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SUMMARYThe Shigella plasmid, pINV, contains a 30 kb pathogenicity island (PAI) encoding a Type III secretion system (T3SS) which is essential for virulence. During growth in the laboratory, avirulent colonies of Shigella (which do not express a T3SS) arise spontaneously. Avirulence in Shigella flexneri mostly follows loss of the PAI, following recombination between insertion sequences (ISs) on pINV; toxin:antitoxin (TA) systems on pINV promote its retention through post-segregational killing (PSK). We show that avirulence in Shigella sonnei mainly results from plasmid loss, consistent with previous findings; IS-mediated PAI deletions can occur in S. sonnei, but through different ISs than in S. flexneri. We investigated the molecular basis for frequent loss of the S. sonnei plasmid, pINVSsonn. Introduction into pINVSsonn of CcdAB and GmvAT, toxin:antitoxin TA systems in pINV from S. flexneri but not S. sonnei, reduced plasmid loss and the emergence of avirulent bacteria. However, plasmid loss remained the leading cause of avirulence. We show that a single amino acid difference in the VapC toxin of the VapBC TA system in pINV also contributes to high frequency plasmid loss in S. sonnei compared to S. flexneri. Our findings demonstrate that the repertoire of ISs, complement of TA systems, and polymorphisms in TA systems influence plasmid dynamics and virulence loss in S. sonnei. Understanding the impact of polymorphisms should be informative about how TA systems contribute to PSK, and could be exploited for generating strains with stable plasmids.

Neisseria meningitidis (the meningococcus) is a member of the normal nasopharyngeal microbiome in healthy individuals, but can cause septicemia and meningitis in susceptible individuals. In this chapter we provide an overview of the disease caused by N. meningitidis and the schemes used to type the meningococcus. We also review the adhesions, virulence factors, and phase variable genes that enable it to successfully colonize the human host. Finally, we outline the history and current status of meningococcal vaccines and highlight the importance of continued molecular investigation of the epidemiology and the structural analysis of the antigens of this pathogen to aid future vaccine development.

Escherichia coli has been engineered to produce chlorophyll., Chlorophylls are essential cofactors for photosynthesis, which sustains global food chains and oxygen production. Billions of tons of chlorophylls are synthesized annually, yet full understanding of chlorophyll biosynthesis has been hindered by the lack of characterization of the Mg–protoporphyrin IX monomethyl ester oxidative cyclase step, which confers the distinctive green color of these pigments. We demonstrate cyclase activity using heterologously expressed enzyme. Next, we assemble a genetic module that encodes the complete chlorophyll biosynthetic pathway and show that it functions in Escherichia coli. Expression of 12 genes converts endogenous protoporphyrin IX into chlorophyll a, turning E. coli cells green. Our results delineate a minimum set of enzymes required to make chlorophyll and establish a platform for engineering photosynthesis in a heterotrophic model organism.

The development of prophylactic vaccines against pathogenic bacteria is a major objective of the World Health Organisation. However, vaccine development is often hindered by antigenic diversity and the difficulties encountered manufacturing immunogenic membrane proteins. Here, we employed structure-based design as a strategy to developChimericAntigens (ChAs) for subunit vaccines. ChAs were generated against serogroup BNeisseria meningitidis(MenB), the predominant cause of meningococcal disease in the Western hemisphere. MenB ChAs exploit the lipoprotein factor H binding protein (fHbp) as a molecular scaffold to display the immunogenic VR2 epitope from the integral membrane protein PorA. Structural analyses demonstrate fHbp is correctly folded and that PorA VR2 epitope adopts an immunogenic conformation. In mice, ChAs elicit antibodies directed against fHbp and PorA, with antibody responses correlating to protection against meningococcal disease. ChAs offer a novel approach for generating multivalent subunit vaccines, containing of epitopes from integral membrane proteins, whose composition can be selected to circumvent pathogen diversity.