Requirements for Septal Localization and Chromosome Segregation Activity of the DNA Translocase SftA from Bacillus subtilis

TY - JOUR

T1 - Requirements for Septal Localization and Chromosome Segregation Activity of the DNA Translocase SftA from Bacillus subtilis

AU - El Najjar, Nina

AU - Kaimer, Christine

AU - Rösch, Thomas

AU - Graumann, Peter L

N1 - © 2017 S. Karger AG, Basel.

PY - 2017

Y1 - 2017

N2 - Bacillus subtilis possesses 2 DNA translocases that affect late stages of chromosome segregation: SftA separates nonsegregated DNA prior to septum closure, while SpoIIIE rescues septum-entrapped DNA. We provide evidence that SftA is associated with the division machinery via a stretch of 47 amino acids within its N-terminus, suggesting that SftA is recruited by protein-protein interactions with a component of the division machinery. SftA was also recruited to mid-cell in the absence of its first 20 amino acids, which are proposed to contain a membrane-binding motif. Cell fractionation experiments showed that SftA can be found in the cytosolic fraction, and to a minor degree in the membrane fraction, showing that it is a soluble protein in vivo. The expression of truncated SftA constructs led to a dominant sftA deletion phenotype, even at very low induction rates of the truncated proteins, indicating that the incorporation of nonfunctional monomers into SftA hexamers abolishes functionality. Mobility shift experiments and surface plasmon binding studies showed that SftA binds to DNA in a cooperative manner, and demonstrated low ATPase activity when binding to short nucleotides rather than to long stretches of DNA.

AB - Bacillus subtilis possesses 2 DNA translocases that affect late stages of chromosome segregation: SftA separates nonsegregated DNA prior to septum closure, while SpoIIIE rescues septum-entrapped DNA. We provide evidence that SftA is associated with the division machinery via a stretch of 47 amino acids within its N-terminus, suggesting that SftA is recruited by protein-protein interactions with a component of the division machinery. SftA was also recruited to mid-cell in the absence of its first 20 amino acids, which are proposed to contain a membrane-binding motif. Cell fractionation experiments showed that SftA can be found in the cytosolic fraction, and to a minor degree in the membrane fraction, showing that it is a soluble protein in vivo. The expression of truncated SftA constructs led to a dominant sftA deletion phenotype, even at very low induction rates of the truncated proteins, indicating that the incorporation of nonfunctional monomers into SftA hexamers abolishes functionality. Mobility shift experiments and surface plasmon binding studies showed that SftA binds to DNA in a cooperative manner, and demonstrated low ATPase activity when binding to short nucleotides rather than to long stretches of DNA.

KW - Bacillus subtilis

KW - Bacterial Proteins

KW - Cell Cycle Proteins

KW - Chromosome Segregation

KW - Chromosomes, Bacterial

KW - Cytosol

KW - DNA, Bacterial

KW - DNA-Binding Proteins

KW - Gene Knockout Techniques

KW - Protein Binding

KW - Sequence Deletion

KW - Journal Article

U2 - 10.1159/000450725

DO - 10.1159/000450725

M3 - SCORING: Journal article

C2 - 28110333

VL - 27

SP - 29

EP - 42

JO - J MOL MICROB BIOTECH

JF - J MOL MICROB BIOTECH

SN - 1464-1801

IS - 1

ER -