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

Abstract

SummaryA 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 N 2 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 s 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-N 5 -hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA-catalysed acylation of the hydroxyornithine, followed by VbsLmediated 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.

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

“…3). As noted previously (Carter et al, 2002;Yeoman et al, 1999), with rpoI-lacZ there is still significant b-galactosidase enzyme activity even in high-Fe medium, but vbsC-lacZ expression is strongly repressed by Fe. Strikingly, the mutations in IRO Ri (in pBIO1335) and IRO Vc (in pBIO1466) caused a large increase in rpoI-lacZ and vbsC-lacZ expression, respectively, and totally abolished Fe-dependent repression (Fig.…”

“…Expression of both rpoI and vbsC is reduced in wild-type, Fe-replete cells, but in rirA mutants this regulation is lost (Todd et al, 2002). Transcription of vbsC and rpoI is normal in rpoI mutants; thus RpoI is not the s factor for either of these genes (Carter et al, 2002).…”

“…Divergently transcribed from rpoI is vbsC, which specifies an enzyme that likely acetylates VB (Carter et al, 2002). Expression of both rpoI and vbsC is reduced in wild-type, Fe-replete cells, but in rirA mutants this regulation is lost (Todd et al, 2002).…”

“…Strains and plasmids are described in Tables 1 and 2. R. leguminosarum and E. coli were grown as described by Beringer (1974), and Paracoccus denitrificans as described by Carter et al (2002). Techniques for conjugal gene transfer and assaying b-galactosidase in Fe-depleted media were as described by Wexler et al (2001) and references therein.…”

“…Mutations in rirA cause deregulated expression of several genes whose transcription is normally repressed in cells grown in Fe-replete medium. These include the vbs and fhu genes, which are involved, respectively, in the synthesis and uptake of the siderophore vicibactin (VB) (Carter et al, 2002;Stevens et al, 1999;Yeoman et al, 2000); hmuPSTUV, which specifies a haem ABC transporter ; tonB ; and rpoI, which encodes an ECF s factor that specifically recognizes promoters of the vbsADL and vbsGSO VB biosynthetic genes (Yeoman et al, 1999(Yeoman et al, , 2003. Transcription of rirA itself is enhanced in Fe-replete conditions and is auto-regulated (Todd et al, 2002).…”