Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-08-24
2004-07-06
Falk, Anne-Marie (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S006120, C435S069800, C435S243000, C435S252300, C435S252310, C435S440000
Reexamination Certificate
active
06759209
ABSTRACT:
Whole-cell assays are known for specific inhibitors of
B. subtilis
proteins involved in chromosome partitioning and cell division. The property, of inhibiting chromosome partitioning and cell division, is indicative of actual or potential anti-microbial properties. The inventor has devised three such assays; they are described in WO 97/00325; WO 98/26087; and WO 98/26088, which are summarised below and to which reference is directed.
New compounds inhibitory for any chromosome partitioning and cell division functions are likely to have a broad spectrum of activity against a wide range of bacteria, including important pathogens, because the functions targeted appear to be highly conserved. However, it is possible that some of the compounds discovered may turn out to be relatively specific for the
B. subtilis
proteins, in which case they would not be useful general purpose antimicrobial agents.
A similar problem arises in any whole-cell assay for an inhibitor of a specific gene of any micro-organism. The problem is that an inhibitor of a specific gene of a particular strain or micro-organism, may be specific to that strain, or alternatively may have inhibitory properties which are exerted over a rather wide range of micro-organisms. The present invention addresses that problem by replacing a target gene in a micro-organism used for a whole cell assay with a homologous gene from a different organism, e.g. a micro-organism of more direct interest.
Thus the invention provides in one aspect a micro-organism having a chromosome in which:
a) at least one gene has been partly or wholly replaced by a homologous gene from another micro-organism, and
b) an artificially introduced reporter gene is present and is expressed in a manner related to a homologous gene expression product.
In another aspect the invention provides a method of assessing an agent for antibiotic activity, which method comprises incubating the micro-organism as defined in the presence of the agent, and observing expression of the reporter gene or genes.
The micro-organism may be for example a yeast or more preferably a bacterium. The bacterium may be a Bacillus species that is capable of growth and sporulation under suitable conditions and for which genetic constructs can be made.
B. subtilis
is conveniently accessible and well characterised and is preferred.
A homologous gene is a functionally equivalent gene from another micro-organism. In the micro-organism of the present invention, at least one gene (the target gene) has been partly or wholly replaced by a homologous gene from another micro-organism. Preferably the target gene is one which is well conserved over many different species of bacteria or other micro-organisms. It is necessary that the homologous gene be functionally incorporated so as to be capable of expression in vivo. When the target gene is partly or wholly replaced by a homologous gene, it is necessary that the homologous gene be capable of forming an expression product that is different in some respect from the expression product of the target gene. Suitable target genes include genes involved in DNA replication, RNA synthesis, protein synthesis, cell wall synthesis, transport and cell division.
For micro-organisms which are Bacillus species e.g.
B. subtilis
, cell division genes include divIB (also called ftsQ), divIC, divIA, ftsA, ftsL (also called mraR), ftsZ, pbpB, as well as spoOJ and spoIIIE, and others, both known and to be discovered. Since these cell division genes are substantially conserved across many bacterial species, it is plausible that these engineered Bacillus strains will grow and sporulate with reasonable efficiency. The homologous gene may be taken from other bacilli or closely related organisms such as clostridia and Listeria. More preferably, the homologous gene may be taken from a pathogenic bacterium such as staphylococci and streptococci.
B. subtilis
molecular genetic methods make it straightforward to replace any gene with a homologous gene from another bacterium,
An artificially introduced reporter gene is one which is not naturally present in the strain in question, and which may have been introduced by genetic manipulation. 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 substrate to give a product that is easily observed e.g. because it is coloured or chemiluminescent of fluorescent. Reporter genes capable of being expressed in Bacillus species and other micro-organisms are well known and documented in the literature. Reporter genes are preferably chosen so that their products can be readily assayed simultaneously. lacZ has been used for more than 10 years with great success in
B. subtilis
and there is a range of useful substrates that generate coloured or fluorescent products upon hydrolysis by &bgr;-galactosidase. The uidA gene of
E. coli
has recently been harnessed for similar purposes, and the range of substrates available for the gene product, &bgr;-glucoronidase is similar to that for &bgr;-galactosidase.
In one example, two different fluorogenic substrates are used to assay the activities of the two reporters simultaneously in a single reaction.
On incubation of the micro-organism, e.g. on cell division or sporulation, a reporter gene is expressed in a manner related to the activity of an expression product e.g. a cell division protein, of the homologous gene. For example, decreased activity of that protein may be associated with either increased expression or reduced expression of the reporter gene. When two reporter genes are used, preferably expression of one is increased, and expression of the other is decreased, in association with a change in the level of activity of that protein.
The preferred assay method of the invention involves inducing the Bacillus strain described to sporulate in the presence of a putative anti-microbial agent. Preferably the Bacillus strain is contacted, just prior to asymmetric cell division with the agent. To screen agents on a large scale, samples of the Bacillus strain may be cultured in an exhaustion medium to stimulate sporulation; either in the wells of a microtitre plate to which the agent is added; or in bulk to be dispensed into the wells of a microtitre plate of which individual wells contain one or more different agents. After suitable incubation, observation is made of expression of the one or more reporter genes. For example, when the expression products of two reporter genes are different enzymes, substrates for the two enzymes may be added to the wells of the microtitre plate, and observation made of e.g. chemiluminescent or fluorescent or coloured products of enzymatic activity.
Use of such strains have several practical consequences:
i) It enables inhibitors which act on the protein product of a pathogen but not on that of a parent micro-organism e.g.
B. subtilis
to be identified.
ii) In the case of an assay for inhibitors of cell division, it may facilitate identification of the specific target of the inhibitor. By screening promising compounds against a series of strains in which cell division genes have been systematically replaced with homologues from other organisms, the specific target of the inhibitory compound becomes evident. Thus, for example, detection of a compound which inhibits the
B. subtilis
parent strain but not a derivative carrying the
S. aureus
homologue of ftsZ, would be strongly suggestive of a compound targeted on the FtsZ protein.
iii) A panel of strains with a given target gene systematically replaced by genes from other organisms also provides information about the spectrum of activity of each potential inhibitor. For example, some of the compounds found to inhibit the
B. subtilis
SpoIIIE protein might not act on the strain bearing its
S. aureus
homologue. Other compounds might show non-species specific inhibition and act on a range of gene products from different organisms. Such tests provide a useful means of ensuring that new inhibitors have a
Falk Anne-Marie
Fulbright & Jaworski LLP
ISIS Innovation Limited
Sullivan Daniel M.
LandOfFree
Bacillus strain and assay methods does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Bacillus strain and assay methods, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Bacillus strain and assay methods will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3225653