The class Alphaproteobacteria includes Gram-negative free-living, symbiotic and obligate intracellular bacteria, as well as important plant, animal and human pathogens. Recent work has established the key antagonistic roles that phosphorylated guanosines, cyclic-di-GMP (c-di-GMP) and the alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p)ppGpp), have in the regulation of the cell cycle in these bacteria. In this Review, we discuss the insights that have been gained into the regulation of the initiation of DNA replication and cytokinesis by these second messengers, with a particular focus on the cell cycle of Caulobacter crescentus. We explore how the fluctuating levels of c-di-GMP and (p)ppGpp during the progression of the cell cycle and under conditions of stress control the synthesis and proteolysis of key regulators of the cell cycle. As these signals also promote bacterial interactions with host cells, the enzymes that control (p)ppGpp and c-di-GMP are attractive antibacterial targets.

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 family Neisseriaceae, which includes Gram-negative coccoid and rod-shaped species. In particular, bacteria of the genera Alysiella, Simonsiella and Conchiformibius, 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 within Neisseriaceae evolved 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 gene amiC2, and amino acid changes in proteins including MreB and FtsA. Introduction of amiC2 and allelic substitution of mreB in 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 the Neisseriaceae family may be good models to study these processes due to their morphological plasticity and genetic tractability.

How DNA-dependent RNA polymerase (RNAP) acts on bacterial cell cycle progression during transcription elongation is poorly investigated. A forward genetic selection for Caulobacter crescentus cell cycle mutants unearthed the uncharacterized DUF1013 protein (TrcR, transcriptional cell cycle regulator). TrcR promotes the accumulation of the essential cell cycle transcriptional activator CtrA in late S-phase but also affects transcription at a global level to protect cells from the quinolone antibiotic nalidixic acid that induces a multidrug efflux pump and from the RNAP inhibitor rifampicin that blocks transcription elongation. We show that TrcR associates with promoters and coding sequences in vivo in a rifampicin-dependent manner and that it interacts physically and genetically with RNAP. We show that TrcR function and its RNAP-dependent chromatin recruitment are conserved in symbiotic Sinorhizobium sp. and pathogenic Brucella spp. Thus, TrcR represents a hitherto unknown antibiotic target and the founding member of the DUF1013 family, an uncharacterized class of transcriptional regulators that track with RNAP during the elongation phase to promote transcription during the cell cycle.

11Many bacteria acquire dissemination and virulence traits in G1-phase. CtrA, an 12 essential and conserved cell cycle transcriptional regulator identified in the dimorphic 13 alpha-proteobacterium Caulobacter crescentus, first activates promoters in late S-14 phase and then mysteriously switches to different target promoters in G1-phase. We 15 uncovered a highly conserved determinant in the DNA-binding domain (DBD) of CtrA 16 uncoupling this promoter switch. We also show that it reprograms CtrA occupancy in 17 stationary cells inducing a (p)ppGpp alarmone signal perceived by the RNA 18 polymerase beta subunit. A simple side chain modification in a critical residue within 19 the core DBD imposes opposing developmental phenotypes and transcriptional 20 activities of CtrA. A naturally occurring polymorphism in the rickettsial DBD resembles 21 a mutation that drives CtrA towards activation of the dispersal (G1-phase) program in 22Caulobacter. Hence, we propose that this determinant dictates promoter 23 reprogramming during the growth transition of obligate intracellular rickettsia 24 differentiating from replicative cells into dispersal cells. 25 26 rod-shaped and dimorphic alpha-proteobacterium that undergoes an asymmetric cell 31 division into two unequally sized and polarized daughter cells: a motile and non-32 replicative dispersal (swarmer, SW) cell residing in G1-phase and a capsulated and 33 replicative (stalked, ST) cell. In C. crescentus, cell cycle progression is intimately tied 34 to polar remodeling via a circuit of transcriptional activators that direct sequential gene 35 expression programs 1 . The G1-phase program is implemented in the SW daughter cell 36 that inherits the new cell pole where the flagellum and adhesive pili are located. By 37 contrast, the old pole is inherited by the replicative ST cell that is engaged in DNA-38replication. As the cell cycle proceeds, the ST cell prepares for division, expresses the 39 late S-phase program and polarizes before dividing asymmetrically into a SW and ST 40 daughter cell ( Figure 1A). The transcriptional programs are not only temporally 41 ordered, but also spatially confined during cytokinesis, with the G1-phase program 42 being activated in the nascent SW chamber during cytokinesis, but not in the ST cell 43 chamber 1,2 . 44 Cell cycle analyses are facile with C. crescentus because the non-capsulated 45 G1-phase (SW) cells can be separated from capsulated S-phase (ST) cells by density 46 gradient centrifugation 3 . Replicative functions are acquired with the obligate G1àS-47 classes and recognized by the C-terminal DNA binding domain (DBD) of CtrA. At the 65 N-terminus, CtrA harbors a receiver domain (RD) with a phosphorylation site at a 66 conserved aspartate (at position 51, D51). Phosphorylation at D51 stimulates DNA 67 binding and is required for viability. The hybrid histidine kinase CckA directs a multi-68 component phosphoryl-transfer reaction to D51 of CtrA 10,11 . Though loss of CckA is 69 lethal, missense mutations in the RD were isolated in ...

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.