due to an altered G3P acyltransferase activity. The plsB promoter was also mapped and the predicted FadR-binding palindrome was found to span positions -19 to -35, upstream of the transcription start site. Gel shift assays confirmed that both V. cholerae FadR and E. coli FadR bound the V. cholerae plsB promoter region and binding was reversed upon addition of long-chain fatty acyl-CoA thioesters. The expression level of the V. cholerae plsB gene was elevated two- to threefold in an E. coli fadR null mutant strain indicating that FadR acts as a repressor of V. cholerae plsB expression. In both E. coli and V. cholerae the β-galactosidase activity of transcriptional fusions of the V. cholerae plsB promoter to lacZ increased two- to threefold upon supplementation of growth media with oleic acid. Therefore, V. cholerae co-ordinates fatty acid metabolism with 1-acyl G3P synthesis.

Effects of substrate nanopatterning on human osteosarcoma cells (SaOs-2) behavior

Engineering of the cellular microenvironment has become an attractive strategy to guide cellular activities such as spreading, motility, proliferation and differentiation. From a technological perspective, the physical crosstalk between a cell and its surrounding represents a design parameter that may be modulated to achieve desired osseointegration in orthopedic and dental implants. In this study we use a surface engineering approach to tap into the interaction between the cell and its surroundings in order to modulate osteogenic adhesion-dependent differentiation and bone tissue formation. The effectiveness of this approach was studied by observing the in vitro cellular behavior of human osteoblastic cells (SaOs-2) seeded on silicon substrates with different nano-scale surface patterns. Our findings suggest that substrate nanopattern geometry can differentially control early cell differentiation decisions. Interestingly, rescue of the differentiation process by supplementation of growth medium with soluble differentiation factors did not appear to compensate for the differences in the early cellular fate decisions observed on the different patterns. Critically, combining surface topography and soluble differentiation factors appeared to modulate the speed of the differentiation process and its shut down at the end of terminal differentiation. Taken together, our findings suggest that cells recognize physical nano-scale topography as an instructive signal that is integrated to fine-tune the multimodal control and guidance of cell behavior and cell fate. This information may find utility in the design of orthopedic and dental implants or innovative bio-materials for regenerative medicine.

PapR peptide maturation: role of the NprB protease inBacillus cereus569 PlcR/PapR global gene regulation

The global transcriptional regulator PlcR controls gene expression in Bacillus cereus and Bacillus thuringiensis. Activity of PlcR is regulated by PapR, the product of an ORF located immediately downstream of plcR. To be active in B. cereus, PapR must be secreted and then processed to the mature peptide by an unknown protease. This peptide is transported by an oligopeptide permease into the cell, where it activates PlcR. In this study, we show that the neutral protease B (NprB) secreted by B. cereus 569 is required for extracellular PapR maturation. Purified recombinant NprB processed the synthetic PapR propeptide to produce a set of peptides derived from the C-terminal domain of PapR. Supplementation of growth media with synthetic PapR-derived C-terminal 5-, 7-, 8- and 27-amino acid (aa) peptides caused activation of intracellular PlcR in a PapR-deficient strain of B. cereus 569 while only the 5- and 7-aa peptides activated PlcR in a nprB mutant. The maximum activity was found for the 7-mer peptide. However, even the 7-mer peptide could not activate PlcR with a C-terminal truncation of as few as 6 aa. This indicates that interactions of the C-terminal regions of both PlcR and PapR are important in transcriptional activation of the B. cereus 569 PlcR regulon.

Phosphatidylethanolamine Is Required for Normal Cell Morphology and Cytokinesis in the Fission Yeast, Schizosaccharomyces pombe

We describe the characterization of three predicted genes, designated psd1‐3, in the fission yeast, Schizosaccharomyces pombe, encoding phosphatidylserine decarboxylases (PSDs), which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine (PE). While S. pombe mutants carrying deletions in any one or two psd genes are viable in both rich and minimal media, psd1‐3δ mutants carrying deletions in all three psd genes grow slowly in rich medium and are inviable in minimal medium, indicating that the psd1‐3 gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to PE by the Kennedy pathway, restores growth to psd1‐3δ cells in minimal medium, indicating that PE is essential for S. pombe cell growth. psd1‐3δ cells appear morphologically indistinguishable from wild type S. pombe cells in medium supplemented with ethanolamine but when cultured in non‐supplemented medium produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that PE is essential for cell growth and for normal morphology and cytokinesis in S. pombe and illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of PE.

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

Nicotinamide phosphoribosyltransferase imparts human endothelial cells with extended replicative lifespan and enhanced angiogenic capacity in a high glucose environment

Endothelial dysfunction is a characteristic of aging-related vascular disease and is worsened during diabetes. High glucose can impair endothelial cell (EC) function through cellular accumulation of reactive oxygen species, an insult that can also limit replicative lifespan. Nicotinamide phosphoribosyltransferase (Nampt), also known as PBEF and visfatin, is rate-limiting for NAD+ salvage from nicotinamide and confers resistance to oxidative stress via SIRT1. We therefore sought to determine if Nampt expression could resist the detrimental effects of high glucose and confer a survival advantage to human vascular EC in this pathologic environment. Human aortic EC were infected with retrovirus encoding eGFP or eGFP-Nampt, and FACS-selected to yield populations with similar, modest transgene expression. Using a chronic glucose exposure model we tracked EC populations to senescence, assessed cellular metabolism, and determined in vitro angiogenic function. Overexpression of Nampt increased proliferation and extended replicative lifespan, and did so preferentially during glucose overload. Nampt expression delayed markers of senescence and limited reactive oxygen species accumulation in high glucose through a modest increase in aerobic glycolysis. Furthermore, tube networks formed by Nampt-overexpressing EC were more extensive and glucose-resistant, in accordance with SIRT1-mediated repression of the anti-angiogenic transcription factor, FoxO1. We conclude that Nampt enables proliferating human EC to resist the oxidative stress of aging and of high glucose, and to productively use excess glucose to support replicative longevity and angiogenic activity. Enhancing endothelial Nampt activity may thus be beneficial in scenarios requiring EC-based vascular repair and regeneration during aging and hyperglycemia, such as atherosclerosis and diabetes-related vascular disease.

Menaquinone synthesis is critical for maintaining mycobacterial viability during exponential growth and recovery from non‐replicating persistence

Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non-replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low microM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non-replicating, persistent M. tuberculosis. Metabolic labelling studies and quantification of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.

Phosphatidylethanolamine Is Required for Normal Cell Morphology and Cytokinesis in the Fission Yeast Schizosaccharomyces pombe

To investigate the contributions of phosphatidylethanolamine to the growth and morphogenesis of the fission yeast Schizosaccharomyces pombe, we have characterized three predicted genes in this organism, designated psd1, psd2, and psd3, encoding phosphatidylserine decarboxylases, which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine in both eukaryotic and prokaryotic organisms. S. pombe mutants carrying deletions in any one or two psd genes are viable in complex rich medium and synthetic defined minimal medium. However, mutants carrying deletions in all three psd genes (psd1-3Delta mutants) grow slowly in rich medium and are inviable in minimal medium, indicating that the psd1 to psd3 gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to phosphatidylethanolamine by the Kennedy pathway, restores growth to psd1-3Delta cells in minimal medium, indicating that phosphatidylethanolamine is essential for S. pombe cell growth. psd1-3Delta cells produce lower levels of phosphatidylethanolamine than wild-type cells, even in medium supplemented with ethanolamine, indicating that the Kennedy pathway can only partially compensate for the loss of phosphatidylserine decarboxylase activity in S. pombe. psd1-3Delta cells appear morphologically indistinguishable from wild-type S. pombe cells in medium supplemented with ethanolamine, but when cultured in nonsupplemented medium, they produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that phosphatidylethanolamine is essential for cell growth and for normal cytokinesis and cellular morphogenesis in S. pombe, and they illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of phosphatidylethanolamine.

Polyamines during somatic embryo development in Norway spruce (Picea abies [L.

Contents of free polyamines (putrescine, spermidine and spermine) were determined in different developmental stages of Norway spruce (<I>Picea abies</I> [L.] Karst.) somatic embryos by means of HPLC. Determinations were performed embryogenic tissue after 4 weeks of the growth on proliferation medium, after 2 and 5 weeks of the culturing on maturation medium, and 2 weeks after desiccation. Maturation of somatic embryos (after 5 weeks) was accompanied by increase of concentrations of putrescine (2.3 times) and spermidine (3.2 times). In comparison with above mentioned polyamines, spermine concentrations were significantly lower (4.3 times). Two weeks after desiccation, the concentrations of putrescine decreased 5.4 times and spermidine 2.2 times in comparison with mature embryos. To improve the efficiency of somatic embryogenesis of less responsive genotypes, the supplementation of growth media by polyamines is discussed.

Novel Modification of Growth Medium Enables Efficient E. coli Expression and Simple Purification of an Endotoxin-Free Recombinant Murine hsp70 Protein

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-D-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite, N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other His.

Chitinolytic activity of the sheep rumen ciliate Diploplastron affine

Supplementation of the rumen ciliate Diploplastron affine growth medium with commercial chitin stimulated growth of ciliates and the density of their population was positively correlated with chitin doses (r = 0.95; p < 0.01). The cell-free extracts prepared from bacteria-free ciliates degraded chitin to N-acetyl-D: -glucosamine and chitobiose. Three exochitinases, two endochitinases and two beta-N-acetylglucosaminidases were identified in the cell-free extract of protozoa. The molar mass of exochitinases was 80, 65 and 30 kDa, and endochitinases 75 and 50 kDa; the molar mass of one of the identified beta-N-acetylglucosaminidases was 45 kDa.

The PduX Enzyme of Salmonella enterica Is an l-Threonine Kinase Used for Coenzyme B12 Synthesis

Here, the PduX enzyme of Salmonella enterica is shown to be an L-threonine kinase used for the de novo synthesis of coenzyme B(12) and the assimilation of cobyric acid (Cby). PduX with a C-terminal His tag (PduX-His(6)) was produced at high levels in Escherichia coli, purified by nickel affinity chromatography, and partially characterized. (31)P NMR spectroscopy established that purified PduX-His(6) catalyzed the conversion of l-threonine and ATP to L-threonine-O-3-phosphate and ADP. Enzyme assays showed that ATP was the preferred substrate compared with GTP, CTP, or UTP. PduX displayed Michaelis-Menten kinetics with respect to both ATP and l-threonine and nonlinear regression was used to determine the following kinetic constants: V(max) = 62.1 +/- 3.6 nmol min(-1) mg of protein(-1); K(m)(, ATP) = 54.7 +/- 5.7 microm and K(m)(,Thr) = 146.1 +/- 8.4 microm. Growth studies showed that pduX mutants were impaired for the synthesis of coenzyme B(12) de novo and from Cby, but not from cobinamide, which was the expected phenotype for an L-threonine kinase mutant. The defect in Cby assimilation was corrected by ectopic expression of pduX or by supplementation of growth medium with L-threonine-O-3-phosphate, providing further support that PduX is an L-threonine kinase. In addition, a bioassay showed that a pduX mutant was impaired for the de novo synthesis of coenzyme B(12) as expected. Collectively, the genetic and biochemical studies presented here show that PduX is an L-threonine kinase used for AdoCbl synthesis. To our knowledge, PduX is the first enzyme shown to phosphorylate free L-threonine and the first L-threonine kinase shown to function in coenzyme B(12) synthesis.

Functional Characterization of the Fission Yeast Phosphatidylserine Synthase Gene, pps1 , Reveals Novel Cellular Functions for Phosphatidylserine

To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1Delta mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that pps1 encodes the major phosphatidylserine synthase activity in S. pombe. Supplementation of growth medium with ethanolamine partially suppresses the growth-defective phenotype of pps1Delta cells, reflecting the likely importance of phosphatidylserine as a precursor for phosphatidylethanolamine in S. pombe. In medium lacking ethanolamine, pps1Delta mutants exhibit striking cell morphology, cytokinesis, actin cytoskeleton, and cell wall remodeling and integrity defects. Overexpression of pps1 likewise leads to defects in cell morphology and cytokinesis, thus implicating phosphatidylserine as a dosage-dependent regulator of these processes. During log-phase growth, green fluorescent protein-Pps1p fusion proteins are concentrated at the cell and nuclear peripheries as well as presumptive endoplasmic reticulum membranes, while in stationary-phase cells, they are redistributed to unusual cytoplasmic structures of unknown origin. Moreover, stationary-phase pps1Delta cultures retain very poor viability relative to wild-type S. pombe cells, even in medium containing ethanolamine, demonstrating a role for phosphatidylserine in the physiological adaptations required for stationary-phase survival. Our findings reveal novel cellular functions for phosphatidylserine and emphasize the usefulness of S. pombe as a model organism for elucidating potentially conserved biological and molecular functions of this phospholipid.

Interaction with mycorrhiza helper bacterium Streptomyces sp. AcH 505 modifies organisation of actin cytoskeleton in the ectomycorrhizal fungus Amanita muscaria (fly agaric)

The actin cytoskeleton (AC) of fungal hyphae is a major determinant of hyphal shape and morphogenesis, implicated in controlling tip structure and secretory vesicle delivery. Hyphal growth of the ectomycorrhizal fungus Amanita muscaria and symbiosis formation with spruce are promoted by the mycorrhiza helper bacterium Streptomyces sp. AcH 505 (AcH 505). To investigate structural requirements of growth promotion, the effect of AcH 505 on A. muscaria hyphal morphology, AC and actin gene expression were studied. Hyphal diameter and mycelial density decreased during dual culture (DC), and indirect immunofluorescence microscopy revealed that the dense and polarised actin cap in hyphal tips of axenic A. muscaria changes to a loosened and dispersed structure in DC. Supplementation of growth medium with cell-free bacterial supernatant confirmed that reduction in hyphal diameter and AC changes occurred at the same stage of growth. Transcript levels of both actin genes isolated from A. muscaria remained unaltered, indicating that AC changes are regulated by reorganisation of the existing actin pool. In conclusion, the AC reorganisation appears to result in altered hyphal morphology and faster apical extension. The thus improved spreading of hyphae and increased probability to encounter plant roots highlights a mechanism behind the mycorrhiza helper effect.

The FRD3-Mediated Efflux of Citrate into the Root Vasculature Is Necessary for Efficient Iron Translocation

Iron, despite being an essential micronutrient, becomes toxic if present at high levels. As a result, plants possess carefully regulated mechanisms to acquire iron from the soil. The ferric reductase defective3 (frd3) mutant of Arabidopsis (Arabidopsis thaliana) is chlorotic and exhibits constitutive expression of its iron uptake responses. Consequently, frd3 mutants overaccumulate iron; yet, paradoxically, the frd3 phenotypes are due to a reduction in the amount of iron present inside frd3 leaf cells. The FRD3 protein belongs to the multidrug and toxin efflux family, members of which are known to export low-M(r) organic molecules. We therefore hypothesized that FRD3 loads an iron chelator necessary for the correct distribution of iron throughout the plant into the xylem. One such potential chelator is citrate. Xylem exudate from frd3 plants contains significantly less citrate and iron than the exudate from wild-type plants. Additionally, supplementation of growth media with citrate rescues the frd3 phenotypes. The ectopic expression of FRD3-GFP results in enhanced tolerance to aluminum in Arabidopsis roots, a hallmark of organic acid exudation. Consistent with this result, approximately 3 times more citrate was detected in root exudate from plants ectopically expressing FRD3-GFP. Finally, heterologous studies in Xenopus laevis oocytes reveal that FRD3 mediates the transport of citrate. These results all strongly support the hypothesis that FRD3 effluxes citrate into the root vasculature, a process important for the translocation of iron to the leaves, as well as confirm previous reports suggesting that iron moves through the xylem as a ferric-citrate complex. Our results provide additional answers to long-standing questions about iron chelation in the vasculature and organic acid transport.

Primary mantle tissue culture from the bivalve mollusc Mytilus galloprovincialis: Investigations on the growth promoting activity of the serum used for medium supplementation

The main objective of the present work was to evaluate with a quantitative approach the effects of different chicken serum (CS) concentrations in the medium used for mussel mantle tissue culturing. Our results showed that a CS level of 20% was optimal. Under these conditions, the cultures reached a maximum mean number of mitotic figures per slide exceeding widely 100. Cultures were also achieved to test whether CS must be heat treated to inactivate complement components before use for medium enrichment. We demonstrated that heat inactivation did not significantly change the promoting activity of the CS. Finally, the growth-stimulatory properties of CS were compared to those of fetal calf serum (FCS). The best results were obtained with 30% FCS. The difference with 20% CS was not statistically significant, but the FCS yielded a much higher level of polyploid metaphases than the CS. Since it had no adverse effect such as polyploid metaphases induction, was readily commercially available and relatively less expensive than other additives, CS at the concentration of 20%, without heat-decomplementation, is routinely used as growth medium supplement for mussel mantle cell culturing in our laboratory. Even though our primary cultures do not have the potency of continuous cell lines, it is possible to use such cultures for in vitro experiments.

Structure and Dynamics of Micelle-bound Human α-Synuclein

Misfolding of the protein alpha-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val3-Val37 and Lys45-Thr92 form curved alpha-helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix.

RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR.

Transposon mutagenesis of rsbU leads to a biofilm-negative phenotype in Staphylococcus epidermidis. However, the pathway of this regulatory mechanism was unknown. To investigate the role of RsbU in the regulation of the alternative sigma factor sigma(B) and biofilm formation, we generated different mutants of the sigma(B) operon in S. epidermidis strains 1457 and 8400. The genes rsbU, rsbV, rsbW, and sigB, as well as the regulatory cascade rsbUVW and the entire sigma(B) operon, were deleted. Transcriptional analysis of sarA and the sigma(B)-dependent gene asp23 revealed the functions of RsbU and RsbV as positive regulators and of RsbW as a negative regulator of sigma(B) activity, indicating regulation of sigma(B) activity similar to that characterized for Bacillus subtilis and Staphylococcus aureus. Phenotypic characterization of the mutants revealed that the dramatic decrease of biofilm formation in rsbU mutants is mediated via sigma(B), indicating a crucial role for sigma(B) in S. epidermidis pathogenesis. However, biofilm formation in mutants defective in sigma(B) or its function could be restored in the presence of subinhibitory ethanol concentrations. Transcriptional analysis revealed that icaR is up-regulated in mutants lacking sigma(B) function but that icaA transcription is down-regulated in these mutants, indicating a sigma(B)-dependent regulatory intermediate negatively regulating IcaR. Supplementation of growth media with ethanol decreased icaR transcription, leading to increased icaA transcription and a biofilm-positive phenotype, indicating that the ethanol-dependent induction of biofilm formation is mediated by IcaR. This icaR-dependent regulation under ethanol induction is mediated in a sigma(B)-independent manner, suggesting at least one additional regulatory intermediate in the biofilm formation of S. epidermidis.

Defenses against oxidative stress in Neisseria gonorrhoeae and Neisseria meningitidis: distinctive systems for different lifestyles.

Defenses against oxidative stress are crucial for the survival of the pathogens Neisseria meningitidis and Neisseria gonorrhoeae. An Mn(II) uptake system is involved in manganese (Mn)-dependent resistance to superoxide radicals in N. gonorrhoeae. Here, we show that accumulation of Mn also confers resistance to hydrogen peroxide killing via a catalase-independent mechanism. An mntC mutant of N. meningitidis is susceptible to oxidative killing, but supplementation of growth media with Mn does not enhance the organism's resistance to oxidative killing. N. meningitidis is able to grow in the presence of millimolar levels of Mn ion, in contrast to N. gonorrhoeae, whose growth is retarded at Mn concentrations >100 micromol/L, indicating that Mn homeostasis in the 2 species is probably quite different. N. meningitidis superoxide dismutase B plays a role in protection against oxidative killing. However, a sodC mutant of N. meningitidis is no more sensitive to oxidative killing than is the wild type. A cytochrome c peroxidase (Ccp) is present in N. gonorrhoeae but not in N. meningitidis. Investigations of a ccp mutant revealed a role for Ccp in protection against hydrogen peroxide killing. These differences in oxidative defenses in the pathogenic Neisseria are most likely a result of their localization in different ecological niches.

Acid-responsive gene induction of ammonia-producing enzymes in Helicobacter pylori is mediated via a metal-responsive repressor cascade.

Although the adaptive mechanisms allowing the gastric pathogen Helicobacter pylori to survive acid shocks have been well documented, the mechanisms allowing growth at mildly acidic conditions (pH approximately 5.5) are still poorly understood. Here we demonstrate that H. pylori strain 26695 increases the transcription and activity of its urease, amidase, and formamidase enzymes four- to ninefold in response to growth at pH 5.5. Supplementation of growth medium with NiCl2 resulted in a similar induction of urease activity (at low NiCl2 concentration) and amidase activity (at > or = 500 micro M NiCl2) but did not affect formamidase activity. Mutation of the fur gene, which encodes an iron-responsive repressor of both amidases, resulted in a constitutively high level of amidase and formamidase activity at either pH but did not affect urease activity at pH 7.0 or pH 5.5. In contrast, mutation of the nikR gene, encoding the nickel-responsive activator of urease expression, resulted in a significant reduction of acid-responsive induction of amidase and formamidase activity. Finally, acid-responsive repression of fur transcription was absent in the H. pylori nikR mutant, whereas transcription of the nikR gene itself was increased at pH 5.5 in wild-type H. pylori. We hypothesize that H. pylori uses a repressor cascade to respond to low pH, with NikR initiating the response directly via the urease operon and indirectly via the members of the Fur regulon.