Ann Vande Broek

Indole-3-acetic acid: a reciprocal signalling molecule in bacteria–plant interactions

uxins were discovered earlyin the twentieth century asplant-regulating substances.Indole-3-acetic acid (IAA) is anaturally occurring auxin withbroad physiological effects. Al-though many plant genes that aretranscriptionally regulated by IAAhave been characterized in recentyears, our understanding of theauxin signal transduction path-way(s) in plants is still incomplete.IAA biosynthesis in plants canoccur via different pathways

Bacterial chemotactic motility is important for the initiation of wheat root colonization by Azospirillum brasilense.

Bacteria of the genus Azospirillum are able to colonize plant roots. Using the beta-glucuronidase (GUS) reporter system, various Azospirillum mutants, including mutants affected in chemotactic motility or extracellular polysaccharide biosynthesis, were investigated for their capacity to initiate wheat root colonization at the root hair zones. Only non-flagellated mutants and a generally non-chemotactic mutant exhibited a strongly reduced colonization ability as compared to the wild-type. No role of the Azospirillum calcofluor-binding polysaccharide in primary wheat root colonization could be observed. This is the first report demonstrating directly, by using different motility mutants, the requirement of bacterial motility in the establishment of the Azospirillum-plant root association.

Review: Genetics of the Azospirillum-Plant Root Association

Abstract Recent advances in four aspects of the Azospririllum-plant root association, namely, the biosynthesis of phytohormones, nitrogen fixation, the mechanisms of plant root attachment, and genetic analysis of Azospirillum brasilense megaplasmids, are presented. At least three biosynthetic pathways are involved in indole-3-acetic acid (IAA) production in A. brasilense, that is, two Trp-dependent pathways identified as the indole-3-acetamide (LAM) pathway, the indole-3-pyruvic acid (IPyA) pathway, and a third as-yet unidentified pathway not using Trp as a precursor. So far, Azospirillum is the only bacterium for which a non-Trp-dependent route for IAA biosynthesis has been reported. The gene encoding the key enzyme in the IPyA pathway, indole-3-pyruvate decarboxylase, has been characterized recently. Azospirillum fixes atmospheric dinitrogen only at low oxygen tensions in media devoid of a combined nitrogen source. Both the synthesis and the activity of the nitrogenase enzyme are regulated according to ...

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

SummaryA recombinant λ phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec− phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.

THE IPDC PROMOTER AUXIN-RESPONSIVE ELEMENT OF AZOSPIRILLUM BRASILENSE, A PROKARYOTIC ANCESTRAL FORM OF THE PLANT AUXRE?

Recently, Novak et al. (1998, Mol Microbiol 29: 1285±1296) reported their investigation on the phenomenon of penicillin tolerance in Streptococcus pneumoniae. A library of mutants in pneumococcal surface proteins was screened for the ability to survive in the presence of 10 ́ the minimum inhibitory concentration of antibiotic. A mutant harbouring an insertion in the known gene psaA was isolated among 10 candidate tolerance mutants. Inactivation of psaA was previously shown to result in reduced virulence of S. pneumoniae (as judged by intranasal or intraperitoneal challenge of mice) and in reduced adherence to A549 cells (type II pneumocytes), leading to the suggestion that PsaA was an adhesin (Berry and Paton, 1996, Infect Immun 64: 5255±5262). This gene is part of the psa locus (Fig. 1) that encodes an ATP-binding cassette (ABC) permease belonging to cluster 9, a family of ABC metal permeases (Dintilhac et al., 1997, Mol Microbiol 25: 727±740). Novak et al. (1998, Mol Microbiol 29: 1285±1296) reported that psa mutants displayed pleiotropic phenotypes: (i) reduced sensitivity to the lytic and killing effects of penicillin; (ii) growth in chains of 40±50 (psaC ) to 200±300 (psaD ) cells; (iii) autolysis defect and loss of sensitivity to low concentrations of deoxycholate (DOC), a species characteristic trait; (iv) absence of LytA, the major autolytic amidase; (v) almost complete loss of choline-binding proteins (ChBPs) (psaC and psaD ) and absence of CbpA; (vi) loss of transformability (except psaA); and (vii) manganese (Mn) requirement for growth in a chemically de®ned medium. Because penicillin tolerance was ®rst associated with an autolysis defect (Tomasz et al., 1970, Nature 227: 138± 140), the absence of LytA (phenotype iv) could itself explain phenotypes i and iii. Dysregulation of lytA could not be investigated because, according to Novak et al. (1998, Mol Microbiol 29: 1285±1296), the dif®culty in lysing psa mutant cells prohibited Northern analysis, although lysates of the psa mutants could be obtained for immunoblot analysis of LytA and of RecA and for Southern con®rmation of the psa mutations. Nevertheless, because expression of the lytA gene has been shown to be driven by three different promoters, including Pb which is the recA basal promoter (Mortier-BarrieÁre et al., 1998, Mol Microbiol 27: 159±170), and because wild-type levels of RecA were detected in the psa mutants (Novak et al., 1998, Mol Microbiol 29: 1285±1296), it seems dif®cult to account for the complete absence of LytA on the basis of altered expression. On the other hand, phenotypes i±iv are reminiscent of alterations observed after the replacement of choline (Ch) by ethanolamine (EA) in the cell wall of pneumococcus (Tomasz, 1968, Proc Natl Acad Sci USA 59: 86±93). Similar phenotypes were also displayed by Ch-independent mutants of S. pneumoniae (Severin et al., 1997, Microb Drug Res 3: 391±400; Yother et al., 1998, J Bacteriol 180: 2093±2101). S. pneumoniae has a nutritional requirement for Ch that is incorporated by covalent bonds into the cell wall teichoic acids (TA) and in the membrane-bound lipoteichoic acid (LTA). Ch residues bound to TA (ChTA) were shown to be absolutely required for LytA activity (Holtje and Tomasz, 1975; J Biol Chem 250: 6072±6076). The action of LytA has long been thought to be restricted to pneumococcal cell walls because of this requirement. However, recent reports suggest that ChTA is required Molecular Microbiology (1999) 32(4), 881±891

A spontaneous chlorate-resistant mutant of Azospirillum brasilense Sp245 displays defects in nitrate reduction and plant root colonization

Abstract. Azospirillum, a soil bacterium capable of colonizing plant roots, can reduce NO3–. In this work, a spontaneous chlorate-resistant mutant of Azospirillum brasilense Sp245, named Sp245chl1, was phenotypically characterized. The mutant is defective in both assimilatory and periplasmic dissimilatory nitrate reductase activity. Using the gusA reporter gene methodology, Sp245chl1 was found to be significantly affected in its ability to colonize roots of wheat and rice seedlings.

Crystallization and preliminary X-ray diffraction analysis of a novel pectate lyase from Azospirillum irakense

The PelA gene from the N(2)-fixing plant-associated bacterium Azospirillum irakense encodes a pectate lyase. Analysis of the corresponding amino-acid sequence revealed no homology to other bacterial, plant and fungal pectinases of known published structure, resulting in the classification of the enzyme in a new pectate lyase family. The A. irakense PelA has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are hexagonal, with unit-cell parameters a = b = 85.55, c = 230.13 A, gamma = 120 degrees, and belong to space group P6(5)22 or P6(1)22, having one molecule per asymmetric unit. Diffraction data to a resolution of 1.97 A were collected at synchrotron facilities, as well as a three-wavelength MAD data set from an Hg-derivative crystal to a resolution of 2.6 A.

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.

Introduction of Plasmids into Azospirillum brasilense by Electroporation

The genus Azospirillum comprises Gram N2 fixing soil bacteria living in close association with the roots of numerous plants. Field trials, carried out at different locations have demonstrated significant plant growth promotion upon Azospirillum inoculation. Based on 16S rRNA similarity, the genus Azospirillum has been classified within the alpha subdivision of Proteobacteria. Plasmids of the IncP group are stably maintained in a wide range of Azospirillum strains and can be succesfully transferred to Azospirillum either by conjugation or by electroporation. As compared to conjugation, the electroporation technique offers the advantages of being much faster and avoiding problems with counterselection of the Escherichia coli donor strain.