|ZHENG, Z - South China Agricultural University|
|XU, M - South China Agricultural University|
|BAO, M - South China Agricultural University|
|WU, F - South China Agricultural University|
|DENG, X - South China Agricultural University|
Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/16/2016
Publication Date: 12/31/2016
Citation: Zheng, Z., Xu, M., Bao, M., Wu, F., Chen, J., Deng, X. 2016. Unusual five copies and dual forms of nrdB in “Candidatus Liberibacter asiaticus”: biological implications and PCR detection. Scientific Reports. 6:39020. doi:10.1038/srep39020.
Interpretive Summary: Citrus Huanglongbing (HLB, yellow shoot disease, also known as citrus greening disease) is currently threatening citrus production worldwide. HLB is associated with an unculturable bacterium “Candidatus Liberibacter asiaticus” (CLas). Research in CLas biology is challenging because the bacterium cannot be cultivated in artificial media, a standard technique for bacteriological research. Effective HLB management relies on comprehensive knowledge of CLas. In this study, the whole genome sequence of CLas strain A4, originating from Guangdong, China, was analyzed. A unique gene (nrdB) was found to have unusually high copy number of five in two forms among bacteria in general. This gene encodes a protein called ribonucleotide reductase (RNR) ß-subunit, which is essential for bacterial growth. Analyses on RNR gene revealed several new biological features of the bacterium. The high copy number feature was further used to develop a sensitive and stable PCR detection system for CLas. A total of 262 HLB samples collected from China and USA were evaluated to test a primer set based on this gene. The RNR system tripled the detection sensitivity of the currently used primer set. It is expected that the RNR detection system will significantly enhance current efforts for early and accurate HLB diagnosis.
Technical Abstract: “Candidatus Liberibacter asiaticus” (CLas), an alpha-proteobacterium, is associated with citrus Huanglongbing (HLB, yellow shoot disease), which is currently threatening citrus production worldwide. Research in CLas biology is challenging because the bacterium cannot be cultivated in vitro. In this study, the whole genome sequence of CLas strain A4 originating from Guangdong of China was analyzed. Five copies of nrdB, encoding ß-subunit of ribonucleotide reductase (RNR), a critical enzyme involving bacterial proliferation, were found. Three copies were in long form (nrdBL, 1,059 bp) and two were in short form (nrdBS, 378 bp). While nrdBL encoded all active sites of the enzyme, nrdBS, sharing >99% identity to 3’ end of nrdBL, did not contain any active site. Multi-copies and dual forms of nrdB were common to all known CLas, but all other known liberibacters (“Ca. L. africanus”, “Ca. L. americanus”, “Ca. L. solanacearum”, and Liberibacter crescent) had only a single copy of nrdB. This unique CLas character was speculated to be involved in regulation of RNR activity at the transcriptional level during cell proliferation. Phylogenetic analyses based on nrdB nucleotide and amino acid sequences showed a distinct monophyletic lineage of CLas in the eubacteria, similar to that based on 16S rRNA gene sequences. The multi-copy feature of nrdB was explored for sensitive and stable CLas detection. A nrdB-based primer set RNRf/RNRr was designed and evaluated in both SYBR Green and TaqMan real-time PCR formats against 262 HLB samples collected from China and U.S.A. Comparing to the current standard primer set HLBas/HLBar based on 16S rRNA gene sequence, RNRf/RNRr had Ct value drops of 1.68 (SYBR Green) and 1.77 (TaqMan), or tripling of detection sensitivity. Meanwhile, RNRf/RNRr was more than double the PCR detection stability of primer set LJ900f/LJ900r derived from multi-copy prophage.