scholarly journals Identification and characterization of a novelpicgene cluster responsible for picolinic acid degradation inAlcaligenes faecalisJQ135

2019 ◽  
Author(s):  
Jiguo Qiu ◽  
Lingling Zhao ◽  
Siqiong Xu ◽  
Qing Chen ◽  
Le Chen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Molecular Mechanisms ◽  
Tricarboxylic Acid Cycle ◽  
Picolinic Acid ◽  
Gene Clusters ◽  
Alcaligenes Faecalis ◽  
Pyridine Derivative ◽  
Complete Degradation ◽  
Important Intermediate ◽  
New Perspective

AbstractPicolinic acid (PA) is a natural toxic pyridine derivative. Microorganisms can degrade and utilize PA for growth. However, the full metabolic pathway and its physiological and genetic foundation remain unknown. In this study, we identified thepicgene cluster responsible for the complete degradation of PA fromAlcaligenes faecalisJQ135. PA was initially 6-hydroxylated into 6-hydroxypicolinic acid (6HPA) by PA dehydrogenase (PicA). 6HPA was then 3-hydroxylated by a four-component 6HPA monooxygenase (PicB) to form 3,6-dihydroxypicolinic acid (3,6DHPA), which was then converted into 2,5-dihydroxypyridine (2,5DHP) by a decarboxylase (PicC). The 2,5DHP was further degraded into fumaric acid, through PicD (2,5DHP dioxygenase), PicE (N-formylmaleamic acid deformylase), PicF (maleamic acid amidohydrolase), and PicG (maleic acid isomerase). Homologouspicgene clusters with diverse organizations were found to be widely distributed inα-,β-, andγ-Proteobacteria. Our findings provide new insights into the microbial metabolism of environmental toxic pyridine derivatives.ImportancePicolinic acid is a common metabolite of L-tryptophan and some aromatic compounds and is an important intermediate of industrial concern. Although the microbial degradation/detoxification of picolinic acid has been studied for over 50 years, the underlying molecular mechanisms are still unknown. Here, we show thepicgene cluster responsible for the complete degradation of picolinic acid into the tricarboxylic acid cycle. This gene cluster was found to be widespread in otherα-,β-, andγ-Proteobacteria. These findings provide new perspective for understanding the mechanisms of picolinic acid biodegradation in bacteria.

scholarly journals Identification and Characterization of a NovelpicGene Cluster Responsible for Picolinic Acid Degradation inAlcaligenes faecalisJQ135

2019 ◽  
Vol 201 (16) ◽  
Author(s):  
Jiguo Qiu ◽  
Lingling Zhao ◽  
Siqiong Xu ◽  
Qing Chen ◽  
Le Chen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Molecular Mechanisms ◽  
Picolinic Acid ◽  
Gene Clusters ◽  
Alcaligenes Faecalis ◽  
Degradation Pathway ◽  
Pyridine Derivative ◽  
Organic Chemical ◽  
Content Type ◽  
New Perspective

ABSTRACTPicolinic acid (PA) is a natural toxic pyridine derivative. Microorganisms can degrade and utilize PA for growth. However, the full catabolic pathway of PA and its physiological and genetic foundation remain unknown. In this study, we identified a gene cluster, designatedpicRCEDFB4B3B2B1A1A2A3, responsible for the degradation of PA fromAlcaligenes faecalisJQ135. Our results suggest that PA degradation pathway occurs as follows: PA was initially 6-hydroxylated to 6-hydroxypicolinic acid (6HPA) by PicA (a PA dehydrogenase). 6HPA was then 3-hydroxylated by PicB, a four-component 6HPA monooxygenase, to form 3,6-dihydroxypicolinic acid (3,6DHPA), which was then converted into 2,5-dihydroxypyridine (2,5DHP) by the decarboxylase PicC. 2,5DHP was further degraded to fumaric acid through PicD (2,5DHP 5,6-dioxygenase), PicE (N-formylmaleamic acid deformylase), PicF (maleamic acid amidohydrolase), and PicG (maleic acid isomerase). Homologouspicgene clusters with diverse organizations were found to be widely distributed inAlpha-,Beta-, andGammaproteobacteria. Our findings provide new insights into the microbial catabolism of environmental toxic pyridine derivatives.IMPORTANCEPicolinic acid is a common metabolite ofl-tryptophan and some aromatic compounds and is an important intermediate in organic chemical synthesis. Although the microbial degradation/detoxification of picolinic acid has been studied for over 50 years, the underlying molecular mechanisms are still unknown. Here, we show that thepicgene cluster is responsible for the complete degradation of picolinic acid. Thepicgene cluster was found to be widespread in otherAlpha-,Beta-, andGammaproteobacteria. These findings provide a new perspective for understanding the catabolic mechanisms of picolinic acid in bacteria.

scholarly journals Catabolism of 3-hydroxypyridine by Ensifer adhaerens HP1: a novel four-component gene encoding 3-hydroxypyridine dehydrogenase HpdA catalyzes the first step of biodegradation

2020 ◽  
Author(s):  
Haixia Wang ◽  
Xiaoyu Wang ◽  
Hao Ren ◽  
Xuejun Wang ◽  
Zhenmei Lu
Keyword(s):  
Gene Cluster ◽  
Microbial Degradation ◽  
Molecular Mechanisms ◽  
Gene Clusters ◽  
Sole Source ◽  
Pyridine Derivative ◽  
Gene Encoding ◽  
Ensifer Adhaerens ◽  
Important Building Block ◽  
Component Gene

Abstract3-Hydroxypyridine (3HP) is an important natural pyridine derivative. Ensifer adhaerens HP1 can utilize 3HP as the sole source of carbon, nitrogen and energy to grow. However, the genes responsible for the degradation of 3HP remain unknown. In this study, we predicted that a gene cluster, designated 3hpd, may be responsible for the degradation of 3HP. The initial hydroxylation of 3HP is catalyzed by a four-component dehydrogenase (HpdA1A2A3A4), leading to the formation of 2,5-dihydroxypyridine (2,5-DHP) in E. adhaerens HP1. In addition, the SRPBCC component in HpdA existed as a separate subunit, which is different from other SRPBCC-containing molybdohydroxylases acting on N-heterocyclic aromatic compounds. Our findings provide a better understanding of the microbial degradation of pyridine derivatives in nature. Additionally, research on the origin of the discovered four-component dehydrogenase with a separate SRPBCC domain may be of great significance.Importance3-Hydroxypyridine is an important building block for synthesizing drugs, herbicides and antibiotics. Although the microbial degradation of 3-hydroxypyridine has been studied for many years, the molecular mechanisms remain unclear. Here, we show that 3hpd is responsible for the catabolism of 3-hydroxypyridine. The 3hpd gene cluster was found to be widespread in Actinobacteria, Rubrobacteria, Thermoleophilia, and Alpha-, Beta-, and Gammaproteobacteria, and the genetic organization of the 3hpd gene clusters in these bacteria showed high diversity. Our findings provide new insight into the catabolism of 3-hydroxypyridine in bacteria.

scholarly journals 3-Hydroxypyridine Dehydrogenase HpdA Is Encoded by a Novel Four-Component Gene Cluster and Catalyzes the First Step of 3-Hydroxypyridine Catabolism in Ensifer adhaerens HP1

2020 ◽  
Vol 86 (19) ◽  
Author(s):  
Haixia Wang ◽  
Xiaoyu Wang ◽  
Hao Ren ◽  
Xuejun Wang ◽  
Zhenmei Lu
Keyword(s):  
Gene Cluster ◽  
Microbial Degradation ◽  
Molecular Mechanisms ◽  
Gene Clusters ◽  
Pyridine Derivative ◽  
Pyruvate Phosphate Dikinase ◽  
Content Type ◽  
Ensifer Adhaerens ◽  
Microbial Strains ◽  
Important Building Block

ABSTRACT 3-Hydroxypyridine (3HP) is an important natural pyridine derivative. Ensifer adhaerens HP1 can utilize 3HP as its sole sources of carbon, nitrogen, and energy to grow, but the genes responsible for the degradation of 3HP remain unknown. In this study, we predicted that a gene cluster, designated 3hpd, might be responsible for the degradation of 3HP. The analysis showed that the initial hydroxylation of 3HP in E. adhaerens HP1 was catalyzed by a four-component dehydrogenase (HpdA1A2A3A4) and led to the formation of 2,5-dihydroxypyridine (2,5-DHP). In addition, the SRPBCC component in HpdA existed as a separate subunit, which is different from other SRPBCC-containing molybdohydroxylases acting on N-heterocyclic aromatic compounds. Moreover, the results demonstrated that the phosphoenolpyruvate (PEP)-utilizing protein and pyruvate-phosphate dikinase were involved in the HpdA activity, and the presence of the gene cluster 3hpd was discovered in the genomes of diverse microbial strains. Our findings provide a better understanding of the microbial degradation of pyridine derivatives in nature and indicated that further research on the origin of the discovered four-component dehydrogenase with a separate SRPBCC domain and the function of PEP-utilizing protein and pyruvate-phosphate dikinase might be of great significance. IMPORTANCE 3-Hydroxypyridine is an important building block for the synthesis of drugs, herbicides, and antibiotics. Although the microbial degradation of 3-hydroxypyridine has been studied for many years, the molecular mechanisms remain unclear. Here, we show that 3hpd is responsible for the catabolism of 3-hydroxypyridine. The 3hpd gene cluster was found to be widespread in Actinobacteria, Rubrobacteria, Thermoleophilia, and Alpha-, Beta-, and Gammaproteobacteria, and the genetic organization of the 3hpd gene clusters in these bacteria shows high diversity. Our findings provide new insight into the catabolism of 3-hydroxypyridine in bacteria.

scholarly journals Novel 3,6-Dihydroxypicolinic Acid Decarboxylase-Mediated Picolinic Acid Catabolism inAlcaligenes faecalisJQ135

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Jiguo Qiu ◽  
Yanting Zhang ◽  
Shigang Yao ◽  
Hao Ren ◽  
Meng Qian ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Picolinic Acid ◽  
Alcaligenes Faecalis ◽  
Degradation Pathway ◽  
Site Directed Mutagenesis ◽  
Pyridine Derivative ◽  
Acid Decarboxylase ◽  
Microbial Cells ◽  
Content Type ◽  
Related Proteins

ABSTRACTPicolinic acid (PA), a typical C-2-carboxylated pyridine derivative, is a metabolite ofl-tryptophan and many other aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize PA for growth. However, the precise mechanism of PA metabolism remains unknown.Alcaligenes faecalisstrain JQ135 utilizes PA as its carbon and nitrogen source for growth. In this study, we screened a 6-hydroxypicolinic acid (6HPA) degradation-deficient mutant through random transposon mutagenesis. The mutant hydroxylated 6HPA into an intermediate, identified as 3,6-dihydroxypicolinic acid (3,6DHPA), with no further degradation. A novel decarboxylase, PicC, was identified to be responsible for the decarboxylation of 3,6DHPA to 2,5-dihydroxypyridine. Although, PicC belonged to the amidohydrolase 2 family, it shows low similarity (<45%) compared to other reported amidohydrolase 2 family decarboxylases. Moreover, PicC was found to form a monophyletic group in the phylogenetic tree constructed using PicC and related proteins. Further, the genetic deletion and complementation results demonstrated thatpicCwas essential for PA degradation. The PicC was Zn2+-dependent nonoxidative decarboxylase that can specifically catalyze the irreversible decarboxylation of 3,6DHPA to 2,5-dihydroxypyridine. TheKmandkcattoward 3,6DHPA were observed to be 13.44 μM and 4.77 s−1, respectively. Site-directed mutagenesis showed that His163 and His216 were essential for PicC activity. This study provides new insights into the microbial metabolism of PA at molecular level.IMPORTANCEPicolinic acid is a natural toxic pyridine derived froml-tryptophan metabolism and other aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize picolinic acid for their growth, and thus a microbial degradation pathway of picolinic acid has been proposed. Picolinic acid is converted into 6-hydroxypicolinic acid, 3,6-dihydroxypicolinic acid, and 2,5-dihydroxypyridine in turn. However, there was no physiological and genetic validation for this pathway. This study demonstrated that 3,6-dihydroxypicolinic acid was an intermediate in picolinic acid catabolism and further identified and characterized a novel amidohydrolase 2 family decarboxylase PicC. PicC was also shown to catalyze the decarboxylation of 3,6-dihydroxypicolinic acid into 2,5-dihydroxypyridine. This study provides a basis for understanding picolinic acid degradation and its underlying molecular mechanism.

scholarly journals The Novel Monocomponent FAD-dependent Monooxygenase HpaM Catalyzes the 2-Decarboxylative Hydroxylation of 5-Hydroxypicolinic Acid inAlcaligenes faecalisJQ135

2017 ◽  
Author(s):  
Jiguo Qiu ◽  
Bin Liu ◽  
Lingling Zhao ◽  
Yanting Zhang ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Microbial Degradation ◽  
Pyridine Ring ◽  
Degradation Process ◽  
Alcaligenes Faecalis ◽  
Pyridine Derivative ◽  
Pyridine Derivatives ◽  
Monooxygenase Activity ◽  
E Coli ◽  
Transformation Mechanisms

Abstract5-hydroxypicolinic acid (5HPA) is a natural pyridine derivative that can be microbially degraded. However, the physiological, biochemical, and genetic foundation of the microbial catabolism of 5HPA remains unknown. In this study, a gene clusterhpa(which is involved in degradation of 5HPA inAlcaligenes faecalisJQ135) was cloned and HpaM was identified as a novel monocomponent FAD-dependent monooxygenase. HpaM shared a sequence only 31% similarity with the most related protein 6-hydroxynicotinate 3-monooxygenase (NicC) ofPseudomonas putidaKT2440.hpaMwas heterologously expressed inE. coliBL21(DE3), and the recombinant HpaM was purified via Ni-affinity chromatography. HpaM catalyzed the 2-decarboxylative hydroxylation of 5-HPA, thus generating 2,5-dihydroxypyridine (2,5-DPH). Monooxygenase activity was only detected in the presence of FAD and NADH, but not of FMN and NADPH. The apparentKmvalues of HpaM toward 5HPA and NADH were 45.4 μ and 37.8 μ, respectively. Results of gene deletion and complementation showed thathpaMwas essential for 5HPA degradation inAlcaligenes faecalisJQ135.ImportancePyridine derivatives are ubiquitous in nature and important chemical materials that are currently widely used in agriculture, pharmaceutical, and chemical industries. Thus, the microbial degradation and transformation mechanisms of pyridine derivatives received considerable attention. Decarboxylative hydroxylation was an important degradation process in pyridine derivatives, and previously reported decarboxylative hydroxylations happened in the C3 of the pyridine ring. In this study, we cloned the gene clusterhpa, which is responsible for 5HPA degradation inAlcaligenes faecalisJQ135, thus identifying a novel monocomponent FAD-dependent monooxygenase HpaM. Unlike 3-decarboxylative monooxygenases, HpaM catalyzed decarboxylative hydroxylation in the C2 of the pyridine ring in 5-hydroxypicolinic acid. These findings deepen our understanding of the molecular mechanism of microbial degradation of pyridine derivatives. Furthermore, HpaM offers potential for applications to transform useful pyridine derivatives.

scholarly journals Cloning of Two Gene Clusters Involved in the Catabolism of 2,4-Dinitrophenol by Paraburkholderia sp. Strain KU-46 and Characterization of the Initial DnpAB Enzymes and a Two-Component Monooxygenase DnpC1C2

2019 ◽  
Author(s):  
Taisei Yamamoto ◽  
Yaxuan Liu ◽  
Nozomi Kohaya ◽  
Yoshie Hasegawa ◽  
Peter C.K. Lau ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Tricarboxylic Acid Cycle ◽  
Gene Clusters ◽  
Degradation Pathway ◽  
Pcr Analysis ◽  
Gram Negative Bacteria ◽  
Protein Database ◽  
Gene Encoding ◽  
Gram Negative ◽  
Two Component

AbstractBesides an industrial pollutant, 2,4-dinitrophenol (DNP) has been used illegally as a weight loss drug that had claimed human lives. Little is known about the metabolism of DNP, particularly among Gram-negative bacteria. In this study, two non-contiguous genetic loci of Paraburkholderia (formerly Burkholderia) sp. strain KU-46 genome were identified and four key initial genes (dnpA, dnpB, and dnpC1C2) were characterized to provide molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription PCR analysis indicated that the dnpA gene encoding the initial hydride transferase (28 kDa), and the dnpB gene encoding a nitrite-eliminating enzyme (33 kDa), are inducible by DNP and the two genes are organized in an operon. Purified DnpA and DnpB from overexpression clones in Escherichia coli effected the transformation of DNP to NP via the formation of hydride-Meisenheimer complex of DNP. The function of DnpB appears new since all homologs of DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER. DnpC1 and DnpC2 were functionally characterized as the respective FAD reductase and oxygenase components of the two-component NP monooxygenase. Both NP and 4-nitrocatechol were shown to be substrates, producing hydroquinone and hydroxyquinol, respectively. Elucidation of the hqdA1A2BCD gene cluster allows the delineation of the final degradation pathway of hydroquinone to ß-ketoadipate prior to its entry to the tricarboxylic acid cycle.ImportanceThis study fills a gap in our knowledge and understanding of the genetic basis and biochemical pathway for the degradation of 2,4-dinitrophenol (DNP) in Gram-negative bacteria, represented by the prototypical Paraburkholderia sp. strain KU-46 that metabolizes DNP through the formation of 4-nitrophenol, a pathway unseen by other DNP utilizers. The newly cloned genes could serve as DNA probes in biomonitoring as well as finding application in new biocatalyst development to access green chemicals. By and large, knowledge of the diverse strategies used by microorganisms to degrade DNP will contribute to the development of bioremediation solutions since DNP is an industrial pollutant used widely in the chemical industry for the synthesis of pesticides, insecticides, sulfur dyes, wood preservatives, and explosives, etc. (119 words)

scholarly journals A Novel Degradation Mechanism for Pyridine Derivatives inAlcaligenes faecalisJQ135

2018 ◽  
Vol 84 (15) ◽  
Author(s):  
Jiguo Qiu ◽  
Bin Liu ◽  
Lingling Zhao ◽  
Yanting Zhang ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Nicotinic Acid ◽  
Maleic Acid ◽  
Degradation Mechanism ◽  
Picolinic Acid ◽  
Alcaligenes Faecalis ◽  
Pyridine Derivative ◽  
Pyridine Derivatives ◽  
Monooxygenase Activity ◽  
Content Type ◽  
Gene Coding

ABSTRACT5-Hydroxypicolinic acid (5HPA), a natural pyridine derivative, is microbially degraded in the environment. However, the physiological, biochemical, and genetic foundations of 5HPA metabolism remain unknown. In this study, an operon (hpa), responsible for 5HPA degradation, was cloned fromAlcaligenes faecalisJQ135. HpaM was a monocomponent flavin adenine dinucleotide (FAD)-dependent monooxygenase and shared low identity (only 28 to 31%) with reported monooxygenases. HpaM catalyzed theorthodecarboxylative hydroxylation of 5HPA, generating 2,5-dihydroxypyridine (2,5DHP). The monooxygenase activity of HpaM was FAD and NADH dependent. The apparentKmvalues of HpaM for 5HPA and NADH were 45.4 μM and 37.8 μM, respectively. The geneshpaX,hpaD, andhpaFwere found to encode 2,5DHP dioxygenase,N-formylmaleamic acid deformylase, and maleamate amidohydrolase, respectively; however, the three genes were not essential for 5HPA degradation inA. faecalisJQ135. Furthermore, the genemaiA, which encodes a maleic acidcis-transisomerase, was essential for the metabolism of 5HPA, nicotinic acid, and picolinic acid inA. faecalisJQ135, indicating that it might be a key gene in the metabolism of pyridine derivatives. The genes and proteins identified in this study showed a novel degradation mechanism of pyridine derivatives.IMPORTANCEUnlike the benzene ring, the uneven distribution of the electron density of the pyridine ring influences the positional reactivity and interaction with enzymes; e.g., theorthoandparaoxidations are more difficult than themetaoxidations. Hydroxylation is an important oxidation process for the pyridine derivative metabolism. In previous reports, theorthohydroxylations of pyridine derivatives were catalyzed by multicomponent molybdenum-containing monooxygenases, while themetahydroxylations were catalyzed by monocomponent FAD-dependent monooxygenases. This study identified the new monocomponent FAD-dependent monooxygenase HpaM that catalyzed theorthodecarboxylative hydroxylation of 5HPA. In addition, we found that themaiAgene coding for maleic acidcis-transisomerase was pivotal for the metabolism of 5HPA, nicotinic acid, and picolinic acid inA. faecalisJQ135. This study provides novel insights into the microbial metabolism of pyridine derivatives.

scholarly journals Fungal chromatin mapping identifies BasR, as the regulatory node of bacteria-induced fungal secondary metabolism

2017 ◽  
Author(s):  
Juliane Fischer ◽  
Sebastian Y. Müller ◽  
Tina Netzker ◽  
Nils Jäger ◽  
Agnieszka Gacek-Matthews ◽  
...  
Keyword(s):  
Secondary Metabolism ◽  
Gene Cluster ◽  
Molecular Mechanisms ◽  
Effective Interaction ◽  
Chromatin Modification ◽  
Gene Clusters ◽  
Fungal Species ◽  
Transcriptional Initiation ◽  
Genome Wide ◽  
Eukaryotic Microorganisms

AbstractThe eukaryotic epigenetic machinery is targeted by bacteria to reprogram the response of eukaryotes during their interaction with microorganisms. In line, we discovered that the bacterium Streptomyces rapamycinicus triggered increased chromatin acetylation and thus activation of the silent secondary metabolism ors gene cluster leading to the production of orsellinic acid in the fungus Aspergillus nidulans. Using this model we aim at understanding molecular mechanisms of communication between bacteria and eukaryotic microorganisms based on bacteria-triggered chromatin modification. By genome-wide ChIP-seq analysis of acetylated histone H3 (H3K9ac, H3K14ac) we uncovered the unique chromatin landscape in A. nidulans upon co-cultivation with S. rapamycinicus. Genome-wide acetylation of H3K9 correlated with increased gene expression, whereas H3K14 appears to function in transcriptional initiation by providing a docking side for regulatory proteins. In total, histones belonging to six secondary metabolism gene clusters showed higher acetylation during co-cultivation including the ors, aspercryptin, cichorine, sterigmatocystin, anthrone and 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde gene cluster with the emericellamide cluster being the only one with reduced acetylation and expression. Differentially acetylated histones were also detected in genes involved in amino acid and nitrogen metabolism, signaling, and genes encoding transcription factors. In conjunction with LC-MS/MS and MALDI-MS imaging, molecular analyses revealed the cross-pathway control and Myb-like transcription factor BasR as regulatory nodes for transduction of the bacterial signal in the fungus. The presence of basR in other fungal species allowed forecasting the inducibility of ors-like gene clusters by S. rapamycinicus in these fungi, and thus their effective interaction with activation of otherwise silent gene clusters.

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 Looking for the X Factor in Bacterial Pathogenesis: Association of orfX-p47 Gene Clusters with Toxin Genes in Clostridial and Non-Clostridial Bacterial Species

Toxins ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 19 ◽  
Author(s):  
Maria B. Nowakowska ◽  
François P. Douillard ◽  
Miia Lindström
Keyword(s):  
Gene Cluster ◽  
In Silico ◽  
Botulinum Neurotoxin ◽  
Bacterial Species ◽  
Gene Clusters ◽  
Bacterial Pathogenesis ◽  
Toxin Gene ◽  
Toxin Complex ◽  
Analytical Tools ◽  
Bacillus Isolate

The botulinum neurotoxin (BoNT) has been extensively researched over the years in regard to its structure, mode of action, and applications. Nevertheless, the biological roles of four proteins encoded from a number of BoNT gene clusters, i.e., OrfX1-3 and P47, are unknown. Here, we investigated the diversity of orfX-p47 gene clusters using in silico analytical tools. We show that the orfX-p47 cluster was not only present in the genomes of BoNT-producing bacteria but also in a substantially wider range of bacterial species across the bacterial phylogenetic tree. Remarkably, the orfX-p47 cluster was consistently located in proximity to genes coding for various toxins, suggesting that OrfX1-3 and P47 may have a conserved function related to toxinogenesis and/or pathogenesis, regardless of the toxin produced by the bacterium. Our work also led to the identification of a putative novel BoNT-like toxin gene cluster in a Bacillus isolate. This gene cluster shares striking similarities to the BoNT cluster, encoding a bont/ntnh-like gene and orfX-p47, but also differs from it markedly, displaying additional genes putatively encoding the components of a polymorphic ABC toxin complex. These findings provide novel insights into the biological roles of OrfX1, OrfX2, OrfX3, and P47 in toxinogenesis and pathogenesis of BoNT-producing and non-producing bacteria.

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