Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Fidaxomicin Inhibits Spore Production in Clostridium difficile

doi: 10.1093/cid/cis453

Figure Lengend Snippet: Development of total viable counts (filled symbols with solid lines) and heat-resistant spores (open symbols with dashed lines) in Clostridium difficile American Type Culture Collection 43255 strain: in the absence of drugs (•, ○), in the presence of fidaxomicin (FDX) (▴, Δ), in the presence of OP-1118 (▾, ▿), and in the presence of vancomycin (▪, □). The data represent the averages of 3 independent runs for FDX, 4 independent runs for vancomycin, and 4 independent runs for OP-1118. Error bars represent the standard error of the mean. Abbreviation: CFUs, colony-forming units.

Article Snippet: There have been conflicting reports of the sporulation efficiency of the ATCC 43255 strain [ , ].

Techniques:

Journal: mSphere

Article Title: The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

doi: 10.1128/mSphere.00007-21

Figure Lengend Snippet: Stages of CotY-SNAP localization during sporulation. Sporulating cells of B. cereus producing CotY-SNAP were labeled with the SNAP substrate TMR-Star and with the membrane dye MTG and were imaged by SR-SIM. Images of a representative cell for each pattern of localization identified at a particular stage of sporulation are shown, with schematic representations (patterns a to h ) below. The white arrow points to the weak signal of CotY-SNAP detected at curved septa; pink arrows point to the exosporium at different stages of its formation; curved black arrows point to the mother cell-proximal (MCP) and -distal (MCD) forespore poles and to the outer forespore membranes (OFM). Bars, 0.5 μm. The stages of ExsY-SNAP, SNAP-CotE, and CotE-SNAP localization during sporulation are shown in Fig. S1A , Fig. S1B , and Fig. S2A , respectively.

Article Snippet: We therefore performed sporulation of ATCC 14579 cells producing CotY-, ExsY-, or CotE-SNAP fusions or SNAP-CotE at 20°C and showed that their patterns of localization during sporulation at 20°C and 37°C were identical (data not shown).

Techniques: Labeling, Membrane

Journal: mSphere

Article Title: The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

doi: 10.1128/mSphere.00007-21

Figure Lengend Snippet: CotE is required for cap formation, allowing CotY and ExsY to localize as caps. Samples were collected from cultures of a cotE mutant producing CotY-SNAP (A) or ExsY-SNAP (B) during sporulation, and the cells were imaged by SR-SIM following staining with TMR-Star and MTG. Cells representative of the different localization patterns observed are shown (panels a to f ). Purple arrows point to the signal from CotY-SNAP or ExsY-SNAP (panels e and f ). In the cotE mutant, CotY-SNAP and ExsY-SNAP never formed the cap or the subsequent patterns identified in the WT ( Fig. 1 and Fig. S1A ) but formed large patches or aggregates in the mother cell cytoplasm. Bars, 0.5 μm.

Article Snippet: We therefore performed sporulation of ATCC 14579 cells producing CotY-, ExsY-, or CotE-SNAP fusions or SNAP-CotE at 20°C and showed that their patterns of localization during sporulation at 20°C and 37°C were identical (data not shown).

Techniques: Mutagenesis, Staining

Journal: mSphere

Article Title: The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

doi: 10.1128/mSphere.00007-21

Figure Lengend Snippet: ExsY is required for encasement by CotY but not by CotE. (A and B) Samples were withdrawn at the indicated times from 37°C cultures of an exsY mutant producing CotY-SNAP (A) or CotE-SNAP (B). The cells were labeled with TMR-Star and MTG and were imaged by phase-contrast and fluorescence microscopy. Localization patterns a to g are identical to patterns a to g observed in WT cells in Fig. 1 . Patterns c *, d *, and j * (white arrows) correspond to patterns c , d , and j observed in sporangia of phase-bright spores or in free spores. Numbers on the right show the percentage of cells with each of the indicated patterns relative to the total number of sporulating cells expressing the different SNAP fusions. ND; no signal detected. (C) Spores of the indicated strains were stained with MTG and TMR-STAR and were imaged by SR-SIM. The strains were ATCC 10876 (WT) and a derivative producing CotE-SNAP, as well as a congenic exsY mutant and derivatives producing CotE-SNAP or CotY-SNAP. Yellow arrows point to exosporia blocked at the two-cap stage. The pink arrow points to a complete exosporium. Cyan arrows point to nonspecific TMR signals. Bars, 1 μm in panels A and B and 0.5 μm in panel C.

Article Snippet: We therefore performed sporulation of ATCC 14579 cells producing CotY-, ExsY-, or CotE-SNAP fusions or SNAP-CotE at 20°C and showed that their patterns of localization during sporulation at 20°C and 37°C were identical (data not shown).

Techniques: Mutagenesis, Labeling, Fluorescence, Microscopy, Expressing, Staining

Journal: mSphere

Article Title: The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

doi: 10.1128/mSphere.00007-21

Figure Lengend Snippet: CotE, CotY, and ExsY form complexes in vivo and interact in vitro . (A) Samples were collected at the indicated times from sporulating cultures of B. cereus strains producing various SNAP fusions. Whole-cell extracts were prepared and subjected to pulldown assays with a SNAP capture resin. Whole-cell extracts, flowthrough, and bound proteins were resolved by SDS-PAGE and subjected to immunoblot analyses with anti-CotE antibodies. A lane corresponding to a pulldown assay performed on proteins extracted from purified CotY-SNAP and ExsY-SNAP spores was added. One red asterisk indicates a monomer of CotE; two red asterisks, a potential dimer of CotE; three red asterisks, multimers; blue asterisks, possible proteolytic products of CotE. A nonspecific signal in the extracts from vegetative cells (hour 0) is indicated by a green asterisk. (B) Heterologous coexpression pulldown assays. E. coli BL21(DE3) cells either producing CotE alone or coproducing CotE with His 6 -CotY or His 6 -ExsY were lysed and subjected to pulldown assays. Proteins were subjected to immunoblot analysis with anti-CotE (top) or anti-His 6 (bottom) antibodies. While CotE produced alone was not eluted from the Ni 2+ beads (6th to 8th lanes, top), His 6 -CotY pulled down CotE in E1 (9th lane, top) and was detected in E1 and E2 (9th and 10th lanes, bottom). CotE was pulled down with His 6 -ExsY in E1 and E2 (12th and 13th lanes, top) and was detected in E1 and E2 (12th and 13th lanes, bottom). CE, cell extract; FT, flowthrough; w 1 to w 3 , washes; E 1 to E 3 , elutions. Red asterisks indicate the different species of CotE (upper panels), His 6 -tagged CotY (9th to 11th lanes, bottom), or His 6 -tagged ExsY (12th to 14th lanes, bottom). The positions of molecular weight (MW) markers (in kDa) are indicated on the left.

Article Snippet: We therefore performed sporulation of ATCC 14579 cells producing CotY-, ExsY-, or CotE-SNAP fusions or SNAP-CotE at 20°C and showed that their patterns of localization during sporulation at 20°C and 37°C were identical (data not shown).

Techniques: In Vivo, In Vitro, SDS Page, Western Blot, Purification, Produced, Molecular Weight

Journal: mSphere

Article Title: The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

doi: 10.1128/mSphere.00007-21

Figure Lengend Snippet: Successive localization, interactions, and interdependence among CotE, CotY, and ExsY during exosporium formation. (A) (Left) Assembly of the indicated spore structures as observed by TEM. Dotted lines show the new structures found in the present study. (Right) Diagrams showing the temporal sequence of localization of CotE, CotY, and ExsY inferred from our results. ( i ) CotE (red) forms a cap in the septal region at the onset of engulfment and recruits CotY (blue), which, in turn, recruits ExsY (gray). Once positioned, CotY and ExsY form the basal layer of the cap (mixed gray and blue). The cap remains unchanged until the completion of engulfment. ( ii ) After the completion of engulfment, the MCP cap is separated from the OFM by the formation of the interspace (IS), and CotE directs the localization of CotY, and therefore of ExsY, as a second cap on the MCD pole (dotted line). ( iii ) CotE progressively encases the spore starting from one longitudinal side of the forespore (red arrows), guiding the simultaneous encasement of ExsY (gray arrows), which, in turn, is required for the encasement of CotY (blue arrows). After the completion of encasement by CotE, CotY and ExsY cover the noncap region of the forespore as an immature morphogenetic scaffold (dotted line). ( iv ) After coat formation and insertion of the late-synthesized proteins, CotE, CotY, and ExsY are found in all the regions of the mature exosporium. (B) Formation of the morphogenetic scaffold and its maturation in B. cereus . Based on their roles in B. subtilis , SpoIVA and SpoVID homologs are good candidates for directing the recruitment and encasement, respectively, of CotE and CotE-controlled proteins. CotO, possibly through its interaction with CotE, may participate in the recruitment of CotY and/or ExsY. ExsA, a SafA homologue that controls B. cereus coat protein deposition, is also present in the morphogenetic scaffold. After sporulation completion, the proteins of the morphogenetic scaffold are positioned in the different layers of the mature spore.

Article Snippet: We therefore performed sporulation of ATCC 14579 cells producing CotY-, ExsY-, or CotE-SNAP fusions or SNAP-CotE at 20°C and showed that their patterns of localization during sporulation at 20°C and 37°C were identical (data not shown).

Techniques: Sequencing, Synthesized

Journal: PLoS Genetics

Article Title: Distinctive Expansion of Potential Virulence Genes in the Genome of the Oomycete Fish Pathogen Saprolegnia parasitica

doi: 10.1371/journal.pgen.1003272

Figure Lengend Snippet: ( A ) 7-day old culture filtrates were capable of degrading rainbow trout IgM after an overnight incubation at 10°C. ( B ) Schematic drawing of the domains present in the protease SPRG_14567 ( C ) Expression pattern of SPRG_14567 in different life stages. The RKPM of RNA-seq data is plotted, and the previously identified effector SpHTP-1 is plotted to show contrasting expression patterns. ( D ) The recombinant subtilisin-like protease SPRG_14567 was partially purified through tandem ion exchange (SO 3 − ) and nickel affinity columns (Fractions 1 to 4) following detection in a Western blot using anti-(His) 5 HRP antibody. ( E ) Fractions 2, 3 and soluble proteins from untransformed E. coli were tested for IgM-degrading activity with only the fraction containing the recombinant SPRG_14567 exhibiting proteolysis.

Article Snippet: 454 and Illumina sequence data were submitted to the NCBI Short Read Archive (SRA) ( http://www.ncbi.nlm.nih.gov/sra ) and can be retrieved using the following accession numbers: 454 fragment reads (SRX007896, SRX007901, SRX007898, SRX007895, SRX005344, SRX007971, SRX007958, SRX005346); 454 3 kb jumping reads (SRX007902, SRX007899, SRX007937, SRX007574, SRX007903); Illumina fragment reads (SRX022535); Illumina RNA-Seq reads (BioProject 164643: mycelium SRX155934, sporulation mycelium SRX155933, cysts SRX155932, germinating cysts SRX155938, infected fish cell-line t = 0 SRX155937, infected fish cell-line t = 8 SRX155936 and infected fish cell-line t = 24 SRX155935.

Techniques: Incubation, Expressing, RNA Sequencing Assay, Recombinant, Purification, Western Blot, Activity Assay