An Adenosine Triphosphate- Dependent 5'-3' DNA Helicase From sk1-Like Lactococcus lactis F13 Phage.

Magdalena Chmielewska-Jeznach,Jacek K Bardowski,Kamil Steczkiewicz,Kamil Kobyłecki,Agnieszka K Szczepankowska

doi:10.3389/fmicb.2022.840219

Magdalena Chmielewska-Jeznach, Jacek K Bardowski + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2022.840219

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Abstract

Here, we describe functional characterization of an early gene (gp46) product of a virulent Lactococcus lactis sk1-like phage, vB_Llc_bIBBF13 (abbr. F13). The GP46F13 protein carries a catalytically active RecA-like domain belonging to the P-loop NTPase superfamily. It also retains features characteristic for ATPases forming oligomers. In order to elucidate its detailed molecular function, we cloned and overexpressed the gp46 gene in Escherichia coli. Purified GP46F13 protein binds to DNA and exhibits DNA unwinding activity on branched substrates in the presence of adenosine triphosphate (ATP). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments demonstrate that GP46F13 forms oligomers, and further pull-down assays show that GP46F13 interacts with host proteins involved in replication (i.e., DnaK, DnaJ, topoisomerase I, and single-strand binding protein). Taking together the localization of the gene and the obtained results, GP46F13 is the first protein encoded in the early-expressed gene region with helicase activity that has been identified among lytic L. lactis phages up to date.

Highlights

Bacteriophages infecting the Lactococcus lactis species pose a great threat to industrial settings by perturbing or arresting milk fermentation processes (Garneau and Moineau, 2011)
RepA and DnaB are replicative helicases (Niedenzu et al, 2001; Bailey et al, 2007), RadA is a DnaB-type helicase interacting with RecA in homologous recombination (Marie et al, 2017), RAD51 and RecA are recombinases forming helical filaments seeking for homology in homologous recombination (Chen et al, 2008; Short et al, 2016), GvpD regulates gas vesicle formation in Archaea, KaiC is an ATPase controlling cyanobacterial circadian clock (Abe et al, 2015) and AAA_25 proteins have no known function described yet
Obtained results suggest that GP46F13 is closely related to the RepA family

Summary

Introduction

Bacteriophages infecting the Lactococcus lactis species pose a great threat to industrial settings by perturbing or arresting milk fermentation processes (Garneau and Moineau, 2011). The knowledge on the replication of lytic lactococcal phages and proteins participating in the process is of great value and may lead to developing new phage resistance mechanisms in lactococcal starter strains for controlling phage multiplication in dairy environments. DNA replication is one of the pivotal processes in bacteriophage development. Distinct phages follow various replication pathways and exploit different sets of own and/or host proteins for multiplication of their genomic content (Weigel and Seitz, 2006 and references within). The Gram(−) phage T4 and the Gram(+) phage phi possess all the necessary components of their replication machinery (i.e., initiator protein, primase, helicase loader, helicase, and DNA polymerase) (Meijer et al, 2001; Kulczyk and Richardson, 2016). Other phages, such as Escherichia coli phage λ or Bacillus subtilis phage SPP1 encode their own replication initiation proteins, but depend on host proteins for further stages

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