Competence remodels the pneumococcal cell wall exposing key surface virulence factors that mediate increased host adherence.

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called "fratricins" such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by the detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae Specifically, we demonstrate an approach for making targeted markerless gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed repair (HDR) is exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid, which is temperature sensitive for replication, by growing it at the nonpermissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The approaches developed here could potentially be adapted for use with other Gram-positive bacteria.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing more than 1 million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts to identify new vaccine candidates. Here, we introduced an easy-to-use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The approaches used here will advance genome editing projects in this important human pathogen.

Shared Ancestry of Symbionts? Sagrinae and Donaciinae (Coleoptera, Chrysomelidae) Harbor Similar Bacteria.

When symbioses between insects and bacteria are discussed, the origin of a given association is regularly of interest. We examined the evolution of the symbiosis between reed beetles (Coleoptera, Chrysomelidae, Donaciinae) and intracellular symbionts belonging to the Enterobacteriaceae. We analyzed the partial sequence of the 16S rRNA to assess the phylogenetic relationships with bacteria we found in other beetle groups (Cerambycidae, Anobiidae, other Chrysomelidae). We discuss the ecology of each association in the context of the phylogenetic analysis. The bacteria in Sagra femorata (Chrysomelidae, Sagrinae) are very closely related to those in the Donaciinae and are located in similar mycetomes. The Sagrinae build a cocoon for pupation like the Donaciinae, in which the bacteria produce the material required for the cocoon. These aspects support the close relationship between Sagrinae and Donaciinae derived in earlier studies and make a common ancestry of the symbioses likely. Using PCR primers specific for fungi, we found Candida sp. in the mycetomes of a cerambycid beetle along with the bacteria.