As the field of synthetic biology is developing, the prospects for de novo design of biosynthetic pathways are becoming more and more realistic. Hence, there is an increasing need for computational tools that can support these efforts. A range of algorithms has been developed that can be used to identify all possible metabolic pathways and their corresponding enzymatic parts. These can then be ranked according to various properties and modelled in an organism-specific context. Finally, design software can aid the biologist in the integration of a selected pathway into smartly regulated transcriptional units. Here, we review key existing tools and offer suggestions for how informatics can help to shape the future of synthetic microbiology.

Accurate spatial and temporal positioning of the tubulin-like protein FtsZ is key for proper bacterial cell division. Streptococcus pneumoniae (pneumococcus) is an oval-shaped, symmetrically dividing opportunistic human pathogen lacking the canonical systems for division site control (nucleoid occlusion and the Min-system). Recently, the early division protein MapZ was identified and implicated in pneumococcal division site selection. We show that MapZ is important for proper division plane selection; thus, the question remains as to what drives pneumococcal division site selection. By mapping the cell cycle in detail, we show that directly after replication both chromosomal origin regions localize to the future cell division sites, before FtsZ. Interestingly, Z-ring formation occurs coincidently with initiation of DNA replication. Perturbing the longitudinal chromosomal organization by mutating the condensin SMC, by CRISPR/Cas9-mediated chromosome cutting, or by poisoning DNA decatenation resulted in mistiming of MapZ and FtsZ positioning and subsequent cell elongation. Together, we demonstrate an intimate relationship between DNA replication, chromosome segregation, and division site selection in the pneumococcus, providing a simple way to ensure equally sized daughter cells.

Highlights d The Bdellovibrio chromosome is polarized, with ori located near the invasive pole d The highly compacted nucleoid excludes cytosolic proteins in non-replicative cells d Replication and segregation of chromosomes are uncoupled from cell division d The centromeric protein ParB localizes at parS in a cell-cycledependent manner

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.