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

Molecular and morphological data suggest weak phylogeographic structure in the fairy shrimp Streptocephalus torvicornis (Branchiopoda, Anostraca)

Inland aquatic organisms almost ubiquitously display a pattern of marked provincialism characterized by substantial population differentiation and genealogical discontinuities. This is the result of strong priority effects and local adaptation following dispersal and colonization of new habitats. We present a case that defies this biogeographic paradigm. We have investigated the phylogeography of the fairy shrimp Streptocephalus torvicornis across its circum-Mediterranean and Eurasian distribution. Based on three independent datasets, namely sequence variation at 12S and 16S rRNA, cyst morphology and male second antenna characters, we discern a pattern of extensive genetic and morphological homogeneity pointing to unhindered gene flow and widespread connectivity among populations. These intriguing findings may provisionally be explained by (i) a high dispersal frequency overwhelming the ability of a population to maintain resource monopolization, (ii) an outbreeding vigour opportunity offered to secondary immigrants, (iii) an ecological equivalence of genotypes generating long-term immigration\u2013extinction equilibria and buffering genetic diversity over spatial scales, (iv) enhanced bird-mediated dispersal in open habitats as opposed to ponds surrounded by forests or shrub, or (v) a shallow population history with little time for substantial genetic differentiation

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