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Abstract
The histone-like protein HU is a conserved nucleoid-associated protein that is involved in the maintenance of the bacterial chromosome architecture. It is the only known nucleoid-associated protein in Streptococcus pneumoniae, but it has not been studied. The pneumococcal gene encoding this protein, hlp, is shown herein to be essential for cell viability. Its disruption was only possible either when it was duplicated in the chromosome and its expression induced from the PZn promoter, or when hlp was cloned into a plasmid under the control of the inducible Pmal promoter. In vitro assays indicated that pneumococcal HU shows a preference for binding to supercoiled DNA rather than to linear or nicked DNA. In vivo experiments in which the amount of HU was manipulated showed a relationship between the amount of HU and the level of DNA supercoiling. A twofold reduction in the amount of HU triggered a 21% increase in DNA relaxation in untreated cells. However, in cells treated with novobiocin, a drug that relaxes DNA by inhibiting DNA gyrase, a 35% increase in DNA relaxation was observed, instead of the expected 20% in cells with a constitutive HU amount. Conversely, a fourfold HU increase caused only 14% of DNA relaxation in the presence of novobiocin. Taken together, these results support an essential role for HU in the maintenance of DNA supercoiling in S. pneumoniae.
Highlights
Streptococcus pneumoniae is the causative agent of respiratory tract infections and invasive serious illness including meningitis and bacteremia (Lynch and Zhanel, 2009)
The identity increased to 75.3 and 89% when compared with HU from Grampositive bacteria such as Staphylococcus aureus (SauHU) and Streptococcus mutans (SmuHU), respectively
Its predicted secondary structure included three α-helices and five β-sheets following the α1-α2-β1-β2-β3β4-β5-α3 organization (Figure 1A). This secondary structure is highly conserved among HU proteins and is divided in three domains: helix-turn-helix (HTH), dimerization signal (DS) and DNA-binding domain (DBD) (Christodoulou and Vorgias, 2002; Christodoulou et al, 2003)
Summary
Streptococcus pneumoniae (the pneumococcus) is the causative agent of respiratory tract infections and invasive serious illness including meningitis and bacteremia (Lynch and Zhanel, 2009). The chromosome of S. pneumoniae is confined within the nucleoid. Chromosome compaction is achieved by the action of several factors, including DNA supercoiling, nucleoid-associated proteins (NAPs) (Pato and Banerjee, 1996) and macromolecular crowding (Wang et al, 2013). DNA is usually negatively supercoiled and it is mainly found in a plectonemic form (Travers and Muskhelishvili, 2007). This plectoneme formation leads to a 10% reduction in the radius of gyration of DNA, which is a measure of the volume (Luijsterburg et al, 2008). Negative supercoiling eases the association of architectural proteins
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