Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone

Tran, Sieu L.; Rao, M. Sadashiva; Simmers, Cameron; Gebhard, Susanne; Olsson, Karen; Cook, Gregory M.

doi:10.1099/mic.0.27624-0

Cited by 37 publications

(30 citation statements)

References 39 publications

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“…The gene encoding 3-oxoacyl-[acylcarrier-protein] synthase was up-regulated 1.7-fold at pH 5.5 (fabH), and a fabH2 mutant was acid sensitive. In agreement with this observation, fatty acid metabolism was found to be an important component of the adaptation of Mycobacterium to growth at low pH (28,83).…”

Section: Discussionsupporting

confidence: 71%

“…In agreement with our observations, methionine biosynthesis appears to play a role in bacterial acid stress responses. In H. pylori, metB expression is activated at low pH by the ArsSR two-component system (67), and a metB-Tn mutant of Mycobacterium smegmatis was recovered in a screen designed to identify mutants unable to grow at low pH in the presence of a proton-motive force uncoupler (83). Finally, the main difference between an acid-resistant mutant of Bifidobacterium longum and its more sensitive parent is the overproduction of MetE, CysD, and MetB in the mutant (75).…”

Section: Discussionmentioning

confidence: 99%

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Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

Reid

Pandey

Palyada

et al. 2008

Appl Environ Microbiol

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In order to cause disease, the food-and waterborne pathogen Campylobacter jejuni must face the extreme acidity of the host stomach as well as cope with pH fluctuations in the intestine. In the present study, C. jejuni NCTC 11168 was grown under mildly acidic conditions mimicking those encountered in the intestine. The resulting transcriptional profiles revealed how this bacterium fine-tunes gene expression in response to acid stress. This adaptation involves the differential expression of respiratory pathways, the induction of genes for phosphate transport, and the repression of energy generation and intermediary metabolism genes. We also generated and screened a transposon-based mutant library to identify genes required for wild-type levels of growth under mildly acidic conditions. This screen highlighted the important role played by cell surface components (flagella, the outer membrane, capsular polysaccharides, and lipooligosaccharides) in the acid stress response of C. jejuni. Our data also revealed that a limited correlation exists between genes required for growth under acidic conditions and genes differentially expressed in response to acid. To gain a comprehensive picture of the acid stress response of C. jejuni, we merged transcriptional profiles obtained from acid-adapted cells and cells subjected to acid shock. Genes encoding the transcriptional regulator PerR and putative oxidoreductase subunits Cj0414 and Cj0415 were among the few up-regulated under both acid stress conditions. As a Cj0415 mutant was acid sensitive, it is likely that these genes are crucial to the acid stress response of C. jejuni and consequently are important for host colonization.

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Section: Discussionsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

Reid

Pandey

Palyada

et al. 2008

Appl Environ Microbiol

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“…Indeed, studies examining the physiology of mycobacteria at low pH indicate that the cell wall plays a critical role in resistance to acid. A large number of the few M. tuberculosis and M. smegmatis acid-sensitive mutants identified so far have defects in genes involved in cell wall functions (92,95,96), and many cell wall or lipid biosynthesis genes are transcriptionally regulated upon exposure to low pH (29,73,80). In addition, mycobacteria do not appear to display a classical acid tolerance response, in which prior exposure to mildly acidic conditions protects the bacteria in a more acidic environment.…”

Section: The Role Of the Cell Envelope In Acid Resistancementioning

confidence: 99%

Acid Resistance inMycobacterium tuberculosis

2009

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“…Such strict coupling would imply that mycobacteria do not support uncoupled respiration: either they lack a conduit for proton reentry in the absence of the F 1 F o -ATP synthase or they are unable to adjust the proton permeability of the cytoplasmic membrane to allow a futile cycle of protons to operate. Recently, we have shown that the cytoplasmic membrane of M. smegmatis is extremely impermeable to protons (27).…”

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

2005

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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...

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Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone

Cited by 37 publications

References 39 publications

Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

Acid Resistance inMycobacterium tuberculosis

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

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Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone

Cited by 37 publications

References 39 publications

Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

Acid Resistance inMycobacterium tuberculosis

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

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