Rifamycin derivatives active against pathogenic rapidly-growing mycobacteria.

Infections due to rapidly growing mycobacteria (RGM), and in particular the RGM species Mycobacterium abscessus (Mab), are very difficult to treat and reports on novel therapeutic options are scarce. A hallmark of all pathogenic RGM species is their resistance to the four first-line drugs used to treat infections with Mycobacterium tuberculosis including rifampicin. This study demonstrates that modification of the rifampicin scaffold can restore rifampicin activity against the three most commonly isolated pathogenic RGM species including Mab. We also note that the structure-activity relationship for Mab is different as compared to the non-pathogenic RGM species Mycobacterium smegmatis.

Rewiring bacterial two-component systems by modular DNA-binding domain swapping.

Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways and valuable sensors for synthetic biology. However, most TCSs remain uncharacterized or difficult to harness for applications. Major challenges are that many TCS output promoters are unknown, subject to cross-regulation, or silent in heterologous hosts. Here, we demonstrate that the two largest families of response regulator DNA-binding domains can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. We exploit this plasticity to eliminate cross-regulation, un-silence a gram-negative TCS in a gram-positive host, and engineer a system with over 1,300-fold activation. Finally, we apply DNA-binding domain swapping to screen uncharacterized Shewanella oneidensis TCSs in Escherichia coli, leading to the discovery of a previously uncharacterized pH sensor. This work should accelerate fundamental TCS studies and enable the engineering of a large family of genetically encoded sensors with diverse applications.

A very luminous magnetar-powered supernova associated with an ultra-long γ-ray burst.

A new class of ultra-long-duration (more than 10,000 seconds) γ-ray bursts has recently been suggested. They may originate in the explosion of stars with much larger radii than those producing normal long-duration γ-ray bursts or in the tidal disruption of a star. No clear supernova has yet been associated with an ultra-long-duration γ-ray burst. Here we report that a supernova (SN 2011kl) was associated with the ultra-long-duration γ-ray burst GRB 111209A, at a redshift z of 0.677. This supernova is more than three times more luminous than type Ic supernovae associated with long-duration γ-ray bursts, and its spectrum is distinctly different. The slope of the continuum resembles those of super-luminous supernovae, but extends further down into the rest-frame ultraviolet implying a low metal content. The light curve evolves much more rapidly than those of super-luminous supernovae. This combination of high luminosity and low metal-line opacity cannot be reconciled with typical type Ic supernovae, but can be reproduced by a model where extra energy is injected by a strongly magnetized neutron star (a magnetar), which has also been proposed as the explanation for super-luminous supernovae.

Refactoring and optimization of light-switchable Escherichia coli two-component systems.

Light-switchable proteins enable unparalleled control of molecular biological processes in live organisms. Previously, we have engineered red/far-red and green/red photoreversible two-component signal transduction systems (TCSs) with transcriptional outputs in E. coli and used them to characterize and control synthetic gene circuits with exceptional quantitative, temporal, and spatial precision. However, the broad utility of these light sensors is limited by bulky DNA encoding, incompatibility with commonly used ligand-responsive transcription factors, leaky output in deactivating light, and less than 10-fold dynamic range. Here, we compress the four genes required for each TCS onto two streamlined plasmids and replace all chemically inducible and evolved promoters with constitutive, engineered versions. Additionally, we systematically optimize the expression of each sensor histidine kinase and response regulator, and redesign both pathway output promoters, resulting in low leakiness and 72- and 117-fold dynamic range, respectively. These second-generation light sensors can be used to program the expression of more genes over a wider range and can be more easily combined with additional plasmids or moved to different host strains. This work demonstrates that bacterial TCSs can be optimized to function as high-performance sensors for scientific and engineering applications.

Mycoplasma pneumoniae thymidine phosphorylase.

Mycoplasma pneumoniae (Mpn) is a human pathogen causing acute respiratory diseases and accounts for approximately 30% cases of community-acquired pneumonia. Co-infection with Mycoplasmas compromises the efficacy of anticancer and antiviral nucleoside analog-based drugs due to the presence of Mycoplasma thymidine phosphorylase (TP). In this study, a TP-deficient strain of Mpn was generated in order to study the effect of Mpn TP in the metabolism of nucleoside analogs. Deficiency in TP activity led to increased uptake and incorporation of radiolabeled deoxyuridine and uracil but thymidine uptake was not affected. The activities of enzymes in the salvage of thymidine and deoxyuridine, e.g., thymidine kinase and uracil phosphoribosyltransferase were upregulated in the TP-deficient mutant, which may explain the increased uptake of deoxyuridine and uracil. Thirty FDA-approved anticancer and antiviral nucleoside and nucleobase analogs were used to screen their inhibitory activity toward the TP mutant and the wild type strain. Seven analogs were found to inhibit strongly the growth of both wild type and TP mutant. Differences in the inhibitory effect of several purine analogs between the two strains were observed. Further study is needed in order to understand the mechanism of inhibition caused by these analogs. Our results indicated that TP is not an essential gene for Mpn survival and TP deficiency affects other enzymes in Mpn nucleotide metabolism, and suggested that Mycoplasma nucleotide biosynthesis pathway enzymes are potential targets for future development of antibiotics.

Implication of glycerol and phospholipid transporters in Mycoplasma pneumoniae growth and virulence.

Mycoplasma pneumoniae, the causative agent of atypical pneumonia, is one of the bacteria with the smallest genomes that are nonetheless capable of independent life. Because of their longstanding close association with their human host, the bacteria have undergone reductive evolution and lost most biosynthetic abilities. Therefore, they depend on nutrients provided by the host that have to be taken up by the cell. Indeed, M. pneumoniae has a large set of hitherto unexplored transporters and lipoproteins that may be implicated in transport processes. Together, these proteins account for about 17% of the protein complement of M. pneumoniae. In the natural habitat of M. pneumoniae, human lung epithelial surfaces, phospholipids are the major available carbon source. Thus, the uptake and utilization of glycerol and glycerophosphodiesters that are generated by the activity of lipases are important for the nutrition of M. pneumoniae in its common habitat. In this study, we have investigated the roles of several potential transport proteins and lipoproteins in the utilization of glycerol and glycerophosphodiesters. On the basis of experiments with the corresponding mutant strains, our results demonstrate that the newly identified GlpU transport protein (MPN421) is responsible for the uptake of the glycerophosphodiester glycerophosphocholine, which is then intracellularly cleaved to glycerol-3-phosphate and choline. In addition, the proteins MPN076 and MPN077 are accessory factors in glycerophosphocholine uptake. Moreover, the lipoproteins MPN133 and MPN284 are essential for the uptake of glycerol. Our data suggest that they may act as binding proteins for glycerol and deliver glycerol molecules to the glycerol facilitator GlpF.

A trigger enzyme in Mycoplasma pneumoniae: impact of the glycerophosphodiesterase GlpQ on virulence and gene expression.

Mycoplasma pneumoniae is a causative agent of atypical pneumonia. The formation of hydrogen peroxide, a product of glycerol metabolism, is essential for host cell cytotoxicity. Phosphatidylcholine is the major carbon source available on lung epithelia, and its utilization requires the cleavage of deacylated phospholipids to glycerol-3-phosphate and choline. M. pneumoniae possesses two potential glycerophosphodiesterases, MPN420 (GlpQ) and MPN566. In this work, the function of these proteins was analyzed by biochemical, genetic, and physiological studies. The results indicate that only GlpQ is an active glycerophosphodiesterase. MPN566 has no enzymatic activity as glycerophosphodiesterase and the inactivation of the gene did not result in any detectable phenotype. Inactivation of the glpQ gene resulted in reduced growth in medium with glucose as the carbon source, in loss of hydrogen peroxide production when phosphatidylcholine was present, and in a complete loss of cytotoxicity towards HeLa cells. All these phenotypes were reverted upon complementation of the mutant. Moreover, the glpQ mutant strain exhibited a reduced gliding velocity. A comparison of the proteomes of the wild type strain and the glpQ mutant revealed that this enzyme is also implicated in the control of gene expression. Several proteins were present in higher or lower amounts in the mutant. This apparent regulation by GlpQ is exerted at the level of transcription as determined by mRNA slot blot analyses. All genes subject to GlpQ-dependent control have a conserved potential cis-acting element upstream of the coding region. This element overlaps the promoter in the case of the genes that are repressed in a GlpQ-dependent manner and it is located upstream of the promoter for GlpQ-activated genes. We may suggest that GlpQ acts as a trigger enzyme that measures the availability of its product glycerol-3-phosphate and uses this information to differentially control gene expression.

Interactions between glycolytic enzymes of Mycoplasma pneumoniae.

With only 688 protein-coding genes, Mycoplasma pneumoniae is one of the smallest self-replicating organisms. These bacteria use glycolysis as the major pathway for ATP production by substrate-level phosphorylation, suggesting that this pathway must be optimized to high efficiency. In this study, we have investigated the interactions between glycolytic enzymes using the bacterial adenylate cyclase-based two-hybrid system. We demonstrate that most of the glycolytic enzymes perform self-interactions, suggesting that they form dimers or other oligomeric forms. In addition, enolase was identified as the central glycolytic enzyme of M. pneumoniae due to its ability to directly interact with all other glycolytic enzymes. Our results support the idea of the formation of a glycolytic complex in M. pneumoniae and we suggest that the formation of this complex might ensure higher fluxes through the glycolytic pathway than would be possible with isolated non-interacting enzymes.

Upregulation of thymidine kinase activity compensates for loss of thymidylate synthase activity in Mycoplasma pneumoniae.

Thymidylate, an essential building block of DNA, is synthesized either from deoxyuridylate by thymidylate synthase (TS) or thymidine (dT) by thymidine kinase (TK). Thymidylate kinase (TMPK) phosphorylates dTMP to dTTP. Thymidine phosphorylase (TP) catalyses reversible phosphorolysis of dT. Using transposon mutagenesis M. pneumoniae TS gene (thyA/MPN320) was interrupted and requirement of these enzymes was studied. We found that TK activity and transcript levels and TP activity, but not TMPK or TS activity, are growth-phase-regulated, with induction at the exponential growth phase and a decline after the stationary phase. Inactivation of thyA results in upregulation of TK transcript and a 10-fold increase in TK activity, reduced TMPK level and it had no effect on TP activity. The level of [3H]-dT uptake and incorporation into DNA in the thyA mutant correlates with increases in TK activity, suggesting that dT uptake and metabolism is TK-dependent and that upregulation of TK activity in the thyA mutant compensates for the lack of ThyA activity. [3H]-dU uptake was low compared with dT, and incorporation of radioactivity into DNA in the thyA mutant indicates the presence of an alternative TS. Our results suggest that TK and TMPK are potential targets for the development of Mycoplasma-specific antibiotics.

In vitro phosphorylation of key metabolic enzymes from Bacillus subtilis: PrkC phosphorylates enzymes from different branches of basic metabolism.

Phosphorylation is an important mechanism of protein modification. In the Gram-positive soil bacterium Bacillus subtilis, about 5% of all proteins are subject to phosphorylation, and a significant portion of these proteins is phosphorylated on serine or threonine residues. We were interested in the regulation of the basic metabolism in B. subtilis. Many enzymes of the central metabolic pathways are phosphorylated in this organism. In an attempt to identify the responsible protein kinase(s), we identified four candidate kinases, among them the previously studied kinase PrkC. We observed that PrkC is indeed able to phosphorylate several metabolic enzymes in vitro. Determination of the phosphorylation sites revealed a remarkable preference of PrkC for threonine residues. Moreover, PrkC often used several phosphorylation sites in one protein. This feature of PrkC-dependent protein phosphorylation resembles the multiple phosphorylations often observed in eukaryotic proteins. The HPr protein of the phosphotransferase system is one of the proteins phosphorylated by PrkC, and PrkC phosphorylates a site (Ser-12) that has recently been found to be phosphorylated in vivo. The agreement between in vivo and in vitro phosphorylation of HPr on Ser-12 suggests that our in vitro observations reflect the events that take place in the cell.

The phosphoproteome of the minimal bacterium Mycoplasma pneumoniae: analysis of the complete known Ser/Thr kinome suggests the existence of novel kinases.

Mycoplasma pneumoniae belongs to the Mollicutes, the group of organisms with the smallest genomes that are capable of host-independent life. These bacteria show little regulation in gene expression, suggesting an important role for the control of protein activities. We have studied protein phosphorylation in M. pneumoniae to identify phosphorylated proteins. Two-dimensional gel electrophoresis and mass spectrometry allowed the detection of 63 phosphorylated proteins, many of them enzymes of central carbon metabolism and proteins related to host cell adhesion. We identified 16 phosphorylation sites, among them 8 serine and 8 threonine residues, respectively. A phosphoproteome analysis with mutants affected in the two annotated protein kinase genes or in the single known protein phosphatase gene suggested that only one protein (HPr) is phosphorylated by the HPr kinase, HPrK, whereas four adhesion-related or surface proteins were targets of the protein kinase C, PrkC. A comparison with the phosphoproteomes of other bacteria revealed that protein phosphorylation is evolutionarily only poorly conserved. Only one single protein with an identified phosphorylation site, a phosphosugar mutase (ManB in M. pneumoniae), is phosphorylated on a conserved serine residue in all studied organisms from archaea and bacteria to man. We demonstrate that this protein undergoes autophosphorylation. This explains the strong conservation of this phosphorylation event. For most other proteins, even if they are phosphorylated in different species, the actual phosphorylation sites are different. This suggests that protein phosphorylation is a form of adaptation of the bacteria to the specific needs of their particular ecological niche.

The stability of cytadherence proteins in Mycoplasma pneumoniae requires activity of the protein kinase PrkC.

Mycoplasma pneumoniae belongs to the mollicutes, a group of bacteria that have strongly reduced genomes but that are nevertheless capable of independent life. With only three transcription factors, the regulatory features of these bacteria are very limited. Thus, posttranslational regulation might be important for M. pneumoniae. In addition to the highly specific HPr kinase, the M. pneumoniae prkC gene encodes the serine/threonine protein kinase C. In order to study the function(s) of this kinase, we isolated an M. pneumoniae mutant affected in PrkC. This mutation resulted in nonadherent growth and loss of cytotoxicity. Examination of the phosphorylation profile of the prkC mutant suggested that phosphorylation of cytadherence proteins was affected by the loss of this kinase. In contrast, inactivation of the prpC gene affecting the protein phosphatase that antagonizes PrkC-dependent phosphorylation resulted in more intensive phosphorylation of the cytadherence proteins HMW1 and HMW3 of the major adhesin P1 and of the surface protein MPN474. Moreover, loss of PrkC affects not only the phosphorylation state of the cytadherence proteins but also their intracellular accumulation. However, the expression of the corresponding genes was not affected by PrkC, suggesting that PrkC-dependent phosphorylation results in stabilization of the cytadherence proteins. The HMW proteins and P1 are part of the so-called terminal organelle of M. pneumoniae that is involved in gliding motility, cell division, and adhesion to host epithelial tissues. Our observations suggest that the posttranslational modification of cytadherence proteins by PrkC is essential for the development and function of the M. pneumoniae terminal organelle.

Expression of Mycoplasma proteins carrying an affinity tag in M. pneumoniae allows rapid purification and circumvents problems related to the aberrant genetic code.

In Mycoplasma pneumoniae and several other mollicutes, the UGA opal codon specifies tryptophan rather than a translation stop. This often makes it difficult to express Mycoplasma proteins in heterologous hosts. In this work, we demonstrate that mollicute proteins can be fused to an affinity tag and be expressed directly in M. pneumoniae. The protein can then be purified by affinity chromatography and be used for biochemical or any other desired analysis.

Regulatory protein phosphorylation in Mycoplasma pneumoniae. A PP2C-type phosphatase serves to dephosphorylate HPr(Ser-P).

Among the few regulatory events in the minimal bacterium Mycoplasma pneumoniae is the phosphorylation of the HPr phosphocarrier protein of the phosphotransferase system. In the presence of glycerol, HPr is phosphorylated in an ATP-dependent manner by the HPr kinase/phosphorylase. The role of the latter enzyme was studied by constructing a M. pneumoniae hprK mutant defective in HPr kinase/phosphorylase. This mutant strain no longer exhibited HPr kinase activity but, surprisingly, still had phosphatase activity toward serine-phosphorylated HPr (HPr(Ser-P)). An inspection of the genome sequence revealed the presence of a gene (prpC) encoding a presumptive protein serine/threonine phosphatase of the PP2C family. The phosphatase PrpC was purified and its biochemical activity in HPr(Ser-P) dephosphorylation demonstrated. Moreover, a prpC mutant strain was isolated and found to be impaired in HPr(Ser-P) dephosphorylation. Homologues of PrpC are present in many bacteria possessing HPr(Ser-P), suggesting that PrpC may play an important role in adjusting the cellular HPr phosphorylation state and thus controlling the diverse regulatory functions exerted by the different forms of HPr.