Journal: The Journal of Biological Chemistry

Article Title: A type II toxin–antitoxin system is responsible for the cell death at low temperature in Pseudomonas syringae Lz4W lacking RNase R

doi: 10.1016/j.jbc.2024.107600

Figure Lengend Snippet: Pulsed-field gel electrophoresis (PFGE) and Southern analysis of cellular DNAs of wild-type and mutant strains. A , PFGE of cellular DNAs of wt , Δ recCBD and Δ rnr cells grown at 22 °C and 4 °C. M1 and M2 are the yeast chromosomal and low-range PFGE markers from New England Biolabs (USA). Ethidium bromide (EtBr)-stained gel showing intact (circular) chromosomal DNA in the well, linearized chromosomal DNA of >2 mb in size, and the broken DNA fragments of sizes around 50 kb are also indicated. Note that the discrete DNA fragments of various sizes are observed only in Δ rnr cells incubated at 4 °C. B , Southern analysis of PFGE resolved cellular DNAs of wt , Δ recCBD, and Δ rnr cells at 4 °C. Southern hybridization using probes specific to the chromosomal DNA, 16S RNA, and the indigenous plasmid pLz4W. A probe specific to pLz4W hybridizes to the discrete DNA fragments of Δ rnr cells incubated at 4 °C. C , time-dependent accumulation of discrete DNA fragments in Δ rnr cells at 4 °C and the Southern analysis using a probe specific to pLz4W. Δrnr cells show a time-dependent accumulation of various forms of pLz4W at 4 °C. pLz4W, plasmid of P. syringae Lz4W.

Article Snippet: M1 and M2 are the yeast chromosomal and low-range PFGE markers from New England Biolabs (USA).

Techniques: Pulsed-Field Gel, Electrophoresis, Mutagenesis, Staining, Incubation, Hybridization, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: A type II toxin–antitoxin system is responsible for the cell death at low temperature in Pseudomonas syringae Lz4W lacking RNase R

doi: 10.1016/j.jbc.2024.107600

Figure Lengend Snippet: Analysis of RNase R deleted P. syringae Lz4W strain complemented with RNase R variants. A , PFGE and Southern analysis of cellular DNAs of Δ rnr cells complemented with RNase R Ps , RNase R Ec , and RNase R D284A mutants at 4 °C. Accumulation of discrete bands pLz4W DNA were observed only in Δ rnr and Δ rnr cells complemented with empty pGL10 vector. B , top panel , a schematic representation of the RNB domain of RNase R Ps , in which, the catalytically important aspartic acids of motif-1 of the RNB domain are shown. Bottom panel , growth profiles of wt and Δ rnr strains expressing RNB domain (pGLRNB) and the RNB domain with D284A mutation (pGLRNB D284A ) at 22 °C and 4 °C. Growth was determined by measuring A 600 at regular time intervals as indicated. C , top panel , a schematic representation of domain organization in RNase R Ps . The domains of RNase R Ps along with amino acids residues’ number and the N and C termini are shown. Bottom panel , PFGE and Southern analysis of cellular DNAs of Δ rnr cells complemented with domain/s deleted mutants of RNase R Ps at 4 °C. The CSD domain (Δ CSD ), the S1 domain (Δ S1 ), and the CSD and S1 domains deleted (RNB) mutants of RNase R Ps do not accumulate pLz4W-derived DNA fragments at 4 °C. D , a schematic representation of the indigenous pLz4W plasmid of P. syringae Lz4W. The psA–psT operon, open-reading frames, and other genetic elements are shown. Organization of the operon having psA and psT genes with overlapping reading frame under a common promoter is also depicted. The starting codons and predicted protein sizes of PsA antitoxin and PsT toxin are indicated. CSD, cold-shock domain; PFGE, pulsed-field gel electrophoresis; pLz4W, plasmid of P. syringae Lz4W; psA–psT, P. syringae antitoxin - P. syringae toxin system; RNase R Ec , Escherichia coli RNase R; RNase R Ps , RNase R of P. syringae .

Article Snippet: M1 and M2 are the yeast chromosomal and low-range PFGE markers from New England Biolabs (USA).

Techniques: Plasmid Preparation, Expressing, Mutagenesis, Derivative Assay, Pulsed-Field Gel, Electrophoresis

Journal: The Journal of Biological Chemistry

Article Title: A type II toxin–antitoxin system is responsible for the cell death at low temperature in Pseudomonas syringae Lz4W lacking RNase R

doi: 10.1016/j.jbc.2024.107600

Figure Lengend Snippet: PsA antitoxin expression alleviates the cold sensitivity of Δ rnr strain. A , growth profiles of wt , Δ rnr stain with an empty expression vector (pGL10), and Δ rnr strain expressing psA antitoxin (pGLpsA) determined at 22 °C and 4 °C. Growth was determined by A 600 at regular time intervals as indicated. B , PsA antitoxin restores the cell viability of the Δ rnr strain similar to wildtype at 4 °C. Cells grown at 22 °C till A 600 is 0.6, then were shifted to 4 °C and incubated for the indicated times. Aliquots of cells were collected at every 24 h, and the colony forming units (cfu/ml) were determined. C , PFGE and Southern analysis of cellular DNA of the Δ rnr strain expressing PsA antitoxin. PsA antitoxin expression reduces the copy number of pLz4W in the Δ rnr strain at 4 °C. D , Western analysis showing the expression of PsA antitoxin protein from the pGLpsA vector in Δ rnr cells. Expression of His-tag PsA antitoxin from the pGLpsA vector was determined by Western analysis using polyclonal antibodies specific to the 6x-His-tag. A protein marker with proteins of sizes 20, 14, and 6.5 are indicated. E , the copy number of pLz4W is reduced to the wildtype levels when RNase R and its derivatives are expressed in trans . Quantification of pLz4W from the PFGE-Southern hybridized gels was carried out using ImageJ2. For the wt , Δ rnr , and Δ rnr (pGL10) strains, the mean ± SD were calculated from the three independent experiments. The relative amount of pLz4W was plotted for the indicated strains compared to wildtype. PFGE, pulsed-field gel electrophoresis; pLz4W, plasmid of P. syringae Lz4W.

Article Snippet: M1 and M2 are the yeast chromosomal and low-range PFGE markers from New England Biolabs (USA).

Techniques: Expressing, Staining, Plasmid Preparation, Incubation, Western Blot, Marker, Pulsed-Field Gel, Electrophoresis

Journal: Microbial genomics

Article Title: Chromosome-scale assembly of the streamlined picoeukaryote Picochlorum sp. SENEW3 genome reveals Rabl-like chromatin structure and potential for C 4 photosynthesis.

doi: 10.1099/mgen.0.001223

Figure Lengend Snippet: Fig. 1. Pulsed-field gel electrophoresis gel slices of P. SENEW3 chromosomal DNA (p) against NEB Yeast Chromosome PFGE marker N0345S (m) at 24, 28, 32 and 36 h time points, using a Bio-Rad CHEF Mapper XA pulsed-field gel electrophoresis system.

Article Snippet: P. SENEW3 chromosomal DNA was separated using a Bio- Rad CHEF Mapper XA PFGE system.

Techniques: Pulsed-Field Gel, Electrophoresis, Marker