Topology of the porin MspA in the outer membrane of Mycobacterium smegmatis

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic beta-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surface-exposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic beta-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nm long part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic alpha-helices or beta-sheets are integrated into a lipid membrane.     

MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis

MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water-filled channels in vitro. Immunogold electron microscopy and an enzyme-linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA-specific primers and reverse-transcribed RNA. Detergent extracts of the DeltamspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild-type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the DeltamspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine- and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the DeltamspA mutant [corrected] This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.     

PapA3 Is an Acyltransferase Required for Polyacyltrehalose Biosynthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans, has a complex cell wall that contains a number of unique glycolipids intimately linked to mycobacterial pathogenesis (1, 2). The biosynthesis of many of these virulence factors, including the trehalose mycolates, phenolic glycolipids, and sulfolipid-1 (SL-1),³ is largely understood (3–5). In contrast, relatively little is known about the biosynthesis of other prominent M. tuberculosis glycolipids, such as di-, tri-, and polyacyltrehaloses. These acyltrehaloses are located in the outer surface of the cell wall and contain di- and tri-methyl branched fatty acids that are only found in pathogenic species of mycobacteria (6, 7). Previous studies suggest a role for these glycolipids in anchoring the bacterial capsule, which impedes phagocytosis by host cells (6).The major polyacyltrehalose (PAT) of M. tuberculosis, also referred to as pentaacyl or polyphthienoyl trehalose, consists of five acyl chains, four mycolipenic (phthienoic) acids and one fully saturated fatty acid, linked to trehalose (Fig. 1A) (8). The mycolipenic acid side chains of PAT are products of the polyketide synthase gene pks3/4 (7). Disruption of pks3/4 (also referred to as msl3 (7)) abolishes PAT biosynthesis and causes cell aggregation. At present, the remaining proteins required for PAT assembly have not been characterized.Open in a separate windowFIGURE 1.PAT and SL-1 share related structures and biosynthetic gene clusters. A, structure of PAT. B, structure of SL-1. C, genomic arrangement of the PAT and SL-1 biosynthetic gene clusters.Interestingly, the PAT biosynthetic gene cluster strongly resembles that of SL-1, which is a structurally similar trehalose-based glycolipid unique to pathogenic mycobacteria (Fig. 1B) (9). Both gene clusters contain polyketide synthase (pks), acyltransferase (pap), and lipid transport (mmpL) genes in a similar genomic arrangement (Fig. 1C). The SL-1 locus encodes two acyltransferase genes, papA1 and papA2, which are required for SL-1 biosynthesis (5, 10). These proteins belong to the mycobacterium-specific polyketide-associated protein (Pap) family of acyltransferases, which share a conserved HX₃DX₁₄Y motif that is required for activity (11). The PapA2 enzyme catalyzes the esterification of the 2′-position of trehalose 2-sulfate with a saturated fatty acid. PapA1 mediates the subsequent esterification of this intermediate with a hydroxyphthioceranoyl group produced by Pks2 (5). Interestingly, the PAT locus contains a gene, Rv1182, that is homologous to both papA1 and papA2 (55 and 53% amino acid identity, respectively). This gene is annotated as papA3 in the genome and was previously shown to encode a protein bearing the signature Pap motif (11).Here we demonstrate that papA3 encodes an acyltransferase essential for the biosynthesis of PAT. Deletion of the papA3 gene resulted in loss of the glycolipid from M. tuberculosis lipid extracts, as determined by high resolution mass spectrometry. Moreover, the purified enzyme was shown to selectively and sequentially acylate trehalose in vitro, generating a diacylated product similar to the 2,3-diacyltrehaloses of M. tuberculosis. Together, these data confirm that PapA3 plays a crucial role in PAT biosynthesis and highlight its potential involvement in the biosynthesis of related M. tuberculosis acyltrehaloses.     

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates

The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosis isolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.     

Chronic Helminth Infection Does Not Exacerbate Mycobacterium tuberculosis Infection

Background

Chronic helminth infections induce a Th2 immune shift and establish an immunoregulatory milieu. As both of these responses can suppress Th1 immunity, which is necessary for control of Mycobacterium tuberculosis (MTB) infection, we hypothesized that chronic helminth infections may exacerbate the course of MTB.

Methodology/Principal Findings

Co-infection studies were conducted in cotton rats as they are the natural host for the filarial nematode Litomosoides sigmodontis and are an excellent model for human MTB. Immunogical responses, histological studies, and quantitative mycobacterial cultures were assessed two months after MTB challenge in cotton rats with and without chronic L. sigmodontis infection. Spleen cell proliferation and interferon gamma production in response to purified protein derivative were similar between co-infected and MTB-only infected animals. In contrast to our hypothesis, MTB loads and occurrence and size of lung granulomas were not increased in co-infected animals.

Conclusions/Significance

These findings suggest that chronic filaria infections do not exacerbate MTB infection in the cotton rat model. While these results suggest that filaria eradication programs may not facilitate MTB control, they indicate that it may be possible to develop worm-derived therapies for autoimmune diseases that do not substantially increase the risk for infections.     

Crystal Structure of the GTPase-activating Protein-related Domain from IQGAP1

IQGAP1 is a 190-kDa molecular scaffold containing several domains required for interaction with numerous proteins. One domain is homologous to Ras GTPase-activating protein (GAP) domains. However, instead of accelerating hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD) binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here the crystal structure of the isolated IQGAP1 GRD. Despite low sequence conservation, the overall structure of the GRD is very similar to the GAP domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the catalytic “arginine finger” seen in functional Ras GAPs, the GRD has a conserved threonine residue. GRD residues 1099–1129 have no structural equivalent in RasGAP and are seen to form an extension at one end of the molecule. Because the sequence of these residues is highly conserved, this region likely confers a functionality particular to IQGAP family GRDs. We have used isothermal titration calorimetry to demonstrate that the isolated GRD binds to active Cdc42. Assuming a mode of interaction similar to that displayed in the Ras-RasGAP complex, we created an energy-minimized model of Cdc42·GTP bound to the GRD. Residues of the GRD that contact Cdc42 map to the surface of the GRD that displays the highest level of sequence conservation. The model indicates that steric clash between threonine 1046 with the phosphate-binding loop and other subtle changes would likely disrupt the proper geometry required for GTP hydrolysis.The small GTPase Ras functions as a binary switch in cell signaling processes. When bound to GTP, Ras is able to interact with effector proteins, including Raf kinase, and alter their activities. Ras signaling is terminated when bound GTP is hydrolyzed to GDP and inorganic phosphate. The basal rate of GTP hydrolysis on Ras is quite slow (∼1.2 × 10^–4 s^–1), but this rate of hydrolysis can be enhanced ∼10⁵-fold by interaction with a GTPase-activating protein (GAP)² (1). Several RasGAPs have been identified to date including p120 RasGAP and neurofibromin (NF1). The Rho family of Ras-related small GTPases also function as binary switches in cell signaling processes. Whereas the intrinsic rate of GTP hydrolysis on Rho proteins is faster than Ras, this rate can also be stimulated by interaction with a RhoGAP. Examination of the structures of the GAP domains of p120RasGAP (2), neurofibromin (3), SynGAP (4), and the GAP domains from the RhoGAPs p50 RhoGAP and the Bcr homology domain of phosphatidylinositol 3-kinase (5, 6) indicates that although ostensibly different, these all-helical domains are structurally related (7).IQGAP1 was discovered by chance during an attempt to isolate novel matrix metalloproteinases (8). Analysis reveals that the protein contains several discrete domains and motifs including a region containing four isoleucine- and glutamine-rich motifs (IQ repeats) and a region with sequence homology to the Ras-specific GAP domains of p120RasGAP, NF1, and SynGAP (2–4, 8). Subsequently, two homologs, IQGAP2 and IQGAP3, have been discovered. The IQ repeats have been shown to mediate binding to calmodulin and calmodulin-like proteins (e.g. S100, myosin essential light chain), whereas the GAP-related domain (GRD) does not appear to bind to Ras but instead is necessary for binding to the Rho family GTPases Cdc42 and Rac1, primarily in their active forms (9–11). However, instead of accelerating hydrolysis of GTP, IQGAP1 preserves the activated states of Cdc42 and Rac1 to the extent that overexpression of IQGAP1 in cells increases the levels of active GTPase (12). Because IQGAP1 expression increases the level of activated Cdc42, initially there was some confusion as to whether the protein might not represent a novel guanine nucleotide exchange factor. However it now appears that IQGAP1 is an effector of Cdc42 and Rac1 and preserves their activated states by tightly binding to the GTPases and stabilizing them in a conformation not conducive to GTP hydrolysis. IQGAP1 appears to be such an important effector for Cdc42 that abrogation of binding to IQGAP1 not only reduces the levels of active Cdc42, it also reduces membrane-localized Cdc42 and the cellular response to bradykinin (12).A growing body of evidence implicates IQGAP1 in carcinogenesis. Expression of IQGAP1 increases during the transition from a minimally to a highly metastastic form of melanoma, and IQGAP1 has been found to be overexpressed in ovarian, breast, lung, and colorectal cancers (13–17). In vitro, overexpressed IQGAP1 enhances cell motility and invasiveness in a process that requires Cdc42 and Rac (18). β-Catenin is one of the many binding partners of IQGAP1 identified to date. IQGAP1 has been shown to bind to β-catenin and interfere with β-catenin binding to α-catenin, an interaction necessary for stable cell-cell adhesion (19). Another study found that IQGAP2 knock-out mice overexpress IQGAP1 and developage-dependent liver cancer and apoptosis (20).To better understand how a protein domain homologous to others that accelerate GTP hydrolysis can function as an effector and preserve the GTP-bound state, we have determined the x-ray structure of the IQGAP1 GRD. Despite low sequence identity, the GRD structure is quite similar to the GAP domains of p120, neurofibromin, and SynGAP; however, unlike those domains, the GRD possesses a conserved threonine in place of the catalytic arginine finger and has a 31-residue insertion that projects from one end of the molecule. Using the coordinates of Ras·GDP·AlF₃ in complex with the GAP domain of p120, we built a model of Cdc42·GTP bound to the GRD. The model indicates that a steric clash between the conserved Thr¹⁰⁴⁶ and the phosphate-binding loop of Cdc42 and other subtle changes within the active site would likely preclude nucleotide hydrolysis. Sequence conservation mapped to the surface of the GRD indicates that the surface with the highest degree of conservation overlaps with the surface that makes contacts to Cdc42 in the model.     

The Glycosylphosphatidylinositol-Anchored Phosphatase from Spirodela oligorrhiza Is a Purple Acid Phosphatase

We recently presented clear evidence that the major low-phosphate-inducible phosphatase of the duckweed Spirodela oligorrhiza is a glycosylphosphatidylinositol (GPI)-anchored protein, and, to our knowledge, is the first described from higher plants (N. Morita, H. Nakazato, H. Okuyama, Y. Kim, G.A. Thompson, Jr. [1996] Biochim Biophys Acta 1290: 53–62). In this report the purified 57-kD phosphatase is shown to be a purple metalloenzyme containing Fe and Mn atoms and having an absorption maximum at 556 nm. The phosphatase activity was only slightly inhibited by tartrate, as expected for a purple acid phosphatase (PAP). Furthermore, the protein cross-reacted with an anti-Arabidopsis PAP antibody on immunoblots. The N-terminal amino acid sequence of the phosphatase was very similar to those of Arabidopsis, red kidney bean (Phaseolus vulgaris), and soybean (Glycine max) PAP. Extracts of S. oligorrhiza plants incubated with the GPI-specific precursor [³H]ethanolamine were treated with antibodies raised against the purified S. oligorrhiza phosphatase. Radioactivity from the resulting immunoprecipitates was specifically associated with a 57-kD band on sodium dodecyl sulfate-polyacrylamide gels. These results, together with previous findings, strongly indicate that the GPI-anchored phosphatase of S. oligorrhiza is a PAP.     

Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP₂), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C₂-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP₂ hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP₂-hydrolyzing activity at 10 μm Ca²⁺ and were inhibited by Al³⁺ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg²⁺. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.     

Drosophila Porin/VDAC Affects Mitochondrial Morphology

Voltage-dependent anion channel (VDAC) has been suggested to be a mediator of mitochondrial-dependent cell death induced by Ca²⁺ overload, oxidative stress and Bax-Bid activation. To confirm this hypothesis in vivo, we generated and characterized Drosophila VDAC (porin) mutants and found that Porin is not required for mitochondrial apoptosis, which is consistent with the previous mouse studies. We also reported a novel physiological role of Porin. Loss of porin resulted in locomotive defects and male sterility. Intriguingly, porin mutants exhibited elongated mitochondria in indirect flight muscle, whereas Porin overexpression produced fragmented mitochondria. Through genetic analysis with the components of mitochondrial fission and fusion, we found that the elongated mitochondria phenotype in porin mutants were suppressed by increased mitochondrial fission, but enhanced by increased mitochondrial fusion. Furthermore, increased mitochondrial fission by Drp1 expression suppressed the flight defects in the porin mutants. Collectively, our study showed that loss of Drosophila Porin results in mitochondrial morphological defects and suggested that the defective mitochondrial function by Porin deficiency affects the mitochondrial remodeling process.     

Structure and Functional Analysis of RifR, the Type II Thioesterase from the Rifamycin Biosynthetic Pathway

Two thioesterases are commonly found in natural product biosynthetic clusters, a type I thioesterase that is responsible for removing the final product from the biosynthetic complex and a type II thioesterase that is believed to perform housekeeping functions such as removing aberrant units from carrier domains. We present the crystal structure and the kinetic analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide synthetases/polyketide synthase rifamycin biosynthetic cluster of Amycolatopsis mediterranei. Steady-state kinetics show that RifR has a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm of the acyl carrier domain over the hydrolysis of acyl units from the phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA and methylmalonyl-CoA. Multiple RifR conformations and structural similarities to other thioesterases suggest that movement of a helical lid controls access of substrates to the active site of RifR.Assembly line complexes, which include modular polyketide synthases (PKS)³ and nonribosomal peptide synthetases (NRPS), are multifunctional proteins composed of modules that work in succession to synthesize secondary metabolites, many of which are precursors of potent antibiotics, immunosuppressants, anti-tumor agents, and other bioactive compounds. Rifamycin, the precursor to the anti-tuberculosis drug rifampicin, is produced by the rifamycin assembly line complex, which is an NRPS/PKS hybrid system composed of one NRPS-like and 10 PKS modules (1). Each module in an assembly line complex extends and modifies the intermediate compound before passing it on to the next module in the series (Fig. 1A). The intermediate compounds are covalently attached through a thioester linkage to the phosphopantetheine arm (Ppant) of carrier domains, one associated with each module, until they are released from the synthase, usually by a type I thioesterase (TEI) (2, 3).Open in a separate windowFIGURE 1.Proposed functions of thioesterase II proteins. A, chain elongation by a PKS module. The chain elongation intermediate is transferred from the ACP of the upstream module to the ketosynthase (KS) domain. The acyltransferase (AT) domain transfers an acyl group building block from CoA to the ACP within the module. The KS domain catalyzes condensation of the new building block with the intermediate, releasing CO₂. B, production of a decarboxylated acyl unit by the ketosynthase domain and the subsequent hydrolysis by a TEII. C, mispriming of a PKS by transfer of an acyl-phosphopantetheine arm by a promiscuous phosphopantetheinyl transferase (Pptase) and the subsequent hydrolysis by a TEII. D, hydrolysis of an amino acid derivative by a TEII from an NRPS module comprising an adenylation domain (A) and a peptide carrier protein (PCP) domain.TEIs are usually integrated into the final module of the assembly line complex and remove the final product through macrocyclization or hydrolysis. Occasionally, tandem type I thioesterases are integrated at the C terminus of the final module of NRPS pathways (4).Although TEIs are covalently attached to the terminal module and generally process only the final product of an assembly line complex, type II thioesterases (TEIIs) are discrete proteins that can remove intermediates from any module in the complex. A variety of functions have been attributed to TEIIs, the most prevalent of which is a “housekeeping function,” the removal of aberrant acyl units from carrier domains. These aberrant acyl units may be due to premature decarboxylation by a PKS ketosynthase domain (5) (Fig. 1B) or to mispriming of the carrier domain by a promiscuous phosphopantetheinyl transferase (6–8) (Fig. 1C). Other proposed functions for TEIIs include the removal of intermediates from the synthase as in the case of the mammary gland rat fatty acid synthase (FAS) TEII in lactating rats, which removes medium chain C₈-C₁₂ fatty acids from the ACP domain (9) and the removal of amino acid derivatives from a carrier domain (10–13), allowing these derivatives to be incorporated into the natural product by a later module in the assembly line complex (Fig. 1D).Disruption of the TEI function results in a complete loss of product, whereas disruption of TEII function results in a significant decrease in product yield (30–95%) (4, 14–24). Removal of the TEII from the rifamycin assembly line resulted in a 60% decrease in product yield (25). Neither TEIs nor TEIIs may rescue the disrupted function of the other (6), but a TEII from another pathway may rescue the function of a disrupted TEII (26).Two models have been proposed for the TEII housekeeping function (5). In the high specificity model, the TEII scans the complex and efficiently removes only aberrant acyl units. In the low specificity model, the TEII removes both correct and incorrect acyl units from the Ppant arm at an inefficient rate. Correct acyl units are quickly incorporated into the growing intermediate compound. In contrast, incorrect acyl units stall the assembly line, providing a longer window of opportunity for removal by a TEII. Thus a slow, low specificity enzyme can be effective.TEIIs from different pathways have differing specificities, but general trends include a preference for decarboxylated acyl units over carboxylated acyl units (5, 6, 27), substrates linked to a carrier domain over substrates linked to CoA or the phosphopantetheine mimic N-acetylcysteamine (7, 28), and single amino acids over di- or tri-peptides (6, 7). TEIIs are able to hydrolyze substrates attached to carrier domains from their native pathway as well as other pathways (6, 20, 28).PKS/NRPS/FAS thioesterases belong to the α/β hydrolase family. Structures are reported for seven PKS/NRPS/FAS thioesterases: crystal structures for the TEIs from the pikromycin (PikTE) PKS (29), 6-deoxyerythronolide B (DEBSTE) PKS (30), surfactin NRPS (SrfTE) (31), fengycin NRPS (FenTE) (32), and human fatty acid synthase (hFasTE) (33) systems, and NMR structures for enterobactin TEI (34), and surfactin TEII (35). Like PKS modules, PKS TEIs are dimers. The dimer interface comprises two N-terminal helices that are unique to the PKS TEIs. NRPS TEIs are monomeric, like NRPS modules. The NRPS TEII of surfactin is also monomeric (31). Although the FAS complex is dimeric, the FAS TEI is a monomer (33). All of the TEs have an α-helical insertion after strand β5 that forms a lid over the active site. Additionally, in the PKS TEIs, the N-terminal dimer-forming helices contribute to the lid structure, forming a fixed channel that runs the length of the TE and contains the active site. In contrast, the active site pocket of monomeric NRPS TEIs and TEIIs is flexible; two conformations of the lid and active site pocket were observed in the surfactin TEI (SrfTEI) crystal structure (7), and chemical shift observations suggested greater flexibility for residues of the lid region in the surfactin TEII (SrfTEII) solution structure (35). These movements seem to be of functional importance, because a movement of a linker peptide in SrfTEI determines the shape of the active site pocket and a movement of the first lid helix appears to modulate access to the active site (31).We report the structure and activity of recombinant RifR, the TEII of the rifamycin biosynthetic cluster. Steady-state kinetic analysis of the hydrolytic activity of RifR on a wide range of acyl-CoA and acyl-ACP substrates demonstrates that acyl-ACP substrates are preferred over the acyl-CoAs. Aberrant, decarboxylated acyl units are processed more efficiently than are the natural rifamycin building blocks. We report the crystal structure of RifR, the first for any hybrid PKS/NRPS TEII. The size and shape of the substrate chamber are variable, because one of the elements forming the chamber, an extended linker segment, is highly flexible, and different crystal forms reveal different shapes for the substrate binding site. Access to the active site is severely restricted, and structural comparisons with other thioesterases suggest that a conformational change in the lid and the flexible linker region is required for access to the substrate pocket.     

Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATX1 from Arabidopsis

A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO₄, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.     

Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina,southern Brazil

Drug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosisclinical isolates were analysed by spoligotyping and a partial region of therpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoB in RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations withinrpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil.     

Using Superfolder Green Fluorescent Protein for Periplasmic Protein Localization Studies

Studies investigating the subcellular localization of periplasmic proteins have been hampered by problems with the export of green fluorescent protein (GFP). Here we show that a superfolding variant of GFP (sfGFP) is fluorescent following Sec-mediated transport and works best when the cotranslational branch of the pathway is employed.     

Escape of Steinernema feltiae from Alginate Capsules Containing Tomato Seeds

The entomogenous nematode Steinernema feltiae was encapsulated in an alginate matrix containing a tomato seed. When these capsules were placed on 0.8% agar for 7 days, the seed germinated and ca. 20% of the nematodes escaped from the capsules, whereas only 0.1% escaped from capsules without seeds. When capsules containing nematodes and a seed were planted into sterilized or nonsterilized soil, nematodes escaped to infect Galleria mellonella larvae. When seed in capsules containing ca. 274 nematodes per capsule were planted in nonsterilized soil, Galleria mortality was 90% 1 week later. Galleria mortality declined to 27%, 23%, and 0% in weeks 2, 4, and 8 postplant, respectively. In sterilized soil, Galleria mortality was 96% and did not differ significantly from the nonsterilized soil in week 1, but was significantly higher in sterilized soil over nonsterilized soil for week 2 (81%) and week 4 (51%). When capsules containing nematodes only were used, Galleria mortality was 71% in sterilized soil 1 week after planting and 58%, 33%, and 12% in weeks 2, 4, and 8 postplant, respectively. In nonsterilized soil, Galleria mortality was 8%, 30%, 21%, and 28% after 1, 2, 4, and 8 weeks, respectively, using only encapsulated nematodes. When the number of nematodes per capsule was increased to ca. 817, Galleria mortality was 92 % or higher in sterilized soil from week 1 to week 4.