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.

ropA encodes a 36-kDa outer membrane protein of Rhizobium leguminosarum bv. viciae strain 248 which constitutes the low-M r part of antigen group III (R. A. de Maagd, I. H. M. Mulders, H. C. J. Canter Cremers, and B. J. J. Lugtenberg, J. Bacteriol. 174:214-221, 1992). We observed that genes homologous to ropA are present in strain 248 as well as in other R. leguminosarum strains, and we describe the cloning and characterization of two of these genes. Sequencing of a 2.2-kb BglII fragment from R. leguminosarum bv. viciae strain 248 that hybridizes with ropA revealed one large open reading frame of 1,074 bp encoding a mature protein of 38.096 kDa. Homology between this gene and ropA is 91.8% on the DNA level. Homology on the amino acid level is only 69.9% as a result of a frameshift. On the basis of homology and immunochemical characteristics, we conclude that this gene encodes the high-M r part of the outer membrane protein antigen group III that is repressed during symbiosis. We named this gene ropA2. The second gene that we cloned was the ropA homologous gene of R. leguminosarum bv. trifolii strain LPR5020. Except for amino acid 43, the N-terminal part of the corresponding protein appeared to be identical to the first 51 amino acids of RopA of strain 248. The transcription start sites of both genes were determined, and the promoter regions were compared with that of ropA of strain 248. No clear consensus sequence could be deduced. The relationship of ropA and ropA2 of R. leguminosarum bv. viciae strain 248 with two similar genes from Brucella abortus is discussed.Rhizobia induce the formation of root nodules on appropriate leguminous host plants (for a review, see reference 20). In alfalfa and pea, the host plants of Rhizobium meliloti and Rhizobium leguminosarum bv. viciae, respectively, the nodules are of the indeterminate type. Five well-defined regions can be distinguished in such nodules. This subdivision in regions is based on the developmental stages of bacteroid differentiation and can be helpful in understanding the regulation of both plant and bacterial genes involved in symbiosis (35).The Rhizobium bacteroids, present in the cytoplasm of root nodule cells, show extensive differences with free-living bacteria at the molecular and biochemical levels. Differences in expression of nod genes have been studied by using antibodies, in situ hybridizations, and gusA fusions. Transcription of the inducible nod genes was still detectable in the invasion zone but not in the early symbiotic zone (30, 31). The synthesis of extracellular polysaccharide, a cell surface component, is also severely decreased in bacteroids. It was shown that exo::TnphoA fusions were mainly expressed in the invasion zone and not in the symbiotic zone of alfalfa nodules (26). This was also shown for the R. leguminosarum bv. phaseoli gene pssA, as detected by using a fusion with the gusA gene. The polysaccharide inhibition gene psiA was expressed throughout the nodule and is probably responsible for the decrease in pssA expression (19).Th...