CRISPR-Cas in Escherichia coli: regulation by H-NS, LeuO and temperature

CRISPR-Cas adaptive immune systems are present in many bacteria and archaea and provide protection against invading DNA such as phages and plasmids. These systems are very versatile and complex in their gene composition and genomic architecture. CRISPR-Cas systems are classified into 2 classes, 6 types and 33 subtypes although this number is not definitive and the research is ongoing. All CRISPR-Cas systems have been thoroughly investigated in order to better understand the mechanism of CRISPR immunity enabling its use as a tool in genome editing and other biotechnological applications. However, regulation of the CRISPR-Cas system is also very complex and still not fully understood; it must provide optimal protection without introducing harmful consequences to the host. In this review we give an overview on the regulation of the CRISPR-Cas system Class 1 Type I-E in Escherichia coli with the emphasis on the role of temperature in regulation of the CRISPR-Cas activity and the interplay of the key regulators H-NS and StpA repressors and LeuO antirepressor in regulation of cas gene expression and HtpG chaperone in maintaining functional levels of Cas3.</p

Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity

CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration

CRISPR-Cas Adaptation in Escherichia coli requires RecBCD helicase but not nuclease activity, is independent of homologous recombination, and is antagonised by 5’ ssDNA exonucleases

Prokaryotic adaptive immunity is established against mobile genetic elements (MGEs) by “naïve adaptation” when DNA fragments from a newly encountered MGE are integrated into CRISPR-Cas systems. In E. coli, DNA integration catalysed by Cas1-Cas2 integrase is well understood in mechanistic and structural detail but much less is known about events prior to integration that generate DNA for capture by Cas1-Cas2. Naïve adaptation in E. coli is thought to depend on the DNA helicase-nuclease RecBCD for generating DNA fragments for capture by Cas1-Cas2. The genetics presented here show that naïve adaptation does not require RecBCD nuclease activity but that helicase activity may be important. RecA loading by RecBCD inhibits adaptation explaining previously observed adaptation phenotypes that implicated RecBCD nuclease activity. Genetic analysis of other E. coli nucleases and naïve adaptation revealed that 5’ ssDNA tailed DNA molecules promote new spacer acquisition. We show that purified E. coli Cas1-Cas2 complex binds to and nicks 5’ ssDNA tailed duplexes and propose that E. coli Cas1-Cas2 nuclease activity on such DNA structures supports naïve adaptation

Usporedba intraplazmidne rekombinacije u bakterijama Agrobacterium tumefaciens i Escherichia coli

In this work we have constructed a plasmid to compare intraplasmid recombination efficiency in Agrobacterium tumefaciens and Escherichia coli. The plasmid contains two directly repeated copies of spectinomycin resistance gene, one lacking 5’ and the other lacking 3’ end. These two copies share a 570-bp region of homology and are separated by the ampicillin resistance gene. Homologous recombination between repeated copies of incomplete spectinomycin resistance genes results in the restoration of spectinomycin resistance. During this process, ampicillin resistance gene is either deleted or incomplete spectinomycin genes are amplified along with the ampicillin resistance gene. This experimental system enabled us to follow for the first time the generation of deletions and amplifications during intraplasmid recombination in A. tumefaciens. We show here that predominantly RecA-independent mechanism contributes to the formation of deletion and amplification products in both, A. tumefaciens and E. coli. Additionally, deletion and amplification products were detected at similar frequencies, suggesting that amplifications and deletions probably occur by a similar mechanism.U ovom smo radu konstruirali plazmid koji nam je omogućio da usporedimo intraplazmidnu rekombinaciju u bakterijama Agrobacterium tumefaciens i Escherichia coli. Plazmid sadržava dvije istosmjerno ponovljene kopije gena odgovornog za rezistenciju na spektinomicin, pri čemu jednoj kopiji nedostaje 5\u27, a drugoj 3\u27 kraj gena, a međusobno su homologne u duljini od 570 pb. Osim toga, DNA koja se nalazi između ove dvije istosmjerno ponovljene sekvencije sadržava gen koji daje rezistenciju na antibiotik ampicilin. Homolognom rekombinacijom između nepotpunih gena za rezistenciju na spektinomicin nastaje funkcionalni gen, odgovoran za pojavu rezistencije. Pritom može doći do delecije gena za rezistenciju na ampicilin ili njegovog umnožavanja, zajedno s nepotpunim genima za otpornost na spektinomicin. Ovaj eksperimentalni sustav omogućio nam je da po prvi put pratimo pojavu delecija i amplifikacija tijekom intraplazmidne rekombinacije u bakteriji A. tumefaciens. Pokazali smo da delecije i amplifikacije u bakterijama Agrobacterium tumefaciens i Escherichia coli nastaju prvenstveno RecA-neovisnim mehanizmom. Osim toga, ustanovili smo da se delecije i amplifikacije pojavljuju s podjednakom učestalošću, što upućuje na to da je mehanizam oba rekombinacijska događaja sličan

CRISPR-Cas Adaptation in Escherichia coli requires RecBCD helicase but not nuclease activity, is independent of homologous recombination, and is antagonised by 5’ ssDNA exonucleases

Prokaryotic adaptive immunity is established against mobile genetic elements (MGEs) by “naïve adaptation” when DNA fragments from a newly encountered MGE are integrated into CRISPR-Cas systems. In E. coli, DNA integration catalysed by Cas1-Cas2 integrase is well understood in mechanistic and structural detail but much less is known about events prior to integration that generate DNA for capture by Cas1-Cas2. Naïve adaptation in E. coli is thought to depend on the DNA helicase-nuclease RecBCD for generating DNA fragments for capture by Cas1-Cas2. The genetics presented here show that naïve adaptation does not require RecBCD nuclease activity but that helicase activity may be important. RecA loading by RecBCD inhibits adaptation explaining previously observed adaptation phenotypes that implicated RecBCD nuclease activity. Genetic analysis of other E. coli nucleases and naïve adaptation revealed that 5’ ssDNA tailed DNA molecules promote new spacer acquisition. We show that purified E. coli Cas1-Cas2 complex binds to and nicks 5’ ssDNA tailed duplexes and propose that E. coli Cas1-Cas2 nuclease activity on such DNA structures supports naïve adaptation

Genetička analiza produkata bakterije Escherichia coli koji sudjeluju u konjugacijskoj rekombinaciji u prisutnosti proteina Gam faga λ

The Gam protein of phage is a well-known inhibitor of the enzymatic activities of the RecBCD enzyme, the major enzyme involved in homologous recombination in bacteria Escherichia coli. In this work, we studied (i) the effect of the RecA loading-deficient recB (recBD1080A) mutation on conjugational recombination in the presence of phage Gam protein and (ii) additional genetic requirements for the RecBCD-Gam-mediated conjugational recombination. For this purpose, we introduced Gam+ and Gam- expressing plasmids into wild type cells and different mutants of E. coli (recJ, recBD1080A, recB, recN, recF, recR, recO, recD), and determined the yields of recombinants after Hfr mediated conjugation. The obtained results suggest that RecA loading activity is not inhibited by Gam and that conjugational recombination in the presence of Gam is partially dependent on recJ and recO gene products.Gam protein bakteriofaga λ je inhibitor enzimskih aktivnosti enzima RecBCD koji sudjeluje u homolognoj genetičkoj rekombinaciji u bakteriji Escherichia coli. U ovom su radu proučavani (i) učinak recB mutacije deficijentne u nanošenju proteina RecA (recBD1080A) na konjugacijsku rekombinaciju u prisutnosti proteina Gam faga λ i (ii) učinak mutacija drugih rekombinacijskih gena na konjugacijsku rekombinaciju u bakterijama s kompleksom RecBCD-Gam. Zbog toga smo unijeli plazmide koji eksprimiraju Gam+ i Gam– u divlji tip i u različite mutante bakterije Escherichia coli (recJ, recBD1080A, recB, recN, recF, recR, recO, recD), te odredili prinos rekombinanata nakon Hfr-konjugacije. Dobiveni su rezultati pokazali da aktivnost nanošenja proteina RecA vjerojatno nije inhibirana proteinom Gam. U prisutnosti proteina Gam konjugacijska rekombinacija djelomično ovisi o produktima gena recJ i recO

CRISPR-Cas adaptation in Escherichia coli requires RecBCD helicase but not nuclease activity, is independent of homologous recombination, and is antagonized by 5' ssDNA exonucleases

© The Author(s) 2018. Prokaryotic adaptive immunity is established against mobile genetic elements (MGEs) by 'naïve adaptation' when DNA fragments from a newly encountered MGE are integrated into CRISPR-Cas systems. In Escherichia coli, DNA integration catalyzed by Cas1- Cas2 integrase is well understood in mechanistic and structural detail butmuch less is known about events prior to integration that generate DNA for capture by Cas1-Cas2. Naïve adaptation in E. coli is thought to depend on the DNA helicase-nuclease RecBCD for generating DNA fragments for capture by Cas1- Cas2. The genetics presented here show that naïve adaptation does not require RecBCD nuclease activity but that helicase activity may be important. RecA loading by RecBCD inhibits adaptation explaining previously observed adaptation phenotypes that implicated RecBCD nuclease activity. Genetic analysis of other E. coli nucleases and naïve adaptation revealed that 5' ssDNA tailed DNA molecules promote new spacer acquisition. We show that purified E. coli Cas1-Cas2 complex binds to and nicks 5' ssDNA tailed duplexes and propose that E. coli Cas1-Cas2 nuclease activity on such DNA structures supports naïve adaptation

A tryptophan ‘gate’ in the CRISPR-Cas3 nuclease controls ssDNA entry into the nuclease site, that when removed results in nuclease hyperactivity

Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30C to 37C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp- 406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells

Cas1–Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation

Prokaryotic Cas1-Cas2 protein complexes generate adaptive immunity to mobile genetic elements (MGEs), by capture and integration of MGE DNA in to CRISPR sites. De novo immunity relies on naive adaptation-Cas1-Cas2 targeting of MGE DNA without the aid of pre-existing immunity 'interference' complexes-by mechanisms that are not clear. Using E. coli we show that the chaperone DnaK inhibits DNA binding and integration by Cas1-Cas2, and inhibits naive adaptation in cells that results from chro-mosomal self-targeting. Inhibition of naive adaptation was reversed by deleting DnaK from cells, by mutation of the DnaK substrate binding domain, and by expression of an MGE (phage) protein. We also imaged fluorescently labelled Cas1 in living cells, observing that Cas1 foci depend on active DNA replica-tion, and are much increased in frequency in cells lacking DnaK. We discuss a model in which DnaK provides a mechanism for restraining naive adaptation from DNA self-targeting, until DnaK is triggered to release Cas1-Cas2 to target MGE DNA