Control of Redox Balance by the Stringent Response Regulatory Protein Promotes Antioxidant Defenses of Salmonella

We report herein a critical role for the stringent response regulatory DnaK suppressor protein (DksA) in the coordination of antioxidant defenses. DksA helps fine-tune the expression of glutathione biosynthetic genes and discrete steps in the pentose phosphate pathway and tricarboxylic acid cycle that are associated with the generation of reducing power. Control of NAD(P)H/NAD(P)⁺ redox balance by DksA fuels downstream antioxidant enzymatic systems in nutritionally starving Salmonella. Conditional expression of the glucose-6-phosphate dehydrogenase-encoding gene zwf, shown here to be under DksA control, increases both the NADPH pool and antioxidant defenses of dksA mutant Salmonella. The DksA-mediated coordination of redox balance boosts the antioxidant defenses of stationary phase bacteria. Not only does DksA increase resistance of Salmonella against hydrogen peroxide (H₂O₂), but it also promotes fitness of this intracellular pathogen when exposed to oxyradicals produced by the NADPH phagocyte oxidase in an acute model of infection. Given the role of DksA in the adjustment of gene expression in most bacteria undergoing nutritional deprivation, our findings raise the possibility that the control of central metabolic pathways by this regulatory protein maintains redox homeostasis essential for antioxidant defenses in phylogenetically diverse bacterial species.     

The role of enteric bacteria in the anerobic metabolism of 5-aminosalicylate

The primary (and inactive) enteric metabolite of 5-aminosalicylate is N-acetyl-5-amino-salicylate. Previous studies have demonstrated acetylation of this anti-inflammatory agent by intestinal and bacterial homogenates. To assess the contribution of anerobic bacteria to the N-acetylation in vivo, we have measured the production of N-acetyl-5-aminosalicylate in anerobic microculture. Our results indicate that enteric bacteria play a minor role in N-acetylation, but may contribute to the production of other metabolites of pharmacologic and toxicological interest.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

The effect of salinity on the survival,growth, sporulation and infection of Phytophthora ramorum

Phytophthora ramorum has been found in waterways outside infested nurseries, but little is known about its behavior in water. This study examined the effect of salinity on survival, growth, sporulation, and infection. P. ramorum survival and growth was negatively correlated with salt concentration (range of 0–45 g l⁻¹), but showed a level of tolerance even at 45 g l⁻¹. No sporangia were observed in cultures with higher than 20 g l⁻¹ of salt and zoospores were not released from sporangia above 14 g l⁻¹. Water sources with different salinity were used to understand the environment where P. ramorum can survive and infect host material. Water from natural bodies and water amended with different salt concentrations were added to P. ramorum-infested sand and baited with rhododendron leaf disks. Infection decreased with increasing salt concentration and increased with higher initial concentration of P. ramorum. This research helps to better understand the effects of water quality on survival and infectivity of P. ramorum, expanding the potential survey range.     

The cooperativity phenomena in a pigment-protein complex of light-harvesting antenna revealed by picosecond absorbance difference spectroscopy

A model of the cooperative changes in optical properties of light-harvesting bacteriochlorophyll molecules of complex B890 in response to the absorption of light quanta is proposed. According to the model, each antenna chromophore may persist in either of two optically non-excited states, R and T. The occurrence of at least one excitation per complex causes all optically non-excited chromophores of the complex to be converted from state R to state T. The theory is shown to be in good agreement with experimental ‘light curves’ (ΔAvs intensity of picosecond excitation pulse) for the ‘minor’ and ‘major’ signals of light-harvesting bacteriochlorophylls of complex B890 from Chromatium minutissimum.     

NleB/SseKs ortholog effectors as a general bacterial monoglycosyltransferase for eukaryotic proteins

Protein glycosylation is the most common post-translational modification as more than 50% of all human proteins are glycosylated. Pathogenic bacteria glycosylation allows adhesion to host cells and manipulates eukaryotic functions. A variety of acceptor proteins in bacterial glycosylation was recently discovered. Especially NleB/SseKs type III effectors unexpectedly glycosylate a poor nucleophile arginine. Other pathogenic toxins modify the unusual tyrosine, as well as canonical serine/threonine residues. And a huge diversity is found in target proteins; Rho/Ras families, death domains and moreover themselves for autoglycosylation. However, in spite of this acceptor diversity, all their sugar donors are only UDP-Glc/-GlcNAc and structural alignments as liganded show their catalytic cores are geometrically conserved, where DRY and DXD motives and W residues equally position to hold the sugar donors and to π-π bind with a uridine ring, respectively. Therefore, bacterial glycosyltransferases have a key for carbohydrate research problems concerning the sugar donors and target proteins recognition.     

Statistical optimization of bacterial cellulose production by Leifsonia soli and its physico-chemical characterization

Bacterial cellulose has multiple applications in various industries such as food, biomedical, textile due to its uniqueness of being a better bio-compatible coating agent, binding material, etc. In this study, optimization of the culture medium for producing BC from Leifsonia soli was carried out by selecting different parameters. Five significant factors such as maltose, pH, incubation days, soy whey and calcium chloride were estimated through ANOVA based response surface methodology. Maximum cellulose production (5.97 g/L) was obtained where maltose 1 % (w/v) supplemented with 0.8 % (v/v) soy whey and calcium chloride 0.8 % (w/v) at pH 6.5 for 7 days of incubation. In addition, assurance of cellulose production from bacteria was done by using High-performance liquid chromatography analysis. Further, the structure and purity of obtained cellulose were examined by SEM and elemental analysis where it was observed that the sample holds the value of carbon 44.1 ± 0.20 % and hydrogen 6.2 ± 0.3 %, respectively. This study concludes that the addition of maltose and soy whey could be used as carbon, nitrogen sources and calcium chloride was used as an additive for the bacterial cellulose production compared to the Hestrin Schramm medium. In addition, the calculated water holding capacity of the sample was found to be 73 %.     

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq)

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.