Microbial Catabolic Pathway Of Dipicolinic Acid And The Function And Distribution Of Catabolic Genes

Pyridine-2,6-dicarboxylic acid（Dipicolinic acid,DPA）,a principal“non-nutrient”component of Bacillus spores for heat resistance,constitutes 5%-15%of the dry weight of spores.DPA is 1:1 chelated with divalent cations(mainly Ca²⁺)in the spore core and connot be catabolized by spores.It is rapidly released into the environment during spore germination.DPA is a major proportion of the global biomass carbon pool.Based on different models,it is estimated that the total DPA amount contained in spores in the uppermost kilometer of global marine sediments is between 5.6×10¹²mol and 4.2×10¹³mol（approximately 936million tons to 7.002 billion tons）.Estimated carbon pool of the DPA is 0.47 Pg C to 3.53 Pg C（1 Pg C is 1 billion tons of carbon）,accounting for about 0.85‰to 6.03‰of the global biomass carbon pool.Although DPA is widely distributed in the environment,it has not received considerable attention so far.The environmental behavior of DPA,especially the microbial-mediated degradation,is rarely studied.The molecular mechanism of microbial degradation of DPA is unknown.DPA is also widely utilized as intermediates for chemosynthesis of pharmaceuticals and chemicals due to its unique compound structure,i.e.,1)the symmetrical carboxyl group substitution on 2’and 6’positions of pyridine ring,2)having both the rigidity of pyridine ring and the flexibility of carboxylate.It can be used to synthesize compounds such as 2,6-diamino-4-chloropyridine,2,6-pyridinedicarbonyl chloride,2,6-pyridine dimethanol,etc.DPA is utilized to inhibit the activity of metallo-β-lactamases and enhance the activity ofβ-lactam antibiotics.Additionally,DPA can against phytopathogens,which can play a role in the field of plant biological control.DPA is a natural compound,but the wide use of DPA in various fields such as medicine and chemical industry,biocontrol,and new material synthesis may lead to intensive emissions and residues in some areas,posing potential threats to the environment.Therefore,it is of great significance to study the molecular mechanism of DPA degradation by bacteria.The study aims to identify the DPA degraders,elucidate the degradation pathway of DPA,clone DPA-degrading gene clusters and characterize the genes,for explaining the molecular mechanism of microbial degradation of DPA.The main results of the paper are as follows:1、Degradation characteristics and pathway of DPA by Alcaligenes faecalis JQ135The multifunctional pyridine degrader Alcaligenes faecalis JQ135 was used for the study of DPA degradation.The degradation characteristics of DPA were firstly studied.It was determined that the strain JQ135 could degrade DPA after induced by the component,and utilize DPA as the sole carbon,nitrogen and energy sources for growth.The highest efficiency of DPA degradation was obtained when pH was 7.0 and temperature was 30℃.In the OD₆₀₀range of 0.1 to 0.5,the larger the inoculum amount,the higher the degradation efficiency.When the OD₆₀₀ of strain JQ135 was 0.25,the strain JQ135 could degrade 1.8 mM(300 mg L^-1)DPA in 28 h.Three DPA catabolic intermediates were identified,which were 3-hydroxydipicolinic acid（3HDPA）,2-oxalyl-imino-5-oxo-pent-3-enoic acid and 4-imino-pent-2-enedioic acid.The oxygen atom source of the hydroxyl group on 3HDPA is from oxygen gas,which was determined by the ¹⁸O₂ stable isotope labeling experiment.It was inferred that the first step that hydroxylating DPA to 3HDPA was catalyzed by monooxygenase.Based on above results and previous studies,the pathway for DPA degradation was proposed,termed as 3HDPA pathway.2、Clone of DPA-degrading gene clusters and characterization of the genesEvidence in this study showed that the molecular mechanism of the 3HDPA pathway is different from maleamate pathway.Maleamate pathway is an important pathway for the catabolism of pyridine derivatives in Gram-negative bacteria.A mutagenesis library was constructed using the p SC123 based transposon to clone the genes responsible for DPA catabolism.Then SEFA-PCR、q PCR and other methods were applied to clone the gene cluster that involved in DPA degradation.The gene cluster was termed as dip gene cluster.The dip gene cluster is induced by DPA and negatively regulated by transcriptional regulator DipR.The genes involved in the first-step hydroxylation of DPA catabolism were studied.It was proposed that the reaction was mediated by a multi-component monooxygenase.The monooxygenase gene dipD was the crucial gene to hydroxylate DPA to 3HDPA.When dipD was inactivated,strain JQ135 completely lost the capacity to degrade DPA,and the complementation of the dipD gene recovered the degradation capacity.DipD was proposed to be the crucial component of the multi-component monooxygenase.Gene knockout and complementation also revealed that the heme-binding protein gene dipF,the ferredoxin reductase gene dipG,and the ferredoxin gene cluster dipJKL were involved in the first hydroxylation reaction either.The genes above was speculated to encode other components of the multi-component monooxygenase.The highest identity of homologous protein sequence of DipD in the Swiss-Prot database is only 29.32%（the coverage is 76%）,indicates that DipD is a novel monooxygenase.Some representative oxygenases for microbial degradation,including 8 heme iron–dependent cytochrome P450 monooxygenases,7Fe（II）/α-ketoglutarate-dependent dioxygenases,12 flavin-dependent monooxygneases,13Rieske non-heme iron dependent oxygenases,and 6 ring cleavage oxygenases were selected for the alignment and phylogenetic analysis with DipD.DipD was distinctly different from the oxygenases above.Relatively,the identity of DipD and Rieske type oxygenases is higher.Some catalytic mechanisms of DipD and Rieske type oxygenases are similar,i.e.,ferredoxin and ferredoxin reductase are required for electron transference.However,the important coenzyme,heme-binding protein,is required for DipD,which is significantly different from the Rieske-type oxygenase.It is proposed that DipD is a novel monooxygenase with a novel catalytic mechanism.In this study,in vitro enzymatic catalysis and heterologous expression in different hosts of the monooxygenase were studied,but no activity was observed.This might be attributed to the lack of knowledge on other crucial cofactors or coenzymes of DipD,and the unsuitable hosts.3、Distribution of dip gene clustersTwo DPA degraders,Bordetella petrii MY10 and Achromobacter sp.MY14,was identified.The homologous dip gene clusters were annotated in the genomes either.Additionally,homologs of dip gene cluster were annotated in the genome of Bordetella bronchiseptica,a pathogen in Bordetella genus.Based on the numbers and distributions of gene elements in dip gene clusters,8 kind of the homologous dip gene clusters were summarized and analyzed.In general,two types of dip gene clusters were observed.For type 1,gene dipD was clustered with dipEFG,while for type 2,gene dipD was clustered with dipJKL.Type 1 of dip gene clusters were annotated in Betaproteobacteria and type 2 of dip gene clusters were annotated in both Alphaproteobacteria and Betaproteobacteria.It is suggested that the dip gene clusters were widely distributed in Alphaproteobacteria and Betaproteobacteria.The dip gene clusters of strains JQ135,MY10 and MY14 belonged to type 1,type 1 and type 2,respectively.All dip gene clusters contain dipR,dipD,dipJKL and dipF genes.It is proposed that the above genes are conserved genes of dip gene cluster.To preliminary reveal the microbial consortium for DPA degradation in environment,two DPA-degrading enrichments were obtained based on the samples from the North city and East city sewage treatment plant of Nanjing.The microbial community and the intergeneric abundance distribution of the crucial gene dipD in the two enrichments were analyzed by metagenomic sequencing.Similarly,in the two enrichments,Pandoraea is the dominant genus.There are nearly 10 major genera such as Cupriavidus and Acinetobacter in the two enrichments either.Four homologous protein sequences of DipD were annotated in the metagenomes of the two enrichments,they are identified in the genera of Comamonas（identity 87%,coverage 93%）,Pandoraea（identity 82%,coverage 91%）,Bordetella（identity 83%,coverage 92%）and Achromobacter（identity 83%,coverage 91%）respectively.In the two enrichments,the dipD gene abundance in Pandoraea is dominant（over 75%）.It is proposed that Pandoraea is the main degrader for DPA degradation.The Comamonas and Pandoraea are belongs to Betaproteobacteria either.In this study,the degradation characteristics and degradation pathways of DPA was studied in strain JQ135,the gene cluster for DPA degradation was cloned and the function of gene elements were elucidated.The homologous dip gene clusters were summarized and analyzed.The work also studied the microbial consortiums of DPA degradation in Nanjing municipal sewage.The study elucidated a novel ring-lysis catabolic pathway for the degradation of pyridine derivatives,cloned a novel monooxygenase gene for the hydroxylation of pyridine ring,provided insights for the degradation of DPA in environment.