A CRISPR with Roles in Myxococcus xanthus Development and Exopolysaccharide Production

Wallace, Regina A.; Black, Wesley P.; Yang, Zhaomin

doi:10.1128/jb.02035-14

Cited by 31 publications

(26 citation statements)

References 45 publications

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“…Because the cas2 mutants exhibit their defect in the absence of added phage, plasmid, or nucleic acid and because L. pneumophila mutants lacking cas9, cas1, cas4, or the CRISPR array are not impaired (15), the infection event mediated by L. pneumophila Cas2 must be distinct from the functions that have been traditionally ascribed to Cas proteins and CRISPR-Cas systems. There are now a variety of studies demonstrating that there are roles for other Cas proteins that are unrelated to phage/plasmid immunity (21,(25)(26)(27)(28)(29); e.g., Cas9 of Francisella novicida promotes virulence in a murine model of disease (30), and Campylobacter jejuni Cas9 enhances invasion of epithelial cells (31).…”

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confidence: 99%

Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

et al. 2015

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Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40-to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts. L egionella pneumophila is a Gram-negative, aquatic bacterium and the agent of a Legionnaires' disease pneumonia (1). The number of reported cases of Legionnaires' disease in the United States has more than tripled since 2001, with similar trends occurring in Canada and Europe (2). Humans acquire L. pneumophila mainly by inhaling contaminated water droplets from aerosolgenerating devices (3). In the lung, the bacterium infects resident macrophages (4). Amoebae are the major replicative niche for L. pneumophila in natural and man-made water systems (5-7). Indeed, L. pneumophila infects Ն20 species of amoebae encompassing the genera Acanthamoeba, Echinamoeba, Hartmannella, Naegleria, Vahlkampfia, and Willaertia (8). L. pneumophila bacteria in amoebae remain viable for long periods of time and are resistant to biocides (9, 10), and internalization by amoebae resuscitates viable-but-not-culturable legionellae (11,12). Also, amoebae may be part of the inoculum that initiates lung infection (3,13,14). Recently, we found that the Cas2 protein of L. pneumophila promotes infection of Acanthamoeba castellanii and Hartmannella vermiformis; i.e., although cas2 mutants grow normally in broth and macrophages, they exhibit a 1,000-fold reduced recovery from A. castellanii and a 20-fold defect i...

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confidence: 99%

Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

et al. 2015

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“…In our hands, the LOF mutant displayed defects in predation but did not display altered motility or sporulation. It has been reported that CRISPR systems can regulate alternative functions, such as biofilm formation and swarming in Pseudomonas aeruginosa, EPS production in M. xanthus, and lipoprotein production in Francisella novicida (56,60,61).…”

Section: Discussionmentioning

confidence: 99%

“…The M. xanthus genome encodes three CRISPR systems, each composed of a typical arrangement of CRIPSR/Cas genes with repeat/spacer regions (44,45,56). The CRIPSR/Cas systems are prokaryotic immune systems that confer resistance to foreign genetic elements, such as plasmids and phages, and provide a form of acquired immunity.…”

Section: Discussionmentioning

confidence: 99%

Identification of Functions Affecting Predator-Prey Interactions between Myxococcus xanthus and Bacillus subtilis

et al. 2016

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Soil bacteria engage each other in competitive and cooperative ways to determine their microenvironments. In this study, we report the identification of a large number of genes required for Myxococcus xanthus to engage Bacillus subtilis in a predatorprey relationship. We generated and tested over 6,000 individual transposon insertion mutants of M. xanthus and found many new factors required to promote efficient predation, including the specialized metabolite myxoprincomide, an ATP-binding cassette (ABC) transporter permease, and a clustered regularly interspaced short palindromic repeat (CRISPR) locus encoding bacterial immunity. We also identified genes known to be involved in predation, including those required for the production of exopolysaccharides and type IV pilus (T4P)-dependent motility, as well as chemosensory and two-component systems. Furthermore, deletion of these genes confirmed their role during predation. Overall, M. xanthus predation appears to be a multifactorial process, with multiple determinants enhancing predation capacity. IMPORTANCESoil bacteria engage each other in complex environments and utilize multiple traits to ensure survival. Here, we report the identification of multiple traits that enable a common soil organism, Myxococcus xanthus, to prey upon and utilize nutrients from another common soil organism, Bacillus subtilis. We mutagenized the predator and carried out a screen to identify genes that were required to either enhance or diminish capacity to consume prey. We identified dozens of genes encoding factors that contribute to the overall repertoire for the predator to successfully engage its prey in the natural environment.

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“…CRISPRi has been shown to work in E. coli 164 , fungi 165 , actinobacteria 55 , and plants 166 . Other organisms relevant to NP biosynthesis, such as cyanobacteria 167 , burkholderia 168 , pseudomonads 169 , and myxobacteria 170 , have endogenous CRISPR systems but have not as of yet been exploited as hosts for CRISPRi regulation. CRISPRi has already been exploited to control metabolic fluxes via multiplexed gene repression of endogenous pathways in E. coli 171 and in heterologous pathways in yeast (Figure 5g) 172 .…”

Section: Potential Applications For Synthetic Genetic Circuitsmentioning

confidence: 99%

Synthetic biology to access and expand nature's chemical diversity

et al. 2016

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Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Economically accessing the potential encoded within sequenced genomes promises to reinvigorate waning drug discovery pipelines and provide novel routes to intricate chemicals. This is a tremendous undertaking, as the pathways often comprise dozens of genes spanning as much as 100+ kiliobases of DNA, are controlled by complex regulatory networks, and the most interesting molecules are made by non-model organisms. Advances in synthetic biology address these issues, including DNA construction technologies, genetic parts for precision expression control, synthetic regulatory circuits, computer aided design, and multiplexed genome engineering. Collectively, these technologies are moving towards an era when chemicals can be accessed en mass based on sequence information alone. This will enable the harnessing of metagenomic data and massive strain banks for high-throughput molecular discovery and, ultimately, the ability to forward design pathways to complex chemicals not found in nature.

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A CRISPR with Roles in Myxococcus xanthus Development and Exopolysaccharide Production

Cited by 31 publications

References 45 publications

Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

Identification of Functions Affecting Predator-Prey Interactions between Myxococcus xanthus and Bacillus subtilis

Synthetic biology to access and expand nature's chemical diversity

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