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Comparative Study
. 2004 Oct;70(10):6197-209.
doi: 10.1128/AEM.70.10.6197-6209.2004.

Characterization of the groEL and groES loci in Bifidobacterium breve UCC 2003: genetic, transcriptional, and phylogenetic analyses

Marco Ventura  1 , Carlos Canchaya, Ralf Zink, Gerald F Fitzgerald, Douwe van Sinderen
Affiliations
Comparative Study

Characterization of the groEL and groES loci in Bifidobacterium breve UCC 2003: genetic, transcriptional, and phylogenetic analyses

Marco Ventura et al. Appl Environ Microbiol. 2004 Oct.

Abstract

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes, including the GroEL and GroES proteins. The groES and groEL genes are highly conserved among eubacteria and are typically arranged as an operon. Genome analysis of Bifidobacterium breve UCC 2003 revealed that the groES and groEL genes are located in different chromosomal regions. The heat inducibility of the groEL and groES genes of B. breve UCC 2003 was verified by slot blot analysis. Northern blot analyses showed that the cspA gene is cotranscribed with the groEL gene, while the groES gene is transcribed as a monocistronic unit. The transcription initiation sites of these two mRNAs were determined by primer extension. Sequence and transcriptional analyses of the region flanking the groEL and groES genes of various bifidobacteria revealed similar groEL-cspA and groES gene units, suggesting a novel genetic organization of these chaperones. Phylogenetic analysis of the available bifidobacterial groES and groEL genes suggested that these genes evolved differently. Discrepancies in the phylogenetic positioning of groES-based trees make this gene an unreliable molecular marker. On the other hand, the bifidobacterial groEL gene sequences can be used as an alternative to current methods for tracing Bifidobacterium species, particularly because they allow a high level of discrimination between closely related species of this genus.

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Figures

FIG. 1.
FIG. 1.
Comparison of the groEL and groES loci in B. breve UCC 2003 with the corresponding loci in different bacteria. Each arrow indicates an ORF. The length of the arrow is proportional to the length of the predicted ORF. Corresponding genes are indicated by the same color. Red indicates the groES gene; blue indicates the groEL gene; brown indicates the cspA gene; dark green indicates a gene encoding a putative response regulator; brilliant green indicates a gene encoding a putative transport regulator; yellow indicates a gene encoding a putative lyase; black indicates a gene encoding the putative ribosomal protein L33; grey indicates a gene encoding a hypothetical protein. The putative function of the protein is indicated above each arrow. Genes exhibiting ≥70% amino acid similarity are linked by blue shading, and genes exhibiting ≤69% amino acid similarity are linked by violet shading. The levels of amino acid identity, expressed as percentages, are indicated.
FIG. 2.
FIG. 2.
Heat shock induction of the B. breve UCC 2003 groEL and groES loci. Total RNA was isolated from B. breve UCC 2003 following exposure to various temperatures for specific times and was analyzed by slot blot hybridization. (a) All slots, each of which contained 25 μg of RNA from cells incubated for up to 150 min at a temperature from 37 to 50°C, were probed with 32P-labeled PCR products corresponding to the groEL gene. (b) All slots, each of which contained 25 μg of RNA from cells incubated for up to 150 min at 43°C, were probed with 32P-labeled PCR products corresponding to the groEL gene. (c) All slots, each of which contained 25 μg of RNA from cells incubated for up to 150 min at 43°C, were probed with 32P-labeled PCR products corresponding to the groES gene. The numbers above the slot blots indicate the incubation times (in minutes), while the temperatures are indicated on the right in panel a.
FIG. 3.
FIG. 3.
Northern blot analysis of the B. breve UCC 2003 groEL (a) and groES (b) loci. The mRNAs isolated from cultures maintained under normal or heat shock conditions were probed with PCR fragments corresponding to the groEL, groES, and cspA genes and genes located in intergenic or upstream regions. Schematic representations of the transcription maps of the groEL and groES loci are included. All predicted ORFs are indicated and are annotated with their database matches. The locations of the probes used are indicated by the lines below the gene maps. The transcripts are indicated by arrows, and the arrows point to the 3′ end of the mRNA. The estimated size of each transcript is indicated. Hairpins indicate possible rho-independent terminators. The transcripts are positioned with respect to the genome map shown above. The DNA probes used for hybridization are indicated as thin lines below the genome map. Each blot contained mRNA extracted from B. breve UCC 2003 maintained under normal or heat shock conditions. Lane 1, RNA isolated from a culture at the beginning of the experiments; lane 2, RNA isolated from a culture at 25 min upon a temperature shift; lane 3, RNA isolated from a culture at 50 min upon a temperature shift; lane 4, RNA isolated from a culture at 100 min upon a temperature shift; lane 5, RNA isolated from a culture at 150 min upon a temperature shift; lane 6, RNA isolated from a culture after 15 h upon a temperature shift. The estimated length of the transcript corresponding to the hybridization signal is indicated. hypoth, hypothetical open reading frame.
FIG. 4.
FIG. 4.
Determination of B. breve UCC 2003 cspA and groES gene transcription start sites by primer extension analysis. (a and b) Primer extension results obtained by using oligonucleotides targeting the 5′ ends of the groES and cspA genes. (c) Computer prediction of the secondary structure of the untranslated leader sequence of mRNA. (d) Comparison of the putative promoter sequences for the cspA and groES genes. Boldface type and underlining indicate the −10 and −35 putative hexamers; boldface type with an asterisk indicates the transcription start point; boldface type without asterisks indicates the start codon. DR, direct repeats; IR1 and IR2, inverted repeats.
FIG. 5.
FIG. 5.
Phylogenetic trees obtained by using the groEL (a) and groES (b) genes. The bar scales indicate phylogenetic distances. Bootstrap values are indicated for a total of 1,000 replicates. The trees were calculated by the neighbor-joining method as implemented in the neighbor module of PHYLIP.
FIG. 6.
FIG. 6.
Histograms showing the distribution of the chaperonin 60 gene (grey bars) and 16S rRNA (solid bars) pairwise DNA sequence identities for bifidobacteria. A total of 135 pairwise comparisons were used for each gene.
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