Purification of p47f,a Necrosis‐Inducing Protein Fraction from the Secretome of Phytophthora capsici

The oomycete Phytophthora capsici causes wilting disease in chilli pepper and another solanaceous plants, with important economic consequences. Although much investigation has been conducted about this pathogen, little is still known about which of its proteins are involved in the infection process. In this study, the bioassay‐guided fractionation of the secretome of P. capsici resulted in the purification of a phytotoxic protein fraction designated as p47f, capable of inducing wilting and necrosis on leaves of Capsicum chinense Jacq, and having a 47 kDa polypeptide with proteolytic activity as the major component. The isolated p47f fraction induced DNA degradation and decreased cell survival of C. chinense cell suspension culture. Sequencing of p47f indicated the presence of 15 proteins, which could be grouped into seven classes including a protease group, cell wall remodelling proteins and the transglutaminase elicitor M81D, among others. This is the first report of P. capsici secreting proteins that modulate cell responses mediated by ROS in the host.     

Unusual pathogenic bacterium isolated from microbial communities of bioaerosols at Chilean Patagonian lakes

Bioaerosols are transported from warm regions and lower latitudes of the planet to colder regions and higher latitudes, such as the Chilean Patagonia. The role of bioaerosols deposition in remote lake ecosystems is a potentially important process, but it has not yet been fully studied. The aim of this study was to detect and characterize potentially pathogenic viable microorganisms in bioaerosols in a pristine area. Samples were collected from the air, at three remote lakes in the Chilean Patagonia, using a sterile filtration system equipped with 0.2-μm-pore-size nitrocellulose filters. The bacterial community present in bioaerosols was studied using scanning electron microscopy (SEM) and denaturing gradient gel electrophoresis. Isolates were identified and characterized for phenotypic and 16S rDNA analysis and antibiotic resistance. SEM observations of samples from each lake showed the presence of bacteria with different morphologies, and after culturing, the identification results revealed that they were strains of Acinetobacter, Alcaligenes, Edwarsiella, Pseudomonas, Burklolderia, Moraxella, Sphingomonas and CDC NO-1. CDC NO-1, uncommonly isolated worldwide, stands out from the rest of the isolates because it is a rarely found bacterium so far associated with dog and cat bites and was found at two out of three pristine lakes studied (Alto and Verde). This work demonstrates, for the first time, the presence of CDC NO-1, a clinically important Gram-negative microorganism, in bioaerosols and first report of CDC-NO1 isolation in Chile. Besides its presence in remote lakes, its antibiotic resistance is worth mentioning.     

Deposition of sediments during a flood event on seasonally flooded forests of the lower Orinoco River and two of its black-water tributaries, Venezuela

Deposition of suspended sediments andtheir associated nutrients were estimated during theflood event of 1995 in the seasonally flooded forestsof the Mapire and Caura Rivers, two black-watertributaries of lower Orinoco, and on two islands ofthis white-water river. The deposition spanned a widerange from 0.07 kg m^–2 in the depositional barforests of the Mapire River to 73.60 kg m^–2 onthe flooded forests of the Orinoco Island site calledJarizo. This variation is associated with the dynamicnature of sediment mobilization, transport anddeposition, as well as with the different geomorphicenvironments and erosion processes upstream from thestudy sites. The deposited sediment in all the studyareas was highly quartzitic with a relatively highcontent of kaolinite and goethite. Only in thesediment of the Orinoco Islands was mica (illite)identified in a relatively high proportion. Thesemineralogical results reflect the intense weatheringprocesses in the catchment areas of the study rivers.The chemical composition of the deposited sedimentsshowed a great variability among the different studyareas, which is in part related to the mineralcomposition of the sediments and their particle sizedistribution. The highest concentrations of K, Caand Mg were found on the clay sediments of the Orinocoagricultural island. The total amount of depositednutrients varied over a wide range, which isinfluenced by the amount of deposited sediments. Inthe Jarizo Island site of lower Orinoco were depositedthe largest amount of nutrients. In the floodedforests of the Mapire River, the nutrient contributionby the deposited sediments to the nutrient cycling isrelatively low in the depositional-bar forests andpractically nonexistent in the forests sites onterraces.     

A new pathway for the synthesis of α‐ribazole‐phosphate in Listeria innocua

The genomes of Listeria spp. encode all but one of 25 enzymes required for the biosynthesis of adenosylcobalamin (AdoCbl; coenzyme B₁₂). Notably, all Listeria genomes lack CobT, the nicotinamide mononucleotide:5,6‐dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) enzyme that synthesizes the unique α‐linked nucleotide N¹‐(5‐phospho‐α‐d ‐ribosyl)‐DMB (α‐ribazole‐5′‐P, α‐RP), a precursor of AdoCbl. We have uncovered a new pathway for the synthesis of α‐RP in Listeria innocua that circumvents the lack of CobT. The cblT and cblS genes (locus tags lin1153 and lin1110) of L. innocua encode an α‐ribazole (α‐R) transporter and an α‐R kinase respectively. Results from in vivo experiments indicate that L. innocua depends on CblT and CblS activities to salvage exogenous α‐R, allowing conversion of the incomplete corrinoid cobinamide (Cbi) into AdoCbl. Expression of the L. innocua cblT and cblS genes restored AdoCbl synthesis from Cbi and α‐R in a Salmonella enterica cobT strain. LinCblT transported α‐R across the cell membrane, but not α‐RP or DMB. UV‐visible spectroscopy and mass spectrometry data identified α‐RP as the product of the ATP‐dependent α‐R kinase activity of LinCblS. Bioinformatics analyses suggest that α‐R salvaging occurs in important Gram‐positive human pathogens.     

Analysis of differentially upregulated proteins in ptsHIcrr− and rppH− mutants in Escherichia coli during an adaptive laboratory evolution experiment

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS⁻ derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH⁻) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH⁻ mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS⁻ and rppH⁻ mutations in the deep of a proteomic approach to understanding the relevance of rppH⁻ phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH⁻, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH⁻ were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS⁻ strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS⁻ derivatives during the ALE experiment. This result suggests that the evolving PTS⁻ population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH⁻ mutation in the PTS⁻ background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.

     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal⁻ ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.     

Spatio‐Temporal Variation of Terpenoids in Wild Plants of Pentalinon andrieuxii

Pentalinon andrieuxii (Müll .Arg .) B.F.Hansen & Wunderlin (Apocynaceae) is a vine native to the Yucatan peninsula, where it is widely used in Mayan traditional medicine to treat, among other ailments, the wounds caused by cutaneous leishmaniasis. Among the secondary metabolites isolated from P. andrieuxii are the triterpene betulinic acid and the chemically unusual tri‐norsesquiterpene urechitol A; however, to date, there is no existing knowledge about the accumulation dynamics of the ubiquitous betulinic acid or the novel urechitol A in the plant. In this article, we report on the accumulation of both secondary metabolites in wild individuals of P. andrieuxii; our results show that while the content of betulinic acid in plant leaves bears no apparent relation to plant ontogeny, the content of urechitol A in root tissue is clearly related to plant development.     

Role of heme oxygenase in heme-mediated inhibition of rat brain Na+−K+-ATPase: Protection by tin-protoporphyrin

Hemoglobin has been shown to inhibit brain Na⁺–K⁺-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na⁺–K⁺-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na⁺–K⁺-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na⁺–K⁺-ATPase. However, in the presence of heme oxygenase, heme inhibited Na⁺–K⁺-ATPase by 75%. Pretreatment of rats with SnCl₂, a known inducer of heme oxygenase, reduced the basal activity of the brain Na⁺–K⁺-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na⁺–K⁺-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage.