The tufB-secE-nusG-rplKAJL-rpoB gene cluster of the liberibacters: sequence comparisons, phylogeny and speciation

Abstract

The rplKAJL-rpoBC operon or b operon is a classic bacterial gene cluster, which codes for proteins K, A, J and L of the large ribosomal subunit, as well as proteins B (b subunit) and C (b9 subunit) of RNA polymerase. In the early 1990s, the operon was obtained as a 2.6 kbp DNA fragment (In-2.6) by random cloning of DNA from periwinkle plants infected with the Poona (India) strain of the huanglongbing agent, later named 'Candidatus (Ca.) Liberibacter asiaticus'. DNA from periwinkle plants infected with the Nelspruit strain (South Africa) of 'Ca. L. africanus' was amplified with a primer pair designed from In-2.6 and yielded, after cloning and sequencing, a 1.7 kbp DNA fragment (AS-1.7) of the b operon of 'Ca. L. africanus'. The b operon of the American liberibacter, as well as the three upstream genes (tufB, secE, nusG), have now also been obtained by the technique of chromosome walking and extend over 4673 bp, comprising the following genes: tufB, secE, nusG, rplK, rplA, rplJ, rplL and rpoB. The sequence of the b operon was also determined for a Brazilian strain of 'Ca. L. asiaticus', from nusG to rpoB (3025 bp), and was found to share 99 % identity with the corresponding b operon sequences of an Indian and a Japanese strain. Finally, the b operon sequence of 'Ca. L. africanus' was extended from 1673 bp (rplA to rpoB) to 3013 bp (nusG to rpoB), making it possible to compare the b operon sequences of the African, Asian and American liberibacters over a length of~3000 bp, from nusG to rpoB. While 'Ca. L. africanus' and 'Ca. L. asiaticus' shared 81.2 % sequence identity, the percentage for 'Ca. L. americanus' and 'Ca. L. africanus' was only 72.2 %, and identity for 'Ca. L. americanus' and 'Ca. L. asiaticus' was only 71.4 %. The~3000 bp nusG-rpoB sequence was also used to construct a phylogenetic tree, and this tree was found to be identical to the known 16S rRNA gene sequence-based tree. These results confirm earlier findings that 'Ca. L. americanus' is a distinct liberibacter, more distantly related to 'Ca. L. africanus' and 'Ca. L. asiaticus' than 'Ca. L. africanus' is to 'Ca. L. asiaticus'. The dates of speciation have also been estimated.

Molecular characterization of ‘Candidatus Liberibacter' species/strains causing huanglongbing disease of citrus in Kenya

“…americanus' and 'Ca. L. asiaticus' was only 71.4% (Teixeira et al 2008). Beyond showing variation on the L10 protein sequence in the species, these results further indicate that the Kenyan strains of the proteobacterium were more closely related to 'Candidatus L. africanus' than the 'Candidatus L. asiaticus' in the amino acid sequences of the L10 and L12 ribosomal proteins.…”

“…Genes encoding the L10 protein of alpha subgroup proteobacteria have been sequenced and characterized in 'Candidatus L. africanus strain Nelspruit' from South Africa (Planet et al 1995), 'Candidatus L. africanus subsp. capensis' (Garnier et al 2000) in 'Candidatus L. asiaticus' (Asiatic strains) (Villechanoux et al 1993;Okuda et al 2005) and in 'Candidatus L. americanus' (Teixeira et al 2008). The L10 and L12 protein are transcribed from the rplKAJL-rpoBC ribosomal protein-RNA polymerase gene cluster.…”

“…Planet et al (1995) cloned and sequenced the rplKAL-rpoBc operon of the African proteobacteria, which was used for characterization of the African strain of HLB causing bacterium. Teixeira et al (2008) sequenced the β operon of the American liberibacter, and compared it with that of the African and Asian liberibacters. Other molecular studies on the proteobacteria have involved PCR detection based on the 16S ribosomal operon followed by digestion of the PCR products to distinguish between the African and Asiatic strain.…”

Incidence of Huanglongbing-Associated ‘CandidatusLiberibacter Asiaticus’ inDiaphorina citri(Hemiptera: Psyllidae) Collected from Plants for Sale in Florida

“…Doddapaneni et al (2008b) considered that speciation may have occurred 110-120 mya, before the breakup of Gondwana. Teixeira et al (2008) estimated that 'Ca. L. africanus and Ca.…”

Intragenomic homogeneity on Liberibacter 16S rDNA confirms phylogeny and explains ecological strategy

“…The insect/plant host species composition varying primarily by liberibacter species [8]. The earliest speciation event by molecular clock technique is 309 Ma [10], supported by biogeography to a putative Pangaean origin [11]. Six species have so far been discovered and named.…”