Cloning and characterization of three hypothetical secretion chaperone proteins from Xanthomonas axonopodis pv. citri.
Xanthomonas axonopodis pv. citri (Xac) causes citrus canker in plantations around the world and is of particular significance in Brazil where its incidence has risen exponentially over the past decade. Approximately one third of the predicted Xac open reading frames show no homology, or homology with very low score with that of known sequences. It is believed that Xac utilizes secretion systems to transfer virulence proteins into susceptible eukaryotic cells. This process is assisted by secretion chaperones that maintain virulence proteins partly or completely unfolded during translocation. We have cloned three of these hypothetical secretion chaperones: XAC0419 and XAC1346 from type III secretion system (TTSS) and XACb0033 from type IV secretion system (TFSS). All proteins were cloned in a pET23a vector (Novagen), expressed at 37 degrees C using a BL21(DE3)pLysS Escherichia coli strain and purified by ion exchange and gel-filtration chromatographic methods. Pure proteins were characterized using spectroscopic measurements: circular dichroism, and both static and lifetime emission fluorescence in the case of XACb0033. The analyzed proteins are stable at elevated temperatures (up to 65 degrees C) and exhibit alpha-helix content from approximately 30% (XACb003) to approximately 87% (XAC1346). XACb0033 exhibits lifetimes in the fluorescence experiments that indicate different neighborhoods for its tryptophan residues. These chaperones have the characteristics of TTSS and TFSS: all are small, with a high alpha-helix content, and without ATP-binding or ATP-hydrolyzing activity.
Tasic
Ljubica
L
Chemistry Institute, UNICAMP, P.O. Box 6154, Campinas, SP, Brazil. ljubica@iqm.unicamp.br
Borin
Paula F L
PF
Khater
Leti Cia
LC
Ramos
Carlos H I
CH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D002942:Circular Dichroism; D002957:Citrus / Q000382:microbiology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010935:Plant Diseases / Q000382:microbiology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D014774:Virulence; D053532:Xanthomonas axonopodis / Q000235:genetics* / Q000472:pathogenicity
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins