The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

Recent studies have demonstrated that the O‐antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N‐formylated sugars (3‐formamido‐3,6‐dideoxy‐d ‐glucose or 4‐formamido‐4,6‐dideoxy‐d ‐glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6‐dehydratase, a pyridoxal 5'‐phosphate or PLP‐dependent aminotransferase, and an N‐formyltransferase. To date, there have been no published reports of N‐formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N‐formyltransferase. Given that M. tuberculosis produces l ‐rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6‐dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N‐formylated sugar in M. tuberculosis, namely a PLP‐dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.     

Assessing the essentiality of the decaprenyl‐phospho‐d‐arabinofuranose pathway in Mycobacterium tuberculosis using conditional mutants

In Mycobacterium tuberculosis the decaprenyl‐phospho‐d ‐arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock‐down mutants of each gene involved using the TET‐PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non‐permissive conditions, whereas rv3807c was not required for survival. In the most vulnerable group, DprE1‐depleted cells died faster in vitro and intracellularly than those lacking UbiA and PrsA. Downregulation of DprE1 and UbiA resulted in similar phenotypes, namely swelling of the bacteria, cell wall damage and lysis as observed at the single cell level, by real time microscopy and electron microscopy. By contrast, depletion of PrsA led to cell elongation and implosion, which was suggestive of a more pleiotropic effect. Drug sensitivity assays with known DPA‐inhibitors supported the use of conditional knock‐down strains for target‐based whole‐cell screens. Together, our work provides strong evidence for the vulnerability of all but one of the enzymes in the DPA pathway and generates valuable tools for the identification of lead compounds targeting the different biosynthetic steps. PrsA, phosphoribosyl‐pyrophosphate synthetase, appears to be a particularly attractive new target for drug discovery.     

One‐plasmid tunable coexpression for mycobacterial protein–protein interaction studies

A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline‐dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two‐protein Mycobacterium tuberculosis stearoyl‐CoA Δ⁹ desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications.     

Dopaminergic and behavioural changes in a loss‐of‐imprinting model of Cdkn1c

The imprinted gene Cdkn1c is expressed exclusively from the maternally inherited allele as a consequences of epigenetic regulation. Cdkn1c exemplifies many of the functional characteristics of imprinted genes, playing a role in foetal growth and placental development. However, Cdkn1c also plays an important role in the brain, being key to the appropriate proliferation and differentiation of midbrain dopaminergic neurons. Using a transgenic model (Cdkn1c^BACx1) with a twofold elevation in Cdkn1c expression that mimics loss‐of‐imprinting, we show that increased expression of Cdkn1c in the brain gives rise to neurobiological and behavioural changes indicative of a functionally altered dopaminergic system. Cdkn1c^BACX1 mice displayed altered expression of dopamine system‐related genes, increased tyrosine hydroxylase (Th) staining and increased tissue content of dopamine in the striatum. In addition, Cdkn1c^BACx1 animals were hypersensitive to amphetamine as showed by c‐fos expression in the nucleus accumbens. Cdkn1c^BACX1 mice had significant changes in behaviours that are dependent on the mesolimbic dopaminergic system. Specifically, increased motivation for palatable food stuffs, as indexed on a progressive ratio task. In addition, Cdkn1c^BACX1 mice displayed enhanced social dominance. These data show, for the first time, the consequence of elevated Cdkn1c expression on dopamine‐related behaviours highlighting the importance of correct dosage of this imprinted gene in the brain. This work has significant relevance for deepening our understanding of the epigenetic factors that can shape neurobiology and behaviour.     

The phage shock protein PspA facilitates divalent metal transport and is required for virulence of Salmonella enterica sv. Typhimurium

The phage shock protein (Psp) system is induced by extracytoplasmic stress and thought to be important for the maintenance of proton motive force. We investigated the contribution of PspA to Salmonella virulence. A pspA deletion mutation significantly attenuates the virulence of Salmonella enterica serovar Typhimurium following intraperitoneal inoculation of C3H/HeN (Ity^r) mice. PspA was found to be specifically required for virulence in mice expressing the natural resistance‐associated macrophage protein 1 (Nramp1) (Slc11a1) divalent metal transporter, which restricts microbial growth by limiting the availability of essential divalent metals within the phagosome. Salmonella competes with Nramp1 by expressing multiple metal uptake systems including the Nramp‐homologue MntH, the ABC transporter SitABCD and the ZIP family transporter ZupT. PspA was found to facilitate Mn²⁺ transport by MntH and SitABCD, as well as Zn²⁺ and Mn²⁺ transport by ZupT. In vitro uptake of ⁵⁴Mn²⁺ by MntH and ZupT was reduced in the absence of PspA. Transport‐deficient mutants exhibit reduced viability in the absence of PspA when grown under metal‐limited conditions. Moreover, the ZupT transporter is required for Salmonella enterica serovar Typhimurium virulence in Nramp1‐expressing mice. We propose that PspA promotes Salmonella virulence by maintaining proton motive force, which is required for the function of multiple transporters mediating bacterial divalent metal acquisition during infection.     

Tamoxifen‐inducible Cre‐mediated recombination in adipocytes

To generate a mouse line which allows inducible, Cre/loxP‐dependent recombination in adipocytes, we used RedE/RedT‐mediated recombineering to insert the CreER^T2‐transgene, which encodes a fusion protein of Cre and a mutated tamoxifen‐responsive estrogen receptor, into the start codon of the adipocyte‐specific Adipoq gene. Adipoq encodes adiponectin, an adipokine specifically expressed in differentiated adipocytes. Tamoxifen treatment induced almost complete recombination in white adipose tissue of the AdipoqCreER^T2 mouse line (97%–99%), while no recombination was seen in vehicle‐treated animals. Recombination in brown adipose tissue was about 15%, whereas other organs and tissues did not undergo recombination. In addition, mice expressing CreER^T2 in adipocytes did not show any alterations of metabolic functions like glucose tolerance, lipolysis, or energy expenditure compared to control mice. Therefore the AdipoqCreER^T2 mouse line will be a valuable tool for studying the consequences of a temporally controlled deletion of floxed genes in white adipose tissue. genesis 48:618–625, 2010. © 2010 Wiley‐Liss, Inc.     

Two‐dimensional proteome reference map for the radiation‐resistant bacterium Deinococcus geothermalis

2‐DE reference maps for Deinococcus geothermalis cytosolic and cell envelope proteomes were constructed. In total, 403 spots were identified as 299 different proteins. Unique in the proteomes were four subunits of V‐type ATPase and Deinococcus specific proteins constituting one‐fourth of cell envelope proteome. The cytoplasmic proteome included enzymes of the central carbon metabolism, chaperones, enzymes of protein and DNA repair, and oxidative stress. A total of 34 abundant proteins with unknown function may relate to the extreme stress tolerance of D. geothermalis.     

A novel F420‐dependent anti‐oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents

Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F₄₂₀‐dependent anti‐oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F₄₂₀‐deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD⁺ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F₄₂₀^? mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin‐dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F₄₂₀H₂‐dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F₄₂₀H₂‐specific obligate two‐electron reduction of endogenous quinones, thereby competing with the one‐electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F₄₂₀ biosynthesis pathway or Fqr‐class proteins as a mechanism to potentiate the action of bactericidal agents.     

The MprB Extracytoplasmic Domain Negatively Regulates Activation of the Mycobacterium tuberculosis MprAB Two-Component System

Mycobacterium tuberculosis is an acid-fast pathogen of humans and the etiological agent of tuberculosis (TB). It is estimated that one-third of the world''s population is latently (persistently) infected with M. tuberculosis. M. tuberculosis persistence is regulated, in part, by the MprAB two-component signal transduction system, which is activated by and mediates resistance to cell envelope stress. Here we identify MprAB as part of an evolutionarily conserved cell envelope stress response network and demonstrate that MprAB-mediated signal transduction is negatively regulated by the MprB extracytoplasmic domain (ECD). In particular, we report that deregulated production of the MprB sensor kinase, or of derivatives of this protein, negatively impacts M. tuberculosis growth. The observed growth attenuation is dependent on MprAB-mediated signal transduction and is exacerbated in strains of M. tuberculosis producing an MprB variant lacking its ECD. Interestingly, full-length MprB, and the ECD of MprB specifically, immunoprecipitates the Hsp70 chaperone DnaK in vivo, while overexpression of dnaK inhibits MprAB-mediated signal transduction in M. tuberculosis grown in the absence or presence of cell envelope stress. We propose that under nonstress conditions, or under conditions in which proteins present in the extracytoplasmic space are properly folded, signaling through the MprAB system is inhibited by the MprB ECD. Following exposure to cell envelope stress, proteins present in the extracytoplasmic space become unfolded or misfolded, leading to removal of the ECD-mediated negative regulation of MprB and subsequent activation of MprAB.     

Hfq reduces envelope stress by controlling expression of envelope‐localized proteins and protein complexes in enteropathogenic Escherichia coli

Gram‐negative bacteria possess several envelope stress responses that detect and respond to damage to this critical cellular compartment. The σ^E envelope stress response senses the misfolding of outer membrane proteins (OMPs), while the Cpx two‐component system is believed to detect the misfolding of periplasmic and inner membrane proteins. Recent studies in several Gram‐negative organisms found that deletion of hfq, encoding a small RNA chaperone protein, activates the σ^E envelope stress response. In this study, we assessed the effects of deleting hfq upon activity of the σ^E and Cpx responses in non‐pathogenic and enteropathogenic (EPEC) strains of Escherichia coli. We found that the σ^E response was activated in Δhfq mutants of all E. coli strains tested, resulting from the misregulation of OMPs. The Cpx response was activated by loss of hfq in EPEC, but not in E. coli K‐12. Cpx pathway activation resulted in part from overexpression of the bundle‐forming pilus (BFP) in EPEC Δhfq. We found that Hfq repressed expression of the BFP via PerA, a master regulator of virulence in EPEC. This study shows that Hfq has a more extensive role in regulating the expression of envelope proteins and horizontally acquired virulence genes in E. coli than previously recognized.     

The three‐component system EsrISR regulates a cell envelope stress response in Corynebacterium glutamicum

When the cell envelope integrity is compromised, bacteria trigger signaling cascades resulting in the production of proteins that counteract these extracytoplasmic stresses. Here, we show that the two‐component system EsrSR regulates a cell envelope stress response in the Actinobacterium Corynebacterium glutamicum. The sensor kinase EsrS possesses an amino‐terminal phage shock protein C (PspC) domain, a property that sets EsrSR apart from all other two‐component systems characterized so far. An integral membrane protein, EsrI, whose gene is divergently transcribed to the esrSR gene locus and which interestingly also possesses a PspC domain, acts as an inhibitor of EsrSR under non‐stress conditions. The resulting EsrISR three‐component system is activated among others by antibiotics inhibiting the lipid II cycle, such as bacitracin and vancomycin, and it orchestrates a broad regulon including the esrI‐esrSR gene locus itself, genes encoding heat shock proteins, ABC transporters, and several putative membrane‐associated or secreted proteins of unknown function. Among those, the ABC transporter encoded by cg3322‐3320 was shown to be directly involved in bacitracin resistance of C. glutamicum. Since similar esrI‐esrSR loci are present in a large number of actinobacterial genomes, EsrISR represents a novel type of stress‐responsive system whose components are highly conserved in the phylum Actinobacteria.     

The σE stress response is required for stress‐induced mutation and amplification in Escherichia coli

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress‐induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double‐strand‐break (DSB) repair and requires DinB error‐prone DNA polymerase and the SOS DNA‐damage‐ and RpoS general‐stress responses. We report that the RpoE envelope‐protein‐stress response is also required. In a screen for mutagenesis‐defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σ^E acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ³², which was postulated to affect mutagenesis. I‐SceI‐induced DSBs alleviated much of the rpoE phenotype, implying that σ^E promoted DSB formation. Thus, a third stress response and stress input regulate DSB‐repair‐associated stress‐induced mutagenesis. This provides the first report of mutagenesis promoted by σ^E, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.     

A Pdgf‐cCreERT2 knock‐in mouse model for tracing PDGF‐C cell lineages during development

PDGF‐C, a member of the platelet‐derived growth factor (PDGF) family, plays important roles in the development of craniofacial structures, the neural system, the vascular system, and tumors. PDGF‐C could also be required for the regulation of certain types of stem or progenitor cells as suggested by its expression in the regions where these cells are located. To further characterize the role of PDGF‐C in development, we generated a Pdgf‐c^CreERT2 mouse strain, in which a tamoxifen‐inducible Cre (CreERT2) cDNA was specifically targeted into the Pdgf‐c genomic locus and controlled by the endogenous Pdgf‐c regulatory elements. We also showed that Cre activity in this mouse strain could be specifically induced by tamoxifen, which allowed the fate of PDGF‐C‐expressing cells to be traced at various stages of development. Using this model system, we demonstrated for the first time that PDGF‐C‐expressing cells could be multipotent, generating multiple cell lineages required for the formation of the cerebellum. Therefore, the Pdgf‐c^CreERT2 mouse strain generated in this study will be a valuable transgenic tool for exploring the function of PDGF‐C in development and stem cell biology.     

The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD‐box RNA helicases are nucleolar‐ and chromocenter‐localized proteins that undergo stress‐mediated relocalization and are involved in epigenetic gene silencing

DEAD‐box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD‐box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up‐regulated stress‐responsive gene expression. Here, we show that Arabidopsis STRS‐overexpressing lines displayed a less tolerant phenotype and reduced expression of stress‐induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP–STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis‐localization in specific gene‐silencing mutants and exhibited RNA‐dependent ATPase and RNA‐unwinding activities. In particular, STRS2 showed mis‐localization in three out of four mutants of the RNA‐directed DNA methylation (RdDM) pathway while STRS1 was mis‐localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response.