The Burkholderia cenocepacia iron starvation σ factor, OrbS, possesses an on-board iron sensor.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board 'anti-σ factor' in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

Distinct Modes of Promoter Recognition by Two Iron Starvation σ Factors with Overlapping Promoter Specificities.

OrbS and PvdS are extracytoplasmic function (ECF) σ factors that regulate transcription of operons required for the biosynthesis of the siderophores ornibactin and pyoverdine in the Burkholderia cepacia complex and Pseudomonas spp., respectively. Here we show that promoter recognition by OrbS requires specific tetrameric -35 and -10 element sequences that are strikingly similar to those of the consensus PvdS-dependent promoter. However, whereas Pseudomonas aeruginosa PvdS can serve OrbS-dependent promoters, OrbS cannot utilize PvdS-dependent promoters. To identify features present at OrbS-dependent promoters that facilitate recognition by OrbS, we carried out a detailed analysis of the nucleotide sequence requirements for promoter recognition by both OrbS and PvdS. This revealed that DNA sequence features located outside the sigma binding elements are required for efficient promoter utilization by OrbS. In particular, the presence of an A-tract extending downstream from the -35 element at OrbS-dependent promoters was shown to be an important contributor to OrbS specificity. Our observations demonstrate that the nature of the spacer sequence can have a major impact on promoter recognition by some ECF σ factors through modulation of the local DNA architecture.IMPORTANCE ECF σ factors regulate subsets of bacterial genes in response to environmental stress signals by directing RNA polymerase to promoter sequences known as the -35 and -10 elements. In this work, we identify the -10 and -35 elements that are recognized by the ECF σ factor OrbS. Furthermore, we demonstrate that efficient promoter utilization by this σ factor also requires a polyadenine tract located downstream of the -35 region. We propose that the unique architecture of A-tract DNA imposes conformational features on the -35 element that facilitates efficient recognition by OrbS. Our results show that sequences located between the core promoter elements can make major contributions to promoter recognition by some ECF σ factors.

Corrigendum: Iron Acquisition Mechanisms and Their Role in the Virulence of Burkholderia Species.

[This corrects the article DOI: 10.3389/fcimb.2017.00460.].

Iron Acquisition Mechanisms and Their Role in the Virulence of Burkholderia Species.

Burkholderia is a genus within the ß-Proteobacteriaceae that contains at least 90 validly named species which can be found in a diverse range of environments. A number of pathogenic species occur within the genus. These include Burkholderia cenocepacia and Burkholderia multivorans, opportunistic pathogens that can infect the lungs of patients with cystic fibrosis, and are members of the Burkholderia cepacia complex (Bcc). Burkholderia pseudomallei is also an opportunistic pathogen, but in contrast to Bcc species it causes the tropical human disease melioidosis, while its close relative Burkholderia mallei is the causative agent of glanders in horses. For these pathogens to survive within a host and cause disease they must be able to acquire iron. This chemical element is essential for nearly all living organisms due to its important role in many enzymes and metabolic processes. In the mammalian host, the amount of accessible free iron is negligible due to the low solubility of the metal ion in its higher oxidation state and the tight binding of this element by host proteins such as ferritin and lactoferrin. As with other pathogenic bacteria, Burkholderia species have evolved an array of iron acquisition mechanisms with which to capture iron from the host environment. These mechanisms include the production and utilization of siderophores and the possession of a haem uptake system. Here, we summarize the known mechanisms of iron acquisition in pathogenic Burkholderia species and discuss the evidence for their importance in the context of virulence and the establishment of infection in the host. We have also carried out an extensive bioinformatic analysis to identify which siderophores are produced by each Burkholderia species that is pathogenic to humans.

An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia.

To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis.

Functional Analysis of the Role of Toxin-Antitoxin (TA) Loci in Bacterial Persistence.

We have developed a method to analyze the functionality of putative TA loci by expressing them in Escherichia coli. Here, we describe the procedure for cloning recombinant TA genes into inducible plasmids and expressing these in E. coli. Following expression, toxicity, resuscitation of growth, and changes in persister cell formation are assayed. This can confirm whether predicted TA loci are active in E. coli and whether expression can affect persister cell formation.