Bacillus strain and antibiotic screening method

Details Bacillus strain and antibiotic screening method Bacillus strain and antibiotic screening method

1999-06-11

2002-02-26

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S006120, C435S041000, C435S069800, C435S243000, C435S252300, C435S252310, C435S440000, C435S029000

Reexamination Certificate

active

06350587

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to a mutant Bacillus strain and a antibotic screening method using the same.
2. Description of the Related Art
We previously showed that the SpoIIIE protein of
Bacillus subtilis
is required for the transfer of the prespore chromosome through the asymmetric division septum that separates the prespore from its larger mother cell (
1
,
2
). spoIIIE mutations lead to a block in sporulation, leaving the prespore with only part of a chromosome, the remainder of the chromosome being trapped in the mother cell compartment. Further analysis of spoIIIE mutants has revealed that the small segment of DNA that is trapped in the prespore is a fairly specific one, centred close to the origin of DNA replication, oriC (
1
,
3
). This implied the existence of a mechanism which imposes a specific orientation on the chromosome destined for the prespore before septation. We have recently shown that the spoOJ gene is required to specify this orientation (
4
). However, specificity is not completely lost in spoOJ mutants (
3
), so it appears that there must be at least one secondary mechanism working to determine chromosome orientation at the onset of sporulation. The assay for inhibitors of SpoOJ function described below exploits some unexpected features of this change of specificity.
The spoOJ gene is highly conserved in a wide range of bacteria (
5
) and it is related to a family of proteins required for accurate partitioning of low-copy-number plasmids found in many diverse bacteria (
6
,
7
). Our recent results strongly suggest that SpoOJ protein has a direct role in segregation of sister chromosomes during both growth and sporulation (
10
). However, the gene is not essential for vegetative growth, although chromosome partitioning is partially impaired (
8
). Most likely, this is because of the presence of a secondary partitioning system in this organism, perhaps the same one that we have detected in the experiments mentioned above. Nevertheless, there is at least one report of a chromosomal spoOJ-like gene being essential (
9
), consistent with the vital importance of chromosome partitioning mechanisms for bacterial viability. Thus, the spoOJ family of proteins may be good targets for antimicrobial agents.
SUMMARY OF THE INVENTION
The effects of spoOJ mutations on prespore chromosome orientation, and the ability to detect this by use of a spoIIIE mutant background, provides the potential for a very specific whole-cell assay for inhibitors of SpoOJ function. The presence of any given segment of chromosomal DNA in the prespore can be detected by use of a reporter gene controlled by a transcription factor, &sgr;
F
, which is activated only in the is small prespore compartment (a process that is not affected by perturbations in chromosome partitioning).
Thus the invention provides in one aspect a Bacillus strain having a chromosome with the following modifications:
a) a mutation of a spoIIIE gene which blocks transfer of the prespore chromosome,
b) a mutation which prevents loss of SpoOJ function from blocking sporulation, together with
c) a first reporter gene having a promoter which is dependent on &sgr;
F
factor and placed at a location where impaired SpoOJ function leads to increased trapping and hence to increased expression in the prespore, and/or
d) a second reporter gene having a promoter which is dependent on &sgr;
F
factor and placed at a location where impaired SpoOJ function leads to reduced trapping and hence to reduced expression in the prespore.
In another aspect, the present invention provides a method of determining whether an agent inhibits SpoOJ function in Bacillus species, which method comprises inducing the Bacillus strain as described to divide asymmetrically, as during sporulation, in the presence of the agent, and observing expression of the first and/or the second reporter gene.
In another aspect, the present invention provides a method which comprises inducing the Bacillus strain as described to sporulate in the presence of an agent, observing expression of the first and/or second reporter gene and thereby determining that the agent inhibits SpoOJ function in the Bacillus species, and using the agent as an antibiotic to kill or inhibit the growth of bacteria.


REFERENCES:
patent: 97 00325 (1997-01-01), None
S. R. Patridge et al., “The importance of morphological events and intercellular interactions in the regulation of prespore-specific gene expression during sporulation inBacillus subtilis”, Molecular Microbiology, vol. 8, No. 5, pp. 945-955, 1993.
D. Sun et al., “Effect of chromosome location ofBacillus subtilisforespore genes on their spo gene dependence and transcription by EoF: Identification of features of good EoF-dependent promoters”, Journal of Bacteriology, vol. 173, No. 24, pp. 7867-7874, Dec. 1991.
K. Ireton et al., “spoOJis required for normal chromosome segregation as well as the initiation of sporulation inBacillus subtilis”, Journal of Bacteriology, vol. 176, No. 17, pp. 5320-5329, Sep. 1994.
M. Sharpe et al., “TheBacillus subtilissoj-spoOJlocus is required for a centromere-like function involved in prespore chromosome partitioning”, Molecular Microbiology, vol. 21, No. 3, pp. 501-509, 1996.
Sharpe et al. Postseptational chromosome partitioning in bacteria, Proc. Natl. Acad. Sci. USA 92: 8630-8634, Sep. 1995.

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