Genome‐wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae. However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn‐seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high‐content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi‐based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp‐proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP‐dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.

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.

β-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of β-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of β-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with β-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by β-lactams or by inhibiting an early step in WTA biosynthesis. The finding that β-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that β-lactams do not kill S. aureus simply by weakening the cell wall.

Summary Inhibition of cell division is critical for viability under DNA‐damaging conditions. DNA damage induces the SOS response that in bacteria inhibits cell division while repairs are being made. In coccoids, such as the human pathogen, Staphylococcus aureus, this process remains poorly studied. Here, we identify SosA as the staphylococcal SOS‐induced cell division inhibitor. Overproduction of SosA inhibits cell division, while sosA inactivation sensitizes cells to genotoxic stress. SosA is a small, predicted membrane protein with an extracellular C‐terminal domain in which point mutation of residues that are conserved in staphylococci and major truncations abolished the inhibitory activity. In contrast, a minor truncation led to SosA accumulation and a strong cell division inhibitory activity, phenotypically similar to expression of wild‐type SosA in a CtpA membrane protease mutant. This suggests that the extracellular C‐terminus of SosA is required both for cell division inhibition and for turnover of the protein. Microscopy analysis revealed that SosA halts cell division and synchronizes the cell population at a point where division proteins such as FtsZ and EzrA are localized at midcell, and the septum formation is initiated but unable to progress to closure. Thus, our findings show that SosA is central in cell division regulation in staphylococci.

Streptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood, and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND gates and IMPLY gates. We demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors. Indeed, we were able to rewire gene expression of the capsule operon, the main pneumococcal virulence factor, to be externally inducible (YES gate) or to act as an IMPLY gate (only expressed in absence of inducer). Importantly, we demonstrate that these synthetic gene-regulatory networks are functional in an influenza A virus superinfection murine model of pneumonia, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

Proteins belonging to the DHH family, a member of the phosphoesterase superfamily, are produced by most bacterial species. While some of these proteins are well studied in Bacillus subtilis and Escherichia coli, their functions in Streptococcus pneumoniae remain unclear. Recently, the highly conserved DHH subfamily 1 protein PapP (SP1298) has been reported to play an important role in virulence. Here, we provide a plausible explanation for the attenuated virulence of the papP mutant. Recombinant PapP specifically hydrolyzed nucleotides 3'-phosphoadenosine-5'-phosphate (pAp) and 5'-phosphoadenylyl-(3'->5')-adenosine (pApA). Deletion of papP, potentially leading to pAp/pApA accumulation, resulted in morphological defects and mis-localization of several cell division proteins. Incubation with both polar solvent and detergent led to robust killing of the papP mutant, indicating that membrane integrity is strongly affected. This is in line with previous studies showing that pAp inhibits the ACP synthase, an essential enzyme involved in lipid precursor production. Remarkably, partial inactivation of the lipid biosynthesis pathway, by inhibition of FabF or depletion of FabH, phenocopied the papP mutant. We conclude that pAp and pApA phosphatase activity of PapP is required for maintenance of membrane lipid homeostasis providing an explanation how inactivation of this protein may attenuate pneumococcal virulence.

Protein phosphorylation is a key post-translational modification required for many cellular functions of the bacterial cell. Recently, we identified a new protein-kinase, named UbK, in Bacillus subtilis that belongs to a new family of protein-kinases widespread in bacteria. In this study, we analyze the function of UbK in Streptococcus pneumoniae. We show that UbK displays a tyrosine-kinase activity and autophosphorylates on a unique tyrosine in vivo. To get insights into its cellular role, we constructed a set of pneumococcal ubk mutants. Using conventional and electron microscopy, we show that the ubk deficient strain, as well as an ubk catalytic dead mutant, display both severe cell-growth and cell-morphology defects. The same defects are observed with a mutant mimicking permanent phosphorylation of UbK whereas they are not detected for a mutant mimicking defective autophosphorylation of UbK. Moreover, we find that UbK phosphorylation promotes its ability to hydrolyze ATP. These observations show that the hydrolysis of ATP by UbK serves not only for its autophosphorylation but also for a distinct purpose essential for the optimal cell growth and cell-morphogenesis of the pneumococcus. We thus propose a model in which the autophosphorylation/dephosphorylation of UbK regulates its cellular function through a negative feedback loop.

Inhibition of cell division is critical for cell viability under DNA damaging conditions. In bacterial cells, DNA damage induces the SOS response, a process that inhibits cell division while repairs are being made. In coccoid bacteria, such as the human pathogen Staphylococcus aureus, the process remains poorly understood. Here we have characterized an SOS-induced cell-division inhibitor, SosA, in S. aureus. We find that in contrast to the wildtype, sosA mutant cells continue division under DNA damaging conditions with decreased viability as a consequence. Conversely, overproduction of SosA leads to cell division inhibition and reduced growth. The SosA protein is localized in the bacterial membrane and mutation of an extracellular amino acid, conserved between homologs of other staphylococcal species, abolished the inhibitory activity as did truncation of the C-terminal 30 amino acids. In contrast, C-terminal truncation of 10 amino acids lead to SosA accumulation and a strong cell division inhibitory activity. A similar phenotype was observed upon expression of wildtype SosA in a mutant lacking the membrane protease, CtpA. Thus, the extracellular C-terminus of SosA is required both for cell-division inhibition and for turnover of the protein. Functional studies showed that SosA is likely to interact with one or more divisome components and, without interfering with early cell-division events, halts cell division at a point where septum formation is initiated yet being unable to progress to septum closure. Our findings provide important insights into cell-division regulation in staphylococci that may foster development of new classes of antibiotics targeting this essential process.ImportanceStaphylococcus aureus is a serious human pathogen and a model organism for cell-division studies in spherical bacteria. We show that SosA is the DNA-damage-inducible cell-division inhibitor in S. aureus that upon expression causes cell swelling and cessation of the cell cycle at a characteristic stage post septum initiation but prior to division plate completion. SosA appears to function via an extracellular activity and is likely to do so by interfering with the essential membrane-associated division proteins, while at the same time being negatively regulated by the membrane protease CtpA. This report represents the first description of the process behind cell-division inhibition in coccoid bacteria. As several pathogens are included in this category, uncovering the molecular details of SosA activity and control can lead to identification of new targets for development of valuable anti-bacterial drugs.