Characterization and expression analysis of the groESL operon of Bartonella bacilliformis.
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.
Callison
Julie A
JA
Division of Biological Sciences, The University of Montana, Missoula, MT, 59812-4824, USA.
Battisti
James M
JM
Sappington
Kate N
KN
Smitherman
Laura S
LS
Minnick
Michael F
MF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D045503:Bartonella bacilliformis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D018833:Chaperonins / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C097970:GroESL protein, Bacteria; D018833:Chaperonins