scholarly journals Characterization of L-Carnitine Metabolism inSinorhizobium meliloti

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Pascal Bazire ◽  
Nadia Perchat ◽  
Ekaterina Darii ◽  
Christophe Lechaplais ◽  
Marcel Salanoubat ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Sinorhizobium Meliloti ◽  
Gene Clusters ◽  
Fatty Acid Transport ◽  
Context Analysis ◽  
Content Type ◽  
Carnitine Metabolism ◽  
Conserved Gene

ABSTRACTl-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from γ-butyrobetaine (GBB) and can be used as a carbon source.l-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosynthetic route analogous to fatty acid degradation. However, this multistep pathway has not been experimentally characterized. In this work, we identified by gene context analysis a cluster ofl-carnitine anabolic genes next to those involved in its catabolism and proceeded to the completein vitrocharacterization ofl-carnitine biosynthesis and degradation inSinorhizobium meliloti. The five enzymes catalyzing the seven steps that convert GBB to glycine betaine are described. Metabolomic analysis confirmed the multistage synthesis ofl-carnitine in GBB-grown cells but also revealed that GBB is synthesized byS. meliloti. To our knowledge, this is the first report of aerobic GBB synthesis in bacteria. The conservation ofl-carnitine metabolism genes in different bacterial taxonomic classes underscores the role ofl-carnitine as a ubiquitous nutrient.IMPORTANCEThe experimental characterization of novel metabolic pathways is essential for realizing the value of genome sequences and improving our knowledge of the enzymatic capabilities of the bacterial world. However, 30% to 40% of genes of a typical genome remain unannotated or associated with a putative function. We used enzyme kinetics, liquid chromatography-mass spectroscopy (LC-MS)-based metabolomics, and mutant phenotyping for the characterization of the metabolism ofl-carnitine inSinorhizobium melilotito provide an accurate annotation of the corresponding genes. The occurrence of conserved gene clusters for carnitine metabolism in soil, plant-associated, and marine bacteria underlines the environmental abundance of carnitine and suggests this molecule might make a significant contribution to ecosystem nitrogen and carbon cycling.

scholarly journals Synthesis, Quantification, and Characterization of Fatty Acid Amides from In Vitro and In Vivo Sources

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2543
Author(s):  
Ruidong Ni ◽  
Suzeeta Bhandari ◽  
Perry R. Mitchell ◽  
Gabriela Suarez ◽  
Neel B. Patel ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fatty Acid ◽  
Apis Mellifera ◽  
Chemical Synthesis ◽  
Acid Amide ◽  
Fatty Acid Amides ◽  
Fatty Acid Amide

Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented.

scholarly journals Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Kkabi Son ◽  
Yangmee Kim
Keyword(s):  
Fatty Acid ◽  
Acinetobacter Baumannii ◽  
Gene Cluster ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Carrier Protein ◽  
Aryl Group ◽  
Visible Absorption

AbstractSome Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure–function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host–pathogen interaction mechanisms and novel antibiotics discovery.

scholarly journals Identification of the molecular regulation of differences in lipid deposition in dedifferentiated preadipocytes from different chicken tissues

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zheng Ma ◽  
Na Luo ◽  
Lu Liu ◽  
Huanxian Cui ◽  
Jing Li ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Metabolism ◽  
Lipid Content ◽  
Regulatory Network ◽  
Total Lipid Content ◽  
Fatty Acid Transport ◽  
Lipid Deposition ◽  
Ppar Signaling ◽  
The Difference

Abstract Background A body distribution with high intramuscular fat and low abdominal fat is the ideal goal for broiler breeding. Preadipocytes with different origins have differences in terms of metabolism and gene expression. The transcriptome analysis performed in this study of intramuscular preadipocytes (DIMFPs) and adipose tissue-derived preadipocytes (DAFPs) aimed to explore the characteristics of lipid deposition in different chicken preadipocytes by dedifferentiation in vitro. Results Compared with DAFPs, the total lipid content in DIMFPs was reduced (P < 0.05). Moreover, 72 DEGs related to lipid metabolism were screened, which were involved in adipocyte differentiation, fatty acid transport and fatty acid synthesis, lipid stabilization, and lipolysis. Among the 72 DEGs, 19 DEGs were enriched in the PPAR signaling pathway, indicating its main contribution to the regulation of the difference in lipid deposition between DAFPs and DIMFPs. Among these 19 genes, the representative APOA1, ADIPOQ, FABP3, FABP4, FABP7, HMGCS2, LPL and RXRG genes were downregulated, but the ACSL1, FABP5, PCK2, PDPK1, PPARG, SCD, SCD5, and SLC27A6 genes were upregulated (P < 0.05 or P < 0.01) in the DIMFPs. In addition, the well-known pathways affecting lipid metabolism (MAPK, TGF-beta and calcium) and the pathways related to cell communication were enriched, which may also contribute to the regulation of lipid deposition. Finally, the regulatory network for the difference in lipid deposition between chicken DAFPs and DIMFPs was proposed based on the above information. Conclusions Our data suggested a difference in lipid deposition between DIMFPs and DAFPs of chickens in vitro and proposed a molecular regulatory network for the difference in lipid deposition between chicken DAFPs and DIMFPs. The lipid content was significantly increased in DAFPs by the direct mediation of PPAR signaling pathways. These findings provide new insights into the regulation of tissue-specific fat deposition and the optimization of body fat distribution in broilers.

scholarly journals Low-Molecular-Weight Metabolites Secreted by Paenibacillus larvae as Potential Virulence Factors of American Foulbrood

2014 ◽  
Vol 80 (8) ◽  
pp. 2484-2492 ◽  
Author(s):  
Hedwig-Annabell Schild ◽  
Sebastian W. Fuchs ◽  
Helge B. Bode ◽  
Bernd Grünewald
Keyword(s):  
Molecular Weight ◽  
Virulence Factors ◽  
Gene Clusters ◽  
Paenibacillus Larvae ◽  
Economic Losses ◽  
Low Molecular Weight ◽  
American Foulbrood ◽  
Toxic Activity ◽  
Content Type

ABSTRACTThe spore-forming bacteriumPaenibacillus larvaecauses a severe and highly infective bee disease, American foulbrood (AFB). Despite the large economic losses induced by AFB, the virulence factors produced byP. larvaeare as yet unknown. To identify such virulence factors, we experimentally infected young, susceptible larvae of the honeybee,Apis mellifera carnica, with differentP. larvaeisolates. Honeybee larvae were rearedin vitroin 24-well plates in the laboratory after isolation from the brood comb. We identified genotype-specific differences in the etiopathology of AFB between the tested isolates ofP. larvae, which were revealed by differences in the median lethal times. Furthermore, we confirmed that extracts ofP. larvaecultures contain low-molecular-weight compounds, which are toxic to honeybee larvae. Our data indicate thatP. larvaesecretes metabolites into the medium with a potent honeybee toxic activity pointing to a novel pathogenic factor(s) ofP. larvae. Genome mining ofP. larvaesubsp.larvaeBRL-230010 led to the identification of several biosynthesis gene clusters putatively involved in natural product biosynthesis, highlighting the potential ofP. larvaeto produce such compounds.

scholarly journals A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection

2015 ◽  
Vol 81 (18) ◽  
pp. 6129-6144 ◽  
Author(s):  
Abdulsamie Hanano ◽  
Ibrahem Almousally ◽  
Mouhnad Shaban ◽  
Elizabeth Blee
Keyword(s):  
Fatty Acid ◽  
Aspergillus Flavus ◽  
Polyunsaturated Fatty Acid ◽  
Calcium Binding ◽  
Antioxidant Activities ◽  
Fungal Growth ◽  
Oxidation Reactions ◽  
Content Type ◽  
Fatty Acid Hydroperoxides

ABSTRACTCaleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein inAspergillus flavusthat is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway,aflRandaflD, were downregulated in the strains in whichA. flavusPXG(AfPXG) was silenced, leading to reduced aflatoxin B1 productionin vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in whichAfPXGwas silenced.PXG-deficientA. flavusstrains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease.

scholarly journals Bacteriocins of Non-aureus Staphylococci Isolated from Bovine Milk

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Domonique A. Carson ◽  
Herman W. Barkema ◽  
Sohail Naushad ◽  
Jeroen De Buck
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Infections ◽  
Genome Mining ◽  
Bovine Milk ◽  
Gene Clusters ◽  
Antibiotic Use ◽  
Content Type ◽  
Treatment And Prevention ◽  
Whole Genomes

ABSTRACT Non-aureus staphylococci (NAS), the bacteria most commonly isolated from the bovine udder, potentially protect the udder against infection by major mastitis pathogens due to bacteriocin production. In this study, we determined the inhibitory capability of 441 bovine NAS isolates (comprising 26 species) against bovine Staphylococcus aureus. Furthermore, inhibiting isolates were tested against a human methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking method. We determined the presence of bacteriocin clusters in NAS whole genomes using genome mining tools, BLAST, and comparison of genomes of closely related inhibiting and noninhibiting isolates and determined the genetic organization of any identified bacteriocin biosynthetic gene clusters. Forty isolates from 9 species (S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus) inhibited growth of S. aureus in vitro, 23 isolates of which, from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus, also inhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing 6 different classes (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. A total of 25 novel bacteriocin precursors were described. In conclusion, NAS from bovine mammary glands are a source of potential bacteriocins, with >21% being possible producers, representing potential for future characterization and prospective clinical applications. IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus) costs Canadian dairy producers $400 million/year and is the leading cause of antibiotic use on dairy farms. With increasing antibiotic resistance and regulations regarding use, there is impetus to explore bacteriocins (bacterially produced antimicrobial peptides) for treatment and prevention of bacterial infections. We examined the ability of 441 NAS bacteria from Canadian bovine milk samples to inhibit growth of S. aureus in the laboratory. Overall, 9% inhibited growth of S. aureus and 58% of those also inhibited MRSA. In NAS whole-genome sequences, we identified >21% of NAS as having bacteriocin genes. Our study represents a foundation to further explore NAS bacteriocins for clinical use.

scholarly journals Restoration of Bioactive Lantibiotic Suicin from a RemnantlanLocus of Pathogenic Streptococcus suis Serotype 2

2013 ◽  
Vol 80 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
Jian Wang ◽  
Yong Gao ◽  
Kunling Teng ◽  
Jie Zhang ◽  
Shutao Sun ◽  
...  
Keyword(s):  
Gene Promoter ◽  
Streptococcus Suis ◽  
Gene Clusters ◽  
Disulfide Bridge ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
System A ◽  
Bactericidal Activities

ABSTRACTLantibiotics are ribosomally synthesized, posttranslationally modified antimicrobial peptides. Their biosynthesis genes are usually organized in gene clusters, which are mainly found in Gram-positive bacteria, including pathogenic streptococci. Three highly virulentStreptococcus suisserotype 2 strains (98HAH33, 05ZYH33, and SC84) have been shown to contain an 89K pathogenicity island. Here, on these islands, we unveiled and reannotated a putative lantibiotic locus designatedsuiwhich contains a virulence-associated two-component regulator,suiK-suiR. In silicoanalysis revealed that the putative lantibiotic modification genesuiMwas interrupted by a 7.9-kb integron and that other biosynthesis-related genes contained various frameshift mutations. By reconstituting the intactsuiMinEscherichia colitogether with a semi-in vitrobiosynthesis system, a putative lantibiotic named suicin was produced with bactericidal activities against a variety of Gram-positive strains, including pathogenic streptococci and vancomycin-resistant enterococci. Ring topology dissection indicated that the 34-amino-acid lantibiotic contained two methyllanthionine residues and one disulfide bridge, which render suicin in an N-terminal linear and C-terminal globular shape. To confirm the function ofsuiK-suiR, SuiR was overexpressed and purified.In vitroanalysis showed that SuiR could specifically bind to thesuiAgene promoter. Its coexpression withsuiKcould activatesuiAgene promoter inLactococcus lactisNZ9000. Conclusively, we obtained a novel lantibiotic suicin by restoring its production from the remnantsuilocus and demonstrated that virulence-associated SuiK-SuiR regulates its production.

scholarly journals Complete Genome Sequences of Three Bacillus amyloliquefaciens Strains That Inhibit the Growth of Listeria monocytogenes In Vitro

2018 ◽  
Vol 6 (25) ◽  
Author(s):  
Thao D. Tran ◽  
Steven Huynh ◽  
Craig T. Parker ◽  
Robert Hnasko ◽  
Lisa Gorski ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Secondary Metabolites ◽  
Growth Inhibition ◽  
Complete Genome ◽  
Bacillus Amyloliquefaciens ◽  
Gene Clusters ◽  
Genome Sequences ◽  
Multiple Gene ◽  
Content Type

ABSTRACT Here, we report the complete genome sequences of three Bacillus amyloliquefaciens strains isolated from alfalfa, almond drupes, and grapes that inhibited the growth of Listeria monocytogenes strain 2011L-2857 in vitro. We also report multiple gene clusters encoding secondary metabolites that may be responsible for the growth inhibition of L. monocytogenes.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti

Microbiology ◽  
2020 ◽  
Vol 166 (5) ◽  
pp. 484-497 ◽  
Author(s):  
Alejandra Arteaga Ide ◽  
Victor M. Hernández ◽  
Liliana Medina-Aparicio ◽  
Edson Carcamo-Noriega ◽  
Lourdes Girard ◽  
...  
Keyword(s):  
Type Species ◽  
Sinorhizobium Meliloti ◽  
Arginase Activity ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Link Type ◽  
Arginine Catabolism

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

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