title: Acquisition of uncharacterized sequences from Candidatus liberibacter, an unculturable bacterium, using an improved genomic walking method.
abstract: An effective PCR-based genomic walking approach is described to discover previously unknown flanking genomic DNA sequences from Candidatus Liberibacter asiaticus, an unculturable, phloem-limited bacterium. Using this technique, 8564bp of new DNA sequences were obtained from three genomic loci; tufB-secE-nusG-rplKAJL-rpoBC gene cluster, omp gene (outer membrane protein, Omp) and 16/23S rRNA gene in Ca. L. asiaticus. These, together with publicly available Ca. Liberibacter sequences, are clustered into five contigs and two singlets representing 24,477 non-redundant base pairs. BLAST annotation predicts 12 full-length genes, two partial genes and one pseudogene among these sequences. The sequences obtained in this study provide new genome information about Ca. Liberibacter that will facilitate development of new genome-based detection tools. The technique described here can also be employed to acquire new genomic information for other unculturable or fastidious organisms for which available sequences are limited or for filling sequence gaps between known flanking genomic DNA sequences.
authors: USDA-ARS, San Joaquin Valley Agricultural Science Center, Parlier, CA 93648, USA. hlin@fresno.ars.usda.gov
authors: Hong
authors: H
authors: Lin
authors: Harshavardhan
authors: H
authors: Doddapaneni
authors: Xianjing
authors: X
authors: Bai
authors: Jiqiang
authors: J
authors: Yao
authors: Xiaolong
authors: X
authors: Zhao
authors: Edwin L
authors: EL
authors: Civerolo
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D016386:Chromosome Walking / Q000379:methods*; D002957:Citrus / Q000382:microbiology; D004275:DNA, Ribosomal; D005798:Genes, Bacterial; D016680:Genome, Bacterial / Q000235:genetics*; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012230:Rhizobiaceae / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004275:DNA, Ribosomal