ABSTRACT
It has previously been shown that the tomato pathogen Clavibacter michiganensis subsp. michiganensis secretes a 14-kDa protein, C. michiganensis subsp. michiganensis AMP-I (CmmAMP-I), that inhibits growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Using sequences obtained from tryptic fragments, we have identified the gene encoding CmmAMP-I and we have recombinantly produced the protein with an N-terminal intein tag. The gene sequence showed that CmmAMP-I contains a typical N-terminal signal peptide for Sec-dependent secretion. The recombinant protein was highly active, with 50% growth inhibition (IC50) of approximately 10 pmol, but was not toxic to potato leaves or tubers. CmmAMP-I does not resemble any known protein and thus represents a completely new type of bacteriocin. Due to its high antimicrobial activity and its very narrow inhibitory spectrum, CmmAMP-1 may be of interest in combating potato ring rot disease.
Subject(s)
Actinomycetales/drug effects , Actinomycetales/metabolism , Anti-Infective Agents/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Actinomycetales/genetics , Anti-Infective Agents/toxicity , Bacterial Proteins/toxicity , Inhibitory Concentration 50 , Solanum lycopersicum , Microbial Sensitivity Tests , Plant Leaves/drug effects , Plant Roots/drug effects , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/toxicity , Solanum tuberosum/drug effectsABSTRACT
Chloroplast division represents a fundamental but complex biological process involving remnants of the ancestral bacterial division machinery and proteins of eukaryotic origin. Moreover, the chloroplast division machinery is divided into stromal and cytosolic sub machineries, which coordinate and control their activities to ensure appropriate division initiation and progression. Dynamin related protein 5B (DRP5B) and plastid division protein 1 and 2 (PDV1 and PDV2) are all plant-derived proteins and represent components of the cytosolic division machinery, where DRP5B is thought to exert constrictional force during division. However, the direct relationship between PDV1, PDV2 and DRP5B, and moreover how DRP5B is regulated during plastid constriction remains unclear. In this study we show that PDV1 and PDV2 can interact with themselves and with each other through their cytosolic domains. We demonstrate that DRP5B interacts with itself and with the cytosolic region of PDV1 and that the two functional isoforms of DRP5B have highly overlapping functions. We further show that DRP5B harbors GTPase activity and moreover that PDV1 and PDV2 inhibits DRP5B-mediated GTP hydrolysis in a ratio dependent manner. Our data suggest that the PDV proteins contribute to the regulation of DRP5B activity thereby enforcing control over the division process during early constriction.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Chloroplasts/genetics , Dynamins/genetics , Gene Expression Regulation, Plant , Amino Acid Sequence , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Binding Sites/genetics , Chloroplasts/metabolism , Cytosol/metabolism , Dynamins/metabolism , Electrophoresis, Polyacrylamide Gel , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Gene Expression Profiling , Guanosine Triphosphate/metabolism , Hydrolysis , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Sequence Data , Mutation , Plants, Genetically Modified , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Two-Hybrid System TechniquesABSTRACT
Many bacteria produce antimicrobial substances such as nonribosomally synthesized antibiotics and ribosomally synthesized proteinaceous compounds referred to as bacteriocins. Secretion of antimicrobials is generally thought to contribute to the competitiveness of the producing organism, but there are indications that these compounds in some cases may have regulatory roles too. Bacteriocins most often act on closely related species only and are thus of interest for application as targeted narrow-spectrum antimicrobials with few side effects. Although the application of bacteriocins in plant disease control is an attractive option, very little is known about the occurrence and roles of these compounds in plant pathogenic bacteria and their natural competitors occurring in the same biotopes. This study presents an overview of current knowledge of bacteriocins from plant pathogenic bacteria.
Subject(s)
Bacteria/chemistry , Bacteriocins/metabolism , Plant Diseases/microbiology , Antibiosis , Bacteria/drug effects , Bacteria/metabolism , Bacteriocins/genetics , Bacteriocins/pharmacology , Peptides/metabolism , Peptides/pharmacologyABSTRACT
The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.
