FadD3 is an acyl‐CoA synthetase that initiates catabolism of cholesterol rings C and D in actinobacteria

The cholesterol catabolic pathway occurs in most mycolic acid‐containing actinobacteria, such as Rhodococcus jostii RHA1, and is critical for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl‐CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild‐type and accumulated 3aα‐H‐4α(3′‐propanoate)‐7aβ‐methylhexahydro‐1,5‐indanedione (HIP), consistent with the catabolism of half the steroid molecule. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3_Mtb catalysed the ATP‐dependent CoA thioesterification of HIP and its hydroxylated analogues, 5α‐OH HIP and 1β‐OH HIP. The apparent specificity constant (k_cat/K_m) of FadD3_Mtb for HIP was 7.3 ± 0.3 × 10⁵ M^?1 s^?1, 165 times higher than for 5α‐OH HIP, while the apparent K_m for CoASH was 110 ± 10 μM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3_Mtb did not detectably transform a metabolite with a partially degraded C17 side‐chain. Overall, these results indicate that FadD3 is a HIP‐CoA synthetase that initiates catabolism of steroid rings C and D after side‐chain degradation is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degradation enzymes.     

Molecular characterization of a new gene cluster for steroid degradation in Mycobacterium smegmatis

The C‐19 steroids 4‐androstene‐3,17‐dione (AD), 1,4‐androstadiene‐3,17‐dione (ADD) or 9α‐hydroxy‐4‐androstene‐3,17‐dione (9OH‐AD), which have been postulated as intermediates of the cholesterol catabolic pathway in Mycobacterium smegmatis, cannot be used as sole carbon and energy sources by this bacterium. Only the ΔkstR mutant which constitutively expresses the genes repressed by the KstR regulator can metabolize AD and ADD with severe difficulties but still cannot metabolize 9OH‐AD, suggesting that these compounds are not true intermediates but side products of the cholesterol pathway. However, we have found that some M. smegmatis spontaneous mutants mapped in the PadR‐like regulator (MSMEG_2868) can efficiently metabolize all C‐19 steroids. We have demonstrated that the PadR mutants allow the expression of a gene cluster named C‐19+ (MSMEG_2851 to MSMEG_2901) encoding steroid degrading enzymes, that are not expressed under standard culture conditions. The C‐19+ cluster has apparently evolved independently from the upper cholesterol kstR‐regulon, but both clusters converge on the lower cholesterol kstR2‐regulon responsible for the metabolism of C and D steroid rings. Homologous C‐19+ clusters have been found only in other actinobacteria that metabolize steroids, but remarkably it is absent in Mycobacterium tuberculosis.     

A patchwork pathway for oxygenase‐independent degradation of side chain containing steroids

The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen‐independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo‐, phyto‐ and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin‐containing steroid dehydrogenases are involved in C25‐hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26‐oic acids. Side chain degradation to androsta‐1,4‐diene‐3,17‐dione (ADD) via aldolytic C–C bond cleavages involves acyl‐CoA synthetases/dehydrogenases specific for the respective 26‐, 24‐ and 22‐oic acids/‐oyl‐CoAs and promiscuous MaoC‐like enoyl‐CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA‐ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase‐dependent reactions.     

Pathogen roid rage: Cholesterol utilization by Mycobacterium tuberculosis

Abstract

The ability of science and medicine to control the pathogen Mycobacterium tuberculosis (Mtb) requires an understanding of the complex host environment within which it resides. Pathological and biological evidence overwhelmingly demonstrate how the mammalian steroid cholesterol is present throughout the course of infection. Better understanding Mtb requires a more complete understanding of how it utilizes molecules like cholesterol in this environment to sustain the infection of the host. Cholesterol uptake, catabolism and broader utilization are important for maintenance of the pathogen in the host and it has been experimentally validated to contribute to virulence and pathogenesis. Cholesterol is catabolized by at least three distinct sub-pathways, two for the ring system and one for the side chain, yielding dozens of steroid intermediates with varying biochemical properties. Our ability to control this worldwide infectious agent requires a greater knowledge of how Mtb uses cholesterol to its advantage throughout the course of infection. Herein, the current state of knowledge of cholesterol metabolism by Mtb is reviewed from a biochemical perspective with a focus on the metabolic genes and pathways responsible for cholesterol steroid catabolism.     

A putative 3‐hydroxyisobutyryl‐CoA hydrolase is required for efficient symbiotic nitrogen fixation in Sinorhizobium meliloti and Sinorhizobium fredii NGR234

We report that the smb20752 gene of the alfalfa symbiont Sinorhizobium meliloti is a novel symbiotic gene required for full N₂‐fixation. Deletion of smb20752 resulted in lower nitrogenase activity and smaller nodules without impacting overall nodule morphology. Orthologs of smb20752 were present in all alpha and beta rhizobia, including the ngr_b20860 gene of Sinorhizobium fredii NGR234. A ngr_b20860 mutant formed Fix^? determinate nodules that developed normally to a late stage of the symbiosis on the host plants Macroptilium atropurpureum and Vigna unguiculata. However an early symbiotic defect was evident during symbiosis with Leucaena leucocephala, producing Fix^? indeterminate nodules. The smb20752 and ngr_b20860 genes encode putative 3‐hydroxyisobutyryl‐CoA (HIB‐CoA) hydrolases. HIB‐CoA hydrolases are required for l ‐valine catabolism and appear to prevent the accumulation of toxic metabolic intermediates, particularly methacrylyl‐CoA. Evidence presented here and elsewhere (Curson et al., 2014 , PLoS ONE 9:e97660) demonstrated that Smb20752 and NGR_b20860 can also prevent metabolic toxicity, are required for l ‐valine metabolism, and play an undefined role in 3‐hydroxybutyrate catabolism. We present evidence that the symbiotic defect of the HIB‐CoA hydrolase mutants is independent of the inability to catabolize l ‐valine and suggest it relates to the toxicity resulting from metabolism of other compounds possibly related to 3‐hydroxybutyric acid.     

The Mycobacterium DosR regulon structure and diversity revealed by comparative genomic analysis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), which claims approximately two million people annually, remains a global health concern. The non‐replicating or dormancy like state of this pathogen which is impervious to anti‐tuberculosis drugs is widely recognized as the culprit for this scenario. The dormancy survival regulator (DosR) regulon, composed of 48 co‐regulated genes, is held as essential for Mtb persistence. The DosR regulon is regulated by a two‐component regulatory system consisting of two sensor kinases—DosS (Rv3132c) and DosT (Rv2027c), and a response regulator DosR (Rv3133c). The underlying regulatory mechanism of DosR regulon expression is very complex. Many factors are involved, particularly the oxygen tension. The DosR regulon enables the pathogen to persist during lengthy hypoxia. Comparative genomic analysis demonstrated that the DosR regulon is widely distributed among the mycobacterial genomes, ranging from the pathogenic strains to the environmental strains. In‐depth studies on the DosR response should provide insights into its role in TB latency in vivo and shape new measures to combat this exceeding recalcitrant pathogen. J. Cell. Biochem. 114: 1–6, 2012. © 2012 Wiley Periodicals, Inc.     

Mechanistic and phylogenetic insights into actinobacteria-mediated oestrogen biodegradation in urban estuarine sediments

Steroidal oestrogens are often accumulated in urban estuarine sediments worldwide at microgram per gram levels. These aromatic steroids have been classified as endocrine disruptors and group 1 carcinogens. Microbial degradation is a naturally occurring mechanism that mineralizes oestrogens in the biosphere; however, the corresponding genes in oestrogen-degrading actinobacteria remain unidentified. In this study, we identified a gene cluster encoding several putative oestrogen-degrading genes (aed; actinobacterial oestrogen degradation) in actinobacterium Rhodococcus sp. strain B50. Among them, the aedA and aedB genes involved in oestrogenic A-ring cleavage were identified through gene-disruption experiments. We demonstrated that actinobacterial oestrone 4-hydroxylase (AedA) is a cytochrome P450-type monooxygenase. We also detected the accumulation of two extracellular oestrogenic metabolites, including pyridinestrone acid (PEA) and 3aα-H-4α(3'-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), in the oestrone-fed strain B50 cultures. Since actinobacterial aedB and proteobacterial edcB shared < 40% sequence identity, 4-hydroxyestrone 4,5-dioxygenase genes (namely aedB and edcB) could serve as a specific biomarker to differentiate the contribution of actinobacteria and proteobacteria in environmental oestrogen degradation. Therefore, 4-hydroxyestrone 4,5-dioxygenase genes and the extracellular metabolites PEA and HIP were used as biomarkers to investigate oestrogen biodegradation in an urban estuarine sediment. Interestingly, our data suggested that actinobacteria are active oestrogen degraders in the urban estuarine sediment.     

Impact of bedaquiline and capreomycin on the gene expression patterns of multidrug-resistant Mycobacterium tuberculosis H37Rv strain and understanding the molecular mechanism of antibiotic resistance

The emergence of multidrug resistance (MDR), extensively drug-resistant, and total drug-resistant Mycobacterium tuberculosis (Mtb) strains have hampered the treatment of tuberculosis (TB). Capreomycin and Bedaquiline are currently used for MDR-TB treatment. To understand the impact of these antibiotics on Mtb genes, we have curated the gene expression data where the Mtb cultures were exposed to the Bedaquiline and Capreomycin. Based on the P value cut off (<0.05) and logFC (<−0.5 and >+0.5) values, we have selected the top differentially expressed genes during the antibiotic exposures. We have observed that the top differentially expressed Mtb genes were related to universal stress genes, two-component regulatory systems, and drug efflux pumps. We have curated the Mtb gene datasets and carried out the functional over-representation analysis using the individual gene expression values. We further, constructed the gene interaction networks of antibiotic resistance genes and virulence genes of Mtb to understand the impact of the antibiotics at the molecular level and thus to understand the antimicrobial resistance and virulence patterns. Our study elucidates the impact of antibiotics on the Mtb genes at the molecular level and the positively enriched pathways, operons, and regulons data are helpful in understanding the resistance patterns in Mtb. The upregulated genes during the exposure of Bedaquiline and Capreomycin can be considered as potent drug targets for the development of new anti-TB drugs.     

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19_Mtb) transformed 3-oxo-4-cholesten-26-oate (k_cat/K_m = 0.33 × 10⁵ ± 0.03 × 10⁵ M⁻¹ s⁻¹) and represents orthologs that activate the C₈ side chain of cholesterol. Both CasG_RHA1 and FadD17_Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C₅ side chain of cholate, while FadD17 and its orthologs appear to activate the C₅ side chain of one or more cholesterol metabolites. CasI_RHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C₃ side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (k_cat/K_m = 2.4 × 10⁵ ± 0.1 × 10⁵ M⁻¹ s⁻¹ and 3.2 × 10⁵ ± 0.3 × 10⁵ M⁻¹ s⁻¹, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes.     

Substrate Uptake and Subcellular Compartmentation of Anoxic Cholesterol Catabolism in Sterolibacterium denitrificans

Cholesterol catabolism by actinobacteria has been extensively studied. In contrast, the uptake and catabolism of cholesterol by Gram-negative species are poorly understood. Here, we investigated microbial cholesterol catabolism at the subcellular level. ¹³C metabolomic analysis revealed that anaerobically grown Sterolibacterium denitrificans, a β-proteobacterium, adopts an oxygenase-independent pathway to degrade cholesterol. S. denitrificans cells did not produce biosurfactants upon growth on cholesterol and exhibited high cell surface hydrophobicity. Moreover, S. denitrificans did not produce extracellular catabolic enzymes to transform cholesterol. Accordingly, S. denitrificans accessed cholesterol by direction adhesion. Cholesterol is imported through the outer membrane via a putative FadL-like transport system, which is induced by neutral sterols. The outer membrane steroid transporter is able to selectively import various C₂₇ sterols into the periplasm. S. denitrificans spheroplasts exhibited a significantly higher efficiency in cholest-4-en-3-one-26-oic acid uptake than in cholesterol uptake. We separated S. denitrificans proteins into four fractions, namely the outer membrane, periplasm, inner membrane, and cytoplasm, and we observed the individual catabolic reactions within them. Our data indicated that, in the periplasm, various periplasmic and peripheral membrane enzymes transform cholesterol into cholest-4-en-3-one-26-oic acid. The C₂₇ acidic steroid is then transported into the cytoplasm, in which side-chain degradation and the subsequent sterane cleavage occur. This study sheds light into microbial cholesterol metabolism under anoxic conditions.     

A single‐stranded DNA aptamer against mannose‐capped lipoarabinomannan enhances anti‐tuberculosis activity of macrophages through downregulation of lipid‐sensing nuclear receptor peroxisome proliferator‐activated receptor γ expression

Mannose‐capped lipoarabinomannan (ManLAM) is an immunomodulatory epitope of Mycobacterium tuberculosis (Mtb). An aptamer (ZXL1) that specifically binds to ManLAM from the virulent Mtb H37Rv strain was previously generated and it was found that ZXL1 functions as an antagonist, inhibiting the ManLAM‐induced immunosuppression of DCs. In the present study, it was found that ZXL1 inhibits Mtb entry into murine macrophages and that ZXL1 enhances IL‐1β and IL‐12 mRNA expression and cytokine production in ManLAM‐treated macrophages but decreases IL‐10 production. Inducible nitric oxide synthase expression in macrophages was upregulated in the presence of ZXL1 after stimulation with ManLAM. ZXL1 was also found to inhibit expression of lipid‐sensing nuclear receptor peroxisome proliferator‐activated receptor γ (PPAR‐γ). These results suggest that ZXL1 promotes anti‐tuberculosis activity through downregulation of PPAR‐γ expression, which may contribute to M1 macrophage polarization and Mtb killing by macrophages.