Network Rewiring: Physiological Consequences of Reciprocally Exchanging the Physical Locations and Growth-Phase-Dependent Expression Patterns of the Salmonella fis and dps Genes

Abstract

The Fis nucleoid-associated protein controls the expression of a large and diverse regulon of genes in Gram-negative bacteria. Fis production is normally maximal in bacteria during the early exponential phase of batch culture growth, becoming almost undetectable by the onset of stationary phase. We tested the effect on the Fis regulatory network in Salmonella of moving the complete fis gene from its usual location near the origin of chromosomal replication to the position normally occupied by the dps gene in the right macrodomain of the chromosome, and vice versa, creating the gene exchange (GX) strain. In a parallel experiment, we tested the effect of rewiring the Fis regulatory network by placing the fis open reading frame under the control of the stationary-phase-activated dps promoter at the dps genetic location within the right macrodomain, and vice versa, creating the open reading frame exchange (OX) strain. Chromatin immunoprecipitation sequencing (ChIP-seq) was used to measure global Fis protein binding levels and to determine gene expression patterns. Strain GX showed few changes compared with the wild type, although we did detect increased Fis binding at Ter, accompanied by reduced binding at Ori. Strain OX displayed a more pronounced version of this distorted Fis protein-binding pattern together with numerous alterations in the expression of genes in the Fis regulon. OX, but not GX, had a reduced ability to infect cultured mammalian cells. These findings illustrate the inherent robustness of the Fis regulatory network with respect to the effects of rewiring based on gene repositioning alone and emphasize the importance of fis expression signals in phenotypic determination. IMPORTANCE We assessed the impact on Salmonella physiology of reciprocally translocating the genes encoding the Fis and Dps nucleoid-associated proteins (NAPs) and of inverting their growth-phase production patterns such that Fis was produced in stationary phase (like Dps) and Dps was produced in exponential phase (like Fis). Changes to peak binding of Fis were detected by ChIP-seq on the chromosome, as were widespread impacts on the transcriptome, especially when Fis production mimicked Dps production. Virulence gene expression and the expression of a virulence phenotype were altered. Overall, these radical changes to NAP gene expression were well tolerated, revealing the robust and well-buffered nature of global gene regulation networks in the bacterium.

Composition of Transcription Machinery and Its Crosstalk with Nucleoid-Associated Proteins and Global Transcription Factors

“…It is thus conceivable that the observed spatial patterns of regulons and couplons are selected to optimize the exposure of target genes to the gradients of diffusing regulators. Furthermore, recent observations that alterations of gene expression and phenotype can be induced by chromosomal position shift of a NAP gene, despite the maintenance of its natural expression pattern [105] and, that the effect of positional shift can be aggravated by altering the expression pattern of the NAP gene [107], support the proposal that the physical structure of chromosome is optimized by direct regulatory interactions involving the NAPs and other DNA structuring proteins [133]. In this respect it is relevant, that the NAPs can distinctly modulate the chromosomal regional dynamics by inducing transient domain boundaries in the genome [134,135].…”

“…So far, the existence of this superhelicity gradient could not be experimentally demonstrated. However, the importance of gene order along the chromosomal OriC-Ter axis has been supported by genetic studies demonstrating that chromosomal position shifts of regulatory genes lead to rewiring of the genetic network as well as alterations of both the growth phase-dependent DNA supercoil dynamics and the bacterial phenotype [105][106][107]. Furthermore, the chromosomal gene order appears to underlie the spatial organization of function in genome.…”

The regulation of DNA supercoiling across evolution

“…That these enzymes exert opposite effects on DNA supercoiling precludes a straightforward relationship between DNA supercoiling and Mg 2+ concentration. In S. Typhimurium , Mg 2+ starvation causes DNA relaxation, 55,121 which may reflect that DNA gyrase is more sensitive to Mg 2+ availability than Topo I. Curiously, excess Mg 2+ also causes relaxation in S. Typhimurium 55 .…”

Consequences of producing DNA gyrase from a syntheticgyrBAoperon inSalmonella entericaserovar Typhimurium

“…Several studies have shown that gene position on the chromosome is physiologically significant in bacteria (Bogue et al., 2020 ; Bryant et al., 2014 ; Gerganova et al., 2015 ; Scholz et al., 2019 ). For example, moving the gene that encodes the nucleoid‐associated protein FIS (Factor for Inversion Stimulation) from its native position, proximal to the origin of chromosome replication in E .…”