Deletion of Lytic Transglycosylases Increases Beta-Lactam Resistance in Shewanella oneidensis

Abstract

Production of chromosome-encoded β-lactamases confers resistance to β-lactams in many Gram-negative bacteria. Some inducible β-lactamases, especially the class C β-lactamase AmpC in Enterobacteriaceae, share a common regulatory mechanism, the ampR-ampC paradigm. Induction of ampC is intimately linked to peptidoglycan recycling, and the LysR-type transcriptional regulator AmpR plays a central role in the process. However, our previous studies have demonstrated that the expression of class D β-lactamase gene blaA in Shewanella oneidensis is distinct from the established paradigm since an AmpR homolog is absent and major peptidoglycan recycling enzymes play opposite roles in β-lactamase expression. Given that lytic transglycosylases (LTs), a class of peptidoglycan hydrolases cleaving the β-1,4 glycosidic linkage in glycan strands of peptidoglycan, can disturb peptidoglycan recycling, and thus may affect induction of blaA. In this study, we investigated impacts of such enzymes on susceptibility to β-lactams. Deletion of three LTs (SltY, MltB and MltB2) increased β-lactam resistance, while four other LTs (MltD, MltD2, MltF, and Slt2) seemed dispensable to β-lactam resistance. The double LT mutants ΔmltBΔmltB2 and ΔsltYΔmltB2 had β-lactam resistance stronger than any of the single mutants. Deletion of ampG (encoding permease AmpG) and mrcA (encoding penicillin binding protein 1a, PBP1a) from both double LT mutants further increased the resistance to β-lactams. Notably, all increased β-lactam resistance phenotypes were in accordance with enhanced blaA expression. Although significant, the increase in β-lactamase activity after inactivating LTs is much lower than that produced by PBP1a inactivation. Our data implicate that LTs play important roles in blaA expression in S. oneidensis.

Cell-Wall Recycling of the Gram-Negative Bacteria and the Nexus to Antibiotic Resistance

“…Gram-negative bacteria possess a family of LTs, few of which have assigned functional roles. Nonetheless, it is evident from studies using LT gene knockouts in both P. aeruginosa 292,400 and S. oneidensis 401 that inhibitor selectivity within the family may be required. The second limitation is the absence of an appropriate high-throughput assay for the LT enzymes to measure both inhibitor selectivity and inhibitor potency.…”

Tuning Redox Potential of Anthraquinone-2-Sulfonate (AQS) by Chemical Modification to Facilitate Electron Transfer From Electrodes in Shewanella oneidensis

“…S. oneidensis MR-1 was used as the wild-type strain for gene disruption. The corresponding mutants of S. oneidensis were generated by an efficient conjugation-based in-frame deletion mutagenesis with pHG1.0 suicide plasmid as described previously ( Yin et al, 2018 ). For routine purposes, S. oneidensis strains were grown in LB medium (0.5% yeast extract, 1% tryptone, 1% NaCl) at 32°C with shaking.…”

Rapid dereplication of microbial isolates using matrix-assisted laser desorption ionization time-of-flight mass spectrometry: A mini-review

“…Previous studies demonstrated the applicability of MALDI-TOF MS for high-throughput dereplication and its applicability for unbiased studies of the cultivable microbial community [36], [37], [38], [39]. The discrimination power of MALDI-TOF MS by combining MS data from both intact proteins and specialized metabolites was recently demonstrated and allowed the characterisation of isolates based on their identity and potential environmental function [10].…”

“…Using additional statistical analysis steps or software with further options can therefore improve the approach in creating a nonredundant set of isolates. Several studies have successfully implemented MALDI-TOF MS-based biotyping to classify mass spectra for dereplication [36], [37], [38], [39], [44]. Generally, highly similar clusters were used to identify identical isolates and select representatives of these clusters for further validation, e.g., via partial 16S rRNA gene sequence analysis, ITS region sequencing or repetitive element-based PCR.…”

“…The evaluation of appropriate cut-off values for cluster delineation was based on threshold values established by the additional validation steps and/or a minimum number of mass peaks shared between isolates. It was shown that a similarity-based MALDI-TOF MS approach can be used for dereplication without additional, costly DNA-based methods [38]. New approaches also include machine learning algorithms such as Random Forest models to automate the identification of isolates in environmental studies [45], [46].

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