Mengsheng Gao

Roles for Azorhizobial Nod Factors and Surface Polysaccharides in Intercellular Invasion and Nodule Penetration, Respectively

In the symbiotic interaction between Azorhizobium caulinodans and Sesbania rostrata root and stem-borne nodules are formed. The bacteria enter the host via intercellular spaces at lateral or adventitious root bases and form infection pockets in outer cortical layers. Infection threads guide the bacteria to nodule primordia where plant cells are invaded. To identify bacterial functions that are required for this infection process, two mutants defective in nodulation were studied; one produced no Nod factors (nodA mutant), the other had altered surface polysaccharides (SPS) and induced the formation of pseudo-nodules. Bacteria were visualized with the help of a nodA-uidA reporter fusion that was functional during nodule development and in bacteroids. In contrast to the SPS mutant, nodA mutants were unable to colonize outer cortical regions. In mixed inoculations with both mutants, functional nodules were formed, the central tissue of which was occupied by the nodA mutant. These observations suggest that SPS...

Knockout of an azorhizobial dTDP-L-rhamnose synthase affects lipopolysaccharide and extracellular polysaccharide production and disables symbiosis with Sesbania rostrata.

A nonpolar mutation was made in the oac2 gene of Azorhizobium caulinodans. oac2 is an ortholog of the Salmonella typhimurium rfbD gene that encodes a dTDP-L-rhamnose synthase. The knockout of oac2 changed the lipopolysaccharide (LPS) pattern and affected the extracellular polysaccharide production but had no effect on bacterial hydrophobicity. Upon hot phenol extraction, the wild-type LPS partitioned in the phenol phase. The LPS fraction of ORS571-oac2 partitioned in the water phase and had a reduced rhamnose content and truncated LPS molecules on the basis of faster migration in detergent gel electrophoresis. Strain ORS571-oac2 induced ineffective nodule-like structures on Sesbania rostrata. There was no clear demarcation between central and peripheral tissues, and neither leghemoglobin nor bacteroids were present. Light and electron microscopy revealed that the mutant bacteria were retained in enlarged, thick-walled infection threads. Infection centers emitted a blue autofluorescence under UV light. The data indicate that rhamnose synthesis is important for the production of surface carbohydrates that are required to sustain the compatible interaction between A. caulinodans and S. rostrata.

Dual control of the nodA operon of Azorhizobium caulinodans ORS571 by a nod box and a NifA-σ54-type promoter

Abstract. Earlier studies have shown that the Azorhizobium caulinodans nodA promoter is controlled by a host plant-derived flavonoid signal via the transcription activator NodD. Here, we report that the transcription of the nodA operon is also under the control of NifA-RpoN. A NifA-σ54-type promoter, P2nodA, is present upstream of the nod-box consensus motif of the nodA gene and directs expression of a nodA-uidA reporter gene both in free-living bacteria under nitrogen fixation conditions and in bacteroids. Mutation of P2nodA reduced, under certain conditions, the efficiency of nodulation and accelerated nodule senescence, suggesting that the dual control may help to optimize nodule initiation and function in the natural context of the symbiosis.

Characterization of an Azorhizobial Mutant with Altered Surface Polysaccharides and Affected in Nodule Invasion

In the symbiosis between Sesbania rostrata and Azorhizobium caulinodans strain ORS571, nitrogen-fixing nodules are formed on roots and stems by intercellular infection at the basis of lateral roots and adventitious root primordia. The nodule development depends on bacterial Nod factors, the lipo-chitin Oligosaccharide signals that are produced as a consequence of nod gene expression (Long, 1996). The bacterial surface Polysaccharides as well are important for nodulation. This is illustrated by the present work, which also demonstrates a role for Nod factors in bacterial entry via intercellular infection.

Identification of Nodulation Genes (nod Locus 5) From Azorhizobium caulinoaans ORS571

An 800 bp AccI-PUCII DNA containing promoter region of nodABC (P1) from A. caulinodans ORS571 was cloned and used as a hybridization probe against the chromosome DNA, which led to the identification of another 8.4-kb EcoRI fragment showing homology to P1. A corresponding clone of 8.4-kb DNA was isolated from a pLAFI gene bank (pRG90). The restriction enzyme analysis and DNA-DNA hybridization of 8.4-kb DNA indicated the P1 homology was located in the 450-bp SmaI-SphI region (P2 region). omega insertion deletion of 8.4 kb DNA resulted in a mutant strain ORS571-5 that delayed to nodulate the stems of S. rostrata. This phenotype was complemented by the introduction of pRK84 carrying nod locus 5 DNA.