Regulation of nitrogen fixation (nif) genes of Azospirillum brasilense by nifA and ntr (gln) type gene products

Abstract Eight Nif⁻ mutants of Azospirillum brasilense were obtained by N -nitrosoguanidine mutagenesis and isolated by growth on glutamate medium. Three of these mutants had no nitrogenase activity, possessed no nitrogenase structural proteins and were complemented by Klebsiella pneumoniae nifA . Evidence will be presented that one of these mutants is defective in a nifA type regulatory gene but the other two were also complemented by K. pneumoniae ntrC and may be ntrC⁻ -type mutants. A fourth mutant was defective in the MoFe component protein of nitrogenase.     

Update on the challenging role of biofilms in peritoneal dialysis

Biofilms are commonly associated with an increased risk of patient infection. In peritoneal dialysis (PD), catheter associated infection, especially peritonitis, remains a clinically relevant problem. Although the presence of a biofilm is recognized in relapsing, repeat, and catheter-related peritonitis, it remains poorly characterized. In this review, an update on the role of biofilms in PD infections is presented. The emerging concept that host cells and tissue associated biofilms, in addition to the biofilms on the catheters themselves, contribute to the recalcitrance of infections is discussed. Furthermore, the evidence of biofilms on PD catheters, their developmental stages, and the possible influence of the PD environment are reviewed. The focus is given to ex vivo and in vitro studies that contribute to the elucidation of the interplay between host, microbial, and dialysis factors. The key issues that are still to be answered and the challenges to clinical practice are discussed.     

Proteomic Analysis of the Action of the Mycobacterium ulcerans Toxin Mycolactone: Targeting Host Cells Cytoskeleton and Collagen

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone''s cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.     

Sequence and structural organization of a nif A-like gene and part of a nifB-like gene of Herbaspirillum seropedicae strain Z78.

The deduced amino acid sequence derived from the sequence of a fragment of DNA from the free-living diazotroph Herbaspirillum seropedicae was aligned to the homologous protein sequences encoded by the nifA genes from Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti and Klebsiella pneumoniae. High similarity was found in the central domain and in the C-terminal region. The H. seropedicae putative NifA sequence was also found to contain an interdomain linker similar to that conserved among rhizobial NifA proteins, but not K. pneumoniae or Azotobacter vinelandii. Analysis of the regulatory sequences found 5' from nifA indicated that the expression of this gene in H. seropedicae is likely to be controlled by NifA, NtrC and RpoN, as judged by the presence of specific NifA- and NtrC-binding sites and characteristic -24/-12 promoters. Possible additional regulatory features included an 'anaerobox' and a site for integration host factor. The N-terminus of another open reading frame was found 3' from nifA and tentatively identified as nifB by amino acid sequence comparison. The putative nifB promoter sequence suggests that expression of H. seropedicae nifB may be activated by NifA and dependent on RpoN.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

Phenotypic and genotypic traits of Shiga toxin-producing Escherichia coli strains isolated from beef cattle from Paraná State, southern Brazil

AIMS: To investigate the prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in cattle from Paraná State, southern Brazil. METHODS AND RESULTS: One hundred and seven faeces cattle samples were cultured on Sorbitol-MacConkey agar. Escherichia coli colonies were tested for production of Shiga toxin using Vero-cell assay. A high prevalence (57%) of STEC was found. Sixty-four STEC were serotyped and examined for the presence of stx(1), stx(2), eae, ehxA and saa genes and stx(2) variants. The isolates belonged to 31 different serotypes, of which three (O152:H8, O175:H21 and O176:H18) had not previously been associated with STEC. A high prevalence of stx(2)-type genes was found (62 strains, 97%). Variant forms found were stx(2), stx(2c), stx(2vhb), stx(2vO111v/OX393) and a form nonclassifiable by PCR-RFLP. The commonest genotypes were stx(2)ehxA saa and stx(1)stx(2)ehxA saa. CONCLUSIONS: A high frequency of STEC was observed. Several strains belong to serotypes previously associated with human disease and carry stx(2) and other virulence factors, thus potentially representing a risk to human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of STEC in Paraná State, and its findings emphasize the need for proper cattle handling to prevent human contamination.     

Synthesis of a novel superabsorbent hydrogel by copolymerization of acrylamide and cashew gum modified with glycidyl methacrylate

Novel superabsorbent hydrogels were manufactured using chemically modified cashew gum (CGMA) and acrylamide (AAm) as reactants. The route for the synthesis was feasible due to the incorporation of glycidyl methacrylate (GMA) into structure of cashew gum (CG) to form the cashew gum-methacrylated (CGMA), in an appropriate mixture water-DMSO, as solvent, and using TEMED as catalyst. Thereafter, the CGMA was copolymerized with AAm yielding (CGMA-co-AAm) hydrogels. The main characteristics of raw and the modified materials are reported in this paper. ¹³C NMR, ¹H NMR and FTIR spectroscopies confirmed the incorporation of vinyl groups, from GMA, into CG structure. By the spectrophotometry analyses, it was found that, ca. 82% of GMA was incorporated to the CG after 24 h of reaction. The cross-linking of CGMA or co-polymerization of CGMA with acrylamide leads to a hydrogel formation. Their gelation was characterized by FT-IR analysis. Alkaline hydrolysis at 40 °C for 3 and 4.5 h increased the water uptake (WU) capacity. Hydrolyzed CGMA-co-AAm hydrogels present higher values of WU (up to 1500) and may be classified as water superabsorbent material. Applications in agriculture, as soil conditioner, and in biomedical field, as biomaterial (scaffold) are being investigated.     

Diet supplementation with beta-carotene improves the serum lipid profile in rats fed a cholesterol-enriched diet

The present study investigated the underlying mechanism associated with the hypocholesterolemic activity of beta-carotene by examining its effects on the serum lipid profile, fecal cholesterol excretion, and gene expression of the major receptors, enzymes, and transporters involved in cholesterol metabolism. Female Fischer rats were divided into three groups and were fed either a control or a hypercholesterolemic diet supplemented or not supplemented with 0.2 % beta-carotene. After 6 weeks of feeding, blood, livers, and feces were collected for analysis, and quantitative real-time polymerase chain reaction (qRT-PCR) was performed. Dietary supplementation with 0.2 % beta-carotene decreased serum total cholesterol, non-HDL cholesterol, the atherogenic index, and hepatic total lipid and cholesterol contents. These changes were accompanied by an increase in the total lipid and cholesterol contents excreted in the feces. The qRT-PCR analyses demonstrated that the hypercholesterolemic diet promoted a decrease in the gene expression of sterol regulatory element-binding protein 2, 3-hydroxy-3-methylglutaryl CoA reductase, and low-density lipoprotein receptor and an increase in the gene expression of peroxisome proliferator-activated receptor α and cholesterol-7a-hydroxylase. The expression of these genes and gene expression of ATP-binding cassette subfamily G transporters 5and 8 were unaffected by beta-carotene supplementation. In conclusion, the decrease in serum cholesterol and the elevation of fecal cholesterol obtained following beta-carotene administration indicate that this substance may decrease cholesterol absorption in the intestine and increase cholesterol excretion into the feces without a direct effect on the expression of cholesterol metabolism genes.