PPE51 Is Involved in the Uptake of Disaccharides by Mycobacterium tuberculosis

We have recently found that selected thio-disaccharides possess bactericidal effects against Mycobacterium tuberculosis but not against Escherichia coli or Staphylococcus aureus. Here, we selected spontaneous mutants displaying resistance against the investigated thio-glycoside. According to next-generation sequencing, four of six analyzed mutants which were resistant to high concentrations of the tested chemical carried nonsynonymous mutations in the gene encoding the PPE51 protein. The complementation of these mutants with an intact ppe51 gene returned their sensitivity to the wild-type level. The uptake of tritiated thio-glycoside was significantly more abundant in wild-type Mycobacterium tuberculosis compared to the strain carrying the mutated ppe51 gene. The ppe51 mutations or CRISPR-Cas9-mediated downregulation of PPE51 expression affected the growth of mutant strains on minimal media supplemented with disaccharides (maltose or lactose) but not with glycerol or glucose as the sole carbon and energy source. Taking the above into account, we postulate that PPE51 participates in the uptake of disaccharides by tubercle bacilli.

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Expression of Mycobacterium smegmatisPyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

Journal of Bacteriology ◽

10.1128/jb.182.19.5479-5485.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5479-5485 ◽

Cited By ~ 34

Author(s):

Helena I. M. Boshoff ◽

Valerie Mizrahi

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Intracellular Localization ◽

Wild Type ◽

Gene Encoding ◽

Allelic Exchange ◽

Mutant Strains ◽

Established Role ◽

Amidase Gene

ABSTRACT A pyrazinamidase (PZase)-deficient pncA mutant ofMycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role ofpncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the M. tuberculosis pncA gene into the pncAmutant complemented its PZase/nicotinamidase defect. In bothpzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. ThepzaA-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type andpncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 μg/ml in all cases) and rendered Escherichia colihypersensitive for growth at low pH.

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IC138 Defines a Subdomain at the Base of the I1 Dynein That Regulates Microtubule Sliding and Flagellar Motility

Molecular Biology of the Cell ◽

10.1091/mbc.e09-04-0277 ◽

2009 ◽

Vol 20 (13) ◽

pp. 3055-3063 ◽

Cited By ~ 43

Author(s):

Raqual Bower ◽

Kristyn VanderWaal ◽

Eileen O'Toole ◽

Laura Fox ◽

Catherine Perrone ◽

...

Keyword(s):

Double Mutant ◽

Essential Role ◽

Null Allele ◽

Flagellar Motility ◽

Wild Type ◽

Gene Encoding ◽

Radial Spoke ◽

Mutant Strains ◽

Dynein Arms ◽

Image Averaging

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120—defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

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Aeromonas Flagella (Polar and Lateral) Are Enterocyte Adhesins That Contribute to Biofilm Formation on Surfaces

Infection and Immunity ◽

10.1128/iai.72.4.1939-1945.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 1939-1945 ◽

Cited By ~ 118

Author(s):

Sylvia M. Kirov ◽

Marika Castrisios ◽

Jonathan G. Shaw

Keyword(s):

Cell Lines ◽

Biofilm Formation ◽

Glass Tube ◽

Intestinal Cell ◽

Wild Type ◽

Wild Type Level ◽

Lateral Flagellum ◽

Mutant Strains ◽

Motility Mutant ◽

Characteristic Features

ABSTRACT Aeromonas spp. (gram-negative, aquatic bacteria which include enteropathogenic strains) have two distinct flagellar systems, namely a polar flagellum for swimming in liquid and multiple lateral flagella for swarming over surfaces. Only ∼60% of mesophilic strains can produce lateral flagella. To evaluate flagellar contributions to Aeromonas intestinal colonization, we compared polar and lateral flagellar mutant strains of a diarrheal isolate of Aeromonas caviae for the ability to adhere to the intestinal cell lines Henle 407 and Caco-2, which have the characteristic features of human intestinal enterocytes. Strains lacking polar flagella were virtually nonadherent to these cell lines, while loss of the lateral flagellum decreased adherence by ∼60% in comparison to the wild-type level. Motility mutants (unable to swim or swarm in agar assays) had adhesion levels of ∼50% of wild-type values, irrespective of their flagellar expression. Flagellar mutant strains were also evaluated for the ability to form biofilms in a borosilicate glass tube model which was optimized for Aeromonas spp. (broth inoculum, with a 16- to 20-h incubation at 37°C). All flagellar mutants showed a decreased ability to form biofilms (at least 30% lower than the wild type). For the chemotactic motility mutant cheA, biofilm formation decreased >80% from the wild-type level. The complementation of flagellar phenotypes (polar flagellar mutants) restored biofilms to wild-type levels. We concluded that both flagellar types are enterocyte adhesins and need to be fully functional for optimal biofilm formation.

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KatG Is the Primary Detoxifier of Hydrogen Peroxide Produced by Aerobic Metabolism in Bradyrhizobium japonicum

Journal of Bacteriology ◽

10.1128/jb.186.23.7874-7880.2004 ◽

2004 ◽

Vol 186 (23) ◽

pp. 7874-7880 ◽

Cited By ~ 36

Author(s):

Heather R. Panek ◽

Mark R. O'Brian

Keyword(s):

Catalase Activity ◽

Bradyrhizobium Japonicum ◽

Aerobic Metabolism ◽

Short Exposure ◽

Wild Type ◽

Gene Encoding ◽

Whole Cells ◽

Cell Extracts ◽

Genes Encoding ◽

High Concentrations

ABSTRACT Bacteria are exposed to reactive oxygen species from the environment and from those generated by aerobic metabolism. Catalases are heme proteins that detoxify H2O2, and many bacteria contain more than one catalase enzyme. Also, the nonheme peroxidase alkyl hydroperoxide reductase (Ahp) is the major scavenger of endogenous H2O2 in Escherichia coli. Here, we show that aerobically grown Bradyrhizobium japonicum cells express a single catalase activity. Four genes encoding putative catalases in the B. japonicum genome were identified, including a katG homolog encoding a catalase-peroxidase. Deletion of the katG gene resulted in loss of catalase activity in cell extracts and of exogenous H2O2 consumption by whole cells. The katG strain had a severe aerobic growth phenotype but showed improved growth in the absence of O2. By contrast, a B. japonicum ahpCD mutant grew well aerobically and consumed H2O2 at wild-type rates. A heme-deficient hemA mutant expressed about one-third of the KatG activity as the wild type but grew well aerobically and scavenged low concentrations of exogenous H2O2. However, cells of the hemA strain were deficient in consumption of high concentrations of H2O2 and were very sensitive to killing by short exposure to H2O2. In addition, KatG activity did not decrease as a result of mutation of the gene encoding the transcriptional activator OxyR. We conclude that aerobic metabolism produces toxic levels of H2O2 in B. japonicum, which is detoxified primarily by KatG. Furthermore, the katG level sufficient for detoxification does not require OxyR.

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Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

Infection and Immunity ◽

10.1128/iai.70.6.3080-3084.2002 ◽

2002 ◽

Vol 70 (6) ◽

pp. 3080-3084 ◽

Cited By ~ 55

Author(s):

Bhavna G. Gordhan ◽

Debbie A. Smith ◽

Heidi Alderton ◽

Ruth A. McAdam ◽

Gregory J. Bancroft ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Auxotrophic Mutant ◽

Phenotypic Characterization ◽

Wild Type ◽

Arginine Biosynthesis ◽

High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

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Transfer of embB Codon 306 Mutations into Clinical Mycobacterium tuberculosis Strains Alters Susceptibility to Ethambutol, Isoniazid, and Rifampin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01486-07 ◽

2008 ◽

Vol 52 (6) ◽

pp. 2027-2034 ◽

Cited By ~ 82

Author(s):

Hassan Safi ◽

Brendan Sayers ◽

Manzour H. Hazbón ◽

David Alland

Keyword(s):

Mycobacterium Tuberculosis ◽

Single Mutation ◽

Variable Degree ◽

Wild Type ◽

Growth Property ◽

Major Mechanism ◽

Mutant Strains ◽

High Level ◽

Competition Assays

ABSTRACT Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMB-resistant clinical M. tuberculosis isolates to identify the role of embB306 mutations in any type of drug resistance. Replacing wild-type embB306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 μg/ml to 7, 7, 8.5, and 14 μg/ml, respectively. Replacing embB306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 μg/ml or 28 μg/ml, respectively, to 3 μg/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB306 CTG mutants had growth advantages compared to strain 210 at sub-MICs of INH or RIF in monocultures and at sub-MICs of INH in competition assays. CTG mutants were also more resistant to the additive or synergistic activities of INH, RIF, or EMB used in different combinations. These results demonstrate that embB306 mutations cause an increase in the EMB MIC, a variable degree of EMB resistance, and are necessary but not sufficient for high-level EMB resistance. The unusual growth property of embB306 mutants in other antibiotics suggests that they may be amplified during treatment in humans and that a single mutation may affect antibiotic susceptibility against multiple first-line antibiotics.

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Propionyl Coenzyme A Is a Common Intermediate in the 1,2-Propanediol and Propionate Catabolic Pathways Needed for Expression of the prpBCDE Operon during Growth of Salmonella enterica on 1,2-Propanediol

Journal of Bacteriology ◽

10.1128/jb.185.9.2802-2810.2003 ◽

2003 ◽

Vol 185 (9) ◽

pp. 2802-2810 ◽

Cited By ~ 44

Author(s):

Sergio Palacios ◽

Vincent J. Starai ◽

Jorge C. Escalante-Semerena

Keyword(s):

Salmonella Enterica ◽

Coenzyme A ◽

Regulatory Protein ◽

Wild Type ◽

Gene Encoding ◽

Catabolic Pathways ◽

Serovar Typhimurium ◽

The Common ◽

High Concentrations ◽

Methylcitrate Synthase

ABSTRACT The studies reported here identify propionyl coenzyme A (propionyl-CoA) as the common intermediate in the 1,2-propanediol and propionate catabolic pathways of Salmonella enterica serovar Typhimurium LT2. Growth on 1,2-propanediol as a carbon and energy source led to the formation and excretion of propionate, whose activation to propionyl-CoA relied on the activities of the propionate kinase (PduW)/phosphotransacetylase (Pta) enzyme system and the CobB sirtuin-controlled acetyl-CoA and propionyl-CoA (Acs, PrpE) synthetases. The different affinities of these systems for propionate ensure sufficient synthesis of propionyl-CoA to support wild-type growth of S. enterica under low or high concentrations of propionate in the environment. These redundant systems of propionyl-CoA synthesis are needed because the prpE gene encoding the propionyl-CoA synthetase enzyme is part of the prpBCDE operon under the control of the PrpR regulatory protein, which needs 2-methylcitrate as a coactivator. Because the synthesis of 2-methylcitrate by PrpC (i.e., the 2-methylcitrate synthase enzyme) requires propionyl-CoA as a substrate, the level of propionyl-CoA needs to be raised by the Acs or PduW-Pta system before 2-methylcitrate can be synthesized and prpBCDE transcription can be activated.

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Novel mutations in katG gene of a clinical isolate of isoniazid-resistant Mycobacterium tuberculosis

Biologia ◽

10.2478/s11756-011-0162-7 ◽

2012 ◽

Vol 67 (1) ◽

Cited By ~ 4

Author(s):

Purkan ◽

Ihsanawati ◽

Yana Syah ◽

Debbie Retnoningrum ◽

Achmad Noer ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Isolate ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Intramolecular Interactions ◽

Wild Type ◽

Katg Gene ◽

Gene Encoding ◽

Oxidation Activity ◽

Catalase Peroxidase

AbstractMost of isoniazid-resistant Mycobacterium tuberculosis evolved due to mutation in the katG gene encoding catalase-peroxidase. A set of new mutations, namely T1310C, G1388T, G1481A, T1553C, and A1660G, which correspond to amino acid substitutions of L437P, R463L, G494D, I518T, and K554E, in the katG gene of the L10 clinical isolate M. tuberculosis was identified. The wild-type and mutant KatG proteins were expressed in Escherichia coli BL21(DE3) as a protein of 80 kDa based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. The mutant KatG protein exhibited catalase and peroxidase activities of 4.6% and 24.8% toward its wild type, respectively, and retained 19.4% isoniazid oxidation activity. The structure modelling study revealed that these C-terminal mutations might have induced formation of a new turn, perturbing the active site environment and also generated new intramolecular interactions, which could be unfavourable for the enzyme activities.

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Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-PTS Show Enhanced Post-Exponential Phase Fitness

10.1101/2021.07.15.452590 ◽

2021 ◽

Author(s):

Lin Zeng ◽

Alejandro R Walker ◽

Kyulim Lee ◽

Zachary A Taylor ◽

Robert A Burne

Keyword(s):

Central Carbon Metabolism ◽

Arginine Deiminase ◽

Transcriptional Analysis ◽

Wild Type ◽

Gene Encoding ◽

Streptococcus Sanguinis ◽

Biochemical Assays ◽

Production Of Hydrogen ◽

Spontaneous Mutants ◽

Production Of Ammonia

Genetic truncations in a gene encoding a putative glucose-PTS protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. Using an engineered mutant of manL and its complemented derivative, we showed that the ManL-deficient strain had improved bacterial viability in stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small, but significant percentage (~10%), of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. Significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e. excess carbohydrates and low pH, are those associated with caries development, we propose that the glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2.

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Clinically relevant mutations of mycobacterial GatCAB inform regulation of translational fidelity

10.1101/2021.03.29.437598 ◽

2021 ◽

Author(s):

Yang-Yang Li ◽

Rong-Jun Cai ◽

Jia-Ying Yang ◽

Tamara L. Hendrickson ◽

Ye Xiang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Second Step ◽

Loss Of Function ◽

Wild Type ◽

Translational Fidelity ◽

Partial Loss ◽

Biophysical Characterization ◽

Minimal Impact ◽

Mutant Strains

AbstractMost bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error-prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampicin tolerance. However, the mechanisms by which these clinically-derived mutations in gatA impact GatCAB function was unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains, but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity.

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