An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S

Abstract

Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene (pheS*). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p-chloro-phenylalanine analog (p-Cl-phe) as a substrate. When pheS* is introduced into L. monocytogenes and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to p-Cl-phe supplemented in the medium. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes. We used this vector to investigate the monocin genomic region in L. monocytogenes strain 10403S by constructing deletion mutants of the region. We have found this region to be active and to cause bacterial lysis upon mitomycin C treatment. The future applications of such an effective counterselection system, which does not require any background genomic alterations, are vast, as it can be modularly used in various selection systems (e.g., genetic screens). We expect this counterselection marker to be a valuable genetic tool in research on L. monocytogenes. IMPORTANCE L. monocytogenes is an opportunistic intracellular pathogen and a widely studied model organism. An efficient counterselection marker is a longstanding need in Listeria research for improving the ability to design and perform various genetic manipulations and screening systems for different purposes. We report the construction and utilization of an efficient suicide vector for allelic exchange which can be conjugated, leaves no marker in the bacterial chromosome, and does not require the use of sometimes leaky inducible promoters. This highly efficient genome editing tool for L. monocytogenes will allow for rapid sequential mutagenesis, introduction of point mutations, and design of screening systems. We anticipate that it will be extensively used by the research community and yield novel insights into the diverse fields studied using this model organism.

A Ubiquitous Platform for Bacterial Nanotube Biogenesis

“… All primers were designed during this study, and synthesized by Integrated DNA Technologies (IDT). NA Recombinant DNA pDR111 ( amyE:: P hyper-spank - spec ) Kindly provided by David Rudner (Harvard U) NA pHB201 ( cat , erm ) Bron et al., 1998 NA pKL168 ( gfp-kan ) Lemon and Grossman, 1998 NA pKD46 (λ RED genes, Amp r ) Datsenko and Wanner, 2000 NA pRP7358 [P tac - CORE-F Ec ( fliOPQRflhBA )] Pal et al., 2019 NA pSH13 ( flhA-gfp-kan ) This study NA pSH17 ( amyE:: P hyper-spank - fliP 2xHA - spec ) This study NA pSH19 ( amyE:: P hyper-spank - fliP 2xHA-LOOP - spec ) This study NA pSH21 [ amyE:: P hyper-spank - CORE-F Ec ( fliOPQRflhBA )- spec ] This study NA pLR16- Phes Kindly provided by Anat Herskovits (Tel Aviv U) ( Argov et al., 2017 ) NA pLR16-AB1 This study NA pSH22 (pLR16- Lm flaA int) This study NA pSH23 (pDG1514- Bm hag int) This study NA Other Carbon film on 300 square mesh copper grids Electron Microscopy Sciences Cat#: CF300-Cu Lysing Matr...…”

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene

“…Construction of a pheS* marker for counterselection in M. capsulatus (Bath). Instead of the sacB marker, we attempted to employ a pheS* marker, which has been established as a counterselectable marker in various bacteria (18,(22)(23)(24)(25), for counterselection in M. capsulatus (Bath). The amino acid sequence of PheS from M. capsulatus (Bath) was aligned with those from E. coli and other representative methanotrophs ( Fig.…”

“…PheS* can be used as a counterselectable marker even in nonnative host strains when PheS* shares a high level of sequence similarity with a host-derived PheS. For instance, Argov et al employed the PheS* marker derived from Bacillus subtilis for counterselection in Listeria monocytogenes (22); pairwise sequence alignment showed 83.1% similarity (69.4% identity) between PheS from B. subtilis and that from L. monocytogenes. Due to the high levels of sequence similarity/identity, the pheS* gene constructed in this study might be available for a wide range of type I methanotrophs, including Methylomicrobium…”

“…Furthermore, Miyazaki recently reported that substitution of T251 in PheS from E. coli with alanine or serine increased the efficiency of counterselection (21). Although individual pheS* genes have been constructed and used for counterselection in various bacteria (18,(22)(23)(24)(25), counterselection using pheS* has not been carried out in methanotrophs.…”

Bacteriocins of Listeria monocytogenes and Their Potential as a Virulence Factor

“…Fusing M35152 effective only against serotype 4b with the receptor-binding function of the A118 (infecting serotypes 1/2a, 1/2b and 1/2c) tail fiber resulted in a recombinant monocin with a wider killing range including serotypes infected by A118 [55]. The ftb cluster was also identified in the serotype 1/2a lab strain 10403S, and the role of the ftb holin-lysin cassette in cell lysis was confirmed using a variety of deletion constructs in 10403S [64] (Figure 4).…”