Kay Yeoman

RirA, an iron-responsive regulator in the symbiotic bacterium Rhizobium leguminosarum

Mutations in a Rhizobium leguminosarum gene, rirA (rhizobial iron regulator), caused high-level, constitutive expression of at least eight operons whose transcription is normally Fe-responsive and whose products are involved in the synthesis or uptake of siderophores, or in the uptake of haem or of other iron sources. Close homologues of RirA exist in other rhizobia and in the pathogen Brucella; many other bacteria have deduced proteins with more limited sequence similarity. None of these homologues had been implicated in Fe-mediated gene regulation. Transcription of rirA itself is about twofold higher in cells grown in Fe-replete than in Fe-deficient growth media. Mutations in rirA reduced growth rates in Fe-replete and -depleted medium, but did not appear to affect symbiotic N(2) fixation.

The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron

In the N2‐fixing bacterium Rhizobium leguminosarum, mutations in a homologue of tonB (tonBRl) block the import of vicibactin and haem as iron sources in free‐living bacteria. TonBRl mutants were normal for growth with ferric dicitrate and slightly reduced for growth with haemoglobin as sole iron sources. The deduced TonBRl product is larger than that of (for example) Escherichia coli, on account of an extended N‐terminal domain. Transcription of tonBRl was enhanced in low‐Fe growth conditions; this was not controlled by Fur, nor RpoI, an Fe‐regulated extracytoplasmic σ factor. Upstream of tonBRl and transcribed divergently is an operon, hmuPSTUV, whose products are homologous to ABC transporters involved in haem uptake in pathogenic bacteria. Expression of hmuPSTUV was enhanced in low‐Fe conditions, and hmu mutants show slightly diminished growth on haem as sole Fe source, suggesting that there is more than one system for the uptake of this molecule. hmuPSTUV expression appears to be from three closely linked promoters. Downstream of hmuPSTUV, a gene that may encode an extracytoplasmic σ factor was identified, but this gene, rpoZ, did not affect the transcription of tonBRl or hmuPSTUV. Mutations in tonBRl, hmu genes and rpoZ did not affect symbiotic N2 fixation in peas.

High affinity iron acquisition in Rhizobium leguminosarum requires the cycHJKL operon and the feuPQ gene products, which belong to the family of two-component transcriptional regulators

The cycHJKL operon of Rhizobium leguminosarum has previously been shown to be involved in the maturation of cytochrome c, possibly by its involvement in the covalent attachment of haem to the apoprotein. Mutations in the cycHJKL genes abolish symbiotic nitrogen fixation. Here, we show that cyc mutants are pleiotropically defective. They have lost a high affinity iron acquisition system due to their failure to make or to export siderophores. They also accumulate protoporphyrin IX, the immediate precursor of haem. A model to account for these phenotypes is presented. Immediately upstream of cycH is a gene, lipA, which is predicted to encode an outer-membrane lipoprotein. Further upstream of lipA, there are two other genes, whose products are similar in sequence to the widespread family of two-component transcriptional regulators. These two genes, feuP and feuQ, did not affect the transcription of lipA, or of the cycHJKL operon. However, a mutation in feuQ also led to the loss of the high affinity iron uptake system, although siderophores were still produced.

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

A Putative ECF σ Factor Gene, rpoI, Regulates Siderophore Production in Rhizobium leguminosarum

A cloned Rhizobium leguminosarum gene, termed rpoI, when transferred to wild-type strains, caused overproduction of the siderophore vicibactin. An rpoI mutant was defective in Fe uptake but was unaffected in symbiotic N2 fixation. The RpoI gene product was similar in sequence to extra-cytoplasmic σ factors of RNA polymerase. Transcription of rpoI was reduced in cells grown in medium that was replete with Fe.

dpp Genes of Rhizobium leguminosarum Specify Uptake of δ-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.

Promoting microbiology education through the iGEM synthetic biology competition

Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM, and all DNA constructs synthesized by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area.

Analysis of a Rhizobium leguminosarum gene encoding a protein homologous to glutathione S-transferases

A novel Rhizobium leguminosarum gene, gstA, the sequence of which indicated that it was a member of the gene family of glutathione S-transferases (GSTs), was identified. The homology was greatest to the GST enzymes of higher plants. The Rhizobium gstA gene was normally expressed at a very low level. The product of gstA was over-expressed and purified from Escherichia coli. It was shown to bind to the affinity matrix glutathione-Sepharose, but no enzymic GST activity with 1-chloro-2,4-dinitrobenzene as substrate was detected. gstA encoded a polypeptide of 203 amino acid residues with a calculated molecular mass of 21990 Da. Transcribed divergently from gstA is another gene, gstR, which was similar in sequence to the LysR family of bacterial transcriptional regulators. A mutation in gstR had no effect on the transcription of itself or gstA under the growth conditions used here. Mutations in gstA and gstR caused no obvious phenotypic defect and the biological functions of these genes remain to be determined.

Investigating the Impact on Skill Development of an Undergraduate Scientific Research Skills Course.

Abstract This paper describes the design and subsequent impact of a scientific research skills course. Student understanding of the university research environment, their confidence in finding and using scientific literature and in scientific writing and presentation pre- and post-course was investigated. The findings suggested that understanding of the research environment and research process which was poor pre-course, improved after its completion. This increase in students’ understanding and confidence was also observed in their understanding of the research literature, and their ability to write scientifically and present scientific material. The students gave the course a high evaluation rating, praising the teaching and the transferable skills that it offered. The research projects carried out by the students were successful, but during the process it was found that those projects which were less well defined at the start and offered the opportunity to design research questions were more successful in the development of higher cognitive skills than projects which were highly defined at the start. The research project allowed the students to become emotionally attached to their work and this substantially increased their motivation to succeed. This enquiry based learning course provided a platform for the meeting of teaching and research and demonstrated a mutualistic symbiosis.

The ECF σ factor RpoI of R. leguminosarum initiates transcription of the vbsGSO and vbsADL siderophore biosynthetic genes in vitro

When complexed with Escherichia coli RNA polymerase core enzyme, purified RpoI protein of Rhizobium leguminosarum initiated transcription in vitro from promoters of the vbsADL and vbsGSO operons, which are needed to synthesise the siderophore vicibactin. There is a single transcription initiation site for rpoI, regardless of whether the cells are grown in Fe-replete or Fe-depleted media, but levels of rpoI mRNA were reduced, though not abolished, in the presence of Fe. Unlike PvdS, a similar Pseudomonas sigma factor needed to transcribe genes involved in pyoverdine synthesis, RpoI transcribes vbsADL and vbsGSO in the absence of the cognate siderophore. The RpoI sigma factor is not required for transcription of rpoI.