Investigation of a regulatory iron-sulfur complex in a bacterial sigma factor

Lead Research Organisation: University of Sheffield
Department Name: Infection Immunity & Cardiovasc Disease


The bacterium Burkholderia thailandensis produces malleobactin which it uses to capture iron. The genes encoding the biosynthesis of malleobactin are under the control of the sigma factor MbaS and are expressed under iron limiting conditions. The characteristics of MbaS suggest that it possesses an iron-sensing domain at its C-terminus. We hypothesise that under iron sufficient conditions, MbaS acquires an iron-sulfur cluster which inhibits association of MbaS-RNA polymerase with promoter DNA.


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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1723623 Studentship BB/M011151/1 30/09/2015 13/10/2019 Christopher Banyard
Description Burkholderia pseudomallei and Burkholderia cenocepacia are pathogenic bacteria that are the causative agents of the deadly diseases melioidosis and 'cepacia syndrome', respectively. Both species utilise extracytoplasmic function (ECF) sigma factors, alternative subunits of bacterial RNA polymerase, to transduce extracellular signals and drive the transcription of specific genes. MbaS, from Burkholderia pseudomallei, and OrbS, from Burkholderia cenocepacia, are two iron starvation (IS) sigma factors that regulate gene clusters associated with the iron-chelating siderophores malleobactin and ornibactin, respectively. OrbS and MbaS have unusual extended C-terminal extensions that are rich in cysteine residues. These are referred to as cysteine-rich extensions (CREs). Given the lack of a cognate anti-sigma factor, the ability of cysteine residues to coordinate metals via their thiol group, and the observation that the cysteine-rich extended C-terminal domains of the ECF44 group sigma factors bind various metals as part of a regulatory mechanism (Gomez-Santos et al., 2011; Marcos-Torres et al., 2016), it was hypothesised that the CRE found in the ECF09 sigma factors OrbS and MbaS may play a role in binding iron. This may regulate the activity of the protein.
OrbS was the selected sigma factor used for the initial investigation into the C-terminal extensions (and also a 35 amino acid N-terminal extension). The effect of deleting these terminal extensions was investigated, with the aims of determining what length of the domains are required for the function of OrbS, and whether they play a role in the ability of OrbS to respond to iron. Furthermore, a previous preliminary analysis of OrbS-dependent promoter affinity had suggested that the substitution of all four C-terminal cysteine residues in OrbS by alanine residues may reduce the sigma factor's responsiveness to iron (K. Agnoli and M. Thomas, unpublished data). A cysteine-substituted mutant, OrbS::CtetraA, was included in this analysis to assess the importance of the four cysteine residues, both in terms of the overall function of OrbS and the sigma factor's ability to respond to iron. A series of pBBR1MCS-based plasmids expressing orbS and derivatives of orbS encoding C-terminal truncations and an N-terminal deletion were constructed. The N-terminus deletion displayed functional ability both in terms of complementation of a ?orbS phenotype and initiation of transcription from the orbH target promoter. Therefore, the 29 residues of the N-terminus present in OrbS are not required for OrbS-dependent transcription. At the C-terminus, deletions extending as far as Cys199 (i.e. deletion of up to 21 amino acids from the C-terminus) also did not affect the ability of the sigma factor to initiate transcription. However, truncations extending N-terminally of Cys199 do abolish this ability, as demonstrated by the inability to complement the orbS mutant or initiate transcription from the orbH promoter. It is likely that the deleted amino acid residues in these cases provide a key role in the overall function of OrbS, particularly as they are located within the conserved region 4.2. Experiments were also performed to analyse the effect of iron upon the activity of OrbS and its N- and C-terminally truncated variants. This was demonstrated with promoter reporter analysis in an E. coli fur null mutant. It was observed that OrbS activity was regulated in response to prevailing iron. There was loss of this iron-dependent regulation of the activity of OrbS variants with C-terminal deletions. This could point to the C-terminal extension having a role in the iron-dependent regulation mechanism. Interestingly, the key disparity between C-terminally truncated variants of OrbS which display some iron regulation, and those which does not display iron regulation, is the deletion of the Cys209 and Pro210 residues. This could imply that these residues, most likely the cysteine residue, have a role in this iron regulation. Furthermore, this loss of iron regulation was observed upon the activity of OrbS::CtetraA. This observation strongly suggests that the four cysteine residues of the CRE play a significant role in the putative direct iron-regulation mechanism of OrbS.
For the analysis of the activity of MbaS in vivo, a series of gene mutations were required in the model organism B. thailandensis E264. Firstly, a ?mbaS mutant was required to remove the genomic copy of MbaS, and allow Fur-independent MbaS (and mutant forms of MbaS) to be supplied in trans. Additionally, the secondary siderophore pyochelin was predicted to interfere with in vivo assays where the production of malleobactin was to be assayed. Therefore, the pyochelin NRPS pchE gene was to be deleted. The transcription of mbaS is regulated by the ferric uptake regulator Fur (Alice et al., 2006); therefore, the fur gene was also selected for deletion to remove the masking effect of Fur-dependent iron regulation. Finally, for the purpose of investigating the effect of the five conserved cysteine residues of the CRE of MbaS, a mutant form of the mbaS gene with five cysteine-to-alanine substitutions (mbaS::CpentaA) was produced. These mbaS substitution mutants were also intended to be introduced into Bth E264. Mutant alleles were amplified by SOE-PCR and cloned into selected allelic exchange vectors. However, several allelic exchange vectors were employed with varying levels of success in Bth E264. Attempts with several different methods of allelic exchange were required to generate mutants, and these were isolated with low efficiency. For the generation of mbaS::CpentaA and fur mutants, usage of these allelic exchange vectors was unsuccessful and it was not possible to isolate the desired mutants. The ?mbaS and ?pchE deletion alleles were successfully introduced to generate Bth E264 ?mbaS, Bth E264 ?pchE and Bth E264 ?mbaS ?pchE. These mutant strains were phenotypically characterised using growth curves, beta-galactosidase assays and chrome azurol-S (CAS) assays to verify the gene deletions and demonstrate the resulting loss of malleobactin and pyochelin production. However, attempts at introducing the ?fur and mbaS::CpentaA alleles was not achieved. The Bth E264 ?mbaS, ?pchE and ?mbaS ?pchE strains had their specific gene deletions verified in vivo by complementation with pBBR2-mbaS and pBBR2-pchE. However, Bth E264 ?pchE displayed no observable phenotypic difference to the wild-type, making it difficult to confirm the pchE deletion and the complementation of this gene deletion. However, in combination with the phenotypic characterisation of the Bth E264 ?mbaS and Bth E264 ?mbaS ?pchE it is unlikely that the gene deletions were not introduced as expected. Additionally, when Bth was grown on CAS agar, the large yellow halo was assigned as being produced due to the secretion of malleobactin, and the large orange halo was assigned as the result of pyochelin production. This is assumption is based upon the known functions of the mbaS and pchE genes.
It has been empirically demonstrated that MbaS is a sigma factor. This has been shown through its capacity to stimulate transcription from target gene promoters in promoter reporter assays and in vitro transcription assays in the presence of core RNAP. Additionally, the association of MbaS and core RNAP has been demonstrated by bio layer interferometry, and a binding affinity for this association has been determined. Although the designation of MbaS as an ECF sigma factor has been established for some time (Alice et al., 2006), this study presents the first empirical demonstration of this. Furthermore, the target promoters of MbaS have been formally identified. Again, based upon homology with the orb gene cluster and RNase protection assays of MbaS-regulated genes, the PmbaH and PmbaE promoters were assumed to be transcribed in an MbaS-dependent manner. A 562 bp DNA region containing the PmbaI promoter DNA was shown to be activated in an MbaS-dependent manner by an in vivo promoter reporter assay (Alice et al., 2006). Through further in vivo promoter reporter assays presented in this study, this has been empirically demonstrated for all three promoters. Moreover, these promoters have been identified with precision to a 49 bp DNA region. It is very likely that the core promoter elements are present within this region, and that they correspond to those precisely identified in the orbH promoter (Agnoli et al., 2018). The mbaS promoter has also been characterised to a greater degree. Through in vivo promoter reporter assays, it has been established that activity from the mbaS promoter does not increase in the presence of MbaS, and therefore it can be concluded MbaS does not autoregulate its own promoter. It is highly likely that the mbaS promoter is regulated in a s70-dependent manner, although this has not been demonstrated unequivocally. Previous work identified that transcription of mbaS was downregulated under high-iron conditions, likely due to a Fur box sequence identified in silico in a DNA region upstream of the translation start codon (Alice et al., 2006). It was also reported that this was demonstrated by FURTA (although the data was not shown and the length of DNA assayed was not specified). Using FURTA, this study has limited the location of the Fur box to an 186 bp DNA of region. Nonetheless, this could be identified with greater precision in the future.
Evidence has been gathered that supports the hypothesis that MbaS and OrbS possess on-board metal-responsive regulatory domains. A series of methods were developed and employed to investigate the influence that the presence of metal ions had on MbaS and OrbS activity upon dependent promoters, and upon association with core RNAP. Through comparison with the cysteine-to-alanine substitution mutants MbaS::CpentaA and OrbS::CtetraA, the dependency of the conserved cysteine residues of the CRE upon this metal regulation could be inferred. In in vivo promoter reporter analyses, inhibition of the activity of OrbS could be observed in the presence of 50 µM iron chloride, copper chloride and zinc chloride. The presence of 10 µM Fe(II) and 25 µM Zn(II) was also demonstrated to inhibit the OrbS-dependent transcription of a target promoter in vitro. Finally, the presence of 25 µM Fe(II) and Zn(II) decreased the binding affinity of core RNAP and OrbS by 435-fold and 934-fold, respectively. This strongly suggests that OrbS is able to respond to the presence of Fe(II), Zn(II), and possibly Cu(II) ions. In the same experiments performed upon MbaS in vivo, the presence of the selected metal showed no effect upon the activity of MbaS upon its target promoter. However, in vitro transcription assays demonstrated that presence of 25 µM Zn(II) and 10 µM Fe(II) resulted in reduced MbaS-dependent transcription from a target promoter, and presence of 25 µM Zn(II) decreased the binding affinity between MbaS and core RNAP 77,544-fold. This suggests that MbaS is able to respond to the presence of Zn(II), and possibly Fe(II) ions. Importantly, these metal inhibition effects were not observed to occur to the same degree upon the activity of MbaS::CpentaA and OrbS::CtetraA. Therefore, it is highly likely that this influence the metal ions exhibit upon the sigma factor activity and association with core RNAP occurs via the thiol groups of some or all of the conserved cysteine residues of the CREs present in both OrbS and MbaS.

References: Agnoli, K., Lowe, C.A., Farmer, K.L., Husnain, S.I., Thomas, M.S. (2006) 'The ornibactin biosynthesis and transport genes of Burkholderia cenocepacia are regulated by an extracytoplasmic function sigma factor which is part of the Fur regulon'. J. Biol., 188(10), p3631-3644. Agnoli, K., Haldipurkar, S. S., Tang, Y., Butt, A. T. and Thomas, M. S. (2018) 'Distinct modes of promoter recognition by two iron starvation sigma factors with overlapping promoter specificities', J Bacteriol. Alice, A.F., Lopez, C.S., Lowe, C.A., Ledesma, M.A., Crosa, J.H. (2006). 'Genetic and transcriptional analysis of the siderophore malleobactin biosynthesis and transport genes in the human pathogen Burkholderia pseudomallei K96243'. J. Biol., 188(4), p1551-1566. Gomez-Santos, N., Perez, J., Sanchez-Sutil, M. C., Moraleda-Munoz, A. and Munoz-Dorado, J. (2011) 'CorE from Myxococcus xanthus is a copper-dependent RNA polymerase sigma factor', PLoS Genet, 7(6), pp. e1002106.
Marcos-Torres, F. J., Pérez, J., Gómez-Santos, N., Moraleda-Muñoz, A. and Muñoz-Dorado, J. (2016) 'In depth analysis of the mechanism of action of metal-dependent sigma factors: characterization of CorE2 from Myxococcus xanthus', Nucleic Acids Research.
Exploitation Route Direct binding of metal ions to OrbS and MbaS has yet to be demonstrated. Using the protein purification protocol developed, and investigating the metal ions that may regulate the sigma factors identified from research funded from this award, one could use several in vitro methods (e.g. isothermal calorimetry, circular dichroism, NMR etc.) to demonstrate direct metal binding. These methods could also be used to determine binding kinetic values. Finally, the protein structures of the two sigma factors could be elucidated.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Title Burkholderia thailandensis E264 mutants 
Description Several mutant strains of the bacteria Burkholderia thailandensis E264 have been developed to investigate the MbaS protein and the production of iron-chelating siderophore compounds. These were generated using either the pEX18Tp-pheS or the pNUFF3Cm allelic exchange vectors. These strains are the following: ?mbaS - in-frame markerless gene deletion of the mbaS gene (malleobactin-deficient phenotype) ?pchE - iniframe markerless gene deletion of the pchE gene (pyochelin-deficient phenotype) ?mbaS ?pchE - in-frame markerless gene deletion of the mbaS and pchE genes (malleobactin and pyochelin deficient phenotype) 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact So far the tools have been used to empirically identify the functions of the mbaS and pchE genes, and used to demonstrate the lack of iron-dependent Fur-independent regulation upon MbaS supplied in trans. Further potential research using these research tools is still ongoing.