Identification of a system for hydroxamate xenosiderophore-mediated iron transport in Burkholderia cenocepacia.

One of the mechanisms employed by the opportunistic pathogen Burkholderia cenocepacia to acquire the essential element iron is the production and release of two ferric iron chelating compounds (siderophores), ornibactin and pyochelin. Here we show that B. cenocepacia is also able to take advantage of a range of siderophores produced by other bacteria and fungi ('xenosiderophores') that chelate iron exclusively by means of hydroxamate groups. These include the tris-hydroxamate siderophores ferrioxamine B, ferrichrome, ferricrocin and triacetylfusarinine C, the bis-hydroxamates alcaligin and rhodotorulic acid, and the monohydroxamate siderophore cepabactin. We also show that of the 24 TonB-dependent transporters encoded by the B. cenocepacia genome, two (FhuA and FeuA) are involved in the uptake of hydroxamate xenosiderophores, with FhuA serving as the exclusive transporter of iron-loaded ferrioxamine B, triacetylfusarinine C, alcaligin and rhodotorulic acid, while both FhuA and FeuA are able to translocate ferrichrome-type siderophores across the outer membrane. Finally, we identified FhuB, a putative cytoplasmic membrane-anchored ferric-siderophore reductase, as being obligatory for utilization of all of the tested bis- and tris-hydroxamate xenosiderophores apart from alcaligin.

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.

A role for tetraspanin proteins in regulating fusion induced by Burkholderia thailandensis.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high morbidity that is endemic in South East Asia and northern Australia. An unusual feature of the bacterium is its ability to induce multinucleated giant cell formation (MNGC), which appears to be related to bacterial pathogenicity. The mechanism of MNGC formation is not fully understood, but host cell factors as well as known bacterial virulence determinants are likely to contribute. Since members of the tetraspanin family of membrane proteins are involved in various types of cell:cell fusion, their role in MNGC formation induced by Burkholderia thailandensis, a mildly pathogenic species closely related to B. pseudomallei, was investigated. The effect of antibodies to tetraspanins CD9, CD81, and CD63 in MNGC formation induced by B. thailandensis in infected mouse J774.2 and RAW macrophage cell lines was assessed along with that of recombinant proteins corresponding to the large extracellular domain (EC2) of the tetraspanins. B. thailandensis-induced fusion was also examined in macrophages derived from CD9 null and corresponding WT mice, and in J774.2 macrophages over-expressing CD9. Antibodies to CD9 and CD81 promoted MNGC formation induced by B. thailandensis, whereas EC2 proteins of CD9, CD81, and CD63 inhibited MNGC formation. Enhanced MNGC formation was observed in CD9 null macrophages, whereas a decrease in MNGC formation was associated with overexpression of CD9. Overall our findings show that tetraspanins are involved in MNGC formation induced by B. thailandensis and by implication, B. pseudomallei, with CD9 and CD81 acting as negative regulators of this process.

Correction to: A role for tetraspanin proteins in regulating fusion induced by Burkholderia thailandensis.

In the original article, incorrect  figures were published with incorrect captions. The correct figures and captions are given below.

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.

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.

Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

Regulation of sulfur assimilation pathways in Burkholderia cenocepacia through control of genes by the SsuR transcription factor.

The genome of Burkholderia cenocepacia contains two genes encoding closely related LysR-type transcriptional regulators, CysB and SsuR, involved in control of sulfur assimilation processes. In this study we show that the function of SsuR is essential for the utilization of a number of organic sulfur sources of either environmental or human origin. Among the genes upregulated by SsuR identified here are the tauABC operon encoding a predicted taurine transporter, three tauD-type genes encoding putative taurine dioxygenases, and atsA encoding a putative arylsulfatase. The role of SsuR in expression of these genes/operons was characterized through (i) construction of transcriptional reporter fusions to candidate promoter regions and analysis of their expression in the presence/absence of SsuR and (ii) testing the ability of SsuR to bind SsuR-responsive promoter regions. We also demonstrate that expression of SsuR-activated genes is not repressed in the presence of inorganic sulfate. A more detailed analysis of four SsuR-responsive promoter regions indicated that ~44 bp of the DNA sequence preceding and/or overlapping the predicted -35 element of such promoters is sufficient for SsuR binding. The DNA sequence homology among SsuR "recognition motifs" at different responsive promoters appears to be limited.

The pobA gene of Burkholderia cenocepacia encodes a group I Sfp-type phosphopantetheinyltransferase required for biosynthesis of the siderophores ornibactin and pyochelin.

The opportunistic pathogen Burkholderia cenocepacia produces the siderophores ornibactin and pyochelin under iron-restricted conditions. Biosynthesis of both siderophores requires the involvement of non-ribosomal peptide synthetases (NRPSs). Using a transposon containing the lacZ reporter gene, two B. cenocepacia mutants were isolated which were deficient in siderophore production. Mutant IW10 was shown to produce normal amounts of ornibactin but only trace amounts of pyochelin, whereas synthesis of both siderophores was abolished in AHA27. Growth of AHA27, but not IW10, was inhibited under iron-restricted conditions. In both mutants, the transposon had integrated into the pobA gene, which encodes a polypeptide exhibiting similarity to the Sfp-type phosphopantetheinyltransferases (PPTases). These enzymes are responsible for activation of NRPSs by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A. Previously characterized PPTase genes from other bacteria were shown to efficiently complement both mutants for siderophore production when provided in trans. The B. cenocepacia pobA gene was also able to efficiently complement an Escherichia coli entD mutant for production of the siderophore enterobactin. Using mutant IW10, in which the lacZ gene carried by the transposon is inserted in the same orientation as pobA, it was shown that pobA is not appreciably iron-regulated. Finally, we confirmed that Sfp-type bacterial PPTases can be subdivided into two distinct groups, and we present the amino acid signature sequences which characterize each of these groups.

Downregulation of the Escherichia coli guaB promoter by upstream-bound cyclic AMP receptor protein.

The Escherichia coli guaB promoter (P(guaB)) is responsible for directing transcription of the guaB and guaA genes, which specify the biosynthesis of the nucleotide GMP. P(guaB) is subject to growth rate-dependent control (GRDC) and possesses an UP element that is required for this regulation. In addition, P(guaB) contains a discriminator, three binding sites for the nucleoid-associated protein FIS, and putative binding sites for the regulatory proteins DnaA, PurR, and cyclic AMP receptor protein (CRP). Here we show that the CRP-cyclic AMP (cAMP) complex binds to a site located over 100 bp upstream of the guaB transcription start site, where it serves to downregulate P(guaB). The CRP-mediated repression of P(guaB) activity increases in media that support lower growth rates. Inactivation of the crp or cyaA gene or ablation/translocation of the CRP site relieves repression by CRP and results in a loss of GRDC of P(guaB). Thus, GRDC of P(guaB) involves a progressive increase in CRP-mediated repression of the promoter as the growth rate decreases. Our results also suggest that the CRP-cAMP complex does not direct GRDC at P(guaB) and that at least one other regulatory factor is required for conferring GRDC on this promoter. However, PurR and DnaA are not required for this regulatory mechanism.

The C-terminal domain of the Escherichia coli RNA polymerase alpha subunit plays a role in the CI-dependent activation of the bacteriophage lambda pM promoter.

The bacteriophage lambda p(M) promoter is required for maintenance of the lambda prophage in Escherichia coli, as it facilitates transcription of the cI gene, encoding the lambda repressor (CI). CI levels are maintained through a transcriptional feedback mechanism whereby CI can serve as an activator or a repressor of p(M). CI activates p(M) through cooperative binding to the O(R)1 and O(R)2 sites within the O(R) operator, with the O(R)2-bound CI dimer making contact with domain 4 of the RNA polymerase sigma subunit (sigma(4)). Here we demonstrate that the 261 and 287 determinants of the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD), as well as the DNA-binding determinant, are important for CI-dependent activation of p(M). We also show that the location of alphaCTD at the p(M) promoter changes in the presence of CI. Thus, in the absence of CI, one alphaCTD is located on the DNA at position -44 relative to the transcription start site, whereas in the presence of CI, alphaCTD is located at position -54, between the CI-binding sites at O(R)1 and O(R)2. These results suggest that contacts between CI and both alphaCTD and sigma are required for efficient CI-dependent activation of p(M).

Burkholderia cenocepacia utilizes a type VI secretion system for bacterial competition.

Burkholderia cenocepacia is an opportunistic bacterial pathogen that poses a significant threat to individuals with cystic fibrosis by provoking a strong inflammatory response within the lung. It possesses a type VI secretion system (T6SS), a secretory apparatus that can perforate the cellular membrane of other bacterial species and/or eukaryotic targets, to deliver an arsenal of effector proteins. The B. cenocepacia T6SS (T6SS-1) has been shown to be implicated in virulence in rats and contributes toward actin rearrangements and inflammasome activation in B. cenocepacia-infected macrophages. Here, we present bioinformatics evidence to suggest that T6SS-1 is the archetype T6SS in the Burkholderia genus. We show that B. cenocepacia T6SS-1 is active under normal laboratory growth conditions and displays antibacterial activity against other Gram-negative bacterial species. Moreover, B. cenocepacia T6SS-1 is not required for virulence in three eukaryotic infection models. Bioinformatics analysis identified several candidate T6SS-dependent effectors that may play a role in the antibacterial activity of B. cenocepacia T6SS-1. We conclude that B. cenocepacia T6SS-1 plays an important role in bacterial competition for this organism, and probably in all Burkholderia species that possess this system, thereby broadening the range of species that utilize the T6SS for this purpose.

The UP element is necessary but not sufficient for growth rate-dependent control of the Escherichia coli guaB promoter.

The Escherichia coli guaB promoter (P(guaB)) regulates the transcription of two genes, guaB and guaA, that are required for de novo synthesis of GMP, a precursor for the synthesis of guanine nucleoside triphosphates. The activity of P(guaB) is subject to growth rate-dependent control (GRDC). Here we show that the A+T-rich sequence located between positions -59 and -38 relative to the guaB transcription start site stimulates transcription from P(guaB) approximately 8- to 10-fold and, in common with other UP elements, requires the C-terminal domain of the RNA polymerase alpha subunit for activity. Like the rrnB P1 UP element, the P(guaB) UP element contains two independently acting subsites located at positions -59 to -47 and -46 to -38 and can stimulate transcription when placed upstream of the lacP1 promoter. We reveal a novel role for the P(guaB) UP element by demonstrating that it is required for GRDC. The involvement of the UP element in GRDC also requires the participation of sequences located at least 100 bp upstream of the guaB transcription start site. These sequences are required for down-regulation of P(guaB) activity at lower growth rates.

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

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

TssA from Burkholderia cenocepacia: expression, purification, crystallization and crystallographic analysis.

TssA is a core component of the type VI secretion system, and phylogenetic analysis of TssA subunits from different species has suggested that these proteins fall into three distinct clades. Whilst representatives of two clades, TssA1 and TssA2, have been the subjects of investigation, no members of the third clade (TssA3) have been studied. Constructs of TssA from Burkholderia cenocepacia, a representative of clade 3, were expressed, purified and subjected to crystallization trials. Data were collected from crystals of constructs of the N-terminal and C-terminal domains. Analysis of the data from the crystals of these constructs and preliminary structure determination indicates that the C-terminal domain forms an assembly of 32 subunits in D16 symmetry, whereas the N-terminal domain is not involved in subunit assocation.

TssA from Aeromonas hydrophila: expression, purification and crystallographic studies.

TssA is a core subunit of the type VI secretion system, which is a major player in interspecies competition in Gram-negative bacteria. Previous studies on enteroaggregative Escherichia coli TssA suggested that it is comprised of three putative domains: a conserved N-terminal domain, a middle domain and a ring-forming C-terminal domain. X-ray studies of the latter two domains have identified their respective structures. Here, the results of the expression and purification of full-length and domain constructs of TssA from Aeromonas hydrophila are reported, resulting in diffraction-quality crystals for the middle domain (Nt2) and a construct including the middle and C-terminal domains (Nt2-CTD).

Structural insights into the function of type VI secretion system TssA subunits.

The type VI secretion system (T6SS) is a multi-protein complex that injects bacterial effector proteins into target cells. It is composed of a cell membrane complex anchored to a contractile bacteriophage tail-like apparatus consisting of a sharpened tube that is ejected by the contraction of a sheath against a baseplate. We present structural and biochemical studies on TssA subunits from two different T6SSs that reveal radically different quaternary structures in comparison to the dodecameric E. coli TssA that arise from differences in their C-terminal sequences. Despite this, the different TssAs retain equivalent interactions with other components of the complex and position their highly conserved N-terminal ImpA_N domain at the same radius from the centre of the sheath as a result of their distinct domain architectures, which includes additional spacer domains and highly mobile interdomain linkers. Together, these variations allow these distinct TssAs to perform a similar function in the complex.

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.

Role of the RNA polymerase alpha subunits in CII-dependent activation of the bacteriophage lambda pE promoter: identification of important residues and positioning of the alpha C-terminal domains.

The bacteriophage lambda CII protein stimulates the activity of three phage promoters, p(E), p(I) and p(aQ), upon binding to a site overlapping the -35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to specific residues in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) to demonstrate that one alphaCTD binds near position -41 at p(E), whilst the other alphaCTD binds further upstream. The alphaCTD bound near position -41 is oriented such that its 261 determinant is in close proximity to sigma(70). The location of alphaCTD in CII-dependent complexes at the p(E) promoter is very similar to that found at many activator-independent promoters, and represents an alternative configuration for alphaCTD at promoters where activators bind sites overlapping the -35 region. We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of alphaCTD is involved in stimulation of the p(E) promoter by CII, and this was confirmed by in vitro transcription assays. We also show that whereas the K271E substitution in alphaCTD results in a drastic decrease in CII-dependent activation of p(E), the p(I) and p(aQ) promoters are less sensitive to this substitution, suggesting that the role of alphaCTD at the three lysogenic promoters may be different.

Escherichia coli can tolerate insertions of up to 16 amino acids in the RNA polymerase alpha subunit inter-domain linker.

The C-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alphaCTD) plays a key role in transcription initiation at many activator-dependent promoters and at UP element-dependent promoters. This domain is connected to the alpha N-terminal domain (alphaNTD) by an unstructured linker. To investigate the requirements of the alpha inter-domain linker to support growth of E. coli, we utilised a recently described technique for the substitution of the chromosomal rpoA gene, encoding alpha, by mutant rpoA alleles. We found that it was possible to replace wild-type rpoA by mutant alleles encoding alpha subunits containing inter-domain linkers that were longer by as many as 16 amino acids. However, using this method, it was not possible to transfer to the chromosome rpoA alleles encoding alpha subunits that contained an insertion of 32 amino acids or short deletions within the inter-domain linker. The effect of lengthening the alpha linker on activator-dependent and UP element-dependent transcription in the "haploid" rpoA system was shown to be qualitatively the same as observed previously in the diploid system. The ability of E. coli to tolerate insertions within the alpha inter-domain linker suggests that lengthening the alpha linker does not severely impair transcription of essential genes.