Patent Application: US-83249707-A

Abstract:
disclosed are an isolated nucleic acid molecule encoding a bacterial autoinducer inactivation protein , the encoded protein , and anti - bacterial uses therefor .

Description:
the present invention is based on the discovery that the seq id no : 2 protein has the effect of reducing or eliminating the activity of bacterial autoinducers ( ais ). consequently , the protein , and any nucleic acid that encodes the protein , may be used in a variety of situations where it is desired to reduce or eliminate the effect of such bacteria . in one preferred aspect , the present invention provides a nucleic acid molecule which is selected from the group consisting of : a ) a nucleic acid having the sequence of seq id no : 1 ; b ) a nucleic acid encoding the amino acid sequence of seq id no : 2 ; and c ) a nucleic acid that hybridizes to a ) or b ) above , wherein a positive hybridization signal is observed after washing with 1 × ssc and 0 . 1 % sds at 55 ° c . for one hour . the nucleic acid optionally further comprises a signal peptide coding region of any sequence . the nucleic acid sequence may be used to confer bacterial resistance in plants or animals . a nucleic acid that encodes a bacterial autoinducer inactivation protein can be introduced into a cell such that the inactivation protein is expressed by the plant or animal . the nucleic acid sequence may be used to confer resistance to diseases where the expression of pathogenic genes are regulated by autoinducers , such as the diseases caused by pseudomonas aeruginosa , erwinia stewartii , xenorhabdus nematophilus , erwinia chrysanthemi , pseudomonas solanacerum , and xanthomonas campestris ( passador , et al ., 1993 ; pirhonen , et al ., 1993 ; pearson , et al ., 1994 ; beck von bodman and farrand , 1995 ; barber , et al ., 1997 ; clough , et al ., 1997 ; costa and loper , 1997 ; dunphy , et al ., 1997 ; nasser , et al ., 1998 ). preferably , in the agricultural setting , the sequence may be used to confer soft rot disease resistance in susceptible plants , such as potato , eggplant , chinese cabbage , carrot and celery . the sequence may be introduced into plant or animal cells by well - known methods . methods for the transformation or transfection of eukaryotic cells with exogenous nucleic acid sequences include transfection , projectile bombardment , electroporation or infection by agrobacterium tumefaciens . these methods are likewise familiar to the person skilled in the area of molecular biology and biotechnology and need not be explained here in detail . as pathogenic bacteria cells are confined to the intercellular area of plant tissues , it is desirable to target the aiia protein into the intercellular spaces . such may be accomplished by fusing a secretion signal peptide to the aiia protein ( sato , et al ., 1995 ; firek , et al ., 1993 ; conrad and fiedler , 1998 ; borisjuk , et al ., 1999 ). alternatively , a plant membrane attachment motif can be incorporated into the peptide sequence of aiia for anchoring the aiia enzyme in the outer surface of plant cell membrane . the present invention provides a new strategy for engineering resistance to diseases . in particular , this strategy targets n - acyl homoserine lactone autoinducers that induce expression of pathogenic genes of many bacterial pathogens at a threshold concentration . this strategy is applicable to all plant , animal or mammal diseases where the expression of pathogenic genes of the bacterial pathogens is inducible by n - acyl homoserine lactone autoinducers . the present invention also contemplates usage of a bacterial autoinducer inactivation protein directly to treat or prevent bacterial damage . for example , the protein may be applied directly to plants in need of such treatment or prevention . in a preferred embodiment , the protein is applied in the form of a composition which comprises an effective amount of the protein and a suitable carrier . the composition may have a wide variety of forms , including solutions , powders , emulsions , dispersions , pastes , aerosols , etc . the bacterial autoinducer inactivation protein may also be used to treat bacterial infections in animals , including humans . in that application , an effective amount of the active ingredient is administered to an animal in need of such treatment . for therapeutic treatment , the active ingredient may be formulated into a pharmaceutical composition , which may include , in addition to an effective amount of the active ingredient , pharmaceutically acceptable carriers , diluents , buffers , preservatives , surface active agents , and the like . compositions may also include one or more other active ingredients if necessary or desirable . the pharmaceutical compositions of the present invention may be administered in a number of ways as will be apparent to one of ordinary skill in the art . administration may be done topically , orally , by inhalation , or parenterally , for example . topical formulations may include ointments , lotions , creams , gels , drops , suppositories , sprays , liquids and powders . oral formulations include powders , granules , suspensions or solution in water or non - aqueous media , capsules or tablets , for example . thickeners , flavorings , diluents , emulsifiers , dispersing aids or binders may be used as needed . parenteral formulations may include sterile aqueous solutions which may also contain buffers , diluents and other suitable additives . the dose regimen will depend on a number of factors which may readily be determined , such as severity and responsiveness of the condition to be treated . aspects of the invention will now be illustrated with reference to the following non - limiting examples . bacterial isolate 240b1 was isolated from soil suspension based on its function for inactivation of n - β - oxo - hexanoyl - l - homoserine lactone ( ohhl ) and n - β - oxooctanoyl - l - homoserine lactone ( oohl ) and n - β - oxodecanoyl - l - homoserine lactone ( odhl ) ( zhang , et al ., 1993 ). unless otherwise stated , ohhl was used for routine bioassay . erwinia carotovora strain scg1 was isolated from chinese cabbage leaf showing soft rot symptoms . it has been confirmed that strain scg1 produces ais and elicits soft rot disease in potato and chinese cabbage . escherichia coli strain dh5α was used as a host for dna cloning and subcloning . agrobacterium tumefaciens strain nt1 ( trar ; tra :: lacz749 ) was used as an indicator in bioassay for ai activity ( piper , et al ., 1993 ). e . coli strain was cultured in luria - bertani ( lb ) medium at 37 ° c . and other strains were cultured in lb ( miller , 1972 ) or yeb medium ( per liter contains : casein hydrolysate 10 g , yeast extract 5 g , nacl 10 g , sucrose 5 g , mgso 4 . 7h 2 o 0 . 5 g , agar 15 g , ph 7 . 2 ) at 28 ° c . the minimal salts medium with mannitol and ( nh 4 ) 2 so 4 as carbon and nitrogen sources was used for bioassay of ohhl ( petit and tempe , 1978 ). appropriate antibiotics were added as indicated at the following concentrations : ampicillin , 100 μg / ml ; tetracycline , 20 μg / ml and kanamycin , 50 μg / ml . the qualitative and quantitative bioassay methods for determination of ais activity has been described previously ( zhang , 1993 ). for determination of the ais production ability of wild - type and genetically modified erwinia strains , the same bioassay procedure was used except that no ohhl was added into the bacterial culture . genomic dna from 240b1 was digested partially with ecori . dna fragments were ligated to the dephosphorylated ecori site of cosmid vector plafr3 ( staskawicz , et al ., 1987 ). ligated dna was packaged with gigapack iii xl packaging extract ( stratagene ) and transfected into e . coli dh5 ∝. cosmid clones with ohhl inactivation activity were identified by using the bioassay method described above . subcloning into sequencing vector pgem - 7zf (+) was carried out by routine techniques ( sambrook , et al ., 1989 ). deletion analysis was carried out by using dnasei method as described by lin , et al . ( 1985 ). the sequencing was performed on both strands using the abi prism ™ drhodamine terminator cycle sequencing ready reaction kit ( pe applied biosystems ). nucleic acid sequence data and deduced amino acid sequences were analyzed with a dnastar ™ sequence analysis software package ( dnastar inc .) and database searches were performed using the blasta search algorithm ( altschul , et al ., 1990 ). the e7 - r3 plasmid , carrying the aiia gene in the cosmid vector plafr3 , was transferred into erwinia stain scg1 by triparental mating with the helper strain rk2013 ( ditta , et al ., 1980 ). transconjugants were selected on the plates containing minimal medium with tetracycline and confirmed by pcr with primers specific to the aiia gene . the virulence of wild - type erw . carolovora strain scg 1 and the aiia gene transformant scg1 ( e7 - r3 ) was evaluated by inoculation . four μl of early stationary phase bacterial suspension ( containing ˜ 2 × 10 9 cell / ml ) or diluted bacteria was added to the cut surfaces or wounding sites of plant tissues . the inoculated plant tissues were incubated in a petri dish at 28 ° c . overnight . the severity of soft rot was examined 48 hours after incubation . bacterial isolates from plant and soil samples were screened for enzymatic inactivation of ais . a bacterial isolate 240b1 , which showed a strong ability to eliminate ais activity , was selected for further study . the total protein extracts from isolate 240b1 eliminated ais activity completely during one - hour incubation ( fig1 ), and the capacity of the protein extract to inactivate ais was abolished by treatment with proteinase k for 1 hour or boiling for 5 min . these observations indicate enzymatic inactivation of ais by bacterial isolate 240b1 . the isolate was taxonomically characterized as bacillus sp ., because of the following characteristics : gram - positive , rod - shaped , catalase positive , facultatively anaerobic , and 16 rrna sequence homology to that of other bacillus bacteria ( data not shown ). the molecular mass of the enzyme for ais inactivation appears to be larger than 30 kda . its activity was lost after passing the protein extract through centricon 30 ( amicon ) but the activity was recovered in the re - suspended fraction that failed to pass the centricon 30 ( fig2 ). to identify the gene encoding ais inactivation , a cosmid library was constructed with the genomic dna of listera sp . strain 240b1 . twelve hundred clones were screened for ais inactivation activity . three clones showing ais inactivating function were identified . restriction analysis showed that the 3 clones shared one common band of 4 . 3 - kb generated by ecori digestion . the bioassay with the subclone e7 - 7 containing this 4 . 3 - kb ecori fragment confirmed that this fragment encodes ais inactivation function ( fig3 ). to identify the minimum size and the location of the ais inactivation gene ( aiia ), a serial of deletion clones was generated by deletion from both ends of this 4 . 3 - kb fragment with the dnasei method ( lin , et al ., 1985 ). the results indicated that the aiia gene is contained in a 1 . 2 kb fragment in clone f41 ( fig3 ). the 1 . 2 - kb dna insert in clone f41 was completely sequenced from both strands . the nucleotide sequence of aiia and the predicted amino acid sequence are shown in fig4 . the complete sequence of the dna insert contains 1 , 222 base pairs and there are 4 potential in - frame open reading frames ( orf ) starting from nucleotide position of 1 , 42 , 156 and 228 respectively ( fig4 ). deletion analysis indicated that only the longest orf encodes ais inactivation function , because the clone r34 , in which the 48 bp promoter region and nucleotides from 1 to 13 in the longest orf were deleted , lost ai inactivation function completely , although the remaining dna insert was placed under the control of a functional ptac promoter ( fig3 ). this is confirmed by fusing the longest orf to the glutathione s - transferase gene in the same orf and testing for ai inactivation activity of the purified fusion protein ( data not shown ). this orf contains 750 bp nucleotide and encodes a protein of 250 amino acids , with a predicted molecular mass of 28 , 036 daltons and an isoelectric point at 4 . 7 , because of 19 strongly basic and 39 strongly acidic amino acids residues . the putative initiation codon is preceded at a spacing of 7 bp by a potential ribosome - binding sequence ( aaggtgg ) which is complementary to the 3 ′ end of the e . coli 16s rrna . the best sequence match ( tattgt ) to the consensus − 10 promoter element ( tataat ) occurs 35 bp upstream of the initiation codon . a tctt box following a t - rich region resembling the potential factor - independent termination site is found downstream of the termination codon ( brendel , 1986 ). the total gc content of the aiia gene is 37 % and gc content in the third position of the codon is 27 . 2 %. database searches showed that the aiia gene has no significant similarity to known sequences in the major databases ( genbank , european molecular biology laboratory , protein information resource , and swiss - prot ) by fasta and blast analysis at either nucleotide or peptide sequence level , suggesting that aiia is a novel protein . consensus protein motif search using the genetics computer group ( madison , wis .) motif program showed that a short peptide sequence , “ ilvdtgmpesav ” ( seq id no : 3 ) from position 47 to 58 in aiia , is similar but not identical to the aspartyl protease active site signature pattern ( rawlings and barrett , 1995 ) ( fig5 ). expression of aiia gene in erwinia carotovora decreases ais releasing and attenuates virulence the cosmid clone e7 - r3 was transferred into erwinia carotovora strain scg1 by triparental mating . the plafr3 vector has been safely maintained in erwinia carotovora without selection pressure . the bioassay showed that the ais released by erwinia carotovora ( e7 - r3 ) was significantly reduced ( fig6 , lane 6 ), while the presence of the cosmid vector plafr3 alone in erwinia carotovora did not affect ais production ( fig6 , lanes 7 ). data suggest that the most of ais produced by erwinia carotovora strain scg1 was inactivated by aiia gene product . the erwinia carotovora scg1 ( e7 - r3 ) that expresses aiia protein failed to or caused only minor soft rot disease symptom in potato , eggplant , chinese cabbage , carrot and celery , while its parental strain caused severe symptoms ( fig7 a , b , c , d , e ). to prevent experimental errors due to genetic variations , four colonies from erwinia carotovora strain scg1 and its aiia gene transformants respectively , were randomly selected for testing ais production and virulence on potato . similar results were obtained in both experiments . the erwinia carotovora strain scgi ( plafr3 ) that contains the cosmid vector only caused the same level of disease severity as its parental strain erwinia carotovora strain scgi ( fig7 f ). bacterial isolate 240b i , which was identified as bacillus sp ., produces an enzyme that can effectively inactivate the three ais tested , i . e ., n - β - oxo - hexanoyl - l - homoserine lactone , n - β - oxo - octanoyl - l - homoserine lactone and n - β - oxo - decanoyl - l - homoserine lactone . the gene ( aiia ) encoding the ai inactivation enzyme has been cloned and fully sequenced . expression of the aiia gene in transformed e . coli and pathogenic bacteria erwinia carolovora confers ability for ai inactivation and significantly reduces the ais release from erwinia carolovora . to our knowledge , it is the first protein identified capable of enzymatic inactivation of n - acyl - homoserine lactones , the autoinducers for global gene regulation in a diverse of bacteria species . the aiia is a novel protein . there is no significant homology to known proteins in major databases . it shares similarities to the consensus pattern of the aspartyl proteases active site ( rawlings and barret , 1995 ). aspartyl proteases , also known as acid proteases , are widely distributed in vertebrates , fungi , plants , retroviruses and some plant viruses . the aspartyl proteases from most retroviruses and some plant viruses are homodimers . the molecular mass of aiia protein is about 28 kda but it failed to pass a molecular sieve with a cut off size of 30 kda , indicating a possibility that aiia protein exists as a homodimer or homomultimer under the natural conditions . however , there is also a possibility that aiia monomer has an irregular three - dimensional structure , which hinders it passing through the molecular sieve . aspartyl proteases are endopeptidases and hydrolyses amide linkages of proteins . crystallographic study has shown that the enzyme of the aspartyl protease family are bilobed molecules with the active - site cleft located between the lobes , and each lobe contributing one of the pair of aspartic acid residues that is responsible for the catalytic activity ( sielecki et al ., 1991 ). erwinia carotovora is a plant pathogen that produces and secretes exoenzymes that act as virulence determinants for soft rot diseases of various plants including potato , cabbages , tomato , chili , carrot , celery , onion , and lettuce ( kotoujansky , 1987 ). mutants that were defective in the producing n - 3 -( oxohexanoyl )- l - homoserine lactone were also defective in systhesis of the pectinase , cellulase and protease exoemzymes . these mutants failed to induce soft rot disease in potato tubers ( jones , et al ., 1993 ). it was found that the expi gene , which is homologous to luxi gene of vibrio fischeri , encodes autoinducer production in erwinia carotovora . the expl mutant was avirulent when it was inoculated to tobacco leaf but the virulence was restored by external antoinducer addition ( pirhonen , et al ., 1993 ). obviously , autoinducers are a potential target for genetic engineering of plant soft rot disease resistance . as an interim test and a concept proving approach , the cosmid clone containing the aiia gene was introduced to erwinia carotovora strain scg1 . expression of the aiia enzyme in erwinia carotovora significantly reduced the release of autoinducers , and the genetically modified erwinia carotovora that expressed aiia failed to induce any or induce only minor soft rot disease symptom on all plants tested , including potato , eggplant , chinese cabbage , carrot and celery . our results further support the important role of autoinducers in the regulation of expression of virulence genes in erwinia carotovora , and the potential of the aiia gene to confer resistance to soft rot disease and other diseases in which the autoinducers are involved in regulation of pathogenic gene expression . the present invention provides a new strategy for engineering resistance to diseases . in particular , this strategy targets n - acyl homoserine lactone autoinducers that induce expression of pathogenic genes of many bacterial pathogens at a threshold concentration . by using the above - mentioned conception - proving approach , the present invention demonstrates that reduction or elimination of autoinducers produced by pathogenic bacteria by an autoinducer inactivation enzyme significantly attenuates pathogenicity of otherwise virulent bacterial pathogen . because the expression of pathogenic genes in pathogenic bacteria requires a threshold concentration , this ai - inactivation strategy is applicable to all plant , animal or mammal diseases where the expression of pathogenic genes of the bacterial pathogens is inducible by n - acyl homoserine lactone autoinducers . the aiia gene could also be a useful tool for investigation of the role of ais in those bacteria where the biological functions regulated by ais has not been established . in recent years , many more bacteria species have been shown to produce ais ( bassler , et al ., 1997 ; dumenyo , et al ., 1998 ; cha , et al ., 1998 ; surette , et al ., 1999 ). some of them are important plant pathogens such as psendomonas and xanthomonas species . the gene knock out approach based on sequence homology could be difficult . the overall levels of sequence similarity of ais synthase and the related regulatory protein from different genera are rather low , often no higher than 28 - 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