Patent Application: US-200913132818-A

Abstract:
the invention provides a method of identifying an inhibitor of ltas comprising : providing bacteria which comprise a mutation in the mbl gene or homologue thereof ; culturing the bacteria of in the presence of a test substance under conditions of low magnesium ; monitoring the growth of the bacteria ; wherein growth or more rapid growth of the bacteria compared to growth in the absence of the test substance is indicative that the test substance is an inhibitor of ltas .

Description:
the present invention provides a method for the identification of an inhibitor of ltas . ltas is a lipoteichoic acid synthase . ltas from staphylococcus aureus has been identified in the prior art , and is described for example in gründling and schneewind ( supra ). homologues of this gene are also known in other bacterial strains . for example , bacillus subtilis carries a homolog previously identified as yfle . the sequence for this gene is set out in seq id no : 1 and 2 . in accordance with the present invention , a bacterial strain of gram positive bacteria , preferably bacillus , preferably b . subtilis is provided . the bacterial strain is selected or modified to include a functional mutation in the mbl gene of b . subtilis or a homolog thereof of other gram positive bacteria . mbl is an actin homolog and has been described previously , for example in abhayawardhane and stewart , 1995 , j . of bacteriol . 177 : 765 - 773 and jones et al ., cell 104 , 2001 , 913 - 922 . the nucleotide and amino acid sequences for mbl are set out in table 4 , and labelled as seq id no 3 and 4 respectively . typically , a homologue of mbl from another bacteria is one having more than about 50 %, 55 % or 65 % identity , preferably at least 70 %, at least 80 %, at least 90 % and particularly preferably at least 95 %, at least 97 % or at least 99 % identity , with the amino acid sequence of seq id no : 4 . such variants may include allelic variants . the identity of variants of seq id no : 4 may be measured over a region of at least 200 , at least 250 , at least 300 , at least 330 or more contiguous amino acids of the sequence shown in seq id no : 4 or more preferably over the full length of seq id no : 4 . amino acid identity may be calculated using any suitable algorithm . for example the uwgcg package provides the bestfit program which can be used to calculate homology ( for example used on its default settings ) ( devereux et al . ( 1984 ) nucleic acids research 12 , 387 - 395 ). the pileup and blast algorithms can be used to calculate homology or line up sequences ( such as identifying equivalent or corresponding sequences ( typically on their default settings ), for example as described in altschul s . f . ( 1993 ) j mol evol 36 : 290 - 300 ; altschul , s . f . et al . ( 1990 ) j mol biol 215 : 403 - 10 . software for performing blast analyses is publicly available through the national center for biotechnology information ( http :// www . ncbi . nlm . nih . gov /). this algorithm involves first identifying high scoring sequence pair ( hsps ) by identifying short words of length w in the query sequence that either match or satisfy some positive - valued threshold score t when aligned with a word of the same length in a database sequence . t is referred to as the neighbourhood word score threshold ( altschul et al ., supra ). these initial neighbourhood word hits act as seeds for initiating searches to find hsps containing them . the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased . extensions for the word hits in each direction are halted when : the cumulative alignment score falls off by the quantity x from its maximum achieved value ; the cumulative score goes to zero or below , due to the accumulation of one or more negative - scoring residue alignments ; or the end of either sequence is reached . the blast algorithm parameters w , t and x determine the sensitivity and speed of the alignment . the blast program uses as defaults a word length ( w ) of 11 , the blosum62 scoring matrix ( see henikoff and henikoff ( 1992 ) proc . natl . acad . sci . usa 89 : 10915 - 10919 ) alignments ( b ) of 50 , expectation ( e ) of 10 , m = 5 , n = 4 , and a comparison of both strands . the blast algorithm performs a statistical analysis of the similarity between two sequences ; see e . g ., karlin and altschul ( 1993 ) proc . natl . acad . sci . usa 90 : 5873 - 5787 . one measure of similarity provided by the blast algorithm is the smallest sum probability ( p ( n )), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance . for example , a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1 , preferably less than about 0 . 1 , more preferably less than about 0 . 01 , and most preferably less than about 0 . 001 . the functional mutation can be any mutation that disrupts the function of the mbl gene . suitable mutations include mutations which disrupt the open reading frame such that a functional mbl protein cannot be expressed . alternatively , the mutation may comprise an insertion , for example by transposon mutagenesis to disrupt expression of the gene . in one embodiment , part or all of the mbl gene is deleted . typically , where the gene is deleted , at least 50 % of the mbl gene is deleted , for example , at least 60 %, 70 %, 80 %, 90 % or 95 %. smaller deletions can be included , for example , single base deletions to disrupt the open reading frame or smaller deletions , for example at the n - terminus encoding region such that a functional protein is no longer be expressed . other mutations that can be incorporated are those mutations causing amino acid substitutions at critical sites in the protein , such as those required for binding of atp . any mutation in or around the mbl gene that generates a phenotype in which the cells become more dependent on high concentrations of mg 2 + in the growth medium can be used . mbl mutants are dependent upon mg 2 + for growth . thus the mbl mutants useful in accordance with the present invention are unviable or grow poorly under low mg 2 + conditions . supplementation of the culture medium with mg 2 + restores cell growth to such bacterial mutants . the functional mutations in the mbl gene can disrupt the function of the gene such that a functional protein is no longer expressed . thus , such mutations may affect chromosome segregation or positioning of the cell wall synthetic machinery . identification of suitable mutants for use in accordance with the present invention can be carried out through a simple analysis of the ability of such mutants to grow under low or normal mg 2 + . as explained above , mbl mutants are dependent on mg 2 + for growth . the assay methods in accordance with the present invention use high levels of mg 2 + , and thus , a suitable mutant for use in accordance with the present invention is one in which a mutation in the mbl gene leads to a bacteria which is unviable , or which grows poorly under low mg 2 + conditions , for example , in which the doubling time of such a mutant under magnesium concentrations of less than 5 mm is typically greater than 12 hours or greater than 24 hours . in accordance with the assay methods of the present application , the mbl mutant strains are cultured under conditions of low mg 2 + in the presence of a test substance . a test substance which acts as an inhibitor of ltas restores viability of the bacterial strains under such low mg 2 + conditions . prior to carrying out the assay methods of the present invention , in the presence of a test substance , the mbl mutant strains can be grown under conditions of high or supplemented mg 2 + , such that the bacteria can grow under these conditions . typically , for bacterial growth of mbl mutants , mg 2 + is present in the range 1 to 100 mm , preferably 3 mm to 50 mm , preferably 5 mm to 30 mm . for example , growth medium can be supplemented with about 20 mm mg 2 + . for the purpose of an assay , and completion of the test in a convenient period of time , any medium that supports reasonable growth rate of the mbl mutant ( e . g . doubling time greater than 120 min at 37 ° c .) can be used . typically , a bacterial culture of an mbl mutant grown under high mg 2 + conditions can be diluted and placed into a sample well . alternatively , such bacteria can be plated on suitable plates with appropriate growth medium such as agar plates , under low mg 2 + conditions . references to low mg 2 + conditions relate to magnesium concentrations of less than 3 mm , typically less than 1 mm . typically , bacteria can be cultured in culture medium which has not been supplemented with mg 2 + . thus once the mbl mutant bacteria have been diluted or plated out in low mg 2 + conditions , their growth will slow or stop . low mg 2 + conditions can also be identified and defined with respect to bacterial cultures supplemented with 20 mm mg 2 + . for example , a mg 2 + concentration which leads to a growth rate of less than 50 %, typically less than 20 % or less than 10 % of the growth rate of mbl mutants grown in 20 mm medium can be used to identify suitable low mg 2 + conditions to conduct the assays in accordance with the present invention . in order to carry out the assays of the present invention , test substances are added to the mbl mutant bacteria growing under low mg 2 + conditions . for example , test substances can be added to the sample wells or spotted on to plates . in accordance with the assays of the present invention , bacterial growth of the mbl mutants is monitored in the presence of the test substance . typically , bacteria are grown under usual temperature and time conditions , for example , between 30 and 45 ° c ., typically 37 ° c . levels of bacterial growth can be measured at a defined time point , for example , after 2 hours , 4 hours , 6 hours , 8 hours , 12 hours or 24 hours . alternatively , bacterial growth can be monitored at regular intervals for example every 15 minutes , 30 minutes , hourly , every 2 hours or every 4 hours . alternatively , bacterial growth can be monitored continuously . bacterial growth can be measured by any suitable method . typically , optical density or a visual assessment of the growth of the bacteria can be carried out . other suitable methods include use of a dye that generates a colour change during growth ( e . g . due to ph change ), centrifugation followed by measurement of wet mass , drying followed by measurement of dry mass , chemical determination of a macromolecular component of cells , such as dna or protein , or counting of cell number microscopically or by an electronic device such as a coulter counter or flow cytometer , viable cell count by dilution and plating on a suitable growth medium , supplemented with mg 2 + . measurement of bacterial growth identifies those mutants whose growth has been rescued despite the low mg 2 + conditions . the ability of a test substance to rescue such growth identifies the test substance as an inhibitor of ltas . once an agent has been identified as an inhibitor of ltas , further studies can be carried out , for example , to assess whether such agent is specific for ltas . typically , such test substances can be formulated as pharmaceutical compositions for subsequent administration as antibiotics . agents identified in accordance with the present invention can be used as antibiotics against gram positive bacteria , and in particular those which comprise ltas or a homologue thereof . in a preferred aspect , such agents are useful in the treatment of staphylococcus aureus infection . such agents can be used alone or in combination with other antibiotics . it will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed , the age , body weight , general health , sex , diet , time of administration , route of administration , rate of excretion , drug combination and the severity of the particular disease undergoing treatment . optimum dose levels and frequency of dosing will be determined by clinical trial , but an exemplary dosage would be 0 . 1 - 1000 mg per day . the compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties . the orally administrable compositions may be in the form of tablets , capsules , powders , granules , lozenges , liquid or gel preparations , such as oral , topical , or sterile parenteral solutions or suspensions . tablets and capsules for oral administration may be in unit dose presentation form , and may contain conventional excipients such as binding agents , for example syrup , acacia , gelatin , sorbitol , tragacanth , or polyvinyl - pyrrolidone ; fillers , for example lactose , sugar , maize - starch , calcium phosphate , sorbitol or glycine ; tabletting lubricant , for example magnesium stearate , talc , polyethylene glycol or silica ; disintegrants , for example potato starch , or acceptable wetting agents such as sodium lauryl sulphate . the tablets may be coated according to methods well known in normal pharmaceutical practice . oral liquid preparations may be in the form of , for example , aqueous or oily suspensions , solutions , emulsions , syrups or elixirs , or may be presented as a dry product for reconstitution with water or other suitable vehicle before use . such liquid preparations may contain conventional additives such as suspending agents , for example sorbitol , syrup , methyl cellulose , glucose syrup , gelatin hydrogenated edible fats ; emulsifying agents , for example lecithin , sorbitan monooleate , or acacia ; non - aqueous vehicles ( which may include edible oils ), for example almond oil , fractionated coconut oil , oily esters such as glycerine , propylene glycol , or ethyl alcohol ; preservatives , for example methyl or propyl p - hydroxybenzoate or sorbic acid , and if desired conventional flavouring or colouring agents . the active ingredient may also be administered parenterally in a sterile medium . depending on the vehicle and concentration used , the drug can either be suspended or dissolved in the vehicle . the invention is hereinafter described in more detail with reference to the following examples . lethal effects of mbl deletion can be rescued by high concentrations of magnesium the actin homologue mbl has been described as non - essential in b . subtilis ( abhayawardhane and stewart , 1995 ; jones et al ., 2001 ), but the former authors had already indicated that mbl mutants are slow growing and tend to pick up mutations that enhance growth . the reported mg 2 + dependency of both mreb ( formstone and errington , 2005 ) and ( though only at low leves ) mrebh mutants ( carballido - lópez et al ., 2006 ) led us to re - construct the mbl deletion strain in the presence of 20 mm mg 2 + . selecting for transformants under these conditions resulted in a 10 - fold increase in plating efficiency giving relatively small but uniformly shaped colonies ( fig1 a ). colonies that were picked continued to grow on nutrient agar plates supplemented with mg 2 + , but failed to grow on unsupplemented plates ( fig3 a ). in liquid culture ( pab medium ) an elevated magnesium concentration again greatly improved the growth rate ( fig1 b ). microscopic examination of mutant and wild type cells revealed the characteristic twisted and bloated morphology of the mutant in the unsupplemented medium ( fig1 c ). however , in the presence of mg 2 + , cell morphology was greatly improved ( fig1 d ). nevertheless , under high mg 2 + conditions the mbl mutant cells still differed from the wild - type in two ways : first , they were slightly bent and irregularly shaped ; second , their average diameter was about 12 % greater ( table 1 ). wild - type cells had their typical straight rod morphology under both conditions ( not shown ). screen for magnesium independent suppressor mutants of b . subtilis δmbl to gain insight into the function of mbl we screened for mutants in which the mg 2 + dependency of the mutant was suppressed . the plasmid pmarb carrying the mariner transposon ( le breton et al ., 2006 ) was introduced into a freshly constructed δmbl strain background in the presence of 20 mm mg 2 + . a library of approximately 60 , 000 mutants was plated with selection for mg 2 + independent growth . loss of the plasmid pmarb ( erm r ), presence of the transposon ( kan r ) and disruption of mbl ( spc r ) were verified by patching on appropriate antibiotic supplemented plates . ten strong suppressor strains were chosen and checked for linkage of the transposon insertion to the suppression phenotype by three consecutive back - crosses into the δmbl mutant background . the sites of transposon insertion were determined by sequencing the products of inverse pcr reactions using primers ipcr1 - 3 ( le breton et al ., 2006 ). in two of the ten suppressor strains , the transposon was found to have independently inserted into the rsgi gene ( previously ykri ). rsgi functions as an anti - sigma factor for sigi ( asai et al ., 2007 ). another hit in the screen was in yfle ( three independent insertions ), encoding a homologue of the lipoteichoic acid synthase ltas from s . aureus ( gründling and schneewind , 2007 ). two independent insertions were found in ylxa ( synonyms yllc or mraw ) which lies in an operon with yllb , ftsl , and pbpb and encodes a protein of unknown function . however , ylxa deletion proved to be not very potent in suppressing the mg 2 + dependency of b . subtilis δmbl ( not shown ). one transposon insertion each was found in yaat encoding a protein of unknown function involved in the phosphorelay cascade during initiation of sporulation ( hosoya et al ., 2002 ), in the gene for the glutamate transporter gltt ( slotboom et al ., 2001 ; tolner et al ., 1992 ), and in pnpa which codes for polynucleotide phosphorylase ( luttinger et al ., 1996 ; mitra et al ., 1996 ; wang and bechhofer , 1996 ). overlapping but distinct function of the actin homologues in b . subtilis the finding that mutants of b . subtilis actin homologues mreb and mrebh are sensitive to a low mg 2 + concentration ( carballido - lópez et al ., 2006 ; formstone and errington , 2005 ) led us to re - construct the mbl mutant in the presence of high concentrations of mg 2 + . the increase in plating efficiency , uniformity of colony shape , and amelioration of the cell morphology recapitulated the earlier findings made for the mreb and mrebh mutants . however , the mutants vary in optimal levels of mg 2 + : the mrebh mutant requires only about 100 - 200 μm mg 2 + for viability and the cells display a reduced cell width ( carballido - lópez et al ., 2006 ); the mreb mutant has a higher requirement for mg 2 + ( 2 . 5 mm ), and depletion of the cation results in an increase in cell diameter and ultimately lysis ( formstone and errington , 2005 ); finally , the newly constructed mbl mutant requires addition of about 3 mm mg 2 + which is in a similar range of the previously described mreb mutant . in unsupplemented medium the strain grows slowly , the cells tend to twist , form chains , swell over their length and are prone to lysis . in an otherwise wild - type background , the only viable double mutant was δmbl δmrebh , which has a phenotype similar to that of an mbl single mutant . combinations with δmreb were lethal , and depletion of mreb in either mbl or mrebh mutant backgrounds led to a loss of rod - shape and cell death ( defeu soufo and graumann , 2006 ; a . formstone and j . errington , unpublished ) irrespective of mg 2 + levels . thus , the three mreb - like proteins appear to have overlapping functions , because mreb is essential in strains deleted for any of the other two homologues . however , although the three mutants share certain characteristics like the mg 2 + dependency and effects on cell shape , the phenotypic differences between the single mutants show that each has a partially differentiated function . b . subtilis strains and plasmids used in this study are listed in table 2 , oligonucleotides in table 3 . liquid cultures of b . subtilis strains were grown in difco antibiotic medium 3 ( pab ) at 37 or 50 ° c . as indicated . nutrient agar ( oxoid ) plates were used for growth on solid medium . minimal concentrations of mg 2 + required for growth were determined on nutrient agar or modified salts medium ( carballido - lópez et al ., 2006 ). to all media mgso 4 was added to the indicated final concentration of mg 2 + . dna manipulations and e . coli dh5α transformations were carried out using standard methods ( sambrook , 1989 ). b . subtilis strains were transformed according to the method of anagnostopoulos and spizizen ( 1961 ) as modified by jenkinson ( 1983 ). selection for b . subtilis transformants was carried out on nutrient agar ( oxoid ), supplemented with antibiotics , as required , with : kanamycin ( 5 mg ml − 1 ) chloramphenicol ( 5 mg ml − 1 ), erythromycin ( 1 mg ml − 1 ), lincomycin ( 25 mg ml − 1 ) and / or spectinomycin ( 50 mg ml − 1 ). iptg ( 1 mm ) was added as indicated . random transposon mutagenesis was performed using the mariner based transposon tnylb - 1 as described before ( le breton et al ., 2006 ). in short , the plasmid pmarb was introduced into an mbl mutant strain ( 2505 ) at 30 ° c . in the presence of high mg 2 + concentrations . individual colonies were picked , grown in lb medium at 37 ° c . for 8 h , and then plated on nutrient agar plates not supplemented with mg 2 + but containing kanamycin to select for the transposon insertions creating mg 2 + independent strains . individual colonies were picked and deletion of mbl ( spe r ), integration of the transposon tnylb - 1 ( neo r ) and loss of the plasmid ( erm s ) were checked by patching on plates containing the appropriate antibiotic . linkage between transposon insertion and mg 2 + independency was verified by back - crossing chromosomal dna of single colonies three times into an mbl mutant background . ten strong suppressors were chosen and the site of transposon insertion was determined by inverse pcr amplification and sequencing as described previously ( le breton et al ., 2006 ). chromosomal regions of about 2 . 5 - 3 kb flanking the gene ( s ) to be deleted were pcr amplified using primers mbl - a / mbl - b and mbl - c / mbl - d for the mbl deletion . these pcr products were then ligated to an antibiotic resistance cassette ( cat from pcotc ; veening et al ., 2006 ) and then reamplified using the outside primers b + d . transformation of the resulting pcr products into b . subtilis 168 with selection for the adequate antibiotic then gave rise to strains where the target gene is substituted by an antibiotic resistance cassette . deletion of the gene and insertion of the resistance cassette was verified by pcr . for microscopy , cells from an overnight liquid or solid culture were diluted into pab medium supplemented with 20 mm mgso 4 when required and grown at 37 ° or 50 ° c . cells were mounted on microscope slides covered with a thin film of 1 % agarose in minimal medium ( glaser et al ., 1997 ). staining of the membrane was achieved by mixing 2 μl of nile red ( molecular probe ) solution ( 12 . 5 mg ml − 1 ) with 600 μl agarose on the slide . nucleoids were stained by mixing 8 μl of the cell suspension with 2 μl of dapi ( sigma ) solution ( 1 mg ml − 1 in 50 % glycerol ) in an eppendorf cup before mounting the sample on the agarose covered slide . images were aquired with a 14 sony coolsnap hq cooled ccd camera ( roper scientific ) camera attached to a zeiss axiovert m135 microscope or to a zeiss 15 axiovert 200m microscope . imagej ( http :// rsb . info . nih . gov / ij /) was used to analyse the images , manipulation was limited to altering brightness and contrast to obtain optimal prints . we have shown above that mbl mutants are not viable at low [ mg 2 + ] and that mutations suppressing this phenotype can be readily obtained . in a collection of transposon induced suppressed mutants were three strains with insertions in the yfle gene . the wild type gene encodes a protein of 649 amino acids with a predicted molecular weight of 74 . 2 kda . dna sequencing showed that each insertion would disrupt the yfle open reading frame , after codons 41 , 72 and 387 , respectively . while the work was in progress , ( gründling and schneewind , 2007 ) showed that a closely related gene ( 79 % identical ) in staphylococcus aureus encodes lta synthase . they also showed that the yfle gene of b . subtilis could complement the lethal phenotype of ltas in s . aureus by restoring lta synthesis . therefore , hereafter we rename the b . subtilis yfle gene as ltas . we constructed a complete deletion of the ltas gene ( strains 4283 ) and confirmed that the ltas mbl double mutant ( strain 4298 ) is not mg 2 + dependent ( fig2 ). both on plates and in liquid medium ( pab or lb ) the double mutant grew without added mg 2 + ( fig2 a and b ), although growth was slower than for the wild type culture . interestingly , deletion of ltas also counteracted the typical swelling and twisting of mbl mutant cells ; instead the double mutant appeared similar to the ltas single mutant ( fig2 c ) ( see below ). the ltas mutant also exhibited impaired growth depending on the growth medium . to understand the consequences of loss of lta synthase activity we characterised the growth of the mutant under a range of conditions . the mutant had a slow growth rate in rich media such as pab ( see below ) and it failed to grow at all in ch or s media . systematic analysis of the effects of components of these media added to pab showed that the mutant strain was particularly sensitive to elevated mn 2 + levels . in the examples shown in fig3 a , addition of 0 . 05 mm mnso 4 to nutrient agar ( na ) abolished growth of the mutant , whereas growth of the wild - type was unaffected . addition of 0 . 5 mm mg 2 + had no effect on growth of the mutant , showing that the effect was not a general sensitivity to divalent cations . on the other hand we noticed that on minimal media plates with defined mg 2 + concentrations the ltas mutant grew at lower mg 2 + concentrations than the wild - type strain ( fig3 b ). the lowered requirement for mg 2 + might be the reason why a deletion of ltas suppresses the mg 2 + dependent phenotype of mbl and mreb ( formstone and errington , 2005 ) mutants . we propose that , consistent with previously suggested functions for lta in scavenging of mg 2 + ions ( neuhaus and baddiley , 2003 ), the absence of lta ( synthesized by ltas ) leads to a loss of a buffering zone around the bacterial envelope . as a consequence ions have more immediate access to the cell , leading to a lower requirement for ions with high affinity such as mg 2 + , which is a co - factor in many bacterial enzymes . at the same time , the toxicity of mn 2 + ions increases : these can replace mg 2 + because of their similar chemical properties but they do not participate correctly in enzyme function ( cowan , 1995 ). these results provide direct evidence that lta has a major role in cell wall physiology and in particular in providing a physicochemical environment that favours the retention of mg 2 + over mn 2 + . in the process of constructing the deletion strain , we noticed that the ltas mutant was also hyper - sensitive to various antibiotics and lysozyme . as an example , growth of the ltas ( strain 4285 ) mutant was abolished at 0 . 5 μg / ml kanamycin , a concentration that had no discernible effect on the growth of wild - type cells . in other experiments on solid medium the zone of inhibition of all antibiotics tested ( kanamycin , ampicillin , vancomycin , penicillin , spectinomycin , erythromycin , lincomycin , carbenicillin ) was wider for the ltas mutant than for the wild - type ( not shown ). finally , the mutant also showed increased susceptibility to lysozyme ( not shown ). the general increase in sensitivity of the mutant to antibiotics and lysozyme is consistent with the notion that lta also provides a protective layer that restricts the access of many bioactive agents to sensitive sites in the cell envelope . b . subtilis strains and plasmids used in this study are listed in table 2 ( supra ). liquid cultures of b . subtilis strains were grown in difco antibiotic medium 3 ( pab ), ch medium ( nicholson & amp ; setlow , 1990 ), or s - medium ( karamata & amp ; gross , 1970 ) at 37 ° c . nutrient agar ( oxoid ) plates were used for growth on solid medium , modified salts medium plates with defined mg 2 + concentrations were prepared as described previously ( carballido - lópez et al ., 2006 ). the given concentration of mg 2 + was achieved by addition of mgso 4 to the medium . dna manipulations and b . subtilis strains were transformed according to the method of anagnostopoulos and spizizen ( 1961 ) as modified by jenkinson ( 1983 ). selection for b . subtilis transformants was carried out on nutrient agar ( oxoid ), supplemented with antibiotics , as required , with : kanamycin ( 5 mg ml − 1 ) chloramphenicol ( 5 mg ml − 1 ), erythromycin ( 1 mg ml − 1 ), lincomycin ( 25 mg ml − 1 ) and / or spectinomycin ( 50 mg ml − 1 ). to test the sensitivity to cations , cultures were grown to mid - exponential growth phase in pab medium , then resuspended in pbs to an od 600 of 1 . 0 . 10 μl of dilutions 10 − 1 to 10 − 6 in pbs were spotted on na plates containing mnso 4 or mgso 4 in the concentrations as indicated . random transposon mutagenesis was performed using the mariner based transposon tnylb - 1 as described before ( le breton et al ., 2006 ). in short , the plasmid pmarb was introduced into an mbl mutant strain ( 2505 ) at 30 ° c . in the presence of high mg 2 + concentrations . individual colonies were picked , grown in lb medium at 37 ° c . for 8 h , and then plated on nutrient agar plates not supplemented with mg 2 + but containing kanamycin to select for the transposon insertions creating mg 2 + independent strains . individual colonies were picked and deletion of mbl ( spe r ), integration of the transposon tnylb - 1 ( neo r ) and loss of the plasmid ( erm s ) were checked by patching on plates containing the appropriate antibiotic . linkage between transposon insertion and mg 2 + independency was verified by back - crossing chromosomal dna of single colonies three times into an mbl mutant background . ten strong suppressors were chosen and the site of transposon insertion was determined by inverse pcr amplification and sequencing as described previously ( le breton et al ., 2006 ). genes were deleted by replacing the coding sequence with antibiotic resistance markers . therefore , approx . 2500 bp up - and downstream of the target genes were amplified using primers yfle - a / yfle - b and yfle - c / yfle - d for the yfle deletion , ligated to the desired resistance cassette and then b . subtilis 168 was transformed with the ligation product , transformants were selected on the appropriate antibiotic and verified by pcr . resistance cassettes were derived by either restriction or pcr amplification from plasmids [ cat from pcotc ( veening et al ., 2006 ); erm from pmutin4 ( vagner et al ., 1998 ); neo from pbest501 ( itaya et al ., 1989 ); spc from ploss * ( claessen et al ., 2008 )]. 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