The F ₁ F _o -ATP Synthase of Mycobacterium smegmatis Is Essential for Growth

Abstract

The F 1 F o -ATP synthase plays an important role in a number of vital cellular processes in plants, animals, and microorganisms. In this study, we constructed a ⌬atpD mutant of Mycobacterium smegmatis and demonstrated that atpD encoding the ␤ subunit of the F 1 F o -ATP synthase is an essential gene in M. smegmatis during growth on nonfermentable and fermentable carbon sources.The F 1 F o -ATP synthases of bacteria play an important role in a number of vital cellular processes (3,22). In aerobic bacteria, these enzymes are responsible for ATP generation via oxidative phosphorylation, leading to large amounts of ATP synthesized per substrate oxidized. In anaerobic bacteria, under nonrespiratory growth conditions, these enzymes work primarily as an ATPase, pumping protons to generate a proton motive force, and in some cases, this is coupled to pH homeostasis to prevent intracellular acidification (5, 10).A recent study reported that the F 1 F o -ATP synthase of mycobacteria is the target of the first new antituberculosis drug family (diarylquinolines) to be discovered in 40 years (1). The authors suggest that the drug leads to ATP depletion and an imbalance in pH homeostasis in mycobacterial species, both contributing to decreased ability to survive. Other studies have implicated a potential role for the F 1 F o -ATP synthase in the physiology of mycobacteria at acidic pH (8,17,18). In this study, we sought to establish a role for this enzyme in the physiology of Mycobacterium smegmatis as a model for understanding its potential role in the biology of other mycobacterial species.The atpD gene encoding the ␤ subunit of the F 1 F o -ATP synthase is an essential gene in M. smegmatis. The putative atp operon coding for the F 1 F o -ATP synthase of M. smegmatis was identified in an unfinished genome database (www.tigr.org). The DNA sequence shows that this operon is similar to those of many bacteria and is colinear to the atp operon of Mycobacterium tuberculosis with the gene order atpBEFHAGDC. In order to disable the F 1 F o -ATP synthase of M. smegmatis, we chose to disrupt the atpD gene by allelic exchange mutagenesis by using a strategy adapted from Pelicic et al. (16).M. smegmatis strain mc 2 155 (23) and derived mutants (Table 1) were grown with agitation at 37°C in either Luria-Bertani medium supplemented with 0.05% (wt/vol) Tween 80 (Sigma Chemicals) (LBT) or Middlebrook 7H9 broth (Difco Laboratories, Detroit, Mich.) supplemented with sterile Middlebrook ADC enrichment (Becton Dickinson, Cockeysville, Md.). For solid medium, Middlebrook 7H11 was supplemented with ADC and glycerol (0.5% vol/vol) or LBT with 1.5% agar. Unless otherwise stated, M. smegmatis transformants were grown at 28°C for temperature-sensitive vector propagation and 40 to 42°C for allelic exchange mutagenesis. All subcloning steps were performed with Escherichia coli strain DH10B (Table 1) with culturing at 37°C in LB broth or 2ϫ YT broth at an initial pH of 7.0 and LB agar (19). The E. coli plasmids used in this study are listed in Table 1...

Novel regulatory roles of cAMP receptor proteins in fast-growing environmental mycobacteria

“…These two PCR products were used as a template for overlap extension PCR (Ho et al, 1989) and the product was then cloned into the SpeI site of the pX33 vector (Gebhard et al, 2006), the pPR23-derived vector (Pelicic et al, 1997), resulting in pHLA13, and transformed into M. smegmatis mc 2 155. The crp1 gene was then deleted using the two-step method for integration and excision of the plasmid as described previously (Tran & Cook, 2005). Similarly, a 1012 bp fragment upstream of Crp2 including a 156 bp coding sequence was amplified with primers HLA39 and HLA40, and a 926 bp fragment downstream of Crp2 including a 102 bp coding sequence was amplified with primers HLA40 and HLA41.…”

An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia

“…It remains unclear whether any Mycobacterium can use fermentation during oxygen limitation. This genus is generally modeled to be strictly respiratory, given their F 1 F o -ATP synthase is required for growth even on fermentable carbon sources (16,17). However, a recent observation of succinate secretion during hypoxia suggests that some mycobacteria may in fact have the capacity to excrete fermentative end products (9,12).…”

The Alanine Racemase of Mycobacterium smegmatis Is Essential for Growth in the Absence of d -Alanine

“…The flanking regions (1,000 bp) of the alr gene were PCR amplified using the primer pair AlrKO1 and AlrKO2 and the pair AlrKO3 and AlrKO4 ( Table 1). The resulting amplicons were then ligated simultaneously with a kanamycin resistance cassette (KpnI-HindIII fragment containing aphA-3 from pUC18K) into the BamHI-XbaI-digested delivery vector pPR23 (19,30), thus creating pDM2 (Fig. 1).…”

“…The alr gene was inactivated in M. smegmatis mc 2 155 (23) by replacement of 99% of its coding sequence with the kanamycin resistance cassette from pUC18K (Table 1) using standard protocols for growth and mutagenesis as previously described (30). All general cloning steps were performed in Escherichia coli DH10B with culturing at 37°C in LB broth at an initial pH of 7.0 or on LB agar plates (20).…”