Charles H. Shaw

Chemotaxis of Rhizobium leguminosarum biovar phaseoli towards Flavonoid Inducers of the Symbiotic Nodulation Genes

Summary: Chemotaxis of Rhizobium leguminosarum biovar phaseoli RP8002 towards a range of carbohydrates, phenolic compounds and flavonoids was assayed. Xylose (peak response 10−4 M), sucrose (peak response 10−6 M) and raffinose (peak response 10−5 M) were strong chemoattractants amongst the carbohydrates, whilst glucose, fructose, galactose and maltose produced little or no detectable response. Of the monocyclic phenolic compounds, vanillyl alcohol, p-hydroxybenzoic acid (both peak responses 10−6 M) and 3,4-dihydroxybenzoic acid (peak response 10−4 M) all evoked strong chemotactic responses. Amongst the nod-inducing flavonoids, apigenin and luteolin were both strong chemoattractants (peaks at 10−5 M) while naringenin produced a very low response. Competition experiments suggest that apigenin and luteolin are recognized by a common receptor, but that there exists a separate receptor for luteolin alone. The inhibitors of nod-induction, umbelliferone and acetosyringone, both produced strong chemotactic responses, with peaks at 10−3 M and 10−2 M respectively. This evidence is indicative of a role for chemotaxis towards nod-inducing flavonoids in the initiation of root nodule formation by rhizobia, and also suggests that chemotaxis may influence the host range of the interaction.

A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins

The lipid-storing tissues of plants contain many small (0.2-1 microns) lipid (normally triacylglycerol) droplets which are surrounded and stabilized by a mixed phospholipid and protein annulus. The proteinaceous components of the lipid storage bodies are termed oleosins and are not associated with any other cellular structures. The major oleosins of rapeseed and radish have been isolated by preparative SDS-PAGE and are respectively classes of 19 kDa and 20 kDa proteins. Both protein classes were N-terminally blocked for direct sequencing, but were partially sequenced following limited proteolytic digestion. The major rapeseed oleosin was made up of at least two 19 kDa polypeptides, termed nap-I and nap-II, which have closely related but different amino acid sequences. A single 20 kDa oleosin, termed rad-I, was found in radish. A near full length cDNA clone for a major rapeseed oleosin was sequenced and found to correspond almost exactly to the sequence of nap-II. The sequences of nap-I and rad-I show very close similarity to one another, as do the sequences of nap-II and the previously determined sequence for the major oleosin from maize. All four oleosins have a large central hydrophobic domain flanked by polar N- and C-terminal domains. Secondary structure predictions for the four oleosins are similar and a novel model is proposed based on a central hydrophobic beta-strand region flanked by an N-terminal polar alpha-helix and a C-terminal amphipathic alpha-helix. The possibility that oleosins exhibit structural and functional similarities with some animal apolipoproteins is discussed.

A general method for the transfer of cloned genes to plant cells

This paper describes a method for the transfer to plant cells of any cloned gene, regardless of its termini or internal restriction enzyme cleavage sites. A broad host-range intermediate vector, pGV1117, was constructed containing HindIII-23, a right-end T-region fragment of the nopaline plasmid pTiC58. Using in vivo protection by EcoRI methylase and EcoRI linker ligation, a fragment of rabbit chromosomal DNA, carrying the beta-globin gene, was inserted into plasmid pGV1117. Following transmission to Agrobacterium tumefaciens, insertion of the gene into the T-region of pTiC58 occurred via in vivo recombination. Infection of axenic tobacco seedlings resulted in the transfer to the plant genome of an intact beta-globin gene, as part of the T-DNA. Although the gene was stably maintained during tissue culture, beta-globin-specific transcripts were not detected in the transformed plant cells.

virA and virG are the Ti‐plasmid functions required for chemotaxis of Agrobacterium tumefaciens towards acetosyringone

Octopine and nopaline Ti‐plasmids confer upon Agrobacterium tumefaciens C58C1 the ability to respond chemotactically to the vir‐inducing phenolic wound exudate, acetosyringone. A. tumefaciens C58C1 containing Ti‐plasmids with Tn5 insertions in virB, C., D or E exhibited marked chemotaxis towards acetosyringone. However, Ti‐plasmids with mutations in virA or VirG were unable to confer the responsive phenotype. Of the cosmid clones pVK219 (virAB) pVK221 (virBGC) pVK225 (virGCDE) and pVK257 (virABGC) mobilized to cured A. tumefaciens C58C1, only pVK257 bestowed acetosyringone chemotaxis. virA and virG are thus required for chemotaxis of A. tumefaciens towards acetosyringone. This suggests a multifunctional role for virA and virG: at low concentrations of acetosyringone they mediate chemotaxis and at higher concentrations they effect vir‐induction.

Attraction of Agrobacterium tumefaciens C58C1 towards Sugars Involves a Highly Sensitive Chemotaxis System

Summary: Motility of Agrobacterium tumefaciens C58C1 consisted of long straight runs, with relatively few tumbles. Speeds of up to 60 μm s−1 and runs of up to 500 μm were recorded. The propulsive mechanism appeared to resemble that of Rhizobium. Chemotaxis towards carbohydrates resolved four groups of sugars: chemoattractants with peaks at 10−6 M (sucrose, glucose and fructose); 10−5 M (maltose, lactulose and galactose); 10−4 M (raffinose, stachyose and arabinose); and weak or non-attractants (palatinose, lactose, cellobiose and xylose). In descending order, the magnitude of the responses was as follows: sucrose ≫ maltose > lactulose > glucose > galactose/fructose > stachyose/arabinose/raffinose. The amino acids valine and arginine were good chemoattractants with peaks at 10−3 M, but no significant attraction was observed with alanine, cysteine, methionine or glycine. These results are indicative of a highly sensitive chemotaxis system towards sugars in A. tumefaciens C58C1, and suggest a role for this process in the ecology of the organism.

Agrobacterium tumefaciens possesses a fourth flagellin gene located in a large gene cluster concerned with flagellar structure, assembly and motility

The authors have identified a fourth flagellin gene in a 21850 bp region of the Agrobacterium tumefaciens C58C1 chromosome containing at least 20 genes concerned with flagellar structure, assembly and function. Three flagellin genes, flaA, flaB and flaC, orientated rightward, are positioned in a tandem array at the right end, with the fourth, substantially larger gene, flaD, in the opposite orientation, at the left end. Between these lie four apparent operons, two transcribed in each direction (motA, flhB leftward; flgF, flgB rightward) from a divergent position approx 7.5 kb from the left end. This unifies the previously published motA, flgB and flaABC sequences into a single region, also containing the homologues of flhB, flgF and fliI. Site-specific mutagenesis of fliI results in a non-flagellate phenotype, while a Tn5-induced flhB mutant possesses abnormal flagella. Mutagenesis and protein profiling demonstrate that all four flagellins contribute to flagellar structure: FlaA is the major protein, FlaB and FlaC are present in lesser amounts, and FlaD is a minor component. FlaA has anomolous electrophoretic mobility, possibly due to glycosylation.

Cloning and characterisation of an oleosin gene from Brassica napus

The sequence of an oleosin gene from Brassica napus has been determined. This gene contains a single intron of 437 bp and encodes a polypeptide of 195 amino acids. The oleosin gene product has an estimated molecular mass of 21.5 kDa and consists of a highly hydrophobic central domain flanked by relatively polar N- and C-terminal domains. The central domain is highly conserved between all oleosins sequenced to date and contains a run of periodically spaced leucine residues similar to that of a leucine-zipper motif. The gene has been shown to be expressed specifically in the embryo, maximally between 9 and 11 weeks after flowering, i.e. during the seed desiccation stage. Two transcriptional start sites have been mapped to -70 and -21 of the ATG and a putative ABA-responsive element and three repeated motifs have been identified in the promoter. These short promoter sequences could correspond to regulatory elements responsible for embryo-specific gene expression. Up to six genes exist in the oleosin gene family.

A CHEMOTAXIS CLUSTER FROM AGROBACTERIUM TUMEFACIENS

We report the DNA sequence of a 9.6-kb region of the Agrobacterium tumefaciens chromosome containing a putative 8-kb chemotaxis operon. The putative operon begins with orf1, whose predicted protein product shows strong sequence identity to methyl-accepting chemotaxis proteins (MCPs), followed by orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, orf10. All of the identified homologues show a high degree of sequence conservation with their counterparts in the che operons from Sinorhizobium meliloti and Rhodobacter sphaeroides, and are arranged in a similar order. Mutations in orf1 and cheA result in impaired chemotaxis, whereas deletion of orf10, appears to have no effect on chemotaxis or motility. Although the putative operon does not contain a cheW homologue, heterologous probing and PCR using consensus primers indicates that cheW maps elsewhere in the Agrobacterium genome.

Sequence of an oleocin cDNA from Brassica napus

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived λgt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 pb and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3′ untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.

The role of VirA and VirG phosphorylation in chemotaxis towards acetosyringone by Agrobacterium tumefaciens

SUMMARY: The Ti-plasmid-encoded two-component sensor-regulator system comprising VirA and VirG confers upon Agrobacterium tumefaciens the ability to respond chemotactically to nanomolar concentrations of vir-inducing phenolics such as acetosyringone. Non-phosphorylatable, mutant VirA and VirG proteins are incapable of replacing their wild-type counterparts in conferring this phenotype. This indicates that, like vir-gene induction in response to acetosyringone, chemotaxis to the same ligand involves phosphorylation of VirA and VirG. However, unlike vir-induction, deletion of the periplasmic domain of VirA severely curtails acetosyringone chemotaxis, suggesting that acetosyringone may mediate effects through more than one region of VirA. When introduced into strains expressing wild-type VirA and VirG, the non-phosphorylatable versions suppress chemotaxis towards acetosyringone, implying that mutant copies of VirA and VirG compete with their wild-type counterparts in interactions between VirA and VirG.