R. Fellay

Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria

We have constructed a series of derivatives of the omega interposon [Prentki and Krisch, Gene 29 (1984) 303-313] that can be used for in vitro insertional mutagenesis. Each of these DNA fragments carries a different antibiotic or Hg2+ resistance gene (ApR, CmR, TcR, KmR or HgR) which is flanked, in inverted orientation, by transcription and translation termination signals and by synthetic polylinkers. The DNA of these interposons can be easily purified and then inserted, by in vitro ligation, into a plasmid linearized either at random by DNase I or at specific sites by restriction enzymes. Plasmid molecules which contain an interposon insertion can be identified by expression of its drug resistance. The position of the interposon can be precisely mapped by the restriction sites in the flanking polylinker. To verify their properties we have used these omega derivatives to mutagenize a broad host range plasmid which contains the entire meta-cleavage pathway of the toluene degradation plasmid pWW0 of Pseudomonas putida. Insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli. We also show that when a plasmid containing an omega interposon is transferred by conjugal mobilization from E. coli to P. putida, Agrobacterium tumefaciens, Erwinia chrysanthemi, Paracoccus denitrificans or Rhizobium leguminosarum, the appropriate interposon drug resistance is usually expressed and, compared to the non-mutated plasmid, much reduced levels of C23O activity are detected. Thus, the selection and/or characterization of omega insertional mutations can be carried out in these bacterial species.

High‐resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad‐host‐range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA‐σ54 regulatory sequences, which are thought to co‐ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)‐like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.

Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria.

To combine the features of the omega interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposon, called Omegon-Km. The Omegon-Km transposon is carried on the plasmid pJFF350 which can be conjugally mobilized into a broad range of Gram-negative bacteria. Omegon-Km is flanked, in inverted orientation, by synthetic 28-bp repeats derived from the ends of IS1. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the omega interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene (alph A) which is expressed well in many Gram-negative bacteria. The IS1 transposition functions are located on the donor plasmid but external to Omegon-Km. Thus, insertions of Omegon-Km are very stable because they lack the capacity for further transposition. Omegon-Km mutagenesis is performed by conjugal transfer of pJFF350 from Escherichia coli into any Gram-negative recipient strain in which this plasmid is unable to replicate. Those cells which have had a transposition event are selected by their resistance to Km. Very high frequencies of Omegon-Km transposition were observed in Pseudomonas putida. Preliminary experiments with other Gram-negative soil and water bacteria (Rhizobium leguminosarum, Paracoccus denitrificans) yielded mutants at reasonable levels. The presence of an E. coli-specific origin of replication (ori) within Omegon-Km allows the rapid and easy cloning, in E. coli, of the nucleotide sequences flanking the site of the transposition event.

Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host‐specificity gene

Rhizobia secrete specific lipo‐chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N‐acetyl‐d‐glucosamine of LCOs is substituted at C6 with 2‐O‐methyl‐l‐fucose which can be acetylated or sulphated. We identified a flavonoid‐inducible locus on the symbiotic plasmid pNGR234a that contains a new nodulation gene, noeEwhich is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non‐sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum. Furthermore, mutation of noeE (NGRΔnoeE ) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus. The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRΔnoeE leads to the production of a mixture of LCOs that are sulphated on C6 of the reducing terminus and sulphated on the 2‐O‐methylfucose residue. Together, these findings show that noeE is a host‐specificity gene which probably encodes a fucose‐specific sulphotransferase.

nodD2 of Rhizobium sp. NGR234 is involved in the repression of the nodABC operon.

Transcriptional regulators of the lysR family largely control the expression of bacterial symbiotic genes. Rhizobium sp. NGR234 contains at least four members of this family: two resemble nodD, while two others are more closely related to syrM. Part of the extremely broad host range of NGR234 can be attributed to nodD1, although the second gene shares a high degree of DNA sequence homology with nodD2 of R. fredii USDA191. A nodD2 mutant of NGR234 was constructed by insertional mutagenesis. This mutant (NGRΩnodD2) was deficient in nitrogen fixation on Vigna unguiculata and induced pseudonodules on Tephrosia vogelii. Several other host plants were tested, but no correlation could be drawn between the phenotype and nodule morphology. Moreover, nodD2 has a negative effect on the production of Nod factors: mutation of this gene results in a fivefold increase in Nod factor production. Surprisingly, while the structure of Nod factors from free‐living cultures of NGRΩnodD2 remained unchanged, those from V. unguiculata nodules induced by the same strain are non‐fucosylated and have a lower degree of oligomerization. In other words, developmental regulation of Nod factor production is also abolished in this mutant. Competitive RNA hybridizations, gene fusions and mobility shift assays confirmed that nodD2 downregulates expression of the nodABC operon.

Organization of host-inducible transcripts on the symbiotic plasmid of Rhizobium sp. NGR234

In a systematic approach to identify genes involved in the early steps of the legume—Rhizobium symbiosis, we studied transcription patterns of symbiotic plasmid‐borne loci. A competitive hybridization procedure was used to identify DNA restriction fragments carrying genes whose expression is enhanced by plant root exudates or by purified flavonoids. Fragments containing induced genes were then located on the physical map of the 500 kb pNGR234a. New inducible loci as well as previously described genes were identified and their time course of induction determined. After initial induction, transcription of loci such as nodABC and the host‐specificity genes nodSU decreased to undetectable levels 24 h after incubation with purified flavonoids. In contrast, expression of other loci is detectable only after several hours of induction. Surprisingly, many genes remained transcribed in the nodD1 mutant suggesting the presence of other flavonoid‐dependent activators in NGR234. The hsnl region, which is involved in host specificity, was shown to carry several inducible but independently regulated transcripts. Sequencing analysis revealed several open reading frames whose products, based on sequence similarities, may be involved in L‐fucose metabolism and its adjunction to the Nod factors.

SyrM1 of Rhizobium sp. NGR234 Activates Transcription of Symbiotic Loci and Controls the Level of Sulfated Nod Factors

One or more transcriptional regulators of the LysR class control transcription of rhizobial nodulation genes. In Rhizobium sp. NGR234, two copies of nodD (nodD1 and nodD2) are present on the symbiotic plasmid pNGR234a. The complete sequence of pNGR234a revealed two additional nodD homologues, syrM1 and syrM2. Competitive RNA hybridization analyses involving a mutant of syrM1 (NGRΔsyrM1) showed that a number of symbiotic genes (e.g., nolXBTUVW) are expressed in an syrM1-dependent manner. Assays in which regions upstream of nolB and nolW were fused to promotorless lacZ confirmed that SyrM1 is required for their late induction. Mutation of syrM1 also drastically reduced production of sulfated Nod factors as shown by reverse phase-thin layer chromatography (RP-TLC). SyrM1 controls sulfation of Nod factors via one of the two chromosomal nodPQ loci. It thus seems likely that syrM1 of NGR234 encodes a transcriptional activator that regulates the expression of genes involved in both the early and late stages of i...

Transposable elements for efficient manipulation of a wide range of Gram-negative bacteria: promoter probes and vectors for foreign genes

We describe here the construction and use of a series of modified transposons based on the insertion sequence IS1. Like their parent, omegon-Km [Fellay et al., Gene 76 (1989) 215-226], these elements permit efficient insertional mutagenesis of a variety of Gram-negative bacteria. The presence of a functional pBR322 origin of replication within the transposable element facilitates subsequent cloning of the mutated gene. The omegon-Km system was previously shown to function in Pseudomonas putida, Rhizobium leguminosarum and Paracoccus denitrificans. The results we present here demonstrate that its use can be extended to Xanthomonas campestris, a plant pathogen, and to the microaeroduric Zymomonas mobilis. Derivative transposons carrying unique restriction sites for ScaI, NdeI, XbaI and XhoI have been constructed, allowing the cloning and introduction of foreign genes. We have also constructed two derivatives which can be used to generate operon fusions upon insertion and are thus useful for isolating and characterising indigenous promoters. One carries a promoterless chloramphenicol acetyl-transferase (CAT)-encoding gene (cat) and the second, the entire promoterless Escherichia coli lac operon. We demonstrate the utility of the cat promoter probe in X. campestris to target conditional promoters inducible by high salt or subject to repression by glucose.

Region II of Rhizobium sp. NGR234 Inhibits Nodulation of Medicago sativa by R. meliloti nodIJ and nodQ1 Mutants

Rhizobium sp. NGR234 contains a plasmid-borne locus that hybridized strongly to region II of R. meliloti. Surprisingly, NGR region II completely inhibited nodulation of Medicago sativa when conjugated into either R. meliloti nodIJ or nodQ1 region II mutants. Further characterization showed that region II of NGR234 contains three putative coding open reading frames (ORFs), which are homologous to, respectively, hypothetical protein A of R. leguminosarum, hypothetical protein C of R. leguminosarum, and ORF2 of Agrobacterium tumefaciens insertion sequence (IS) 66, as well as an ORF of unknown function located downstream of nodQ1 of R. meliloti. A site-directed mutation in the IS66 homologue improved nodulation efficiency on some NGR234 hosts, but the structure and composition of the Nod-factor family produced by the mutant were unchanged. The quantity of Nod factors secreted was reduced by two-thirds, however. Insertions or deletions of the genes encoding either the hypothetical protein C or the IS66 homolog...

Molecular Analysis of the Symbiotic Replicon of Rhizobium sp. NGR234

Most symbiotic determinants of the broad host-range Rhizobium species NGR234 are carried by pNGR234a, a self-transmissible plasmid of 536 kb. The DNA sequence of this replicon was predicted to encode 416 proteins (Freiberg et al., 1997). As befits a symbiotic plasmid which can be cured by heat treatment (Morrison et al., 1983), no essential gene was found on pNGR234a. More than a third of the putative products (139) showed no obvious similarities to any known protein. Of the remaining 276 gene products, 31 (8%) correspond to proteins for which no biochemical or phenotypic role has been assigned, 11 (3%) are similar to proteins for which limited biological data is available, and 234 (56%) resemble proteins with a more precise function. Another way of gaining insights into the function of predicted genes is to follow their expression under different conditions. To do this, RNA was hybridized against Southern filters containing 441 amplified DNA fragments designed to represent each of the 416 predicted ORF’s as well as intergenic regions. Various conditions were tested by using labelled RNA purified from bacterial cultures (with or without flavonoid induction) as well as from bacteroids found in determinate or indeterminate nodules.