The PII protein interacts with the Amt ammonium transport and modulates nitrate/nitrite assimilation in mycobacteria.

PII proteins are signal transduction proteins that belong to a widely distributed family of proteins involved in the modulation of different metabolisms in bacteria. These proteins are homotrimers carrying a flexible loop, named T-loop, which changes its conformation due to the recognition of diverse key metabolites, ADP, ATP, and 2-oxoglutarate. PII proteins interact with different partners to primarily regulate a set of nitrogen pathways. In some organisms, PII proteins can also control carbon metabolism by interacting with the biotin carboxyl carrier protein (BCCP), a key component of the acetyl-CoA carboxylase (ACC) enzyme complex, inhibiting its activity with the consequent reduction of fatty acid biosynthesis. Most bacteria contain at least two PII proteins, named GlnB and GlnK, with different regulatory roles. In mycobacteria, only one PII protein was identified, and the three-dimensional structure was solved, however, its physiological role is unknown. In this study we purified the Mycobacterium tuberculosis (M. tb) PII protein, named GlnB, and showed that it weakly interacts with the AccA3 protein, the α subunit shared by the three different, and essential, Acyl-CoA carboxylase complexes (ACCase 4, 5, and 6) present in M. tb. A M. smegmatis deletion mutant, ∆MsPII, exhibited a growth deficiency on nitrate and nitrite as unique nitrogen sources, and accumulated nitrite in the culture supernatant. In addition, M. tb PII protein was able to interact with the C-terminal domain of the ammonium transporter Amt establishing the ancestral role for this PII protein as a GlnK functioning protein.

Complete genome sequence of the Microbacterium foliorum bacteriophage Garey24.

In this work, we report the discovery and characterization of Garey24, a bacteriophage that forms medium-size plaques with halo rings isolated from a soil sample in Funes, Argentina. Its 41,522 bp circularly permuted genome contains 63 putative protein-coding genes. Based on gene content similarity, Garey24 was assigned to subcluster EA1.

Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum.

Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole or how it is organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. In this study we used alanine scanning mutagenesis to identify Wag31 residues involved in cell morphogenesis. Our data show that Wag31 helps to control proper septation as well as new and old pole elongation. We have identified key amino acid residues involved in these essential functions. Enzyme assays revealed that Wag31 interacts with lipid metabolism by modulating acyl-CoA carboxylase (ACCase) activity. We show that Wag31 does not control polar growth by regulating the localization of cell wall precursor enzymes to the Intracellular Membrane Domain, and we also demonstrate that phosphorylation of Wag31 does not substantively regulate peptidoglycan metabolism. This work establishes new regulatory functions of Wag31 in the mycobacterial cell cycle and clarifies the need for new molecular models of Wag31 function.

Blue light directly modulates the quorum network in the human pathogen Acinetobacter baumannii.

Quorum sensing modulates bacterial collective behaviors including biofilm formation, motility and virulence in the important human pathogen Acinetobacter baumannii. Disruption of quorum sensing has emerged as a promising strategy with important therapeutic potential. In this work, we show that light modulates the production of acyl-homoserine lactones (AHLs), which were produced in higher levels in the dark than under blue light at environmental temperatures, a response that depends on the AHL synthase, AbaI, and on the photoreceptor BlsA. BlsA interacts with the transcriptional regulator AbaR in the dark at environmental temperatures, inducing abaI expression. Under blue light, BlsA does not interact with AbaR, but induces expression of the lactonase aidA and quorum quenching, consistently with lack of motility at this condition. At temperatures found in warm-blooded hosts, the production of AHLs, quorum quenching as well as abaI and aidA expression were also modulated by light, though in this case higher levels of AHLs were detected under blue light than in the dark, in a BlsA-independent manner. Finally, AbaI reduces A. baumannii's ability to kill C. albicans only in the dark both at environmental as well as at temperatures found in warm-blooded hosts. The overall data indicate that light directly modulates quorum network in A. baumannii.

trans-3-Methylglutaconyl CoA isomerization-dependent protein acylation.

3-methylglutaconic (3MGC) aciduria is associated with a growing number of discrete inborn errors of metabolism. Herein, an antibody-based approach to detection/quantitation of 3MGC acid has been pursued. When trans-3MGC acid conjugated keyhole limpet hemocyanin (KLH) was inoculated into rabbits a strong immune response was elicited. Western blot analysis provided evidence that immune serum, but not pre-immune serum, recognized 3MGC-conjugated bovine serum albumin (BSA). In competition ELISAs using isolated immune IgG, the limit of detection for free trans-3MGC acid was compared to that for cis-3MGC acid and four structurally related short-chain dicarboxylic acids. Surprisingly, cis-3MGC acid yielded a much lower limit of detection (∼0.1 mg/ml) than trans-3MGC acid (∼1.0 mg/ml) while all other dicarboxylic acids tested were poor competitors. The data suggest trans-3MGC- isomerized during, or after, conjugation to KLH such that the immunogen was actually comprised of KLH harboring a mixture of cis- and trans-3MGC haptens. To investigate this unexpected isomerization reaction, trans-3MGC CoA was prepared and incubated at 37 °C in the presence of BSA. Evidence was obtained that non-enzymatic isomerization of trans-3MGC CoA to cis-3MGC CoA precedes intramolecular catalysis to form cis-3MGC anhydride plus CoASH. Anhydride-dependent acylation of BSA generated 3MGCylated BSA, as detected by anti-3MGC immunoblot. The results presented provide an explanation for the unanticipated detection of 3MGCylated proteins in a murine model of primary 3MGC aciduria. Furthermore, non-enzymatic hydrolysis of cis-3MGC anhydride represents a potential source of cis-3MGC acid found in urine of subjects with 3MGC aciduria.

Correction: Pleiotropic Effect of AccD5 and AccE5 Depletion in Acyl-Coenzyme A Carboxylase Activity and in Lipid Biosynthesis in Mycobacteria.

[This corrects the article DOI: 10.1371/journal.pone.0099853.].

Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel and a hydrophobic transmission spine.

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.

KDM2B regulates choline kinase expression and neuronal differentiation of neuroblastoma cells.

The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells.

3-methylcrotonyl Coenzyme A (CoA) carboxylase complex is involved in the Xanthomonas citri subsp. citri lifestyle during citrus infection.

Citrus canker is a disease caused by the phytopathogen Xanthomonas citri subsp. citri (Xcc), bacterium which is unable to survive out of the host for extended periods of time. Once established inside the plant, the pathogen must compete for resources and evade the defenses of the host cell. However, a number of aspects of Xcc metabolic and nutritional state, during the epiphytic stage and at different phases of infection, are poorly characterized. The 3-methylcrotonyl-CoA carboxylase complex (MCC) is an essential enzyme for the catabolism of the branched-chain amino acid leucine, which prevents the accumulation of toxic intermediaries, facilitates the generation of branched chain fatty acids and/or provides energy to the cell. The MCC complexes belong to a group of acyl-CoA carboxylases (ACCase) enzymes dependent of biotin. In this work, we have identified two ORFs (XAC0263 and XAC0264) encoding for the α and ß subunits of an acyl-CoA carboxylase complex from Xanthomonas and demonstrated that this enzyme has MCC activity both in vitro and in vivo. We also found that this MCC complex is conserved in a group of pathogenic gram negative bacteria. The generation and analysis of an Xcc mutant strain deficient in MCC showed less canker lesions in the interaction with the host plant, suggesting that the expression of these proteins is necessary for Xcc fitness during infection.

Lipid metabolism and its implication in mycobacteria-host interaction.

The complex lipids present in the cell wall of Mycobacterium tuberculosis (Mtb) act as major effector molecules that actively interact with the host, modulating its metabolism and stimulating the immune response, which in turn affects the physiology of both, the host cell and the bacilli. Lipids from the host are also nutrient sources for the pathogen and define the fate of the infection by modulating lipid homeostasis. Although new technologies and experimental models of infection have greatly helped understanding the different aspects of the host-pathogen interactions at the lipid level, the impact of this interaction in the Mtb lipid regulation is still incipient, mainly because of the low background knowledge in this area of research.

Expression and purification of untagged GlnK proteins from actinobacteria.

The PII protein family constitutes one of the most conserved and well distributed family of signal transduction proteins in nature. These proteins play key roles in nitrogen and carbon metabolism. PII function has been well documented in Gram-negative bacteria. However, there are very few reports describing the in vitro properties and function of PII derived from Gram-positive bacteria. Here we present the heterologous expression and efficient purification protocols for untagged PII from three Actinobacteria of medical and biotechnological interest namely: Mycobacterium tuberculosis, Rhodococcus jostii and Streptomyces coelicolor. Circular dichroism and gel filtration analysis supported that the purified proteins are correctly folded. The purification protocol described here will facilitate biochemical studies and help to uncover the biochemical functions of PII proteins in Actinobacteria.

Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two ß subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen.

The infectious intracellular lifestyle of Salmonella enterica relies on the adaptation to nutritional conditions within the Salmonella-containing vacuole.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that causes various host-specific diseases. During their life cycle, Salmonellae survive frequent exposures to a variety of environmental stresses, e.g. carbon-source starvation. The virulence of this pathogen relies on its ability to establish a replicative niche, named Salmonella-containing vacuole, inside host cells. However, the microenvironment of the SCV and the bacterial metabolic pathways required during infection are largely undefined. In this work we developed different biological probes whose expression is modulated by the environment and the physiological state of the bacterium. We constructed transcriptional reporters by fusing promoter regions to the gfpmut3a gene to monitor the expression profile of genes involved in glucose utilization and lipid catabolism. The induction of these probes by a specific metabolic change was first tested in vitro, and then during different conditions of infection in macrophages. We were able to determine that Entner-Doudoroff is the main metabolic pathway utilized by Salmonella during infection in mouse macrophages. Furthermore, we found sub-populations of bacteria expressing genes involved in pathways for the utilization of different sources of carbon. These populations are modified in presence of different metabolizable substrates, suggesting the coexistence of Salmonella with diverse metabolic states during the infection.

Pleiotropic effect of AccD5 and AccE5 depletion in acyl-coenzyme A carboxylase activity and in lipid biosynthesis in mycobacteria.

Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs.

Fatty acid biosynthesis in actinomycetes.

All organisms that produce fatty acids do so via a repeated cycle of reactions. In mammals and other animals, these reactions are catalyzed by a type I fatty acid synthase (FAS), a large multifunctional protein to which the growing chain is covalently attached. In contrast, most bacteria (and plants) contain a type II system in which each reaction is catalyzed by a discrete protein. The pathway of fatty acid biosynthesis in Escherichia coli is well established and has provided a foundation for elucidating the type II FAS pathways in other bacteria (White et al., 2005). However, fatty acid biosynthesis is more diverse in the phylum Actinobacteria: Mycobacterium, possess both FAS systems while Streptomyces species have only the multienzyme FAS II system and Corynebacterium species exclusively FAS I. In this review, we present an overview of the genome organization, biochemical properties and physiological relevance of the two FAS systems in the three genera of actinomycetes mentioned above. We also address in detail the biochemical and structural properties of the acyl-CoA carboxylases (ACCases) that catalyzes the first committed step of fatty acid synthesis in actinomycetes, and discuss the molecular bases of their substrate specificity and the structure-based identification of new ACCase inhibitors with antimycobacterial properties.

Crystal structures and mutational analyses of acyl-CoA carboxylase beta subunit of Streptomyces coelicolor.

The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities toward these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.

Bacterial manipulation of innate immunity to promote infection.

The mammalian innate immune response provides a barrier against invading pathogens. Innate immune mechanisms are used by the host to respond to a range of bacterial pathogens in an acute and conserved fashion. Host cells express pattern recognition receptors that sense pathogen-associated molecular patterns. After detection, an arsenal of antimicrobial mechanisms is deployed to kill bacteria in infected cells. Innate immunity also stimulates antigen-specific responses mediated by the adaptive immune system. In response, pathogens manipulate host defence mechanisms to survive and eventually replicate. This Review focuses on the control of host innate immune responses by pathogenic intracellular bacteria.

Interaction between the SifA virulence factor and its host target SKIP is essential for Salmonella pathogenesis.

SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a DeltasifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.

Biochemical and structural characterization of an essential acyl coenzyme A carboxylase from Mycobacterium tuberculosis.

Pathogenic mycobacteria contain a variety of unique fatty acids that have methyl branches at an even-numbered position at the carboxyl end and a long n-aliphatic chain. One such group of acids, called mycocerosic acids, is found uniquely in the cell wall of pathogenic mycobacteria, and their biosynthesis is essential for growth and pathogenesis. Therefore, the biosynthetic pathway of the unique precursor of such lipids, methylmalonyl coenzyme A (CoA), represents an attractive target for developing new antituberculous drugs. Heterologous protein expression and purification of the individual subunits allowed the successful reconstitution of an essential acyl-CoA carboxylase from Mycobacterium tuberculosis, whose main role appears to be the synthesis of methylmalonyl-CoA. The enzyme complex was reconstituted from the alpha biotinylated subunit AccA3, the carboxyltransferase beta subunit AccD5, and the epsilon subunit AccE5 (Rv3281). The kinetic properties of this enzyme showed a clear substrate preference for propionyl-CoA compared with acetyl-CoA (specificity constant fivefold higher), indicating that the main physiological role of this enzyme complex is to generate methylmalonyl-CoA for the biosynthesis of branched-chain fatty acids. The alpha and beta subunits are capable of forming a stable alpha6-beta6 subcomplex but with very low specific activity. The addition of the epsilon subunit, which binds tightly to the alpha-beta subcomplex, is essential for gaining maximal enzyme activity.

Crystal structure of the beta-subunit of acyl-CoA carboxylase: structure-based engineering of substrate specificity.

Acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) catalyze the carboxylation of acetyl- and propionyl-CoA to generate malonyl- and methylmalonyl-CoA, respectively. Understanding the substrate specificity of ACC and PCC will (1) help in the development of novel structure-based inhibitors that are potential therapeutics against obesity, cancer, and infectious disease and (2) facilitate bioengineering to provide novel extender units for polyketide biosynthesis. ACC and PCC in Streptomyces coelicolor are multisubunit complexes. The core catalytic beta-subunits, PccB and AccB, are 360 kDa homohexamers, catalyzing the transcarboxylation between biotin and acyl-CoAs. Apo and substrate-bound crystal structures of PccB hexamers were determined to 2.0-2.8 A. The hexamer assembly forms a ring-shaped complex. The hydrophobic, highly conserved biotin-binding pocket was identified for the first time. Biotin and propionyl-CoA bind perpendicular to each other in the active site, where two oxyanion holes were identified. N1 of biotin is proposed to be the active site base. Structure-based mutagenesis at a single residue of PccB and AccB allowed interconversion of the substrate specificity of ACC and PCC. The di-domain, dimeric interaction is crucial for enzyme catalysis, stability, and substrate specificity; these features are also highly conserved among biotin-dependent carboxyltransferases. Our findings enable bioengineering of the acyl-CoA carboxylase (ACCase) substrate specificity to provide novel extender units for the combinatorial biosynthesis of polyketides.