Bacteria swim and swarm by rotating the micrometers long, helical filaments of their flagella. They change direction by reversing their flagellar rotation, which switches the handedness of the filament’s supercoil. So far, all studied functional filaments are composed of a mixture of L- and R-state flagellin monomers. Here we show in a study of the wild type Firmicute Kurthia sp., that curved, functional filaments can adopt a conformation in vivo that is closely related to a uniform, all-L-state. This sheds additional light on transitions of the flagellar supercoil and uniquely reveals the atomic structure of a wild-type flagellar filament in vivo, including six residues showing clearly densities of O-linked glycosylation.

The production of specialized resting cells is a remarkable strategy developed by several organisms to survive unfavorable environmental conditions. Spores are specialized resting cells that are characterized by low to absent metabolic activity and higher resistance. Spore-like cells are known from multiple groups of bacteria, which can form spores under suboptimal growth conditions (e.g., starvation). In contrast, little is known about the production of specialized resting cells in archaea. In this study, we applied a culture-independent method that uses physical and chemical lysis, to assess the diversity of lysis-resistant bacteria and archaea and compare it to the overall prokaryotic diversity (direct DNA extraction). The diversity of lysis-resistant cells was studied in the polyextreme environment of the Salar de Huasco. The Salar de Huasco is a high-altitude athalassohaline wetland in the Chilean Altiplano. Previous studies have shown a high diversity of bacteria and archaea in the Salar de Huasco, but the diversity of lysis-resistant microorganisms has never been investigated. The underlying hypothesis was that the combination of extreme abiotic conditions might favor the production of specialized resting cells. Samples were collected from sediment cores along a saline gradient and microbial mats were collected in small surrounding ponds. A significantly different diversity and composition were found in the sediment cores or microbial mats. Furthermore, our results show a high diversity of lysis-resistant cells not only in bacteria but also in archaea. The bacterial lysis-resistant fraction was distinct in comparison to the overall community. Also, the ability to survive the lysis-resistant treatment was restricted to a few groups, including known spore-forming phyla such as Firmicutes and Actinobacteria. In contrast to bacteria, lysis resistance was widely spread in archaea, hinting at a generalized resistance to lysis, which is at least comparable to the resistance of dormant cells in bacteria. The enrichment of Natrinema and Halarchaeum in the lysis-resistant fraction could hint at the production of cyst-like cells or other resistant cells. These results can guide future studies aiming to isolate and broaden the characterization of lysis-resistant archaea.

In most habitats, fluctuating environmental conditions create periods of compromised survival for metabolically active organisms. In response, various survival strategies have evolved, including the formation of resilient resting cells. We assessed the biodiversity of the lysis‐resistant bacteria in three different environments by applying a harsh physicochemical treatment to the samples. The bacterial diversity of the lysis‐resistant fraction was compared with the bacterial diversity from the same environmental samples without the application of the enrichment method. As expected, in the lysis‐resistant fraction, a significantly higher relative abundance of endospore‐forming Firmicutes (for instance, Bacillus, Clostridium and Paenisporosarcina) was observed in comparison with the untreated samples. However, genera from which the existence of a resistant cell form is not yet reported were also highly enriched in comparison with the untreated samples. Our results suggest a more diversified repertoire of bacterial resistant cellular structures than previously thought.

38Sporulation is a complex morphophysiological process resulting in a cellular structure that is more 39 resistant than the vegetative form. In Firmicutes, this structure is produced within the mother cell, 40 and is called an endospore. Endospore formation is thought to have evolved in the common 41 ancestor of Firmicutes. However, sporulation has apparently been lost in some extant lineages that 42 are defined as asporogenic. We isolated strain 11kri321, a representative of the genus Kurthia, 43 from an oligotrophic geothermal reservoir. While Kurthia spp. is considered to comprise only 44 asporogenic species, strain 11kri321 produced spores. Genomic reconstruction of the sporulation 45 pathway shows elements typical of sporulation in Bacilli, including the signaling for sporulation 46 onset. However, key genes were missing, including those involved in engulfment and dipicolinic 47 Importance 58Endospore-forming Firmicutes include many environmental and medical relevant bacterial clades. 59In these microorganisms, the ability to produce endospores is essential for survival in the 60 environment and even for pathogenesis. The minimum core of genes required to produce a viable 61 and resistant spore, the distinction between endospore-forming and asporogenic groups, as well as 62 the evolution of sporulation have been a subject of investigation and debate for decades. Here, we 63 demonstrate endosporulation in the genus Kurthia, considered as asporogenic. Morphological, 64 physiological and genomic analyses were undertaken to demonstrate that sporulation is not lost 65 within this lineage. Based on our results we propose a re-examination of the minimal genetic 66 requirements of sporulation and the use of the term cryptosporulant to describe lineages of 67

Endosporulation is a complex morphophysiological process resulting in a more resistant cellular structure that is produced within the mother cell and is called endospore. Endosporulation evolved in the common ancestor of Firmicutes, but it is lost in descendant lineages classified as asporogenic. While Kurthia spp. is considered to comprise only asporogenic species, we show here that strain 11kri321, which was isolated from an oligotrophic geothermal reservoir, produces phase-bright spore-like structures. Phylogenomics of strain 11kri321 and other Kurthia strains reveals little similarity to genetic determinants of sporulation known from endosporulating Bacilli. However, morphological hallmarks of endosporulation were observed in two of the four Kurthia strains tested, resulting in spore-like structures (cryptospores). In contrast to classic endospores, these cryptospores did not protect against heat or UV damage and successive sub-culturing led to the loss of the cryptosporulating phenotype. Our findings imply that a cryptosporulation Mathilda Fatton and Sevasti Filippidou contributed equally to this study.