Cell-wall synthesis in Chlamydomonas reinhardtii: an immunological study on the wild type and wall-less mutants cw2 and cw15

ABSTRACTVancomycin-intermediateStaphylococcus aureus(VISA) strains present an increasingly difficult problem in terms of public health. However, the molecular mechanism for this resistance is not yet understood. In this study, we define the role of themsaABCRoperon in vancomycin resistance in three clinical VISA strains, i.e., Mu50, HIP6297, and LIM2. Deletion of themsaABCRoperon resulted in significant decreases in the vancomycin MIC (from 6.25 to 1.56 μg/ml) and significant reductions of cell wall thickness in strains Mu50 and HIP6297. Growth of the mutants in medium containing vancomycin at concentrations greater than 2 μg/ml resulted in decreases in the growth rate, compared with the wild-type strains. Mutation of themsaABCRoperon also reduced the binding capacity for vancomycin. We conclude that themsaABCRoperon contributes to resistance to vancomycin and cell wall synthesis inS. aureus.

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A novel and conserved cell wall enzyme that can substitute for the Lipid II synthase MurG

10.1101/2020.10.12.336396 ◽

2020 ◽

Author(s):

L. Zhang ◽

K. Ramijan ◽

V.J. Carrión ◽

L.T van der Aart ◽

J. Willemse ◽

...

Keyword(s):

Cell Wall ◽

Streptomyces Coelicolor ◽

Genomic Analysis ◽

Cell Wall Synthesis ◽

Wild Type ◽

Precursor Molecule ◽

Sequence Identity ◽

Lipid Ii ◽

Bona Fide ◽

Bearing Structure

ABSTRACTThe cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule Lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here we present the discovery of a novel cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster including murG surprisingly produced Lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG paralogue, which encodes a putative enzyme that shares only around 31% aa sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MurG2. Orthologues of murG2 are present in 38% of all genomes of Kitasatosporae and members of the sister genus Streptomyces. CRISPRi experiments showed that K. viridifaciens murG2 can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. Altogether, these results identify MurG2 as a bona fide Lipid II synthase, thus demonstrating plasticity in cell wall synthesis.

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A Defective Undecaprenyl Pyrophosphate Synthase Induces Growth and Morphological Defects That Are Suppressed by Mutations in the Isoprenoid Pathway ofEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00255-18 ◽

2018 ◽

Vol 200 (18) ◽

Cited By ~ 7

Author(s):

William J. MacCain ◽

Suresh Kannan ◽

Dannah Z. Jameel ◽

Jerry M. Troutman ◽

Kevin D. Young

Keyword(s):

Cell Wall ◽

Bacterial Survival ◽

Cell Wall Synthesis ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Peptidoglycan Biosynthesis ◽

Bacterial Morphology ◽

Morphological Defects ◽

Undecaprenyl Phosphate

ABSTRACTThe peptidoglycan exoskeleton shapes bacteria and protects them against osmotic forces, making its synthesis the target of many current antibiotics. Peptidoglycan precursors are attached to a lipid carrier and flipped from the cytoplasm into the periplasm to be incorporated into the cell wall. InEscherichia coli, this carrier is undecaprenyl phosphate (Und-P), which is synthesized as a diphosphate by the enzyme undecaprenyl pyrophosphate synthase (UppS).E. coliMG1655 exhibits wild-type morphology at all temperatures, but one of our laboratory strains (CS109) was highly aberrant when grown at 42°C. This strain contained mutations affecting the Und-P synthetic pathway genesuppS,ispH, andidi. Normal morphology was restored by overexpressinguppSor by replacing the mutant (uppS31) with the wild-type allele. Importantly, movinguppS31into MG1655 was lethal even at 30°C, indicating that the altered enzyme was highly deleterious, but growth was restored by adding the CS109 versions ofispHandidi. Purified UppSW31Rwas enzymatically defective at all temperatures, suggesting that it could not supply enough Und-P during rapid growth unless suppressor mutations were present. We conclude that cell wall synthesis is profoundly sensitive to changes in the pool of polyisoprenoids and that isoprenoid homeostasis exerts a particularly strong evolutionary pressure.IMPORTANCEBacterial morphology is determined primarily by the overall structure of the semirigid macromolecule peptidoglycan. Not only does peptidoglycan contribute to cell shape, but it also protects cells against lysis caused by excess osmotic pressure. Because it is critical for bacterial survival, it is no surprise that many antibiotics target peptidoglycan biosynthesis. However, important gaps remain in our understanding about how this process is affected by peptidoglycan precursor availability. Here, we report that a mutation altering the enzyme that synthesizes Und-P prevents cells from growing at high temperatures and that compensatory mutations in enzymes functioning upstream ofuppScan reverse this phenotype. The results highlight the importance of Und-P metabolism for maintaining normal cell wall synthesis and shape.

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Gametic differentiation in Chlamydomonas reinhardtii. III. Cell wall lysis and microfilament-associated mating structure activation in wild-type and mutant strains.

The Journal of Cell Biology ◽

10.1083/jcb.67.3.623 ◽

1975 ◽

Vol 67 (3) ◽

pp. 623-637 ◽

Cited By ~ 76

Author(s):

U W Goodenough ◽

R L Weiss

Keyword(s):

Cell Wall ◽

Chlamydomonas Reinhardtii ◽

Mating Type ◽

Experimental Manipulation ◽

Wild Type ◽

Opposite Mating Type ◽

Mating Structure ◽

Cell Wall Lysis ◽

Gamete Formation ◽

Mutant Strains

Cell fusion between mating type plus (mt+) and minus (mt-) gametes of Chlamydomonas reinhardtii is analyzed structurally and subjected to experimental manipulation. Cell wall lysis, a necessary prelude to fusion, is shown to require flagellar agglutination between competent gametes; glutaraldehyde-fixed gametes ("corpses") of one mating type will elicit both agglutination and cell wall lysis in the opposite mating type, whereas nonagglutinating impotent (imp) mutant strains are without effect. The fusion process is mediated by a narrow fertilization tubule which extends from the mt+ gamete and establishes contact with the mt- gamete. Formation of the tubule requires the "activation" of a specialized mating structure associated with the ml+ cell membrane; activation causes microfilaments to polymerize from the mating structure into the growing fertilization tubule. Mating structure activation is shown to depend on gametic flagellar agglutination; isoagglutination mediated by the lectin concanavalin A has no effect. Gametes carrying the imp-l mt+ mutation are able to agglutinate but not fuse with mt- cells; the imp-l gametes are shown to have structurally defective mating structures that do not generate microfilaments in response to gametic agglutination.

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Ultrastructure and Morphology of the Neurospora Crassa Cell Wall Mutants, Slime And Slime X, Using Tem and Sem

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090257 ◽

1976 ◽

Vol 34 ◽

pp. 54-55

Author(s):

Karen S. Howard ◽

H. D. Braymer ◽

M. D. Socolofsky ◽

S. A. Milligan

Keyword(s):

Cell Wall ◽

Neurospora Crassa ◽

Wild Type ◽

Morphological Comparison ◽

Small Areas ◽

Solid Media ◽

Thin Sectioning ◽

X Cells ◽

Mutant Cells ◽

Isolated Cell

The recently isolated cell wall mutant slime X of Neurospora crassa was prepared for ultrastructural and morphological comparison with the cell wall mutant slime. The purpose of this article is to discuss the methods of preparation for TEM and SEM observations, as well as to make a preliminary comparison of the two mutants.TEM: Cells of the slime mutant were prepared for thin sectioning by the method of Bigger, et al. Slime X cells were prepared in the same manner with the following two exceptions: the cells were embedded in 3% agar prior to fixation and the buffered solutions contained 5% sucrose throughout the procedure.SEM: Two methods were used to prepare mutant and wild type Neurospora for the SEM. First, single colonies of mutant cells and small areas of wild type hyphae were cut from solid media and fixed with OSO4 vapors similar to the procedure used by Harris, et al. with one alteration. The cell-containing agar blocks were dehydrated by immersion in 2,2-dimethoxypropane (DMP).

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