The Glycerol-3-Phosphate Permease GlpT Is the Only Fosfomycin Transporter in Pseudomonas aeruginosa

Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10⁻⁶. However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, ≤5 × 10⁻¹¹). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.Pseudomonas aeruginosa is an opportunistic, life-threatening bacterial pathogen that especially affects critically ill patients in intensive care units or those suffering from chronic respiratory diseases such as cystic fibrosis (19, 40). Its 6.3-Mb genome supports its enormous metabolic versatility and, consequently, its adaptability to almost any challenging environment. One of the consequences of this versatility is the rapid adaptation to stressful environmental conditions, including starvation, desiccation, and antibiotic treatments (14, 40). Mutants resistant to one or several antibiotics will evolve during sufficiently prolonged treatments, this being a process facilitated by the presence of hypermutable alleles (31, 32). After years of treating cystic fibrosis patients with antibiotics, P. aeruginosa became unavoidably resistant to many or all of them (5). Multidrug-resistant strains of P. aeruginosa are an important problem for the treatment of nosocomial outbreaks and cystic fibrosis patients (27, 37). Currently, the treatment of multidrug-resistant P. aeruginosa requires the combination of various antimicrobial agents. Fosfomycin (Fos) has been reported to be effective in combination with other antipseudomonal agents (6, 29, 42, 44). The proportion of Fos-resistant (Fos-R) strains in clinical isolates of P. aeruginosa currently is not well known, and even the mechanisms that support Fos resistance in P. aeruginosa are not clear. Thus, the knowledge of the molecular bases involved in the development of spontaneous Fos resistance in P. aeruginosa is of particular interest.Fos is a unique broad-spectrum bactericidal antibiotic that is chemically unrelated to any other known antimicrobial agent used to treat urinary tract and gastrointestinal infections in humans (9, 35). It binds UDP-GlcNAc enol-pyruvyltransferase (MurA), acting as a phosphoenolpyruvate analogue and avoiding the formation of UDP-N-acetylglucosamine-3-O-enolpyruvate from UDP-N-acetylglucosamine and phosphoenolpyruvate (12, 33). Fos is taken up actively into bacterial cells via transport systems. In Escherichia coli, Fos is imported through two nutrient transport systems, the glycerol-3-phosphate (glycerol-3-P) transporter (GlpT) and glucose-6-phosphate (glucose-6-P) transporter (UhpT), to achieve its target and inhibits the initial step in cell wall synthesis (12, 17). The expression of these transport systems is induced by their substrates (glycerol-3-P and glucose-6P) and requires the presence of the cyclic AMP receptor protein (cAMP-CRP) complex (23, 30). Additionally, the high-level expression of UhpT requires the regulatory genes uhpA, uhpB, and uhpC (12, 30). Therefore, Fos-R strains isolated in E. coli contain mutations that prevent Fos transport using GlpT or UhpT (23, 30). Plasmid-encoded resistance also has been described previously (4, 41).In this paper, we describe the screening and analysis of Fos-R clones in a P. aeruginosa PA14 ordered insertional library (18). In addition, we studied the mutations responsible for the spontaneous resistance to Fos in P. aeruginosa PA14, the effect of these mutations on the in vitro growth rate, and the uniqueness of the mutational target.     

Comparative Structural and Molecular Characterization of Ribitol-5-Phosphate-Containing Streptococcus oralis Coaggregation Receptor Polysaccharides

The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfβ1-6GalNAcβ1-3Galα1-2ribitol-5-PO₄⁻-6Galfβ1-3Galβ1]_n, thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and ribitol-5-phosphate, which is α1-2 in strain SK144 versus α1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.The coaggregations observed between different viridans group streptococci and Actinomyces naeslundii (6) provided early evidence for the role of interbacterial adhesion in dental plaque biofilm formation. Interactions between these bacteria were subsequently attributed to binding of A. naeslundii type 2 fimbriae to specific Gal and GalNAc-containing cell wall polysaccharides, referred to as receptor polysaccharides (RPS), on strains of Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii (7, 9, 14). These streptococci inhabit the tooth surface (23), where they grow in close association with type 2 fimbriated A. naeslundii (26) and other members of the dental plaque biofilm community. Growth and biofilm formation were not observed in flow cells when coaggregating strains of S. oralis and A. naeslundii were cultured separately in dilute saliva (27). However, when cultured together, the two strains grew as a mixed-species community, thereby supporting a recognition role for cell surface RPS in biofilm development.Six structural types of RPS have been identified by high resolution nuclear magnetic resonance (NMR) of the cell wall polysaccharides isolated from over 20 coaggregating strains of S. sanguinis, S. gordonii, and S. oralis (8). These polysaccharides are composed of structurally distinct repeating units that contain conserved Galf linked β1-6 to a host-like recognition motif, which is GalNAcβ1-3Gal (Gn) in certain types of RPS and Galβ1-3GalNAc (G) in others. The flexible β1-6 linkage from Galf (34) is thought to function as a hinge, exposing the adjacent host-like motif for adhesin-mediated recognition (21). Whereas both Gn and G types of RPS are recognized by type 2 fimbriated A. naeslundii, only Gn types are recognized by the GalNAc-binding adhesins present on non-RPS-bearing strains of S. sanguinis and S. gordonii (8). Conversely, only G types are coaggregation receptors of certain Veillonella spp. (25). The host-like features of these polysaccharides, although critical for interbacterial adhesion, contribute little to RPS serotype specificity, which instead reflects the immunogenic features of these molecules (21). As a result, the identification of RPS-bearing streptococci requires both serotyping (i.e., serotypes 1, 2, 3, 4, or 5) and receptor typing (i.e., types Gn or G) of these bacteria.A possible molecular approach for the identification of these bacteria is evident from comparative studies of the chromosomal loci (rps) for RPS biosynthesis in different strains (33, 35-37). In this regard, the genes wchA and wchF, which were first identified in Streptococcus pneumoniae (5, 15), encode the first two transferases for synthesis of RPS serotypes 1, 2, and 3. WchA transfers Glc-1-phosphate from UDP-Glc to a carrier lipid, and WchF adds Rha β1-4 to Glc. Subsequent synthesis of both the antigenic and receptor regions in these polysaccharides depends on other encoded transferases (35-37), many of which are distinguishable from those identified in S. pneumoniae. In addition to Glc- and Rha-containing types of RPS, other types have been described that lack these sugars but contain ribitol-5-phosphate (3), in addition to GalNAc, Galp, and Galf, which are common constituents of all types. The ribitol-5-phosphate-containing group, represented by type 4Gn RPS of S. oralis C104 and type 5Gn RPS of S. oralis SK144, is the subject of the present study. The results define the structural and genetic basis of the antigenic difference noted between these polysaccharides. They also reveal a close molecular relationship between these types of RPS and certain capsular polysaccharides (CPS) of S. pneumoniae, most notably those in CPS serogroup 10.     

Functional Role of Bradyrhizobium japonicum Trehalose Biosynthesis and Metabolism Genes during Physiological Stress and Nodulation

Trehalose, a disaccharide accumulated by many microorganisms, acts as a protectant during periods of physiological stress, such as salinity and desiccation. Previous studies reported that the trehalose biosynthetic genes (otsA, treS, and treY) in Bradyrhizobium japonicum were induced by salinity and desiccation stresses. Functional mutational analyses indicated that disruption of otsA decreased trehalose accumulation in cells and that an otsA treY double mutant accumulated an extremely low level of trehalose. In contrast, trehalose accumulated to a greater extent in a treS mutant, and maltose levels decreased relative to that seen with the wild-type strain. Mutant strains lacking the OtsA pathway, including the single, double, and triple ΔotsA, ΔotsA ΔtreS and ΔotsA ΔtreY, and ΔotsA ΔtreS ΔtreY mutants, were inhibited for growth on 60 mM NaCl. While mutants lacking functional OtsAB and TreYZ pathways failed to grow on complex medium containing 60 mM NaCl, there was no difference in the viability of the double mutant strain when cells were grown under conditions of desiccation stress. In contrast, mutants lacking a functional TreS pathway were less tolerant of desiccation stress than the wild-type strain. Soybean plants inoculated with mutants lacking the OtsAB and TreYZ pathways produced fewer mature nodules and a greater number of immature nodules relative to those produced by the wild-type strain. Taken together, results of these studies indicate that stress-induced trehalose biosynthesis in B. japonicum is due mainly to the OtsAB pathway and that the TreS pathway is likely involved in the degradation of trehalose to maltose. Trehalose accumulation in B. japonicum enhances survival under conditions of salinity stress and plays a role in the development of symbiotic nitrogen-fixing root nodules on soybean plants.Rhizobia induce the formation of nodules on the roots of legume plants, in which atmospheric nitrogen is fixed and supplied to the host plant, thereby enhancing growth under nitrogen-limiting conditions. The symbiotic interaction between rhizobia and their cognate leguminous plants is important for agricultural productivity, especially in less developed countries. However, physiological stresses, such as desiccation and salinity, negatively affect these symbiotic interactions by limiting nitrogen fixation (44). The osmotic environment within the rhizosphere may affect root colonization, infection thread development, nodule development, and the formation of effective N₂-fixing nodules (21). Moreover, when legume seeds are inoculated with appropriate rhizobial strains prior to planting in the field, the vast majority of nodules produced are often not formed by the inoculant bacteria but rather by indigenous strains in the soil (36). This is in part due to the death of inoculant strains from rapid seed coat-mediated desiccation. Therefore, improvement of the survival of rhizobia under conditions of physiological stresses may promote biological nitrogen fixation and enhance plant growth.Rhizobia synthesize and accumulate compatible solutes, including trehalose, in response to desiccation and solute-mediated physiological stresses (5, 21, 42). Trehalose, a nonreducing disaccharide with an α,α-1,1 linkage between the two glucose molecules, has been shown to protect cell membranes and proteins from stress-induced inactivation and denaturation (8, 23, 24). The relationship between trehalose accumulation and symbiotic phenotype is dependent on rhizobial species and host genotype. Suarez et al. (39) reported an increase in root nodule number and nitrogen fixation by Phaseolus vulgaris inoculated with a trehalose-6-phosphate synthase-overexpressing strain of Rhizobium etli. In contrast, trehalose accumulation in Rhizobium leguminosarum and Sinorhizobium meliloti cells did not result in an increase in nitrogen-fixing nodules but led to enhancement of competitiveness on clover and on certain alfalfa genotypes, respectively (1, 16, 20).Four trehalose biosynthetic pathways, mediated by OtsAB, TreS, TreYZ, and TreT, have been reported thus far for prokaryotes (8, 25). The OtsAB pathway results in the condensation of glucose-6-phosphate with UDP-glucose by trehalose-6-phosphate synthase (OtsA) to form trehalose-6-phosphate. Trehalose is subsequently formed from trehalose-6-phosphate by the action of trehalose-6-phosphate phosphatase (OtsB). The TreS pathway involves a reversible transglycosylation reaction in which trehalose synthase (TreS) converts maltose, a disaccharide with α,α-1,4 linkage between the two glucose molecules, to trehalose. The third pathway, mediated by TreYZ, involves the conversion of maltodextrins into trehalose. The terminal α-1,1-glycosylic bond at the end of the maltodextrin polymer is hydrolyzed by maltooligosyltrehalose synthase (TreY), and trehalose is subsequently released from the end of the polymer via hydrolysis by maltooligosyltrehalose trehalohydrolase (TreZ). More recently, a trehalose glycosyltransferring synthase (TreT) was shown to catalyze the reversible formation of trehalose from ADP-glucose and glucose (25).In addition to biosynthesis, Gram-negative bacteria have also been reported to have trehalose degradation systems. Typically, trehalose is hydrolyzed into two glucose moieties by periplasmic and cytoplasmic trehalase enzymes, TreA and TreF, respectively (13, 15). However, Sinorhizobium meliloti also uses ThuA and ThuB for trehalose utilization (16).Bradyrhizobium japonicum, the root nodule symbiont of soybeans, accumulates trehalose in cultured cells and bacteroids (34, 35). Biochemical studies indicated that B. japonicum has three independent trehalose biosynthetic pathways involving trehalose synthase (TreS), maltooligosyltrehalose synthase (TreYZ), and trehalose-6-phosphate synthetase (OtsAB) (38). Sequence analysis of the B. japonicum USDA 110 genome identified the genes that encode these biosynthetic pathways: otsAB (bll0322 to bll0323), two homologs of treS (blr6767 and bll0902), and treYZ (blr6770 to blr6771), but not treT (17). Orthologous gene sequences to the trehalose degradation genes treA, treF, and thuAB have not been found in the genome of B. japonicum USDA 110. Cytryn et al. (6) reported that expression of otsA, treS (blr6767), and treY genes were highly induced by desiccation stress. Moreover, the concentrations of these three enzymes increased when B. japonicum was cultured in the presence of salt (38). Trehalose concentration in B. japonicum has been reported to increase due to desiccation stress (6), and this sugar is purported to act as an osmoprotectant. The addition of exogenously supplied trehalose has been reported to enhance the survival of B. japonicum in response to desiccation and salinity stresses (9, 37). Despite this information, little is known about how the various trehalose biosynthetic pathways modulate stress tolerance and symbiotic performance in B. japonicum.The purpose of this study was to examine the functional role(s) of the B. japonicum trehalose biosynthetic pathways on stress survival by constructing single, double, and triple mutants and by producing strains that overexpress the trehalose biosynthesis enzymes. Here we report on the relationship between trehalose accumulation and physiological responses to salinity and desiccation stresses in mutant and overexpression strains and that mutations in the trehalose biosynthesis pathways altered the symbiotic performance of B. japonicum USDA 110 on soybeans. Results of these studies indicate that trehalose accumulation in B. japonicum plays a prominent role in the saprophytic and symbiotic competence of this agriculturally important soil bacterium.     

Glycerol-3-Phosphate Acyltransferases Gat1p and Gat2p Are Microsomal Phosphoproteins with Differential Contributions to Polarized Cell Growth

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in the synthesis of all glycerolipids. It is the committed and rate-limiting step and is redundant in Saccharomyces cerevisiae, mammals, and plants. GPAT controls the formation of lipid intermediates that serve not only as precursors of more-complex lipids but also as intracellular signaling molecules. Saccharomyces cerevisiae possesses two GPATs, encoded by the GAT1 and GAT2 genes. Metabolic analysis of yeast lacking either GAT1 or GAT2 indicated partitioning of the two main branches of phospholipid synthesis at the initial and rate-limiting GPAT step. We are particularly interested in identifying molecular determinants mediating lipid metabolic pathway partitioning; therefore, as a starting point, we have performed a detailed study of Gat1p and Gat2p cellular localization. We have compared Gat1p and Gat2p localization by fluorescence microscopy and subcellular fractionation using equilibrium density gradients. Our results indicate Gat1p and Gat2p overlap mostly in their localization and are in fact microsomal GPATs, localized to both perinuclear and cortical endoplasmic reticula in actively proliferating cells. A more detailed analysis suggests a differential enrichment of Gat1p and Gat2p in distinct ER fractions. Furthermore, overexpression of these enzymes in the absence of endogenous GPATs induces proliferation of distinct ER arrays, differentially affecting cortical ER morphology and polarized cell growth. In addition, our studies also uncovered a dynamic posttranslational regulation of Gat1p and Gat2p and a compensation mechanism through phosphorylation that responds to a cellular GPAT imbalance.The first step in the synthesis of almost all membrane phospholipids and neutral glycerolipids is catalyzed by glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15). This enzyme transfers a fatty acid from fatty acyl coenzyme A to the sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LysoPA). LysoPA is further acylated at the sn-2 position by a separate acyltransferase to produce phosphatidic acid (PA). PA can be either (i) dephosphorylated to produce diacylglycerol (DAG) or (ii) converted to CDP-DAG. These lipids not only are precursors of all glycerolipids but also are dynamic components of signal transduction systems that control cell physiology. Regulated interconversion of signaling lipids like LysoPA, PA, and DAG transmits information in part by their biophysical properties (5) and through lipid-lipid and lipid-protein interactions (18, 23, 29). The mechanisms of the regulation of PA biosynthesis, of the rate-limiting GPAT step, and of lipid metabolic pathway partitioning are not known (8, 12).GPATs are present in bacteria, fungi, plants, and animals. We and others have previously identified a unique gene pair in Saccharomyces cerevisiae, YKR067W (GAT1/GPT2) and YBL011W (GAT2/SCT1), and demonstrated that they code for the major GPATs in this organism (32, 34). Bioinformatic approaches, using a region conserved between the yeast GPATs and other fatty acid acyltransferases as a query, identified seven members of the GPAT family in the model organism Arabidopsis thaliana (33). A substantial level of redundancy is also found in animals. Four mammalian GPAT isoforms have been identified to date, each encoded by a different gene. Two are localized in the mitochondria (mitochondrial GPAT1 [mtGPAT1] and mtGPAT2) (4, 20) and two in the endoplasmic reticulum (ER) (microsomal GPAT3 and GPAT4) (4, 24). The existence of additional genes encoding proteins with GPAT activity has been suggested (12).Thus, the emerging picture indicates that the traditional PA biosynthetic pathway in most eukaryotes is divided into many more parts that were recently believed and opens the possibility of each GPAT having a differential contribution to specific pools of LysoPA, PA, and DAG. In this regard, metabolic analysis of yeast containing an inactivated GAT1 gene or an inactivated GAT2 gene indicated that Gat2p is the primary supplier of DAG, used mainly in triacylglycerol synthesis and phosphatidylcholine synthesis through the CDP-choline pathway (32). These results indicated partitioning of the two main branches of phospholipid synthesis at the initial and rate-limiting GPAT step (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Differential partitioning of glycerolipids metabolized by separate GPATs in yeast. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; TAG, triacylglycerol; LPAAT, LysoPA acyltransferase; CoA, coenzyme A.We are particularly interested in identifying molecular determinants mediating lipid metabolic pathway partitioning. Elucidation of how lipid metabolic systems are spatiotemporally regulated is a major challenge for the field (29).It is well known that within eukaryotic cells, the synthesis of lipids is restricted, and localization of biosynthetic systems is in fact the first determinant of the distinct compositions of organelles. One plausible explanation for the differential contribution of Gat1p and Gat2p to lipid metabolic pathway partitioning is that they are localized to different subcellular compartments.To explore this possibility, we have compared Gat1p and Gat2p subcellular localization by fluorescence microscopy and subcellular fractionation using equilibrium density gradients. Biochemical assays have previously pointed out that GPAT activity in yeast is distributed between microsomal fractions and lipid particles (1, 2). Furthermore, a global green fluorescent protein (GFP) localization study in yeast indicated that Gat1p and Gat2p localize primarily to the ER, but it was not determined whether the Gat1-GFP and Gat2-GFP proteins were functional (1, 2, 11). Our results indicate that Gat1p and Gat2p are in fact microsomal GPATs, localized to both perinuclear and cortical ER in exponentially growing cells. Although they overlap mostly in their localization, a detailed analysis of their distribution using equilibrium density gradients suggests a differential enrichment of Gat1p and Gat2p in distinct ER fractions. Moreover, overexpression of Gat1p or Gat2p in the absence of endogenous GPATs induces proliferation of distinct ER arrays, differentially affecting cortical ER morphology. Our studies also revealed a dynamic posttranslational regulation of Gat1p and Gat2p through phosphorylation that responds to Gat1p/Gat2p cellular imbalance.     

Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor

In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺), was expressed in the gpd1Δ gpd2Δ strain, anaerobic growth was restored by supplementation with 2.0 g liter⁻¹ acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).Bioethanol production by Saccharomyces cerevisiae is currently, by volume, the single largest fermentation process in industrial biotechnology. A global research effort is under way to expand the substrate range of S. cerevisiae to include lignocellulosic hydrolysates of nonfood feedstocks (e.g., energy crops and agricultural residues) and to increase productivity, robustness, and product yield (for reviews see references 20 and 35). A major challenge relating to the stoichiometry of yeast-based ethanol production is that substantial amounts of glycerol are invariably formed as a by-product (24). It has been estimated that, in typical industrial ethanol processes, up to 4% of the sugar feedstock is converted into glycerol (24). Although glycerol also serves as a compatible solute at high extracellular osmolarity (10), glycerol production under anaerobic conditions is primarily linked to redox metabolism (34).During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via alcoholic fermentation. In this process, the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate to ethanol via NAD⁺-dependent alcohol dehydrogenase. The fixed stoichiometry of this redox-neutral dissimilatory pathway causes problems when a net reduction of NAD⁺ to NADH occurs elsewhere in the metabolism. Such a net production of NADH occurs in assimilation when yeast biomass is synthesized from glucose and ammonia (34). Under anaerobic conditions, NADH reoxidation in S. cerevisiae is strictly dependent on reduction of sugar to glycerol (34). Glycerol formation is initiated by reduction of the glycolytic intermediate dihydroxyacetone phosphate to glycerol-3-phosphate, a reaction catalyzed by NAD⁺-dependent glycerol-3-phosphate dehydrogenase. Subsequently, the glycerol-3-phosphate formed in this reaction is hydrolyzed by glycerol-3-phosphatase to yield glycerol and inorganic phosphate.The importance of glycerol production for fermentative growth of yeasts was already observed in the 1960s during studies of non-Saccharomyces yeasts that exhibit a so-called “Custers effect.” In such yeast species, which are naturally unable to produce glycerol, fermentative growth on glucose is possible only in the presence of an external electron acceptor that can be reduced via an NADH-dependent reaction (e.g., the reduction of acetoin to butanediol via NAD⁺-dependent butanediol dehydrogenase) (29). It was later shown that gpd1Δ gpd2Δ strains of S. cerevisiae, which are also unable to produce glycerol, are similarly unable to grow under anaerobic conditions unless provided with acetoin as an external electron acceptor (8).In view of its large economic significance, several metabolic engineering strategies have been explored to reduce or eliminate glycerol production in anaerobic cultures of S. cerevisiae. Nissen et al. (25) changed the cofactor specificity of glutamate dehydrogenase, the major ammonia-fixing enzyme of S. cerevisiae, thereby increasing NADH consumption in biosynthesis. This approach significantly reduced glycerol production in anaerobic cultures grown with ammonia as the nitrogen source. Attempts to further reduce glycerol production by expression of a heterologous transhydrogenase, with the aim to convert NADH and NADP⁺ into NAD⁺ and NADPH, were unsuccessful (24) because intracellular concentrations of these pyridine nucleotide cofactor couples favor the reverse reaction (23).The goal of the present study was to investigate whether the engineering of a linear pathway for the NADH-dependent reduction of acetic acid to ethanol can replace glycerol formation as a redox sink in anaerobic, glucose-grown cultures of S. cerevisiae and thus provide a stoichiometric basis for elimination of glycerol production during industrial ethanol production. Significant amounts of acetic acid are released upon hydrolysis of lignocellulosic biomass, and, in fact, acetic acid is studied as an inhibitor of yeast metabolism in lignocellulosic hydrolysates (5, 7, 26). The S. cerevisiae genome already contains genes encoding acetyl coenzyme A (acetyl-CoA) synthetase (32) and NAD⁺-dependent alcohol dehydrogenases (ADH1-5 [12]). To complete the linear pathway for acetic acid reduction, we expressed an NAD⁺-dependent, acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) from Escherichia coli into a gpd1Δ gpd2Δ strain of S. cerevisiae. This enzyme, encoded by the E. coli mhpF gene (15), catalyzes the reaction acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺. Growth and product formation of the engineered strain were then compared in the presence and absence of acetic acid and compared to those of a congenic reference strain.     

Comprehensive Investigation of Marine Actinobacteria Associated with the Sponge Halichondria panicea

Representatives of Actinobacteria were isolated from the marine sponge Halichondria panicea collected from the Baltic Sea (Germany). For the first time, a comprehensive investigation was performed with regard to phylogenetic strain identification, secondary metabolite profiling, bioactivity determination, and genetic exploration of biosynthetic genes, especially concerning the relationships of the abundance of biosynthesis gene fragments to the number and diversity of produced secondary metabolites. All strains were phylogenetically identified by 16S rRNA gene sequence analyses and were found to belong to the genera Actinoalloteichus, Micrococcus, Micromonospora, Nocardiopsis, and Streptomyces. Secondary metabolite profiles of 46 actinobacterial strains were evaluated, 122 different substances were identified, and 88 so far unidentified compounds were detected. The extracts from most of the cultures showed biological activities. In addition, the presence of biosynthesis genes encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in 30 strains was established. It was shown that strains in which either PKS or NRPS genes were identified produced a significantly higher number of metabolites and exhibited a larger number of unidentified, possibly new metabolites than other strains. Therefore, the presence of PKS and NRPS genes is a good indicator for the selection of strains to isolate new natural products.Sponges are multicellular invertebrates and sessile filter feeders which are abundant in the oceans as well as in freshwater habitats (41). They gained great interest due to their association with a wide variety of microorganisms. These microorganisms are known to be a rich source of secondary metabolites (108), which exhibit a broad range of bioactivities such as inhibition of enzyme activities and cell division and antiviral, antimicrobial, anti-inflammatory, antitumor, cytotoxic, and cardiovascular properties (77).Numerous studies concerning specific aspects of sponge-bacterium associations were accomplished using distinct methods for the evaluation of the microbial diversity (mostly molecular approaches) or the bioactivities (culture-dependent methods) or biosynthetic aspects (chemical analyses and molecular approaches) of secondary metabolites of the associated bacteria (19, 47, 51, 54, 110, 122, 126). So far, there is less comprehensive information about the integration of this knowledge into concepts for sponge-bacterium interactions based on small molecules.We focused on Actinobacteria associated with Halichondria panicea Pallas (Porifera, Demospongiae, Halichondriida, Halichondriidae), a sponge species living in coastal habitats worldwide (9). Previous work demonstrated a phylogenetically diverse array of bacterial groups present in this sponge: representatives of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Cytophaga/Flavobacteria, the Deinococcus group, low-G+C-content Gram-positive bacteria, Actinobacteria, and Planctomycetales were identified by means of a genetic approach (47, 122). Among these, though representing only 3 to 20% of the sponge-associated bacterial community (41, 47, 103), Actinobacteria are the most promising bacterial group regarding secondary metabolite production. Members of this phylum account for approximately half of the bioactive secondary metabolites that have so far been discovered in bacteria (64). Although the majority of secondary metabolite-producing Actinobacteria originate from terrestrial habitats (101), recent studies of marine Actinobacteria have revealed many new chemical entities and bioactive metabolites (13, 30, 50, 100). Among these, only a few substances were isolated from Actinobacteria associated with H. panicea (85, 123), e.g., the antimicrobially active substances 2,4,4′-trichloro-2′-hydroxydiphenylether and acyl-1-(acyl-6′-mannobiosyl)-3-glycerol produced by Micrococcus luteus (17). By combining data about the phylogenetic characterization of the Actinobacteria associated with H. panicea, their biosynthetic potential for secondary metabolite production, and their chemical profiles, we present comprehensive insights into a great variety of produced natural products as well as their bioactivities. By means of these results, we attempt to close the gap of knowledge about Actinobacteria associated with H. panicea and discuss the biological roles of identified small molecules in the sponge-associated community.     

Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate

Applying the genomic library construction process and colony screening, a novel aroA gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) is the sixth enzyme in the shikimate pathway, which is essential for the synthesis of aromatic amino acids and many aromatic metabolites in plants, fungi, and microorganisms (2, 11, 16), including apicomplexan parasites (22). It converts shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. Interest in the characterization of EPSPS has increased significantly since the enzyme was identified as the primary target of the broad-spectrum, nonselective herbicide glyphosate [N-(phosphonomethyl)glycine] (25). Glyphosate is a competitive inhibitor with respect to PEP and binds adjacent to S3P in the active site of EPSPS, thereby mimicking an intermediate state of the ternary enzyme-substrate complex (23).Two classes of EPSPS, class I and II enzymes, sharing less than 30% amino acid similarity have been reported (9). Class I includes those found in plants and bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium, whose catalytic activity is inhibited at low micromolar concentrations of glyphosate (8). Class II EPSPS, found in Pseudomonas sp. strain PG2982, Agrobacterium tumefaciens strain CP4, Streptococcus pneumoniae, and Staphylococcus aureus, was distinguished by its ability to sustain efficient catalysis in the presence of high glyphosate concentrations (6, 9).Although a large number of AroA enzymes (EPSPS) have been cloned, identified, and tested as glyphosate resistant, only AroA variants derived from the A. tumefaciens strain CP4 have been successfully used commercially (9). To find a new enzyme similar to that of the AroA_{A. tumefaciens CP4}, in this study a highly glyphosate-tolerant strain from the rhizosphere of rice in a field where glyphosate is frequently used has been selected and identified on M9 minimal medium containing 200 mM glyphosate, and its 16S rRNA gene sequence confirmed that this strain was strongly related to Ochrobactrum anthropi (99.9%). Additionally, the aroA_{O. anthropi} gene was isolated and kinetic characteristics of the Ochrobactrum anthropi strain EPSP synthase were determined in this study.     

Human Immunodeficiency Virus Type 1 Nef Protein Targets CD4 to the Multivesicular Body Pathway

The Nef protein of human immunodeficiency virus type 1 downregulates the CD4 coreceptor from the surface of host cells by accelerating the rate of CD4 endocytosis through a clathrin/AP-2 pathway. Herein, we report that Nef has the additional function of targeting CD4 to the multivesicular body (MVB) pathway for eventual delivery to lysosomes. This targeting involves the endosomal sorting complex required for transport (ESCRT) machinery. Perturbation of this machinery does not prevent removal of CD4 from the cell surface but precludes its lysosomal degradation, indicating that accelerated endocytosis and targeting to the MVB pathway are separate functions of Nef. We also show that both CD4 and Nef are ubiquitinated on lysine residues, but this modification is dispensable for Nef-induced targeting of CD4 to the MVB pathway.Primate immunodeficiency viruses infect helper T lymphocytes and cells of the macrophage/monocyte lineage by binding of their viral envelope glycoprotein, Env, to a combination of two host cell-specific surface proteins, CD4 and either the CCR5 or CXCR4 chemokine receptors (reviewed in reference 62). Ensuing fusion of the viral envelope with the host cell plasma membrane delivers the viral genetic material into the cytoplasm. Remarkably, the most highly transcribed viral gene in the early phase of infection does not encode an enzyme or structural protein but an accessory protein named Nef. Early expression of Nef is thought to reprogram the host cell for optimal replication of the virus. Indeed, Nef has been shown to enhance virus production (19, 24, 59, 74) and to promote progression to AIDS (23, 47, 48), making it an attractive candidate for pharmacologic intervention.Nef is an N-terminally myristoylated protein with a molecular mass of 27 kDa for human immunodeficiency virus type 1 (HIV-1) and 35 kDa for HIV-2 and simian immunodeficiency virus (27, 29, 50, 65). Nef has been ascribed many functions, the best characterized of which is the downregulation of the CD4 coreceptor from the surface of infected cells (28, 35, 57). CD4 downregulation is believed to prevent superinfection (8, 52) and to preclude the cellular retention of newly synthesized Env (8, 49), thus allowing the establishment of a robust infection (30, 71).The molecular mechanism by which Nef downregulates CD4 has been extensively studied. A consensus has emerged that Nef accelerates the endocytosis of cell surface CD4 (2, 64) by linking the cytosolic tail of CD4 to the heterotetrameric (α-β2-μ2-σ2) adaptor protein-2 (AP-2) complex (17, 25, 34, 45, 67). Determinants in the CD4 tail bind to a hydrophobic pocket comprising tryptophan-57 and leucine-58 on the folded core domain of Nef (34). On the other hand, a dileucine motif (i.e., ENTSLL, residues 160 to 165) (14, 22, 32) and a diacidic motif (i.e., DD, residues 174 and 175) (3) (residues correspond to the NL4-3 clone of HIV-1) within a C-terminal, flexible loop of Nef bind to the α and σ2 subunits of AP-2 (17, 18, 25, 51). AP-2, in turn, binds to clathrin, leading to the concentration of CD4 within clathrin-coated pits (15, 33). These pits eventually bud from the plasma membrane as clathrin-coated vesicles that deliver internalized CD4 to endosomes. In essence, then, Nef acts as a connector that confers on CD4 the ability to be rapidly internalized in a manner similar to endocytic receptors (75).Unlike typical endocytic recycling receptors like the transferrin receptor or the low-density lipoprotein receptor, however, CD4 that is forcibly internalized by Nef does not return to the cell surface but is delivered to lysosomes for degradation (4, 64, 68). Thus, expression of Nef decreases both the surface and total levels of CD4. What keeps internalized CD4 from returning to the plasma membrane? We hypothesized that Nef might additionally act on endosomes to direct CD4 to lysosomes. This is precisely the fate followed by signaling receptors, transporters, and other transmembrane proteins that undergo ubiquitination-mediated internalization and targeting to the multivesicular body (MVB) pathway (40, 46). This targeting involves the endosomal sorting complex required for transport (ESCRT), including the ESCRT-0, -I, -II, and -III complexes, which function to sort ubiquitinated cargoes into intraluminal vesicles of MVBs for eventual degradation in lysosomes (40, 46). Herein, we show that Nef indeed plays a novel role in targeting internalized CD4 from endosomes to the MVB pathway in an ESCRT-dependent manner. We also show that both Nef and CD4 undergo ubiquitination on lysine residues, but, strikingly, this modification is not required for CD4 targeting to the MVB pathway.     

Role of Conjugative Elements in the Evolution of the Multidrug-Resistant Pandemic Clone Streptococcus pneumoniaeSpain23F ST81

Streptococcus pneumoniae is a human commensal and pathogen able to cause a variety of diseases that annually result in over a million deaths worldwide. The S. pneumoniae^Spain23F sequence type 81 lineage was among the first recognized pandemic clones and was responsible for almost 40% of penicillin-resistant pneumococcal infections in the United States in the late 1990s. Analysis of the chromosome sequence of a representative strain, and comparison with other available genomes, indicates roles for integrative and conjugative elements in the evolution of pneumococci and, more particularly, the emergence of the multidrug-resistant Spain 23F ST81 lineage. A number of recently acquired loci within the chromosome appear to encode proteins involved in the production of, or immunity to, antimicrobial compounds, which may contribute to the proficiency of this strain at nasopharyngeal colonization. However, further sequencing of other pandemic clones will be required to establish whether there are any general attributes shared by these strains that are responsible for their international success.Streptococcus pneumoniae (the pneumococcus) is a human commensal and pathogen that represents a major cause of otitis media, pneumonia, and meningitis (8). Worldwide, pneumococcal disease is thought to be responsible for over a million fatalities annually, including more than 800,000 deaths in children under 5 years of age living in developing countries (64). While the introduction of the heptavalent polysaccharide conjugate vaccine (PCV7) has dramatically reduced the incidence of pneumococcal disease in some areas (37), limited serotype coverage, strain replacement, and capsule switching have resulted in a smaller, and decreasing, impact in other communities (66).S. pneumoniae is a naturally competent, genetically diverse species, with less than half of the pan-genome conserved between all strains thus far studied (33). The pneumococcal population is normally confined to the human nasopharynx, with rates of asymptomatic carriage varying with demographics, region, and season: surveys of colonization in healthy children generally estimate between 20 and 97% of younger individuals carry pneumococci (9, 32), with levels falling with age. Epidemiological data and animal models of infection indicate that strains exhibit differing propensities for causing invasive disease (13, 29, 63). The invasive disease potential odds ratio, which takes into account the relative frequencies of invasive disease and asymptomatic carriage observed in the human population, varies 80-fold between serotypes (13). However, the functional genetic variation to which this differing ability to cause disease is attributable remains largely unknown. Genome sequencing efforts have mainly focused on clinical pneumococcal isolates; the complete genomes of two highly invasive strains, TIGR4 (70) and D39 (38) (and the laboratory-adapted D39 derivative R6) (34), have been published, along with draft sequences for serotype 19F strain G54 (26) and eight clinical isolates from a hospital in Pittsburgh (65). However, in order to understand the bacterial population structure, and the reasons underlying the variation in pathogenicity, genomic studies of strains that only rarely invade past the mucosal surfaces are required.S. pneumoniae^Spain23F sequence type 81 (ST81) was one of the first pandemic penicillin-resistant clones identified (47). Initially characterized among isolates from Spain in the 1980s, it spread globally, and by the late 1990s it was estimated to constitute almost 40% of penicillin-resistant disease isolates in the United States (21). The clone is also resistant to chloramphenicol and tetracycline and is one of those most frequently associated with the emergence of fluoroquinolone and macrolide resistance (58, 60). This lack of susceptibility to the major classes of antimicrobial chemotherapies used to treat pneumococcal infections has undoubtedly aided the spread of the strain and lead to the inclusion of the 23F serotype in PCV7. This has resulted in a reduction in the prevalence of the S. pneumoniae^Spain23F ST81 clone in some regions (35). However, the lineage has undergone capsule switching to alternative capsule types on at least three occasions (from serotype 23F to 14 (36), 19A (20), and 19F (19), suggesting it is liable to eventually evade the vaccine. Despite its high carriage prevalence (22), it has a low propensity for causing invasive disease (67) (odds ratio of 0.4 [13]), suggesting its intercontinental distribution has been facilitated by adaptations to colonization of, and survival within, the human nasopharynx. Here we report our analysis of the complete genome of S. pneumoniae ATCC 700669, a member of the serotype 23F ST81 lineage that was isolated in a hospital in Barcelona in 1984 (18).     

Glycerol Metabolism Is Important for Cytotoxicity of Mycoplasma pneumoniae

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH₂. The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.Mycoplasma pneumoniae causes infections of the upper and lower respiratory tracts. These bacteria are responsible for a large fraction of community-acquired pneumonias. Although usually harmless for adult patients, M. pneumoniae may cause severe disease in children or elderly people. In addition, M. pneumoniae is involved in extrapulmonary complications such as pediatric encephalitis and erythema multiforme (for reviews, see references 15, 21, and 34).M. pneumoniae and its relatives, the Mollicutes, are all characterized by the lack of a cell wall and a very close adaptation to a life within a eukaryotic host. This close adaptation is reflected by degenerative genome evolution that resulted in an extreme genome reduction. As a result, the Mollicutes are the organisms that are capable of independent life with the smallest known genome. M. pneumoniae has a genome of 816 kb and encodes only 688 proteins (18). This genome reduction is taken even further in the close relative Mycoplasma genitalium, which has only 482 protein-coding genes (18). Thus, the analysis of the Mollicutes allows us to study a minimal form of natural life. This question has recently attracted much interest and resulted in the determination of the essential gene sets of M. pneumoniae, M. genitalium, and, more recently, Mycoplasma pulmonis (6, 20). In M. genitalium, with the most reduced genomes, only 100 out of the 482 protein-coding genes are dispensable, suggesting that the remaining 382 genes form the essential gene set (7).Reductive genome evolution in M. pneumoniae is still under way: the genes for the utilization of mannitol as a carbon source seem to be present in M. pneumoniae; however, this substrate cannot be used by the bacteria. M. genitalium, which is further advanced in genome reduction, has lost the genes for mannitol transport and oxidation. It was therefore suggested that the genes for mannitol utilization in M. pneumoniae either are not expressed or encode inactive proteins (12).In M. pneumoniae as well as in other Mollicutes, pathogenicity is closely linked to carbon metabolism (13). M. pneumoniae can use glucose, fructose, and glycerol as the only carbon sources (12). Studies with Mycoplasma mycoides revealed that glycerol metabolism has a major impact on the pathogenicity of these bacteria. Oxidation of glycerol involves the glycerol 3-phosphate oxidase, which produces hydrogen peroxide rather than NADH₂, which is generated by the glycerol 3-phosphate dehydrogenase in most other bacteria (28). In addition to the induction of autoimmune responses, the formation of hydrogen peroxide is the only established mechanism by which mycoplasmas cause damage to their hosts (31, 34). Pathogenic strains of M. mycoides possess a highly active ABC transport system for glycerol in addition to the ubiquitous glycerol facilitator (33). The efficient formation of hydrogen peroxide by the membrane-bound glycerol 3-phosphate oxidase is the major virulence factor of the highly pathogenic strains of M. mycoides (28).M. pneumoniae possesses the complete set of genes for glycerol utilization, and the bacteria do indeed use this carbon source (12). The first component in glycerol metabolism is the glycerol facilitator encoded by the glpF gene. The transported glycerol is then phosphorylated by the glycerol kinase (product of glpK), and glycerol 3-phosphate is subsequently oxidized to dihydroxyacetone phosphate, a glycolytic intermediate. The relevant enzyme is annotated as glycerol 3-phosphate dehydrogenase (encoded by the gene glpD) in M. pneumoniae (17).In all organisms studied so far, glycerol metabolism is under dual control: the genes involved in glycerol utilization are expressed only if glycerol or glycerol 3-phosphate is present in the medium, and they are not expressed in the presence of glucose, the preferred carbon source (3, 4). This second mode of regulation, carbon catabolite repression, involves two distinct mechanisms in the Firmicutes, from which the Mollicutes evolved. In the presence of preferred sugars, the CcpA repressor protein binds in the promoter regions of glycerol utilization genes and prevents their expression. Moreover, the molecular inducer of the system, glycerol 3-phosphate, is formed only in the absence of glucose. This results from the low activity of the glycerol kinase. This enzyme is activated upon phosphorylation by HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). HPr can phosphorylate other proteins only in the absence of glucose, thus providing a link between glucose availability, the activity of the glycerol kinase, and the induction of the glycerol utilization genes (3). Nothing is known about the regulation of glycerol utilization in any member of the Mollicutes; however, regulatory events seem to be rare in these organisms due to the lack of regulatory proteins, among them CcpA.In this work, we studied the mechanisms of glycerol utilization in M. pneumoniae, its regulation, and its contribution to cytotoxicity. We demonstrate constitutive expression of the genes for glycerol utilization in M. pneumoniae. As observed in M. mycoides, glycerol 3-phosphate oxidation involves the formation of hydrogen peroxide and is important for damaging the host cells.     

Coxsackievirus Infection Induces Autophagy-Like Vesicles and Megaphagosomes in Pancreatic Acinar Cells In Vivo

Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.Macroautophagy—henceforth referred to as autophagy—is an intracellular process that is important for cellular differentiation, homeostasis, and survival. Through autophagy, long-lived cytosolic proteins and organelles become encapsulated within double-membraned vesicles, called autophagosomes, which fuse with lysosomes to facilitate degradation of protein and cellular organelles and to promote nutrient recycling/regeneration. Autophagy plays a key role in the host immune response to infection by viruses, bacteria, fungi, and parasites (reviewed in references 10 and 62). Within virus-infected cells, whole virions and/or viral proteins and nucleic acids are captured inside autophagosomes and degraded (following lysosomal fusion) through the process of xenophagy. Moreover, autophagosome fusion with the endosomal/lysosomal pathway facilitates Toll-like receptor recognition of viral materials and delivers endogenous cytosolic viral proteins to the major histocompatibility complex (MHC) class II antigen presentation pathway, which in turn may help to trigger activation of innate immunity (and type I interferon production) and promote antigen presentation to virus-specific CD4⁺ T cells (reviewed in references 9, 41, 44, 47, 72, and 90). A recent study has shown that autophagy is also involved in the processing and presentation of MHC class I-restricted viral epitopes (13).Given the importance of autophagy in antiviral immunity, it is perhaps not surprising that viruses have evolved mechanisms to evade and/or subvert this pathway (reviewed in references 9, 11, 14, 35, 37, 60, 61, and 77). Several members of the herpesvirus family, most notably herpes simplex virus type 1, inhibit autophagy within an infected cell and encode proteins that block and/or target intracellular signaling pathways that regulate autophagy (reviewed in references 60 and 61). However, some viruses not only evade autophagy but also appear to take advantage of the process; several RNA viruses induce autophagy and exploit the pathway during their replication (1, 12, 15, 31, 40, 43, 76, 93, 96). Viruses belonging to the Picornaviridae family and the Nidovirales order replicate their genomes on double-membraned vesicles that resemble autophagosomes; these vesicles are notably smaller in size than cellular autophagosomes and are decorated with proteins derived from the autophagic pathway (19, 21, 31, 37, 67, 68, 71, 92). Viral proteins encoded by poliovirus and equine arterivirus can trigger the formation of these autophagy-like vesicles (79, 80), and the expression of a single poliovirus protein, 2BC, is sufficient to induce lipidation of the host autophagy protein light chain 3 (LC3), encoded by the Atg8 gene (87). Taken together, these studies suggest that some viruses subvert the autophagy pathway to generate double-membraned vesicles that provide a surface for RNA replication (8, 37, 88). In addition, these vesicles may permit newly formed virions to escape from infected cells via a nonlytic route (36, 85).Although studies have demonstrated that the autophagic pathway may play an important role in virus infection in vitro, either to promote or to restrict viral replication, we are just beginning to appreciate and understand the function and effects of autophagy for virus infections in vivo. Autophagy acts in an antiviral fashion to limit tobacco mosaic virus replication and programmed cell death in plants (46), to prevent a pathogenic infection with vesicular stomatitis virus in flies (73), and to protect against fatal encephalitis in Sindbis virus- or herpes simplex virus type 1-infected mice (45, 59, 63). Nonetheless, to date there is a dearth of in vivo studies; animal models of virus infection are needed in order to better define the antiviral role of autophagy in vivo (41, 62). In addition, studies that address the role of viral subversion of autophagy in vivo are warranted. Does this process occur within infected animals, and is it required for viral replication in particular cell types or for viral pathogenesis? Recent studies have shown that autophagy not only promotes the replication of hepatitis B virus and enterovirus 71 in vitro but also may be induced by infection in vivo, potentially to benefit the virus rather than the host (28, 78).Type B coxsackieviruses (CVBs) are members of the Picornaviridae family and Enterovirus genus and, as such, are closely related to polioviruses. CVBs are important human pathogens that often induce severe acute and chronic diseases and cause morbidity and mortality (69, 91). CVBs are the most common cause of infectious myocarditis (38, 82) and frequently trigger pancreatitis and aseptic meningitis (7, 16, 29, 51). Tissue culture studies (93) have shown that CVB type 3 (CVB3) promotes LC3 conversion and autophagosome accumulation in virus-infected cells in vitro and that modulation of the autophagic pathway (using chemicals or small interfering RNA-mediated knockdown) to enhance or dampen autophagy results in an increase and a decrease, respectively, in viral protein expression and/or viral titers; however, the reported changes in viral titers were modest (2- to 4-fold). In the present study, we examine whether CVB3 activates the autophagic pathway in vivo, specifically in pancreatic acinar cells, which are a natural primary target for this virus. Using a mouse model of CVB3 infection, which faithfully recapitulates most aspects of CVB disease in humans, we demonstrate that this virus triggers LC3 conversion and also modulates other components of the autophagy machinery. In addition, using a recombinant CVB3 (rCVB3) that expresses Discosoma sp. red fluorescent protein (DsRed-CVB3), we identify virus-infected cells in situ and show that CVB3 infection increases autophagosome abundance in vivo. Lysosomal-associated membrane protein 1 (LAMP-1) immunostaining confirmed that amphisomes are generated in virus-infected cells but that autophagic flux was not substantially enhanced as the infection progressed; rather, there appears to be a substantial blockade in fusion with lysosomes. Finally, transmission electron microscopy (TEM) ultrastructural analysis of the infected pancreas confirmed that double-membraned autophagy-like vesicles as well as very large autophagic compartments (for which we have coined the term “megaphagosomes”) were generated in acinar cells following virus infection. Overall, these data provide compelling evidence that CVB3 induces autophagy in vivo and suggest that this picornavirus may subvert this process in a mammalian host.