The priB gene of Klebsiella pneumoniae encodes a 104-amino acid protein that is similar in structure and function to Escherichia coli PriB

Abstract

Primosome protein PriB is a single-stranded DNA-binding protein that serves as an accessory factor for PriA helicase-catalyzed origin-independent reinitiation of DNA replication in bacteria. A recent report describes the identification of a novel PriB protein in Klebsiella pneumoniae that is significantly shorter than most sequenced PriB homologs. The K. pneumoniae PriB protein is proposed to comprise 55 amino acid residues, in contrast to E. coli PriB which comprises 104 amino acid residues and has a length that is typical of most sequenced PriB homologs. Here, we report results of a sequence analysis that suggests that the priB gene of K. pneumoniae encodes a 104-amino acid PriB protein, akin to its E. coli counterpart. Furthermore, we have cloned the K. pneumoniae priB gene and purified the 104-amino acid K. pneumoniae PriB protein. Gel filtration experiments reveal that the K. pneumoniae PriB protein is a dimer, and equilibrium DNA binding experiments demonstrate that K. pneumoniae PriB's single-stranded DNA-binding activity is similar to that of E. coli PriB. These results indicate that the PriB homolog of K. pneumoniae is similar in structure and in function to that of E. coli.

Figure 1. Multiple amino acid sequence alignment of PriB homologs.

Amino acid sequences of Klebsiella pneumoniae PriB (GenBank ID:YP_001338213), Pectobacterium carotovorum PriB (GenBank ID:C6DE14), Yersinia ruckeri PriB (GenBank ID:ZP_04617249), Salmonella enterica PriB (GenBank ID:AAL23212), Escherichia coli PriB (GenBank ID:NP_418622), and Neisseria gonorrhoeae PriB (GenBank ID:YP_207725) were aligned using the program ClustalX . Amino acid residues that are identical in at least five of the six aligned PriB proteins are highlighted in blue.

Figure 2. Gel filtration of PriB proteins from K. pneumoniae and E. coli.

Equivalent amounts of (a) K. pneumoniae PriB, and (b) E. coli PriB, each at approximately 3.4 g/l, were individually resolved through a sephacryl S-100 size-exclusion chromatography column under identical experimental conditions as described in Materials and Methods.

Figure 3. Single-stranded DNA-binding activity of K. pneumoniae PriB.

PriB protein was diluted serially and incubated with fluorescein-labeled 15-base (circles), 30-base (squares), or 45-base (triangles) ssDNA oligonucleotides as described in Materials and Methods. Measurements are reported in triplicate and error bars represent one standard deviation of the mean.