Identification of C-terminal hydrophobic residues important for dimerization and all known functions of ParB of Pseudomonas aeruginosa.
The ParB protein of Pseudomonas aeruginosa is important for growth, cell division, nucleoid segregation and different types of motility. To further understand its function we have demonstrated a vital role of the hydrophobic residues in the C terminus of ParB(P.a.). By in silico modelling of the C-terminal domain (amino acids 242-290) the hydrophobic residues L282, V285 and I289 (but not L286) are engaged in leucine-zipper-like structure formation, whereas the charged residues R290 and Q266 are implicated in forming a salt bridge involved in protein stabilization. Five parB mutant alleles were constructed and their functionality was defined in vivo and in vitro. In agreement with model predictions, the substitution L286A had no effect on mutant protein activities. Two ParBs with single substitutions L282A or V285A and deletions of two or seven C-terminal amino acids were impaired in both dimerization and DNA binding and were not able to silence genes adjacent to parS, suggesting that dimerization through the C terminus is a prerequisite for spreading on DNA. The defect in dimerization also correlated with loss of ability to interact with partner protein ParA. Reverse genetics demonstrated that a parB mutant producing ParB lacking the two C-terminal amino acids as well as mutants producing ParB with single substitution L282A or V285A had defects similar to those of a parB null mutant. Thus so far all the properties of ParB seem to depend on dimerization.
The Institute of Biochemistry and Biophysics, PAS, Pawinskiego 5A, 02-106 Warsaw, Poland.
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Mierzejewska
The Institute of Biochemistry and Biophysics, PAS, Pawinskiego 5A, 02-106 Warsaw, Poland.
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Bartosik
The Institute of Biochemistry and Biophysics, PAS, Pawinskiego 5A, 02-106 Warsaw, Poland.
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Macioszek
The Institute of Biochemistry and Biophysics, PAS, Pawinskiego 5A, 02-106 Warsaw, Poland.
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Płochocka
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
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Thomas
The Institute of Biochemistry and Biophysics, PAS, Pawinskiego 5A, 02-106 Warsaw, Poland.
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Jagura-Burdzy
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D016350:Leucine Zippers; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D059386:Reverse Genetics; D017384:Sequence Deletion
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C052883:chromosome partition proteins, bacterial