Discovery of the actinoplanic acid pathway in Streptomyces rapamycinicus reveals a genetically conserved synergism with rapamycin.

Actinobacteria possess a great wealth of pathways for production of bioactive compounds. Following advances in genome mining, dozens of natural product (NP) gene clusters are routinely found in each actinobacterial genome; however, the modus operandi of this large arsenal is poorly understood. During investigations of the secondary metabolome of Streptomyces rapamycinicus, the producer of rapamycin, we observed accumulation of two compounds never before reported from this organism. Structural elucidation revealed actinoplanic acid A and its demethyl analogue. Actinoplanic acids (APLs) are potent inhibitors of Ras farnesyltransferase and therefore represent bioactive compounds of medicinal interest. Supported with the unique structure of these polyketides and using genome mining, we identified a gene cluster responsible for their biosynthesis in S. rapamycinicus Based on experimental evidence and genetic organization of the cluster, we propose a stepwise biosynthesis of APL, the first bacterial example of a pathway incorporating the rare tricarballylic moiety into an NP. Although phylogenetically distant, the pathway shares some of the biosynthetic principles with the mycotoxins fumonisins. Namely, the core polyketide is acylated with the tricarballylate by an atypical nonribosomal peptide synthetase-catalyzed ester formation. Finally, motivated by the conserved colocalization of the rapamycin and APL pathway clusters in S. rapamycinicus and all other rapamycin-producing actinobacteria, we confirmed a strong synergism of these compounds in antifungal assays. Mining for such evolutionarily conserved coharboring of pathways would likely reveal further examples of NP sets, attacking multiple targets on the same foe. These could then serve as a guide for development of new combination therapies.

Isofuranonaphthoquinone produced by an Actinoplanes isolate.

A new isofuranonaphthoquinone, 7,8-dihydroxy-1-methylnaphtho[2,3-c]furan-4,9-dione, was isolated from cultures of an Actinoplanes isolate obtained using an in situ diffusion technology that facilitates the isolation of soil microorganisms. This compound was demonstrated to have the ability to complex Fe(III). The structure was determined on the basis of spectroscopic data.

Neocitreamicins I and II, novel antibiotics with activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci.

Two novel antibiotics, neocitreamicins I and II, were isolated from a fermentation broth of a Nocardia strain. This producing strain was obtained using an in situ diffusion chamber that facilitates the cultivation of soil microorganisms. The structures of neocitreamicins I and II were elucidated using UV, MS, and NMR data, and found to be related to the polycyclic xanthone antibiotics of the citreamicin class. The neocitreamicins showed in vitro activity against Gram-positive bacteria including strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis.

Metal ion homeostasis in Bacillus subtilis.

Bacillus subtilis, a Gram-positive soil bacterium, provides a model system for the study of metal ion homeostasis. Metalloregulatory proteins serve as the arbiters of metal ion sufficiency and regulate the expression of metal homeostasis pathways. In B. subtilis, uptake systems are regulated by the highly selective metal-sensing repressors Fur (iron), Zur (zinc), and MntR (manganese). Metal efflux systems are regulated by MerR and ArsR family homologs which, by contrast, can be rather non-specific with regard to metal selectivity. A Fur homolog, PerR, functions as an Fe(II)-dependent peroxide stress sensor and regulates putative metal transport and storage functions.

Natural products as catalysts for innovation: a pharmaceutical industry perspective.

Natural products are evolutionarily designed and chemically distinct from most synthetic library molecules. In addition to their role as drugs, they are successfully used as molecular probes to identify disease relevant targets. Novel natural products are still routinely discovered from traditional sources through cultivation of microorganisms. Complementary approaches based on genome sequence information and subsequent annotation of biosynthetic pathways are emerging technologies. However, to be of practical use for drug discovery, these concepts must be advanced beyond their current state.

Bacillus subtilis paraquat resistance is directed by sigmaM, an extracytoplasmic function sigma factor, and is conferred by YqjL and BcrC.

A Bacillus subtilis sigM null mutant, lacking the extracytoplasmic function sigma(M) protein, was sensitive to paraquat (PQ), a superoxide-generating reagent, but not to the redox stress-inducing compounds hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or diamide. Surprisingly, a sigM mutant was only sensitive to superoxide-generating compounds with a dipyridyl ring such as PQ, ethyl viologen, benzyl viologen, and diquat but not to menadione, plumbagin, pyrogallol, or nitrofurantoin. Mutational analysis of candidate sigma(M)-regulated genes revealed that both YqjL, a putative hydrolase, and BcrC, a bacitracin resistance protein, were involved in PQ resistance. Expression of yqjL, but not bcrC, from a xylose-inducible promoter restored PQ resistance to the sigM mutant.

Genetic and physiological responses of Bacillus subtilis to metal ion stress.

Metal ion homeostasis is regulated principally by metalloregulatory proteins that control metal ion uptake, storage and efflux genes. We have used transcriptional profiling to survey Bacillus subtilis for genes that are rapidly induced by exposure to high levels of metal ions including Ag(I), Cd(II), Cu(II), Ni(II) and Zn(II) and the metalloid As(V). Many of the genes affected by metal stress were controlled by known metalloregulatory proteins (Fur, MntR, PerR, ArsR and CueR). Additional metal-induced genes are regulated by two newly defined metal-sensing ArsR/SmtB family repressors: CzrA and AseR. CzrA represses the CadA efflux ATPase and the cation diffusion facilitator CzcD and this repression is alleviated by Zn(II), Cd(II), Co(II), Ni(II) and Cu. CadA is the major determinant for Cd(II) resistance, while CzcD protects the cell against elevated levels of Zn(II), Cu, Co(II) and Ni(II). AseR negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. Our results extend the range of identified effectors for the As(III)-sensor ArsR to include Cd(II) and Ag(I) and for the Cu-sensor CueR to include Ag(I) and, weakly, Cd(II) and Zn(II). In addition to systems dedicated to metal homeostasis, specific metal stresses also strongly induced pathways related to cysteine, histidine and arginine metabolism.

Dissimilatory Fe(III) and Mn(IV) reduction by Shewanella putrefaciens requires ferE, a homolog of the pulE (gspE) type II protein secretion gene.

Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468-1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer-) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).

Response of Bacillus subtilis to nitric oxide and the nitrosating agent sodium nitroprusside.

We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression. In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival. DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B. subtilis to NO and SNP. Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress. Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur. In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II). Aerobically, NO gas induced hmp, the sigmaB general stress regulon, and, to a lesser extent, the Fur and PerR regulons. Surprisingly, NO gas induced the sigmaB regulon via the energy branch of the sigmaB regulatory cascade while induction by SNP was mediated by the environmental stress branch. This emphasizes that NO and SNP elicit genetically distinct stress responses.

The global transcriptional response of Bacillus subtilis to manganese involves the MntR, Fur, TnrA and sigmaB regulons.

We have used DNA microarrays to monitor the global transcriptional response of Bacillus subtilis to changes in manganese availability. Mn(II) leads to the MntR-dependent repression of both the mntH and mntABCD operons encoding Mn(II) uptake systems. Mn(II) also represses the Fur regulon. This repression is unlikely to be a direct effect of Mn(II) on Fur as repression is sensitive to 2,2'-dipyridyl, an iron-selective chelator. We suggest that elevated Mn(II) displaces iron from cellular-binding sites and the resulting rise in free iron levels leads to repression of the Fur regulon. Many of the genes induced by Mn(II) are activated by sigmaB or TnrA. Both of these regulators are controlled by Mn(II)-dependent enzymes. Induction of the sigmaB-dependent general stress response by Mn(II) is largely dependent on RsbU, a Mn(II)-dependent phosphatase that dephosphorylates RsbV, ultimately leading to release of active sigmaB from its antisigma, RsbW. The activity of TnrA is inhibited when it forms an inactive complex with feedback-inhibited glutamine synthetase. Elevated Mn(II) reduces the sensitivity of glutamine synthetase to feedback inhibitors, and we suggest that this leads to the observed increase in TnrA activity. In sum, three distinct mechanisms can account for most of the transcriptional effects elicited by manganese: (i) direct binding of Mn(II) to metalloregulators such as MntR, (ii) perturbation of cellular iron pools leading to increased Fur activity and (iii) altered activity of Mn(II)-dependent enzymes that regulate the activity of sigmaB and TnrA.