Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

Abstract

Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7?0-9?0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9?0. Growth still occurred at pH 9?5 but at a reduced rate. The expression of the pH-regulated F 0 F 1 ATPase operon (containing the eight genes atpBEFHAGDC) was induced at alkaline pH. A 7?5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9?0. The same occurred with a 1?2 kb transcript corresponding to the atpB gene. RT-PCR studies confirmed the alkaline pH induction of the F 0 F 1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F 0 F 1 operon, was expressed at a lower level than the polycistronic 7?5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F 0 F 1 operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional in C. glutamicum and Escherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetative s factor of C. glutamicum, whereas the "35 and "10 boxes of P-atp2 fitted the consensus sequence for s H -recognized Mycobacterium tuberculosis promoters C C / G GG A / G AC 17-22 nt C / G GTT C / G , known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F 0 F 1 operon is highly expressed at alkaline pH, probably using a s H RNA polymerase.

A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli

“…WLY78 nifB promoter is apparently recognized by E. coli σ 70 RNA polymerase in vivo . These data concur with previous studies where promoters of gram-positive bacteria, for example, Bacillus stereothermophilus [22] and Corynebacterium glutamicum [23], were shown to be functional in E. coli .…”

Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis

“…Among them are genes encoding subunits of the succinate dehydrogenase, the F 1 F 0 -ATPase, and rRNA genes. For several of them the expression was shown to be dependent on the growth rate which is in agreement with a lower expression at pH 6 and pH 9 at which we observed lower growth rates than at neutral pH [10]. The evaluation of differential transcript and protein patterns by the comparison with targets of transcriptional regulators in C. glutamicum unraveled numerous regulatory modules that are activated during pH acclimatization.…”

“…The F 1 F 0 ATPase was found to function as a proton exporter under acidic conditions in Enterococcus hirae and its role in pH homeostasis in other bacteria was discussed [7,9]. In C. glutamicum the atp gene cluster encoding the F 1 F 0 ATPase was found to be transcriptionally induced at alkaline pH under the control of the sigma factor SigH and subsequent studies indicated that the expression is correlated with growth rate rather than the pH value of the medium [10,11]. Furthermore a putative cobalt transporter encoded by the gene cg1447 was found to be important under alkaline conditions [12].…”

F1F0-ATP synthases of alkaliphilic bacteria: Lessons from their adaptations

“…This is an atpI gene that encodes a putative hydrophobic protein predicted to have 4 TMH. The AtpI protein sequence is not highly conserved across species [76]. In some instances atpI is under control of a separate promoter from the other atp genes [76], in some instances atpI is absent entirely [77] and in some instances atpI is absent but a different gene of unknown function takes its usual place [78].…”

“…The AtpI protein sequence is not highly conserved across species [76]. In some instances atpI is under control of a separate promoter from the other atp genes [76], in some instances atpI is absent entirely [77] and in some instances atpI is absent but a different gene of unknown function takes its usual place [78]. Gay and Walker suggested a chaperone/assembly type of function for the AtpI protein when they first described the atpI gene [79].…”