Regulation of ytfK by cAMP-CRP Contributes to SpoT-Dependent Accumulation of (p)ppGpp in Response to Carbon Starvation YtfK Responds to Glucose Exhaustion

Guanosine penta- or tetraphosphate (known as (p)ppGpp) serves as second messenger to respond to nutrient downshift and other environmental stresses, a phenomenon called stringent response. Accumulation of (p)ppGpp promotes the coordinated inhibition of macromolecule synthesis, as well as the activation of stress response pathways to cope and adapt to harmful conditions. In Escherichia coli, the (p)ppGpp level is tightly regulated by two enzymes, the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified the small protein YtfK as a key regulator of SpoT-mediated activation of stringent response in E. coli. Here, we further characterized the regulation of ytfK. We observed that ytfK is subjected to catabolite repression and is positively regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Importantly, YtfK contributes to SpoT-dependent accumulation of (p)ppGpp and cell survival in response to glucose starvation. Therefore, regulation of ytfK by the cAMP-CRP appears important to adjust (p)ppGpp level and coordinate cellular metabolism in response to glucose availability.

cAMP Receptor Protein Positively Regulates the Expression of Genes Involved in the Biosynthesis of Klebsiella oxytoca Tilivalline Cytotoxin

Klebsiella oxytoca is a resident of the human gut. However, certain K. oxytoca toxigenic strains exist that secrete the nonribosomal peptide tilivalline (TV) cytotoxin. TV is a pyrrolobenzodiazepine that causes antibiotic-associated hemorrhagic colitis (AAHC). The biosynthesis of TV is driven by enzymes encoded by the aroX and NRPS operons. In this study, we determined the effect of environmental signals such as carbon sources, osmolarity, and divalent cations on the transcription of both TV biosynthetic operons. Gene expression was enhanced when bacteria were cultivated in tryptone lactose broth. Glucose, high osmolarity, and depletion of calcium and magnesium diminished gene expression, whereas glycerol increased transcription of both TV biosynthetic operons. The cAMP receptor protein (CRP) is a major transcriptional regulator in bacteria that plays a key role in metabolic regulation. To investigate the role of CRP on the cytotoxicity of K. oxytoca, we compared levels of expression of TV biosynthetic operons and synthesis of TV in wild-type strain MIT 09-7231 and a Δcrp isogenic mutant. In summary, we found that CRP directly activates the transcription of the aroX and NRPS operons and that the absence of CRP reduced cytotoxicity of K. oxytoca on HeLa cells, due to a significant reduction in TV production. This study highlights the importance of the CRP protein in the regulation of virulence genes in enteric bacteria and broadens our knowledge on the regulatory mechanisms of the TV cytotoxin.

Fluorescence Spectroscopic Analysis of ppGpp Binding to cAMP Receptor Protein and Histone-Like Nucleoid Structuring Protein

The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.

Cra and cAMP Receptor Protein Have Opposing Roles in the Regulation of fruB in Vibrio cholerae

ABSTRACT The Gram-negative bacterium Vibrio cholerae adapts to changes in the environment by selectively producing the necessary machinery to take up and metabolize available carbohydrates. The import of fructose by the fructose-specific phosphoenolpyruvate (PEP) phosphotransferase system (PTS) is of particular interest because of its putative connection to cholera pathogenesis and persistence. Here, we describe the expression and regulation of fruB, which encodes an EIIA-FPr fusion protein as part of the fructose-specific PTS in V. cholerae. Using a series of transcriptional reporter fusions and additional biochemical and genetic assays, we identified Cra (catabolite repressor/activator) and cAMP receptor protein (CRP) as regulators of fruB expression and determined that this regulation is dependent upon the presence or absence of PTS sugars. Cra functions as a repressor, downregulating fruB expression in the absence of fructose when components of PTSFru are not needed. CRP functions as an activator of fruB expression. We also report that Cra and CRP can affect fruB expression independently; however, CRP can modulate cra expression in the presence of fructose and glucose. Evidence from this work provides the foundation for continued investigations into PTSFru and its relationship to the V. cholerae life cycle. IMPORTANCE Vibrio cholerae is the causative agent of cholera disease. While current treatments of care are accessible, we still lack an understanding of the molecular mechanisms that allow V. cholerae to survive in both aquatic reservoirs and the human small intestine, where pathogenesis occurs. Central to V. cholerae’s survival is its ability to use available carbon sources. Here, we investigate the regulation of fruB, which encodes a protein central to the import and metabolism of fructose. We show that fruB expression is controlled by the transcriptional regulators Cra and CRP. This work contributes toward a clearer understanding of how carbon source availability impacts the physiology and, potentially, the persistence of the pathogen.

Induction of the cydAB Operon Encoding the bd Quinol Oxidase Under Respiration-Inhibitory Conditions by the Major cAMP Receptor Protein MSMEG_6189 in Mycobacterium smegmatis

The respiratory electron transport chain (ETC) of Mycobacterium smegmatis is terminated with two terminal oxidases, the aa3 cytochrome c oxidase and the cytochrome bd quinol oxidase. The bd quinol oxidase with a higher binding affinity for O2 than the aa3 oxidase is known to play an important role in aerobic respiration under oxygen-limiting conditions. Using relevant crp1 (MSMEG_6189) and crp2 (MSMEG_0539) mutant strains of M. smegmatis, we demonstrated that Crp1 plays a predominant role in induction of the cydAB operon under ETC-inhibitory conditions. Two Crp-binding sequences were identified upstream of the cydA gene, both of which are necessary for induction of cydAB expression under ETC-inhibitory conditions. The intracellular level of cAMP in M. smegmatis was found to be increased under ETC-inhibitory conditions. The crp2 gene was found to be negatively regulated by Crp1 and Crp2, which appears to lead to significantly low cellular abundance of Crp2 relative to Crp1 in M. smegmatis. Our RNA sequencing analyses suggest that in addition to the SigF partner switching system, Crp1 is involved in induction of gene expression in M. smegmatis exposed to ETC-inhibitory conditions.

Phenotype, Virulence and Immunogenicity of Edwardsiella piscicida Cyclic AMP Receptor Protein (Crp) Mutants in Catfish Host

Edwardsiella piscicida, a facultative aerobic pathogen belonging to the Enterobacteriaceae family, is the etiological agent of edwardsiellosis that causes significant economic loses in the aquaculture industry. cAMP receptor protein (CRP) is one of the most important transcriptional regulators, which can regulate large quantities of operons in different bacteria. Here we characterize the crp gene and report the effect of a crp deletion in E. piscicida. The crp-deficient mutant lost the capacity to utilize maltose, and showed significantly reduced motility due to the lack of flagella synthesis. We further constructed a ΔPcrp mutant to support that the phenotype above was caused by the crp deletion. Evidence obtained in fish serum killing assay and competitive infection assay strongly indicated that the inactivation of crp impaired the ability of E. piscicida to evade host immune clearance. More importantly, the virulence of the crp mutant was attenuated in both zebrafish and channel catfish, with reductions in mortality rates. In the end, we found that crp mutant could confer immune protection against E. piscicida infection to zebrafish and channel catfish, indicating its potential as a live attenuated vaccine.