We describe the characterization of a novel Tn5lacZ colonization mutant of the efficiently colonizing Pseudomonas fluorescens strain WCS365, mutant strain PCL1210, which is at least 300- to 1,000-fold impaired in colonization of the potato root tip after co-inoculation of potato stem cuttings with a 1:1 mixture of mutant and parental cells. Similarly, the mutant is also impaired in colonization of tomato, wheat, and radish, indicating that the gene involved plays a role in the ability of P. fluorescens WCS365 to colonize a wide range of plant species. A 3.1-kb DNA fragment was found to be able to complement the observed mutation. The nucleotide sequence of the region around the Tn5lacZ insertion showed three open reading frames (ORFs). The transcriptional start site was determined. The operon is preceded by an integration host factor (IHF) binding site consensus sequence whereas no clear -10 and -35 sequences are present. The deduced amino acid sequences of the first two genes of the operon, designated as colR and colS, show strong similarity with known members of two-component regulatory systems. ColR has homology with the response regulators of the OmpR-PhoB subclass whereas ColS, the product of the gene in which the mutation resides, shows similarity to the sensor kinase members of these two-component systems. Hydrophobicity plots show that this hypothetical sensor kinase has two transmembrane domains, as is also known for other sensor kinases. The product of the third ORF, Orf222, shows no homology with known proteins. Only part of the orf222 gene is present in the colonization-complementing, 3.1-kb region, and it therefore does not play a role in complementation. No experimental evidence for a role of the ColR/ColS two-component system in the suspected colonization traits chemotaxis and transport of exudate compounds could be obtained. The function of this novel two-component system therefore remains to be elucidated. We conclude that colonization is an active process in which an environmental stimulus, through this two-component system, activates a so far unknown trait that is crucial for colonization.

Cell 60: [281][282][283][284][285][286][287][288][289][290][291][292][293][294] 1990). The nodFE region of Rhizobium kguminosarum has been cloned into a multicopy plasmid and has been shown in R. leguminosarum to code for a flavonoid-inducible protein that is effectively labeled by radioactive I-alanine added to the growth medium. After purification, the labeled protein migrates as a single band with an apparent molecular weight of 5,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, more rapidly than E. coli ACP. In contrast, in native gels the protein is resolved into two bands, both identified as NodF by analysis of the amino terminus and both migrating more slowly than E. coli ACP. Pulse-chase experiments with labeled ji-alanine suggested that the slower-moving band may be the precursor of the faster band. The NodF protein carries a 4'-phosphopantetheine as a prosthetic group. A NodF fusion protein under the control of the lac promoter is expressed in E. coli and is labeled with Il-alanine, indicating that it is recognized by the ACP synthase of E. coli. The ACP phosphodiesterase of E. coli, which catalyzes the release of phosphopantetheine from E. coli ACP, does not remove phosphopantetheine from NodF.Bacteria of the genus Rhizobium are able to interact symbiotically with specific plant hosts, leading to the formation of nitrogen-fixing root nodules. In the exchange of signals prior to nodulation, flavonoids secreted by the plant cause the induction of rhizobial nodulation (nod) genes. The nodABCIJ genes are termed common nod genes because they are functionally interchangeable among different Rhizobium species. In contrast, genes determining the host range of nodulation are termed host specific. Among them are genes of the nodFEL operon (7,22,37) (Fig. 1). NodF functions together with NodE in the production of a hostspecific rhizobial signal (31).Some indication of the function of NodFEL proteins can be derived from studying their homology, deduced from the gene sequence, to known proteins. The NodL protein appears to be homologous to the acetyl transferases LacA and CysE (9, 44), and NodE shares homology with a group of ,-ketoacyl synthases, such as FabB (the condensing enzyme of fatty acid biosynthesis of Escherichia coli), or those presumed to be involved in the synthesis of ,B-ketide antibiotics in Streptomyces species (2, 34). The NodF sequence was found to resemble that of the acyl carrier protein (ACP) of E. coli in the region near the serine to which the phosphopantetheine is attached, although amino acid identity to the ACP was very low in the rest of the molecule (see Fig. 6A tutive ACP, which presumably does function in the synthesis of essential membrane lipids, has been recently isolated from R. meliloti and studied in this laboratory (27).The periplasmic cyclic P-1,2-glucans of Rhizobium species are also involved in the cell signalling leading to nodulation (13) in a way that is so far not understood. These glucans are very similar to the periplasmic membrane-derived oligosa...

Ton Bisseling,'At a nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot cell mutant tsll only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type embryos. The causative component in the conditioned medium has previously been identified as a 32-kD acidic endochitinase. In search of a function for this enzyme in plant embryogenesis, several compounds that contain oligomers of N-acetylglucosamine were tested for their ability to promote tsll embryo formation. Of these compounds, only the Rhizobium lipooligosaccharides or nodulation (Nod) factors were found to be effective in rescuing the formation of tsll embryos. These results suggest that N-acetylglucosamine-containing lipooligosaccharides from bacterial origin can mimic the effect of the carrot endochitinase. This endochitinase may therefore be involved in the generation of plant analogs of the Rhizobium Nod factors.

Rhizobia, bacterial symbionts of leguminous plants, produce lipo-oligosaccharide signal molecules after induction of their nod genes by the plant host. The effects of these signals on plant developmental processes are discussed. Considering the similarity of the lipo-oligosaccharides with chitin, we discuss the possible basis of these effects and show some preliminary results which indicate that analogous molecules could occur in uninfected plants.

No abstract