Promoter Characterization in the AT-Rich Genome of the Obligate Endosymbiont “ Candidatus Blochmannia floridanus”

Abstract

The characterization of transcriptional start sites of 14 genes encoded by the extremely AT-rich genome of "Candidatus Blochmannia floridanus" revealed a high degree of conservation with the RpoD promoter consensus sequence of the free-living relative Escherichia coli. Moreover, in agreement with the presence of the alternative heat shock sigma factor RpoH in "Ca. Blochmannia," typical RpoH-dependent promoters were identified. However, no heat shock response resembling that of E. coli could be detected in "Ca. Blochmannia."In many cases obligate intracellular pathogens or mutualists have strongly size-reduced genomes which are also characterized by a very high AT content ranging from 70 to 80% (9, 16). "Candidatus Blochmannia" species are endosymbionts of carpenter ants which are required by the host for normal development and mainly contribute to nutritional upgrading of the host during larval and pupal stages (4, 13). With only 705 kb, the genome of "Candidatus Blochmannia floridanus," the endosymbiont of Camponotus floridanus, is about six times smaller than that of the free-living relative Escherichia coli and it has an AT content of 74%, characteristic of endosymbiotic bacteria (5). Due to the obligate intracellular location in the cytosol of specialized cells, the bacteriocytes, in the midgut or in the ovaries, the bacteria are considered to encounter little environmental change. However, very little is known about gene-regulatory phenomena in "Ca. Blochmannia." In a recent survey of transcription profiles of the endosymbiont during different developmental stages of the holometabolous host animals, only minor transcriptional changes were noted (15). This is in line with the fact that most of the transcription regulators present in free-living relatives within the Enterobacteriaceae have been lost in "Ca. Blochmannia floridanus" and only four dedicated transcription regulators and two sigma factors, RpoD and RpoH, were annotated (5).Similarly, virtually nothing is known about the cis-acting regulatory sequences in "Ca. Blochmannia floridanus." In other obligate cell-associated or intracellular bacteria exhibiting an extremely high AT content and reduced genomes such as pathogenic Mycoplasma pneumoniae or symbiotic "Candidatus Carsonella ruddii," interesting genomic features were observed. In M. pneumoniae the majority of all genes are arranged in long convergent gene clusters with only very short intergenic distances, suggesting very long transcription units (7). In "Ca. Carsonella ruddii," almost no intergenic regions are present and very long transcripts and massive translational coupling of the genes were suggested (1, 10). In contrast, in "Ca. Blochmannia floridanus" as well as in Buchnera aphidicola, the closely related obligate endosymbiont of aphids, overall gene organization resembles that of the free-living relative E. coli (5, 14). Here, numerous intergenic sequences of considerable length are present, and in many cases genes can be assigned to specific operons or consist of single transcription u...

Genomic analysis of the regulatory elements and links with intrinsic DNA structural properties in the shrunken genome of Buchnera

“…Moreover, when comparing the putative σ 32 regulons of these four Buchnera strains, they appeared to be very divergent, revealing that predicted σ 32 promoters are not tractable in Buchnera without any experimental data (Additional file 6). These results are quite important as regards the possible role of σ 32 in the AT-rich genomes of insect endosymbionts, characterised by mild transcriptional changes in response to heat shock [98,99]. It has been proposed that, in symbiotic genomes, this transcriptional regulator might control currently unknown stress signals, or it may have been transformed into an alternative vegetative σ factor in order to replace the lost σ factors (σ 24 , σ 28 , σ 38 and σ 54 ).…”

Versatile roles of the chaperonin GroEL in microorganism-insect interactions

“…These bacteria only retain the single vegetative sigma factor RpoD, which in these microorganisms must have taken over the task to express the groELS operon. This is in line with the observation that in Buchnera and Blochmannia , both possessing relatively large genomes between 650 and 800 Kb, there appears to be a tendency to replace RpoH‐dependent promoters by RpoD‐dependent promoters (Wilcox et al ., ; Stoll et al ., ). Thus, while the groESL operon was retained in all primary endosymbionts, the transcription machinery required for its expression appears to be changing with increasing genome reduction.…”

“…SOPE is able to respond to elevated temperature since induction of the groEL gene was observed under heat-shock conditions (Charles et al, 1997b). In contrast, despite the presence of RpoH, in Blochmannia and Buchnera, no typical heat-shock response is mounted any longer and expression of the groEL gene is not or relatively mildly influenced by high temperature (Wilcox et al, 2003;Stoll et al, 2009b). In fact, in several cases, it was shown that primary endosymbionts can be eliminated by rearing the respective animals at elevated temperature for some time (Ohtaka & Ishikawa, 1991;Sacchi et al, 1993;Heddi et al, 1999;Montllor et al, 2002;Fan & Wernegreen, 2013), and consequences of climate change for endosymbiont-bearing insect populations are discussed (Wernegreen, 2012).…”

Functional characterization of the principal sigma factor RpoD of phytoplasmas via an in vitro transcription assay

“…In addition, the upstream region of the molecular chaperone gene ( ibpA ) was used as a template (P ibpA ) because a previous study suggested that it is regulated by RpoD in an AT-rich bacterium ‘ Ca. Blochmannia floridanus’ 30 , while in some other bacteria, ibpA is reported to be regulated by an alternative heat shock sigma factor RpoH 31 32 . Our in vitro transcription assays revealed that RNAP Ec –RpoD OY produced specific transcripts from the four templates (P infC , P rplM , P rpsD , and P ibpA ), but did not produce a transcript from P rpoD ( Fig.…”