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.

Staphylococci, like Staphylococcus aureus and S. epidermidis, are common colonizers of the human microbiota. While being harmless in many cases, many virulence factors result in them being opportunistic pathogens and one of the major causes of hospital-acquired infections worldwide. One of these virulence factors is the ability to form biofilms—three-dimensional communities of microorganisms embedded in an extracellular polymeric matrix (EPS). The EPS is composed of polysaccharides, proteins and extracellular DNA, and is finely regulated in response to environmental conditions. This structured environment protects the embedded bacteria from the human immune system and decreases their susceptibility to antimicrobials, making infections caused by staphylococci particularly difficult to treat. With the rise of antibiotic-resistant staphylococci, together with difficulty in removing biofilms, there is a great need for new treatment strategies. The purpose of this review is to provide an overview of our current knowledge of the stages of biofilm development and what difficulties may arise when trying to eradicate staphylococcal biofilms. Furthermore, we look into promising targets and therapeutic methods, including bacteriocins and phage-derived antibiofilm approaches.