Identification of a key functional region in harpins from Xanthomonas that suppresses protein aggregation and mediates harpin expression in E. coli.
In the current study, we identified a key functional region in harpins from Xanthomonas that suppressed protein aggregation and mediated its expression in E. coli. Our data suggested that the presence of two common features in harpins [Wei et al. (1992) Science 257:85-88], namely, high glycine content and lack of cysteine residues, were not sufficient for Xanthomonas to elicit hypersensitive response (HR) activity or heat stability. Additionally, bioinformatic analyses revealed that the secondary structure of a conserved N-terminal region consisting of 12 highly hydrophilic amino acids (QGISEKQLDQLL) was alpha-helical. Following site-directed mutagenesis deletion of this region, the three mutated harpin proteins, in cultures induced at 37 degrees C, failed to elicit a HR in tobacco leaves. However, at 24 degrees C, two mutated harpins retained the ability to elicit HR, albeit with lower expression levels than that noted with the wild-type. SDS-PAGE and Western blot data suggested the HpaG mutant protein was found almost entirely in the inclusion body. These data demonstrated that these conserved amino acid residues played a critical role in protein aggregation and inclusion body formation in harpins from Xanthomonas.
Wang
Xiaoyu
X
Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China.
Li
Ming
M
Zhang
Jiahuan
J
Zhang
Yan
Y
Zhang
Guiying
G
Wang
Jinsheng
J
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D002479:Inclusion Bodies / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014026:Nicotiana / Q000235:genetics; D014974:Xanthomonas / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D050505:Mutant Proteins; C075462:harpin protein, Erwinia amylovora