Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES

Abstract

In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene. The complete DNA sequence of this groESL operon was determined. The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85-90% and 59-61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins. Southern blotting analyses with M. leprae groES- and groEL-specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis. This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene. Using five T-cell clones from two leprosy patients as probes, expression of the M. leprae GroES protein in Escherichia coli after heat shock was demonstrated. Four of these clones recognized the same M. leprae-specific GroES-derived peptide in a DR2-restricted fashion. No expression of the groEL gene from this operon was detected in E. coli after heat shock, as tested with a panel of T-cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.

Mycobacteria induce CD4+ T cells that are cytotoxic and display Th1‐like cytokine secretion profile: Heterogeneity in cytotoxic activity and cytokine secretion levels

“…These clones had been generated by in vitro stimulation of PBMC with M. leprue or M. tuberculosis bacilli [16,171. Subsequent analysis of the precise antigen specificities of these clones revealed that they recognized a wide range of mycobacterial antigens including hsp65, hsp70, and hsplO [16,19,21], non heat shock proteins (30/31 kDafibronectin binding protein, 15 kDa protein) [20,221 and other antigens of M. leprue or M. tuberculosis that have been defined using the "immunoblot" technique [19].…”

Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔCexpression strain

“…Unlike most other bacteria, mycobacteria possess two Hsp60 chaperone groEL genes, one of which is arranged in the bicistronic groESL operon [4]. M. smegmatis also encodes a third Hsp60 protein (Msmeg1978), which is more distantly related to GroEL1 (Msmeg1583) and GroEL2 (Msmeg0880) [3].…”

Identification and characterization of human B‐cell epitopes in recombinant antigens of Leishmania aethiopica

“…DNA sequence analysis were performed either by subcloning of the inserts into the Eco RI sites of pUC13 with subsequent double stranded dideoxy DNA sequencing using 35 S-dATP (Amersham) and a Taq polymerase sequencing kit (Promega), supplemented with deaza dideoxynucleotides triphosphates and pUC forward and reverse primers (Rinke de Wit et al 1992), or by using single stranded template obtained from PCR amplified inserts with biotinylated gt11 forward or reverse primers (obtained from the Biotechnology Centre, University of Oslo, Norway). Biotinylated single stranded template was isolated using streptavidin coupled to magnetic beads (Dynal A/S, Oslo, Norway) as described by the manufacturer.…”