Identification of the pH-inducible, proton-translocating F1F0-ATPase (atpBEFHAGDC) operon of Lactobacillus acidophilus by differential display: gene structure, cloning and characterization.
The influence of low pH on inducible gene expression in Lactobacillus acidophilus was investigated by the use of differential display. Logarithmic phase cultures were exposed to pH 3.5 for various intervals, and RNA was isolated and reverse transcribed. The resultant cDNAs were subjected to PCR and the products were resolved by electrophoresis. Several cDNA products were induced after exposure to pH 3.5. One of these products, a 0.7 kb fragment, showed sequence similarity to bacterial atpBEF genes of the atp operon, whose genes encode the various subunits of the F1F0-ATPase. With the 0.7 kb differential display product as a probe, hybridizations with total RNA from untreated and acid-treated L. acidophilus verified the acid inducibility of this operon. The increase in atp mRNA induced by low pH was accompanied by an increase in the activity of the enzyme in membrane extracts. The full-length atp operon was sequenced, and its genes were in the order of atpBEFHAGDC, coding for the a, c, b, delta, alpha, gamma, beta and epsilon subunits respectively. The operon contained no i gene, but was preceded by a 122 bp intergenic space, which contained putative extended -10 and -35 promoter regions. Primer extension analysis of RNA from cultures that were shifted from pH 5.6 to pH 3. 5, and held for 0, 30 or 45 min, revealed that the transcriptional start site did not change position as a function of culture pH or time after exposure to pH 3.5. The primary structure and genetic organization indicated that the H+-ATPase of L. acidophilus is a typical F1F0-type ATPase. The similarity to streptococcal ATPases and the acid inducibility of this operon suggest that it may function in the ATP-dependent extrusion of protons and maintenance of cytoplasmic pH. Finally, the use of differential display RT-PCR was an effective approach to identify genes in L. acidophilus induced by an environmental stimulus.
Kullen
M J
MJ
Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, NC 27695-7624, USA.
Klaenhammer
T R
TR
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007779:Lactobacillus acidophilus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D006180:Proton-Translocating ATPases