Reversible metamorphosis in a bacterium

The cell wall is a shape-defining and protective structure that envelops virtually all bacteria. Wall-less variants, called L-forms, have been generated in laboratories for many decades under highly specialized conditions, invariably aimed at interrupting cell wall synthesis. As such, the relevance of these cells has remained obscure. Here we show that the filamentous actinomycete Kitasatospora viridifaciens has the natural ability to switch between a wall-less state and the canonical mycelial mode-of-growth. We show that this organism thrives in a cell wall-less form, and identify the polar growth determinant DivIVA as an essential regulator required for reversible metamorphosis. This is the first report of a reversible metamorphosis in a bacterium that includes wall-less cells as a natural stage in bacterial development.

many decades under highly specialized conditions, invariably aimed at interrupting 23 cell wall synthesis. As such, the relevance of these cells has remained obscure. Here 24 we show that the filamentous actinomycete Kitasatospora viridifaciens has the natural 25 ability to switch between a wall-less state and the canonical mycelial mode-of-growth. 26 We show that this organism thrives in a cell wall-less form, and identify the polar 27 growth determinant DivIVA as an essential regulator required for reversible 28 metamorphosis. This is the first report of a reversible metamorphosis in a bacterium 29 that includes wall-less cells as a natural stage in bacterial development.

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The cell wall, an essential component of virtually all bacteria, is a dynamic structure 32 that largely determines bacterial cell shape 1 . It provides structural rigidity and 33 protection to osmotic stresses and forms the barrier between the bacterium and its 34 environment 2,3 . Given its protective role, the cell wall and its biosynthetic enzymes minutes after the first appearance of these cells (Extended Data Video S1, t= 640 107 min). Notably, such branches frequently also extruded L-form-like cells, similarly to 108 the leading hypha (Extended Data Video S2). This showed that the L-form-like cells 109 are produced at hyphal tips. 110 To study the viability of these L-form-like cells, we separated them from the 111 mycelia by filtration, and used the filtrate to inoculate fresh LPB medium. Strikingly, 112 these cells were able to proliferate in a manner reminiscent of the extrusion-resolution 113 mechanism that was previously described for proliferation of B. subtilis L-forms 20 114 ( Fig. 1F; Extended Data Video S3). Apparently, these wall-less cells are natural L-115 forms that can proliferate without the need for any genetic mutations. To discriminate 116 these natural L-forms from other stable and unstable L-forms generated by inducing 117 agents, we hereinafter refer to them as N-forms (for natural L-forms).

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When N-forms were plated on solid LPMA medium, we noticed that some of 119 the cells generated colonies consisting of both mycelia and N-form cells, implying 120 that these N-forms can revert to mycelial growth ( Fig. 1G; Extended Data Video S4).

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To exclude that mycelial growth was caused by outgrowing spores present in the 122 filtrate, we also analysed a non-sporulating ΔssgB mutant of K. viridifaciens  Altogether, this work thus presents the first example of a bacterium with the 127 natural ability to generate wall-less cells as a natural stage in bacterial life. These cells 128 can propagate without their cell wall, but can also revert to mycelial growth (Fig. 1H).

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Generation of the stable L-form lineage alpha 131 Although N-forms could propagate in the wall-less state, their phenotype was 132 reversible leading to mixtures of both hyphae and N-forms. To understand the 133 mechanism of this reversible metamorphosis, we aimed to generate a strain that could 134 reproducibly switch between an all-walled and completely wall-less state. To this end, 135 we exposed the wild-type strain to penicillin and lysozyme following a weekly sub-136 culturing regime to obtain a derivative strain, which we designated alpha. As  However, unlike the N-forms generated by the wild-type strain, the L-forms formed 147 by alpha indefinitely propagated in the wall-less state and did not revert to mycelial 148 growth on osmo-protective media.

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To see if we could identify the mutations that predisposed proliferation in the 150 L-form state, we performed SNP analysis by comparing the genome sequence of 151 alpha to that of the parent DMS40239 33 . This revealed that alpha had lost the 1.7MB 152 linear plasmid KVP1 that is present in the wild-type strain. In addition, three SNPs 153 were identified in the genome (Extended Data Table 1). One of the mutations mapped 154 to a non-coding DNA region between two genes encoding a putative glutamine-    Importantly, the DivIVA-eGFP fusion protein also restored filamentous growth to the 203 divIVA mutant, implying that the chimeric protein is largely functional in vivo. 204 However, some hyphae showed apical branching or tip-splitting indicative of polar 205 effects on growth and development (Extended Data Fig. S4). Altogether, these results 206 identify DivIVA as a key protein that is essential for reversible metamorphosis.

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For general cloning purposes, E. coli strains DH5α and JM109 were used, 308 while E. coli ET12567 and SCS110 were used to obtain unmethylated DNA 309 (Extended Data Table 2). E. coli strains were grown at 37 °C in LB medium, 310 supplemented with chloramphenicol (25 µg ml -1 ), ampicillin (100 µg ml -1 ), apramycin 311 (50 µg ml -1 ), kanamycin (50 µg ml -1 ), or viomycin (30 µg ml -1 ), where necessary. All plasmids and primers used in this work are shown in Extended Data Table 3 and   315 Extended Data Table 4, respectively.  Finally, the promoter and divIVA coding sequence were cloned into pKR2 as a 327 BglII/XbaI and XbaI/NdeI fragment respectively, yielding plasmid pKR3. Construction of the deletion constructs pKR1, pKR4 and pKR5 330 The ssgB mutant was created in K. viridifaciens using pKR1, which is a derivative of 331 the unstable plasmid pWHM3 47 . In the ssgB mutant, nucleotides +20 to +261 relative 332 to the start codon of ssgB were replaced with the loxP-apra resistance cassette as               Extended Data Figure S2