Isolation of Tn917 insertional mutants of Bacillus subtilis that are resistant to the protonophore carbonyl cyanide m-chlorophenylhydrazone

Quirk, Philip G.; Guffanti, Arthur A.; Clejan, Sanda; Cheng, Jianbo; Krulwich, Terry A.

doi:10.1016/0005-2728(94)90131-7

Cited by 23 publications

(21 citation statements)

References 39 publications

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“…B. subtilis response regulator YcbB has been originally described as one of the genes whose disruption increased cell resistance to protonophores (105). This effect remained enigmatic until a recent study showed that YcbB controls the glutamine utilization operon (113).…”

Section: Vol 188 2006 Domain Architectures Of Bacterial Response Rementioning

confidence: 99%

Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations

2006

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Section: Vol 188 2006 Domain Architectures Of Bacterial Response Rementioning

confidence: 99%

Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations

2006

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“…For example, some mutations may increase membrane permeability to this protonophore. Mutants of Gram-positive bacteria that are resistant to CCCP exhibit changes in fatty acid composition of membrane lipids (Krulwich et al, 1990;Quirk et al, 1994). Notwithstanding this, a screen using the protonophore DNP identified genes important for acid resistance in S. typhimurium (Foster & Bearson, 1994).…”

Section: Identification Of Tn611-interrupted Genes In Acid-sensitive mentioning

confidence: 99%

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

et al. 2005

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Mycobacterium smegmatis is able to grow and survive at acidic pH, and exhibits intracellular pH homeostasis under these conditions. In this study, the authors have identified low proton permeability of the cytoplasmic membrane, and high cytoplasmic buffering capacity, as determinants of intrinsic acid resistance of M. smegmatis. To identify genes encoding proteins involved in protecting cells from acid stress, a screening method was developed using the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP was used to suppress intrinsic acid resistance of M. smegmatis. The screen involved exposing cells to pH 5·0 in the presence of CCCP, and survivors were rescued at various time intervals on solid medium at pH 7·5. Cells capable of responding to intracellular acidification (due to CCCP-induced proton equilibration) will survive longer under these conditions than acid-sensitive cells. From a total pool of 5000 transposon (Tn611) insertion mutants screened, eight acid-sensitive M. smegmatis mutants were isolated. These acid-sensitive mutants were unable to grow at pH 5·0 in the presence of 1–5 μM CCCP, a concentration not lethal to the wild-type strain mc2155. The DNA flanking the site of Tn611 was identified using marker rescue in Escherichia coli, and DNA sequencing to identify the disrupted locus. Acid-sensitive mutants of M. smegmatis were disrupted in genes involved in phosphonate/phosphite assimilation, methionine biosynthesis, the PPE multigene family, xenobiotic-response regulation and lipid biosynthesis. Several of the acid-sensitive mutants were also defective in stationary-phase survival, suggesting that overlapping stress protection systems exist in M. smegmatis.

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“…This protein is related to the large family of thiol-specific antioxidant proteins (Chae et al, 1994), which includes alkylhydroperoxide reductase, a protein whose synthesis was also shown to be partially controlled by sulfate in E. coli (Quadroni et al, 1996). The N-terminal sequence of protein PA13 revealed 61 YO identity to the N-terminus of the orfM gene product from Bacillus subtilis (Quirk et al, 1994), and 70% identity to that of the Ssi6 protein in E. coli (Quadroni et al., 1996), both of unknown function. All of the remaining SSI proteins yielded sequences with no significant similarity to sequences in the databases.…”

Section: Sulfate Starvation-induced (551) Proteins Of P Aeruginosa Pa0mentioning

confidence: 99%

Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates

et al. 1998

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Pseudomonas aemginosa PA01 grew in defined synthetic medium with any of a broad variety of single sulfur sources, including sulfate, cysteine, thiocyanate, alkanesulfonates, organosulfate esters and methionine, but not with aromatic sulfonates, thiophenols or organothiocyanates or isothiocyanates. During growth with any of these compounds except sulfate, cysteine or thiocyanate, a set of I 0 sulfate starvation-induced (SSI) proteins was strongly upregulated, as observed by two-dimensional protein electrophoresis of total cell extracts. A comparable level of up-regulation was found for the hydrolytic enzyme arylsulfatase, which has previously been used as a marker enzyme for the sulfate starvation response. One of the SSI proteins was identified by N-terminal sequencing as a high-affinity periplasmic sulfate-binding protein, and another was related to thiol-specif ic antioxidants, but the N-terminal sequences of the other SSI proteins revealed no similarity to N-termini of proteins of known function, and they probably represent uncharacterized enzymes involved in sulfur scavenging when preferred sulfur sources are absent. T o study the role that cysteine biosynthetic intermediates play in the synthesis of these proteins in wiwo, we isolated mini-Tn5 transposon mutants of P. aeruginosa with insertions in the cysN and cysl genes, which encode subunits of ATP-sulfurylase and sulf ite reductase, respectively. These two genes were cloned and sequenced. cysl showed high similarity to the cognate gene in Escherichia coli, whereas cysN encoded a 693 kDa protein with two domains corresponding to the E. coli CysN and CysC proteins. Sulfate no longer repressed synthesis of the SSI proteins in cysN mutants, but repression was restored by sulfite; in the cysl mutant, sulfate, sulfite and sulfide all led to repression of SSI protein synthesis. This suggests that there are at least t w o independent corepressors of the sulfate starvation response in this species.Keywords : Pseudornonas aeruginosa, sulfate starvation, cysteine biosynthesis INTRODUCTIONCysteine biosynthesis by the sulfate assimilation pathway has been well-characterized in both plants and micro-organisms (Kredich, 1996 ; Leustek, 1996). It Abbreviations: 551, sulfate starvation-induced; SSIS, sulfate starvationinduced sti mu Ion.The GenBank accession numbers for the sequences reported in this paper are AF026066 (cysl) and AF035608 (cysDN).proceeds by activation of inorganic sulfate, followed by reduction of the intermediate phosphoadenosyl phosphosulfate to sulfite and hence to sulfide, and transfer of this molecule onto an organic backbone. In limnic and enteric environments, sulfate is present in excess of microbial requirements, but in aerobic soils sulfur is available almost exclusively ( > 95 % ) in organically bound form, and inorganic sulfate is rare (Autry & Fitzgerald, 1990 al., 1986). This cycling is assumed to be due to microbial activity in the soil, or to microbially released soil enzymes, but the mechanisms involved in sulfur soil cycling and in th...

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Isolation of Tn917 insertional mutants of Bacillus subtilis that are resistant to the protonophore carbonyl cyanide m-chlorophenylhydrazone

Cited by 23 publications

References 39 publications

Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations

Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations

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

Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates

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