Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), one of the deadliest infectious diseases. The alarming health context coupled with the emergence of resistant M. tuberculosis strains highlights the urgent need to expand the range of anti-TB antibiotics. A subset of anti-TB drugs in use are prodrugs that require bioactivation by a class of M. tuberculosis enzymes called Baeyer-Villiger monooxygenases (BVMOs), which remain understudied. To examine the prevalence and the molecular function of BVMOs in mycobacteria, we applied a comprehensive bioinformatic analysis that identified six BVMOs in M. tuberculosis, including Rv3083 (MymA), Rv3854c (EthA), Rv0565c, and Rv0892, which were selected for further characterization. Homology modeling and substrate docking analysis, performed on this subset, suggested that Rv0892 is closer to the cyclohexanone BVMO, while Rv0565c and EthA are structurally and functionally similar to MymA, which is by far the most prominent type I BVMO enzyme. Thanks to an unprecedented purification and assay optimization, biochemical studies confirmed that all four BVMOs display BV-oxygenation activity. We also showed that MymA displays a distinctive substrate preference that we further investigated by kinetic parameter determination and that correlates with in silico modeling. We provide insights into distribution of BVMOs and the structural basis of their substrate profiling, and we discuss their possible redundancy in M. tuberculosis, raising questions about their versatility in prodrug activation and their role in physiology and infection. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of death worldwide. The rise in drug resistance highlights the urgent need for innovation in anti-TB drug development. Many anti-TB drugs require bioactivation by Baeyer-Villiger monooxygenases (BVMOs). Despite their emerging importance, BVMO structural and functional features remain enigmatic. We applied a comprehensive bioinformatic analysis and confirmed the presence of six BVMOs in M. tuberculosis, including MymA, EthA, and Rv0565c-activators of the second-line prodrug ethionamide-and the novel BVMO Rv0892. Combining in silico characterization with in vitro validation, we outlined their structural framework and substrate preference. Markedly, MymA displayed an enhanced capacity and a distinct selectivity profile toward ligands, in agreement with its catalytic site topology. These features ground the molecular basis for structure-function comprehension of the specificity in these enzymes and expand the repertoire of BVMOs with selective and/or overlapping activity for application in the context of improving anti-TB therapy.
The emergence of B. cereus as an opportunistic food-borne pathogen has intensified the need to distinguish strains of public health concern. The heterogeneity of the diseases associated with B. cereus infections emphasizes the versatility of these bacteria strains to colonize their host. Nevertheless, the molecular basis of these differences remains unclear. Several toxins are involved in virulence, particularly in gastrointestinal disorders, but there are currently no biological markers able to differentiate pathogenic from harmless strains. We have previously shown that CwpFM is a cell wall peptidase involved in B. cereus virulence. Here, we report a sequence/structure/function characterization of 39 CwpFM sequences, chosen from a collection of B. cereus with diverse virulence phenotypes, from harmless to highly pathogenic strains. CwpFM is homology-modeled in silico as an exported papain-like endopeptidase, with an N-terminal end composed of three successive bacterial Src Homology 3 domains (SH3b1–3) likely to control protein–protein interactions in signaling pathways, and a C-terminal end that contains a catalytic NLPC_P60 domain primed to form a competent active site. We confirmed in vitro that CwpFM is an endopeptidase with a moderate peptidoglycan hydrolase activity. Remarkably, CwpFMs from pathogenic strains harbor a specific stretch of twenty residues intrinsically disordered, inserted between the SH3b3 and the catalytic NLPC_P60 domain. This strongly suggests this linker as a marker of differentiation between B. cereus strains. We believe that our findings improve our understanding of the pathogenicity of B. cereus while advancing both clinical diagnosis and food safety.
The Cover Feature shows the active site of an amine dehydrogenase (AmDH), an enzyme which catalyzes the transformation of ketones into chiral amines. The protein has been modified by protein engineering, creating a variant that can synthesizee longer amines than the wild-type. In their Research Article, C. Vergne-Vaxelaire and co-workers explain how, inspired by natural sequence biodiversity, the key amino acids of different enzymes active sites have been selected and then mutated into less bulky residues, successfully leading to variants that are able to transform unprecedented bulky aldehydes and ketones into amines. The improved performance of the variants has been investigated using both X-ray crystal structures and molecular studies, helping to inform further engineering of these important sustainable tools for amine synthesis. The credit for the picture belongs to Kim-Beverly Ajavon and Laurine Ducrot. More information can be found in the Research Article by C. Vergne-Vaxelaire and co-workers.
Lactococcus lactis and Lactococcus cremoris are Gram-positive lactic acid bacteria widely used as starter in milk fermentations. Lactococcal cells are covered with a polysaccharide pellicle (PSP) that was previously shown to act as the receptor for numerous bacteriophages of the Caudoviricetes class. Thus, mutant strains lacking PSP are phage resistant. However, because PSP is a key cell wall component, PSP-negative mutants exhibit dramatic alterations of cell shape and severe growth defects, which limit their technological value. In the present study, we isolated spontaneous mutants with improved growth, from L. cremoris PSP-negative mutants. These mutants grow at rates similar to the wild-type strain, and based on transmission electron microscopy analysis, they exhibit improved cell morphology compared to their parental PSP-negative mutants. In addition, the selected mutants maintain their phage resistance. Whole-genome sequencing of several such mutants showed that they carried a mutation in pbp2b, a gene encoding a penicillin-binding protein involved in peptidoglycan biosynthesis. Our results indicate that lowering or turning off PBP2b activity suppresses the requirement for PSP and ameliorates substantially bacterial fitness and morphology. IMPORTANCE Lactococcus lactis and Lactococcus cremoris are widely used in the dairy industry as a starter culture. As such, they are consistently challenged by bacteriophage infections which may result in reduced or failed milk acidification with associated economic losses. Bacteriophage infection starts with the recognition of a receptor at the cell surface, which was shown to be a cell wall polysaccharide (the polysaccharide pellicle [PSP]) for the majority of lactococcal phages. Lactococcal mutants devoid of PSP exhibit phage resistance but also reduced fitness, since their morphology and division are severely impaired. Here, we isolated spontaneous, food-grade non-PSP-producing L. cremoris mutants resistant to bacteriophage infection with a restored fitness. This study provides an approach to isolate non-GMO phage-resistant L. cremoris and L. lactis strains, which can be applied to strains with technological functionalities. Also, our results highlight for the first time the link between peptidoglycan and cell wall polysaccharide biosynthesis.
Abstract Background : Yarrowia lipolytica , a non-conventional oleaginous yeast species, has attracted attention due to its high lipid degradation and accumulation capacity. Y lipolytica is used as a chassis for the production of usual and unusual lipids and lipids derivatives. While genes involved in the intracellular transport and activation of fatty acids in the different cellular compartments have been characterized, no genes involved in fatty acid transport from the extracellular medium into the cell have been identified so far. In this study, we have identified secreted proteins involved in extracellular fatty acid binding. Results : The recent analysis of the Y. lipolytica secretome leads to the identification of a multi-gene family composed of four secreted proteins hereafter named UP1 to UP4. The protein products were efficiently over-expressed individually in native and multi-deletant strain (Q4: Δup1Δup2Δup3Δup4 ) backgrounds. Phenotype analysis demonstrated the involvement of those proteins in the binding of extracellular fatty acid. Also, deletion of these genes could prevent octanoic acid (C8) toxicity; while their individual over-expression increased sensitivity to its toxic action. The results suggested binding according to aliphatic chain length- and fatty acid concentration-dependent manner. 3D structure modelling supports at a molecular level their role in fatty acid accommodation. Conclusions : Extracellular fatty acid binding proteins were identified for the first time in Y. lipolytica . The new gene family names are proposed eFbp1 to eFbp4. The exact mode of eFbps action remains to be deciphered individually and synergistically, nevertheless, it is expected that the proteins may be relevant in lipid biotechnology, such as improving fatty acid production and/or bioconversion.
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