Bacillus strain and antibiotic screening method

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

1999-06-11

2001-07-03

Mosher, Mary E. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S004000, C435S252500, C435S252310

Reexamination Certificate

active

06255065

ABSTRACT:

The assay method described herein targets a group of related activities functioning in cell division. The assay is based on the observation that activation of the sporulation-specific transcription factor &sgr;
F
, which has been extensively studied in several laboratories (reviewed by Errington, 1996
, Trends in Genetics
12, 31-34), requires the completion of cell division.
Synthesis of the sigma factor begins at the onset of sporulation but its product is initially held in an inactive state by the action of an anti-sigma factor, SpollAB. Release from inhibition requires the concerted action of at least two other proteins, SpollAA and SpollE, through a series of biochemical interactions that are now well characterised (Errington, 1996). These proteins serve to allow release of &sgr;
F
activity only after the sporulating cell has undergone asymmetric cell division and to restrict the &sgr;
F
activity to the smaller prespore cell type. This mechanism works in such a way that it renders &sgr;
F
activation dependent on septation. Thus, mutants or genetically engineered strains of
B. subtilis
that are prevented from undergoing septation because of the absence of essential cell division gene products such as ftsZ (Beall and Lutkenhaus, 1991
, Genes Devel
. 5, 447-455), divlC (Levin and Losick, 1994
, J. Bacteriol
. 176, 716-722) or ftsL (Daniel, R. A. and Errington, J., unpublished results), synthesise but do not activate &sgr;
F
. The dependence of &sgr;
F
activation on septation is herein used as the basis for a sensitive assay for inhibitors of cell division. Although the assay is based on inhibition of the specialised asymmetric cell division which occurs at the onset of sporulation, there is ample evidence that this process is functionally very similar to cell division in vegetative cells.
In one aspect the invention provides a Bacillus strain having a chromosome with two artificially introduced reporter genes, a first reporter gene having a promoter which is dependent on active &sgr;
F
or &sgr;
E
factors, and a second reporter gene which provides a measure of the synthesis of the (inactive) &sgr;
F
or &sgr;
E
factor.
In another aspect the invention provides a method of determining whether an agent inhibits cell division in Bacillus species, which method comprises inducing the Bacillus strain as described to sporulate in the presence of the agent, and observing expression of the first and second reporter genes. It is thought that the property of inhibiting cell division, is indicative of actual or potential anti-microbial properties in the agent. The method is thus expected to be useful for screening possible anti-microbial agents.
In another aspect the 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 second reporter genes and thereby determining that the agent inhibits cell division in the Bacillus species, and using the agent as an antibiotic to kill or inhibit the growth of bacteria.
The assay described below is based on use of &sgr;
F
activation but it could also have used &sgr;
E
, another sporulation specific sigma factor that is dependent on asymmetric septation (Stragier et al, 1988, Cell, 52, 697-704). The dependence of &sgr;
E
on septation is now thought to be an indirect effect caused by the dependence of &sgr;
E
activity on &sgr;
F
activation (see Errington, 1996). Use of an &sgr;
E
-dependent reporter gene would be less desirable as it would probably detect more non-specific inhibitors than with &sgr;
F
.
Any Bacillus species may be used that is capable of sporulating under suitable conditions and for which genetic constructions can be made.
B. subtilis
is conveniently accessible and well characterised and is preferred.
The Bacillus strain constructed has a chromosome with two reporter genes each linked to a different promoter. A reporter gene is one which on expression gives rise to an easily detected or observed phenotype. For example, the expressed protein may be an enzyme which acts on a substance to give a product that is easily observed e.g. because it is coloured or chemiluminescent or fluorescent. Reporter genes capable of being expressed in Bacillus species are well known and documented in the literature. The two reporter genes are preferably chosen so that their products can be readily assayed simultaneously. lacZ has been used for more than ten years with great success in
B. subtilis
. There are a range of useful substrates that generate coloured or fluorescent products upon hydrolysis by &bgr;-galactosidase. The uidA gene of
E. coli
, also known as the gusA gene, has recently been harnessed for similar purposes, and the range of substrates available for the gene product, &bgr;-glucuronidase, is similar to that of &bgr;-galactosidase.
In a preferred form, the assay uses a specific strain of
B. subtilis
containing two reporter genes. The first (gpr-uidA) provides a means of monitoring &sgr;
F
(or &sgr;
E
) activity: its promoter is &sgr;
F
(or &sgr;
E
)-dependent and it directs the production of an enzyme, &bgr;-glucuronidase, the activity of which can be readily measured by spectrophotometry or spectrofluorimetry. The second reporter gene (spollA-lacZ), which monitors expression of the gene encoding &sgr;
F
(or &sgr;
E
), e.g. by virtue of having a promoter regulated similarly to the gene encoding sigma factor, provides a check for non-specific effects on sporulation or general inhibitors of gene expression. Again the product of the reporter gene is an enzyme, &bgr;-galactosidase, that can readily be measured. By using appropriate (enzyme) substrates, the two enzyme activities could be measured simultaneously.
To use the assay, this
B. subtilis
strain would be induced to sporulate by the resuspension method (Sterlini and Mandelstam, 1969
, Biochem. J
. 113, 29-37; Partridge and Errington, 1993
, Mol Microbiol
8, 945-955). The culture would be dispensed into the wells of a microtitre plate just before the onset of asymmetric cell division (e.g. after 1 h at 37° C.). Individual wells would contain one or more potential inhibitors. After a period of incubation sufficient for induction of the spollA operon and activation of &sgr;
F
(or &sgr;
E
), the microtitre plate cultures would be assayed for the two reporter activities by standard methods. Potential “hits” would show inhibition of &bgr;-glucuronidase activity but normal &bgr;-galactosidase activity (indicating synthesis but not activation of &sgr;
F
(or &sgr;
E
))
Alternatively, test compounds can be dropped onto a lawn of sporulating cells on a solid surface (e.g. agar). In this case, the effect of the test compounds on reporter gene activity will be assessed by the colour or fluorescence produced by hydrolysis of colourigenic or fluorogenic substrates incorporated into the solid medium.
Two kinds of compounds might be expected to be detected by the assay. First, the desired compounds that inhibit asymmetric cell division. Second, compounds that interfere in some way with the protein—protein interactions, or the kinase or phosphatase activities known to be involved in &sgr;
F
(or &sgr;
E
) regulation. These would be of purely academic interest (at least in the short term). The two classes could be readily distinguished by light microscopy because the latter class should form normal asymmetric septa.
Irrespective of the specific biomolecule affected in the screen, any compounds identified would be good potential candidates for development as antimicrobial agents because cell division is such a central target. Moreover, since cell division proteins tend to be highly conserved in bacteria, it is likely that broad spectrum inhibitors could be obtained.


REFERENCES:
patent: 0 174 477 (1986-03-01), None
patent: 96/35804 (1996-11-01), None
patent: 97/00325 (1997-01-01), None
W. Haldenwang et al., “The sigma factors ofBacillus subtilis”, Microbiological Reviews, vol. 59, No. 1, pp. 1-30, Mar. 1995.
D. Sun et al., “Effect of chromosome location ofBacillus subtilis

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