Department of Microbiology, Southern Illinois University, Carbondale, IL 62901-6508, USA1
Author for correspondence: David P. Clark. Tel: +1 618 453 3737. Fax: +1 618 453 8036. e-mail: clark{at}micro.siu.edu
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ABSTRACT |
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Keywords: lactic acid, fermentation, anaerobic growth, CsrAB system, Mlc regulator, ArcAB system
Abbreviations: LDH, lactate dehydrogenase; PTS, phosphoenolpyruvate-dependent sugar phosphotransferase system
a Present address: Valentis, Inc., 863A Mitten Road, Burlingame, CA 94010, USA.
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INTRODUCTION |
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Our unpublished work has shown that ldhA is not regulated by Fnr, which activates most of the genes involved in anaerobic respiration. Neither is ldhA affected by the AdhR regulator that activates alcohol fermentation in response to the level of reduced NADH (Clark, 1989 ; Clark & Rod, 1987
; Dailly, 1998
). Here we show that ldhA is affected by the ArcAB system and by several genes involved in the control of carbohydrate metabolism. In particular, ldhA appears to be under control of both CsrAB (Liu et al., 1997
; Romeo, 1998
) and Mlc (Kim et al., 1999
; Plumbridge, 1999
). The CsrAB system oversees the balance between glycolysis and sugar storage as glycogen. In particular, CsrA binds to glgCAP mRNA and prevents translation, hence inhibiting glycogen synthesis (Liu et al., 1997
). CsrB is a non-translated RNA molecule that acts as a dock for CsrA. Consequently, mutations inactivating CsrB have opposite effects to those in CsrA (Romeo, 1998
). Mlc acts as a repressor for several operons encoding proteins of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS), including ptsHI, ptsG and manXYZ as well as genes involved in the metabolism of certain non-PTS sugars, such as maltose (Plumbridge, 1998a
, b
).
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METHODS |
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DNA procedures.
Chromosomal DNA was isolated and purified as described by Sato & Miura (1963) . Lambda DNA was isolated as described by Sambrook et al. (1989)
. Plasmids were isolated by alkaline lysis followed by ethidium bromide/CsCl density centrifugation (Sambrook et al., 1989
). The rapid plasmid isolation technique of Birnboim & Doly (1979)
was used to screen plasmid constructs. Ligations using T4 DNA ligase and restriction enzyme digests were performed under conditions recommended by the manufacturer (Bethesda Research Laboratories). DNA fragments were separated by electrophoresis on 0·7% agarose gels in TBE (89 mM Tris, 89 mM boric acid and 0·2 mM EDTA) (Sambrook et al., 1989
). DNA restriction fragments and PCR products were purified from agarose gels by Gene Clean (Bio 101). Transformation procedures were essentially as described by Hanahan (1983)
. Both strands of DNA were completely sequenced using the technique of Sanger et al. (1977)
.
Construction of modified lac-fusion vectors.
The fusion vectors pRS414 and pRS415 are widely used in studies of gene expression (Simons et al., 1987 ). However, they have only three restriction sites available for cloning, which limits their use. Based on the presence of useful restriction sites in the ldhA upstream region, we designed two complementary 50-mer oligonucleotides containing recognition sequences for eight common restriction enzymes (5'-EcoRI, PstI, SphI, ClaI, MluI, SalI, HindIII, BamHI-3'). The two oligonucleotides were mixed at equal molarity, heated at 76 °C for 10 min to eliminate possible intramolecular base pairing and incubated at 48 °C overnight. The polylinker and pRS414 and pRS415 were digested with EcoRI plus BamHI. DNA fragments were purified by agarose gel electrophoresis and DEAE membrane elution. The polylinker was then ligated into the pRS plasmids and the products transformed into strain JK597. As no selection was available for the presence of the insert, plasmid minipreps were made from 48 ampicillin-resistant colonies and cut with PstI and with SphI plus ScaI. Presence of the polylinker insert was confirmed by sequencing. A derivative of pRS414 with the polylinker was designated pRJ1 and a polylinker-containing derivative of pRS415 was named pRJ2 (Fig. 2
).
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RESULTS |
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A 369 bp segment of the ldhA upstream is sufficient for expression
As described in Methods, we constructed a series of ldhAlacZ fusions containing different lengths of the ldhA upstream region, ranging from 1170 to 369 bp. Derivatives of P90C carrying such a series of ldhAlacZ operon fusions were grown anaerobically on rich broth plus glucose at both pH 5·5 and pH 9·0. The 1170 bp fragment is present in RJ53, whose ß-galactosidase levels are shown in Table 2. Almost the same levels of expression were seen in all the other shorter constructs (data not shown). Moreover, the ldhA gene was induced by low pH conditions in all four constructs, as seen in previous work. The identical response of the four constructs implied that the 369 bp MluI/HindIII fragment of the ldhA upstream region was sufficient for normal expression of the ldhA gene.
Effect of temperature on expression of ldhA
It has been suggested that ldhA might correspond to hslI, a poorly characterized heat-shock gene, identified only by restriction site and mapping data (Chuang & Blattner, 1993 ). We therefore assayed our ldhAlacZ fusions after shifting from 30 °C to 37 °C, 42 °C and 50 °C. However, we did not see any increased expression of ldhAlacZ in response to elevated temperature, either aerobically or anaerobically, whether at neutral or acidic pH (data not shown). We also assayed LDH from cultures grown for 12 h at 37 °C and 42 °C and again found no difference.
Effect of anaerobic regulators on expression of ldhA
Several regulatory genes are known that control the response to anaerobiosis, acidity and/or entry into stationary phase. We transduced insertion mutations in fnr, narL, rpoS, osmZ, appY, arcB and arcA into the fusion strains RJ43, RJ47, RJ53 and RJ57. The derivatives were grown aerobically and anaerobically in rich broth plus glucose at both pH 5·5 and pH 9·0 and their levels of ß-galactosidase were assayed and compared to the parental ldhAlacZ fusion strains. The arcB ::Kan insertion caused a four- to fivefold drop in ldhA expression under all conditions tested in both protein and operon fusion strains (Table 3). Insertions in arcA had essentially the same results as those in arcB (data not shown). However, none of the other regulatory defects had any significant effect on ldhAlacZ expression (data not shown). The ldhAlacZ fusion strains and their arcB ::Kan derivatives were grown on a range of carbon sources, examples of which are shown in Table 3
. Expression of the ldhAlacZ fusions was highest on glucose, lower on other PTS sugars (e.g. mannose), and lowest on glycerol plus fumarate where growth is by anaerobic respiration rather than sugar fermentation. However, the relative drop in expression due to arcB ::Kan was approximately the same on all growth susbstrates. These results suggested that expression of the ldhA gene might be affected by the nature of the carbon source and/or the metabolic products of sugar breakdown.
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Effect of metabolites on expression of ldhA
Our previous work showed minor effects on LDH levels of mutations blocking acetate formation (Mat-Jan et al., 1989 ; Bunch et al., 1997
). We therefore tested the possibility that metabolic blocks in pyruvate, lactate or acetate metabolism might affect the expression of ldhA. However, introduction of defects in ack, pta,
(ack pta),
aceEF, pfl and ldhA had no effect on the expression of ldhAlacZ (data not shown). Thus these earlier observations were presumably due to indirect effects of changes in the accumulation of acidic fermentation products in poorly buffered media.
We also measured the effect of adding exogenous fermentation acids on the expression of the ldhAlacZ fusions. These acids were tested at pH 9 and pH 7 to assess whether they might mimic low pH conditions by increasing the expression of ldhA. However, succinate, acetate, lactate and formate had no significant effect (data not shown). Addition of pyruvate (20 or 40 mM) resulted in a two- to fourfold increase in the expression of the ldhAlacZ fusions (Table 4). Conversely, the pyruvate analogue ketobutyrate had a slight negative effect, of doubtful significance, on the expression of ldhA, although only at pH 7. Oxamate, another pyruvate analogue that inhibits the fermentative LDH of E. coli to a moderate degree, had no significant effect (Table 4
).
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The CsrAB and Mlc regulatory systems affect expression of ldhA
Insertions in regulatory genes csrA and csrB were tested for effects on the anaerobic expression of the ldhAlacZ fusions. The csrA ::Kan mutation decreased expression of ldhAlacZ by two- to threefold in both operon and protein fusions at both pH 5·5 and pH 9·0 for cultures grown with several different carbon sources (Table 5). The csrB ::Cam mutation had little effect under most conditions; however, it did increase expression by about twofold for cultures grown on maltose (a non-PTS sugar causing only mild catabolite repression). CsrA binds to the mRNA of target genes and CsrB provides an RNA molecule that competes for binding of CsrA (Liu et al., 1997
). Consequently, a deficiency of CsrB will result in a higher level of free CsrA and so the effects of defects in CsrA and CsrB will be opposite, as observed.
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We transduced the mlc ::Tet mutation into the ldhAlacZ fusion strains RJ43, RJ47, RJ53 and RJ57. The mlc ::Tet derivatives were grown at pH 5·5 and pH 9·0, and under both aerobic and anaerobic conditions on rich broth supplemented with glucose, fructose, mannose, maltose, xylose or glycerol plus fumarate. For both the operon and the protein ldhAlacZ fusions, and under all conditions tested, the presence of mlc ::Tet almost abolished expression of ldhA (representative data are shown in Table 6). We also tested the effects of ptsG and cra, which are also involved in the regulation of sugar metabolism. Derivatives of RJ43, RJ47, RJ53 and RJ57 carrying ptsG ::Kan or cra ::Tn10 insertions were tested under the same conditions as the mlc::Tet derivatives. However, neither ptsG::Kan nor cra ::Tn10 had any significant effect on ldhA expression (data not shown).
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DISCUSSION |
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A survey of regulatory mutations demonstrated that ldhA was affected both by the ArcAB regulatory system and by the CsrAB and Mlc carbohydrate metabolism regulators. Introduction of defects in arcA or arcB lowered expression of ldhA by three- to fourfold both aerobically and anaerobically; both at pH 5·5 and at pH 9·0. Thus, although the ArcAB system promotes expression of ldhA, it is not apparently responsible for the response of ldhA to these environmental conditions. A similar response to defects in the ArcAB system has been seen for cyd (Iuchi et al., 1990 ). Expression of cyd decreased upon introduction of mutations in arcA or arcB under both aerobic and anaerobic conditions. The cytochrome d complex is induced under micro-oxic conditions where the pO2 is too low for the cytochrome o complex to function yet too high for alcohol dehydrogenase and fumarate reductase (Becker et al., 1997
; Tseng et al., 1996
). The LDH is also functional under such micro-oxic conditions and, like cyd, is regulated by ArcAB. Unlike cyd, ldhA is not affected by Fnr, perhaps because LDH is also functional under fully anaerobic and fermentative conditions.
Inactivation of the mlc regulatory gene (Kim et al., 1999 ; Plumbridge, 1999
) had by far the greatest effect on the expression of the ldhAlacZ fusions that we observed. Expression of both protein and operon fusions, under all growth conditions tested, was almost totally abolished by the presence of mlc ::Tet. The Mlc (=DgsA) protein is a repressor of several genes of the phosphotransferase system, including ptsHIcrr, ptsG and manXYZ (Kimata et al., 1998
; Plumbridge, 1998a
, b
). The presence of glucose and other sugars derepresses these transport genes. The mechanism involves the sequestration of Mlc by non-phosphorylated PtsG (Lee et al., 2000
; Tanaka et al., 2000
). In ptsG mutants, Mlc-controlled genes become non-inducible as no PtsG is available to bind Mlc. The presence of PtsG is also required for induction of the glycolytic genes gapA and gapB-pgk (Charpentier et al., 1998
) by glucose.
In the present case, Mlc exerts a positive regulatory effect on ldhA. Examination of the DNA upstream of ldhA shows no sequence which agrees with the deduced consensus sequence for Mlc-binding sites (Plumbridge, 2001 ). Moreover, no binding of Mlc to the ldhA upstream region was detected in vitro, at least under standard conditions (J. Plumbridge, personal communication). Thus the regulatory effect of Mlc on ldhA appears to be indirect. Mlc is known to repress the MalT activator, so perhaps it might activate transcription of an ldhA-specific activator that is yet to be identified. Hence mlc-negative strains would behave as activator minus. Using an mlclacZ fusion, we found that mlc itself was expressed at a two- to threefold higher level anaerobically, although no effect of pH was seen. Though small, this effect was consistent and was seen during growth on several different carbon sources. What induces mlc anaerobically remains unknown. Overall, the anaerobic component of ldhA induction may be accounted for, at least in part, by the increased transcription of mlc.
The CsrAB regulatory system controls the balance between carbon storage (as glycogen, which accumulates in stationary phase) and the breakdown of sugars by glycolysis (Liu et al., 1997 ; Romeo, 1998
). The Csr system is unusual in consisting of an RNA-binding protein, CsrA, plus an RNA molecule, CsrB (Liu et al., 1997
; Romeo, 1998
). CsrA is primarily an activator of glycolysis and a repressor of gluconeogenesis and glycogen synthesis (Romeo et al., 1993
). The binding of CsrA promotes decay of glg mRNA (Liu et al., 1995
) and in a csrA ::Kan mutant, glycogen is overproduced. Whether CsrB is merely a dock for CsrA or is actively involved in combating the effects of CsrA is still debatable. Although overproduction of CsrB antagonizes the effect of CsrA by sequestration, insertional inactivation of csrB has relatively minor effects (Liu et al., 1997
).
In the present case, introduction of csrA ::Kan reduced expression of ldhA approximately fivefold. The csrB ::Cam knockout had a positive though small effect on ldhA expression. However, a glgA ::Kan knockout increased ldhA expression significantly. This suggested that the effect of CsrA on ldhA might be indirect, via its effect on glycogen accumulation. If glycogen synthesis is slow (as in glgA ::Kan strains), more sugar would be available for glycolysis and fermentation. Conversely, if glycogen accumulates rapidly (as with csrA ::Kan), there is less free hexose. If the expression of ldhA responds to the availability of hexoses and/or derived glycolytic intermediates such as pyruvate, this would explain the present results.
Pyruvate builds up in the absence of respiratory metabolism and is the substrate for LDH. The fermentative D-LDH of E. coli is known to be allosterically activated by pyruvate (Tarmy & Kaplan, 1968b ). Here we have shown that expression of ldhA was also increased by pyruvate by two- to fourfold. The effect was specific for pyruvate as none of the fermentation acids acetate, formate, lactate or succinate had any effect on ldhA expression. The pyruvate analogue 2-ketobutyrate had an opposite but marginal effect. Benzoate, a weak acid that lowers the internal pH (Rosner et al., 1994
; White et al., 1992
), did not induce the ldhAlacZ fusions; in fact, higher concentrations of benzoate decreased expression somewhat. This implies that the response of ldhA to acidity is not due to lowering of the internal pH. We also found that the induction of ldhA by low pH only occurred when sugars were the carbon source. Cultures grown anaerobically on glycerol plus fumarate showed little or no effect of pH. Conceivably, the pH effect on ldhA expression may be due to the internal accumulation of pyruvate.
Unlike the fermentative alcohol dehydrogenase, whose levels respond to the accumulation of reducing equivalents in the form of NADH (Leonardo et al., 1993 , 1996
), LDH levels respond to carbon flow. In view of its regulation by Mlc, one might almost regard LDH as the final step in the PTS. After phosphoenolpyruvate donates its phosphate to the incoming sugar, the pyruvate formed is disposed of by conversion to lactate by LDH, under fermentative conditions.
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ACKNOWLEDGEMENTS |
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Received 22 February 2001;
revised 16 May 2001;
accepted 22 May 2001.