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

Gene ◽  
1990 ◽  
Vol 94 (1) ◽  
pp. 121-124 ◽  
Author(s):  
Heinrich Leonhardt
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Plasmid Stability ◽  
Low Copy Number

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

2004 ◽  
Vol 70 (4) ◽  
pp. 2508-2513 ◽  
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.

scholarly journals A vector for studying plasmid stability functions in Streptomyces

1988 ◽  
Vol 51 (1) ◽  
pp. 71-74 ◽  
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.

Construction of high, intermediate and low-copy-number promoter-probe plasmids for Bacillus subtilis

Gene ◽  
1986 ◽  
Vol 43 (3) ◽  
pp. 231-236 ◽  
Author(s):  
Imanaka Tadayuki ◽  
Takagaki Kazuchika ◽  
Aiba Shuichi
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Low Copy Number ◽  
Promoter Probe

scholarly journals Characterization of the Replication Region of Plasmid pLS32 from the Natto Strain of Bacillus subtilis

2005 ◽  
Vol 187 (13) ◽  
pp. 4315-4326 ◽  
Author(s):  
Teruo Tanaka ◽  
Hirofumi Ishida ◽  
Tomoko Maehara
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Tandem Repeats ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Gram Positive Bacteria ◽  
Low Copy Number ◽  
Replication Region ◽  
Translational Start

ABSTRACT Plasmid pL32 from the Natto strain of Bacillus subtilis belongs to a group of low-copy-number plasmids in gram-positive bacteria that replicate via a theta mechanism of replication. We studied the DNA region encoding the replication protein, RepN, of pLS32, and obtained the following results. Transcription of the repN gene starts 167 nucleotides upstream from the translational start site of repN. The copy number of repN-coding plasmid pHDCS2, in which the repN gene was placed downstream of the IPTG (isopropyl-1-thio-β-d-galactopyranoside)-inducible Pspac promoter, was increased 100 fold by the addition of IPTG. Histidine-tagged RepN bound to a specific region in the repN gene containing five 22-bp tandem repeats (iterons) with partial mismatches, as shown by gel retardation and foot printing analyses. Sequence alterations in the first three iterons resulted in an increase in plasmid copy number, whereas those in either the forth or fifth iteron resulted in the failure of plasmid replication. The iterons expressed various degrees of incompatibility with an incoming repN-driven replicon pSEQ243, with the first three showing the strongest incompatibility. Finally, by using a plasmid, pHDMAEC21, carrying the sequence alterations in all the five iterons in repN and thus unable to replicate but encoding intact RepN, the region necessary for replication was confined to a 96-bp sequence spanning the 3′-terminal half of the fourth iteron to an A+T-rich region located downstream of the fifth iteron. From these results, we conclude that the iterons in repN are involved in both the control of plasmid copy number and incompatibility, and we suggest that the binding of RepN to the last two iterons triggers replication by melting the A+T-rich DNA sequence.

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

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.

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

2018 ◽  
Vol 115 (13) ◽  
pp. 3458-3463 ◽  
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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