Mark Lambrecht

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

Bi-functional gfp-and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies

The gfp gene, encoding the green fluorescent protein, was combined with the gusA gene, coding for the beta-glucuronidase enzyme, in mini-Tn5 transposon derivatives for use in Gram-negative bacteria. These mini-Tn5 elements allow simultaneously monitoring of gene expression and localization of the marked bacteria. Introduction of the resultant mini-Tn5 transposons into Rhizobium etli, Azospirillum brasilense and Pseudomonas stutzeri allowed us to visualise the interaction of these bacteria with their host plant. The dual-marker mini-Tn5 transposons constitute a powerful new tool for studying gene expression and ecology of bacteria in the environment and during the interaction with plants.

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.

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.

Annotation of the pRhico plasmid of Azospirillum brasilense reveals its role in determining the outer surface composition.

The plant growth-promoting soil bacterium Azospirillum brasilense enhances growth of economically important crops, such as wheat, corn and rice. In order to improve plant growth, a close bacterial association with the plant roots is needed. Genes encoded on a 90-MDa plasmid, denoted pRhico plasmid, present in A. brasilense Sp7, play an important role in plant root interaction. Sequencing, annotation and in silico analysis of this 90-MDa plasmid revealed the presence of a large collection of genes encoding enzymes involved in surface polysaccharide biosynthesis. Analysis of the 90-MDa plasmid genome provided evidence for its essential role in the viability of the bacterial cell.

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

Isolation and sequence analysis of the trpBA gene cluster, encoding tryptophan synthase, from Azospirillum brasilense.

The trpBA ene cluster of Azospirillum brasilense Sp7 was isolated by complementation of an Escherichia coli trpBA mutant. Both genes code for the two subunits of tryptophan synthase, which catalyzes the last step in tryptophan biosynthesis. No structural features indicating transcriptional relation could be identified. Upstream of the trpBA cluster an open reading frame encoding a putative periplasmic binding protein, involve in amino acid transport, was identified. Analysis of the downstream region of the trpBA cluster revealed the presence of a utative open reading frame encodin a subunit of the acetl-coenzyme A carboxylase carboxyl transferase complex.

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.

Evidence for a Physiological Role of IAA in Azospirillum brasilense Sp245

Regarding direct plant growth promotion, the ability of Azospirillum brasilense to produce indole-3-acetic acid (IAA) is now intensively studied. IAA biosynthesis in A. brasilense proved to be surprisingly complex, since at least three biosynthetic pathways are present (Prinsen et al, 1993). For one biosynthesis pathway, we have cloned and sequenced the A. brasilense ipdC gene, encoding the indole-3-pyruvic acid decarboxylase (Costacurta et al, 1994).