Journal: Synthetic and Systems Biotechnology

Article Title: Determination of the chromosomal position effects for plug-and-play application in the Myxococcus xanthus chassis cells

doi: 10.1016/j.synbio.2024.04.007

Figure Lengend Snippet: The dimension and transcription of M. xanthus DK1622 chromosome. (a) The genetic compartmentalization of DK1622 chromosome. Green rectangle, oriC (21,268–21,643 bp); yellow rectangles, rRNA operons; red rectangle, replication terminal Ter; blue rectangle, attB site for site-specific recombination. (b) The GC content of M. xanthus DK1622 chromosome. (c) The distribution of essential genes in DK1622 chromosome. Red rectangles, genes not inserted in mutant libraries. (d) The distribution of smBGCs in DK1622 chromosome. (e) Global transcription of DK1622 chromosome. RPKM, reads per kilobase per million mapped reads.

Article Snippet: The M. xanthus DK1622 cells were collected after 28 h of incubation, followed with total RNA extraction with the TRIzol® Reagents according to the manufacturer's instructions (Invitrogen) and genomic DNA was removed using DNase I (Takara).

Techniques: Mutagenesis

Journal: Synthetic and Systems Biotechnology

Article Title: Determination of the chromosomal position effects for plug-and-play application in the Myxococcus xanthus chassis cells

doi: 10.1016/j.synbio.2024.04.007

Figure Lengend Snippet: Flowchart depicting Tn-seq, from library construction to massively parallel sequencing of transposon-chromosome junctions. (a) Construction of transposition mutation library of M. xanthus DK1622. The GK cassette integrated into DK1622 genome was marked with green square. IR, inverted repeat; TT, transcription terminator, km r , kanamycin resistance gene. The mutants were screened into three libraries with different concentrations of kanamycin. (b) Preparation of library for Tn-seq sequencing. (c) Processing of Tn-seq sequencing data. Detailed information was described in Methods.

Article Snippet: The M. xanthus DK1622 cells were collected after 28 h of incubation, followed with total RNA extraction with the TRIzol® Reagents according to the manufacturer's instructions (Invitrogen) and genomic DNA was removed using DNase I (Takara).

Techniques: Sequencing, Mutagenesis

Journal: Synthetic and Systems Biotechnology

Article Title: Determination of the chromosomal position effects for plug-and-play application in the Myxococcus xanthus chassis cells

doi: 10.1016/j.synbio.2024.04.007

Figure Lengend Snippet: Description of insertion sites in mutant libraries. (a) Venn diagram of the insertion sites in libraries km40, km200, and km1000. (b) Distribution of insertion sites with corresponding insertion index across the DK1622 genome in different libraries. The km1000 was also enlarged and displayed at the same scale of km40 and km200. (c) Comparation of the insertion index of each insertion site in different libraries. The median of insertion indexes was indicated.

Article Snippet: The M. xanthus DK1622 cells were collected after 28 h of incubation, followed with total RNA extraction with the TRIzol® Reagents according to the manufacturer's instructions (Invitrogen) and genomic DNA was removed using DNase I (Takara).

Techniques: Mutagenesis

Journal: Synthetic and Systems Biotechnology

Article Title: Determination of the chromosomal position effects for plug-and-play application in the Myxococcus xanthus chassis cells

doi: 10.1016/j.synbio.2024.04.007

Figure Lengend Snippet: Screening of enrichment sites. (a) Classification of insertion sites shared by three libraries. (b) Distribution of enrichment and dilution sites on the circular DK1622 genome. From the inside out, circle 1, genomic location information; circle 2 (black), GC content of DK1622 genome; circle 3 (red), all enrichment sites in DK1622 genome; circle 4 (red), the top 100 enrichment sites with large insertion index; circle 5 (green), horizontal transfer genes in DK1622 genome; circle 6 (purple), dilution sites in DK1622 genome. The vacuum regions of the top 100 enrichment sites are marked by the outer bold red arrows. The enrichment sites and dilution sites for verification are marked with inner red and purple thin arrows, respectively. (c) Comparison of the local GC content of theoretical TA sites, enrichment sites and dilution sites. *** Wilcoxon test, p < 0.001.

Article Snippet: The M. xanthus DK1622 cells were collected after 28 h of incubation, followed with total RNA extraction with the TRIzol® Reagents according to the manufacturer's instructions (Invitrogen) and genomic DNA was removed using DNase I (Takara).

Techniques: Comparison

Journal: Synthetic and Systems Biotechnology

Article Title: Determination of the chromosomal position effects for plug-and-play application in the Myxococcus xanthus chassis cells

doi: 10.1016/j.synbio.2024.04.007

Figure Lengend Snippet: Verification of the enrichment sites. (a) Analysis of the egfp expression at representative enrichment sites and dilution sites. The expression of egfp integrated at attB site was used as control (the red and green dotted lines represent the transcription and expression of egfp in DK-19- egfp , respectively). The error bars represent the standard deviation of three independent experiments. (b) Detection of the indigoidine in M. xanthus mutants expressing the idgS . The error bars represent the standard deviation of three independent experiments. (c) The growth of DK1622 and different M. xanthus mutants expressing the idgS at representative enrichment sites and attB site.

Article Snippet: The M. xanthus DK1622 cells were collected after 28 h of incubation, followed with total RNA extraction with the TRIzol® Reagents according to the manufacturer's instructions (Invitrogen) and genomic DNA was removed using DNase I (Takara).

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: A lytic transglycosylase connects bacterial focal adhesion complexes to the peptidoglycan cell wall

doi: 10.1101/2024.04.04.588103

Figure Lengend Snippet: A) AgmT shows significant similarity to a widely conserved PG transglycosylase in YceG/MltG family. The conserved glutamine residue is marked by an asterisk. M_xant, M. xanthus ; E_coli, E. coli ; M_tube, Mycobacterium tuberculosis ; S_coel, Streptomyces coelicolor ; A_calc, Acinetobacter calcoaceticus ; X_albi, Xanthomonas albilineans ; N_meni, Neisseria menningitidis ; R_prow, Rickettsia prowazekii ; T_aqua, Thermus aquaticus . B) AgmT is required for M. xanthus gliding. Colony edges were imaged after incubating cells on 1.5% agar surface for 24 h. Deleting or Disabling the active site of AgmT abolishes gliding but fusing an PAmCherry (PAmCh) to its C-terminus does not. Heterologous Expression of E. coli MltG restores gliding of 1agmT cells but not the cells that express AgmT EAEA .

Article Snippet: Vegetative M. xanthus cells were grown in liquid CYE medium (10 mM MOPS pH 7.6, 1% (w/v) Bacto™ casitone (BD Biosciences), 0.5% yeast extract and 8 mM MgSO 4 ) at 32 °C, in 125-ml flasks with rigorous shaking, or on CYE plates that contains 1.5% agar.

Techniques: Residue, Expressing

Journal: bioRxiv

Article Title: A lytic transglycosylase connects bacterial focal adhesion complexes to the peptidoglycan cell wall

doi: 10.1101/2024.04.04.588103

Figure Lengend Snippet: A) Immunoblotting using M. xanthus cell lysates and an anti-mCherry antibody shows that PAmCherry-labeled AgmT and AgmT EAEA accumulate as full-length proteins. AgmT is significantly more abundant than the motor protein AglR. The bacterial actin homolog MreB visualized using an MreB antibody is shown as a loading control. B) AgmT does not aggregate into bFACs. C) The LTG activity of AgmT is required for connecting bFACs to PG and expressing the E. coli LTG MltG (MltG Ec ) restores PG binding by bFACs in cells that lack AgmT but not in the ones that express an inactive AgmT variant (AgmT EAEA ). AglR-PAmCherry was detected using an anti-mCherry antibody to mark the presence of bFACs that co-precipitate with PG-containing (lysozyme-) pellets. Lysates from the cells that express AglR-PAmCherry in different genetic backgrounds were pelleted by centrifugation in the presence and absence of lysozyme.

Article Snippet: Vegetative M. xanthus cells were grown in liquid CYE medium (10 mM MOPS pH 7.6, 1% (w/v) Bacto™ casitone (BD Biosciences), 0.5% yeast extract and 8 mM MgSO 4 ) at 32 °C, in 125-ml flasks with rigorous shaking, or on CYE plates that contains 1.5% agar.

Techniques: Western Blot, Labeling, Activity Assay, Expressing, Binding Assay, Variant Assay, Centrifugation