Search

This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.

Aims: To examine stool specimens from children with diarrhea from Paraná State, southern Brazil, for presence of STEC., Methods and Results: A PCR screening assay for stx genes was used to examine a loopful of confluent colonies of 306 stool samples cultures. In six (1.96%) of them, DNA fragments of the expected size were observed, and the presence of stx was confirmed by DNA sequencing. Then up to 100 single colonies from each of the six stool cultures were analyzed using the same PCR protocol. However, stx-positive colonies were found only in two of the cultures. The E. coli strains belonged to serotypes O69:H11 and O178:H19, and presented genotypes stx(1)eae ehxA and stx(1) respectively. Shiga toxin production was confirmed using the VTEC Screen Seiken. Except ampicillin, they were susceptible to all the antimicrobials tested., Conclusions: These results show that STEC may be an important cause of diarrhea in children of Paraná State, and that they are present in low numbers in stools. The strains belonged to serotypes not commonly found associated with STEC and probably present low virulence., Significance and Impact of Study: These results indicate that molecular methods are required to diagnosis of STEC infections.

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.

The Azospirillum brasilense PII and PZ proteins, encoded by the glnB and glnZ genes respectively, are intracellular transducers of nitrogen levels with distinct functions. The PII protein participates in nif regulation by controlling the activity of the transcriptional regulator NifA. PII is also involved in transducing the prevailing nitrogen levels to the Fe-protein ADP-ribosylation system. PZ regulates negatively ammonium transport and is involved in nitrogenase reactivation. To further investigate the role of PII and PZ in the regulation of nitrogen fixation, broad-host-range plasmids capable of over-expressing the glnB and glnZ genes under control of the ptac promoter were constructed and introduced into A. brasilense. The nitrogenase activity and nitrate-dependent growth was impaired in A. brasilense cells over-expressing the PII protein. Using immunoblot analysis we observed that the reduction of nitrogenase activity in cells over-expressing PII was due to partial ADP-ribosylation of the Fe-protein under derepressing conditions and a reduction in the amount of Fe-protein. These results support the hypothesis that the unmodified PII protein act as a signal to the DraT enzyme to ADP-ribosylate the Fe-protein in response to ammonium shock, and that it also inhibits nif gene expression. In cells over-expressing the PZ protein the nitrogenase reactivation after an ammonium shock was delayed indicating that the PZ protein is involved in regulation of DraG activity.

Abstract The interactions between maize, sorghum, wheat and rice plants and Herbaspirillum seropedicae were examined microscopically following inoculation with the H. seropedicae LR15 strain, a Nif(+) (Pnif::gusA) mutant obtained by the insertion of a gusA-kanamycin cassette into the nifH gene of the H. seropedicae wild-type strain. The expression of the Pnif::gusA fusion was followed during the association of the diazotroph with the gramineous species. Histochemical analysis of seedlings of maize, sorghum, wheat and rice grown in vermiculite showed that strain LR15 colonized root surfaces and inner tissues. In early steps of the endophytic association, H. seropedicae colonized root exudation sites, such as axils of secondary roots and intercellular spaces of the root cortex; it then occupied the vascular tissue and there expressed nif genes. The expression of nif genes occurred in roots, stems and leaves as detected by the GUS reporter system. The expression of nif genes was also observed in bacterial colonies located in the external mucilaginous root material, 8 days after inoculation. Moreover, the colonization of plant tissue by H. seropedicae did not depend on the nitrogen-fixing ability, since similar numbers of cells were isolated from roots or shoots of the plants inoculated with Nif(+) or Nif(-) strains.

In Azospirillum brasilense the glnB and glnA genes are clustered in an operon regulated by three different promoters: two located upstream of glnB (glnBp1-sigma(70), and glnBp2-sigma(N)) and one as yet unidentified promoter, in the glnBA intergenic region. We have investigated the expression of the glnB gene promoter using glnB-lacZ gene fusions, mutation analysis, heterologous expression and DNA band-shift assays. Deletion of the glnB promoter region showed that NtrC-binding sequences were essential for glnB expression under nitrogen limitation. The A. brasilense NtrC protein activated transcription of glnB-lacZ fusions in the heterologous genetic background of Escherichia coli. Expression of glnB-lacZ fusions in two A. brasilense ntrC mutants differed from that in the wild-type strain. In vitro studies also indicated that the purified NtrC protein from E. coli was able to bind to the glnB promoter region of A. brasilense. Our results show that the NtrC protein activates glnBglnA expression under nitrogen limitation in A. brasilense.

Three Azospirillum brasilense mutants constitutive for nitrogen fixation (Nif(C)) in the presence of NH4(+) and deficient in nitrate-dependent growth were used as tools to define the roles of the glnB and ntrYX genes in this organism. Mutant HM14 was complemented for nitrate-dependent growth and NH4(+) regulation of nitrogenase by plasmid pL46 which contains the ntrYX genes of A. brasilense. Mutant HM26 was restored for NH4(+) regulation and nitrate-dependent growth by plasmid pJC1, carrying the A. brasilense glnB gene expressed from a constitutive promoter. Mutant HM053, on the other hand, was not complemented for NH4(+) regulation of nitrogenase and nitrate-dependent growth by both plasmids pJCI and pL46. The levels and control of glutamine synthetase activity of all mutants were not affected by both plasmids pL46 (ntrYX) and pJC1 (glnB). These results support the characterization of strains HM14 as an ntrYX mutant and strain HM26 as a glnB mutant and the involvement of ntrYX and glnB in the regulation of the general nitrogen metabolism in A. brasilense.

The role of the Ntr system in Herbaspirillum seropedicae was determined via ntrB and ntrC mutants. Three phenotypes were identified in these mutants: Nif(-), deficiency in growth using nitrate, and low glutamine synthetase (GS) activity. All phenotypes were restored by the plasmid pKRT1 containing the intact glnA, ntrB and ntrC genes of H. seropedicae. The promoter region of glnA was subcloned into a beta-galactosidase fusion vector and the results suggested that NtrC positively regulates the glnA promoter in response to low nitrogen. The H. seropedicae ntrC and ntrB mutant strains showed a deficiency of adenylylation/deadenylylation of GS, indicating that NtrC and NtrB are involved in both transcription and activity control of GS in this organism.