Most bacterial cells are surrounded by a peptidoglycan (PG) cell wall that is essential for their integrity. Major synthases of this exoskeleton are called penicillin-binding-proteins (PBPs) 1,2. Surprisingly little is known about how cells control these enzymes given their importance as drug targets. In the model gram-negative bacterium Escherichia coli, outer membrane lipoproteins are critical activators of the class A PBPs (aPBPs) 3,4, bifunctional synthases capable of polymerizing and crosslinking PG to build the exoskeletal matrix 1. Regulators of PBP activity in gram-positive bacteria have yet to be discovered but are likely to be distinct due to the absence of an outer membrane. To uncover gram-positive PBP regulatory factors, we used transposon-sequencing (Tn-Seq) 5 to screen for mutations affecting the growth of Streptococcus pneumoniae cells when the aPBP synthase PBP1a was inactivated. Our analysis revealed a set of genes that were essential for growth in wild-type cells yet dispensable when pbp1a was deleted. The proteins encoded by these genes included the conserved cell wall elongation factors MreC and MreD 2,6,7 as well as a membrane protein of unknown function (SPD_0768) that we have named CozE (coordinator of zonal elongation). Our results indicate that CozE is a novel member of the MreCD complex of S. pneumoniae that directs the activity of PBP1a to the midcell plane where it promotes zonal cell elongation and normal morphology. CozE homologues are widely distributed among bacteria, suggesting they represent a new family of morphogenic proteins controlling cell wall biogenesis by the PBPs.

Accurate chromosome segregation depends on the kinetochore, the complex of proteins that link microtubules to centromeric DNA1. The budding yeast kinetochore consists of more than 80 proteins assembled on a 125bp region of DNA1. We studied the assembly and function of kinetochore components by fusing individual kinetochore proteins to the lactose repressor (LacI) and testing their ability to improve the segregation of a plasmid carrying tandem repeats of the lactose operator (LacO). Targeting Ask1, a member of the Dam1-DASH microtubule-binding complex, creates a synthetic kinetochore that performs many functions of a natural kinetochore: it can replace an endogenous kinetochore on a chromosome, biorient sister kinetochores at metaphase of mitosis, segregate sister chromatids, and repair errors in chromosome attachment. We show the synthetic kinetochore’s functions do not depend on the DNA-binding components of the natural kinetochore but do require other kinetochore proteins. We conclude that tethering a single kinetochore protein to DNA triggers the assembly of the complex structure that directs mitotic chromosome segregation.

Summary Background The spindle checkpoint ensures accurate chromosome transmission by delaying chromosome segregation until all chromosomes are correctly aligned on the mitotic spindle. The checkpoint is activated by kinetochores that are not attached to microtubules or are attached but not under tension and arrests cells at metaphase by inhibiting the anaphase-promoting complex (APC) and its co-activator Cdc20. Despite numerous studies, we still do not understand how the checkpoint proteins coordinate with each other to inhibit APCCdc20 activity. Results To ask how the checkpoint components induce metaphase arrest, we constructed fusions of checkpoint proteins and expressed them in the budding yeast, Saccharomyces cerevisiae, to mimic possible protein interactions during checkpoint activation. We found that expression of a Mad2-Mad3 protein fusion or non-covalently linked Mad2 and Mad3, but not the overexpression of the two separate proteins, induces metaphase arrest that is independent of functional kinetochores or other checkpoint proteins. We further showed that artificially tethering Mad2 to Cdc20 also arrests cells in metaphase independently of other checkpoint components. Conclusion Our results suggest that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, which is sufficient to inhibit APC activity and is the most downstream event in spindle checkpoint activation.

© 2 0 1 7 M a c m i l l a n P u b l i s h e r s L i m i t e d , p a r t o f S p r i n g e r N a t u r e . A l l r i g h t s r e s e r v e d .