title: Intracellular accumulation of high levels of gamma-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: uncoupling of gamma-aminobutyrate synthesis from efflux in a chemically defined medium.
abstract: It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular gamma-aminobutyrate (GABA(i)). The glutamate is then decarboxylated to GABA(i), a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA(e)) in response to acidification. In addition, high levels of GABA(i) (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA(i) in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA(i). These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.
authors.affiliation: Bacterial Stress Response Group, Microbiology, School of Natural Sciences, National University of Ireland-Galway, Ireland.
authors.forename: Kimon-Andreas G
authors.initials: KA
authors.lastname: Karatzas
authors.forename: Orla
authors.initials: O
authors.lastname: Brennan
authors.forename: Sinéad
authors.initials: S
authors.lastname: Heavin
authors.forename: John
authors.initials: J
authors.lastname: Morrissey
authors.forename: Conor P
authors.initials: CP
authors.lastname: O'Byrne
mesh_terms: D003470:Culture Media / Q000737:chemistry*; D003600:Cytosol / Q000737:chemistry*; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008089:Listeria monocytogenes / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D050296:Microbial Viability; D013312:Stress, Physiological; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D003470:Culture Media; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid
references.citation: Appl Environ Microbiol. 1992 Dec;58(12):3959-63
references.pmid: 1476439
references.citation: Appl Environ Microbiol. 2008 Feb;74(3):594-604
references.pmid: 18065622
references.citation: Nature. 1976 Jul 15;262(5565):231-4
references.pmid: 778641
references.citation: Int J Food Microbiol. 2007 Dec 15;120(3):250-8
references.pmid: 17935812
references.citation: J Bacteriol. 2006 Jan;188(2):556-68
references.pmid: 16385046
references.citation: Mol Microbiol. 1998 Jan;27(2):299-310
references.pmid: 9484886
references.citation: Antonie Van Leeuwenhoek. 2002 Aug;81(1-4):33-42
references.pmid: 12448703
references.citation: Nat Rev Microbiol. 2004 Nov;2(11):898-907
references.pmid: 15494746
references.citation: Biochem J. 1972 Nov;130(2):569-73
references.pmid: 4664581
references.citation: Mol Microbiol. 2001 Apr;40(2):465-75
references.pmid: 11309128
references.citation: J Bacteriol. 1996 Jul;178(13):3978-81
references.pmid: 8682809
references.citation: Mol Microbiol. 2002 Jul;45(2):569-83
references.pmid: 12123465
references.citation: Microbiol Mol Biol Rev. 2003 Sep;67(3):429-53, table of contents
references.pmid: 12966143
references.citation: Mol Microbiol. 1996 Sep;21(5):925-40
references.pmid: 8885264
references.citation: Microbiology (Reading). 1995 Jan;141 ( Pt 1):41-9
references.pmid: 7894718
references.citation: Appl Environ Microbiol. 2008 Nov;74(22):6848-58
references.pmid: 18806006
references.citation: J Food Prot. 2001 Sep;64(9):1362-8
references.pmid: 11563513
references.citation: Appl Environ Microbiol. 2005 Jun;71(6):2832-9
references.pmid: 15932974
references.citation: J Bacteriol. 1999 Jun;181(11):3525-35
references.pmid: 10348866