scholarly journals Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA

2012 ◽  
Vol 194 (18) ◽  
pp. 4933-4940 ◽  
Author(s):  
Lauren J. Rajakovich ◽  
John Tomlinson ◽  
Patricia C. Dos Santos
Keyword(s):  
Functional Analysis ◽  
Bacillus Subtilis ◽  
Gene Encoding ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
Sulfur Transfer ◽  
Essential Enzyme ◽  
Sulfur Trafficking

ABSTRACTThiI has been identified as an essential enzyme involved in the biosynthesis of thiamine and the tRNA thionucleoside modification, 4-thiouridine. InEscherichia coliandSalmonella enterica, ThiI acts as a sulfurtransferase, receiving the sulfur donated from the cysteine desulfurase IscS and transferring it to the target molecule or additional sulfur carrier proteins. However, inBacillus subtilisand most species from theFirmicutesphylum, ThiI lacks the rhodanese domain that contains the site responsible for the sulfurtransferase activity. The lack of the gene encoding for a canonical IscS cysteine desulfurase and the presence of a short sequence of ThiI in these bacteria pointed to mechanistic differences involving sulfur trafficking reactions in both biosynthetic pathways. Here, we have carried out functional analysis ofB. subtilisthiIand the adjacent gene,nifZ, encoding for a cysteine desulfurase. Gene inactivation experiments inB. subtilisindicate the requirement of ThiI and NifZ for the biosynthesis of 4-thiouridine, but not thiamine.In vitrosynthesis of 4-thiouridine by ThiI and NifZ, along with labeling experiments, suggests the occurrence of an alternate transient site for sulfur transfer, thus obviating the need for a rhodanese domain.In vivocomplementation studies inE. coliIscS- or ThiI-deficient strains provide further support for specific interactions between NifZ and ThiI. These results are compatible with the proposal thatB. subtilisNifZ and ThiI utilize mechanistically distinct and mutually specific sulfur transfer reactions.

scholarly journals Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

2015 ◽  
Vol 197 (11) ◽  
pp. 1952-1962 ◽  
Author(s):  
Katherine A. Black ◽  
Patricia C. Dos Santos
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Lysine Trna ◽  
Domains Of Life

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

scholarly journals Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans

2015 ◽  
Vol 197 (16) ◽  
pp. 2675-2684 ◽  
Author(s):  
Seram Nganbiton Devi ◽  
Brittany Kiehler ◽  
Lindsey Haggett ◽  
Masaya Fujita
Keyword(s):  
Bacillus Subtilis ◽  
Mutant Protein ◽  
Phosphoryl Group ◽  
Atp Binding ◽  
Histidine Kinases ◽  
Content Type ◽  
Point Mutant ◽  
Terminal Domain

ABSTRACTEntry into sporulation inBacillus subtilisis governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur intrans. To test this hypothesis, we generated a series ofB. subtilisstrains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in atransfashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressedin vivo. Taken together, thesein vitroandin vivoresults reinforce the evidence that KinA autophosphorylation is able to occur in atransfashion.IMPORTANCEAutophosphorylation of histidine kinases is known to occur by either thecis(one subunit of kinase phosphorylating itself within the multimer) or thetrans(one subunit of the multimer phosphorylates the other subunit) mechanism. The present study provided directin vivoandin vitroevidence that autophosphorylation of the major sporulation histidine kinase (KinA) is able to occur intranswithin the homotetramer complex. While the physiological and mechanistic significance of thetransautophosphorylation reaction remains obscure, understanding the detailed reaction mechanism of the sporulation kinase is the first step toward gaining insight into the molecular mechanisms of the initiation of sporulation, which is believed to be triggered by unknown factors produced under conditions of nutrient depletion.

scholarly journals ModifiedmarinerTransposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

2011 ◽  
Vol 78 (3) ◽  
pp. 778-785 ◽  
Author(s):  
Eric R. Pozsgai ◽  
Kris M. Blair ◽  
Daniel B. Kearns
Keyword(s):  
Bacillus Subtilis ◽  
Insertional Mutagenesis ◽  
Inducible Promoter ◽  
Insertion Sequences ◽  
Content Type ◽  
Transcriptional Reporter ◽  
Species Specific ◽  
Genetically Manipulated

ABSTRACTTransposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA.marineris a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existingmarinertransposon, TnYLB, such that it can easily be genetically manipulated and introduced intoBacillus subtilis. We generate a series of three newmarinerderivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterlesslacZgene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted toB. subtilisand should be applicable to anymariner-compatible host organism, provided thatin vitromutagenesis or anin vivospecies-specific delivery vector is employed.

scholarly journals Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in Bacillus subtilis 168

2006 ◽  
Vol 72 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Kazuhiko Kurosawa ◽  
Takeshi Hosaka ◽  
Norimasa Tamehiro ◽  
Takashi Inaoka ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Antibiotic Production ◽  
Point Mutations ◽  
Protease Production ◽  
Gene Encoding ◽  
Translational Activity ◽  
Amylase Production ◽  
Ribosomal Protein S12

ABSTRACT The capacity of ribosomal modification to improve antibiotic production by Streptomyces spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (str) also enhances α-amylase (and protease) production by a strain of Bacillus subtilis as estimated by measuring the enzyme activity. The str mutations are point mutations within rpsL, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various rpsL mutations K56R (which corresponds to position 42 in E. coli) was particularly effective at enhancing α-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg2+ concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in α-amylase production seen in the affected strain.

scholarly journals Identification of a Mutation in the Bacillus subtilis S-Adenosylmethionine Synthetase Gene That Results in Derepression of S-Box Gene Expression

2006 ◽  
Vol 188 (10) ◽  
pp. 3674-3681 ◽  
Author(s):  
Brooke A. McDaniel ◽  
Frank J. Grundy ◽  
Vineeta P. Kurlekar ◽  
Jerneja Tomsic ◽  
Tina M. Henkin
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Rna Structure ◽  
Synthetase Activity ◽  
Gene Encoding ◽  
Sam Synthetase ◽  
Transcription In Vitro ◽  
Adenosylmethionine Synthetase

ABSTRACT Genes in the S-box family are regulated by binding of S-adenosylmethionine (SAM) to the 5′ region of the mRNA of the regulated gene. SAM binding was previously shown to promote a rearrangement of the RNA structure that results in premature termination of transcription in vitro and repression of expression of the downstream coding sequence. The S-box RNA element therefore acts as a SAM-binding riboswitch in vitro. In an effort to identify factors other than SAM that could be involved in the S-box regulatory mechanism in vivo, we searched for trans-acting mutations in Bacillus subtilis that act to disrupt repression of S-box gene expression during growth under conditions where SAM pools are elevated. We identified a single mutant that proved to have one nucleotide substitution in the metK gene, encoding SAM synthetase. This mutation, designated metK10, resulted in a 15-fold decrease in SAM synthetase activity and a 4-fold decrease in SAM concentration in vivo. The metK10 mutation specifically affected S-box gene expression, and the increase in expression under repressing conditions was dependent on the presence of a functional transcriptional antiterminator element. The observation that the mutation identified in this search affects SAM production supports the model that the S-box RNAs directly monitor SAM in vivo, without a requirement for additional factors.

scholarly journals CodY Deletion EnhancesIn VivoVirulence of Community-Associated Methicillin-Resistant Staphylococcus aureus Clone USA300

2012 ◽  
Vol 80 (7) ◽  
pp. 2382-2389 ◽  
Author(s):  
Christopher P. Montgomery ◽  
Susan Boyle-Vavra ◽  
Agnès Roux ◽  
Kazumi Ebine ◽  
Abraham L. Sonenshein ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Genes ◽  
Target Genes ◽  
Gene Encoding ◽  
Methicillin Resistant ◽  
Content Type ◽  
Global Regulators ◽  
Alpha Hemolysin

ABSTRACTTheStaphylococcus aureusglobal regulator CodY responds to nutrient availability by controlling the expression of target genes.In vitro, CodY represses the transcription of virulence genes, but it is not known if CodY also represses virulencein vivo. The dominant community-associated methicillin-resistantS. aureus(CA-MRSA) clone, USA300, is hypervirulent and has increased transcription of global regulators and virulence genes; these features are reminiscent of a strain defective in CodY. Sequence analysis revealed, however, that thecodYgenes of USA300 and other sequencedS. aureusisolates are not significantly different from thecodYgenes in strains known to have active CodY.codYwas expressed in USA300, as well as in other pulsotypes assessed. Deletion ofcodYfrom a USA300 clinical isolate resulted in modestly increased expression of the global regulatorsagrandsaeRS, as well as the gene encoding the toxin alpha-hemolysin (hla). A substantial increase (>30-fold) in expression of thelukF-PVgene, encoding part of the Panton-Valentine leukocidin (PVL), was observed in thecodYmutant. All of these expression differences were reversed by complementation with a functionalcodYgene. Moreover, purified CodY protein bound upstream of thelukSF-PVoperon, indicating that CodY directly represses expression oflukSF-PV. Deletion ofcodYincreased the virulence of USA300 in necrotizing pneumonia and skin infection. Interestingly, deletion oflukSF-PVfrom thecodYmutant did not attenuate virulence, indicating that the hypervirulence of thecodYmutant was not explained by overexpression of PVL. These results demonstrate that CodY is active in USA300 and that CodY-mediated repression restrains the virulence of USA300.

scholarly journals Strain-Specific Regulatory Role of Eukaryote-Like Serine/Threonine Phosphatase in Pneumococcal Adherence

2012 ◽  
Vol 80 (4) ◽  
pp. 1361-1372 ◽  
Author(s):  
Shivangi Agarwal ◽  
Shivani Agarwal ◽  
Preeti Pancholi ◽  
Vijay Pancholi
Keyword(s):  
High Efficiency ◽  
Regulatory System ◽  
Gene Encoding ◽  
Content Type ◽  
Knockout Mutants ◽  
Catalytically Active

ABSTRACTStreptococcus pneumoniaeexploits a battery of virulence factors to colonize the host. Although the eukaryote-like Ser/Thr kinase ofS. pneumoniae(StkP) has been implicated in physiology and virulence, the role of its cotranscribing phosphatase (PhpP) has remained elusive. The construction of nonpolar markerlessphpPknockout mutants (ΔphpP) in two pathogenic strains, D39 (type 2) and 6A-EF3114 (type 6A), indicated that PhpP is not indispensable for pneumococcal survival. Further, PhpP also participates in the regulation of cell wall biosynthesis/division, adherence, and biofilm formation in a strain-specific manner. Additionally, we provide hitherto-unknownin vitroandin vivoevidence of a physiologically relevant biochemical link between the StkP/PhpP-mediated cognate regulation and the two-component regulatory system TCS06 (RR06/HK06) that regulates the expression of the gene encoding an important pneumococcal surface adhesin, CbpA, which was found to be significantly upregulated in ΔphpPmutants. In particular, StkP (threonine)-phosphorylated RR06 bound to thecbpApromoter with high efficiency even in the absence of the HK06-responsive and catalytically active aspartate 51 residue. Together, our findings unravel the significant contributions of PhpP in pneumococcal physiology and adherence.

scholarly journals Comparative Analysis of the Capacity of the Candida Species To Elicit Vaginal Immunopathology

2018 ◽  
Vol 86 (12) ◽  
Author(s):  
Hubertine M. E. Willems ◽  
David J. Lowes ◽  
Katherine S. Barker ◽  
Glen E. Palmer ◽  
Brian M. Peters
Keyword(s):  
Comparative Analysis ◽  
Mucosal Damage ◽  
Neutrophil Recruitment ◽  
Cytokine Signaling ◽  
Vaginal Mucosa ◽  
Gene Encoding ◽  
Fungal Virulence ◽  
Content Type

ABSTRACT The human fungal pathogen Candida albicans is the major etiological agent of vulvovaginal candidiasis (VVC). Despite this fact, other non-albicans Candida (NAC) species have frequently been reported, as well. Despite their presence in the vaginal environment, little is known about their capacities to elicit immune responses classically associated with C. albicans-mediated immunopathology, including neutrophil recruitment and proinflammatory cytokine signaling. Therefore, using a combination of in vitro and in vivo approaches, we undertook a comparative analysis to determine whether a representative panel of NAC species could colonize, induce immunopathological markers, or cause damage at the vaginal mucosa. Using a murine model of VVC, C. albicans was found to induce robust immunopathology (neutrophils and interleukin 1β [IL-1β]) and elicit mucosal damage. However, all the NAC species tested (including C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, C. glabrata, and C. auris) induced significantly less damage and neutrophil recruitment than C. albicans, despite achieving similar early colonization levels. These results largely correlated with a notable lack of ability by the NAC species (including C. dubliniensis and C. tropicalis) to form hyphae both in vitro and in vivo. Furthermore, both C. dubliniensis and C. tropicalis induced significantly less expression of the ECE1 gene encoding candidalysin, a key fungal virulence determinant driving VVC immunopathology. In order to determine the relative capacities of these species to elicit inflammasome-dependent IL-1β release, both wild-type and NLRP3−/− THP-1 cells were challenged in vitro. While most species tested elicited only modest amounts of IL-1β, challenge with C. albicans led to significantly elevated levels that were largely NLRP3 dependent. Collectively, our findings demonstrate that although NAC species are increasingly reported as causative agents of VVC, C. albicans appears to be exceedingly vaginopathogenic, exhibiting robust immunopathology, hypha formation, and candidalysin expression. Thus, this study provides mechanistic insight into why C. albicans is overwhelmingly the major pathogen reported during VVC.

scholarly journals The Major Chromosome Condensation Factors Smc, HBsu, and Gyrase in Bacillus subtilis Operate via Strikingly Different Patterns of Motion

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Sonja Schibany ◽  
Rebecca Hinrichs ◽  
Rogelio Hernández-Tamayo ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Single Molecule ◽  
High Mobility ◽  
Single Molecule Tracking ◽  
Content Type ◽  
Dna Compaction ◽  
Transient Interactions

ABSTRACT Although DNA-compacting proteins have been extensively characterized in vitro, knowledge of their DNA binding dynamics in vivo is greatly lacking. We have employed single-molecule tracking to characterize the motion of the three major chromosome compaction factors in Bacillus subtilis, Smc (structural maintenance of chromosomes) proteins, topoisomerase DNA gyrase, and histone-like protein HBsu. We show that these three proteins display strikingly different patterns of interaction with DNA; while Smc displays two mobility fractions, one static and one moving through the chromosome in a constrained manner, gyrase operates as a single slow-mobility fraction, suggesting that all gyrase molecules are catalytically actively engaged in DNA binding. Conversely, bacterial histone-like protein HBsu moves through the nucleoid as a larger, slow-mobility fraction and a smaller, high-mobility fraction, with both fractions having relatively short dwell times. Turnover within the SMC complex that makes up the static fraction is shown to be important for its function in chromosome compaction. Our report reveals that chromosome compaction in bacteria can occur via fast, transient interactions in vivo, avoiding clashes with RNA and DNA polymerases. IMPORTANCE All types of cells need to compact their chromosomes containing their genomic information several-thousand-fold in order to fit into the cell. In eukaryotes, histones achieve a major degree of compaction and bind very tightly to DNA such that they need to be actively removed to allow access of polymerases to the DNA. Bacteria have evolved a basic, highly dynamic system of DNA compaction, accommodating rapid adaptability to changes in environmental conditions. We show that the Bacillus subtilis histone-like protein HBsu exchanges on DNA on a millisecond scale and moves through the entire nucleoid containing the genome as a slow-mobility fraction and a dynamic fraction, both having short dwell times. Thus, HBsu achieves compaction via short and transient DNA binding, thereby allowing rapid access of DNA replication or transcription factors to DNA. Topoisomerase gyrase and B. subtilis Smc show different interactions with DNA in vivo, displaying continuous loading or unloading from DNA, or using two fractions, one moving through the genome and one statically bound on a time scale of minutes, respectively, revealing three different modes of DNA compaction in vivo.

scholarly journals Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases from Mycobacterium smegmatis Lack Flavin Coenzyme

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
Shivjee Sah ◽  
Kuldeep Lahry ◽  
Chandana Talwar ◽  
Sudhir Singh ◽  
Umesh Varshney
Keyword(s):  
Mycobacterium Smegmatis ◽  
De Novo ◽  
Aminosalicylic Acid ◽  
Methionine Biosynthesis ◽  
Content Type ◽  
Folate Pathway ◽  
Essential Enzyme ◽  
Antifolate Resistance

ABSTRACT 5,10-Methylenetetrahydrofolate reductase (MetF/MTHFR) is an essential enzyme in one-carbon metabolism for de novo biosynthesis of methionine. Our in vivo and in vitro analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR in Mycobacterium smegmatis, failed to reveal their function as MTHFRs. However, we identified two hypothetical proteins, MSMEG_6596 and MSMEG_6649, as noncanonical MTHFRs in the bacterium. MTHFRs are known to be oligomeric flavoproteins. Both MSMEG_6596 and MSMEG_6649 are monomeric proteins and lack flavin coenzymes. In vitro, the catalytic efficiency (kcat/Km) of MSMEG_6596 (MTHFR1) for 5,10-CH2-THF and NADH was ∼13.5- and 15.3-fold higher than that of MSMEG_6649 (MTHFR2). Thus, MSMEG_6596 is the major MTHFR. This interpretation was further supported by better rescue of the E. coli Δmthfr strain by MTHFR1 than by MTHFR2. As identified by liquid chromatography-tandem mass spectrometry, the product of MTHFR1- or MTHFR2-catalyzed reactions was 5-CH3-THF. The M. smegmatis Δmsmeg_6596 strain was partially auxotrophic for methionine and grew only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the Δmsmeg_6596 strain was more sensitive to folate pathway inhibitors (sulfachloropyridazine, p-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are two noncanonical MTHFR proteins that are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria. IMPORTANCE MTHFR/MetF is an essential enzyme in a one-carbon metabolic pathway for de novo biosynthesis of methionine. MTHFRs are known to be oligomeric flavoproteins. Our in vivo and in vitro analyses of Mycobacterium smegmatis MSMEG_6664/MSMEI_6484, annotated as putative MTHFR, failed to reveal their function as MTHFRs. However, we identified two of the hypothetical proteins, MSMEG_6596 and MSMEG_6649, as MTHFR1 and MTHFR2, respectively. Interestingly, both MTHFRs are monomeric and lack flavin coenzymes. M. smegmatis deleted for the major mthfr (mthfr1) was partially auxotroph for methionine and more sensitive to folate pathway inhibitors (sulfachloropyridazine, para-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are novel MTHFRs involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.

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