Systematic Discovery of a New Catalogue of Tyrosine-Type Integrases in Bacterial Genomic Islands.

Site-specific recombinases (integrases) can mediate the horizontal transfer of genomic islands. The ability to integrate large DNA sequences into target sites is very important for genetic engineering in prokaryotic and eukaryotic cells. Here, we characterized an unprecedented catalogue of 530 tyrosine-type integrases by examining genes potentially encoding tyrosine integrases in bacterial genomic islands. The phylogeny of putative tyrosine integrases revealed that these integrases form an evolutionary clade that is distinct from those already known and are affiliated with novel integrase groups. We systematically searched for candidate integrase genes, and their integration activities were validated in a bacterial model. We verified the integration functions of six representative novel integrases by using a two-plasmid integration system consisting of a donor plasmid carrying the integrase gene and attP site and a recipient plasmid harboring an attB site in recA-deficient Escherichia coli. Further quantitative reverse transcription-PCR (qRT-PCR) assays validated that the six selected integrases can be expressed with their native promoters in E. coli. The attP region reductions showed that the extent of attP sites of integrases is approximately 200 bp for integration capacity. In addition, mutational analysis showed that the conserved tyrosine at the C terminus is essential for catalysis, confirming that these candidate proteins belong to the tyrosine-type recombinase superfamily, i.e., tyrosine integrases. This study revealed that the novel integrases from bacterial genomic islands have site-specific recombination functions, which is of physiological significance for their genomic islands in bacterial chromosomes. More importantly, our discovery expands the toolbox for genetic engineering, especially for efficient integration activity. IMPORTANCE Site-specific recombinases or integrases have high specificity for DNA large fragment integration, which is urgently needed for gene editing. However, known integrases are not sufficient for meeting multiple integrations. In this work, we discovered an array of integrases through bioinformatics analysis in bacterial genomes. Phylogeny and functional assays revealed that these new integrases belong to tyrosine-type integrases and have the ability to conduct site-specific recombination. Moreover, attP region extent and catalysis site analysis were characterized. Our study provides the methodology for discovery of novel integrases and increases the capacity of weapon pool for genetic engineering in bacteria.

Efficient Suppression of Natural Plasmid-Borne Gene Expression in Carbapenem-Resistant Klebsiella pneumoniae Using a Compact CRISPR Interference System.

There is an urgent need for efficient tools for genetic manipulation to assess plasmid function in clinical drug-resistant bacterial strains. To address this need, we developed an all-in-one CRISPR interference (CRISPRi) system that easily inhibited the gene expression of a natural multidrug-resistant plasmid in an sequence type 23 (ST23) Klebsiella pneumoniae isolate. We established an integrative CRISPRi system plasmid, pdCas9gRNA, harboring a dcas9 gene and a single guide RNA (sgRNA) unit under the control of anhydrotetracycline-induced and J23119 promoters, respectively, using a one-step cloning method. This system can repress the single resistance gene blaNDM-1, with a >1,000-fold reduction in the meropenem MIC, or simultaneously silence the resistance genes blaNDM-1 and blaSHV-12, with a 16-fold and 8-fold respective reduction in the meropenem and aztreonam MIC on a large natural multidrug-resistant pNK01067-NDM-1 plasmid in an ST23 K. pneumoniae isolate. Furthermore, an sgRNA targeting the blaNDM-1 promoter region can silence the entire blaNDM-1-bleMBL-trpF operon, confirming the existence of the operon. We also used this tool to knock down the multicopy resistance gene blaKPC-2 in pathogenic Escherichia coli, increasing the susceptibility to meropenem. In a word, the all-in-one CRISPRi system can be used for efficient interrogation of indigenous plasmid-borne gene functions, providing a rapid, easy genetic manipulation tool for clinical K. pneumoniae isolates.

Novel 3,6-Dihydroxypicolinic Acid Decarboxylase-Mediated Picolinic Acid Catabolism in Alcaligenes faecalis JQ135.

Picolinic acid (PA), a typical C-2-carboxylated pyridine derivative, is a metabolite of l-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 faecalis strain 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 that picC was 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. The Km and kcat toward 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.IMPORTANCE Picolinic acid is a natural toxic pyridine derived from l-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.

The Properties of 5-Methyltetrahydrofolate Dehydrogenase (MetF1) and Its Role in the Tetrahydrofolate-Dependent Dicamba Demethylation System in Rhizorhabdus dicambivorans Ndbn-20.

The herbicide dicamba is initially degraded via the tetrahydrofolate (THF)-dependent demethylation system in Rhizorhabdus dicambivorans Ndbn-20. Two THF-dependent dicamba methyltransferase gene clusters, scaffold 50 and scaffold 66, were found in the genome of strain Ndbn-20. Each cluster contains a dicamba methyltransferase gene and three THF metabolism-related genes, namely, metF (coding for 5,10-CH2-THF reductase), folD (coding for 5,10-CH2-THF dehydrogenase-5,10-methenyl-THF cyclohydrolase), and purU (coding for 10-formyl-THF deformylase). In this study, reverse transcription-PCR (RT-PCR) results showed that only genes in scaffold 66, not those in scaffold 50, were transcribed in dicamba-cultured cells. The metF gene of scaffold 66 (metF1) was expressed in Escherichia coli BL21(DE3), and the product was purified as a His6-tagged protein. Purified MetF1 was found to be a monomer and exhibited 5-CH3-THF dehydrogenase activity in vitro The kcat and Km for 5-CH3-THF were 0.23 s-1 and 16.48 µM, respectively. However, 5,10-CH2-THF reductase activity was not detected for MetF1 under the conditions tested. Gene disruption results showed that metF1 is essential for dicamba degradation, whereas folD1 is dispensable.IMPORTANCE There are several THF-dependent methyltransferase genes and THF-metabolic genes in the genome of R. dicambivorans Ndbn-20; however, which genes are involved in dicamba demethylation and the mechanism underlying THF regeneration remain unknown. This study revealed that scaffold 66 is responsible for dicamba demethylation and that MetF1 physiologically catalyzes the dehydrogenation of 5-CH3-THF to 5,10-CH2-THF in the THF-dependent dicamba demethylation system in R. dicambivorans Ndbn-20. Furthermore, the results showed that MetF1 differs from previously characterized MetF in phylogenesis, biochemical properties, and catalytic activity; e.g., MetF1 in vitro did not show 5,10-CH2-THF reductase activity, which is the physiological function of Escherichia coli MetF. This study provides new insights into the mechanism of the THF-dependent methyltransferase system.

A Novel Aerobic Degradation Pathway for Thiobencarb Is Initiated by the TmoAB Two-Component Flavin Mononucleotide-Dependent Monooxygenase System in Acidovorax sp. Strain T1.

Thiobencarb is a thiocarbamate herbicide used in rice paddies worldwide. Microbial degradation plays a crucial role in the dissipation of thiobencarb in the environment. However, the physiological and genetic mechanisms underlying thiobencarb degradation remain unknown. In this study, a novel thiobencarb degradation pathway was proposed in Acidovorax sp. strain T1. Thiobencarb was oxidized and cleaved at the C-S bond, generating diethylcarbamothioic S-acid and 4-chlorobenzaldehyde (4CDA). 4CDA was then oxidized to 4-chlorobenzoic acid (4CBA) and hydrolytically dechlorinated to 4-hydroxybenzoic acid (4HBA). The identification of catabolic genes suggested further hydroxylation to protocatechuic acid (PCA) and finally degradation through the protocatechuate 4,5-dioxygenase pathway. A novel two-component monooxygenase system identified in the strain, TmoAB, was responsible for the initial catabolic reaction. TmoA shared 28 to 32% identity with the oxygenase components of pyrimidine monooxygenase from Agrobacterium fabrum, alkanesulfonate monooxygenase from Pseudomonas savastanoi, and dibenzothiophene monooxygenase from Rhodococcus sp. TmoB shared 25 to 37% identity with reported flavin reductases and oxidized NADH but not NADPH. TmoAB is a flavin mononucleotide (FMN)-dependent monooxygenase and catalyzed the C-S bond cleavage of thiobencarb. Introduction of tmoAB into cells of the thiobencarb degradation-deficient mutant T1m restored its ability to degrade and utilize thiobencarb. A dehydrogenase gene, tmoC, was located 7,129 bp downstream of tmoAB, and its transcription was clearly induced by thiobencarb. The purified TmoC catalyzed the dehydrogenation of 4CDA to 4CBA using NAD+ as a cofactor. A gene cluster responsible for the complete 4CBA metabolic pathway was also cloned, and its involvement in thiobencarb degradation was preliminarily verified by transcriptional analysis.IMPORTANCE Microbial degradation is the main factor in thiobencarb dissipation in soil. In previous studies, thiobencarb was degraded initially via N-deethylation, sulfoxidation, hydroxylation, and dechlorination. However, enzymes and genes involved in the microbial degradation of thiobencarb have not been studied. This study revealed a new thiobencarb degradation pathway in Acidovorax sp. strain T1 and identified a novel two-component FMN-dependent monooxygenase system, TmoAB. Under TmoAB-mediated catalysis, thiobencarb was cleaved at the C-S bond, producing diethylcarbamothioic S-acid and 4CDA. Furthermore, the downstream degradation pathway of thiobencarb was proposed. Our study provides the physiological, biochemical, and genetic foundation of thiobencarb degradation in this microorganism.

Harnessing the Native Type I-F CRISPR-Cas System of Acinetobacter baumannii for Genome Editing and Gene Repression.

INTRODUCTION: The rapid emergence of infections caused by multidrug-resistant Acinetobacter baumannii (A. baumannii) has posed a serious threat to global public health. It has therefore become important to obtain a deeper understanding of the mechanisms of multidrug resistance and pathogenesis of A. baumannii; however, there are still relatively few genetic engineering tools for this. Although A. baumannii possesses Type I-F CRISPR-Cas systems, they have not yet been used for genetic modifications. METHODS: A single plasmid-mediated native Type I-F CRISPR-Cas system for gene editing and gene regulation in A. baumannii was developed. The protospacer adjacent motif sequence was identified as 5'-NCC-3' by analysis of the CRISPR array. RESULTS: Through introduction of the RecAb homologous recombination system, the knockout efficiency of the oxyR gene significantly increased from 12.5% to 75.0% in A. baumannii. To investigate transcriptional inhibition by the Type I-F CRISPR system, the gene encoding its Cas2-3 nuclease was deleted and the native Type I-F Cascade effector was repurposed to regulate transcription of alcohol dehydrogenase gene adh4. The level of adh4 transcription was inhibited by up to 900-fold compared with the control. The Cascade transcriptional module was also successfully applied in a clinical Klebsiella pneumoniae isolate. CONCLUSION: This study proposed a tool for future exploration of the genetic characteristics of A. baumannii or other clinical strains.

Epidemicity and clonal replacement of hypervirulent carbapenem-resistant Klebsiella pneumoniae with diverse pathotypes and resistance profiles in a hospital.

OBJECTIVES: The emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) poses a great threat to public health. There is a paramount need to increase awareness of the epidemiology, evolution, and pathogenesis of CR-hvKP. METHODS: We collected strains of K. pneumoniae for over two years in a hospital. CR-hvKP strains were screened by polymerase chain reaction (PCR) with primers targeting the virulence genes. Genome sequencing was used to determine phylogenetic relationships and genetic characterization of virulence elements. The population dynamics within these strains were analyzed through epidemiological data. The string test, siderophore secretion, and murine infection experiments were performed to investigate virulence potential of different clones. RESULTS: A total of 1172 K. pneumoniae strains were isolated from 817 patients, and 125 isolates were identified as CR-hvKP. In all, 102 CR-hvKP strains belonged to sequence type (ST) 11. Genomic analysis demonstrated that three clones of ST11 successively replaced each other in the hospital. Among them, the strains of clade A and clade B acquired virulence plasmids and the strains of clade C acquired a new integrating conjugative element (ICE). Phenotypic experiments revealed enhanced virulence potential of the recent epidemic clone from clade B. Sequence type 11 strains were favorable hosts for the convergence of virulence and resistance, indicated by clonal replacement and acquisition patterns of virulence elements. CONCLUSION: The emergence of the enhanced virulence potential of ST11 CR-hvKP suggests that coevolution between hosts and exogenous factors can produce super-virulent CR-hvKP strains, highlighting the need to closely monitor changes in the virulence characteristics of CR-hvKP.

Coexpression of Methyltransferase Gene dmt50 and Methylene Tetrahydrofolate Reductase Gene Increases Arabidopsis thaliana Dicamba Resistance.

Dicamba, a broad-spectrum and highly efficient herbicide, is an excellent target herbicide for the engineering of herbicide-resistant crops. In this study, a new tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt50, was cloned from the dicamba-degrading strain Rhizorhabdus dicambivorans Ndbn-20. Dmt50 catalyzed the methyl transfer from dicamba to THF, generating the herbicidally inactive product 3,6-dichlorosalicylic acid (3,6-DCSA) and 5-methyl-THF. A dmt50 transgenic Arabidopsis thaliana clearly showed dicamba resistance (560 g/ha, the normal field application rate). However, Dmt50 demethylation activity was inhibited by the product 5-methyl-THF. Mthfr66, encoded by the 5,10-methylene-THF reductase gene mthfr66 could relieve the inhibition by removing 5-methyl-THF in vitro. Compared with expression of dmt50 alone, simultaneous expression of dmt50 and mthfr66 further improved the dicamba resistance (1120 g/ha) of transgenic A. thaliana. This study provides new genes for dicamba detoxification and a strategy for the engineering of dicamba-resistant crops.