Method and probes for the identification of microbial genes specifically induced during host infection

A method of identifying microbial coding sequences which are specifically induced in a pathogenic microorganism during infection of a host and which may be used as probes to detect and identify pathogens, comprising the steps of:

• (a) providing a one to four kilobase fragment of size fractionated chromosomal DNA that shares homology to a genomic DNA of the pathogenic microorganism;
• (b) infecting a host with a pool of fusion strains wherein said fusion strains are constructed by integrating an expression plasmid into the genomic DNA of a pathogenic microorganism that is either:
• (i) an auxotrophic mutant strain of said pathogenic microorganism that lacks a transposition competent element, or
• (ii) sensitive to an antibiotic, and wherein said expression plasmid comprises:
• A) a promoterless synthetic operon comprising two genes, wherein the first gene complements the mutation of the pathogenic microorganism or confers resistance to said antibiotic, and the second gene functions as a reporter gene, and
• B) a one to four kilobase fragment of chromosomal DNA that shares homology to said genomic DNA of the pathogenic microorganism;
• (c) treating said host with said antibiotic if said first gene of said synthetic operon confers resistance to said antibiotic;
• (d) harvesting from said host said fusion strains that survive and propagate in said host after step (b);
• (e) detecting expression of said one to four kilobase fragments by identifying harvested fusion strains from step (d) that fail to express said reporter gene in vitro;
• (f) sequencing the one to four kilobase fragments from step (e);
• (g) identifying one or more aberrant fragments from step (f) having a total guanine and cytosine content of less than about 49% or greater than about 59%;
• and
• (h) detecting and identifying a microorganism by hybridizing one or more of said aberrant fragments to genomic DNA derived from said microorganism.

In a method of identifying microbial coding sequences which are specifically induced in a pathogenic microorganism during infection of a host, wherein:

• (a) a host is infected with a pool of fusion strains wherein said fusion strains are constructed by integrating an expression plasmid into the genomic DNA of a pathogenic microorganism that is either:
• (i) an auxotrophic mutant strain of said pathogenic microorganism, or
• (ii) sensitive to an antibiotic, and wherein said expression plasmid comprises:
• A) a promoterless synthetic operon comprising two genes, wherein the first gene complements the mutation of the pathogenic microorganism or confers resistance to said antibiotic, and the second gene functions as a reporter gene, and
• B) a fragment of chromosomal DNA that shares homology to said genomic DNA of the pathogenic microorganism;
• (b) treating said host with said antibiotic if said first gene of said synthetic operon confers resistance to said antibiotic;
• (c) harvesting from said host fusion strains that survive and propagate in said host after step (a);
• and
• (d) detecting expression of said fragment by identifying harvested fusion strains from step (c) that fail to express said reporter gene in vitro, wherein the improvement comprises:
• providing a one to four kilobase fragment of size fractionated chromosomal DNA that shares homology to a genomic DNA of the pathogenic microorganism;
• and
• selecting an auxotrophic mutant strain of said pathogenic microorganism which lacks a transportation competent element.