Carel A. Wijffelman

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.

Role of the O-Antigen of Lipopolysaccharide, and Possible Roles of Growth Rate and of NADH:ubiquinone Oxidoreductase (nuo) in Competitive Tomato Root-Tip Colonization by Pseudomonas fluorescens WCS365

Colonization-defective, transposon-induced mutants of the efficient root colonizer Pseudomonas fluorescens WCS365 were identified with a gnotobiotic system. Most mutants were impaired in known colonization traits, i.e., prototrophy for amino acids, motility, and synthesis of the O-antigen of LPS (lipopolysaccharide). Mutants lacking the O-antigen of LPS were impaired in both colonization and competitive growth whereas one mutant (PCL1205) with a shorter O-antigen chain was defective only in colonization ability, suggesting a role for the intact O-antigen of LPS in colonization. Eight competitive colonization mutants that were not defective in the above-mentioned traits colonized the tomato root tip well when inoculated alone, but were defective in competitive root colonization of tomato, radish, and wheat, indicating they contained mutations affecting host range. One of these eight mutants (PCL1201) was further characterized and contains a mutation in a gene that shows homology to the Escherichia coli nuo4 gene, which encodes a subunit of one of two known NADH:ubiquinone oxidoreductases. Competition experiments in an oxygen-poor medium between mutant PCL1201 and its parental strain showed a decreased growth rate of mutant PCL1201. The requirement of the nuo4 gene homolog for optimal growth under conditions of oxygen limitation suggests that the root-tip environment is micro-aerobic. A mutant characterized by a slow growth rate (PCL1216) was analyzed further and contained a mutation in a gene with similarity to the E. coli HtrB protein, a lauroyl transferase that functions in lipid A biosynthesis.

A two-component system plays an important role in the root-colonizing ability of Pseudomonas fluorescens strain WCS365

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.

On the control of transcription of bacteriophage Mu.

SummaryThe transcription pattern of bacteriophage Mu has been studied with the use of Mu-1 cts62, a thermo-inducible derivative of wild-type Mu. The rate of transcription at various times after induction was measured by pulse-labeling the RNA during synthesis and determining the fraction of Mu-specific RNA by hybridization with the separated strands of Mu-DNA. Transcription was found to take place predominantly from the heavy strand of Mu-DNA, as was found previously by Bade (1972). A study of the kinetics of this process revealed four phases. Initially after the induction the rate of transcription increases and reaches a maximum after four minutes. In the second phase during five minutes the rate falls down. During the third phase, up to 25 minutes after induction, the rate of transcription rises slowly, followed by a very rapid increase in the final phase, at the end of the lytic cycle. Phage Mu can be integrated in the host chromosome in two opposite orientations. The strand specificity, rate and time-course of transcription appeared not to be influenced by the orientation. The presence of chloramphenicol during the induction of the phage does not have an effect on the initial phase of transcription, but it prevents the decrease in the second phase. This suggests that in the early phase a Mu-specific protein is synthesized which acts as a negative regulator of trancription. In non-permissive strains, lysogenic for a phage with an amber mutation in gene A or B, the transcription during the first and the second phase is the same as with wild-type phage; in the third phase, however, there is much less transcription than with wild type phage, whereas in the final phase the increase of the transcription rate is completely absent.Control experiments showed that DNA synthesis does not take place when a non-permissive strain is infected with a phage with an amber mutation in gene A or B. Therefore we conclude that the products of the genes A and B are required, directly or indirectly, for the autonomous replication of phage DNA. Since these amber mutants are also impaired in the integration process, we conclude that the genes A and B code for regulator proteins with a crucial role in the development of bacteriophage Mu.

Analysis of the major inducers of the Rhizobium nodA promoter from Vicia sativa root exudate and their activity with different nodD genes

Root exudate of Vicia sativa contains 7 inducers for the nodA promoter of Rhizobium leguminosarum biovar viciae. Six of these inducers are flavanones. One inducer was identified as 3,5,7,3′-tetrahydroxy-4′-methoxyflavanone, and a second inducer most likely is 7,3′-dihydroxy-4′-methoxyflavanone. The inducing activity of these compounds and the other inducers depends on the nodD gene present in the test strains, which orginated either from R. leguminosarum biovars viciae or trifolii, or from R. meliloti. Three inducers are ‘common’, three others almost exclusively induce the nodA promoter in the presence of the R. leguminosarum biovar viciae nodD gene, and the last one is active with the noD genes of either R. leguminosarum biovar viciae or that of R. meliloti. Testing of a large number of flavonoids revealed two classes of structural features required for inducing ability: (i) features required for induction in general, and (ii), features restricting the inducing ability to (a) specific nodD gene(s). These features are discussed in relation to current models of the process of nodD-mediated transcription activation of the inducible nod genes.

Transcription of bacteriophage Mu

SummaryIt has previously been shown that the transcription of Mu is asymmetric and takes place on the heavy DNA strand (Bade, 1972; Wijffelman et al., 1974). The direction of transcription of Mu has now been determined by RNA-DNA hybridizations between purified Mu-RNA and the separated strands of λ-Mu hybrid phages. The direction of transcription is from the c-gene (immunity gene) end of the heavy strand to the β-end (immunity distal end) (Fig. 1). Thermo-inducible, defective Mu lysogens, in which the prophage is deleted from the β-end, have a normal early transcription pattern, but the increase of RNA at later times is absent. A defective lysogen, which contains only the immunity gene c and the genes A and B, still has an early transcription pattern similar to that of the wild-type. Therefore, we conclude that the early RNA is transcribed from that region of the Mu genome.The early Mu-RNA synthesis is negatively regulated with a minimum rate of transcription at 9 minutes after induction. Before the onset of the late RNA synthesis, at about 22 minutes there is a rather long period in which the rate of Mu-RNA synthesis slowly increases. Using DNA strands of λ-Mu hybrids which contain only that part of the Mu-DNA on which the early RNA synthesis takes place, we have determined that during the first half in the intermediate phase only early genes are transcribed.The amount of Mu-RNA synthesized by a Mu prophage carrying the X-mutation, which influences the excision of Mu, is greatly reduced. Negative regulation of early transcription occurs normally in this mutant.

Kinetics of Mu DNA synthesis.

SummaryMu specific DNA synthesis starts at 10 min after infection. All essential amber mutants of Mu were tested for the ability to replicate in a non permissive host. Except for the amber mutants A and B, which were already known to be blocked in Mu DNA synthesis (Wijffelman et al., 1974), all the other mutants showed normal Mu DNA replication.Using mitomycin C-treated cells Mu DNA synthesis was found to start at about 20 min after induction. However using the much more sensitive method of DNA-RNA hybridization, it was found that the DNA synthesis starts already at 10 min after induction, and that at 20 min after induction about 7 copies of the Mu DNA are present per cell.

Nodulation of specific legumes is controlled by several distinct loci in Rhizobium trifolii

SummaryThree distinct loci (designated regions III, IV and V) were identified in the 14 kb Nod region of Rhizobium trifolii strain ANU843 and were found to determine the host range characteristics of this strain. Deletion of region III or region V only from the 14 kb Nod region affected clover nodulation capacity. The introduction to R. Leguminosarum of DNA fragments on multicopy vectors carrying regions III, IV and V (but not smaller fragments) extended the host range of R. leguminosarum so that infection threads and nodules occurred on white clover plants. The same DNA fragments were introduced to the Sym plasmid-cured strain (ANU845) carrying the R. meliloti recombinant nodulation plasmid pRmSL26. Plasmid pRmSL26 alone does not confer root hair curling or nodulation on clover plants. However, the introduction to ANU845 (pRmSL26) of a 1.4 kb fragment carrying R. trifolii region IV only, resulted in the phenotypic activation of marked root hair curling ability to this strain on clovers but no infection events or nodules resulted. Only the transfer of regions III, IV and V to strain ANU845 (pRmSL26) conferred normal nodulation and host range ability of the original wild type R. trifolii strain. These results indicate that the host range genes determine the outcome of early plant-bacterial interactions primarily at the stage of root hair curling and infection.

Similarity of vegetative map and prophage map of bacteriophage Mu-1

Abstract The vegetative map of bacteriophage Mu was extended by the localization of three more amber mutations belonging to different complementation groups, and the position of the c gene on the vegetative map was approximately determined. The data confirm the previous finding that the vegetative map is linear (Wijffelman et al. , 1972). Defective Mu lysogens were isolated from an Escherichia coli K12 strain, which has an insertion of Mu in the trp operon, by selecting for TonB colonies. All these defective lysogens were no longer immune to Mu infection, suggesting that the gene controlling immunity is located near to the end of the prophage proximal to tonB . By analyzing the remaining prophage genes in the defective lysogens, it was possible to determine the order of 16 amber mutations belonging to different complementation groups. The results show that the position of the genes on both the vegetative map and the prophage map is the same. The prophage map of Mu was determined by using the same method in another 9 independently isolated insertion mutants. In 7 of these mutants the gene order in the prophage is the same as found for the above-mentioned strain. The other two contain the prophage genes in the reversed order. All defective lysogens derived from these two insertion mutants by selection for TonB colonies were immune. The results show that Mu is integrated in a unique way and that both orientations may occur. The similarity between prophage map and vegetative map and the consequence for the mechanism of the integration process are discussed.

Structural polypeptides and products of late genes of bacteriophage Mu: Characterization and functional aspects☆

Abstract This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion. In vitro reconstitution of infectious phage particles has shown that four genes ( E , D , I , J ) control the formation of phage heads and that a cluster of eight genes ( K , L , M , N , P , Q , R , S ) controls the formation of phage tails. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10 3 M r ); H (64 × 10 3 M r ); F (54 × 10 3 M r ); G (16 × 10 3 M r ); L (55 × 10 3 M r ); N (60 × 10 3 M r ); P (43 × 10 3 M r ) and S (56 × 10 3 M r ). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V , W or R (41.5 × 10 3 M r , and 45 × 10 3 M r ); U (20.5 × 10 3 M r ) and of genes located in the β region (24 × 10 3 M r (gp gin ) and 37 × 10 3 M r (possibly gp mom )). Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10 3 , a second head polypeptide of 54 × 10 3 (gp F ) and two major tail polypeptides with molecular weights of 55 × 10 3 and 12.5 × 10 3 (gp L and gp Y , respectively). In addition, there are five minor components of the tail (including gp N , gp S and gp U ) and approximately seven minor components of the head structure of the virion (including gp H ).