Abstract Is There An Answer? is intended to serve as a forum in which readers to IUBMB Life may pose questions of the type that intrigue biochemists but for which there may be no obvious answer or one may be available but not widely known or easily accessible. Readers are invited to e‐mail ascenzi@uniroma3.it if they have questions to contribute or if they can provide answers to questions that are provided here from time to time. In the latter case, instructions will be sent to interested readers. Answers should be, whenever possible, evidence‐based and provide relevant references. Paolo Ascenzi
Cyanide is a serious environmental pollutant and a biocontrol metabolite in plant growth-promoting Pseudomonas species. Here we report on the presence of multiple sulfurtransferases in the cyanogenic bacterium Pseudomonas aeruginosa PAO1 and investigate in detail RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) which converts cyanide to less toxic thiocyanate. RhdA is a cytoplasmic enzyme acting as the principal rhodanese in P. aeruginosa. The rhdA gene forms a transcriptional unit with the PA4955 and psd genes and is controlled by two promoters located upstream of PA4955 and rhdA. Both promoters direct constitutive RhdA expression and show similar patterns of activity, involving moderate down-regulation at the stationary phase or in the presence of exogenous cyanide. We previously observed that RhdA overproduction protects Escherichia coli against cyanide toxicity, and here we show that physiological RhdA levels contribute to P. aeruginosa survival under cyanogenic conditions. The growth of a DeltarhdA mutant is impaired under cyanogenic conditions and fully restored upon complementation with rhdA. Wild-type P. aeruginosa outcompetes the DeltarhdA mutant in cyanogenic coculture assays. Hence, RhdA could be regarded as an effector of P. aeruginosa intrinsic resistance to cyanide, insofar as it provides the bacterium with a defense mechanism against endogenous cyanide toxicity, in addition to cyanide-resistant respiration.
The level of environmental oxygen (EO) within various Pseudomonas aeruginosa infection sites is low (microaerobic), and this can affect the production of different virulence factors. Expression of the toxA gene, encoding exotoxin A (ETA), is regulated by regA, ptxR and pvdS. Moreover, the iron-starvation sigma factor PvdS directs the transcription of pyoverdine siderophore genes (e.g. pvdD). DNA-protein binding analysis using recombinant PvdS showed that the PvdS-RNA polymerase holoenzyme complex specifically bound the toxA, regA and ptxR promoter regions. All three promoters contain a PvdS-binding site, the iron-starvation box. To determine the relationship between these different genes and PvdS, we conducted a comparative analysis of toxA, regA, ptxR and pvdD transcription throughout the growth cycle of wild-type P. aeruginosa and its pvdS mutant in iron-deficient medium under aerobic-shaking (A-sh) and microaerobic-static (M-st) conditions. Under both EO conditions, optimal toxA, regA and pvdD expression and pyoverdine production required PvdS, while ptxR expression was moderately dependent on PvdS only under A-sh conditions. Expression of regA, pvdD and pyoverdine production in wild-type P. aeruginosa was significantly lower under M-st in comparison with A-sh conditions, while the opposite was observed for toxA and ptxR. Although low, the level of toxA expression and ETA production in the pvdS mutant were higher under M-st than under A-sh conditions. Transcription of pvdS and PvdS expression were also reduced by low EO. We propose that the regulation of toxA expression under aerobic conditions primarily involves PvdS, while an additional EO-responsive regulator(s) besides PvdS is required under low EO levels. Thus, PvdS may control the transcription of the ptxR, regA and toxA genes, and respond to EO by acting at different levels of the toxA regulatory cascade.
The l-ornithine N δ -oxygenase PvdA catalyses the N δ -hydroxylation of l-ornithine in many Pseudomonas spp., and thus provides an essential enzymic function in the biogenesis of the pyoverdine siderophore. Here, we report a detailed analysis of the membrane topology of the PvdA enzyme from the bacterial pathogen Pseudomonas aeruginosa. Membrane topogenic determinants of PvdA were identified by computational analysis, and verified in Escherichia coli by constructing a series of translational fusions between PvdA and the PhoA (alkaline phosphatase) reporter enzyme. The inferred topological model resembled a eukaryotic reverse signal-anchor (type III) protein, with a single N-terminal domain anchored to the inner membrane, and the bulk of the protein spanning the cytosol. According to this model, the predicted transmembrane region should overlap the putative FAD-binding site. Cell fractionation and proteinase K accessibility experiments in P. aeruginosa confirmed the membrane-bound nature of PvdA, but excluded the transmembrane topology of its N-terminal hydrophobic region. Mutational analysis of PvdA, and complementation assays in a P. aeruginosa ΔpvdA mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain.
In response to iron limitation, Pseudomonas aeruginosa produces the fluorescent siderophore pyoverdine. Transcription of pyoverdine biosynthetic (pvd) genes is driven by the iron starvation sigma factor PvdS, which is negatively regulated by the Fur-Fe(II) holorepressor. We studied the effect of AlgQ, the Escherichia coli Rsd orthologue, on pyoverdine production by P. aeruginosa PAO1. AlgQ is a global regulatory protein which activates alginate, ppGpp, and inorganic polyphosphate synthesis through a cascade involving nucleoside diphosphate kinase (Ndk). AlgQ is also capable of interacting with region 4 of RpoD. In a reconstituted E. coli system, PvdS-dependent transcription from the pvdA promoter was doubled by the multicopy algQ gene. The P. aeruginosa DeltaalgQ mutant exhibited a moderate but reproducible reduction in pyoverdine production compared with wild-type PAO1, as a result of a decline in transcription of pvd genes. PvdS expression was not affected by the algQ mutation. Single-copy algQ fully restored pyoverdine production and expression of pvd genes in the DeltaalgQ mutant, while ndk did not. An increased intracellular concentration of RpoD mimicked the DeltaalgQ phenotype, whereas PvdS overexpression suppressed the algQ mutation. E. coli rsd could partially substitute for algQ in transcriptional modulation of pvd genes. We propose that AlgQ acts as an anti-sigma factor for RpoD, eliciting core RNA polymerase recruitment by PvdS and transcription initiation at pvd promoters. AlgQ provides a link between the pyoverdine and alginate regulatory networks. These systems have similarities in responsiveness and physiological function: both depend on alternative sigma factors, respond to nutrient starvation, and act as virulence determinants for P. aeruginosa.
This chapter contains sections titled: Introduction Siderophores Used by Pseudomonads Chemical Diversity of Siderophores Endogenous Siderophores of Pseudomonads Pyoverdines Pyochelin Other Endogenous Siderophores Exogenous Siderophores Utilized by Pseudomonads Enterobactin Desferrioxamines Ferrichromes Aerobactin Citrate Siderophore Synthesis Nonribosomal Peptide Synthesis Pyoverdine Biosynthetic Pathways Synthesis of other Pseudomonas Siderophores Ferri-Siderophore Transport Overview Outer Membrane Ferri-Siderophore Receptors TonB Complex Transport of Ferri-Siderophores into the Periplasm Iron Transport Across the Cytoplasmic Membrane Regulation of Siderophore Synthesis and Transport Fur Protein as Master Repressor ECF σ Factors QS and Expression of Iron Transport Genes Introduction to Signaling Pyoverdine-Mediated Signaling FpvA and Pyoverdine Transfer of Signal: Role of FpvR Pyochelin: An Alternative Signaling Mechanism Enterobactin Multiplicity of Heterologous Siderophore Signaling Systems in Pseudomonads Concluding Remarks and Future Perspectives References
Is There an Answer? is intended to serve as a forum in which readers to IUBMB Life may pose questions of the type that intrigue biochemists but for which there may be no obvious answer or one may be available but not widely known or easily accessible. Readers are invited to e-mail [email protected] if they have questions to contribute or if they can provide answers to questions that are provided here from time to time. In the latter case, instructions will be sent to interested readers. Answers should be, whenever possible, evidence-based and provide relevant references. Paolo Ascenzi Heme or iron-protoporphyrin IX (PP-IX) is an ubiquitous biologic catalyst involved in a wide array of essential functions in prokaryotes and eukaryotes. Heme has long been known to be required for respiration, oxygen metabolism, and electron transfer as the prosthetic group of hemoglobins, hydroxylases, catalases, peroxidases, and cytochromes. Moreover, heme acts as a regulatory molecule that controls cellular processes at the level of DNA transcription, RNA translation, protein stability and targeting, and cell differentiation(1, 2). In addition to its function as cofactor for numerous enzymes, heme is a useful iron source for nutrition of both microorganisms and higher organisms. Humans generally absorb heme-iron more easily than inorganic iron due to the insolubility of oxidized iron salts(3). The same holds true for bacteria, especially for human pathogenic bacteria, given that ∼95% of iron within the host is present in the form of heme, primarily as hemoglobin, hemopexin, heme-albumin, and heme-lipoproteins(4, 5). To acquire heme from host heme-proteins, bacteria have evolved sophisticated strategies. The common theme in all bacterial heme assimilation systems is that the intact heme molecule is internalized into the cell. Gram-negative bacteria acquire heme from host heme-proteins through either direct binding to specific outer membrane receptor or secretion of high-affinity heme-binding protein, called hemophores, which take up heme from a variety of host heme carriers and shuttle it back to specific receptors. Heme is then actively transported into the periplasm, where it is transferred by a periplasmic-binding protein (PBP) to a cytoplasmic ATP-binding cassette (ABC) importer, which, in turn, promotes heme translocation across the membrane (Fig. 1A). In gram-positive bacteria, heme is recognized by lipoproteins anchored onto the plasma membrane and then transferred to ABC permeases. This transport device is similar to the cytoplasmic membrane systems of gram-negative organisms, which mediates heme translocation into the cytoplasm, where the macrocycle is further been utilized (Fig. 1B) (6, 7). The intracellular fate of heme in bacteria. (Panel A) Gram-negative bacteria utilize specific outer membrane (OM) receptors to translocate heme in the periplasmic space (PP). Once in the periplasm, a PBP shuttles heme to an ABC permease, which, in turn, promote heme translocation in the cytosol (C) at expenses of ATP. (Panel B) In gram-positive bacteria, a lipoprotein receptor, anchored to the cytoplasmic membrane (CM), sequesters heme from the extracellular environment and transfers it to the ATPase/permease for internalization. Once in the cell of both gram-negative (panel A) and gram-positive bacteria (panel B), the heme molecule could have one of four possible fates: degradation to release iron for use as a nutrient source, incorporation into trafficking or storage proteins, incorporation into heme-cofactored proteins, and secretion by an ABC transporter. In contrast to the breadth of knowledge about heme transport processes in microorganisms, relatively little is known about the fate of heme inside the cell. In eukaryotes, monooxygenases, known as heme oxygenases (HOs), catalyze the oxidative cleavage of heme to biliverdin, iron, and carbon monoxide(8). In prokaryotes such as Corynebacterium diptheriae, Neisseria meningitidis, and Pseudomonas aeruginosa, the existence of enzymes homologous to eukaryotic HOs has been demonstrated, providing evidence that oxidative cleavage of heme is a mechanism by which some pathogenic bacteria can acquire iron(7). However, the analysis of more than 70 complete bacterial genomes identified an unexpectedly small number of bacterial HO orthologs, strongly suggesting that heme degradation could also involve enzymes that do not share significant homology with eukaryotic HOs(9). Recently, a new family of monooxygenases that degrade heme by a pathway differing from canonical HOs has been reported in some pathogenic gram-positive bacteria, including Staphyloccoccus aureus, Listeria monocytogenes, and Bacillus anthracis(10). Although more enzymes implicated in heme degradation are likely be discovered in the future, the paucity of identifiable heme-degrading enzymes in bacteria that utilize extracellular heme raises the possibility that heme degradation is only one of the possible pathways of heme processing in the bacterial cytoplasm(4). An alternative hypothesis, also known as "heme hijacking hypothesis", proposes that heme acquired from the host can be incorporated as it is into bacterial heme-proteins(10), thus raising the intriguing possibility that the main role of bacterial heme-uptake systems is to provide the cell with heme rather than with iron. In principle, direct utilization of exogenous heme as an enzyme cofactor rather than as an iron source is advantageous for a bacterial pathogen given that a large proportion of iron is likely to be directed to the endogenous heme synthesis. Moreover, it would be energetically advantageous to import the host heme molecule rather than synthesize the porphyrin ring de novo. Heme biosynthesis poses a major cost to the bacterial cell, involving multiple enzymatic steps(11), and some experimental observations support the molecular hijacking hypothesis. It is well known that, even if most organisms can synthesize their own heme, some others, like Porphyromonas gingivalis and Haemophilus influenzae, which have lost one or more of the enzymes essential for the PP-IX biosynthesis, have an absolute requirement for exogenous heme/PP-IX to activate the entire pool of their intracellular heme-cofactored enzymes(4, 10). Moreover, the same need for exogenous heme has been observed in heme biosynthesis mutants belonging to several bacterial species(12-14), which suggests that the ability to shuttle the host heme into endogenous heme-proteins is a characteristic shared by almost all microorganisms that acquire extracellular heme. However, even if heme incorporation into heme-proteins could be the main fate of host heme, the great majority of reports have focused on the role of heme as an iron source, often neglecting alternative roles. Coherently, only few reports addressing this issue can be retrieved from the literature. In 1997, Schiott et al.(15) showed that heme, added exogenously to Bacillus subtilis cultures, is incorporated into four distinct membrane proteins identified as c-type cytochromes. Furthermore, Skaar et al.(16) investigated the role and fate of exogenously acquired heme in S. aureus. They demonstrated that, in the presence of both heme and transferrin, the transferrin-iron is mainly sorted to the cytoplasm, most likely for iron storage or use in metalloproteins, whereas the heme-iron is found largely associated with the cytoplasmic membrane. Given that the gram-positive cytoplasmic membrane is the primary site of heme-binding proteins such as cytochromes(15, 17), the intact heme molecule has been assumed to be sorted to the membrane and assembled as a cofactor for redox active proteins, corroborating the heme hijacking hypothesis(10, 16). As a whole, the data just presented depict a dichotomic route of heme inside the bacterial cell (Fig. 1). Heme could directly be incorporated into heme-proteins or degraded to release iron. Conceivably, the ultimate decision is dependent on the level of iron and heme available for the bacterium. Under iron-depleted conditions, most internalized heme could be degraded in order to provide the cell with iron, and this is consistent with the evidence that most characterized microbial HOs are induced by iron shortage(7). Conversely, under iron-replete conditions or, alternatively, when more readily available iron sources are present in the environment, the intact host heme could be incorporated in bacterial heme-proteins thereby preventing energy loss due to the de novo synthesis of the heme-porphyrin ring. A still open question is how bacteria maintain intracellular free heme pool below hazardous levels. As free heme acts as a catalyst to generate reactive oxygen species, bacteria must avoid free heme overload. The existence of proteins controlling the intracellular heme homeostasis has long been hypothesized in bacteria to explain the balance between exogenous heme uptake and heme degradation or incorporation rates. In this regard, a heme-responsive transporter predicted to be involved in the efflux of heme or heme metabolites has been identified in S. aureus and its homologous can be retrieved in many pathogenic bacteria, including B. anthracis and L. monocytogenes(18). Moreover, members of a new family of cytoplasmic heme-binding proteins, previously identified as HOs, have been recently proposed to be involved in heme-storage and/or heme trafficking(7). Although the actual role of these proteins is still under investigation, their existence suggests additional fates for intracellular heme. This molecule could be pumped out when present in excess to prevent oxidative damage, or it could be stored in inactive form for future use (Fig. 1) (7, 10). New Questions 1. When paper is cut with scissors, are covalent bonds in cellulose being broken, and if so, what is the chemical reaction? 2. Who first used the term "double helix" and where and when? This work was supported by grants from the Ministry of University and Research of Italy (PRIN-2006) and the Fondazione per la Ricerca sulla Fibrosi Cistica (Grant FFC#10/2007) to P.V.
Summary In the Gram‐negative pathogen Pseudomonas aeruginosa , the alternative sigma factor PvdS acts as a key regulator of the response to iron starvation. PvdS also controls P. aeruginosa virulence, as it drives the expression of a large set of genes primarily implicated in biogenesis and transport of the pyoverdine siderophore and synthesis of extracellular factors, such as protease PrpL and exotoxin A. Besides the ferric uptake regulatory protein Fur, which shuts off pvdS transcription under iron‐replete conditions, no additional regulatory factor(s) controlling the pvdS promoter activity have been characterized so far. Here, we used the promoter region of pvdS as bait to tentatively capture, by DNA‐protein affinity purification, P. aeruginosa proteins that are able to bind specifically to the pvdS promoter. This led to the identification and functional characterization of the LysR‐like transcription factor CysB as a novel regulator of pvdS transcription. The CysB protein directly binds to the pvdS promoter in vitro and acts as a positive regulator of PvdS expression in vivo . The absence of a functional CysB protein results in about 50% reduction of expression of PvdS‐dependent virulence phenotypes. Given the role of CysB as master regulator of sulfur metabolism, our findings establish a novel molecular link between the iron and sulfur regulons in P. aeruginosa .
Summary In Pseudomonas aeruginosa the iron starvation sigma factor PvdS directs the transcription of pyoverdine and virulence genes under iron limitation. PvdS activity is modulated by pyoverdine through the surface signalling cascade involving the FpvA receptor and the inner membrane‐spanning sensor FpvR. To gain insight into the molecular mechanisms enabling PvdS to compete with the major sigma RpoD for RNA polymerase (RNAP) binding, we determined the intracellular levels of RNAP, RpoD and PvdS in P. aeruginosa PAO1, and the effect of pyoverdine signalling on PvdS activity. Under iron limitation, P. aeruginosa contains 2221 and 933 molecules of RNAP and RpoD per cell respectively. PvdS attains 62% of RpoD levels. The high PvdS content is partly offset by retention of 30% of PvdS on the membrane, lowering the concentration of cytosolic PvdS to 45% of RpoD levels. RNAP purification from iron‐starved P. aeruginosa cells demonstrated that PvdS–RNAP is poorly represented compared with RpoD–RNAP (1 and 27% of total RNAP respectively). Pyoverdine signalling does not affect the PvdS cellular content but facilitates PvdS release from the membrane, increasing its cytosolic concentration from 35% in both pvdF and fpvA signalling mutants to 70% in the wild type and 83% in the fpvR mutant.
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