Multifaceted Roles of AFG3L2, a Mitochondrial ATPase in Relation to Neurological Disorders.

AFG3L2 is a zinc metalloprotease and an ATPase localized in an inner mitochondrial membrane involved in mitochondrial quality control of several nuclear- and mitochondrial-encoded proteins. Mutations in AFG3L2 lead to diseases like slow progressive ataxia, which is a neurological disorder. This review delineates the cellular functions of AFG3L2 and its dysfunction that leads to major clinical outcomes, which include spinocerebellar ataxia type 28, spastic ataxia type 5, and optic atrophy type 12. It summarizes all relevant AFG3L2 mutations associated with the clinical outcomes to understand the detailed mechanisms attributable to its structure-related multifaceted roles in proteostasis and quality control. We face early diagnostic challenges of ataxia and optic neuropathy due to asymptomatic parents and variable clinical manifestations due to heterozygosity/homozygosity of AFG3L2 mutations. This review intends to promote AFG3L2 as a putative prognostic or diagnostic marker. Functions, mutations, and clinical manifestations in AFG3L2, a mitochondrial AAA + ATPases.

Creation of Markerless Genome Modifications in a Nonmodel Bacterium by Fluorescence-Aided Recombineering.

Metabolic engineering of nonmodel bacteria is often challenging because of the paucity of genetic tools for iterative genome modification necessary to equip bacteria with pathways to produce high-value products. Here, we outline a homologous recombination-based method developed to delete or add genes to the genome of a nonmodel bacterium, Zymomonas mobilis, at the desired locus using a suicide plasmid that contains gfp as a fluorescence marker to track its presence in cells. The suicide plasmid is engineered to contain two 500 bp regions homologous to the DNA sequence immediately flanking the target locus. A single crossover event at one of the two homologous regions facilitates insertion of the plasmid into the genome and subsequent homologous recombination events excise the plasmid from the genome, leaving either the original genotype or the desired modified genotype. A key feature of this plasmid is that Green Fluorescent Protein (GFP) expressed from the suicide plasmid allows easy identification and sorting of cells that have lost the plasmid by use of a fluorescence activated cell sorter. Subsequent PCR amplification of genomic DNA from strains lacking GFP allows rapid identification of the desired genotype, which is confirmed by DNA sequencing. This method provides an efficient and flexible platform for improved genetic engineering of Z. mobilis, which can be easily adapted to other nonmodel bacteria.

Corrigendum: A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4.

[This corrects the article DOI: 10.3389/fmicb.2019.02216.].

Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems.

Zymomonas mobilis has emerged as a promising candidate for production of high-value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr-like type IV restriction modification (RM) system, a type I-F CRISPR system, a chromosomal type I RM system (hsdMSc), and a previously uncharacterized type I RM system, located on an endogenous plasmid (hsdRMSp). The DNA recognition motif of HsdRMSp was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMSc and hsdRMSp systems to that of the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMSc and HsdRMSp recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction-negative strains that we constructed and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products. IMPORTANCE Zymomonas mobilis is equipped with a number of traits that make it a desirable platform organism for metabolic engineering to produce valuable bioproducts. Engineering strains equipped with synthetic pathways for biosynthesis of new molecules requires integration of foreign genes. In this study, we developed an all-purpose strain, devoid of known host restriction systems and free of any antibiotic resistance markers, which dramatically improves the uptake efficiency of heterologous DNA into Z. mobilis ZM4. We also confirmed the role of a previously known restriction system as well as identifying a previously unknown type I RM system on an endogenous plasmid. Elimination of the barriers to DNA uptake as shown here will allow facile genetic engineering of Z. mobilis.

A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4.

Metabolic engineering of the biofuel-producing Zymomonas mobilis is necessary if we are to unlock the metabolic potential present in this non-model microbe. Manipulation of such organisms can be challenging because of the limited genetic tools for iterative genome modification. Here, we have developed an efficient method for generating markerless genomic deletions or additions in Z. mobilis. This is a two-step process that involves homologous recombination of an engineered suicide plasmid bearing Z. mobilis targeting sequences and a subsequent recombination event that leads to loss of the suicide plasmid and a genome modification. A key feature of this strategy is that GFP expressed from the suicide plasmid allows easy identification of cells that have lost the plasmid by using a fluorescence activated cell sorter. Using this method, we demonstrated deletion of the gene encoding lactate dehydrogenase (ldh) and the operon for cellulose synthase (bcsABC). In addition, by modifying the plasmid design, we demonstrated targeted insertion of the crtIBE operon encoding a neurosporene biosynthetic pathway into the Z. mobilis genome without addition of any antibiotic resistance genes. We propose this approach will provide an efficient and flexible platform for improved genetic engineering of Z. mobilis.

The redundant aminotransferases in lysine and arginine synthesis and the extent of aminotransferase redundancy in Escherichia coli.

Aminotransferases can be redundant or promiscuous, but the extent and significance of these properties is not known in any organism, even in Escherichia coli. To determine the extent of redundancy, it was first necessary to identify the redundant aminotransferases in arginine and lysine synthesis, and then complement all aminotransferase-deficient mutants with genes for all aminotransferases. The enzymes with N-acetylornithine aminotransferase (ACOAT) activity in arginine synthesis were ArgD, AstC, GabT and PuuE; the major anaerobic ACOAT was ArgD. The major enzymes with N-succinyl-l,l-diaminopimelate aminotransferase (SDAP-AT) activity in lysine synthesis were ArgD, AstC, and SerC. Seven other aminotransferases, when overproduced, complemented the defect in a triple mutant. Lysine availability did not regulate synthesis of the major SDAP-ATs. Complementation analysis of mutants lacking aminotransferases showed that the SDAP-ATs and alanine aminotransferases were exceptionally redundant, and it is proposed that this redundancy may ensure peptidoglycan synthesis. An overview of all aminotransferase reactions indicates that redundancy and broad specificity are common properties of aminotransferases.