Identification of a nifW-like gene in Azospirillum brasilense.

A small ORF, 5' upstream of the fixABC operon in Azospirillum brasilense Sp7, has been identified. Sequence comparison shows significant homology to the Azotobacter vinelandii and Azorhizobium caulinodans nifW gene.

Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

The Sinorhizobium meliloti nutrient-deprivation-induced tyrosine degradation gene hmgA is controlled by a novel member of the arsR family of regulatory genes.

The regulation of the nutrient-deprivation-induced Sinorhizobium meliloti homogentisate dioxygenase (hmgA) gene, involved in tyrosine degradation, was examined. hmgA expression was found to be independent of the canonical nitrogen regulation (ntr) system. To identify regulators of hmgA, secondary mutagenesis of an S. meliloti strain harboring a hmgA-luxAB reporter gene fusion (N4) was carried out using transposon Tn1721. Two independent Tn1721 insertions were found to be located in a positive regulatory gene (nitR), encoding a protein sharing amino acid sequence similarity with proteins of the ArsR family of regulators. NitR was found to be a regulator of S. meliloti hmgA expression under nitrogen deprivation conditions, suggesting the presence of a ntr-independent nitrogen deprivation regulatory system. nitR insertion mutations were shown not to affect bacterial growth, nodulation of Medicago sativa (alfalfa) plants, or symbiotic nitrogen fixation under the physiological conditions examined. Further analysis of the nitR locus revealed the presence of open reading frames encoding proteins sharing amino acid sequence similarities with an ATP-binding phosphonate transport protein (PhnN), as well as transmembrane efflux proteins.

Transcription of the Azospirillum brasilense nifH gene is positively regulated by NifA and NtrA and is negatively controlled by the cellular nitrogen status.

The expression of a translational Azospirillum brasilense nifH-uidA fusion was studied in A. brasilense and in Rhizobium meliloti strains with mutations in nifA, ntrA and ntrC. Induction of the fusion was observed in the R. meliloti wild-type and NtrC- strains on incubation under microaerobic conditions but not in the NifA- and NtrA- strains, showing the absolute requirement of both sigma 54 and NifA for activation of the nifH promoter. Histochemical analysis of the root nodules elicited by R. meliloti wild-type showed expression of the fusion in the late symbiotic zone but not in the meristematic and the early symbiotic zones. No induction of the nifH-uidA fusion was observed in the R. meliloti wild-type or NifA- strains incubated aerobically in nitrogen-free medium, indicating that, in contrast to R. meliloti nifH, A. brasilense nifH cannot be activated directly by NtrC. Expression of the nifH gene in A. brasilense only occurs under nitrogen-limiting, microaerobic conditions, suggesting the presence of a nitrogen-dependent control system for nif gene expression.

The Azospirillum brasilense rpoN gene is involved in nitrogen fixation, nitrate assimilation, ammonium uptake, and flagellar biosynthesis.

The rpoN (ntrA) gene (encoding sigma 54) of Azospirillum brasilense Sp7 was isolated by using conserved rpoN primers and the polymerase chain reaction, and its nucleotide sequence was determined. The deduced amino acid sequence of the RpoN protein was found to share a high degree of homology with other members of the sigma 54 family. Two additional open reading frames were found in the Azospirillum brasilense rpoN region, with significant similarity to equivalent regions surrounding the rpoN locus in other bacteria. An rpoN mutant of Azospirillum brasilense Sp7 was constructed by gene replacement and found to be defective in nitrogen fixation, nitrate assimilation, and ammonium uptake. Lack of ammonium uptake was also found in previously isolated Azospirillum brasilense ntrB and ntrC mutants, further supporting the role of the ntr system in this process. In addition, the rpoN mutant was found to be nonmotile, suggesting a role of RpoN in Azospirillum brasilense flagellar biosynthesis.