V. Keijers

The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri

The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.

Characterization of a sugar-binding protein from Azospirillum brasilense mediating chemotaxis to and uptake of sugars

Our approach to the isolation of plant‐inducible bacterial genes of Azospirillum brasilense, based on the analysis of protein patterns of bacteria grown in the presence and in the absence of plant root exudates, led to the identification of an acidic 40 kDa protein. Cloning and sequencing analysis of the corresponding coding DNA region revealed the presence of two open reading frames transcribed in the same orientation. The deduced ORF1 protein, which corresponds to the 40 kDa protein, is very similar to the periplasmic ChvE protein, identified in Agrobacterium tumefaciens and involved in enhanced virulence. The deduced ORF2 protein shows homology to members of the LysR family of transcriptional regulators. The function of the ChvE‐like protein in A. brasilense was investigated further. The protein, designated as SbpA (sugar binding protein A), is involved in the uptake of D‐galactose and functions in the chemotaxis of A. brasilense towards several sugars, including D‐galactose, L‐arabinose and D‐fucose. Expression of the sbpA gene requires the presence of the same sugars in the growth medium and is enhanced further in combination with carbon starvation of A. brasilense cells.

Cloning and characterisation of the Azospirillum brasilense glnD gene and analysis of a glnD mutant

Abstract. Nitrogen regulation in bacteria involves the capacity to sense the availability of fixed nitrogen and to translate a signal indicating nitrogen deficiency or nitrogen excess into a cellular response. One of the key enzymes in this complex regulation process, the uridylyltransferase/uridylyl-removing (UTase) enzyme, encoded by the glnD gene, was characterised in the diazotroph Azospirillum brasilense, which promotes plant growth. The glnD gene product is responsible for the uridylylation of both PII-like nitrogen regulatory proteins, PII and PZ, depending on the nitrogen status of the cell. The nitrogen-regulated activity of the main ammonium-assimilating enzyme, glutamine synthetase, is not altered in a glnD-Tn5-B30 insertion mutant. UTase influences processes that are regulated by the NtrB-NtrC two-component histidine protein kinase system, such as ammonium uptake and nitrate assimilation. Moreover, the glnD gene product is indispensable for the activation of nitrogen fixation. Transcription of glnD is up-regulated under nitrogen-fixing conditions. This regulation is only partially dependent on the global nitrogen regulation (Ntr) system.

Isolation and characterization of the Azospirillum brasilense trpE(G) gene, encoding anthranilate synthase.

Abstract. The Azospirillum brasilense trpE gene has been isolated by DNA hybridization and by genetic complementation of an Escherichia coli trpE deletion mutant. DNA sequence analysis of a 3.1-kb PstI restriction fragment of A. brasilense revealed the presence of an open reading frame encoding a putative TrpE(G) fusion protein. Previously an A. brasilense clone containing trpGDC was identified (Zimmer et al. Mol Gen Genet 229:41–51, 1991). It can, therefore, be concluded that A. brasilense contains two trpG genes. A putative leader peptide is found upstream of trpE(G), containing three consecutive tryptophan residues. Putative terminator and anti-terminator loops have also been identified. The LLESX10S motif, which is responsible for feedback inhibition by tryptophan in other TrpE proteins, is absent in the A. brasilense TrpE(G) fused protein.

Genetics of Azospirillum brasilense with respect to ammonium transport, sugar uptake, and chemotaxis

This paper describes molecular aspects of Azospirillum-plant root association with respect to nitrogen flux and carbon utilization. In the first part, biochemical and genetic data are reported on the transport of ammonium and methylammonium in A. brasilense cells. Ammonium excreting A. brasilense mutants reported so far appear to result from alterations in genes encoding for enzymes involved in ammonium assimilation. Solid genetic evidence is given on the occurrence of a postulated ammonium transporter in A. brasilense. In the second part, biochemical and genetic evidence is likewise given for the occurrence of a high-affinity uptake system for D-galactose in A. brasilense. A sugar- binding protein that is part of this uptake system is required for chemotaxis of A. brasilense towards particular sugars, including D-galactose.

Isolation and sequence analysis of the GlnKamtB1amtB2 gene cluster, encoding a PII homologue and two putative ammonium transporters, from Pseudomonas stutzeri A15.

By PCR, using primers based on heterologous amtB genes, an amtB sequence of Pseudomonas stutzeri A15 was amplified. This DNA fragment was used as a probe in Southern hybridisation experiments and resulted in the isolation and sequence analysis of a 6017 u bp genomic fragment of P. stutzeri A15 containing glnKamtB 1 amtB 2. GlnK codes for a homologue of the nitrogen regulatory P II protein, amtB 1 and amtB 2 encode putative ammonium transporters. Whereas a glnKamtB gene cluster is common among bacteria, a tandem repeat of ammonium transporter genes has not been reported before. Apart from the presence of a second amtB gene, the gene organisation on this 6 u kbp fragment is very similar to a particular region in the genome of Pseudomonas aeruginosa PAO1, relatively closely related to P. stutzeri. Furthermore, the amtB 1 gene shows the highest similarity with P. aeruginosa amtB, whereas the amtB 2 gene is more closely related to cyanobacterial amtB genes, which are reported to be monocistronically transcribed and not clustered with glnK homologues. Upstream of glnK, NtrC and RpoN recognition sites can be observed. In the intergenic region of glnKamtB 1 amtB 2 no terminators nor extra promoter sequences were observed, indicating that glnKamtB 1 amtB 2 is possibly transcribed as a nitrogen regulated operon.

The Azospirillum brasilense amtB Gene is Responsible for Nitrogen Regulated Ammonium Uptake

Azospirillum species are plant growth promoting rhizosphere bacteria with a considerable potential as biofertiliser for non-leguminous plants (Vande Broek, Vanderleyden, 1995). Ammonium is not only the key factor in the nitrogen regulation in bacteria, but it is also the preferred nitrogen source for many, if not all bacteria, and the end product of the nitrogen fixing process in diazotrophic bacteria. In order to improve the beneficial effect of nitrogen fixing bacteria associated with plants, mechanisms that enable the bacteria to retain or release NH4 + are being studied. The characterisation of a (methyl)ammonium transporter contributes to this investigation.

Azospirillum-Plant Root Associations: Genetics of IAA Biosynthesis and Plant Cell Wall Degradation

The genus Azospirillum comprises free-living N2 fixing rhizosphere bacteria that have been isolated from different soil types and from the roots of numerous wild and cultivated plants all over the world. Field trials, carried out at different locations, have demonstrated that under certain environmental and soil conditions, inoculation with Azospirillum has beneficial effects on plant yields. Bacterial phytohormone biosynthesis has often been proposed as being responsible for the observed plant growth promotion upon Azospirillum inoculation.

Azospirillum Genes Involved in Chemotaxis and Adhesion to Plant Roots

Bacteria of the genus Azospirillum are diazotrophs that colonize the roots of plants. Colonization patterns can be visualized by using strains equipped with a reporter gene (Vande Broek et al., 1993; Arsene et al., 1994). The initial steps of bacterial colonization are chemotaxis and adhesion to the root surface. In order to characterize the bacterial genes and signals that determine these processes genetic and biochemical approaches were used.

Azospirillum-Cereals: An Intriguing Partnership

Azospirillum is probably the best studied example of beneficial plant rhizosphere bacteria. Studies in our laboratory focus on the identification of bacterial genes and gene products that are of importance in the physical and metabolic interaction of Azospirillum brasilense with plant roots. Here we report for Azospirillum brasilense, flagellation, motility, the physical interaction with plant roots, the synthesis of indole-3-acetic acid, the expression of nif genes in plant-root associated bacteria, and the induction of gene expression with plant root exudates.