Rod-shaped bacteria typically elongate and divide by transverse fission. However, several bacterial species can form rod-shaped cells that divide longitudinally. Here, we study the evolution of cell shape and division mode within the familyNeisseriaceae, which includes Gram-negative coccoid and rod-shaped species. In particular, bacteria of the generaAlysiella,SimonsiellaandConchiformibius, which can be found in the oral cavity of mammals, are multicellular and divide longitudinally. We use comparative genomics and ultrastructural microscopy to infer that longitudinal division withinNeisseriaceaeevolved from a rod-shaped ancestor. In multicellular longitudinally-dividing species, neighbouring cells within multicellular filaments are attached by their lateral peptidoglycan. In these bacteria, peptidoglycan insertion does not appear concentric, i.e. from the cell periphery to its centre, but as a medial sheet guillotining each cell. Finally, we identify genes and alleles associated with multicellularity and longitudinal division, including the acquisition of amidase-encoding geneamiC2, and amino acid changes in proteins including MreB and FtsA. Introduction ofamiC2and allelic substitution ofmreBin a rod-shaped species that divides by transverse fission results in shorter cells with longer septa. Our work sheds light on the evolution of multicellularity and longitudinal division in bacteria, and suggests that members of theNeisseriaceaefamily may be good models to study these processes due to their morphological plasticity and genetic tractability.

In a previous study, differential proteomic analysis was used to identify membrane proteins of the human enteropathogen Yersinia enterocolitica, whose levels are influenced by OmpR, the transcriptional regulator in the two-component EnvZ/OmpR system. Interestingly, this analysis demonstrated that at 37 °C, OmpR negatively affects the level of over a dozen Ysc-Yop proteins, which constitute a type III secretion system (T3SS) that is essential for the pathogenicity of Y. enterocolitica. Here, we focused our analysis on the role of OmpR in the expression and secretion of Yops (translocators and effectors). Western blotting with anti-Yops antiserum and specific anti-YopD, -YopE and -YopH antibodies, confirmed that the production of Yops is down-regulated by OmpR with the greatest negative effect on YopD. The RT-qPCR analysis demonstrated that, while OmpR had a negligible effect on the activity of regulatory genes virF and yscM1, it highly repressed the expression of yopD. OmpR was found to bind to the promoter of the lcrGVsycD-yopBD operon, suggesting a direct regulatory effect. In addition, we demonstrated that the negative regulatory influence of OmpR on the Ysc-Yop T3SS correlated with its positive role in the expression of flhDC, the master regulator of the flagellar-associated T3SS.

Yersinia enterocolitica exhibits a dual lifestyle, existing as both a saprophyte and a pathogen colonizing different niches within a host organism. OmpR has been recognized as a regulator that controls the expression of genes involved in many different cellular processes and the virulence of pathogenic bacteria. Here, we have examined the influence of OmpR and varying temperature (26°C vs. 37°C) on the cytoplasmic proteome of Y. enterocolitica Ye9N (bio-serotype 2/O:9, low pathogenicity). Differential label-free quantitative proteomic analysis indicated that OmpR affects the cellular abundance of a number of proteins including subunits of urease, an enzyme that plays a significant role in acid tolerance and the pathogenicity of Y. enterocolitica. The impact of OmpR on the expression of urease under different growth conditions was studied in more detail by comparing urease activity and the transcription of ure genes in Y. enterocolitica strains Ye9N and Ye8N (highly pathogenic bio-serotype 1B/O:8). Urease expression was higher in strain Ye9N than in Ye8N and in cells grown at 26°C compared to 37°C. However, low pH, high osmolarity and the presence of urea did not have a clear effect on urease expression in either strain. Further analysis showed that OmpR participates in the positive regulation of three transcriptional units encoding the multi-subunit urease (ureABC, ureEF, and ureGD) in strain Ye9N, but this was not the case in strain Ye8N. Binding of OmpR to the ureABC and ureEF promoter regions was confirmed using an electrophoretic mobility shift assay, suggesting that this factor plays a direct role in regulating the transcription of these operons. In addition, we determined that OmpR modulates the expression of a ureR-like gene encoding a putative regulator of the ure gene cluster, but in the opposite manner, i.e., positively in Ye9N and negatively in Ye8N. These findings provide some novel insights into the function of OmpR in adaptation strategies of Y. enterocolitica.

Iron is both essential for and potentially toxic to bacteria, so the precise maintenance of iron homeostasis is necessary for their survival. Our previous study indicated that in the human enteropathogen Yersinia enterocolitica, the regulator OmpR directly controls the transcription of the fur, fecA and fepA genes, encoding the ferric uptake repressor and two transporters of ferric siderophores, respectively. This study was undertaken to determine the significance of the RNA chaperone Hfq and the small RNAs OmrA and RyhB1 in the post-transcriptional control of the expression of these OmpR targets. We show that Hfq silences fur, fecA and fepA expression post-transcriptionally and negatively affects the production of FLAG-tagged Fur, FecA and FepA proteins. In addition, we found that the fur gene is under the negative control of the sRNA RyhB1, while fecA and fepA are negatively regulated by the sRNA OmrA. Finally, our data revealed that the role of OmrA results from a complex interplay of transcriptional and post-transcriptional effects in the feedback circuit between the regulator OmpR and the sRNA OmrA. Thus, the expression of fur, fecA and fepA is subject to complex transcriptional and post-transcriptional regulation in order to maintain iron homeostasis in Y. enterocolitica.

In spite of the staggering number of bacteria that live associated with animals, the growth mode of only a few symbionts has been studied so far. Here, we focused on multicellular longitudinally dividing (MuLDi) Neisseriaceae occurring in the oral cavity of mammals and belonging to the genera Alysiella, Simonsiella and Conchiformibius. Firstly, by applying comparative genomics coupled with ultrastructural analysis, we inferred that longitudinal division evolved from a rod-shaped ancestor of the Neisseriaceae family. Secondly, transmission electron microscopy on cells and sacculi showed that, within each A. filiformis, S. muelleri or C. steedae filament, neighbouring cells are attached by their lateral cell walls. Thirdly, by applying a palette of peptidoglycan metabolic precursors to track their growth, we showed that A. filiformis septates in a distal-to-proximal fashion. In S. muelleri and C. steedae, instead, septation proceeds synchronously from the host-attached poles to midcell. Strikingly, based on confocal-based 3D reconstructions, PG did not appear to be inserted concentrically from the cell periphery to its centre, but as a medial sheet guillotining each cell. Finally, comparative genomics revealed MuLDi-specific differences that set them apart from rod-shaped members of the Neisseriaceae. These MuLDi-specific genetic differences comprise the acquisition of the amidase-encoding gene amiC2, the loss of dgt, gloB, mraZ (an activator of the dcw cluster), rapZ, and amino acids changes in 7 proteins, including the actin homolog MreB and FtsA. Strikingly, introduction of amiC2 and allelic substitution of mreB in the rod-shaped Neisseria elongata resulted in cells with longer septa. In conclusion, we identified genetic events that may have allowed rod-shaped Neisseriaceae to evolve multicellularity and longitudinal division. The morphological plasticity of Neisseriaceae together with their genetic tractability, make them archetypal models for understanding the evolution of bacterial shape, as well as that of animal-bacterium symbioses.