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

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716424115 ◽

2018 ◽

Vol 115 (13) ◽

pp. 3458-3463 ◽

Cited By ~ 5

Author(s):

Andrzej Szewczak-Harris ◽

Jan Löwe

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Copy Number ◽

Adaptor Protein ◽

Nucleotide Hydrolysis ◽

Left Handed ◽

Plasmid R1 ◽

Copy Number Plasmid ◽

Centromeric Sequence ◽

Low Copy Number

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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Analysis of a DNA region from low-copy-number plasmid pYAN-1 ofSphingobium yanoikuyaeresponsible for plasmid stability

Bioscience Biotechnology and Biochemistry ◽

10.1080/09168451.2014.890029 ◽

2014 ◽

Vol 78 (3) ◽

pp. 510-515 ◽

Cited By ~ 2

Author(s):

Hiroe Hayashi ◽

Yasuroh Kurusu

Keyword(s):

Copy Number ◽

Plasmid Stability ◽

Copy Number Plasmid ◽

Low Copy Number

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Detection of displacement (“D”) loops with the properties of a replicating intermediate synthesized by a DNA/membrane complex derived from the low-copy-number plasmid RK2

Plasmid ◽

10.1016/0147-619x(84)90051-9 ◽

1984 ◽

Vol 12 (3) ◽

pp. 227-232 ◽

Cited By ~ 2

Author(s):

William Firshein ◽

Lucien Caro

Keyword(s):

Copy Number ◽

Membrane Complex ◽

Copy Number Plasmid ◽

Low Copy Number ◽

Plasmid Rk2

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Identification of a low-copy-number mutation within the pUB110 replicon and its effect on plasmid stability in Bacillus subtilis

Gene ◽

10.1016/0378-1119(90)90477-9 ◽

1990 ◽

Vol 94 (1) ◽

pp. 121-124 ◽

Cited By ~ 10

Author(s):

Heinrich Leonhardt

Keyword(s):

Bacillus Subtilis ◽

Copy Number ◽

Plasmid Stability ◽

Low Copy Number

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A vector for studying plasmid stability functions in Streptomyces

Genetics Research ◽

10.1017/s0016672300023971 ◽

1988 ◽

Vol 51 (1) ◽

pp. 71-74 ◽

Cited By ~ 4

Author(s):

Kevin Kendall ◽

John Cullum

Keyword(s):

Copy Number ◽

Stability Region ◽

Selectable Marker ◽

Plasmid Stability ◽

Stability Function ◽

Melanin Production ◽

Stability Functions ◽

Tyrosinase Gene ◽

Copy Number Plasmid ◽

Low Copy Number

SummaryWe constructed a cloning vector (pMT603) based on the low copy number plasmid SCP2*. pMT6O3 is unstable because it lacks the SCP2* stability region and carries the selectable marker thiostrepton-resistance and a tyrosinase gene which results in melanin production. This allows easy testing of plasmid stability and we demonstrated its usefulness by cloning a plasmid stability function.

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