Hydrogenase-3 contributes to anaerobic acid resistance of Escherichia coli.
BACKGROUND
Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H(2) production involves consumption of 2H(+), hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2-2.5) that are three pH units lower than the pH limit of growth (pH 5-6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms.


RESULTS
We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H(2) to 2H(+). Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H(2) production and consumption was tested using a H(2)-specific Clark-type electrode. Hyd-3-dependent H(2) production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H(2) consumption was maximal at alkaline pH. H(2) production, was unaffected by a shift in external or internal pH. H(2) production was associated with hycE expression levels as a function of external pH.


CONCLUSIONS
Anaerobic growing cultures of E. coli generate H(2) via Hyd-3 at low external pH, and consume H(2) via Hyd-2 at high external pH. Hyd-3 proton conversion to H(2) is required for acid resistance in anaerobic cultures of E. coli.
Noguchi
Ken
K
Department of Biology, Kenyon College, Gambier, Ohio, United States of America.
Riggins
Daniel P
DP
Eldahan
Khalid C
KC
Kitko
Ryan D
RD
Slonczewski
Joan L
JL
D000143:Acids / Q000378:metabolism* / Q000494:pharmacology; D000693:Anaerobiosis; D056804:Biofuels; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005285:Fermentation; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000378:metabolism*; D011522:Protons
D000143:Acids; D056804:Biofuels; D029968:Escherichia coli Proteins; D011522:Protons; D006859:Hydrogen; D006864:Hydrogenase
J Bacteriol. 1988 Dec;170(12):5440-5
3056900
Appl Environ Microbiol. 2005 Nov;71(11):6762-8
16269707
Biochemistry (Mosc). 2003 Nov;68(11):1159-70
14640957
J Bacteriol. 2007 Aug;189(15):5601-7
17545292
Mol Syst Biol. 2006;2:2006.0008
16738554
Clinics (Sao Paulo). 2007 Feb;62(1):47-54
17334549
Appl Microbiol Biotechnol. 2007 Oct;76(5):1035-42
17668201
BMC Microbiol. 2006 Oct 06;6:89
17026754
Appl Environ Microbiol. 1995 Apr;61(4):1669-72
7747983
J Bacteriol. 1995 Jul;177(14):4097-104
7608084
J Bacteriol. 2005 Jan;187(1):304-19
15601715
J Bacteriol. 1999 Apr;181(7):2209-16
10094700
Eur J Biochem. 1986 Apr 15;156(2):277-84
3516690
Microbiology (Reading). 1997 Nov;143 ( Pt 11):3633-3647
9387241
Science. 2002 Nov 29;298(5599):1788-90
12459589
Antonie Van Leeuwenhoek. 1994;66(1-3):57-88
7747941
Infect Immun. 1993 Jan;61(1):364-7
8418063
Mol Microbiol. 1991 Jan;5(1):123-35
1849603
Eur J Biochem. 1969 Dec;11(2):316-27
4902610
Eur J Biochem. 1986 Apr 15;156(2):265-75
3516689
J Bacteriol. 2004 Jan;186(2):580-7
14702328
J Microbiol Methods. 2003 Nov;55(2):399-409
14529961
Nat Rev Microbiol. 2004 Nov;2(11):898-907
15494746
Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4586-91
10200306
J Bacteriol. 1983 Apr;154(1):336-43
6339476
Mol Microbiol. 1989 May;3(5):609-20
2527331
FEMS Microbiol Lett. 2008 Jan;278(1):48-55
17995952
J Infect Dis. 1969 Jun;119(6):641-9
4893893
J Appl Microbiol. 2007 Feb;102(2):352-62
17241340
J Bacteriol. 1957 Jun;73(6):706-21
13449036
J Bacteriol. 2004 Jan;186(1):192-9
14679238
J Bacteriol. 1985 Dec;164(3):1324-31
3905769
J Biol Chem. 2002 Dec 20;277(51):49945-51
12377778
Infect Immun. 2004 Nov;72(11):6294-9
15501756
J Bacteriol. 2006 Aug;188(15):5650-3
16855258
Mol Microbiol. 1991 Nov;5(11):2807-14
1779767
FEMS Microbiol Lett. 2001 Aug 7;202(1):121-4
11506918
Appl Environ Microbiol. 2004 Aug;70(8):4792-9
15294816
Cell Biochem Biophys. 2004;41(3):357-66
15509886
J Bacteriol. 1994 Mar;176(6):1729-37
8132468
J Bacteriol. 2007 Apr;189(7):2759-68
17259322
J Bacteriol. 1999 Jun;181(11):3525-35
10348866