Symbiosis between the bobtail squid Euprymna scolopes (Mollusca: Cephalopoda) and Vibrio fischeri bacteria has been a well-studied model for understanding the molecular mechanisms of colonization and adherence to host cells. For example, pilin expression has been observed to cause subtle variation in colonization for a number of Gram-negative bacteria with eukaryotic hosts. To investigate variation amongst pil genes of closely related strains of vibrios, we amplified pil genes A, B, C and D to determine orientation and sequence similarity to other symbiotic vibrios. The pilA gene was found to be upstream from all other pil genes, and not contiguous with the rest of the operon. The pilB, pilC and pilD loci were flanked at the 3′ end by yacE, followed by a conserved hypothetical gene. DNA sequences of each pil gene were aligned and analysed phylogenetically using parsimony for both individual and combined gene trees. Results demonstrate that certain pil loci (pilB and pilD) are conserved among strains of V. fischeri, but pilC differs in sequence between symbiotic and free-living strains. Phylogenetic analysis of all pil genes gives better resolution of Indo-west Pacific V. fischeri symbionts compared with analysis of the 16S rRNA gene. Hawaiian and Australian symbiotic strains form one monophyletic tree, supporting the hypothesis that V. fischeri strain specificity is selected by the geographical location of their hosts and is not related to specific squid species.
The rplKAJL–rpoBC operon or β 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 (β subunit) and C (β′ 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 β operon of ‘Ca. L. africanus’. The β 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 β 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 β operon sequences of an Indian and a Japanese strain. Finally, the β 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 β 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.
This is the first study that estimates mycobacterial phylogeny using the maximum-likelihood method (PhyML-aLRT) on a seven-gene concatenate (hsp65, rpoB, 16S rRNA, smpB, sodA, tmRNA and tuf) and the super distance matrix (SDM) supertree method. Two sets of sequences were studied: a complete seven gene sequence set (set R, type strains of 87 species) and an incomplete set (set W, 132 species) with some missing data. Congruencies were computed by using the consense program (phylip package). The evolution rate of each gene was determined, as was the evolution rate of each strain for a given gene. Maximum-likelihood trees resulting from concatenation of the R and W sets resulted in a similar phylogeny, usually showing an early separation between slow-growing (SG) and rapidly growing (RG) mycobacteria. The SDM tree for the W set resulted in a different phylogeny. The separation of SG and RG was still evident, but it was located later in the nodes. The SG were therefore positioned as a subgroup of RG. Maximum-likelihood phylogenetic reconstruction was less affected by increasing the number of strains (with incomplete data), but did seem to cushion the variability of the evolution rate (ER), whereas the SDM method seemed to be more accurate and took into account both the differing ER values and the incomplete data. With regard to ER, it was observed that the 16S rRNA gene was the gene that displayed the slowest evolution, whereas smpB was the most rapidly evolving gene. Surprisingly, these two genes alone accurately separated the SG from the RG on the basis of their ER values. This study focused on the differences in ER between genes and in some cases linked the ER to the phenotypic classification of the mycobacteria.
Phytoplasmas were detected in cactus (Opuntia species) plants exhibiting witches'-broom disease symptoms in Yunnan Province, south-western China. Comparative and phylogenetic analyses of 16S rRNA gene sequences indicated that an overwhelming majority of the cactus-infecting phytoplasmas under study belonged to the peanut witches'-broom phytoplasma group (16SrII). Genotyping through use of computer-simulated restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed a remarkable genetic diversity among these cactus-infecting phytoplasma strains. Based on calculated coefficients of RFLP pattern similarities, seven new 16SrII subgroups were recognized, bringing the total of described group 16SrII subgroups to 12 worldwide. Geographical areas differed from one another in the extent of genetic diversity among cactus-infecting phytoplasma strains. The findings have implications for relationships between ecosystem distribution and the emergence of group 16SrII subgroup diversity.