ena-STUDY-UW MADISON-07-12-2013-20:13:27:816-23
The CRISPR/cas system of Xenorhabdus nematophila contributes to colonization of symbiotic host nematodes
The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes, and provides functions that facilitate the killing and degradation of insect hosts.  X. nematophila is carried between insects in the intestine of a specialized transmission stage of S. carpocapsae, known as the infective juvenile (IJ). In a previously identified X. nematophila colonization-defective mutant, nilD6::Tn5 the transposon inserted in a region lacking obvious coding potential.  In this study we demonstrate that the transposon insertion disrupts expression of a single CRISPR RNA, NilD, which has the same 5' start site and length as Escherichia coli K12 CRISPR RNAs.  Nematode colonization was restored by inserting a copy of the nilD locus in trans on the X. nematophila chromosome, indicating that NilD contributes to the colonization process. Furthermore, we show that NilD expression is dependent on the Cas (¬CRISPR-associated) machinery.  However, while deletion of the casE gene in the complemented strain abolished nematode colonization, disruption of casE in the wild type parental background had no effect on this process.  Likewise, deletion of the nilD locus in the parental strain did not inhibit colonization of the nematode host, indicating that the requirement for NilD is evident only in specific genetic backgrounds.  Our data demonstrate that NilD CRISPR RNA is conditionally necessary for host colonization and suggest a model that its function is to regulate expression of endogenous sequences.
The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes, and provides functions that facilitate the killing and degradation of insect hosts.  X. nematophila is carried between insects in the intestine of a specialized transmission stage of S. carpocapsae, known as the infective juvenile (IJ). In a previously identified X. nematophila colonization-defective mutant, nilD6::Tn5 the transposon inserted in a region lacking obvious coding potential.  In this study we demonstrate that the transposon insertion disrupts expression of a single CRISPR RNA, NilD, which has the same 5' start site and length as Escherichia coli K12 CRISPR RNAs.  Nematode colonization was restored by inserting a copy of the nilD locus in trans on the X. nematophila chromosome, indicating that NilD contributes to the colonization process. Furthermore, we show that NilD expression is dependent on the Cas (¬CRISPR-associated) machinery.  However, while deletion of the casE gene in the complemented strain abolished nematode colonization, disruption of casE in the wild type parental background had no effect on this process.  Likewise, deletion of the nilD locus in the parental strain did not inhibit colonization of the nematode host, indicating that the requirement for NilD is evident only in specific genetic backgrounds.  Our data demonstrate that NilD CRISPR RNA is conditionally necessary for host colonization and suggest a model that its function is to regulate expression of endogenous sequences.