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Anti-DnaA antibodies specificity validation by mass spectrometry

ABSTRACT: The ubiquitous protein DnaA is a chromosomal DNA replication initiator and transcription factor. It binds to 9-bp DNA sites called "DnaA-boxes" that are localized within the replication origin (oriC) and throughout chromosomal DNA, usually in the proximity of the promoter regions. During stress, bacteria undergo metabolic shifts that halt their growth and activate survival mechanisms. One outcome of these shifts is the accumulation of long chains of polyphosphate (polyP), an evolutionarily conserved linear polymer composed of up to 1,000 phosphate groups. It was previously identified that DnaA and the Lon protease bind to polyP, which stimulates Lon for the polyP-dependent DnaA proteolysis. We assumed that it have an impact not only on DnaA intracellular concentration but might also affect DNA binding pattern. To investigate this hypothesis and to analyze the DNA interaction profile of DnaA molecules remaining in stressed cells, we performed Chromatin Immunoprecipitation coupled with DNA sequencing (ChIP-seq). To exclude the presence of nonspecifically bound proteins in the sample after immunoprecipitation using anti-DnaA antibodies, mass spectrometry analysis was performed.

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ORGANISM(S): Escherichia Coli

SUBMITTER: Natalia Musiał

LAB HEAD: prof. dr hab. Igor Konieczny

PROVIDER: PXD061973 | Pride | 2026-03-02

REPOSITORIES: Pride

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Project description:Polyphosphate (polyP) is a ubiquitous and abundant compound found in bacteria, fungi, algae, plant, and animals. Among roles of intracellular polyP in bacteria are resistance and survival in the stationary phase of the growth against stress and stringent condition. Therefore, intracellular polyP is considered as a virulence factor of bacteria. In contrast, exogenous polyP has an antimicrobial activity against a variety of microorganisms. To date, much has been numerous studies of antibacterial effect of polyP against gram positive bacteria and fungi while relatively little reports have been published concerning gram negative bacteria. Here we describe bactericidal effect of polyP against gram negative periodontopathic bacterium, Porphyromonas gingivalis, and the transcriptional change by polyP in this bacterium. The bacterial growth was inhibited by polyP (chain length of P3~P75) at concentration of 0.02~0.03%, but not by Pi and PPi at the concentrations. polyP75 was chosen for further experiments, which suppressed the further growth of P. gingivalis as low as 0.03%. polyP75 completely killed the bacterial cells at the concentration of 0.035%. Microarray analysis was employed to identify genes that showed a greater than 1.5-fold difference in the expression by polyP75 at the concentration of 0.03%. It was found that 155 genes were up-regulated and 173 were down-regulated. Down-regulated genes include groups of energy metabolism-related genes, cell envelope-related genes, and genes in relation to biosynthesis of cofactors, prosthetic groups and carriers. Among the down-regulated genes were several involved in DNA replication, cell division protein, and biosynthesis of purines, pyrimidines, nucleosides and nucleotides. In contrast, a large number of ribosomal proteins, transciptional regulators and transposases were up-regulated. Oxidative stress-related genes and iron storage proteins also appeared to be increased in the expression. Real-time PCR confirmed up- and down-regulation of some selected genes. The overall results suggest that polyP has a bactericidal activity against P. gingivalis, interfering with translation, energy metabolism, DNA replication, cell division, and biosynthesis of purines, pyrimidines, nucleoside and nucleotides. polyP may be used as an agent for prevention and treatment of periodontal infections. Polyp-treated vs. untreated intensity ratio data linked below as a supplementary file. Keywords: agent response

			Action	DRS
	1.group	Other
	1.wiff	Wiff
	1.wiff.scan	Wiff
	2.group	Other
	2.wiff	Wiff

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Anti-DnaA antibodies specificity validation by mass spectrometry

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