The dddP gene, encoding a novel enzyme that converts dimethylsulfoniopropionate into dimethyl sulfide, is widespread in ocean metagenomes and marine bacteria and also occurs in some Ascomycete fungi.

The marine alphaproteobacterium Roseovarius nubinhibens ISM can produce the gas dimethyl sulfide (DMS) from dimethylsulfoniopropionate (DMSP), a widespread secondary metabolite that occurs in many phytoplankton. Roseovarius possesses a novel gene, termed dddP, which when cloned, confers on Escherichia coli the ability to produce DMS. The DddP polypeptide is in the large family of M24 metallopeptidases and is wholly different from two other enzymes, DddD and DddL, which were previously shown to generate DMS from dimethylsulfoniopropionate. Close homologues of DddP occur in other alphaproteobacteria and more surprisingly, in some Ascomycete fungi. These were the biotechnologically important Aspergillus oryzae and the plant pathogen, Fusarium graminearum. The dddP gene is abundant in the bacterial metagenomic sequences in the Global Ocean Sampling Expedition. Thus, dddP has several novel features and is widely dispersed, both taxonomically and geographically.

Molecular genetic analysis of a dimethylsulfoniopropionate lyase that liberates the climate-changing gas dimethylsulfide in several marine alpha-proteobacteria and Rhodobacter sphaeroides.

The alpha-proteobacterium Sulfitobacter EE-36 makes the gas dimethylsulfide (DMS) from dimethylsulfoniopropionate (DMSP), an abundant antistress molecule made by many marine phytoplankton. We screened a cosmid library of Sulfitobacter for clones that conferred to other bacteria the ability to make DMS. One gene, termed dddL, was sufficient for this phenotype when cloned in pET21a and introduced into Escherichia coli. Close DddL homologues exist in the marine alpha-proteobacteria Fulvimarina, Loktanella Oceanicola and Stappia, all of which made DMS when grown on DMSP. There was also a dddL homologue in Rhodobacter sphaeroides strain 2.4.1, but not in strain ATCC 17025; significantly, the former, but not the latter, emits DMS when grown with DMSP. Escherichia coli containing the cloned, overexpressed dddL genes of R. sphaeroides 2.4.1 and Sulfitobacter could convert DMSP to acrylate plus DMS. This is the first identification of such a 'DMSP lyase'. Thus, DMS can be made either by this DddL lyase or by a DMSP acyl CoA transferase, specified by dddD, a gene that we had identified in several other marine bacteria.

dpp genes of Rhizobium leguminosarum specify uptake of delta-aminolevulinic acid.

An operon with homology to the dppABCDF genes required to transport dipeptides in bacteria was identified in the N2-fixing symbiont, Rhizobium leguminosarum. As in other bacteria, dpp mutants were severely affected in the import of delta-aminolevulinic acid (ALA), a heme precursor. ALA uptake was antagonized by adding dipeptides, indicating that these two classes of molecule share the same transporter. Mutations in dppABCDF did not affect symbiotic N2 fixation on peas, suggesting that the ALA needed for heme synthesis is not supplied by the plant or that another uptake system functions in the bacteroids. The dppABCDF operon of R. leguminosarum resembles that in other bacteria, with a gap between dppA and dppB containing inverted repeats that may stabilize mRNA and may explain why transcription of dppA alone was higher than that of dppBCDF. The dppABCDF promoter was mapped and is most likely recognized by sigma70.

Evidence that the Rhizobium regulatory protein RirA binds to cis-acting iron-responsive operators (IROs) at promoters of some Fe-regulated genes.

Mutations in rirA of Rhizobium have been shown to deregulate expression of several genes that are normally repressed by iron. A conserved sequence, the iron-responsive operator (IRO), was identified near promoters of vbsC (involved in the synthesis of the siderophore vicibactin), rpoI (specifies an ECF sigma factor needed for vicibactin synthesis) and the two fhuA genes (encoding vicibactin receptor). Removal of these IRO sequences abolished Fe-responsive repression. Most of these genes were constitutively expressed in the heterologous host, Paracoccus denitrificans, but introduction of the cloned rirA gene repressed expression of these Rhizobium genes in this heterologous host if the corresponding IRO sequences were also intact. These observations are the first to examine the mechanisms of RirA, which has no sequence similarity to well-known iron-responsive regulators such as Fur or DtxR. They provide strong circumstantial evidence that RirA is a transcriptional regulator that binds to cis-acting regulatory sequences near the promoters of at least some of the genes whose expression it controls in response to Fe availability.

The Fur-like protein Mur of Rhizobium leguminosarum is a Mn(2+)-responsive transcriptional regulator.

In wild-type Rhizobium leguminosarum, the sitABCD operon specifies a Mn(2+) transporter whose expression is severely reduced in cells grown in the presence of this metal. Mutations in the R. leguminosarum gene, mur (manganese uptake regulator), whose product resembles the Fur transcriptional regulator, cause high-level expression of sitABCD in the presence of Mn(2+). In gel-shift mobility assays, purified R. leguminosarum Mur protein bound to at least two regions near the sitABCD promoter region, although this DNA has no conventional consensus Fur-binding sequences (fur boxes). Thus, in contrast to gamma-proteobacteria, where Fur binds Fe(2+), the R. leguminosarum Fur homologue, Mur, act as a Mn(2)-responsive transcriptional regulator.

Fur is not the global regulator of iron uptake genes in Rhizobium leguminosarum.

Rhizobium leguminosarum fur mutants were unaffected in Fe-dependent regulation of several operons that specify different Fe uptake systems, yet cloned R. leguminosarum fur partially corrected an Escherichia coli fur mutant and R. leguminosarum Fur protein bound to canonical fur boxes. The lack of a phenotype in fur mutants is not due to functional redundancy with Irr, another member of the Fur superfamily found in the rhizobia, since irr fur double mutants are also unaffected in Fe-responsive regulation of several operons involved in Fe uptake. Neither Irr nor Fur is needed for symbiotic N(2) fixation on peas. As in Bradyrhizobium japonicum, irr mutants accumulated protoporphyrin IX. R. leguminosarum irr is not regulated by Fur and its Irr protein lacks the motif needed for haem-dependent post-translational modification that occurs in B. japonicum Irr. The similarities and differences in the Fur superfamily in the rhizobia and other Gram-negative bacteria are discussed.

Structural studies of the Fur protein from Rhizobium leguminosarum.

The X-ray crystal structure of the apo-form of the Fur protein from Rhizobium leguminosarum has been solved at 2.7 A resolution. Small-angle X-ray scattering was used to give information on the solution conformation of the protein. The Fur homodimer folds into two domains. The N-terminal domain is formed from the packing of two helix-turn-helix motifs while the C-terminal domain appears primarily to stabilize the dimeric state of the protein.

The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF sigma factor RpoI.

A cluster of eight genes, vbsGSO, vbsADL, vbsC and vbsP, are involved in the synthesis of vicibactin, a cyclic, trihydroxamate siderophore made by the symbiotic bacterium Rhizobium leguminosarum. None of these vbs genes was required for symbiotic N2 fixation on peas or Vicia. Transcription of vbsC, vbsGSO and vbsADL (but not vbsP) was enhanced by growth in low levels of Fe. Transcription of vbsGSO and vbsADL, but not vbsP or vbsC, required the closely linked gene rpoI, which encodes an ECF sigma factor of RNA polymerase. Transfer of the cloned vbs genes, plus rpoI, to Rhodobacter, Paracoccus and Sinorhizobium conferred the ability to make vicibactin on these other genera. We present a biochemical genetic model of vicibactin synthesis, which accommodates the phenotypes of different vbs mutants and the homologies of the vbs gene products. In this model, VbsS, which is similar to many non-ribosomal peptide synthetase multienzymes, has a central role. It is proposed that VbsS activates L-N5-hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA-catalysed acylation of the hydroxyornithine, followed by VbsL-mediated epimerization and acetylation catalysed by VbsC, yields the vicibactin subunit, which is then trimerized and cyclized by the thioesterase domain of VbsS to give the completed siderophore.