Fusion proteins, recombinant bacteria, and methods for using recombinant bacteria

A recombinant Bacillus cereus family member that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member, wherein the protein or peptide of interest comprises:

• a phospholipase, the phospholipase comprising a phospholipase A1, a phospholipase A2, a phospholipase C, a phospholipase D, or a lysophospholipase;
• a phosphomonoesterase comprising PhoA4;
• a phytase, wherein the phytase comprises Bacillus subtilis EE148 phytase or Bacillus thuringiensis BT013A phytase;
• a chitosanase comprising an amino acid sequence having at least 85% identity with SEQ ID NO:
• 313;
• a Cry toxin from Bacillus thuringiensis, wherein the Cry toxin from Bacillus thuringiensis comprises a Cry5B protein or a Cry21A protein;
• a superoxide dismutase, the superoxide dismutase comprising superoxide dismutase 1 (SODA1) or superoxide dismutase 2 (SODA2);
• a protein or peptide that protects a plant from an environmental stress, wherein the protein or peptide that protects the plant from an environmental stress comprises an ice nucleation protein, a prolinase, a phenylalanine ammonia lyase, an isochorismate synthase, an isochorismate pyruvate lyase, or a choline dehydrogenase;
• an enzyme that catalyzes the production of nitric oxide;
• a nucleic acid binding protein or peptide, wherein the nucleic acid binding protein or peptide comprises an Hfq protein or a small acid-soluble spore protein (SASP);
• or
• a nucleic acid binding protein or peptide, wherein a nucleic acid molecule is bound to the nucleic acid binding protein or peptide, the nucleic acid molecule comprising a modulating RNA molecule;
• an RNAi molecule;
• a microRNA;
• an aptamer;
• or a DNA molecule that encodes a modulating RNA molecule, an RNAi molecule, a microRNA, or an aptamer;
• and wherein the recombinant Bacillus cereus family member comprises a mutation that results in Bacillus cereus family member spores having an exosporium that is easier to remove from the spore as compared to the exosporium of a wild-type spore.

A method for stimulating germination of a plant seed comprising:

• introducing exosporium fragments into a plant growth medium;
• or
• applying exosporium fragments to a plant seed or an area surrounding a plant or a plant seed;
• wherein the exosporium fragments are derived from spores of a recombinant Bacillus cereus family member, wherein the recombinant Bacillus cereus family member:
• expresses a fusion protein comprising:
• (a) a superoxide dismutase or an enzyme that catalyzes the production of nitric oxide;
• and (b) a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member;
• and
• comprises a mutation that results in Bacillus cereus family member spores having an exosporium that is easier to remove from the spore as compared to the exosporium of a wild-type spore.

A method for delivering nucleic acids to a plant comprising:

• introducing exosporium fragments into a plant growth medium;
• or
• applying exosporium fragments to a plant, a plant seed, or an area surrounding a plant or a plant seed;
• wherein the exosporium fragments are derived from spores of a recombinant Bacillus cereus family member, wherein the recombinant Bacillus cereus family member:
• expresses a fusion protein comprising:
• (a) a nucleic acid binding protein or peptide, wherein the nucleic acid binding protein or peptide is bound to a nucleic acid molecule;
• and (b) a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member;
• and
• comprises a mutation that results in Bacillus cereus family member spores having an exosporium that is easier to remove from the spore as compared to the exosporium of a wild-type spore.