Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon and analysis of its genetic structure, characteristics, and phylogeny

Abstract

The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F(1)F(0)-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.

FIG. 1.

Comparison of the atp operon of B. lactis DSM 10140 with the corresponding operons of different bacteria. The putative function of the protein is indicated above each arrow. Related proteins are linked by light blue shading (≥52%) and dark blue shading (≤51%) according to the different levels of amino acid similarity; the levels of amino acid identity (expressed as percentages) are indicated. The length of each arrow is proportional to the length of the predicted ORF. Corresponding genes are indicated by arrows that are the same color.

FIG. 2.

Northern hybridization analysis of B. lactis RNA and transcription unit mapping of the atp operon. All predicted ORFs are indicated and are annotated with their database matches. The transcripts are indicated by arrows, and the arrows point toward the 3′ end of the mRNA. The estimated size of the mRNA is indicated. Hairpins indicate possible rho-independent terminators. The transcripts are positioned with respect to the genome map shown at the top. The gels show the results of Northern blot hybridization of RNA isolated from B. lactis DSM 10140 upon exposure to low pH at 100 min. Lane 1, RNA isolated from a culture after exposure to pH 6.0; lane 2, RNA isolated from a culture after exposure to pH 5.5; lane 3, RNA isolated from a culture after exposure to pH 4.0; lane 4, RNA isolated from a culture after exposure to pH 3.5. Hybridization was performed by using the probes corresponding to the ORFs shown in the gene map at the top. The molecular size calculated for the hybridization signal is indicated next to each autoradiograph.

FIG. 3.

(a) Primer extension analysis and comparison of the putative promoter sequences for the atp operon. Boldface type and underlining indicate −10 and −35 putative hexamers; the box indicates a TG doublet of an extended −10 sequence; and the asterisks indicate transcription start points. The start codon is at the right end of the sequence. (b) Slot blot hybridization of mRNA from cells incubated at pH 3.5 for various times. Each slot contained 20 μg of RNA, and all slots were probed with ³²P-labeled PCR products corresponding to the atpD gene. The graph shows the results of two different experiments. The numbers above the slot blot indicate the times of exposure of the B. lactis DSM 10140 culture at pH 3.5.

FIG. 4.

Neighbor-joining phylogenetic trees obtained by using the 16S rRNA genes (a) and the atpD genes (b). The scale bars indicate phylogenetic distances. Bootstrap values are indicated at the nodes for a total of 1,000 replicates. Boldface type indicates species in which horizontal gene transfer of the atpD gene is suspected. The strains and the accession numbers of the atpD genes and 16S rDNA sequences are shown in Table 1.

FIG. 5.

Factorial correspondence analysis of codon usage in various ORFs in Streptococcus (a), Lactococcus (b), and Listeria (c). The position of atpD is indicated by a cross.