Mutants in the nodFEL promoter of Rhizobium leguminosarum bv. viciae reveal a role of individual nucleotides in transcriptional activation and protein binding.

The highly conserved nod box sequence in the promoters of the inducible nodulation genes of rhizobia is required for transcription activation together with NodD, a LysR-type transcriptional regulator, and a flavonoid as a coinducer. DNA fragments containing nod box sequences form two binding complexes when crude preparations of Rhizobium leguminosarum bv. viciae are used: a NodD-dependent and an additional, NodD-independent complex.

The NodD protein does not bind to the promoters of inducible nodulation genes in extracts of bacteroids of Rhizobium leguminosarum biovar viciae.

In a previous study, we showed that in bacteroids, transcription of the inducible nod genes does not occur and expression of nodD is decreased by 65% (H. R. M.

Suppression of nodulation gene expression in bacteroids of Rhizobium leguminosarum biovar viciae.

The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was investigated at both the translational and transcriptional levels. By using immunoblots, it was found that the levels of NodA, NodI, NodE, and NodO proteins were reduced at least 14-fold in bacteriods compared with cultured cells, whereas NodD protein was reduced only 3-fold.

Subcellular localization of the Rhizobium leguminosarum nodI gene product.

By the use of antibodies raised against a fusion protein of lacZ'-nodI (produced in Escherichia coli) which specifically react with NodI protein, it was shown that in wild-type Rhizobium leguminosarum biovar viciae NodI protein (i) is recovered with the cytoplasmic membrane fraction and (ii) is translated as part of the nodABCIJ operon. In addition, it was found that the bacterial chromosomal background strongly influences the expression of several nod genes.

nodO, a new nod gene of the Rhizobium leguminosarum biovar viciae sym plasmid pRL1JI, encodes a secreted protein.

The region of the Rhizobium leguminosarum biovar viciae Sym plasmid pRL1JI, responsible for the production and secretion of a previously described 50-kilodalton protein (R. A. de Maagd, C.

Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein.

The nucleotide sequence of the nodE gene of Rhizobium trifolii strain ANU843 was determined. Like the nodE gene of R. leguminosarum strain 248 it encodes a protein with a predicted mol.

Subcellular localization of the nodD gene product in Rhizobium leguminosarum.

In Rhizobium strains the transcription of symbiosis plasmid-localized nod genes, except nodD, is induced by plant flavonoids and requires the nodD gene product. In order to localize NodD protein in R. leguminosarum, a NodD protein-specific antiserum was raised against a lacZ'-'nodD gene fusion product.

Additional nodulation genes on the Sym plasmid of Rhizobium leguminosarum biovar viciae.

A Rhizobium leguminosarum biovar viciae strain lacking a 40 kb DNA region of the Sym plasmid pRL1IJ to the left (3' side) of gene nodE failed to nodulate Vicia sativa plants. Therefore this DNA region was investigated for the presence of additional nodulation genes. Complementation experiments indicated that the DNA region to the left (3' side) of nodE is functionally homologous between R.

Symbiotic properties of rhizobia containing a flavonoid-independent hybrid nodD product.

A hybrid nodD gene consisting of 75% of the nodD1 gene of Rhizobium meliloti at the 5' end and 27% of the nodD gene of Rhizobium trifolii at the 3' end activates the six tested inducible nod promoters of Rhizobium leguminosarum, R. trifolii, or R. meliloti to maximal levels, even in the absence of flavonoids.

Localization and symbiotic function of a region on the Rhizobium leguminosarum Sym plasmid pRL1JI responsible for a secreted, flavonoid-inducible 50-kilodalton protein.

A previously described (R. A. de Maagd, C.

Localization of functional regions of the Rhizobium nodD product using hybrid nodD genes.

The flavonoid-inducible nod promoters of Rhizobium are positively regulated by the nodD gene which is highly conserved in various Rhizobium species. The nodD gene are functionally different in (i) their response to various exogenously added flavonoid inducers, (ii) the extent to which they mediate the activation of the flavonoid-inducible promoters, and (iii) the extent to which they repress their own constitutive transcription. In order to localize the regions of the nodD product which determine these differences, two series of nodD hybrid genes have been constructed.

Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI.

A region of 16.8 kb of the Sym(biosis) plasmid pRL1JI of Rhizobium leguminosarum, consisting of the established 9.7 kb nodulation region which confers nodulation ability on Vicia hirsuta and a region of 7.

Induction of the nodA promoter of Rhizobium leguminosarum Sym plasmid pRL1JI by plant flavanones and flavones.

An expression vector containing the Rhizobium leguminosarum nodA promoter cloned in front of the Escherichia coli lacZ gene was used to characterize the properties of the R. leguminosarum nodA gene-inducing compound(s) present in sterile root exudate of the host plant Vicia sativa L. subsp.

Plant-inducible virulence promoter of the Agrobacterium tumefaciens Ti plasmid.

Agrobacterium tumefaciens is the causative agent of crown gall, a plant tumour that can arise on most species of dicotyledonous plants. The tumour-inducing capacity of the bacterium requires the presence of a large plasmid, designated the Ti plasmid, which itself contains two regions essential for tumour formation-the T(umour)-region and the Vir(ulence)-region. The T-region is transferred to plant cells by an unknown mechanism, and becomes stably integrated into the plant genome.

Partial exclusion of bacteriophage T2 by bacteriophage T4: induction of early enzymes by excluded T2.

In crosses between bacteriophages T2 and T4 most early genes of T2 are partially excluded from the progeny. Six genes of T4 affect the exclusion of six corresponding exclusion-sensitive sites in T2, each gene being specific for the exclusion of one site. Mutants of T4 in these genes have been isolated (ex mutants).

Partial exclusion of bacteriophage T2 by bacteriophage T4: an exclusion-resistant mutation in gene 56 of T2.

The early genes of bacteriophage T2 are partially excluded from the progeny of crosses between the related bacteriophages T2 and T4. This is due to complete exclusion from the progeny of six exclusion-sensitive sites in T2. A mutation [exr(56)1] in the sensitive site near T2 gene 56 renders the site partially resistant against exclusion.

Specificity of partial exclusion of bacteriophage T2 in crosses with T4.

An attempt was made to isolate a bacteriophage T4 mutant generally disturbed in its ability to exclude the localized exclusion sensitivity determinants of T2 from the progeny of crosses. The method used was a modification of that used previously, which revealed T4 mutants unable to exclude more than one of the T2 sensitive sites specifically. One out of 1000 isolates from a mutagenized T4 am56:am32 stock was found to be mutated in its exclusion properties.