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Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene.

ABSTRACT: The dnaK gene region of Clostridium acetobutylicum was cloned in Escherichia coli by using the pBluescript SK+ and pUC18 vectors. By using the E. coli dnaK gene as a probe and by in vivo chromosome walking, three positive clones harboring the recombinant plasmids pKG1, pKG2, and pKG3 containing 1.2-kbp HindIII, 3.55-kbp EcoRV, and 1.2-kbp PstI fragments of the chromosome of C. acetobutylicum, respectively, were isolated. The cloned fragments partially overlapped, and together they spanned 4,083 bp of the clostridial genome that were completely sequenced. On one strand, four open reading frames of which the last was obviously truncated were identified. The last three genes showed high homology to the grpE, dnaK, and dnaJ heat shock genes of E. coli, respectively. They were preceded by an open reading frame (orfA) without any homology to sequences available in the EMBL or GenBank data bases. Typical translational start sites could be found in front of all four genes. Northern (RNA) blot analysis revealed transcripts of this region with a maximum length of 5.0 kb. Thus, these genes are probably organized in an operon. A transcription terminator could be found between the dnaK and dnaJ genes. By primer extension analysis, a major heat-inducible transcription start site was identified 49 bases upstream of orfA. This site was preceded by a region (5'-TTGACA[17 bp]TATTTT) that exhibited high homology to the consensus promoter sequences of gram-positive bacteria as well as sigma 70-dependent E. coli. Between this promoter and the initiation codon of orfA, a hairpin-loop structure with a possible regulatory role in the expression of these genes was found. Additional heat-inducible transcription start sites were located 69 bases upstream of orfA and 87 bases upstream of grpE; the corresponding promoter regions showed less similarity to other known promoter sequences. Maximum mRNA levels of this heat shock operon were found about 15 min after a heat shock from 30 to 42 degrees C. Our results indicate that orfA codes for an unknown heat shock protein.

SUBMITTER: Narberhaus F

PROVIDER: S-EPMC205998 | biostudies-other | 1992 May

REPOSITORIES: biostudies-other

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Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene.

Narberhaus F F Giebeler K K Bahl H H

Journal of bacteriology 19920501 10

The dnaK gene region of Clostridium acetobutylicum was cloned in Escherichia coli by using the pBluescript SK+ and pUC18 vectors. By using the E. coli dnaK gene as a probe and by in vivo chromosome walking, three positive clones harboring the recombinant plasmids pKG1, pKG2, and pKG3 containing 1.2-kbp HindIII, 3.55-kbp EcoRV, and 1.2-kbp PstI fragments of the chromosome of C. acetobutylicum, respectively, were isolated. The cloned fragments partially overlapped, and together they spanned 4,083 ...[more]

PMID: 1577695

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Project description:Antigens (Ags) in Mycobacterium tuberculosis (Mtb) that are constitutively expressed, overexpressed during growth, essential for survival, and highly conserved may be good vaccine targets if they induce the appropriate anti-Mtb Th1 immune response. In this context, stress response-related antigens of Mtb might serve as attractive targets for vaccine development as they are rapidly expressed and are up-regulated during Mtb infection in vivo. Our group recently demonstrated that GrpE, encoded by rv0351 as a cofactor of heat-shock protein 70 (HSP70) in the DnaK operon, is a novel immune activator that interacts with DCs to generate Th1-biased memory T cells in an antigen-specific manner. In this study, GrpE was evaluated as a subunit vaccine in comparison with the well-known HSP70 against the hyper-virulent Mtb Beijing K-strain. Both HSP70- and GrpE-specific effector/memory T cells expanded to a similar extent as those stimulated with ESAT-6 in the lung and spleen of Mtb-infected mice, but GrpE only produced a similar level of IFN-γ to that produced by ESAT-6 stimulation during the late phase and the early phase of Mtb K infection, indicating that GrpE is highly-well recognised by the host immune system as a T cell antigen. Mice immunised with the GrpE subunit vaccine displayed enhanced antigen-specific IFN-γ and serum IgG2c responses along with antigen-specific effector/memory T cell expansion in the lungs. In addition, GrpE-immunisation markedly induced multifunctional Th1-type CD4+ T cells co-expressing IFN-γ, TNF-α, and IL-2 in the lungs of Mtb K-infected mice, whereas HSP70-immunisation induced mixed Th1/Th2 immune responses. GrpE-immunisation conferred a more significant protective effect than that of HSP70-immunisation in terms of bacterial reduction and improved inflammation, accompanied by the remarkable persistence of GrpE-specific multifunctional CD4+ T cells. These results suggest that GrpE is an excellent vaccine antigen component for the development of a multi-antigenic Mtb subunit vaccine by generating Th1-biased memory T cells with multifunctional capacity, and confers durable protection against the highly virulent Mtb K.

Dataset Information

Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene.

Publications

Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene.

Similar Datasets

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