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Microbiology Spectrum May 2024Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and...
Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. subspecies () growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including . While C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for .IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for . This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of subspecies produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of . This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid.
Topics: Mycobacterium avium subsp. paratuberculosis; Animals; Paratuberculosis; Cattle; Bacterial Proteins; Sheep; Fatty Acids; Polymorphism, Single Nucleotide; Methyltransferases; Stearic Acids
PubMed: 38501867
DOI: 10.1128/spectrum.00508-24 -
Heliyon Mar 2024( is the pathogen of human tuberculosis (TB). Resistance to numerous stresses, including oxidative stress, is determinant for intracellular survival, and...
( is the pathogen of human tuberculosis (TB). Resistance to numerous stresses, including oxidative stress, is determinant for intracellular survival, and understanding associated mechanisms is crucial for developing new therapeutic strategies. Rv2617c has been associated with oxidative stress response when interacting with other proteins in ; however, its functional promiscuity and underlying molecular mechanisms remain elusive. In this study, we investigated the phenotypic changes of () expressing Rv2617c (Ms_Rv2617c) and its behavior in the presence of various stresses and phage infections. We found that Rv2617c conferred resistance to SDS and diamide while sensitizing to oxidative stress (HO) and altered mycobacterial phenotypic properties (single-cell clone and motility), suggestive of reprogrammed mycobacterial cell wall lipid contents exemplified by increased cell wall permeability. Interestingly, we also found that Rv2617c promoted resistance to infection by phages (SWU1, SWU2, D29, and TM4) and kept phage TM4 from destroying mycobacterial biofilms. Our findings provide new insights into the role of Rv2617c in resistance to oxide and acid stresses and report for the first time on its role in phage resistance in
PubMed: 38495141
DOI: 10.1016/j.heliyon.2024.e27400 -
International Journal of Molecular... Mar 2024The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for...
The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this study, a series of -alkylcytidines, including 5- and 6-methylcytidine derivatives, with extended alkyl substituents, were obtained in order to develop a new generation of antibacterial and antifungal biocides based on nucleoside derivatives. It has been shown that -alkyl 5- or 6-methylcytidines effectively inhibit the growth of molds, isolated from the paintings in the halls of the Ancient Russian Paintings of the State Tretyakov Gallery, Russia, Moscow. The novel compounds showed activity similar to antiseptics commonly used to protect works of art, such as benzalkonium chloride, to which a number of microorganisms have acquired resistance. It was also shown that the activity of -alkylcytidines is comparable to that of some antibiotics used in medicine to fight Gram-positive bacteria, including resistant strains of and . -dodecyl-5- and 6-methylcytidines turned out to be the best. This compound seems promising for expanding the palette of antiseptics used in painting, since quite often the destruction of painting materials is caused by joint fungi and bacteria infection.
Topics: Humans; Disinfectants; Bacteria; Paintings; Fungi; Anti-Bacterial Agents; Anti-Infective Agents, Local
PubMed: 38474298
DOI: 10.3390/ijms25053053 -
Nature Communications Mar 2024The growth and division of mycobacteria, which include clinically relevant pathogens, deviate from that of canonical bacterial models. Despite their Gram-positive...
The growth and division of mycobacteria, which include clinically relevant pathogens, deviate from that of canonical bacterial models. Despite their Gram-positive ancestry, mycobacteria synthesize and elongate a diderm envelope asymmetrically from the poles, with the old pole elongating more robustly than the new pole. The phosphatidylinositol-anchored lipoglycans lipomannan (LM) and lipoarabinomannan (LAM) are cell envelope components critical for host-pathogen interactions, but their physiological functions in mycobacteria remained elusive. In this work, using biosynthetic mutants of these lipoglycans, we examine their roles in maintaining cell envelope integrity in Mycobacterium smegmatis and Mycobacterium tuberculosis. We find that mutants defective in producing mature LAM fail to maintain rod cell shape specifically at the new pole and para-septal regions whereas a mutant that produces a larger LAM becomes multi-septated. Therefore, LAM plays critical and distinct roles at subcellular locations associated with division in mycobacteria, including maintenance of local cell wall integrity and septal placement.
Topics: Lipopolysaccharides; Mycobacterium smegmatis; Cell Wall; Mycobacterium tuberculosis
PubMed: 38467648
DOI: 10.1038/s41467-024-46565-5 -
Microbiology Resource Announcements Apr 2024Bacteriophage Rummer is a siphovirus morphology actinophage isolated from . Rummer has a 50,908 base pair genome encoding 89 predicted protein-coding genes and three...
Bacteriophage Rummer is a siphovirus morphology actinophage isolated from . Rummer has a 50,908 base pair genome encoding 89 predicted protein-coding genes and three tRNAs. Based on gene content similarity to sequenced actinobacteriophages, Rummer is assigned to phage subcluster A3.
PubMed: 38466105
DOI: 10.1128/mra.01268-23 -
G3 (Bethesda, Md.) May 2024Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and...
Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacterium Mycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes (~28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic no known functions proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts.
Topics: Mycobacterium smegmatis; Mycobacteriophages; Genome, Viral; Viral Proteins
PubMed: 38456318
DOI: 10.1093/g3journal/jkae049 -
Microbiology Resource Announcements Apr 2024JorRay, Blocker23, Nibbles, and OlgasClover are actinobacteriophages belonging to clusters G1, B2, CT, and DJ, respectively. JorRay and Blocker23 were identified in host...
JorRay, Blocker23, Nibbles, and OlgasClover are actinobacteriophages belonging to clusters G1, B2, CT, and DJ, respectively. JorRay and Blocker23 were identified in host bacterium . Nibbles and OlgasClover were identified in host bacterium NRRL B-16540.
PubMed: 38445868
DOI: 10.1128/mra.01256-23 -
Nature Methods Apr 2024Precise identification and quantification of amino acids is crucial for many biological applications. Here we report a copper(II)-functionalized Mycobacterium smegmatis...
Precise identification and quantification of amino acids is crucial for many biological applications. Here we report a copper(II)-functionalized Mycobacterium smegmatis porin A (MspA) nanopore with the N91H substitution, which enables direct identification of all 20 proteinogenic amino acids when combined with a machine-learning algorithm. The validation accuracy reaches 99.1%, with 30.9% signal recovery. The feasibility of ultrasensitive quantification of amino acids was also demonstrated at the nanomolar range. Furthermore, the capability of this system for real-time analyses of two representative post-translational modifications (PTMs), one unnatural amino acid and ten synthetic peptides using exopeptidases, including clinically relevant peptides associated with Alzheimer's disease and cancer neoantigens, was demonstrated. Notably, our strategy successfully distinguishes peptides with only one amino acid difference from the hydrolysate and provides the possibility to infer the peptide sequence.
Topics: Nanopores; Amino Acids; Peptides; Amino Acid Sequence; Porins
PubMed: 38443507
DOI: 10.1038/s41592-024-02208-7 -
Nature Communications Mar 2024Natural fruits contain a large variety of cis-diols. However, due to the lack of a high-resolution sensor that can simultaneously identify all cis-diols without a need...
Natural fruits contain a large variety of cis-diols. However, due to the lack of a high-resolution sensor that can simultaneously identify all cis-diols without a need of complex sample pretreatment, direct and rapid analysis of fruits in a hand-held device has never been previously reported. Nanopore, a versatile single molecule sensor, can be specially engineered to perform this task. A hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore modified with a sole phenylboronic acid (PBA) adapter is prepared. This engineered MspA accurately recognizes 1,2-diphenols, alditols, α-hydroxy acids and saccharides in prune, grape, lemon, different varieties of kiwifruits and commercial juice products. Assisted with a custom machine learning program, an accuracy of 99.3% is reported and the sample pretreatment is significantly simplified. Enantiomers such as DL-malic acids can also be directly identified, enabling sensing of synthetic food additives. Though demonstrated with fruits, these results suggest wide applications of nanopore in food and drug administration uses.
Topics: United States; Fruit; Nanopores; Sugar Alcohols; Carboxylic Acids; Citrus; Mycobacterium smegmatis; Porins
PubMed: 38443434
DOI: 10.1038/s41467-024-46303-x -
Nature Communications Mar 2024Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be...
Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.
Topics: Mycobacterium smegmatis; Mycobacterium tuberculosis; Proteolysis; Dimerization; Drug Discovery
PubMed: 38443338
DOI: 10.1038/s41467-024-46218-7