Species-specific mutation rates for ampC derepression in Enterobacterales with chromosomally encoded inducible AmpC β-lactamase

Lee

Open Forum Infectious Diseases

et al. 2019

The optimal treatment for potential AmpC-producing Enterobacteriaceae, including Serratia, Providencia, Citrobacter, Enterobacter, and Morganella species, remains unknown. An updated systematic review and meta-analysis of studies comparing beta-lactam/beta-lactamase inhibitors with carbapenems in the treatment of bloodstream infections with these pathogens found no significant difference in 30-day mortality (OR, 1.13; 95% CI, 0.58 – 2.20).

Section: Discussionmentioning

confidence: 58%

Beta-Lactam/Beta-Lactamase Inhibitor Therapy for Potential AmpC-Producing Organisms: A Systematic Review and Meta-Analysis

Lee

Open Forum Infectious Diseases

et al. 2019

“…What data do exist emphasize that true on-treatment emergence of beta-lactam resistance is probably rare, at least in S. marcescens and in Morganella morganii (37) . In vitro experiments also hint at important heterogeneity among these pathogens; in this setting, the development of spontaneous cephalosporin resistance has been reported to be nearly 100-fold lower in S. marcescens compared to E. cloacae and C. freundii , and 10-fold lower still in M. morganii (38).…”

Section: Discussionmentioning

confidence: 99%

A New Suite of Allelic Exchange Vectors for the Scarless Modification of Proteobacterial Genomes

Lazarus

Warr

Kuehl

et al. 2019

Preprint

20Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through 21 homologous recombination remains an important tool for genetic analysis. Currently, sacB-based 22 Importance 40Targeted modification of bacterial genomes is critical for genetic analyses of microorganisms. 41Allelic exchange is a technique that relies on homologous recombination to substitute native loci for 42 engineered sequences. However, current allelic exchange vectors often enable only weak 43 selection for successful homologous recombination. We developed a suite of new allelic exchange 44 vectors, pTOX, which were validated in several medically important Proteobacteria. They encode 45 visible non-fluorescent chromoproteins that enable easy identification of colonies bearing 46 integrated vector, and permit stringent selection for the second step of homologous recombination, 47 yielding modified loci. We demonstrate the utility of these vectors by using them to investigate the 48 effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on beta-lactam 49 antibiotic resistance. 50 51The ever-increasing availability of bacterial genome sequence data has driven the demand for 52 widely applicable and facile techniques enabling site-specific targeted mutagenesis. In general, 53 such techniques can be divided into those that rely on exogenous enzymes versus those that 54 depend exclusively on endogenous enzymes. Examples of methods in the former category include 55 those utilizing the Lambda Red recombinase ("recombineering" (1)), those employing clustered 56 regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems (2), or a combination of the 57 two (3, 4). These systems can be fast and reliable, but often require organism-specific 58 modifications, rely on efficient transformation, and can leave genetic scars or result in off-target 59 mutations. 60 61In contrast, "allelic exchange" utilizes endogenous homologous recombination enzymes to facilitate 62 the replacement of a native genomic region with a foreign sequence of interest. This is a versatile 63 technique that can routinely yield mutations ranging from kilobase-scale deletions or insertions to 64 the generation of precise point mutations. The early allele exchange vectors resulted in antibiotic-65 marked strains (5, 6); subsequent advances using counter-selectable cassettes allowed the 66 generation of truly scarless, unmarked mutant strains. However, many genes used in counter-67 selection strategies (e.g. rpsL, pheS, thyA, ccdB) require a specific host genotype, limiting their 68 widespread utility (7). Background-independent counter-selection strategies utilizing tetAR (8), 69 sacB (9), or a combination of the two (10) are valuable but often require considerable optimization. 70Moreover, counter-selection escape, where the integrated allelic exchange vector remains lodged 71 in the genome, preventing isolation of the desired mutant, remains common with such schemes 72 even after optimization. This has been a key tech...

“…The Can r mutant frequencies were calculated by dividing the Can r mutant count by the viable cell count. The Drake equation was applied to the mutation frequencies to calculate the mutation rates (50). The median mutation rates and frequencies were used to compare spontaneous and induced mutagenesis in different strains.…”

Section: Methodsmentioning

confidence: 99%

Sml1 Inhibits the DNA Repair Activity of Rev1 in Saccharomyces cerevisiae during Oxidative Stress

Yao

Zhou

et al. 2020

Appl Environ Microbiol

In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress. IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.