Patent Publication Number: US-2004055040-A1

Title: Genetically modified plants and plant cells comprising heterologous heavy metal transport and complexation proteins

Description:
FIELD OF THE INVENTION  
       [0001] The present invention is in the field of genetically modified plants and plants cells having improved heavy metal tolerance and accumulation due to increased plant growth and biomass production based upon the expression of exo-cytoplasmic heavy metal resistance system (efflux and complexation).  
       [0002] More particularly, the present invention is related to genetically modified plants and plant cells, comprising nucleotide sequences encoding heterologous heavy metal transport proteins and exocytoplasmic metal binding proteins of various origins.  
       BACKGROUND OF THE INVENTION AND STATE OF THE ART  
       [0003] Heterologous nucleic acid sequences, coding for heavy metal resistance, were functionally expressed in plants, to improve their tolerance against these toxic elements. The heterologous heavy metal resistance genes, in casu represent either heavy metal efflux systems or functions involved in heavy metal sequestration.  
       [0004] Until present, only cytoplasmic functions that provide increased heavy metal resistance were expressed in plants.  
       [0005] 1. Expression of Heterologous Metallothionein and Phytochelatines in Plants  
       [0006] Metallothioneins and phytochelatines, which are rich in cystein sulfhydryl residues that bind and sequester heavy metal ions in very stable complexes (Karin, 1985), are found in eukaryotic organisms, but recently also in Synechococcus. Various MT genes—mouse MTI, human MTIA (alpha domain), human MTII, Chinese hamster MTII, yeast CUP1, pea PsMTA—have been transferred to tobacco, cauliflower or  Arabidopsis thaliana  (Lefebre et al., 1987; Maiti et al., 1988, 1989, 1991; Misra and Gedamu, 1989; Evans et al., 1992; Yeargan et al., 1992; Brandle et al., 1993; Pan et al., 1993; Elmayan and Tepfer, 1994; Hattori et al., 1994; Pan et al., 1994a, b; Hasegawa et al., 1997). As a result, varying degrees of enhanced Cd tolerance have been achieved, being maximally 20-fold compared with the control. Metal uptake levels were not dramatically changed; in some cases there were no differences, in others maximally 70% less or 60% more Cd was taken up by the shoots or leaves. Only one study has been reported on a transgenic plant generated with MT of plant origin. When pea ( Pisum sativum ) MT-like gene PsMTA was expressed in  Arabidopsis thaliana , more Cu (several-fold in some plants) accumulated in transformed than in control plants (Evans et al., 1992).  
       [0007] 2. Heterologous Expression of Heavy Metal Reduction  
       [0008] The only example known is the mer operon of Tn21 of  Shigella flexneri,  whose expression in plants results in the reduction mercury (Hg 2+ ) in its metallic form (Hg 0 ). This metallic mercury is volatilized out of the cell (Rugh et al. 1996).  
       AIMS OF THE INVENTION  
       [0009] The present invention aims to provide a new way in obtaining plants and plant cells with improved heavy metal tolerance characteristics, and possibly heavy metal accumulation.  
       [0010] Another aim of the present invention is to provide such plants and plant cells which allow increased heavy metal resistance for revegetation and phytostabilisation of heavy metal contaminated sites.  
       [0011] A further aim of the present invention is to provide plants and plant cells, characterised by increased heavy metal accumulation combined with increased heavy metal tolerance which allow phytoextraction of heavy metals (inclusive rhizofiltration).  
       [0012] A last aim of the present invention is to provide a method which results in the possibility to improve important agriculture crop species with high biomass production in their heavy metal tolerance and accumulation.  
       SUMMARY OF THE INVENTION  
       [0013] The present invention is related to genetically modified plant and plant cell having improved (induced or increased) heavy metal resistance, comprising at least one nucleotide sequence encoding one or more heterologous heavy metal transport and/or sequestration proteins of various prokaryotic or eukaryotic origins.  
       [0014] Said transporters are preferably membrane proteins, which result in reduced toxicity due to the efflux of heavy metals from the cells and being preferably selected from the group consisting of P-type ATPases, 3 component efflux pumps, ABC transporters and CDF proteins (Cation Diffusion Facilitator proteins).  
       [0015] The family of the P-type ATPases is preferred, because of their advantage that for functional resistance only one protein is required.  
       [0016] Said proteins are found in both prokaryotic and eukaryotic organisms including plants.  
       [0017] Another advantage of said transporters is found as resistance mechanisms against many toxic trace elements of environmental concern, such as copper, cadmium, lead, zinc and silver.  
       [0018] Unexpectedly, it was not necessary to make structural changes in the coding sequence of said proteins, like it is necessary for the merA gene in order to obtain functional expression in plants (Rugh et al., 1996).  
       [0019] Preferably, the gene incorporated in the plants or plant cells is a gene encoding a bacterial P-type ATPase, preferably the cadmium ATPase, such as the cadA gene.  
       [0020] Another aspect of the present invention is related to a method for inducing (or improving) increased heavy metal resistance into a plant or a plant cell, said method comprising the following steps:  
       [0021] preparing at least one nucleotide sequence encoding one or more heterologous heavy metal transport and/or sequestration proteins, operably linked to one or more regulatory sequences active into a plant,  
       [0022] transforming a plant or plant cell with said nucleotide sequence and,  
       [0023] possibly regenerating a (transgenic) plant from the transformed plant or plant cell.  
       [0024] According to a second embodiment of the present invention, the system is based upon a prokaryotic heavy metal sequestration system, such as the pcoA family protein (more preferably the pcoA gene).  
       [0025] The various nucleotide sequences encoding heterologous heavy metal transport proteins can be deleted partially from non-specific nucleotide sequences which are not involved in efficient heavy metal transport or accumulation.  
       [0026] Said genetic sequences could be incorporated in a vector for the transfection of said plants or plant cells, such as the pBI121 vector, as described in the FIG. 1, said vector being advantageously an  E. coli /Agrobacterium/plant shuttle vector, said vector comprising preferably a CaMV 35S promoter (a strong promoter constitutively expressed in plants).  
       [0027] Preferably, the system was introduced in the plants, such system allowing the transformation of plants with the  Agrobacterium tumefaciens  technology.  
       [0028] The present invention can be used for phytoremediation of contaminated sites, or for the preparation of a medicament or food/feed supplements containing trace elements.  
       [0029] The plant or plant cell according to the invention has various applications, especially in the field of phytoremediation of contaminated sites with heavy metal (such as areas having grounds contaminated with heavy metals or aquatic or semi-aquatic areas contaminated with heavy metals). The phytoremediation of contaminated sites comprises usually the step of revegetation, phytostabilisation, phytoextraction of soil and/or water contaminated with trace elements of said heavy metal.  
       [0030] Another application of the plant or plant cell according to the invention is a medical and agro-industrial application, wherein said plant or plant cell are natural sources for trace elements.  
       [0031] Therefore, a last aspect of the present invention is related to a pharmaceutical composition of food or feed compositions or additives, containing said plant or plant cells with heavy metal trace elements. 
     
    
    
     SHORT DESCRIPTION OF THE DRAWINGS  
     [0032]FIG. 1 is a schematic representation of the cloning of cadA in pBI121.  
     [0033]FIG. 2 is a leaf disk-test with Nt WT SR1 (wild type), Nt PBI14 (pBI121) and Nt Cd 309 (pBI121-cadA) on 350 μM Cd and control medium without Cd.  
     [0034]FIG. 3 represents the regeneration and growth of Nt WT SR1 (wild type), Nt PBI14 (pBI121) and Nt Cu122 (pBI121-pcoA) on 100 μM Cu, the plant growth being shown from above (left) and top (right). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0035] Heterologous Expression of cadA  
     [0036] The heavy metal efflux system was cadA, a member of the P-type heavy metal efflux ATPase family of proteins found both in prokaryotic and eukaryotic organisms. P-type ATPases are all cation pumps, either for uptake, for efflux or for cation exchange. These enzymes have a conserved aspartate residue that is transiently phosphorylated from ATP during the transport cycle, hence the name ‘P-type’ ATPase (Silver et al., 1993).  
     [0037] The cadA gene from  Staphylococcus aureus  was amplified by PCR and cloned in the pBI121 vector.  
     [0038] During PCR, appropriate plant specific translation signals were added as well as XbaI and BamHI restriction sites, allowing cloning of the insert in the correct orientation.  
     [0039] The cadA fragment was cloned in the  Escherichia coli /Agrobacterium/plant shuttle vector pBI121. In this vector, cadA expression is derived from the CaMV35S promotor, a strong promoter constitutively expressed in plants. The system was introduced in the plant  Nicotiana tabacum  cv. Petit Havana line SR1 via an  Agrobacterium tumefaciens  transformation (Horsch et al., 1985). The selection marker used was kanamycine.  
     [0040] Kanamycine resistant transformants were obtained after transformation. All the kanamycine resistant transformants tested showed an increased resistance to cadmium (tested by a leaf disk assay) compared to the wild type and transformant with the pBI121 vector without gene (FIG. 1). This proves that the CadA P-type ATPase can be functionally expressed in plants, resulting in an increased resistance of the plant to the trace element (in casu cadmium).  
     [0041] It can be expected that for other members of the P-type ATPase family, which form a family of closely related proteins (both structural and functional) the same positive effect on resistance to specific trace elements will be found. Until present, P-type ATPases from both prokaryotic and eukaryotic have been identified that were found to interact with Zn, Cd, Pb, Cu and Ag (see table 1). It can not be excluded that P-type ATPases, encoding resistance to other trace elements including radioisotopes, will be identified.  
               TABLE 1                          different representatives of the family of P-type       ATPases, from prokaryotic and eukariotic origin, which       encode resistance against trace elements       such as Zn, Cd, Pb, Cu and Ag.                                 Sequence               Gene   ID   Metals   Reference               CadA   P20021   Cd, Zn and   Nucifora et al. 1989               Pb   Rensing et al. 1998       ZntA   P37617   Zn and Pb   Rensing et al. 1997                   Rensing et al. 1998       CopF   Non   Cu   van der Lelie and           available       Borremans                   unpublished       PbrA   Not   Pb   Borremans et al, 2000           available       SilP   AF067954,   Ag   Gupta et al, 1999           nucleotide           sequence           sil operon       Menkes&#39;   Q04656   Cu   Vulpe et al. 1993       disease       Wilsons&#39;   1J08344   Cu   Pethrukin et al. 1993       disease                  
 
     [0042] Heterologous Expression of pcoA  
     [0043] The other heavy metal resistance system is involved in exo-cytoplasmic heavy metal sequestration. The tested gene here was pcoA from  Escherichia coli  (Brown et al., 1995), which was also cloned in pBI121 and introduced in  Nicotiana tabacum  through an  Agrobacterium tumefaciens  transformation in a way similar as described for cadA. Kanamycine resistant transformants were obtained after transformation. All the kanamycine resistant transformants tested showed an increased resistance to copper (tested by a leaf disk assay) compared to the wild type and transformant with the pBI121 vector without gene (FIG. 3).  
     [0044] The pcoA protein has many closely related members, found to be involved in resistance against Cu. In addition, other proteins of these copper resistance determinants have also been shown to be involved in Cu sequestration, such as PcoC/CopC and CopE. These proteins, although different in structure, are also active in the bacterial periplasm and possess similar heavy metal binding sites as pcoA. In addition, a CopE like protein, referred to as SilE, was identified in the  Salmonella sil  operon encoding for Ag-resistance. The potential genes whose heterologous expression can result in improved resistance, are summarised in table 2.  
                                           Genes   Sequence ID   Metals   References                  cop operon (copA, C)   M19930   Cu   Mellano and       e.g. of  Pseudomonas             Cooksey         syringae             (1988)       pco operon (pcoA, C)   G619126   Cu   Brown et al.,       of e.g.  E. coli             1995       PcoE   X83541   Cu   Brown et al.,                   1995       sil operon of   AF067954,   Ag   Gupta et al.,       Salmonella   nucleotide       1999           sequence           sil operon                  
 
     [0045] Use of the Genetically Modified Plants or Plant Cells According to the Invention:  
     [0046] Plants or plant cells according to the present invention can be used in several applications. It is clear that a plant according to the invention can be used for phytoremediation of contaminated sites, for revegetation, phytostabilisation, phytoextraction of contaminated soils and/or water. While the genetic modification allow the plants to grow in such contaminated environments, they will accumulate trace elements and remove them from the soil and/or water.  
     [0047] Another possible application is to grow said genetically modified plants or plant cells on a medium (solid or fluid) containing certain trace elements necessary or beneficial for human and animal health.  
     [0048] It would then be possible to prepare food/feed supplements from these plants containing beneficial trace elements which can be readily adsorbed. Further, the plants could serve as a basis for the preparation of a medicament.  
     REFERENCES  
     [0049] Silver S. et al. (1993). Molecular Microbiology 10(1): 7-12.  
     [0050] Horsch R. B. et al. (1985). Science 227: 1229-1231.  
     [0051] Karin M (1985). Metallothioneins: Proteins in search of function; Cell 41, 9-10.  
     [0052] Brown N. L. et al. (1995). Molecular Microbiology 17(6): 1153-1166.  
     [0053] Mellano, M. A. et al. (1988). J. Bacteriol. 170: 2879-2883.  
     [0054] Vulpe C. D., et al. (1993). Nature genet. 3: 7-13.  
     [0055] Petrukhin, K. et al. (1993). Nature Genet. 5 (4): 338-343.  
     [0056] Rugh C. L. et al. (1996). Proc. Natl. Acad. Sci. USA 93: 3182-3187.  
     [0057] Lefebvre, D. D. et al, 1987. Bio/Technology 5, 1053-1056.  
     [0058] Maiti, I. B. et al, 1988. Biochemical and Biophysical Research Communications 150, 640-647.  
     [0059] Maiti, I. B. et al, 1989. Plant Physiology 91, 1020-1024.  
     [0060] Maiti, I. B. et al. 1991. Plant Science 76, 99-107.  
     [0061] Misra, S. et al. 1989. Theoretical and Applied Genetics 78, 161-168.  
     [0062] Evans, K. M. et al 1992. Implications for PsMTA function. Plant Molecular Biology 20, 1019-1028.  
     [0063] Yeargan, R. et al 1992. Transgenic Research 1, 261-267.  
     [0064] Nucifora G. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86 (10): 3544-3548.  
     [0065] Rensing C., et al. (1998). J. Biol. Chem. 273: 32614-32617.  
     [0066] Rensing C., et al. (1997). Proc. Natl. Acad. Sci. U.S.A. 94 (26): 14326-14331.  
     [0067] Gupta, A., et al. 1999. Nature Medicine 5: 183-188.  
     [0068] 
    
     
       
         1 
         
           
             2  
           
           
             1  
             2217  
             DNA  
             Artificial Sequence  
             
               Staphylococcus aureus cadA gene with BamH I and 
      Xba I restriction sites and plant specific translation signals  
             
           
            1 

tctagattta ccacc atg tct gaa caa aag gtt aaa cta atg gaa gaa gaa      51 
                 Met Ser Glu Gln Lys Val Lys Leu Met Glu Glu Glu 
                 1               5                   10 

atg aac gtc tat cgg gtc caa gga ttt aca tgt gca aat tgt gca gga       99 
Met Asn Val Tyr Arg Val Gln Gly Phe Thr Cys Ala Asn Cys Ala Gly 
        15                  20                  25 

aag ttt gag aaa aat gtt aaa aag att cca ggc gtt cag gac gca aaa      147 
Lys Phe Glu Lys Asn Val Lys Lys Ile Pro Gly Val Gln Asp Ala Lys 
    30                  35                  40 

gta aac ttt ggc gct tct aaa att gat gta tat gga aat gca tcg gtt      195 
Val Asn Phe Gly Ala Ser Lys Ile Asp Val Tyr Gly Asn Ala Ser Val 
45                  50                  55                  60 

gaa gaa ctt gaa aaa gca ggt gct ttt gag aat cta aaa gta tct cct      243 
Glu Glu Leu Glu Lys Ala Gly Ala Phe Glu Asn Leu Lys Val Ser Pro 
                65                  70                  75 

gaa aaa cta gcg aat caa acg ata caa agg gtt aaa gat gac act aag      291 
Glu Lys Leu Ala Asn Gln Thr Ile Gln Arg Val Lys Asp Asp Thr Lys 
            80                  85                  90 

gct cat aaa gaa gag aaa aca cca ttt tat aaa aaa cat agt aca ttg      339 
Ala His Lys Glu Glu Lys Thr Pro Phe Tyr Lys Lys His Ser Thr Leu 
        95                  100                 105 

ctg ttt gcc aca cta cta att gct ttt ggt tac ctt tct cac ttt gta      387 
Leu Phe Ala Thr Leu Leu Ile Ala Phe Gly Tyr Leu Ser His Phe Val 
    110                 115                 120 

aat gga gaa gat aac ctc gta act tcc atg tta ttt gta ggt tct att      435 
Asn Gly Glu Asp Asn Leu Val Thr Ser Met Leu Phe Val Gly Ser Ile 
125                 130                 135                 140 

gta att ggc gga tat tca tta ttt aaa gtc ggt ttt caa aat ttg ata      483 
Val Ile Gly Gly Tyr Ser Leu Phe Lys Val Gly Phe Gln Asn Leu Ile 
                145                 150                 155 

cgc ttt gat ttc gac atg aaa acc ctg atg acc gtt gcc gtt att gga      531 
Arg Phe Asp Phe Asp Met Lys Thr Leu Met Thr Val Ala Val Ile Gly 
            160                 165                 170 

gct acc att att ggt aaa tgg gca gag gca tct att gtt gtt att ctc      579 
Ala Thr Ile Ile Gly Lys Trp Ala Glu Ala Ser Ile Val Val Ile Leu 
        175                 180                 185 

ttt gca atc agt gaa gca ctt gaa cgc ttc tct atg gac aga tca aga      627 
Phe Ala Ile Ser Glu Ala Leu Glu Arg Phe Ser Met Asp Arg Ser Arg 
    190                 195                 200 

caa tcc ata cgt tca ttg atg gat atc gcc cca aaa gaa gca cta gtt      675 
Gln Ser Ile Arg Ser Leu Met Asp Ile Ala Pro Lys Glu Ala Leu Val 
205                 210                 215                 220 

aga cga aat ggt cag gaa ata ata atc cat gtg gac gat atc gct gtg      723 
Arg Arg Asn Gly Gln Glu Ile Ile Ile His Val Asp Asp Ile Ala Val 
                225                 230                 235 

ggt gat atc atg att gtc aaa cca ggg gag aaa att gcc atg gat gga      771 
Gly Asp Ile Met Ile Val Lys Pro Gly Glu Lys Ile Ala Met Asp Gly 
            240                 245                 250 

atc att gtg aat ggc ttg tcg gct gtc aat cag gca gct ata aca gga      819 
Ile Ile Val Asn Gly Leu Ser Ala Val Asn Gln Ala Ala Ile Thr Gly 
        255                 260                 265 

gaa tct gtt ccc gtc tcc aaa gcg gta gat gac gaa gta ttt gca ggt      867 
Glu Ser Val Pro Val Ser Lys Ala Val Asp Asp Glu Val Phe Ala Gly 
    270                 275                 280 

acg ctt aac gaa gag gga cta att gaa gta aaa atc acc aaa tac gta      915 
Thr Leu Asn Glu Glu Gly Leu Ile Glu Val Lys Ile Thr Lys Tyr Val 
285                 290                 295                 300 

gaa gat aca acc att acc aag att att cat ctt gtt gaa gaa gca caa      963 
Glu Asp Thr Thr Ile Thr Lys Ile Ile His Leu Val Glu Glu Ala Gln 
                305                 310                 315 

ggg gag cgt gct cca gcc caa gca ttc gtt gat aaa ttt gcg aaa tac     1011 
Gly Glu Arg Ala Pro Ala Gln Ala Phe Val Asp Lys Phe Ala Lys Tyr 
            320                 325                 330 

tac act ccg atc att atg gtt att gca gcc ttg gtt gca gtc gtt cca     1059 
Tyr Thr Pro Ile Ile Met Val Ile Ala Ala Leu Val Ala Val Val Pro 
        335                 340                 345 

ccc cta ttc ttt ggt ggc agt tgg gat aca tgg gtt tat caa gga tta     1107 
Pro Leu Phe Phe Gly Gly Ser Trp Asp Thr Trp Val Tyr Gln Gly Leu 
    350                 355                 360 

gca gtt ctt gta gtt gga tgt cct tgt gca tta gtt att tct act cca     1155 
Ala Val Leu Val Val Gly Cys Pro Cys Ala Leu Val Ile Ser Thr Pro 
365                 370                 375                 380 

atc tcg att gtc tcg gca att gga aat gca gcg aaa aaa ggt gtg ttg     1203 
Ile Ser Ile Val Ser Ala Ile Gly Asn Ala Ala Lys Lys Gly Val Leu 
                385                 390                 395 

gtt aaa ggt ggt gtc tat ctc gag aaa tta gga gcc att aag aca gtc     1251 
Val Lys Gly Gly Val Tyr Leu Glu Lys Leu Gly Ala Ile Lys Thr Val 
            400                 405                 410 

gca ttt gat aaa aca gga aca ctg aca aaa ggt gta cca gtg gta aca     1299 
Ala Phe Asp Lys Thr Gly Thr Leu Thr Lys Gly Val Pro Val Val Thr 
        415                 420                 425 

gat ttt gaa gta tta aat gac caa gtg gaa gaa aaa gag cta ttc tct     1347 
Asp Phe Glu Val Leu Asn Asp Gln Val Glu Glu Lys Glu Leu Phe Ser 
    430                 435                 440 

atc att aca gct tta gaa tat cgt tca caa cat cca ctt gct tca gca     1395 
Ile Ile Thr Ala Leu Glu Tyr Arg Ser Gln His Pro Leu Ala Ser Ala 
445                 450                 455                 460 

ata atg aaa aag gca gag caa gat aat atc cct tat tct aat gta caa     1443 
Ile Met Lys Lys Ala Glu Gln Asp Asn Ile Pro Tyr Ser Asn Val Gln 
                465                 470                 475 

gtg gaa gaa ttc act tcg att act ggg cga ggt ata aaa ggg att gta     1491 
Val Glu Glu Phe Thr Ser Ile Thr Gly Arg Gly Ile Lys Gly Ile Val 
            480                 485                 490 

aac gga act act tac tat att gga agc cca aaa ctt ttc aag gaa tta     1539 
Asn Gly Thr Thr Tyr Tyr Ile Gly Ser Pro Lys Leu Phe Lys Glu Leu 
        495                 500                 505 

aat gtt tcc gat ttt agc ctt ggg ttt gaa aac aat gtg aaa atc cta     1587 
Asn Val Ser Asp Phe Ser Leu Gly Phe Glu Asn Asn Val Lys Ile Leu 
    510                 515                 520 

caa aac caa gga aaa aca gcc atg att att gga acg gaa aaa aca att     1635 
Gln Asn Gln Gly Lys Thr Ala Met Ile Ile Gly Thr Glu Lys Thr Ile 
525                 530                 535                 540 

ctc ggc gta att gcc gtt gca gat gag gtt cgt gaa aca agt aaa aat     1683 
Leu Gly Val Ile Ala Val Ala Asp Glu Val Arg Glu Thr Ser Lys Asn 
                545                 550                 555 

gtg att caa aaa ctt cat cag tta ggt atc aag caa aca att atg ctg     1731 
Val Ile Gln Lys Leu His Gln Leu Gly Ile Lys Gln Thr Ile Met Leu 
            560                 565                 570 

aca ggt gat aat caa ggt act gca aat gca atc ggt aca cat gta ggc     1779 
Thr Gly Asp Asn Gln Gly Thr Ala Asn Ala Ile Gly Thr His Val Gly 
        575                 580                 585 

gtt tct gat att cag tct gaa ttg atg cca cag gat aaa tta gat tat     1827 
Val Ser Asp Ile Gln Ser Glu Leu Met Pro Gln Asp Lys Leu Asp Tyr 
    590                 595                 600 

att aaa aaa atg caa tcg gag tat gat aat gta gct atg att ggc gat     1875 
Ile Lys Lys Met Gln Ser Glu Tyr Asp Asn Val Ala Met Ile Gly Asp 
605                 610                 615                 620 

ggc gtt aat gat gct cca gca ctt gct gca tct act gtt gga att gca     1923 
Gly Val Asn Asp Ala Pro Ala Leu Ala Ala Ser Thr Val Gly Ile Ala 
                625                 630                 635 

atg ggc ggt gct gga acg gat act gca att gaa aca gct gat att gca     1971 
Met Gly Gly Ala Gly Thr Asp Thr Ala Ile Glu Thr Ala Asp Ile Ala 
            640                 645                 650 

tta atg gga gat gat tta agt aag ctt cca ttt gca gta aga ctc agt     2019 
Leu Met Gly Asp Asp Leu Ser Lys Leu Pro Phe Ala Val Arg Leu Ser 
        655                 660                 665 

cga aaa act tta aat atc att aaa gct aac atc act ttt gct atc gga     2067 
Arg Lys Thr Leu Asn Ile Ile Lys Ala Asn Ile Thr Phe Ala Ile Gly 
    670                 675                 680 

att aaa ata att gcc tta cta tta gtt atc ccg gga tgg tta acc ctt     2115 
Ile Lys Ile Ile Ala Leu Leu Leu Val Ile Pro Gly Trp Leu Thr Leu 
685                 690                 695                 700 

tgg ata gcg att ctt tcc gat atg gga gct act att ttg gta gca tta     2163 
Trp Ile Ala Ile Leu Ser Asp Met Gly Ala Thr Ile Leu Val Ala Leu 
                705                 710                 715 

aat agt tta cga ctg atg aga gtg aag gat aaa taggtaatga tgtttggatc   2216 
Asn Ser Leu Arg Leu Met Arg Val Lys Asp Lys 
            720                 725 

c                                                                   2217 

 
           
             2  
             727  
             PRT  
             Artificial Sequence  
             
               Staphylococcus aureus cadA gene with BamH I and 
      Xba I restriction sites and plant specific translation signals  
             
           
            2 

Met Ser Glu Gln Lys Val Lys Leu Met Glu Glu Glu Met Asn Val Tyr 
1               5                   10                  15 

Arg Val Gln Gly Phe Thr Cys Ala Asn Cys Ala Gly Lys Phe Glu Lys 
            20                  25                  30 

Asn Val Lys Lys Ile Pro Gly Val Gln Asp Ala Lys Val Asn Phe Gly 
        35                  40                  45 

Ala Ser Lys Ile Asp Val Tyr Gly Asn Ala Ser Val Glu Glu Leu Glu 
    50                  55                  60 

Lys Ala Gly Ala Phe Glu Asn Leu Lys Val Ser Pro Glu Lys Leu Ala 
65                  70                  75                  80 

Asn Gln Thr Ile Gln Arg Val Lys Asp Asp Thr Lys Ala His Lys Glu 
                85                  90                  95 

Glu Lys Thr Pro Phe Tyr Lys Lys His Ser Thr Leu Leu Phe Ala Thr 
            100                 105                 110 

Leu Leu Ile Ala Phe Gly Tyr Leu Ser His Phe Val Asn Gly Glu Asp 
        115                 120                 125 

Asn Leu Val Thr Ser Met Leu Phe Val Gly Ser Ile Val Ile Gly Gly 
    130                 135                 140 

Tyr Ser Leu Phe Lys Val Gly Phe Gln Asn Leu Ile Arg Phe Asp Phe 
145                 150                 155                 160 

Asp Met Lys Thr Leu Met Thr Val Ala Val Ile Gly Ala Thr Ile Ile 
                165                 170                 175 

Gly Lys Trp Ala Glu Ala Ser Ile Val Val Ile Leu Phe Ala Ile Ser 
            180                 185                 190 

Glu Ala Leu Glu Arg Phe Ser Met Asp Arg Ser Arg Gln Ser Ile Arg 
        195                 200                 205 

Ser Leu Met Asp Ile Ala Pro Lys Glu Ala Leu Val Arg Arg Asn Gly 
    210                 215                 220 

Gln Glu Ile Ile Ile His Val Asp Asp Ile Ala Val Gly Asp Ile Met 
225                 230                 235                 240 

Ile Val Lys Pro Gly Glu Lys Ile Ala Met Asp Gly Ile Ile Val Asn 
                245                 250                 255 

Gly Leu Ser Ala Val Asn Gln Ala Ala Ile Thr Gly Glu Ser Val Pro 
            260                 265                 270 

Val Ser Lys Ala Val Asp Asp Glu Val Phe Ala Gly Thr Leu Asn Glu 
        275                 280                 285 

Glu Gly Leu Ile Glu Val Lys Ile Thr Lys Tyr Val Glu Asp Thr Thr 
    290                 295                 300 

Ile Thr Lys Ile Ile His Leu Val Glu Glu Ala Gln Gly Glu Arg Ala 
305                 310                 315                 320 

Pro Ala Gln Ala Phe Val Asp Lys Phe Ala Lys Tyr Tyr Thr Pro Ile 
                325                 330                 335 

Ile Met Val Ile Ala Ala Leu Val Ala Val Val Pro Pro Leu Phe Phe 
            340                 345                 350 

Gly Gly Ser Trp Asp Thr Trp Val Tyr Gln Gly Leu Ala Val Leu Val 
        355                 360                 365 

Val Gly Cys Pro Cys Ala Leu Val Ile Ser Thr Pro Ile Ser Ile Val 
    370                 375                 380 

Ser Ala Ile Gly Asn Ala Ala Lys Lys Gly Val Leu Val Lys Gly Gly 
385                 390                 395                 400 

Val Tyr Leu Glu Lys Leu Gly Ala Ile Lys Thr Val Ala Phe Asp Lys 
                405                 410                 415 

Thr Gly Thr Leu Thr Lys Gly Val Pro Val Val Thr Asp Phe Glu Val 
            420                 425                 430 

Leu Asn Asp Gln Val Glu Glu Lys Glu Leu Phe Ser Ile Ile Thr Ala 
        435                 440                 445 

Leu Glu Tyr Arg Ser Gln His Pro Leu Ala Ser Ala Ile Met Lys Lys 
    450                 455                 460 

Ala Glu Gln Asp Asn Ile Pro Tyr Ser Asn Val Gln Val Glu Glu Phe 
465                 470                 475                 480 

Thr Ser Ile Thr Gly Arg Gly Ile Lys Gly Ile Val Asn Gly Thr Thr 
                485                 490                 495 

Tyr Tyr Ile Gly Ser Pro Lys Leu Phe Lys Glu Leu Asn Val Ser Asp 
            500                 505                 510 

Phe Ser Leu Gly Phe Glu Asn Asn Val Lys Ile Leu Gln Asn Gln Gly 
        515                 520                 525 

Lys Thr Ala Met Ile Ile Gly Thr Glu Lys Thr Ile Leu Gly Val Ile 
    530                 535                 540 

Ala Val Ala Asp Glu Val Arg Glu Thr Ser Lys Asn Val Ile Gln Lys 
545                 550                 555                 560 

Leu His Gln Leu Gly Ile Lys Gln Thr Ile Met Leu Thr Gly Asp Asn 
                565                 570                 575 

Gln Gly Thr Ala Asn Ala Ile Gly Thr His Val Gly Val Ser Asp Ile 
            580                 585                 590 

Gln Ser Glu Leu Met Pro Gln Asp Lys Leu Asp Tyr Ile Lys Lys Met 
        595                 600                 605 

Gln Ser Glu Tyr Asp Asn Val Ala Met Ile Gly Asp Gly Val Asn Asp 
    610                 615                 620 

Ala Pro Ala Leu Ala Ala Ser Thr Val Gly Ile Ala Met Gly Gly Ala 
625                 630                 635                 640 

Gly Thr Asp Thr Ala Ile Glu Thr Ala Asp Ile Ala Leu Met Gly Asp 
                645                 650                 655 

Asp Leu Ser Lys Leu Pro Phe Ala Val Arg Leu Ser Arg Lys Thr Leu 
            660                 665                 670 

Asn Ile Ile Lys Ala Asn Ile Thr Phe Ala Ile Gly Ile Lys Ile Ile 
        675                 680                 685 

Ala Leu Leu Leu Val Ile Pro Gly Trp Leu Thr Leu Trp Ile Ala Ile 
    690                 695                 700 

Leu Ser Asp Met Gly Ala Thr Ile Leu Val Ala Leu Asn Ser Leu Arg 
705                 710                 715                 720 

Leu Met Arg Val Lys Asp Lys 
                725