Robert J. H. Okker

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 7.1 kb which appeared to be necessary for nodulation on V. sativa and Trifolium subterraneum, was subcloned as fragments of maximally 2.5 kb in a newly developed IncQ transcriptional fusion vector. The expression of these fragments was studied in Rhizobium. One constitutive promoter, pr.nodD, and three plant-exudate inducible promoters were found, namely the known pr.nodA and pr.nodF as well as a new promoter designated pr.nodM. The latter promoters were localized within 114 bp, 330 bp and 630 bp respectively and they regulate the transcription of the operons nodA, B, C, I, J, nodF, E and of an operon of at least 2.5 kb located in the 7.1 kb region. Induction of the three inducible operons required plant exudate and a functional nodD product. The flavanone naringenin could replace plant exudate. Each of the three inducible promoters contained a nod-box. A consensus for the nod-box sequence, based on known sequences, is proposed. The 114 bp fragment which contains pr.nodA activity was used to localize pr.nodA by means of deletion mapping. The fragment which appeared necessary for complete pr.nodA activity is 72 bp in size, contains the complete nod-box and in addition a region of 21 bp downstream of the nod-box, in which the loosely conserved sequence AT(T)AG appears to be important for promoter activity.

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. wt of 42.0 kd. The predicted NodE proteins of R.trifolii and R.leguminosarum have a homology of 78%. Using antibodies raised against the NodE protein of R.trifolii it was shown that the NodE products of R.leguminosarum and R.trifolii are localized in the cytoplasmic membrane. Furthermore, these NodE proteins are predicted to contain at least two non‐polar transbilayer alpha‐helices. The nodE genes of R.trifolii and R.leguminosarum were separated from the nodF genes that precede them in the respective nodFE operons. Using the resulting clones, in which NodE was produced under control of the flavonoid‐inducible nodABCIJ promoter of R.leguminosarum, it was shown that the NodE product is the main factor that distinguishes the host range of nodulation of R.trifolii and R.leguminosarum. Hybrid nodE genes, which consist of a 5′ part of the R.leguminosarum nodE gene and a 3′ part of the R.trifolii gene, were constructed. From the properties of these hybrid genes it was concluded that a central region of 185 amino acids at the most, containing only 44 non‐identical amino acids, determines the difference in the host‐specific characteristics of the two NodE proteins.

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

Abstract. 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 role of individual nucleotides in the conserved nod box sequence in complex formation and in nodulation gene expression was investigated by introducing 13 individual base-pair substitutions in the nodF nod box of R. leguminosarum bv. viciae and studying their effect on promoter activity and protein–DNA complex formation. Two mutants showed decreased NodD binding and decreased promoter activity. Five mutants showed a NodD-dependent complex as with the wild-type nodF nod box, whereas their promoter activity was severely reduced after induction. This result is in agreement with earlier observations that NodD DNA binding also occurs in the absence of inducer. Four mutants were impaired in the formation of the NodD-independent retardation complex. Three of them showed no alterations in promoter activity, meaning that no specific role for the protein forming the NodD-independent complex could be established. The two mutants in the highly conserved LysR motif of the nod box were unable to direct coinducer-dependent promoter activity but, unexpectedly, their retardation patterns were not altered. The remaining two mutants showed constitutive promoter activity. The results are discussed in terms of the relevance of conserved nucleotides and motifs identified in the nod box.

Promoters and Operon Structure of the Nodulation Region of the Rhizobium Leguminosarum Symbiosis Plasmid pRL1JI

Bacteria of the genus Rhizobium, which are able to establish a symbiosis with leguminous plants, invade the roots of their hosts where they induce the formation of nodules in which they fix atmospheric nitrogen. Each Rhizobium strain has only a narrow range of host plants on which it is successfully able to establish a symbiosis. In fast-growing Rhizobia, which include R. leguminosarum with peas and vetches as their hosts, R. trifolii with clovers as their hosts, and R. meliloti with alfalfa as a host, many genes involved in nodulation (nod) and nitrogen fixation (nif) are localized on large plasmids designated symbiosis (Sym) plasmids (Johnston et al 1978; Hooykaas et al 1981; Banfalvi et al 1981; Rosenberg et al 1981). The nod genes, which constitute only a relatively small portion of the Sym plasmid (Downie et al 1983; Schofield et al 1984), appear to be of prime importance in the determination of the host-specificity.

Flavonoid Compounds as Molecular Signals in Rhizobium — Legume Symbiosis

The bacterium Rhizobium nodulates leguminous and some non -leguminous plants and establishes a symbiotic relationship with its host plant in which the bacterium fixes nitrogen, after differentiation to bacteroids. The symbiosis is host-specific, i.e. each species of Rhizobium nodulates only a limited set of host plants. Rhizobium leguminosarum nodulates plants like Pisum and Vicia, R.trifolii nodulates only Trifolium, and R.meliloti nodulates Melilotus and Medicago. Nodulation is a complex process in which probably many signals from the plant to the bacterium and vice versa are involved. The molecular mechanism of most of the steps in this process are still unknown, but recent results provide some insight into the first signals necessary for nodulation.

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. This paper describes the mapping of the exr(56)1 mutation. The mutation was mapped between two clusters of amber 56 mutations in T2, but mapping was not completely unequivocal. Additional evidence for location of exr(56)1 within gene 56 was provided by the decrease in the activity of the gene 56 product (dCTPase: EC 3.6.1.12) induced by T2 exr(56)1 strains. The location of exr(56)1 within an essential gene contradicts the exclusion model proposed by Russell & Huskey (1974).

Regulation of Bacterial Genes Involved in Bacterium-Plant Interactions by Plant Signal Molecules

Agrobacterium tumefaciens is the causative agent of the plant tumour, crown gall. The molecular basis of the tumour induction by A. tumefaciens is the transfer and subsequent expression of a defined part, the T-DNA, of the Ti-plasmid, a large plasmid, from the bacterium to the plant cell [1]. The processes occurring between the first bacterium-plant contact and the expression of the T-DNA in planta are almost unknown. At least eight virulence (Vir) operons are involved, whose functions are essential for tumour formation on all plants or which influence the host range. Many of the Vir genes are located on the Ti-plasmid [2] but some Vir genes are located on the chromosome [3].

A Model System to Detect Rhizobium Promotor Activity

The genes of fast growing Rhizobia that code for the nodulation properties (Nod genes) are only expressed in bacterial cells that are involved in symbiosis. Only a small number of cells is involved in the formation of each nodule. A special approach is therefore needed to study the expression of rhizobial Nod genes during the development of symbiosis.This paper describes the development of a method to determine the time of onset of each of the Nod promotors. The method uses bacteriophage Mu d (ApR, lac) (Casadaban, Cohen, 1979) as an indicator of promotor activity and a specific histochemical staining to detect the Mu-lac coded β-galactosidase (b-gal.).

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). The induction of the gene product (deoxycytidine triphosphate nucleotidohydrolase, dCTPase) of the strongly excluded T2 gene 56 was determined. The dCTPase was induced in the presence of the replication inhibitor oxolinic acid to prevent possible artefacts from unequal replicaton rates of T2 and T4. The rate of T2 dCTPase induction was 30% of the control in T2 X T4 infections in which all six exclusion-sensitive sites were excluded and was 57% in infections in which the sites except 56 were excluded. The dCTPase induction was 67% of the control with exclusion of the 56 site only and equalled the control when no T2 site was excluded. Inhibition of dCTPase induction under conditions of exclusion is partly due to exclusion of neighboring sites and partly due to exclusion of the 56 site.