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PubMed2006

AmiC functions as an N-acetylmuramyl-l-alanine amidase necessary for cell separation and can promote autolysis in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is prone to undergo autolysis under many conditions not conducive to growth. The role of autolysis during gonococcal infection is not known, but possible advantages for the bacterial population include provision of nutrients to a starving population, modulation of the host immune response by released cell components, and donation of DNA for natural transformation. Biochemical studies indicated that an N-acetylmuramyl-l-alanine amidase is responsible for cell wall breakdown during autolysis. In order to better understand autolysis and in hopes of creating a nonautolytic mutant, we mutated amiC, the gene for a putative peptidoglycan-degrading amidase in N. gonorrhoeae. Characterization of peptidoglycan fragments released during growth showed that an amiC mutant did not produce free disaccharide, consistent with a role for AmiC as an N-acetylmuramyl-l-alanine amidase. Compared to the wild-type parent, the mutant exhibited altered growth characteristics, including slowed exponential-phase growth, increased turbidity in stationary phase, and increased colony opacity. Thin-section electron micrographs showed that mutant cells did not fully separate but grew as clumps. Complementation of the amiC deletion mutant with wild-type amiC restored wild-type growth characteristics and transparent colony morphology. Overexpression of amiC resulted in increased cell lysis, supporting AmiC's purported function as a gonococcal autolysin. However, amiC mutants still underwent autolysis in stationary phase, indicating that other gonococcal enzymes are also involved in this process.

Bacterial Proteins↗
PubMed2006

Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide.

The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H(2)O(2)) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H(2)O(2) damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2).

Anti-Bacterial Agents↗
PubMed2006

Survival and growth in the presence of elevated copper: transcriptional profiling of copper-stressed Pseudomonas aeruginosa.

Transcriptional profiles of Pseudomonas aeruginosa exposed to two separate copper stress conditions were determined. Actively growing bacteria subjected to a pulse of elevated copper for a short period of time was defined as a "copper-shocked" culture. Conversely, copper-adapted populations were defined as cells actively growing in the presence of elevated copper. Expression of 405 genes changed in the copper-shocked culture, compared to 331 genes for the copper-adapted cultures. Not surprisingly, there were genes identified in common to both conditions. For example, both stress conditions resulted in up-regulation of genes encoding several active transport functions. However, there were some interesting differences between the two types of stress. Only copper-adapted cells significantly altered expression of passive transport functions, down-regulating expression of several porins belonging to the OprD family. Copper shock produced expression profiles suggestive of an oxidative stress response, probably due to the participation of copper in Fenton-like chemistry. Copper-adapted populations did not show such a response. Transcriptional profiles also indicated that iron acquisition is fine-tuned in the presence of copper. Several genes induced under iron-limiting conditions, such as the siderophore pyoverdine, were up-regulated in copper-adapted populations. Interesting exceptions were the genes involved in the production of the siderophore pyochelin, which were down-regulated. Analysis of the copper sensitivity of select mutant strains confirmed the array data. These studies suggest that two resistance nodulation division efflux systems, a P-type ATPase, and a two-component regulator were particularly important for copper tolerance in P. aeruginosa.

Adaptation, Physiological↗
PubMed2006

Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae.

Toxin-antitoxin (TA) loci, which were initially characterized as plasmid stabilization agents, have in recent years been detected on the chromosomes of numerous free-living bacteria. Vibrio cholerae, the causative agent of cholera, contains 13 putative TA loci, all of which are clustered within the superintegron on chromosome II. Here we report the characterization of the V. cholerae higBA locus, also known as VCA0391/2. Deletion of higA alone was not possible, consistent with predictions that it encodes an antitoxin, and biochemical analyses confirmed that HigA interacts with HigB. Transient exogenous expression of the toxin HigB dramatically slowed growth of V. cholerae and Escherichia coli and reduced the numbers of CFU by several orders of magnitude. HigB toxicity could be counteracted by simultaneous or delayed production of HigA, although HigA's effect diminished as the delay lengthened. Transcripts from endogenous higBA increased following treatment of V. cholerae with translational inhibitors, presumably due to reduced levels of HigA, which represses the higBA locus. However, no higBA-dependent cell death was observed in response to such stimuli. Thus, at least under the conditions tested, activation of endogenous HigB does not appear to be bactericidal.

Amino Acid Sequence↗
PubMed2006

Minimum length requirement of the flexible N-terminal translocation subdomain of colicin E3.

The 315-residue N-terminal T domain of colicin E3 functions in translocation of the colicin across the outer membrane through its interaction with outer membrane proteins including the OmpF porin. The first 83 residues of the T domain are known from structure studies to be disordered. This flexible translocation subdomain contains the TolB box (residues 34 to 46) that must cross the outer membrane in an early translocation event, allowing the colicin to bind to the TolB protein in the periplasm. In the present study, it was found that cytotoxicity of the colicin requires a minimum length of 19 to 23 residues between the C terminus (residue 46) of the TolB box and the end of the flexible subdomain (residue 83). Colicin E3 molecules of sufficient length display normal binding to TolB and occlusion of OmpF channels in vitro. The length of the N-terminal subdomain is critical because it allows the TolB box to cross the outer membrane and interact with TolB. It is proposed that the length constraint is a consequence of ordered structure in the downstream segment of the T domain (residues 84 to 315) that prevents its insertion through the outer membrane via a translocation pore that includes OmpF.

Bacterial Outer Membrane Proteins↗
PubMed2006

Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus.

Oxidative-stress resistance in Staphylococcus aureus is linked to metal ion homeostasis via several interacting regulators. In particular, PerR controls the expression of a regulon of genes, many of which encode antioxidants. Two PerR regulon members, ahpC (alkylhydroperoxide reductase) and katA (catalase), show compensatory regulation, with independent and linked functions. An ahpC mutation leads to increased H2O2 resistance due to greater katA expression via relief of PerR repression. Moreover, AhpC provides residual catalase activity present in a katA mutant. Mutation of both katA and ahpC leads to a severe growth defect under aerobic conditions in defined media (attributable to lack of catalase activity). This results in the inability to scavenge exogenous or endogenously produced H2O2, resulting in accumulation of H2O2 in the medium. This leads to DNA damage, the likely cause of the growth defect. Surprisingly, the katA ahpC mutant is not attenuated in two independent models of infection, which implies reduced oxygen availability during infection. In contrast, both AhpC and KatA are required for environmental persistence (desiccation) and nasal colonization. Thus, oxidative-stress resistance is an important factor in the ability of S. aureus to persist in the hospital environment and so contribute to the spread of human disease.

Animals↗
PubMed2007

Role of the coronavirus E viroporin protein transmembrane domain in virus assembly.

Coronavirus envelope (E) proteins are small (approximately 75- to 110-amino-acid) membrane proteins that have a short hydrophilic amino terminus, a relatively long hydrophobic membrane domain, and a long hydrophilic carboxy-terminal domain. The protein is a minor virion structural component that plays an important, not fully understood role in virus production. It was recently demonstrated that the protein forms ion channels. We investigated the importance of the hydrophobic domain of the mouse hepatitis coronavirus (MHV) A59 E protein. Alanine scanning insertion mutagenesis was used to examine the effect of disruption of the domain on virus production in the context of the virus genome by using a MHV A59 infectious clone. Mutant viruses exhibited smaller plaque phenotypes, and virus production was significantly crippled. Analysis of recovered viruses suggested that the structure of the presumed alpha-helical structure and positioning of polar hydrophilic residues within the predicted transmembrane domain are important for virus production. Generation of viruses with restored wild-type helical pitch resulted in increased virus production, but some exhibited decreased virus release. Viruses with the restored helical pitch were more sensitive to treatment with the ion channel inhibitor hexamethylene amiloride than were the more crippled parental viruses with the single alanine insertions, suggesting that disruption of the transmembrane domain affects the functional activity of the protein. Overall the results indicate that the transmembrane domain plays a crucial role during biogenesis of virions.

Alanine↗
PubMed2007

AMPK-mediated inhibition of mTOR kinase is circumvented during immediate-early times of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) infection increases synthetic rates in infected cells. The resulting increase in energy utilization could potentially increase the AMP:ATP ratio, causing activation of 5'-AMP-activated protein kinase (AMPK). Activated AMPK promotes inhibition of mammalian target of rapamycin (mTOR) kinase, which could be deleterious to the viral infection. Using the AMPK-activating drug 5-amino-4-imidazolecarboxamide ribose (AICAR), we showed that, by 12 h post-HCMV infection, inhibition of mTOR by AMPK is circumvented. However, growth curves showed that progeny virion production is inhibited when AICAR is added, suggesting other inhibitory effects of AICAR or activated AMPK.

AMP-Activated Protein Kinases↗
PubMed2006

Activities and survival of endophytic bacteria in white clover (Trifolium repens L.).

In this study, the genera, abundance, and activities of endophytic bacteria in field-grown white clover (Trifolium repens) and the fate of introduced antibiotic-tolerant bacteria in white clover tissues were investigated. Pseudomonas, Pantoea, and Corynebacterium were the most frequently isolated endophytic bacteria genera, whereas Xanthomonas, Microbacterium, and Cellulomonas occurred less frequently. The average bacterial populations in stolons and roots were approximately 100,000 colony-forming units (CFU) (g wet mass)-1. Of the 28 strains tested for activity, none were chitinolytic or able to inhibit the root pathogen Codinaea fertilis in vitro. However, Fusarium oxysporum and Cylindrocladium scoparium were inhibited by one and five strains, respectively. Four of seven strains tested depressed clover seedling growth. In pot experiments, colonization and recovery of spontaneous rifampicin-tolerant mutants (Rif+) of bacteria were studied in clover plants for periods up to 20 weeks. The strains used, sourced from white clover (endophytic and rhizoplane) and organic compost, had previously shown growth promotion potential of white clover seedlings by increasing plant mass and decreasing nematode numbers. In one experiment in this present study, five Rif+ strains were individually inoculated onto white clover seedlings, all five were re-isolated from shoots after 6 weeks and four strains were re-isolated after 20 weeks (numbers of Rif+ bacteria ranged from 51 to 200 CFU (g wet mass)-1). No Rif+ bacteria were isolated from root tissue at either time. In the second experiment, conducted with two strains of Rif+ bacteria, the population was highest in the shoots (range>500 CFU of Rif+ bacteria (g shoot fresh mass)-1) in weeks 2 and 3, declining to <200 CFU in week 5. Again, no Rif+ bacteria could be detected in roots. No Rif+ bacteria were recovered after 14 weeks for one of the strains. It appears that the main route of bacterial entry into seedlings was through stomata and that bacteria remained in the aerial parts of plants rather than migrating to the roots.

Bacteria↗
PubMed2006

Inhibition of cultivable bacteria by chlorhexidine treatment of dentin lesions treated with the ART technique.

The aim of this study was to examine the changes in the cultivable microflora of carious dentin before and after atraumatic restorative treatment (ART) and investigate the inhibitory effect of chlorhexidine-gluconate-based cavity disinfectant in the microflora. Using a split mouth design, 35 primary molar pairs with class II carious lesions in 35 patients (mean age 7.31+/-0.47 years) were selected. The total viable counts (TVC), Streptococcus mutans and lactobacilli were first measured in the center of the infected demineralized lesion and then from the hard dentine after caries removal by the ART technique. Chlorhexidine-gluconate (2%)-based cavity disinfectant was applied to one of the molar pairs and the other molar received no disinfectant treatment. Thereafter, all of the teeth were restored with glass ionomer cement (GIC). Cavities were reassessed after 6 months and again dentine samples were microbiologically investigated. Removal of carious dentine by ART significantly reduced TVC, S. mutans and lactobacilli. After 6 months, application of chlorhexidine exhibited a greater significant reduction in TVC (p=0.013), and a significant reduction in S. mutans compared to the nondisinfected group (p<0.001). A significant reduction in lactobacilli counts was observed in both groups after 6 months, but the difference between the disinfected and nondisinfected groups was not significant (p=0.056). ART was found to be effective in reducing the cultivable microflora and chlorhexidine-gluconate-based cavity disinfectant might serve as a suitable additional agent in inhibiting the residual bacteria in the dentine.

Analysis of Variance↗
PubMed2005

Abnormalities in the pulmonary innate immune system in cystic fibrosis.

Pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the basis for this susceptibility remains incompletely understood. One hypothesis is that CF airway surface liquid (ASL) is abnormal and interferes with neutrophil function. To study this possibility, we developed an in vitro system in which we collected ASL from primary cultures of normal and CF airway epithelial cells. Microbial killing was less efficient when bacteria were incubated with neutrophils in the presence of ASL from CF epithelia compared with normal ASL. Antimicrobial functions of human neutrophils were assessed in ASL from CF and normal epithelia using a combination of quantitative bacterial culture, flow cytometry, and microfluorescence imaging. The results of these assays of neutrophil function were indistinguishable in CF and normal ASL. In contrast, the direct bactericidal activity of ASL to Escherichia coli and to clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa was substantially less in CF than in normal ASL, even when highly diluted in media of identical ionic strength. Together, these observations indicate that the antimicrobial properties of ASL in CF are compromised in a manner independent of ionic strength of the ASL, and that this effect is not mediated through a direct effect of the ASL on phagocyte function.

Bronchi↗
PubMed2006

Antioxidant activity of L-ascorbic acid in wild-type and superoxide dismutase deficient strains of Saccharomyces cerevisiae.

Much has been published on the non-enzymatic antioxidant L-ascorbic acid (vitamin C), but even so its interaction with endogenous cellular defense systems has not yet been fully elucidated. Our study investigated the antioxidant activity of L-ascorbic acid in wild-type strain EG103 (SOD) Saccharomyces cerevisiae and isogenic mutant strains deficient in cytosolic superoxide dismutase (sod1delta), mitochondrial superoxide dismutase (sod2delta) or both (sod1delta sod2delta), metabolizing aerobically or anaerobically with and without the stressing agent paraquat. The results show that during both aerobic and anaerobic metabolism there was a significant increase in the survival of both wild-type S. cerevisiae cells and the mutant cells (sod1delta, sod2delta and sod1delta sod2delta) when pretreated with L-ascorbic acid before exposure to paraquat. Exposure to paraquat resulted in higher catalase activity but this significantly decreased when the cells were pre-treated with L-ascorbic acid. These results demonstrate that due to the damage caused by paraquat, the antioxidant protection of L-ascorbic acid seems to be mediated by catalase levels in yeast cells.

Aerobiosis↗