Characterization and expression analysis of the groESL operon of Bartonella bacilliformis

Abstract

The groESL operon of Bartonella bacilliformis, a facultative intracellular, Gram-negative bacterium and etiologic agent of Oroya Fever, was characterized. Sequence analysis revealed an operon containing two genes of 294 (groES) and 1632 nucleotides (groEL) separated by a 55-nt intergenic spacer. The operon is preceded by a 72-nt ORF (ORF1) that encodes a hypothetical protein with homology to a portion of the HrcA repressor for groESL. A divergent fumarate hydratase C (fumC) gene lies further upstream. Deduced amino acid sequences for B. bacilliformis GroEL and GroES revealed a high degree of identity with homologues from other Bartonella and alpha-Protebacteria. A single transcriptional start site (TSS) was mapped 79 nucleotides upstream of the groES start codon, regardless of incubation temperature. The TSS was located immediately 5' to a potential controlling inverted repeat of chaperonin expression (CIRCE) element and is preceded by a sigma70-like promoter. The operon is followed by a predicted rho-independent transcriptional terminator. Northern blot analysis indicated that groES and groEL are co-transcribed as a single mRNA of approximately 2.4 kb. A 6-h time course analysis by qRT-PCR showed that groEL expression increases 1.3-fold within 30 min of a temperature upshift from 30 to 37 degrees C, with maximum transcription reached after 60 min (approximately 4.3-fold), followed by a steady decrease to background (30 degrees C) transcription levels by 6 h. Western blot analysis revealed a 1.4- and 1.5-fold increase in GroEL synthesis following a temperature upshift or by inhibiting DNA supercoiling with coumermycin A1, respectively. Functional expression and complementation of temperature-sensitive Escherichia coli groES or groEL mutants with the cloned operon allowed them to grow at otherwise restrictive temperatures.

Intruders below the Radar: Molecular Pathogenesis of Bartonella spp

“…GroEL was further found to localize to the outer membrane not only of B. bacilliformis but also of B. henselae, and it is highly immunogenic in various Bartonella species (43,84,229,357,432). In addition, it was recognized that Bartonella GroEL harbors a C-terminal phenylalanine that is a general characteristic of outer membrane proteins, indicating that GroEL of Bartonella would be adapted to exposure on the bacterial surface (69,409).…”

Carrion's Disease: the Sound of Silence

“…Cell proliferation is inhibited in the presence of anti-GroEL antibodies (177). The levels of expression of this protein are regulated by different factors, such as temperature or DNA supercoiling relaxation (178), which may be produced at different levels during the different infection phases. Furthermore, the increase in cell numbers could be caused by either increased cell division or reduced cell death.…”

Carrion’s disease: more than a neglected disease

“…Secretion of GroEL has also been described in Helicobacter pylori to protect secreted ureases [ 53 , 54 ]. The groESL operon is upregulated in response to thermal stress resulting in a ~4-fold induction of groEL expression by a temperature upshift from 30 °C to 37 °C comparable to the temperature shift occurring at the transmission event from sand fly vectors to the human host [ 55 ]. GroEL of B. bacilliformis increases apoptosis of human umbilical vein endothelial cells (HUVEC) [ 56 ] thereby possibly regulating the growth of endothelial cells.…”