DE60219077T2 - RALSTONIA AHL ACYLASE GEN - Google Patents

Description

• The This invention relates to the field of molecular biology. In particular, the invention relates to an N-acyl homoserine lactone acylase gene from Ralstonia sp. XJ12B.
• N-acylhomoserine lactones (AHLs), also known as autoinducers, are found in many gram-negative bacteria in a big one Scope used as quorum-sensitive signaling molecules. These connections regulate certain classes of target genes in bacteria, such as virulence genes or biofilm differentiation genes. In general, are quorum sensitive molecules highly conserved and share an identical homoserine lactone component. The length and the structure of their acyl side chains, however, are different. Although the AHLs regulated target genes in different Bacterial species vary, are the basic mechanisms of AHL biosynthesis and gene regulation within the various bacterial species preserved.
• The general feature of AHL-mediated gene regulation is its dependence from the cell population (quorum sensitivity). Bacteria enter AHLs their environment and the extracellular concentration of AHLs decreases with the growth of bacterial cell populations too. If AHL up to an extracellular Accumulated threshold concentration, the expression of certain Triggered groups of target genes in the bacteria.
• Bacteria, which use, detect and respond to this signal release on the accumulation of AHL signaling molecules with synchronization the expression of a particular group of genes as well as with the coordination of cellular activities within the bacterial cell population. AHLs are in regulation involved in a number of biological functions, including the Bioluminescence in Vibrio species (13, 4), the conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens (31), induction of virulence genes in Burkholderia cepacia, Erwinia carotovora, Erw. chrysanthemi, Erw. stewartii, Pseudomonas aeruginosa and Xenorhabdus nematophilus (3, 6, 12, 17, 19, 22, 23, 24, 26), regulation of Antibiotic production in P. aureofaciens and Erw. Carotovora (6, 26), swarm motility in Serratia liquifaciens (14) and biofilm formation in P. fluorescens and P. aeruginosa (1, 8). In many other types of bacteria have to the relevant biological functions controlled by AHLs to be investigated (2, 5, 11).
• A Series of plant, animal and human bacterial Pathogens use quorum-sensitive AHL signaling to increase expression of pathogenic genes and in the formation of biofilms to help. Therefore, the quorum-sensitive AHL signaling molecules are one Group of molecular targets for the genetic and chemical manipulation, since the interruption of these signaling mechanisms the ability of these bacteria, to infect plant and animal tissues or biofilms too form, prevented or reduced.
• The Gene encoding an AHL inactivating enzyme (AiiA) was obtained from a Gram-positive bacterium (Bacillus strain 240B1) cloned (9). AiiA (also known as AHL lactonase) inactivates AHL activity by Hydrolysis of the lactone bond of the AHLs (10). The expression of AiiA in transformed adults carotovora (a pathogenic strain that is found in many plants root rot caused) significantly reduces the release of AHL, lowers the extracellular pectolytic enzyme activities off and weakens the pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower and tobacco (9). Transgenic plants expressing AHL lactonase showed a significantly increased resistance to one Erw. Carotovora infection and a delayed development of symptoms the root rot (10). AHL-inactivation seem to be widely used. It was, for example, one Variovorax paradoxus bacterial isolate reported using the AHL molecules as its Energy and nitrogen source uses what the possible present of AHL-degrading enzymes (18).
• Further Procedures against an AHL-mediated herbal, animal and human disease and against plant pathogen virulence disorder at the bacterial intercellular Interaction would be very desirable.
• Accordingly, in this study over the cloning and characterization of a gene is reported, which for one AHL acylase from the bacterial isolate Ralstonia sp. JX12B coded.
• To an embodiment the invention provides a nucleic acid according to SEQ ID NO: 1. After a another embodiment the invention provides a nucleic acid containing the nucleotides 1234-3618 of SEQ ID NO: 1.
• To another embodiment the invention provides a peptide having a sequence according to SEQ ID NO. 2 or with a sequence comprising amino acids 36-217 of SEQ ID NO. 2, or a peptide having a sequence which comprises the amino acids 233-794 of SEQ ID NO: 2.
• According to one another embodiment the invention provides a peptide having a sequence derived from a nucleic acid which consists of nucleotides 1234-3618 of SEQ ID NO. 1 exists.
• According to one another embodiment the invention provides a peptide as described above which AHL disabled.
• According to one another embodiment the invention provides an in vitro method for modulating the AHL signal activity ready to contact AHL with one described above Peptide comprises.
• According to one another embodiment the invention provides a transgenic plant or a non-human one mammal which comprises a nucleic acid as described above.
• According to one another embodiment the invention provides a nucleic acid or a peptide as above described for use in therapy and the use of this nucleic acid and / or this peptide for the preparation of a drug for the control of a bacterial Disease in a mammal ready, with the expression of pathogenic genes from these bacteria be regulated by AHL signals.
• According to one another embodiment the invention provides a method for controlling a bacterial Disease in a plant, which is the administration of a Peptides, as described above, to this plant, wherein the expression of pathogenic genes from these bacteria is regulated by AHL signals become. Alternatively, the method comprises the transformation of Plant with a nucleic acid described above.
• The 1 Figure 3 is a photograph showing bioassay results of AHL inactivation for bacterial cultures and bacterial proteins from the bacterial clones shown. The 1A shows the results of a bioassay with bacterial cultures of E. coli DH5α strains 13H10 (row 1), 2B10 (row 2), MUB3 (row 3), MUC6 (row 4), GST-QsbA (row 5) and GST (row 6 ) containing the plasmid clones or constructs pI3H10, p2B10, pMUB3, pMUC6, pGST-QsbA and pGST, respectively. The 1B shows the results of the bioassay for the indicated bacterial proteins GST-QsbA and GST.
• 2 Figure 2 is a graph showing the temperature and pH optimum profiles of the AHL acylase.
• The 3 Figure 9 is a graph showing the time course of OOHL inactivation by purified AHL acylase.
• One bacterial isolate of Ralstonia sp. XJ12B from a biofilm sample in a water treatment system was for enzymatic inactivation of AHLs, bacterial quorum-sensitive molecules, in a biotest below Use of the Agrobacterium tumefaciens strain Nt1 (traR; tra :: lacZ749) as an indicator of the AHL activity in a position. The gene for encoded the protein expressing this enzyme activity for AHL inactivation (qsbA) was cloned from a bacterial strain that came from a Biofilm sample was isolated. The gene encodes a 794 peptide Amino acids.
• Bacterial cultures and bacterial proteins were tested for their ability to inactivate AHL using Agrobacterium tumefaciens indicator cells. A. tumefaciens was cultured at 28 ° C in an MM medium as described in Zhang et al. (31). The bacteria or protein to be tested are first tested with an AHL substrate, for example N-β-oxooctanoyl-L-homoserine lactone (OOHL), and the reaction (inactivation of AHL) is allowed to proceed. If an AHL inactivating activity is present in the sample (that is, AHL is cut and inactivated), then the inactivated AHL products will not be able to induce expression of the lacZ reporter gene, which is under the control of a TraR-dependent promoter. The A. tumefaciens NT1 strain containing the lacZ reporter system will thus not turn blue in the presence of the substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). See Example 2 for the details of this bioassay. Any AHL can be used in the test, if desired. Of course, any suitable assay for the cleavage of AHL, including traditional in vitro enzyme assays, can be used to detect AHL inactivating activity. professionals will be able to modify or devise tests to detect and / or quantify AHL inactivation.
• Of the Escherichia coli strain DH5α was as a host for the DNA manipulation used. Ralstonia sp. and E. coli were both in an LB medium (Tryptone: 10 g / l, yeast extract: 5 g / l and NaCl: 10 g / l, pH 7.0) at 37 ° C. If necessary, appropriate antibiotics were obtained with the following concentrations added: ampicillin, 100 μg / ml; Tetracycline, 10 μg / ml and kanamycin, 20 μg / ml.
• The Gene, which for the protein that codes for the proven AHL inactivation is responsible Use of a cosmid library of 1,600 clones with the genomic DNA from Ralstonia sp. Strain XJ12B, constructed in E. coli, isolated. The E. coli transfectants were assessed for their ability tested for inactivation of AHL. One clone, p13H10, was made the ability found to inactivate AHL. The cosmid DNA from p13H10 was cut, fused into a cloning vector, ligated and transformed in E. coli. The E. coli clones were again examined their activity tested for AHL inactivation. A clone containing a 4 kb insert had the AHL inactivation activity.
• The Plasmids were subsequently for the Purified sequencing. The 4 kb fragment from clone p2B10 was Completely according to known Method Using ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystems) sequenced. See the following Table I. The sequence contained an open reading frame of 2,385 nucleotides containing the AHL inactivation gene, qsbA, represented and for the predicted polypeptide encoded 794 amino acids (85,373 daltons).
• Table I. QsbA gene (Ralstonia sp.) Nucleotide sequence (SEQ ID NO: 1).

  
• Of the predicted open reading frame qsbA gene is in capital letters with the start codon and the stop codon shown in bold are. A suspected Ribosome binding site (AGGAGA) is underlined.
• Sequence analysis of this peptide showed that QsbA shows no significant homology with the known, quorum-sensitive AHL lactonase molecule activator derived from the aiiA gene from Bacillus sp. 240B1 is encoded. However, the deduced peptide sequence was typical of the primary structure of aculeacin A acylases (AACs) and penicillin G acylases with a signal peptide-α subunit-spacer-β subunit organization (16, 30). The Ralstonia sequence shares a substantial identity with the AACs from Deinococcus radiodurans strain R1, Actinoplanes utahensis, and a putative Pseudomonas aeruginosa acylase, all of which catalyze the deacylation of their substrates. These AAC genes are translated as a single precursor polypeptide and then processed to the active form, which has two subunits. Aculeacin A is a fungal echinocandin type antibiotic with a long fatty acid side chain. Aculeacin A acylases purified from A. utahensis catalyze the hydrolysis of the amide bond on the palmitoyl side chain of aculeacin A (29). The primary structure of the protein as well as the enzyme activity analysis with various substrates discussed below thus demonstrate that qsbA encodes an AHL acylase which cleaves the amide linkage between the acyl side chain and the homoserine lactone moiety of the AHLs.
• The putative α and β subunits of QsbA are located at amino acid positions 36-217 and 233-794, respectively, of SEQ ID NO: 2, with a spacer of 15 amino acids between them, as determined by comparison with the D. radiodurans peptide sequences Strain R1, A. utahensis and P. aeruginosa. See Table II. Table II. Aligned amino acid sequences of QsbA from Ralstonia sp. XJ12B (SEQ ID NO: 2), D. radiodurans strain R1 acylase (SEQ ID NO: 3), A. utahensis acylase (SEQ ID NO: 4) and P. aeruginosa acylase (SEQ ID NO: 5).

  
• 
• * = identical residues; : = conserved substitutions; , = semi-conserved substitutions;
• | = post-translational processing sites for signal peptide and subunits; - = spacer.
• The coding region of the qsbA gene was amplified by PCR. The amplified PCR products were cut, placed in the coding sequence of the glutathione S transferase (GST) gene in frame and expressed in E. coli. The protein extracts from the recombinant E. coli cells were tested for their ability to inactivate AHL. Proteins from E. coli expressing GST alone served as controls. The results show that the GST-QsbA fusion protein effectively eliminated AHL activity. Please refer 1B ,
• The substrate specificity of QsbA was assessed by assaying the total soluble protein, which was derived from re Combinant E. coli (pGST-QsbA) was extracted for inactivation of AHLs using substrates with acyl chains of different lengths. QsbA was capable of completely inactivating 3-oxo group acyl HSLs with acyl chains of 8, 10, and 12 carbon atoms. QsbA also strongly inactivated methylene group acyl HSLs with acyl chains of 8 and 10 carbon atoms. QsbA also inactivated the butyl and hexyl esters of N-β-octanoyl-L-homoserine while the AHL lactonase encoded by aiiA was unable to inactivate them. Substrate specificity data show that QsbA is an AHL acylase.
• QsbA and qsbA introduce new down-regulation tools AHL mediated biological activities, such as expression of virulence genes and biofilm differentiation into pathogenic Bacteria, both in vitro and in vivo. The qsbA gene, which is responsible for the AHL Inactivation enzyme (QsbA), or a functional fragment, a subunit or a substantially homologous variant thereof can introduced into a plant genome Become a genetically modified plant with the ability to quench a quorum-sensitive pathogenic signaling agent. transgenic Plants expressing an enzyme that inactivates AHLs may have a significantly increased resistance to infection by bacterial Pathogens show even when the bacteria are in high concentrations available.