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July 7, 2019  |  

The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para-hydroxybenzoate catabolism.

The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration, primarily during an enzymatic hydroxylation reaction. The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for 40 years. Here, we elucidate the molecular mechanism of the reaction in the conversion of para-hydroxybenzoate (PHB) to gentisate (GA, 2, 5-dihydroxybenzoate). Three genes (phgABC) from the PHB utilizer Brevibacillus laterosporus PHB-7a encode enzymes (p-hydroxybenzoyl-CoA ligase, p-hydroxybenzoyl-CoA hydroxylase and gentisyl-CoA thioesterase, respectively) catalyzing the conversion of PHB to GA via a route involving CoA thioester formation, hydroxylation concomitant with a 1, 2-shift of the acetyl CoA moiety and thioester hydrolysis. The shift of the carboxyl group was established rigorously by stable isotopic experiments with heterologously expressed phgABC, converting 2, 3, 5, 6-tetradeutero-PHB and [carboxyl-13 C]-PHB to 3, 4, 6-trideutero-GA and [carboxyl-13 C]-GA respectively. This is distinct from the NIH shifts of hydrogen and aceto substituents, where a single oxygenase catalyzes the reaction without the involvement of a thioester. The discovery of this three-step strategy for carboxyl group migration reveals a novel role of the CoA thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.© 2018 John Wiley & Sons Ltd.

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July 7, 2019  |  

Deciphering mixotrophic Clostridium formicoaceticum metabolism and energy conservation: Genomic analysis and experimental studies.

Clostridium formicoaceticum, a Gram-negative mixotrophic homoacetogen, produces acetic acid as the sole metabolic product from various carbon sources, including fructose, glycerol, formate, and CO2. Its genome of 4.59-Mbp contains a highly conserved Wood-Ljungdahl pathway gene cluster with the same layout as that in other mixotrophic acetogens, including Clostridium aceticum, Clostridium carboxidivorans, and Clostridium ljungdahlii. For energy conservation, C. formicoaceticum does not have all the genes required for the synthesis of cytochrome or quinone used for generating proton gradient in H+-dependent acetogens such as Moorella thermoacetica; instead, it has the Rnf system and a Na+-translocating ATPase similar to the one in Acetobacterium woodii. Its growth in both heterotrophic and autotrophic media were dependent on the sodium concentration. C. formicoaceticum has genes encoding acetaldehyde dehydrogenases, alcohol dehydrogenases, and aldehyde oxidoreductases, which could convert acetyl-CoA and acetate to ethanol and butyrate to butanol under excessive reducing equivalent conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

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July 7, 2019  |  

Methanogenic and bacterial endosymbionts of free-living anaerobic ciliates

Trimyema compressum thrives in anoxic freshwater environments in which it preys on bacteria and grows with fermentative metabolisms. Like many anaerobic protozoa, instead of mitochondria, T. compressum possess hydrogenosomes, which are hydrogen-producing, energy-generating organelles characteristic of anaerobic protozoa and fungi. The cytoplasm of T. compressum harbours hydrogenotrophic methanogens that consume the hydrogen produced by hydrogenosome, which confers an energetic advantage to the host ciliate. Symbiotic associations between methanogenic archaea and Trimyema ciliates are thought to be established independently and/or repeatedly in their evolutional history. In addition to methanogenic symbionts, T. compressum houses bacterial symbiont TC1 whose function is unknown in its cytoplasm. Recently, we analysed whole-genome sequence of TC1 symbiont to investigate its physiological function in the tripartite symbiosis and found that fatty acid synthesis fab operon of TC1 symbiont lacked typical transcriptional repressor, which is normally coded on the upstream of the fab operon. The sequence data suggested that TC1 symbiont contributes to host Trimyema by the synthesis of fatty acid or its derivative. In this review, we summarize the early works and recent progress of the studies on Trimyema ciliates, including a stably cultivable model protozoa T. compressum, and discuss about symbiotic associations in oxygen-scarce environments.

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July 7, 2019  |  

Emergence of tigecycline resistance in Escherichia coli co-producing MCR-1 and NDM-5 during tigecycline salvage treatment.

Here, we report a case of severe infection caused by Escherichia coli that harbored mcr-1, blaNDM-5, and acquired resistance to tigecycline during tigecycline salvage therapy.Antimicrobial susceptibility testing, Southern blot hybridization, and complete genome sequence of the strains were carried out. The genetic characteristics of the mcr-1 and blaNDM-5 plasmids were analyzed. The whole genome sequencing of mcr-1-containing plasmid was completed. Finally, putative single nucleotide polymorphisms and deletion mutations in the tigecycline-resistant strain were predicted.Three E. coli isolates were obtained from ascites, pleural effusion, and stool of a patient; they were resistant to almost all the tested antibiotics. The first two strains separated from ascites (E-FQ) and hydrothorax (E-XS) were susceptible to amikacin and tigecycline; however, the third strain from stool (E-DB) was resistant to tigecycline after nearly 3 weeks’ treatment with tigecycline. All three isolates possessed both mcr-1 and blaNDM-5. The blaNDM-5 gene was found on the IncX3 plasmid, whereas the mcr-1, fosA3 and blaCTX-M-14 were located on the IncHI2 plasmid. Mutations in acrB and lon were the reason for the resistance to tigecycline.This is the first report of a colistin-, carbapenem-, and tigecycline-resistant E. coli in China. Tigecycline resistance acquired during tigecycline therapy is of great concern for us because tigecycline is a drug of last resort to treat carbapenem-resistant Gram-negative bacterial infections. Furthermore, the transmission of such extensively drug-resistant isolates may pose a great threat to public health.

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July 7, 2019  |  

The ß-lactamase gene profile and a plasmid-carrying multiple heavy metal resistance genes of Enterobacter cloacae.

In this work, by high-throughput sequencing, antibiotic resistance genes, including class A (blaCTX-M, blaZ, blaTEM, blaVEB, blaKLUC, and blaSFO), class C (blaSHV, blaDHA, blaMIR, blaAZECL-29, and blaACT), and class D (blaOXA) ß-lactamase genes, were identified among the pooled genomic DNA from 212 clinical Enterobacter cloacae isolates. Six blaMIR-positive E. cloacae strains were identified, and pulsed-field gel electrophoresis (PFGE) showed that these strains were not clonally related. The complete genome of the blaMIR-positive strain (Y546) consisted of both a chromosome (4.78?Mb) and a large plasmid pY546 (208.74?kb). The extended-spectrum ß-lactamases (ESBLs) (blaSHV-12 and blaCTX-M-9a) and AmpC (blaMIR) were encoded on the chromosome, and the pY546 plasmid contained several clusters of genes conferring resistance to metals, such as copper (pco), arsenic (ars), tellurite (ter), and tetrathionate (ttr), and genes encoding many divalent cation transporter proteins. The comparative genomic analyses of the whole plasmid sequence and of the heavy metal resistance gene-encoding regions revealed that the plasmid sequences of Klebsiella pneumoniae (such as pKPN-332, pKPN-3967, and pKPN-262) shared the highest similarity with those of pY546. It may be concluded that a variety of ß-lactamase genes present in E. cloacae which confer resistance to ß-lactam antibiotics and the emergence of plasmids carrying heavy metal resistance genes in clinical isolates are alarming and need further surveillance.

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