Consequences of producing DNA gyrase from a synthetic gyrBA operon in Salmonella enterica serovar Typhimurium.

German Pozdeev,Charles J Dorman,Aalap Mogre

doi:10.1111/mmi.14689

Abstract

DNA gyrase is an essential type II topoisomerase that is composed of two subunits, GyrA and GyrB, and has an A2B2 structure. Although the A and B subunits are required in equal proportions to form DNA gyrase, the gyrA and gyrB genes that encode them in Salmonella (and in many other bacteria) are at separate locations on the chromosome, are under separate transcriptional control, and are present in different copy numbers in rapidly growing bacteria. In wild‐type Salmonella, gyrA is near the chromosome's replication terminus, while gyrB is near the origin. We generated a synthetic gyrBA operon at the oriC‐proximal location of gyrB to test the significance of the gyrase gene position for Salmonella physiology. Although the strain producing gyrase from an operon had a modest alteration to its DNA supercoiling set points, most housekeeping functions were unaffected. However, its SPI‐2 virulence genes were expressed at a reduced level and its survival was reduced in macrophage. Our data reveal that the horizontally acquired SPI‐2 genes have a greater sensitivity to disturbance of DNA topology than the core genome and we discuss its significance in the context of Salmonella genome evolution and the gyrA and gyrB gene arrangements found in other bacteria.

Highlights

DNA gyrase is an essential type II topoisomerase that introduces negative supercoils into DNA through an ATP-dependent mechanism (Gellert et al., 1976a; Higgins et al, 1978; Nöllmann et al, 2007); it can relax negatively supercoiled DNA via an ATP-independent mechanism (Gellert et al, 1977; Higgins et al, 1978; Williams and Maxwell, 1999)
Our data reveal that the horizontally acquired Salmonella pathogenicity island (SPI)-2 genes have a greater sensitivity to disturbance of DNA topology than the core genome and we discuss its significance in the context Salmonella genome evolution and the gyrA and gyrB gene arrangements found in other bacteria. 37 Key words: Salmonella enterica serovar Typhimurium, DNA gyrase, gyrA, gyrB, DNA supercoiling, SPI-1, SPI-2
Typhimurium strain SL1344 to serve as a selectable marker 162 (Experimental procedures)

Summary

Introduction

DNA gyrase is an essential type II topoisomerase that introduces negative supercoils into DNA through an ATP-dependent mechanism (Gellert et al., 1976a; Higgins et al, 1978; Nöllmann et al, 2007); it can relax negatively supercoiled DNA via an ATP-independent mechanism (Gellert et al, 1977; Higgins et al, 1978; Williams and Maxwell, 1999). The GyrA subunits form covalent bonds to the single-stranded DNA overhangs via tyrosine amino acids in their active sites while the GyrB subunits bind and hydrolyse ATP (Corbett and Berger, 2004). Topoisomerase activity is required to eliminate the over-wound (positively supercoiled) and under-wound (negatively supercoiled) zones of the DNA template that are generated by transcription and DNA replication (Liu and Wang, 1987; Stracy et al, 2019). DNA gyrase relaxes the positively supercoiled DNA by introducing negative supercoils in an ATP-dependent manner (Ashley et al, 2017). The changes to local DNA supercoiling that are caused by transcription and DNA replication affect the activities of some transcription promoters (Ahmed et al, 2016; 2017; Chong et al, 2014; Dorman, 2019; Higgins, 2014; Rahmouni and Wells 1992; Rani and Nagaraja, 2019; Wu et al, 1988; Tobe et al, 1995)

Results

Discussion

Conclusion