Anđela Rodić1*, Marko Tumbas1, Jane Kondev2, Magdalena Đorđević3 and Marko Đorđević1
1 Faculty of Biology, University of Belgrade, Belgrade, Serbia
2 Martin A. Fisher School of Physics, Brandeis University, Waltham, MA, USA
3 Institute of Physics Belgrade, University of Belgrade, Belgrade, Serbia
andjela.rodic [at] bio.bg.ac.rs
Abstract
CRISPR-Cas systems protect bacteria from viruses by using spacers, viral DNA fragments stored within the CRISPR array in the bacterial genome. These spacers are transcribed and then processed into crRNAs, guiding Cas proteins to eliminate complementary viral DNA sequences. Despite CRISPR-Cas’s extensive use in biotechnology, its natural function in bacteria, especially Escherichia coli, is not fully understood. In E. coli, CRISPR-Cas activity is silenced by cooperatively bound H-NS proteins to the cas genes promoter. Viral DNA with higher AT content might sequester some H-NS, alleviating this repression. The transcriptional regulator LeuO, whose transcription is activated by BglJ-RcsB, can further activate cas genes transcription.
This study explores whether a slight reduction in H-NS levels can provide initial transcription derepression, and trigger a positive feedback loop activating the CRISPR-Cas system expression through BglJ-RcsB and LeuO, leading to rapid crRNA generation for defense against fast-replicating bacteriophages. We developed a dynamical model for crRNA expression upon infection by foreign DNA and used the Random Forest machine learning technique to identify parameters essential for achieving a sufficient crRNA increase within 30 minutes of foreign DNA entry.
A bioinformatics analysis of 16,388 viruses and their host bacteria revealed a consistent, slight increase in viral genomic AT content compared to hosts, suggesting reduced H-NS levels available for repression upon bacteriophage infection. Significant reductions in H-NS levels can rapidly induce crRNA expression, while smaller reductions require high H-NS binding cooperativity and the baseline cellular H-NS level near to the H-NS equilibrium binding constant.
Our findings indicate that CRISPR-Cas can quickly respond to fast-replicating bacteriophages. The kinetic features, which are essential for understanding the natural function of CRISPR-Cas and enhancing biotechnological applications, should inform future experimental work.
Keywords: CRISPR-Cas, Escherichia coli, H-NS, dynamic modeling, machine learning
Acknowledgement: This work is supported by the Science Fund of the Republic of Serbia (projects No. 7750294, q-bioBDS, and No. 6417603, CRISPR modelling).