Fate of the H-NS–Repressed bgl Operon in Evolution of Escherichia coli

In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS–repressed locus is the bgl (aryl-β,D-glucoside) operon of E. coli. This locus is “cryptic,” as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli

Nonlinear Fitness Landscape of a Molecular Pathway

Genes are regulated because their expression involves a fitness cost to the organism. The production of proteins by transcription and translation is a well-known cost factor, but the enzymatic activity of the proteins produced can also reduce fitness, depending on the internal state and the environment of the cell. Here, we map the fitness costs of a key metabolic network, the lactose utilization pathway in Escherichia coli. We measure the growth of several regulatory lac operon mutants in different environments inducing expression of the lac genes. We find a strikingly nonlinear fitness landscape, which depends on the production rate and on the activity rate of the lac proteins. A simple fitness model of the lac pathway, based on elementary biophysical processes, predicts the growth rate of all observed strains. The nonlinearity of fitness is explained by a feedback loop: production and activity of the lac proteins reduce growth, but growth also affects the density of these molecules. This nonlinearity has important consequences for molecular function and evolution. It generates a cliff in the fitness landscape, beyond which populations cannot maintain growth. In viable populations, there is an expression barrier of the lac genes, which cannot be exceeded in any stationary growth process. Furthermore, the nonlinearity determines how the fitness of operon mutants depends on the inducer environment. We argue that fitness nonlinearities, expression barriers, and gene–environment interactions are generic features of fitness landscapes for metabolic pathways, and we discuss their implications for the evolution of regulation

Activation of leuO by LrhA in Escherichia coli

LeuO is a conserved LysR-type transcription factor of pleiotropic function in Enterobacteria. Regulation of the leuO gene has been best studied in Escherichia coli and Salmonella enterica. Its expression is silenced by the nucleoid-associated proteins H-NS and StpA, autoregulated by LeuO, and in E. coli activated by the transcription regulator BglJ-RcsB. However, signals which induce leuO expression remain unknown. Here we show that LrhA, a conserved LysR-type transcription regulator, activates leuO in E. coli. LrhA specifically binds the leuO regulatory region and activates expression of leuO from three promoters. Activation of leuO by LrhA is synergistic with activation by BglJ-RcsB, while co-regulation by LrhA, LeuO and H-NS/StpA suggests a complex regulatory interplay. In addition, hyperactive LrhA mutants including LrhA-12DN, 221TA, 61HR/221TA and 303DG were identified. Regulation of leuO by LrhA reveals a connection between the two pleiotropic regulators LeuO and LrhA in E. coli

Correlation of Antagonistic Regulation of leuO Transcription with the Cellular Levels of BgIJ-RcsB and LeuO in Escherichia coil

LeuO is a conserved and pleiotropic transcription regulator, antagonist of the nucleoid-associated silencer protein H-NS, and important for pathogenicity and multidrug resistance in Enterobacteriaceae. Regulation of transcription of the leuo gene is complex. It is silenced by H-NS and its paralog StpA, and it is autoregulated. In addition, in Escherichia colt leuO is antagonistically regulated by the heterodimeric transcription regulator BgIJ-RcsB and by LeuO. BgIJ-RcsB activates leuO, while LeuO inhibits activation by BgIJ-RcsB. Furthermore, LeuO activates expression of bg/J, which is likewise H-NS repressed. Mutual activation of leuO and bglJ resembles a double-positive feedback network, which theoretically can result in bi-stability and heterogeneity, or be maintained in a stable OFF or ON states by an additional signal. Here we performed quantitative and single-cell expression analyses to address the antagonistic regulation and feedback control of leuO transcription by BgIJ-RcsB and LeuO using a leuO promoter m Venus reporter fusion and finely tunable bgIJ and leuO expression plasmids. The data revealed uniform regulation of leuO expression in the population that correlates with the relative cellular concentration of BgIJ and LeuO. The data are in agreement with a straightforward model of antagonistic regulation of leuO expression by the two regulators, LeuO and BgIJ-RcsB, by independent mechanisms. Further, the data suggest that at standard laboratory growth conditions feedback regulation of leuO is of minor relevance and that silencing of feu and bglJ by H-NS (and StpA) keeps these loci in the OFF state

High Abundance of Transcription Regulators Compacts the Nucleoid in Escherichia coli

In enteric bacteria organization of the circular chromosomal DNA into a highly dynamic and toroidal-shaped nucleoid involves various factors, such as DNA supercoiling, nucleoid-associated proteins (NAPs), the structural maintenance of chromatin (SMC) complex, and macrodomain organizing proteins. Here, we show that ectopic expression of transcription regulators at high levels leads to nucleoid compaction. This serendipitous result was obtained by fluorescence microscopy upon ectopic expression of the transcription regulator and phosphodiesterase PdeL of Escherichia coll. Nucleoid compaction by PdeL depends on DNA-binding, but not on its enzymatic phosphodiesterase activity. Nucleoid compaction was also observed upon high-level ectopic expression of the transcription regulators Lad, RutR, RcsB, LeuO, and Cra, which range from singletarget gene regulators to global regulators. In the case of Lad, its high-level expression in the presence of the gratuitous inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) also led to nucleoid compaction, indicating that compaction is caused by unspecific DNAbinding. In all cases nucleoid compaction correlated with misplacement of the FtsZ ring and loss of MukB foci, a subunit of the SMC complex Thus, high levels of several transcription regulators cause nucleoid compaction with consequences for replication and cell division. IMPORTANCE The bacterial nucleoid is a highly organized and dynamic structure for simultaneous transcription, replication, and segregation of the bacterial genome. Compaction of the nucleoid and disturbance of DNA segregation and cell division by artificially high levels of transcription regulators, as described here, reveals that an excess of DNA-binding protein disturbs nucleoid structuring. The results suggest that ectopic expression levels of DNA-binding proteins for genetic studies of their function but also for their purification should be carefully controlled and adjusted

Interference of transcription across H-NS binding sites and repression by H-NS

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression

(The Transcription Regulator and c-di-GMP Phosphodiesterase PdeL Represses Motility in Escherichia coli)

PdeL is a transcription regulator and catalytically active c-di-GMP phosphodiesterase (PDE) in Escherichia coli. PdeL has been shown to be a transcription autoregulator, while no other target genes have been identified so far. Here, we show that PdeL represses the transcription of the flagellar class II operon fliFGHIJK and activates sslE, encoding an extracellularly anchored metalloprotease, among additional loci. DNA-binding studies and expression analyses using plasmidic reporters suggest that the regulation of the fliF and ssIE promoters by PdeL is direct. Transcription repression of the fliFGHIJK operon, encoding proteins required for the assembly of the flagellar basal body, results in inhibition of motility on soh-agar plates and reduction of flagellum assembly, as shown by fluorescence staining of the flagellar hook protein FlgE. PdeL-mediated repression of motility is independent of its phosphodiesterase activity. Thus, in motility control, the transcription regulator function of PdeL in reducing the number of assembled flagella is apparently epistatic to its phosphodiesterase function, which can indirectly promote the activity of the flagellar motor by lowering the c-di-GMP concentration. IMPORTANCE Bacteria adopt different lifestyles depending on their environment and physiological conditions. In Escherichia coli and other enteric bacteria, the transition between the motile and the sessile states is controlled at multiple levels from the regulation of gene expression to the modulation of various processes by the second messenger c-di-GMP as a signaling molecule. The significance of our research is in identifying PdeL, a protein of dual function that hydrolyzes c-di-GMP and regulates the transcription of genes, as a repressor of flagellar gene expression and an inhibitor of motility, which adds an additional regulatory switch to the control of motility

Regulation of the yjjQ-bglJ Operon, Encoding LuxR-Type Transcription Factors, and the Divergent yjjP Gene by H-NS and LeuO▿ †

The yjjQ and bglJ genes encode LuxR-type transcription factors conserved in several enterobacterial species. YjjQ is a potential virulence factor in avian pathogenic Escherichia coli. BglJ counteracts the silencing of the bgl (β-glucoside) operon by H-NS in E. coli K-12. Here we show that yjjQ and bglJ form an operon carried by E. coli K-12, whose expression is repressed by the histone-like nucleoid structuring (H-NS) protein. The LysR-type transcription factor LeuO counteracts this repression. Furthermore, the yjjP gene, encoding a membrane protein of unknown function and located upstream in divergent orientation to the yjjQ-bglJ operon, is likewise repressed by H-NS. Mapping of the promoters as well as the H-NS and LeuO binding sites within the 555-bp intergenic region revealed that H-NS binds to the center of the AT-rich regulatory region and distal to the divergent promoters. LeuO sites map to the center and to positions distal to the yjjQ promoters, while one LeuO binding site overlaps with the divergent yjjP promoter. This latter LeuO site is required for full derepression of the yjjQ promoters. The arrangement of regulatory sites suggests that LeuO restructures the nucleoprotein complex formed by H-NS. Furthermore, the data support the conclusion that LeuO, whose expression is likewise repressed by H-NS and which is a virulence factor in Salmonella enterica, is a master regulator that among other loci, also controls the yjjQ-bglJ operon and thus indirectly the presumptive targets of YjjQ and BglJ