SbnG,a Citrate Synthase in Staphylococcus aureus: A NEW FOLD ON AN OLD ENZYME*

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production.     

Involvement of reductases IruO and NtrA in iron acquisition by Staphylococcus aureus

To obtain host iron, Staphylococcus aureus secretes siderophores staphyloferrin A (SA) or staphyloferrin B (SB), and accesses heme iron through use of iron‐regulated surface determinant proteins. While iron transport in S. aureus is well documented, there is scant information about proteins required to access iron from complexes in the cytoplasm. In vitro studies identified a pyridine nucleotide‐disulfide oxidoreductase, named IruO, as an electron donor for the heme monooxygenases IsdG and IsdI, promoting heme degradation. Here, we show that an iruO mutant was not debilitated for growth on heme, suggesting involvement of another reductase. NtrA is an iron‐regulated nitroreductase and, as with the iruO mutant, a ntrA mutant grew on heme comparable with wild type (WT). In contrast, a iruO ntrA double mutant was severely debilitated for growth on heme, a phenotype that was complemented by expression of either iruO or ntrA in trans, demonstrating their overlapping role in heme‐iron utilization. Contrasting the involvement of multiple reductases for heme iron utilization, ntrA was shown essential for iron utilization using SA, although not SB or other siderophores tested, and an iruO mutant was incapable of deferoxamine‐mediated growth. Accordingly, virulence of WT S. aureus, but not an iruO mutant, was enhanced in mice receiving deferoxamine.     

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.     

Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus

Iron is critical for virtually all forms of life. The production of high-affinity iron chelators, siderophores, and the subsequent uptake of iron–siderophore complexes are a common strategy employed by microorganisms to acquire iron. Staphylococcus aureus produces siderophores but genetic information underlying their synthesis and transport is limited. Previous work implicated the sbn operon in siderophore synthesis and the sirABC operon in uptake. Here we characterize a second siderophore biosynthetic locus in S. aureus ; the locus consists of four genes (in strain Newman these open reading frames are designated NWMN_2079–2082) which, together, are responsible for the synthesis and export of staphyloferrin A, a polycarboxylate siderophore. While deletion of the NWMN_2079–2082 locus did not affect iron-restricted growth of S. aureus , strains bearing combined sbn and NWMN_2079–2082 locus deletions produced no detectable siderophore and demonstrated severely attenuated iron-restricted growth. Adjacent to NWMN_2079–2082 resides the htsABC operon, encoding an ABC transporter previously implicated in haem acquisition. We provide evidence here that HtsABC, along with the FhuC ATPase, is required for the uptake of staphyloferrin A. The crystal structure of apo-HtsA was determined and identified a large positively charged region in the substrate-binding pocket, in agreement with a role in binding of anionic staphyloferrin A.     

Specificity of Staphyloferrin B Recognition by the SirA Receptor from Staphylococcus aureus

Many organisms use sophisticated systems to acquire growth-limiting iron. Iron limitation is especially apparent in bacterial pathogens of mammalian hosts where free iron concentrations are physiologically negligible. A common strategy is to secrete low molecular weight iron chelators, termed siderophores, and express high affinity receptors for the siderophore-iron complex. Staphylococcus aureus, a widespread pathogen, produces two siderophores, staphyloferrin A (SA) and staphyloferrin B (SB). We have determined the crystal structure of the staphyloferrin B receptor, SirA, at high resolution in both the apo and Fe(III)-SB (FeSB)-bound forms. SirA, a member of the class III binding protein family of metal receptors, has N- and C-terminal domains, each composed of mainly a β-stranded core and α-helical periphery. The domains are bridged by a single α-helix and together form the FeSB binding site. SB coordinates Fe(III) through five oxygen atoms and one nitrogen atom in distorted octahedral geometry. SirA undergoes conformational change upon siderophore binding, largely securing two loops from the C-terminal domain to enclose FeSB with a low nanomolar dissociation constant. The staphyloferrin A receptor, HtsA, homologous to SirA, also encloses its cognate siderophore (FeSA); however, the largest conformational rearrangements involve a different region of the C-terminal domain. FeSB is uniquely situated in the binding pocket of SirA with few of the contacting residues being conserved with those of HtsA interacting with FeSA. Although both SirA and HtsA bind siderophores from the same α-hydroxycarboxylate class, the unique structural features of each receptor provides an explanation for their distinct specificity.     

Purification and chemical characterization of staphyloferrin B,a hydrophilic siderophore from staphylococci

This paper describes the chemical characterization of staphyloferrin B, a new complexone type siderophore isolated from low iron cultures of Staphylococcus hyicus DSM 20459. Purification of the very hydrophilic metabolite was achieved by anion exchange high performance liquid chromatography HPLC. Mass spectrometry showed a molecular mass of 448 amu. Hydrolysis with 8 mHCl revealed the presence of l-2,3-diaminopropionic acid, citrate, ethylenediamine and succinic semialdehyde. The connections between the four building blocks were determined by two-dimensional nuclear magnetic resonance measurements. UV/Vis and circular dichroism spectra are consistent with the proposed structure, which could also be confirmed by precursor feeding. The siderophore activity of staphyloferrin B was demonstrated by iron transport measurements.     

The influence of pH on Staphylococcus saprophyticus iron metabolism and the production of siderophores

Staphylococcus saprophyticus is a gram-positive coagulase negative bacteria which shows clinical importance due to its capability of causing urinary tract infections (UTI), as well as its ability to persist in this environment. Little is known about how S. saprophyticus adapts to the pH shift that occurs during infection. Thus, in this study we aim to use a proteomic approach to analyze the metabolic adaptations which occur as a response by S. saprophyticus when exposed to acid (5.5) and alkaline (9.0) pH environments. Proteins related to iron storage are overexpressed in acid pH, whilst iron acquisition proteins are overexpressed in alkaline pH. It likely occurs because iron is soluble at acid pH and insoluble at alkaline pH. To evaluate if S. saprophyticus synthesizes siderophores, CAS assays were performed, and the results confirmed their production. The chemical characterization of siderophores demonstrates that S. saprophyticus produces carboxylates derived from citrate. Of special note is the fact that citrate synthase (CS) is down-regulated during incubation at acid pH, corroborating this result. This data was also confirmed by enzymatic assay. Our results demonstrate that iron metabolism regulation is influenced by different pH levels, and show, for the first time, the production of siderophores by S. saprophyticus. Enzymatic assays suggest that citrate from the tricarboxylic acid cycle (TCA) is used as substrate for siderophore production.     

Structural Insights into Substrate Recognition and Activity Regulation of the Key Decarboxylase SbnH in Staphyloferrin B Biosynthesis

Staphyloferrin B is a hydroxycarboxylate siderophore that is crucial for the invasion and virulence of Staphylococcus aureus in mammalian hosts where free iron ions are scarce. The assembly of staphyloferrin B involves four enzymatic steps, in which SbnH, a pyridoxal 5′-phosphate (PLP)-dependent decarboxylase, catalyzes the second step. Here, we report the X-ray crystal structures of S. aureus SbnH (SaSbnH) in complex with PLP, citrate, and the decarboxylation product citryl-diaminoethane (citryl-Dae). The overall structure of SaSbnH resembles those of the previously reported PLP-dependent amino acid decarboxylases, but the active site of SaSbnH showed unique structural features. Structural and mutagenesis analysis revealed that the citryl moiety of the substrate citryl-l-2,3-diaminopropionic acid (citryl-l-Dap) inserts into a narrow groove at the dimer interface of SaSbnH and forms hydrogen bonding interactions with both subunits. In the active site, a conserved lysine residue forms an aldimine linkage with the cofactor PLP, and a phenylalanine residue is essential for accommodating the l-configuration Dap of the substrate. Interestingly, the freestanding citrate molecule was found to bind to SaSbnH in a conformation inverse to that of the citryl group of citryl-Dae and efficiently inhibit SaSbnH. As an intermediate in the tricarboxylic acid (TCA) cycle, citrate is highly abundant in bacterial cells until iron depletion; thus, its inhibition of SaSbnH may serve as an iron-dependent regulatory mechanism in staphyloferrin B biosynthesis.     

Molecular characterization of staphyloferrin B biosynthesis in Staphylococcus aureus

Siderophores are iron-scavenging molecules produced by many microbes. In general, they are synthesized using either non-ribosomal peptide synthetase (NRPS) or NRPS-independent siderophore (NIS) pathways. Staphylococcus aureus produces siderophores, of which the structures of staphyloferrin A and staphyloferrin B are known. Recently, the NIS biosynthetic pathway for staphyloferrin A was characterized. Here we show that, in S. aureus , the previously identified sbn ( s iderophore b iosy n thesis) locus encodes enzymes required for the synthesis of staphyloferrin B, an α-hydroxycarboxylate siderophore comprised of l -2,3-diaminopropionic acid, citric acid, 1,2-diaminoethane and α-ketoglutaric acid. Staphyloferrin B NIS biosynthesis was recapitulated in vitro , using purified recombinant Sbn enzymes and the component substrates. In vitro synthesized staphyloferrin B readily promoted the growth of iron-starved S. aureus , via the ABC transporter SirABC. The SbnCEF synthetases and a decarboxylase, SbnH, were necessary and sufficient to produce staphyloferrin B in reactions containing component substrates l -2,3-diaminopropionic acid, citric acid and α-ketoglutaric acid. Since 1,2-diaminoethane was not required, this component of the siderophore arises from the SbnH-dependent decarboxylation of a 2,3-diaminoproprionic acid-containing intermediate. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analyses of a series of enzyme reactions identified mass ions corresponding to biosynthetic intermediates, allowing for the first proposed biosynthetic pathway for staphyloferrin B.     

c‐di‐AMP modulates Listeria monocytogenes central metabolism to regulate growth,antibiotic resistance and osmoregulation

Cyclic diadenosine monophosphate (c‐di‐AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall‐active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the β‐lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl‐CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100‐fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c‐di‐AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down‐stream enzymes suppressed ΔdacA phenotypes. These data suggested that c‐di‐AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild‐type bacteria.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains

The Type VII protein secretion system, found in Gram‐positive bacteria, secretes small proteins, containing a conserved W‐x‐G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.     

Azotobacter vinelandii gene clusters for two types of peptidic and catechol siderophores produced in response to molybdenum

Aim: To characterize the complementary production of two types of siderophores in Azotobacter vinelandii. Methods and Results: In an iron‐insufficient environment, nitrogen‐fixing A. vinelandii produces peptidic (azotobactin) and catechol siderophores for iron uptake to be used as a nitrogenase cofactor. Molybdenum, another nitrogenase cofactor, was also found to affect the production level of siderophores. Wild‐type cells excreted azotobactin into molybdenum‐supplemented and iron‐insufficient medium, although catechol siderophores predominate in molybdenum‐free environments. Two gene clusters were identified to be involved in the production of azotobactin and catechol siderophores through gene annotation and disruption. Azotobactin‐deficient mutant cells produced catechol siderophores under the molybdenum‐supplemented and iron‐insufficient conditions, whereas catechol siderophore–deficient mutant cells extracellularly secreted excess azotobactin under iron‐deficient condition independent of the concentration of molybdenum. This evidence suggests that a complementary siderophore production system exists in A. vinelandii. Conclusions: Molybdenum was found to regulate the production level of two types of siderophores. Azotobacter vinelandii cells are equipped with a complementary production system for nitrogen fixation in response to a limited quantity of metals. Significance and Impact of the Study: This is the first study identifying A. vinelandii gene clusters for the biosynthesis of two types of siderophores and clarifying the relationship between them.     

Growth and siderophores production in <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> is regulated by metal ions

During stationary phase of growth under low stress of iron in succinic acid medium, Alcaligenes feacalis BCCM ID 2374 produced microbial iron chelators. Increase in iron concentration supported bacterial growth but suppressed siderophores production, 1 μM and 2 μM of iron was optimum for maximum siderophore yield, i.e. 354 and 360 μg/ml in untreated and deferrated medium, respectively. Threshold level of iron, which suppressed siderophores production in A. feacalis BCCM ID 2374, was 20 μM. Ten micromoles and above concentration of CuCl₂ and CoCl₂, and 20 μM of MgCl₂, MgSO₄, ZnCl₂ and ZnSO₄ severely affected siderophores production.     

Multiple modes of iron uptake by the filamentous,siderophore‐producing cyanobacterium,Anabaena sp. PCC 7120

Iron is a member of a small group of nutrients that limits aquatic primary production. Mechanisms for utilizing iron have to be efficient and adapted according to the ecological niche. In respect to iron acquisition cyanobacteria, prokaryotic oxygen evolving photosynthetic organisms can be divided into siderophore‐ and non‐siderophore‐producing strains. The results presented in this paper suggest that the situation is far more complex. To understand the bioavailability of different iron substrates and the advantages of various uptake strategies, we examined iron uptake mechanisms in the siderophore‐producing cyanobacterium Anabaena sp. PCC 7120. Comparison of the uptake of iron complexed with exogenous (desferrioxamine B, DFB) or to self‐secreted (schizokinen) siderophores by Anabaena sp. revealed that uptake of the endogenous produced siderophore complexed to iron is more efficient. In addition, Anabaena sp. is able to take up dissolved, ferric iron hydroxide species (Fe′) via a reductive mechanism. Thus, Anabaena sp. exhibits both, siderophore‐ and non‐siderophore‐mediated iron uptake. While assimilation of Fe′ and FeDFB are not induced by iron starvation, FeSchizokinen uptake rates increase with increasing iron starvation. Consequently, we suggest that Fe′ reduction and uptake is advantageous for low‐density cultures, while at higher densities siderophore uptake is preferred.     

The configuration of the chiral carbon atoms in staphyloferrin A and analysis of the transport properties in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Staphyloferrin A, the iron-transporting siderophore of Staphylococci, contains two citric acid residues linked to a D-ornithine backbone, having thus three chiral centers. While the chirality of the backbone can be determined after hydrolysis, the chirality of the two citryl residues can only be determined from the intact staphyloferrin A molecule by circular dichroism spectra. The chirality of the quarternary carbon atoms of citryl residues in fungal rhizoferrin and bacterial enantio-rhizoferrin have been determined previously to be R,R and S,S respectively. The present investigation shows that of the three chiral centers in staphyloferrin A, the citryl residues can be assigned an S,S-configuration by comparison with synthetic analogs, confirming a common chirality among the bacterial enantio-rhizoferrin and staphyloferrin A. This suggests that the bacterial carboxylates originate from a common biosynthetic pathway leading to an S,S-configuration, while the fungal rhizoferrin possessing an R,R-configuration must have a different biosynthetic origin. Growth promotion tests with staphylococci revealed that the S,S-configuration of staphyloferrin A and enantio-rhizoferrin enabled iron uptake, while the fungal rhizoferrin with R,R-configuration was not utilized. Published online December 2004