Consequences of producing DNA gyrase from a syntheticgyrBAoperon inSalmonella entericaserovar Typhimurium

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.

Reciprocally rewiring and repositioning the Integration Host Factor (IHF) subunit genes in Salmonella enterica serovar Typhimurium: impacts on physiology and virulence

“…All three of these are part of the H-NS regulon [120–123] while SPI-1 and SPI-2 consist of genetic elements that have been acquired by horizontal gene transfer [122, 123]. Their encoded T3SS exhibit a marked hypersensitivity to changes to the expression of NAPs and the control of DNA topology [44, 61, 67, 98, 114, 124, 125], indicating that they belong to a genomic category that has an acute dependency on WT patterns of global gene control. This may be because they specify gene products that are physiologically expensive to produce.…”

“…Is the physical location of each gene significant for the life of the bacterium, or might other arrangements work just as well? Several lines of evidence suggest that gene location on bacterial chromosomes is important [61][62][63][64][65][66][67]. For example, gene distance from the origin of chromosome replication (oriC) influences gene copy number during rapid growth.…”

The consequences of reciprocally exchanging the genomic sites of Integration Host Factor (IHF) subunit production for subunit stoichiometry and bacterial physiology in Salmonella enterica serovar Typhimurium

“…All three of these are part of the H-NS regulon [118][119][120][121] while SPI-1 and SPI-2 consist of genetic elements that have been acquired by horizontal gene transfer [120,121]. Their encoded T3SS exhibit a marked hypersensitivity to changes to the expression of NAPs and the control of DNA topology [44,60,66,96,112,122,123], indicating that they belong to a genomic category that has an acute dependency on wildtype patterns of global gene control. This may be because they specify gene products that are physiologically expensive to produce.…”

“…Is the physical location of each gene significant for the life of the bacterium, or might other arrangements work just as well? Several lines of evidence suggest that gene location on bacterial chromosomes is important [60][61][62][63][64][65][66]. For example, gene distance from the origin of chromosome replication (oriC) influences gene copy number during rapid growth.…”

Physiological Robustness of Model Gram-Negative Bacteria in Response to Genome Rewiring

“…Topoisomerase I topA Type IA topoisomerase (monomeric) Removes negative supercoils from double-stranded DNA by breaking one strand and using an intact passage mechanism; ATP-independent; suppresses R-loop formation; restarts stalled transcription complexes [Dekker et al, 2002;Crozat et al, 2010;Cameron et al, 2011] DNA gyrase gyrA gyrB Type II topoisomerase (A 2 B 2 heterotetramer) Introduces negative supercoils into relaxed DNA and removes positive supercoils using a double-strand breakage, intact-duplex-segment passage, and religation mechanism. Negative supercoiling is ATPdependent; DNA relaxation is ATP-independent [Gellert et al, 1976[Gellert et al, , 1977Higgins et al, 1978;Williams and Maxwell, 1999;Nöllmann et al, 2007;Pozdeev et al, 2021] Topoisomerase III topB Type IA topoisomerase (monomeric) A decatenase concerned with the smooth operation of chromosome segregation; suppresses R-loop formation; ATP-independent [Nurse et al, 2003;Perez-Cheeks et al, 2012;Brochu et al, 2018] Topoisomerase IV…”