Definition of the single integration site of the pathogenicity locus in Clostridium difficile.
We determined the nucleotide sequence 3.8 kb upstream and 5.2 kb downstream of the toxin genes A and B of Clostridium difficile. Nine ORFs were discovered. Based on PCR-directed approaches, two were attributed to the pathogenicity locus (PaLoc). The other seven were found in every C. difficile isolate obtained from the human gastrointestinal tract, respectless of their toxinogenicity. The ORFs cdu1 and cdu2/2' upstream of the PaLoc displayed similarity to repressors of Gram-positive bacteria (cdu1), and to an Na+/H+ antiporter described for Enterococcus hirae (cdu2/2'). Downstream of the locus a putative ABC transporter (cdd2-4) was identified. With a set of three paired primers used in polymerase chain reactions we succeeded in delineating the PaLoc. Sequencing of the appropriate stretch of DNA in C. difficile VPI10463 and four additional toxinogenic strains proved a high conservation of the borders of the PaLoc in all these strains. Our data define the locus as a distinct genetic element. Comparing the sequences of five toxinogenic and five non-toxinogenic strains the integration site of the PaLoc was defined. This showed that a stretch of 115 bp found in non-toxinogenic strains is replaced by the 19-kb locus in toxinogenic strains. Analysis of the boundary sequences showed that the locus is obviously not a mobile genetic element by itself. Instead we propose that it is the independent pathogenic part of a more extended genetic element associated with virulence. The 115 bp of non-toxinogenic strains replaced by the locus in toxinogenic strains carry the putative transcription terminator of the cdu1, a predicted repressor protein. A possible polar effect of the loss of this terminator on transcription of the TcdABCDE genes is discussed. Such an effect would explain the unidirectional insertion of the PaLoc at a single site of the C. difficile genome and might give a rationale for the development of the disease which is induced after antibiotical treatment.
Braun
V
V
Verfügungsgebäude für Forschung und Entwicklung, Institut für Medizinische Mikrobiologie und Hygiene, Johannes-Gutenberg-Universität, Mainz, Germany.
Hundsberger
T
T
Leukel
P
P
Sauerborn
M
M
von Eichel-Streiber
C
C
D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D016360:Clostridioides difficile / Q000235:genetics / Q000472:pathogenicity*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004768:Enterotoxins / Q000235:genetics*; D006801:Humans; D007422:Intestines / Q000382:microbiology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014774:Virulence / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile