Control of morphogenesis in the budding Alphaproteobacterium Hyphomonas neptunium
The size and shape of bacteria are manifold just as their modes of propagation. The cell wall, composed of peptidoglycan (PG), is the major cell shape determinant in most bacteria. So far research on spatiotemporal coordination of morphology and cell division has mainly focused on rod-shaped bacteri...
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|Summary:||The size and shape of bacteria are manifold just as their modes of propagation. The cell wall, composed of peptidoglycan (PG), is the major cell shape determinant in most bacteria. So far research on spatiotemporal coordination of morphology and cell division has mainly focused on rod-shaped bacteria like Escherichia coli and Bacillus subtilis. In this study, we investigate the dimorphic Alphaproteobacteria Hyphomonas neptunium as a new model organism for the study of asymmetric morphology and reproduction by budding at the distal end of a stalk.
Our goal was to comprehensively analyse the growth mode and budding mechanism of H. neptunium. Detailed electron cryo-tomography images revealed that, unlike previously suggested, the stalk and the bud form a continuum with the mother cell up until cell division. We show that during budding the daughter cell incorporates part of the stalk belonging to the mother cell to complete its own growth. Furthermore, we demonstrate that H. neptunium can accomplish more replicative cycles than previously proposed. By monitoring the incorporation of nascent PG with HADA, we identified four different growth phases in H. neptunium that can be divided into dispersed (swarmer cell growth and bud formation) and zonal growth (stalk biogenesis and cell division). PG composition analysis revealed a very high PG turnover rate as well as an unusually high incorporation of glycine instead of alanine at the 5th position of the stem peptide. A comprehensive analysis of the PG biosynthesis machinery in H. neptunium shows that the conserved actin homologue MreB, the PG synthases PBP2 and PBP3, and the PG hydrolase LmdC play a vital role in cell growth in H. neptunium. Polar PG biogenesis seems to be modulated by an array of mostly redundant synthases and hydrolases, in which LD-transpeptidases do not partake. We postulate that the morphological asymmetry of H. neptunium underlies a much more complex intracellular asymmetry determined by distinct, multiple sites of dispersed and zonal growth.
To maintain its correct cell shape, H. neptunium requires MreB as well as a coiled-coil rich protein termed CCRP and the non-canonical bactofilins, which are a new class of nucleotide-independent polymer-forming cytoskeletal elements. Upon inhibition of MreB with A22 or MP265, cells become increasingly spherical and eventually cease growing. The deletion of ccrp causes elongated stalks accompanied by slight cell chaining and irregular cell shape. In the absence of both bactofilin para-logues BacA and BacB, H. neptunium cells adopt a severely distorted cell morphology with multiple and branched stalks. In addition to bud formation at the distal end of the stalk, these mutants can generate buds directly from the cell body of the mother cell. Both bactofilins localize dynamically at the future stalked pole throughout the cell cycle and within the stalk just adjacent to the tip and later at the future division site. Time-lapse microscopy of the double deletion mutant revealed that the first step which leads to loss of cell morphology is the relinquishment of the stalk as a reproductive organelle, which is unimpededly incorporated by the emerging bud. Thus the stalk is lost, which leads to deregulation of cell wall biogenesis within the complete cell, generating amorphous cell bodies. However, further experiments indicate that bactofilins are not essential for stalk biogenesis, they merely seem to pay a role in confining cell growth to the terminal region of the stalk. In short, bactofilins play a vital role in the maintenance of PG incorporation at the stalked pole and consequently ensure proper cell morphology.|
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