Cryo-EM reconstruction of AlfA fromBacillus subtilisreveals the structure of a simplified actin-like filament at 3.4-Å resolution

Low copy-number plasmid pLS32 ofBacillus subtilissubsp.nattocontains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequenceparN. Similar to the ParMRC partitioning system fromEscherichia coliplasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB andparN. Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system’s biological raison d’être.

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Cryo-EM reconstruction of AlfA fromBacillus subtilisreveals the structure of a simplified actin-like filament at 3.4 Å resolution

10.1101/190744 ◽

2017 ◽

Author(s):

Andrzej Szewczak-Harris ◽

Jan Löwe

Keyword(s):

Atomic Structure ◽

Adaptor Protein ◽

Nucleotide Hydrolysis ◽

E Coli ◽

Left Handed ◽

Copy Number Plasmid ◽

Resolution Electron ◽

Centromeric Sequence ◽

Low Copy Number ◽

Almost All

AbstractLow copy-number plasmid pLS32 ofBacillus subtilissubsp.nattocontains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB and the centromeric sequenceparN. Similar to the ParMRC partitioning system fromE. coliplasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids, through interactions with AlfB andparN. Since, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of non-bundling AlfA filaments to 3.4 Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin-fold has been accommodated by unique longitudinal and lateral contacts, whilst still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments we obtained a pseudo-atomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly anti-phasic with 8-fold integer helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, signs of the strong evolutionary pressure for simplicity, placed on plasmids: deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerisation, nucleotide hydrolysis and antiparallel doublet formation are retained to fulfil the system's biological raison d'être.Significance StatementProtein filaments perform a vast array of functions inside almost all living cells. Actin-like proteins in archaea and bacteria have previously been found to form a surprising diversity of filament architectures, reflecting their divergent cellular roles. Actin-like AlfA is unique in that it is much smaller than all other filament forming actin-like proteins. With an atomic structure of the AlfA filament, obtained by high-resolution electron cryo-microscopy, we have revealed—at atomic level of detail—how AlfA filaments form dynamic filaments capable of transporting plasmid DNA in cells and how these filaments arrange into antiparallel bundles required for the segregation mechanism.

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Escherichia coli low-copy-number plasmid R1 centromere parC forms a U-shaped complex with its binding protein ParR

Nucleic Acids Research ◽

10.1093/nar/gkm672 ◽

2007 ◽

Vol 36 (2) ◽

pp. 607-615 ◽

Cited By ~ 11

Author(s):

C. Hoischen ◽

M. Bussiek ◽

J. Langowski ◽

S. Diekmann

Keyword(s):

Escherichia Coli ◽

Copy Number ◽

Binding Protein ◽

Plasmid R1 ◽

Copy Number Plasmid ◽

Low Copy Number

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Targeted Isolation of a Designated Region of the Bacillus subtilis Genome by Recombinational Transfer

Applied and Environmental Microbiology ◽

10.1128/aem.70.4.2508-2513.2004 ◽

2004 ◽

Vol 70 (4) ◽

pp. 2508-2513 ◽

Cited By ~ 12

Author(s):

Satoshi Tomita ◽

Kenji Tsuge ◽

Yo Kikuchi ◽

Mitsuhiro Itaya

Keyword(s):

Bacillus Subtilis ◽

Copy Number ◽

Positional Cloning ◽

Deletion Mutant ◽

Essential Gene ◽

Transfer System ◽

Copy Number Plasmid ◽

Low Copy Number ◽

Subtilis Genome

ABSTRACT A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

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Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

Journal of Bacteriology ◽

10.1128/jb.150.3.1234-1243.1982 ◽

1982 ◽

Vol 150 (3) ◽

pp. 1234-1243 ◽

Cited By ~ 7

Author(s):

W Firshein ◽

P Strumph ◽

P Benjamin ◽

K Burnstein ◽

J Kornacki

Keyword(s):

Escherichia Coli ◽

Plasmid Dna ◽

Copy Number ◽

Membrane Complex ◽

Copy Number Plasmid ◽

Low Copy Number

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Versatile low-copy-number plasmid vectors for cloning in Escherichia coli

Gene ◽

10.1016/0378-1119(82)90172-x ◽

1982 ◽

Vol 18 (3) ◽

pp. 335-341 ◽

Cited By ~ 203

Author(s):

Neil G. Stoker ◽

Neu F. Fairweathe ◽

Brian G. Spratt

Keyword(s):

Escherichia Coli ◽

Copy Number ◽

Plasmid Vectors ◽

Copy Number Plasmid ◽

Low Copy Number

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Lentivirus envelope sequences and pro viral genomes are stabilized in Escherichia coli when cloned in low-copy-number plasmid vectors

Gene ◽

10.1016/0378-1119(93)90766-v ◽

1993 ◽

Vol 124 (1) ◽

pp. 93-98 ◽

Cited By ~ 36

Author(s):

Thomas P. Cunningham ◽

Ronald C. Montelaro ◽

Keith E. Rushlow

Keyword(s):

Escherichia Coli ◽

Copy Number ◽

Plasmid Vectors ◽

Viral Genomes ◽

Copy Number Plasmid ◽

Low Copy Number

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Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid.

Journal of Bacteriology ◽

10.1128/jb.169.9.4141-4146.1987 ◽

1987 ◽

Vol 169 (9) ◽

pp. 4141-4146 ◽

Cited By ~ 8

Author(s):

K Watabe ◽

R Forough

Keyword(s):

Bacillus Subtilis ◽

Copy Number ◽

Temperature Sensitive ◽

Copy Number Plasmid ◽

Low Copy Number ◽

Effects Of Temperature

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Polymer dynamics of Alp7A reveals two ‘critical’ concentrations that govern dynamically unstable actin-like proteins

10.1101/098954 ◽

2017 ◽

Author(s):

Natalie A. Petek ◽

Alan I. Derman ◽

Jason A. Royal ◽

Joe Pogliano ◽

R. Dyche Mullins

Keyword(s):

Bacillus Subtilis ◽

Copy Number ◽

Atp Hydrolysis ◽

Critical Concentration ◽

Polymer Dynamics ◽

Fundamental Parameters ◽

Critical Concentrations ◽

Copy Number Plasmid ◽

Accessory Factor ◽

Low Copy Number

ABSTRACT:Dynamically unstable polymers capture and move cellular cargos in both bacteria and eukaryotes, but the regulation of their assembly remains poorly understood. Here we describe polymerization of Alp7A, a bacterial Actin-Like Protein (ALP) that segregates the low copy-number plasmid pLS20 inBacillus subtilis.Purified Alp7A forms dynamically unstable polymers with two critical points: an intrinsic critical concentration (0.6 μM), observed when ATP hydrolysis is blocked, and a dynamic critical concentration (10.3 μM), observed when ATP hydrolysis occurs. From biochemical and kinetic analysis, the intrinsic critical concentration reflects a balance between filament elongation and shortening, while the dynamic critical concentration reflects a balance between filament nucleation and catastrophic disassembly. Although Alp7A does not form stable polymers at physiological concentrations, rapid nucleation by an accessory factor, Alp7R, decreases the dynamic critical concentration into the physiological range. Intrinsic and dynamic critical concentrations are fundamental parameters that can be used to describe the behavior of all dynamically unstable polymers.

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