Genome editing in bacteria encompasses a wide array of laborious and multi-step methods such as suicide plasmids. The discovery and applications of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas based technologies have revolutionized genome editing in eukaryotic organisms due to its simplicity and programmability. Nevertheless, this system has not been as widely favored for bacterial genome editing. In this review, we summarize the main approaches and difficulties associated with CRISPR-Cas-mediated genome editing in bacteria and present some alternatives to circumvent these issues, including CRISPR nickases, Cas12a, base editors, CRISPR-associated transposases, prime-editing, endogenous CRISPR systems, and the use of pre-made ribonucleoprotein complexes of Cas proteins and guide RNAs. Finally, we also address fluorescent-protein-based methods to evaluate the efficacy of CRISPR-based systems for genome editing in bacteria. CRISPR-Cas still holds promise as a generalized genome-editing tool in bacteria and is developing further optimization for an expanded application in these organisms. This review provides a rarely offered comprehensive view of genome editing. It also aims to familiarize the microbiology community with an ever-growing genome-editing toolbox for bacteria.

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer. Lipids are essential constituents of biological membranes, and most organisms across the tree of life use a relatively limited repertoire of lipids in their membranes. This work reveals the natural and abundant presence of an exclusive lipid phosphatidylthreonine (PtdThr) in Toxoplasma gondii, a ubiquitous protozoan parasite of humans and animals. PtdThr is made by a novel parasite-specific enzyme, PtdThr synthase, which has evolved from the widespread enzyme phosphatidylserine synthase. The study shows that PtdThr is required for asexual reproduction and virulence of the parasite in vivo, and a metabolically attenuated mutant strain of Toxoplasma lacking PtdThr can protect vaccinated mice against acute and currently incurable chronic infection. This discovery demonstrates adaptive “speciation” of PtdThr from an otherwise near-universal membrane lipid phosphatidylserine and reveals de novo PtdThr synthesis in T. gondii as a potential drug target.

Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models.

The journal for unicellular biology and modeling of human disease

Dynein light chain 8 (DLC8) is a ubiquitous eukaryotic protein regulating diverse cellular functions. We show that the obligate intracellular parasite Toxoplasma gondii harbors 4 DLC8 proteins ( Tg DLC8a–d), of which only Tg DLC8a clusters in the mainstream LC8 class. Tg DLC8b–d proteins form a divergent and alveolate‐specific clade. Tg DLC8b–d proteins are largely cytosolic, whereas Tg DLC8a resides in the conoid at the apical end of T. gondii The apical location of Tg DLC8a is also not shared by its nearly identical Eimeria ( Et DLC8a), Plasmodium ( Pf DLC8), or human ( Hs DLC8) orthologs. Notwithstanding an exclusive conoid targeting, Tg DLC8a exhibits a classical LC8 structure. It forms a homodimer by swapping of the β ₂ strands that interact with the antiparallel β ₁ ′ strands of the opposing monomers. The Tg DLC8a dimer contains two identical binding grooves and appears to be adapted for multitarget recognition. By contrast, the previously reported Pf DLC8 homodimer is shaped by binding of the β ₀ strand with the parallel β ₂ ′ strand and lacks such a distinct binding interface. Our comparisons suggest an unexpected structural and functional divergence of the two otherwise conserved proteins from apicomplexan parasites. Finally, we demonstrate that a phosphomimetic S88E mutation renders the Tg DLC8a‐S88E mutant monomeric and cytosolic in T. gondii , and its overexpression inhibits the parasite growth in human fibroblasts.—Qureshi, B. M., Hofmann, N. E., Arroyo‐Olarte, R. D., Nickl, B., Hoehne, W., Jungblut, P. R., Lucius, R., Scheerer, P., Gupta, N. Dynein light chain 8a of Toxoplasma gondii , a unique conoid‐localized β‐strand‐swapped homodimer, is required for an efficient parasite growth. FASEB J. 27, 1034–1047 (2013). www.fasebj.org

Key words: complement system; Trypanosoma cruzi; infection