Genome rearrangements and megaplasmid loss in the filamentous bacteriumKitasatospora viridifaciensare associated with protoplast formation and regeneration

Abstract

20Filamentous Actinobacteria are multicellular bacteria with linear replicons. Kitasatospora 21 viridifaciens DSM 40239 contains a linear 7.8 Mb chromosome and an autonomously 22 replicating plasmid KVP1 of 1.7 Mb. Here we show that lysozyme-induced protoplast 23 formation of the multinucleated mycelium of K. viridifaciens drives morphological diversity. 24Characterization and sequencing of an individual revertant colony that had lost the ability to 25 differentiate revealed that the strain had not only lost most of KVP1 but also carried lesions 26 in the right arm of the chromosome. Strikingly, the lesion sites were preceded by insertion 27 sequence elements, suggesting that the rearrangements may have been caused by 28 replicative transposition and homologous recombination between both replicons. These 29 data indicate that protoplast formation is a stressful process that can lead to profound 30 genetic changes. 31 34 metabolites are mostly used as weapons that provide protection against other 35 microorganisms and phages in the environment [1][2][3]. This is particularly useful for 36 filamentous organisms, given that they generally lack the ability to make flagella for escaping 37 dangerous situations. In addition, these bacteria are able to generate resistant spores that 38 can invade new environments after their dispersal. Germination of spores leads to the 39 formation of 1-2 germ tubes, which grow by tip extension, thereby establishing filamentous 40 cells called hyphae. Branching of hyphae leads to the formation of a multinucleated 41 vegetative mycelium, which forages and acquires nutrients by decomposing polymeric 42 substances. Stressful conditions (such as nutrient depletion) induce program cell death 43 (PCD) of the mycelium, which in turn triggers morphological and chemical differentiation [4].44 This developmental transition leads to the formation of specialized hyphae that grow into the 45 air, and the onset of production of a suite of bioactive compounds [5]. Eventually, the aerial 46 hyphae metamorphose into chains of grey-pigmented spores. Mutants that are unable to 47 establish an aerial mycelium are called bald (bld), while those that are not capable to form 48 spores are called white (whi) after their whitish color [6, 7]. 49Genome mining has been instrumental for the revival of drug discovery [8, 9]. Many 50 of the biosynthetic gene clusters that specify bioactive natural products are contained on 51 giant linear plasmids [10-13]. Although linear replicons are rare in many bacterial taxa, they 52 are common in Actinobacteria [14, 15]. In fact, Streptomyces chromosomes (between 8 and 53 10 Mb in size) are also linear and typically comprise a "core region" containing the essential 54 genes, and two variable "arms" with lengths ranging from 1.5 Mb to 2.3 Mb [16]. Like linear 55 plasmids, the linear chromosomes are capped by terminal proteins bound to the 5' end of 56 the DNA [17]. The chromosomal ends are genetically unstable, and readily undergo large 57 (up to 2 Mb) DNA rearrangements. Such...

Cell Wall Deficiency as a Coping Strategy for Stress

“…This is perhaps most evident in reverting S-cells of actinomycetes, which revealed colonies with a wide range of different phenotypes. These phenotypic differences are in part explained by dramatic chromosomal rearrangements and lesions, perhaps due to transposase activity triggered by osmotic stress [16,77]. And while these massive genetic changes will often be deleterious, they may in some cases lead to the evolution of new beneficial traits [78].…”