Comparison of Partial Citrate Synthase Gene (gltA) Sequences for Phylogenetic Analysis of Bartonella Species

Abstract

Nucleotide base sequence data were obtained for a 940-bp fragment of the citrate synthase-encoding gene @.A) of representatives of the eight validly described Burtonella species and seven uncharacterized Burtonella strains obtained from small mammals. Complete 16s rRNA gene sequences were also determined for the uncharacterized strains, and these sequences revealed that each strain had a unique sequence which was very similar to the sequences of the previously recognized Bartonellu species. A comparison of the gltA sequences of the different Burtonella species revealed that the levels of similarity between sequences were 83.8 to 93.5%, whereas comparisons of sequences obtained from different strains of the same species revealed that the levels of similarity were more than 99.8%. One of the uncharacterized strains had a gZtA sequence that matched the sequence of Burtonelk elizabethue, three uncharacterized strains had sequences which were more than 99.6% similar to each other (but less than 93.5% similar to any other sequence), and the remaining three uncharacterized strains each exhibited less than 93.5% sequence similarity to other Burtonella species or isolates. Phylogenetic trees were inferred from multiple alignments of both g U and 16s ribosomal DNA (rDNA) sequences. Whereas the proposed intra-Burtonella architecture of trees inferred from 16s rDNA sequence data by using both distance matrix and parsimony methods had virtually no statistical support, the trees inferred from the gk-4 sequence data contained four well-supported lineages in the genus. The gU-derived phylogeny appears to be more useful than the phylogeny derived from 16s rDNA sequence data for investigating the evolutionary relationships of Burtonella species, and the validity of the lineages identified by the gltA analysis is discussed in this paper.The family Bartonellaceae has recently been subjected to considerable taxonomic reassessment, which has resulted in (i) the transfer of members of the genus Rochalirnaea (formerly placed in the Rickettsiaceae) to the genus Bartonella (7), (ii) the unification of the two established genera in the family, the genera Bartonella and Grahamella, with the name of the former taking precedence (5), (iii) the proposal of several new species and subspecies (5,6), and (iv) the removal of the family from the order Rickettsiales (7). The restructured family now includes the single genus Bartonella, which contains eight species. A polyphasic approach was used to develop all of these proposals, and phylogenetic data derived from a comparison of 16s rRNA gene sequences provided important supporting evidence. These studies also revealed that the Bartonella species are very closely related and that they exhibit very high levels of 16s rRNA gene sequence similarity (>97.8%) and significant levels of DNA-DNA homology (16 to 67%) (5, 7, 8). The paucity of 16s rRNA gene sequence differences between species limits the usefulness of this tool for inferring intragenus phylogeny and, as in other closely related taxa, does n...

BARTONELLA ROCHALIMAEANDB. VINSONIISUBSP.BERKHOFFIIIN WILD CARNIVORES FROM COLORADO, USA

“…Any ITS-positive samples were further analyzed by two additional genes, citrate synthase gene (gltA) and cell division gene (ftsZ), using a nested PCR platform. Two pairs of specific primers were applied for each target (e.g., primers CS443F (GCT ATG TCT GCA TTC TAT CA; Birtles et al 1996) and CS1210R (GAT CYT CAA TCA TTT CTT TCC A; Gundi et al 2012) as the outer set and Bhcs.781p (GGG GAC CAG CTC ATG GTG G) and Bhcs.1137n (AAT GCA AAA AGA ACA GTA AAC A; Norman et al 1995) as the inner set for gltA; primers Bfp1 (ATT AAT CTG CAY CGG CCA GA) and Bfp2 (ACV GAD ACA CGA ATA ACA CC; Zeaiter et al 2002) were used as the outer set and the newly designed primers R83 (ATA TCG CGG AAT TGA AGC C); and L83 (CGC ATA GAA GTA TCA TCC C) as the inner set for ftsZ. Positive and negative controls were included in each PCR run to evaluate the presence of appropriately sized amplicons and contamination, respectively.…”

Phylogenetic Analysis of Bartonella Detected in Rodent Fleas in Yunnan, China

“…The citrate synthase sequence (gltA) is acknowledged as the phylogenetic marker for Bartonella (Birtles and Raoult, 1996;La Scola et al, 2003). To detect and identify Bartonella DNA, we used PCR assays and sequencing of the amplicons derived from the gltA gene of Bartonella species and compared the Bartonella species from the rodent fleas with the available Bartonella strains in GenBank.…”

“…Areas with a high prevalence of Bartonella infections in rodents are often characterized by the presence of a diversity of Bartonella species and strains (Birtles et al, 1994(Birtles et al, , 2001Kosoy et al, 1997). During the period 2001-2005, a high prevalence of Bartonella was demonstrated through isolation from rodents (Apodemus species, Eothenomys species, and Rattus species) from the west, northwest, southwest, south, and middle of Yunnan Province, China; isolates typically clustered according to the genus of rodent hosts (Ying et al, 2002;Li et al, 2004a;Bai et al, 2005).…”

The effect of ecological and temporal factors on the composition of Bartonella infection in rodents and their fleas

“…Bartonella organisms are fastidious, facultative intracellular Gram-negative bacteria belonging to the alpha-2-Proteobacteria class (Birtles and Raoult, 1996). Bartonellae establish long-term and often subclinical infections in their mammalian reservoirs, as an outcome of an apparent co-evolution between the bacteria and their hosts (Breitschwerdt and Kordick, 2000;Harms and Dehio, 2012).…”

“…Accordingly, the 454-pyrosequencing assay enables the detection of individual sequences obtained from the same source (Dowd et al, 2008), and through an adequate target sequence, it allows the characterization of a specific bacterial group. Thus, the citrate synthase gene (gltA) offers a reliable and specific target for distinguishing between closely and distantly related Bartonella lineages in mixed infections, due to its potent discriminatory power (Birtles and Raoult, 1996;La Scola et al, 2003), its stability (as a housekeeping gene), its extensive GenBank database and its single copy presence in the Bartonella genome (Guy et al, 2013).…”