Elsevier

Veterinary Microbiology

Volume 250, November 2020, 108868
Veterinary Microbiology

Targeted mutagenesis of Mycoplasma gallisepticum using its endogenous CRISPR/Cas system

https://doi.org/10.1016/j.vetmic.2020.108868Get rights and content

Highlights

  • The endogenous CRISPR system of Mycoplasma gallisepticum is active.

  • It can be used to perform targeted mutagenesis in Mycoplasma gallisepticum.

  • The Mycoplasma gallisepticum Cas protein MgaCas9 has site-specific activity.

  • MgaCas9 activity has a low dependency on sequences adjacent to its target.

Abstract

New, more efficient methods are needed to facilitate studies of gene function in the mycoplasmas. CRISPR/Cas systems, which provide bacteria with acquired immunity against invading nucleic acids, have been developed as tools for genomic editing in a wide range of organisms. We explored the potential for using the endogenous Mycoplasma gallisepticum CRISPR/Cas system to introduce targeted mutations into the chromosome of this important animal pathogen. Three constructs carrying different CRISPR arrays targeting regions in the ksgA gene (pK1-CRISPR, pK-CRISPR-1 and pK-CRISPR-2) were assembled and introduced into M. gallisepticum on an oriC plasmid. The loss of KsgA prevents ribosomal methylation, which in turn confers resistance to the aminoglycoside antimicrobial kasugamycin, enabling selection for ksgA mutants. Analyses of the complete sequence of the ksgA gene in 78 resistant transformants revealed various modifications of the target region, presumably caused by the directed CRISPR/Cas activity of M. gallisepticum. The analyses suggested that M. gallisepticum may utilize a non-homologous end joining (NHEJ) repair system, which can result in deletion or duplication of a short DNA segment in the presence of double-stranded breaks. This study has generated an improved understanding of the M. gallisepticum CRISPR/Cas system, and may also facilitate further development of tools to genetically modify this important pathogen.

Introduction

Mycoplasma gallisepticum causes respiratory disease and production loss in poultry, resulting in considerable economic loss (Browning et al., 2010; Mohammed et al., 1987). More effective control of the disease it causes requires more detailed understanding of the basis of its pathogenicity and the functions of genes involved in virulence. Although its genome sequence was determined some time ago (Papazisi et al., 2003), the functions of many of its genes remain unknown, in part because of the limited array of genetic tools available for manipulating it.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a bacterial acquired immunity system that has been adapted for use as a tool for genome editing (Bolotin et al., 2005). The system is comprised of the Cas genes and the CRISPR arrays, which consist of short direct repeats of sequences (typically 30–40 nt) interspersed with spacers, which are variable sequences of the same size as the direct repeats. The spacers are short sequences of mobile genetic elements or phages that have been integrated into the chromosome after previous exposure to that invading nucleic acid (Bolotin et al., 2005). The Cas9 protein, which is a signature of type II CRISPR/Cas systems, has two nuclease domains homologous to HNH and RuvC, and is targeted to the invading DNA by crRNAs transcribed from the CRISPR arrays (Makarova et al., 2011). The HNH domain then cleaves the complementary DNA strand and the RuvC-like domain cleaves the non-complementary DNA strand (Jinek et al., 2012). It is possible to direct Cas9 function (recognition and targeting) by introducing guide RNAs complementary to a target sequence into a bacterial cell (Cong et al., 2013; Jiang et al., 2013; Jinek et al., 2012). The recognition and cleavage of the invading nucleic acid sequence in many type II systems depend on a short sequence close to the protospacer in the invading DNA, called the Protospacer Adjacent Motif (PAM) (Jiang et al., 2013; Jinek et al., 2012). There are a few Cas proteins capable of targeting both DNA and RNA, including the Cas9s of Staphylococcus aureus (SauCas9), Campylobacter jejuni (CjeCas9) and Neisseria meningitidis (NmeCas9). RNA cleavage by SauCas9, CjeCas9 and NmeCas9 is site-specific and independent of the PAM sequence (Rousseau et al., 2018; Strutt et al., 2018). Repair of the double-stranded breaks in can result in insertion/deletion (indel) mutations (Cong et al., 2013; Shuman and Glickman, 2007). These endogenous CRISPR/Cas systems have now been used as genomic engineering tools in many bacteria, including Streptococcus pneumoniae (Jiang et al., 2013), Escherichia coli (Yang et al., 2014), Clostridium pasteurianum (Pyne et al., 2016), Clostridium difficile (Maikova et al., 2019) and Heliobacterium modesticaldum (Baker et al., 2019), but there are no reports of their use to manipulate mollicute genomes.

The genome of M. gallisepticum contains CRISPR arrays and predicted cas1, cas2 and csn1 genes, the signature cas genes for CRISPR type II systems. The M. gallisepticum CRISPR/Cas system is categorized as type II-A, based on its cas operon composition and CRISPR repeat sequences, with additional putative cas genes encoding proteins with no obvious sequence similarity with Csn2 variants in other bacteria (Chylinski et al., 2013). While the streptococcal CRISPR/Cas system has been used to generate mutations in a Mycoplasma mycoides subspecies capri genome cloned in yeast, and the effects of the mutations studied using genome transplantation techniques (Tsarmpopoulos et al., 2015), not all mycoplasmas have been amenable to genome transplantation, so there remains a need for targeted mutagenesis methods that can be applied within mycoplasma cells to improve our capacity to create specific novel mutants to enable gene function to be better understood and to facilitate studies on the molecular pathogenesis of this pathogen and other mycoplasmas.

The ksgA gene encodes a ribosomal RNA adenine dimethylase family protein that methylates the 16S rRNA. Loss of KsgA methyltransferase activity modifies the ribosomal binding site for the aminoglycoside antimicrobial kasugamycin, resulting in resistance to it (Duffin and Seifert, 2009). The orthologues of the ksgA gene can be found in some mycoplasma genomes, but the role of the product of these orthologues in resistance to kasugamycin has not been confirmed experimentally (Li et al., 2017).

The aims of the studies described here were to determine whether the CRISPR/Cas9 system in M. gallisepticum was functional and whether it could be utilised to introduce mutations into the M. gallisepticum genome using synthetic CRISPR arrays carried on an oriC plasmid. The ksgA gene of M. gallisepticum strain S6 (GenBank CP006916.3 locus_tag: GCW_00045) was targeted as a selectable marker of successful mutagenesis, with the ultimate goal of establishing whether this endogenous CRISPR/Cas9 system may have potential for use in genomic engineering of this important mycoplasma pathogen.

Section snippets

Bacterial strains and culture conditions

Mycoplasma gallisepticum strain S6 for these experiments was kindly provided by Anna Kanci Condello from the Asia-Pacific Centre for Animal Health, University of Melbourne. The pMIori plasmid, which is replicable in M. gallisepticum and contains the oriC region of M. imitans (Lee et al., 2008), was used as a negative control plasmid. M. gallisepticum strain S6 was cultured in Mycoplasma Broth (MB) at 37 °C for 12 h. MB contained 0.75 % trypticase peptones, 0.25 % phytone peptones, 0.05 %

Evaluation of resistance to kasugamycin on Mycoplasma Agar

The analyses of the MIC of kasugamycin for M. gallisepticum strain S6 showed that at a concentration of 2.1 × 108 CCU/mL strain S6 was inhibited by kasugamycin at 400 μg/mL and not by any concentration of kasugamycin below 400 μg/mL. M. gallisepticum strain S6 transformed with different plasmids were cultured on Mycoplasma Agar (MA) containing the antibiotic kasugamycin. The colony counts after transformation with pK-CRISPR-1, the plasmid containing a CRISPR array with spacers complementary to

Discussion

Although the genomes of many mollicutes, including that of M gallisepticum, were sequenced some time ago, the functions of many genes remain unknown because of the limited tools available for targeted mutagenesis. The established CRISPR/Cas system (derived from that of Streptococcus pyogenes), which has been used as a genomic engineering tool in many species, has not been used successfully for targeted manipulation of the genomes of mycoplasmas. While the endogenous CRISPR/Cas systems have been

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

S.M. was supported by a Research Training Program (RTP) Scholarship from the University of Melbourne, Australia.

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      To achieve this we assembled a CRISPR array targeting the mnuA gene of M. gallisepticum in the plasmid pKM_CRISPR and incorporated a second spacer in the CRISPR array targeting ksgA gene. Our previous work suggested that mutations in the ksgA gene (GenBank CP006916.3 locus_tag: GCW_00045) result in resistance of M. gallisepticum to the antibiotic kasugamycin (Mahdizadeh et al., 2020). We hypothesised that the combination of these two spacers in one CRISPR array would enable us to select for transformants, which might be expected to have concurrent CRISPR/Cas-induced mutations in their mnuA and ksgA genes.

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    These authors contributed equally to this work.

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