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Buffering capacity and H+ membrane conductance of Gram-negative bacteria
ELSEVIER
FEMS Microbiology
Letters 130 (1995) 103-l 10
Buffering capacity and H+ membrane conductance of Gram-negative bacteria Nhia Rius, Montserrat Sol& Alicia Francis, Jo& G. L&n Departument de Microbiologia
i Parasitologia Sanitirries. Dit isi de Ci&cies de la S&t, 08028 Barcelona, Spain
*
Uniuersitat de Barcelona, Auda Joan XXIII s/n,
Received 24 January 1995; accepted 15 May 1995
Abstract Buffering capacity and membrane H’ conductance were examined in seven Gram-negative species: Aquaspirillum serpens, Pseudomonas aeruginosa, Alcaligenes faecalis, Escherichia coli, Salmonella typhimurium, Proteus mirabilis and Aeromonas hydrophila. All strains of Enterobacteriaceaestudied here showed a decreasein both parametersas the external pH increased,over the pH range studied. The other four speciespresentedan increasein buffering capacity and membrane conductanceto protons as the external pH increasedfrom 5.5 to 7.0. Keywords:
Buffering capacity: H+ membrane conductance; Gram-negative bacteria
1. Introduction Bacteria must respond to environmental stress during life in the biosphere. In particular, they can adapt to changes in external pH [1,2]. The ability of food-borne pathogens to adapt to acidic conditions is a concern in food safety [3-61. It has also been reported that the susceptibility of some enterobacteria to antibiotics change under acidic conditions [7,8]. Foster and Hall [9] proposed that acid adaptation may alter metabolism and enhance internal pH homeostasis. Krulwich et al. [lo] examined the buffering capacity of E. coli and of Gram-positive bacilli that grow at different pH ranges, but there are no comparative measurements of buffering capacity
or membrane conductance to protons of Gram-negative bacteria. We report here measurements of buffering capacity and passive H+ conductance over a wide range of pH, from 3.5 to 7.5, for the following seven Gramnegative species: three Enterobacteriaceae, E. coli, S. typhimurium and P. mirabilis, and four bacterial species distributed in water and the environment, A. serpens, P. aeruginosa, A. faecalis and A. hydrophila. We used the method in which the decay of an acid pulse is measured to determine both parameters [ll-131. 2. Materials and methods 2. I. Bacterial
strains and growth conditions
* Corresponding author. Tel: + 34-3-402 4497: Fax: + 34-3-402 1886.
Bacterial strains, media and growth conditions used in this study are listed in Table 1. All the
0378-1097/95/$09.50 0 199.5 Federation of European Microbiological SSDI 0378.1097(95)00191-3
Societies. All rights reserved
104
N. Rius et al. /FEMS Microbiology
Letters 130 (1995) 103-110
N. Rius et al./FEMS
Mrcrobioiogy Letters 130 (19951 103-110
105
1 . . .
.
P ,1.5Ol-
: \
00
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Aqtmspirlllum
5m-
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“+ 700 J
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6
6,5
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7.5
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3.5
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43
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7
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Fig. 2. B, and B, values for Aquaspirillum serpens. Pseudomonas aeruginosa, Alcaligenes faecalis and Aeromonas hydrophila over a range of external pH values. Aquaspirilfum serpens cell protein was 1.2 mg ml - * , Pseudomonas aeruginosa cell protein was 2.1 mg ml ’ , Alcaligenes faedis cell protein was 6.6 mg ml-’ and Aeromonas hydrophila cell protein was 6.9 mg ml-‘. The smooth cmves were obtained from a polynomic regression.
106
N. Rius et al./FEMS
Microbiology
Table 1 Bacterial species used and their growth conditions Organism
source
Temp. (“C)
Medium [ref]
A. serpens P. aeruginosa A. faecnlis E. coli S. typhimurium P. mirubilis A. hydrophila
ATCC 11335 PA0 I ATCC 19018 ATCC 10536 Mutton NCTC 5887 NCIB 9233
27 37 37 37 37 37 27
MPSS [16] TSB NB TSB TSB TSB TSB
Letters 130 (1995) 103-l 10
of these bacteria were measured by an acid-pulse technique, according to [13]. The pH recordings were analyzed graphically, as described elsewhere [ll-131. Measurements of buffering capacity and the observed half-time of approach to final equilibrium were used to calculate membrane Hf conductance 1111. Buffering capacity and passive H+ conductance are presented as functions of external pH.
TSB: Trypticase Soy Broth (BBL, Becton Dickinson and Co., Cockeysville, USA); NB: Nutrient Broth (Oxoid, Unipath Ltd.. Basingstoke, UK).
2.3. Measurement of cell protein Protein content was determined according to [14].
bacteria were grown with shaking except A. serpens and A. fueculis. The cells, grown to early stationary phase, were collected and washed three times with 300 mM KCl. The washed cells were treated with 3 mM EDTA and resuspended in 300 mM KC1 [12]. The final concentration of cell protein was 1-13 mg ml-’ (1 X lOlO- X 10” cfu ml-‘).
3. Results and discussion Fig. 1 and Fig. 2 show the measurements of buffering capacity of the cell surface (B,) and total buffering capacity (B,) of the seven bacterial species studied. Both the B, and the B, values were quite different from species to species. Three of the organisms exhibited B, values up to about 1200 nmol H+/pH unit per mg of protein, whereas the four others exhibited values that were at least double. It is also interesting that the Enterobacteriaceae showed different curves that described the behaviour of B, and B,, compared with the other Gram-negative species studied. The B, and B, of E. coli, S. ty-
2.2. Measurement of buffering capacity and membrane H + conductance Experiments were conducted on 7-ml samples of cell suspensions in lo-ml glass vials, which were magnetically stirred at room temperature. The pH of such suspensions was between 6.2 and 7.2. The buffering capacity and membrane conductance to H+
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
6
PM
Fig. 3. Cytoplasmic buffering capacity of the bacterial species studied. B, values were calculated from smooth curves that described the behavior of B, and B,.
N. Rim et al./FEMS
Microbiology
Letters 130 (1995) 103-110
.I-.... s l
l.
107
N. Rius et al. / FEMS Microbiology
108
Letters 130 (1995) 103-I 10
16
Aquarplrlllum
sorpenr
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Prudomorvr
16
.
314 3 h $2 2 f 110 5 ; ii6 5 t fe % 84
2
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PH
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. ,
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P"
Fig. 5. Membrane conductance to H+ of Aquaspirillum hydrophila.
6
3
3.5
4
435
5
5,s
6
6.5
7
PH
serpens, Pseudomonas aeruginosa, Alcaligenes faecalis
and Aeromonas
N. Rius et al. / FEMS Microbiology
phimurium and P. mirabilis generally increased with decreasing pH in the acid range of pH studied (Fig. 1). P. mirabilis presented higher values of these parameters than E. coli and S. typhimurium. The cell wall composition of P. mirubilis cells differs from that of other Enterobacteriaceae in that (i) a large percentage of the N-acetylmuramic residues of the peptidoglycan are substituted by Oacetyl groups and (ii) the lipopolysaccharide molecules have negatively charged components [ 151. These differences can explain the high B, and B, values of P. mirabilis cells. The other four bacterial species are oxidase positive Gram-negative rods that are distributed in water and the environment. Aquaspirillum serpens, Pseudomonas aeruginosa and Alcaligenes faecalis are aerobic, with a strictly respiratory type of metabolism, with oxygen as a terminal electron acceptor. P. aeruginosa can also use nitrate as an alternate electron acceptor. A. hydrophila is a facultatively anaerobic bacterium physiologically, genetically and taxonomically related to the enterobacteria studied here but their buffering capacity and H+ membrane conductance were comparable to the other three bacterial species found in water and the environment. All of them presented higher values of B, at pH 6.5 than at pH 5 (Fig. 2). With all the species, the B, values were appreciable and varied over the range of external pH values examined. Except for A. serpens, the B, of the cells decreased as external pH increased. There were no significant differences between cytoplasmic buffering capacity values for the Enterobacteriaceae and A. fuecalis studied here and the values found for Serratia marcescens [12] or for E. coli cells permeabilized with n-butanol [lo], over the pH range from 5.0 to 7.5 (Fig. 3). A. serpens, P. aeruginosa and A. hydrophila presented high values of Bi at their optimal pH for growth, but A. serpens showed Bi values that were 3-fold greater than those of the other two species. Passive H+ conductance of these species, as well as buffering capacity, was sensitive to proton concentration at the external surface, over the pH range studied. The membrane conductance to protons of the three enterobacteria increased as pH became more acidic at pH below 5.5 (Fig. 4). The smooth curves that described the behavior of Ci were com-
Letters 130 (1995) 103-l 10
109
parable to the curve reported for a pigmented strain of Serratia marcescens [12]. However, this pigmented strain showed CE values that were 2-fold greater than E. coli andS. typhimurium. The membrane H’ conductance and buffering capacity of P. mirabilis was higher than those found for the other Enterobacteriaceae. Ci values of the rest of the species studied were markedly different from data reported here for the Enterobacteriaceae. Our results of Cz values of A. faecalis were comparable to those found for a nonpigmented strain of Serratia marcescens [12]. A. serpens and P. aeruginosa exhibited a maximum value of CE at pH 6.5 and the Cz of A. serpens was 2-fold greater than that of P. aeruginosa. A. hydrophilu had a maximum at pH 4 and a minimum at pH 5.5 (Fig. 5). The experiments described in this paper show that buffering capacities and membrane H+ conductance of Enterobacteriaceae were higher at acidic external pH than at neutral pH, and that non-Enterobacteriaceae species presented higher values of CE at pH near neutrality than at pH 5.5. These two groups of bacteria can be found in different natural habitats, intestinal and urinary tract and water respectively, where they adapt to environmental changes. It has been reported that adaptative responses to acid for E. coli and S. typhimurium, involve changes in cell surface properties in addition to the enhancement of intracellular pH homeostasis [1,2,9]. Our results of buffering capacity for the Enterobacteriaceae agree well with these reports. There are no reports about or pH-homeostasis of chemopH-response heterotrophic Gram-negative bacteria, other than Enterobacteriaceae, over the pH range from 4 to 7.5, available in the literature. The results reported here contribute to the study of the response of bacteria to external pH changes. Acknowledgements We are grateful to Robin Rycroft for expert criticism during the preparation of this manuscript. References [ll Heyde, M. and Portalier, R. (1990) Acid shock proteins of Escherichia coii. FEMS Microbial. Lett. 69, 19-26.
110
N Rius et al./FEMS
Microbiology
[21 Leyer, G.J. and Johnson, E.A. (1993) Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl. Environ. Microbial. 59, 1842-1847. [3] Brown, M.H. and Booth, I.R. (19911 Acidulants and low pH. In: Food Preservatives (Russell, N.J. and Gould, G.W., Eds.), pp. 22-43. Blackie, Glasgow, Scotland. [4] Foster, J.W. and Hall, H.K. (19901 Adaptative acidification tolerance response of Salmonella typhimurium. J. Bacterial. 172, 771-778. [5] Goodson, M. and Rowbury. R.J. (19891 Habituation to normally lethal acidity by prior growth of Escherichia coli at a sub-lethal acid pH value. Appl. Microbial. Len. 8, 77-79. [6] Kroll, R.G. and Patchett. R.A. (1992) Induced acid tolerance in Lisreria monocytogenes. J. Appl. Microbial. 14, 224-227. [7] Laub, R., Schneider Y-J. and Trouet, A. (19891 Antibiotic susceptibility of Salmonella spp. at different pH values. T. Gen. Microbial. 135, 1407-1416. IS] Sawai, T., Hiruma, R., Kawana, N., Kaneko, M., Taniyasu, F. and Inami, A. (1982) Outer membrane permeation of /3-lactam antibiotics in Escherichia coli, Profeus mirabilis and Enterobacter cloacae. Antimicrob. Agents Chemother. 22, 585-592. [9] Foster, J.W. and Hall, H.K. (19911 Inducible pH homeostasis
Letters 130 (19951 103-110
ilO]
[l I] [121
[13]
[14]
[15]
[16]
and the acid tolerance response of Salmonella typhimurium. J. Bacterial. 173, 5129-5135. Krulwich, T.A., Agus, R., Schneier, M. and Guffanti, A.A. (1985) Buffering capacity of bacilli that grow at different pH ranges. J. Bacterial. 162, 768-772. Maloney, P. (1979) Membrane H+ Conductance of Streptococcus la&s. J. Bacterial. 140, 197-205. Rius, N., Sole, M., Francis, A. and Loren, J.G. (19941 Buffering capacity of pigmented and non-pigmented strains of Serraria marcescens. Appl. Environ. Microbial. 60, 2152-2154. Rius, N., Sole, M., Francis, A. and L&n, J.G. (1994) Buffering capacity and membrane H+ conductance of lactic acid bacteria. FEMS Microbial. L&t. 120, 291-296. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Kotelko, K. (1986) Proteus mirabilis: taxonomic position, peculiarities of growth, components of the cell envelope. Curr. Topics Microbial. Immunol. 129, 181-215. Krieg, N.R. (1984) Aerobic/microaerophilic motile, helical/vibrioid Gram negative bacteria. In: Bergey’s Manual of Systematic Bacteriology (Krieg, N.R. and Holt, J.G., Eds.), pp. 71-93. The Williams & Wilkins Co., Baltimore.
FEMS Microbiology
Letters 130 (1995) 103-l 10
Buffering capacity and H+ membrane conductance of Gram-negative bacteria Nhia Rius, Montserrat Sol& Alicia Francis, Jo& G. L&n Departument de Microbiologia
i Parasitologia Sanitirries. Dit isi de Ci&cies de la S&t, 08028 Barcelona, Spain
*
Uniuersitat de Barcelona, Auda Joan XXIII s/n,
Received 24 January 1995; accepted 15 May 1995
Abstract Buffering capacity and membrane H’ conductance were examined in seven Gram-negative species: Aquaspirillum serpens, Pseudomonas aeruginosa, Alcaligenes faecalis, Escherichia coli, Salmonella typhimurium, Proteus mirabilis and Aeromonas hydrophila. All strains of Enterobacteriaceaestudied here showed a decreasein both parametersas the external pH increased,over the pH range studied. The other four speciespresentedan increasein buffering capacity and membrane conductanceto protons as the external pH increasedfrom 5.5 to 7.0. Keywords:
Buffering capacity: H+ membrane conductance; Gram-negative bacteria
1. Introduction Bacteria must respond to environmental stress during life in the biosphere. In particular, they can adapt to changes in external pH [1,2]. The ability of food-borne pathogens to adapt to acidic conditions is a concern in food safety [3-61. It has also been reported that the susceptibility of some enterobacteria to antibiotics change under acidic conditions [7,8]. Foster and Hall [9] proposed that acid adaptation may alter metabolism and enhance internal pH homeostasis. Krulwich et al. [lo] examined the buffering capacity of E. coli and of Gram-positive bacilli that grow at different pH ranges, but there are no comparative measurements of buffering capacity
or membrane conductance to protons of Gram-negative bacteria. We report here measurements of buffering capacity and passive H+ conductance over a wide range of pH, from 3.5 to 7.5, for the following seven Gramnegative species: three Enterobacteriaceae, E. coli, S. typhimurium and P. mirabilis, and four bacterial species distributed in water and the environment, A. serpens, P. aeruginosa, A. faecalis and A. hydrophila. We used the method in which the decay of an acid pulse is measured to determine both parameters [ll-131. 2. Materials and methods 2. I. Bacterial
strains and growth conditions
* Corresponding author. Tel: + 34-3-402 4497: Fax: + 34-3-402 1886.
Bacterial strains, media and growth conditions used in this study are listed in Table 1. All the
0378-1097/95/$09.50 0 199.5 Federation of European Microbiological SSDI 0378.1097(95)00191-3
Societies. All rights reserved
104
N. Rius et al. /FEMS Microbiology
Letters 130 (1995) 103-110
N. Rius et al./FEMS
Mrcrobioiogy Letters 130 (19951 103-110
105
1 . . .
.
P ,1.5Ol-
: \
00
I ml.OOC-
Aqtmspirlllum
5m-
0) 33
“+ 700 J
wrponr
P88UdOmOM8
]
/
,
i
,
,
/
4
43
5
5,s
6
6.5
7
7,s
5
a
88rUghO88
8 65
5.5
7
73
6
.
100
1 3
$5
4
4,s
5
$5 PM
6
6,5
7
7.5
a
3
3.5
4
43
5
5,s
6
6.5
7
7.5
6
PM
Fig. 2. B, and B, values for Aquaspirillum serpens. Pseudomonas aeruginosa, Alcaligenes faecalis and Aeromonas hydrophila over a range of external pH values. Aquaspirilfum serpens cell protein was 1.2 mg ml - * , Pseudomonas aeruginosa cell protein was 2.1 mg ml ’ , Alcaligenes faedis cell protein was 6.6 mg ml-’ and Aeromonas hydrophila cell protein was 6.9 mg ml-‘. The smooth cmves were obtained from a polynomic regression.
106
N. Rius et al./FEMS
Microbiology
Table 1 Bacterial species used and their growth conditions Organism
source
Temp. (“C)
Medium [ref]
A. serpens P. aeruginosa A. faecnlis E. coli S. typhimurium P. mirubilis A. hydrophila
ATCC 11335 PA0 I ATCC 19018 ATCC 10536 Mutton NCTC 5887 NCIB 9233
27 37 37 37 37 37 27
MPSS [16] TSB NB TSB TSB TSB TSB
Letters 130 (1995) 103-l 10
of these bacteria were measured by an acid-pulse technique, according to [13]. The pH recordings were analyzed graphically, as described elsewhere [ll-131. Measurements of buffering capacity and the observed half-time of approach to final equilibrium were used to calculate membrane Hf conductance 1111. Buffering capacity and passive H+ conductance are presented as functions of external pH.
TSB: Trypticase Soy Broth (BBL, Becton Dickinson and Co., Cockeysville, USA); NB: Nutrient Broth (Oxoid, Unipath Ltd.. Basingstoke, UK).
2.3. Measurement of cell protein Protein content was determined according to [14].
bacteria were grown with shaking except A. serpens and A. fueculis. The cells, grown to early stationary phase, were collected and washed three times with 300 mM KCl. The washed cells were treated with 3 mM EDTA and resuspended in 300 mM KC1 [12]. The final concentration of cell protein was 1-13 mg ml-’ (1 X lOlO- X 10” cfu ml-‘).
3. Results and discussion Fig. 1 and Fig. 2 show the measurements of buffering capacity of the cell surface (B,) and total buffering capacity (B,) of the seven bacterial species studied. Both the B, and the B, values were quite different from species to species. Three of the organisms exhibited B, values up to about 1200 nmol H+/pH unit per mg of protein, whereas the four others exhibited values that were at least double. It is also interesting that the Enterobacteriaceae showed different curves that described the behaviour of B, and B,, compared with the other Gram-negative species studied. The B, and B, of E. coli, S. ty-
2.2. Measurement of buffering capacity and membrane H + conductance Experiments were conducted on 7-ml samples of cell suspensions in lo-ml glass vials, which were magnetically stirred at room temperature. The pH of such suspensions was between 6.2 and 7.2. The buffering capacity and membrane conductance to H+
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
6
PM
Fig. 3. Cytoplasmic buffering capacity of the bacterial species studied. B, values were calculated from smooth curves that described the behavior of B, and B,.
N. Rim et al./FEMS
Microbiology
Letters 130 (1995) 103-110
.I-.... s l
l.
107
N. Rius et al. / FEMS Microbiology
108
Letters 130 (1995) 103-I 10
16
Aquarplrlllum
sorpenr
l eruglno~
Prudomorvr
16
.
314 3 h $2 2 f 110 5 ; ii6 5 t fe % 84
2
0
I
I
1
I
: 3,5
I
I
I
I
I
I
I
I
4
4.5
5
5,5
6
6,5
1
7,s
I
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6
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PH
.
.
. ,
..=
I
ot
3
3,s
I
/
I
I
,
I
I
4
4,s
5
45
6
6,s
7
7.5
P"
Fig. 5. Membrane conductance to H+ of Aquaspirillum hydrophila.
6
3
3.5
4
435
5
5,s
6
6.5
7
PH
serpens, Pseudomonas aeruginosa, Alcaligenes faecalis
and Aeromonas
N. Rius et al. / FEMS Microbiology
phimurium and P. mirabilis generally increased with decreasing pH in the acid range of pH studied (Fig. 1). P. mirabilis presented higher values of these parameters than E. coli and S. typhimurium. The cell wall composition of P. mirubilis cells differs from that of other Enterobacteriaceae in that (i) a large percentage of the N-acetylmuramic residues of the peptidoglycan are substituted by Oacetyl groups and (ii) the lipopolysaccharide molecules have negatively charged components [ 151. These differences can explain the high B, and B, values of P. mirabilis cells. The other four bacterial species are oxidase positive Gram-negative rods that are distributed in water and the environment. Aquaspirillum serpens, Pseudomonas aeruginosa and Alcaligenes faecalis are aerobic, with a strictly respiratory type of metabolism, with oxygen as a terminal electron acceptor. P. aeruginosa can also use nitrate as an alternate electron acceptor. A. hydrophila is a facultatively anaerobic bacterium physiologically, genetically and taxonomically related to the enterobacteria studied here but their buffering capacity and H+ membrane conductance were comparable to the other three bacterial species found in water and the environment. All of them presented higher values of B, at pH 6.5 than at pH 5 (Fig. 2). With all the species, the B, values were appreciable and varied over the range of external pH values examined. Except for A. serpens, the B, of the cells decreased as external pH increased. There were no significant differences between cytoplasmic buffering capacity values for the Enterobacteriaceae and A. fuecalis studied here and the values found for Serratia marcescens [12] or for E. coli cells permeabilized with n-butanol [lo], over the pH range from 5.0 to 7.5 (Fig. 3). A. serpens, P. aeruginosa and A. hydrophila presented high values of Bi at their optimal pH for growth, but A. serpens showed Bi values that were 3-fold greater than those of the other two species. Passive H+ conductance of these species, as well as buffering capacity, was sensitive to proton concentration at the external surface, over the pH range studied. The membrane conductance to protons of the three enterobacteria increased as pH became more acidic at pH below 5.5 (Fig. 4). The smooth curves that described the behavior of Ci were com-
Letters 130 (1995) 103-l 10
109
parable to the curve reported for a pigmented strain of Serratia marcescens [12]. However, this pigmented strain showed CE values that were 2-fold greater than E. coli andS. typhimurium. The membrane H’ conductance and buffering capacity of P. mirabilis was higher than those found for the other Enterobacteriaceae. Ci values of the rest of the species studied were markedly different from data reported here for the Enterobacteriaceae. Our results of Cz values of A. faecalis were comparable to those found for a nonpigmented strain of Serratia marcescens [12]. A. serpens and P. aeruginosa exhibited a maximum value of CE at pH 6.5 and the Cz of A. serpens was 2-fold greater than that of P. aeruginosa. A. hydrophilu had a maximum at pH 4 and a minimum at pH 5.5 (Fig. 5). The experiments described in this paper show that buffering capacities and membrane H+ conductance of Enterobacteriaceae were higher at acidic external pH than at neutral pH, and that non-Enterobacteriaceae species presented higher values of CE at pH near neutrality than at pH 5.5. These two groups of bacteria can be found in different natural habitats, intestinal and urinary tract and water respectively, where they adapt to environmental changes. It has been reported that adaptative responses to acid for E. coli and S. typhimurium, involve changes in cell surface properties in addition to the enhancement of intracellular pH homeostasis [1,2,9]. Our results of buffering capacity for the Enterobacteriaceae agree well with these reports. There are no reports about or pH-homeostasis of chemopH-response heterotrophic Gram-negative bacteria, other than Enterobacteriaceae, over the pH range from 4 to 7.5, available in the literature. The results reported here contribute to the study of the response of bacteria to external pH changes. Acknowledgements We are grateful to Robin Rycroft for expert criticism during the preparation of this manuscript. References [ll Heyde, M. and Portalier, R. (1990) Acid shock proteins of Escherichia coii. FEMS Microbial. Lett. 69, 19-26.
110
N Rius et al./FEMS
Microbiology
[21 Leyer, G.J. and Johnson, E.A. (1993) Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl. Environ. Microbial. 59, 1842-1847. [3] Brown, M.H. and Booth, I.R. (19911 Acidulants and low pH. In: Food Preservatives (Russell, N.J. and Gould, G.W., Eds.), pp. 22-43. Blackie, Glasgow, Scotland. [4] Foster, J.W. and Hall, H.K. (19901 Adaptative acidification tolerance response of Salmonella typhimurium. J. Bacterial. 172, 771-778. [5] Goodson, M. and Rowbury. R.J. (19891 Habituation to normally lethal acidity by prior growth of Escherichia coli at a sub-lethal acid pH value. Appl. Microbial. Len. 8, 77-79. [6] Kroll, R.G. and Patchett. R.A. (1992) Induced acid tolerance in Lisreria monocytogenes. J. Appl. Microbial. 14, 224-227. [7] Laub, R., Schneider Y-J. and Trouet, A. (19891 Antibiotic susceptibility of Salmonella spp. at different pH values. T. Gen. Microbial. 135, 1407-1416. IS] Sawai, T., Hiruma, R., Kawana, N., Kaneko, M., Taniyasu, F. and Inami, A. (1982) Outer membrane permeation of /3-lactam antibiotics in Escherichia coli, Profeus mirabilis and Enterobacter cloacae. Antimicrob. Agents Chemother. 22, 585-592. [9] Foster, J.W. and Hall, H.K. (19911 Inducible pH homeostasis
Letters 130 (19951 103-110
ilO]
[l I] [121
[13]
[14]
[15]
[16]
and the acid tolerance response of Salmonella typhimurium. J. Bacterial. 173, 5129-5135. Krulwich, T.A., Agus, R., Schneier, M. and Guffanti, A.A. (1985) Buffering capacity of bacilli that grow at different pH ranges. J. Bacterial. 162, 768-772. Maloney, P. (1979) Membrane H+ Conductance of Streptococcus la&s. J. Bacterial. 140, 197-205. Rius, N., Sole, M., Francis, A. and Loren, J.G. (19941 Buffering capacity of pigmented and non-pigmented strains of Serraria marcescens. Appl. Environ. Microbial. 60, 2152-2154. Rius, N., Sole, M., Francis, A. and L&n, J.G. (1994) Buffering capacity and membrane H+ conductance of lactic acid bacteria. FEMS Microbial. L&t. 120, 291-296. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Kotelko, K. (1986) Proteus mirabilis: taxonomic position, peculiarities of growth, components of the cell envelope. Curr. Topics Microbial. Immunol. 129, 181-215. Krieg, N.R. (1984) Aerobic/microaerophilic motile, helical/vibrioid Gram negative bacteria. In: Bergey’s Manual of Systematic Bacteriology (Krieg, N.R. and Holt, J.G., Eds.), pp. 71-93. The Williams & Wilkins Co., Baltimore.