Institut de Biologie Physico-chimique (UPR9073), 13 rue Pierre et Marie Curie, 75005 Paris, France1
Tel: +33 1 58 41 50 00. Fax: +33 1 58 41 50 20. e-mail: plumbridge{at}ibpc.fr
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ABSTRACT |
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Keywords: Mlc, glucosamine, glucose induction, phosphotransferase system, catabolite repression
Abbreviations: DT, doubling time; Glc, glucose; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; Man, mannose; PTS, phosphoenolpyruvate (PEP)-dependent phosphotransferase system; Cm, chloramphenicol; Km, kanamycin; Tc, tetracycline
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INTRODUCTION |
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The EIIs can be specific for one sugar or capable of transporting several different sugars. For example, the only known sugar substrate of EIINag from Escherichia coli, encoded by nagE, is N-acetylglucosamine (GlcNAc), although EIINag does transport the antibiotic streptozotocin (Lengeler, 1980 ). GlcNAc is also transported by the mannose (Man) PTS, EIIABCDMan, encoded by the manXYZ operon. This transporter has a wide substrate specificity, transporting a number of hexoses, including Man, glucose (Glc), fructose and the amino sugars, glucosamine (GlcN) and GlcNAc. On the other hand, the Glc PTS (EIICBGlc), encoded by ptsG, has a more limited range of substrates; it is the major transporter for Glc but it is also capable of transporting Man and the non-metabolizable Glc analogue
-methylglucoside (Curtis & Epstein, 1975
; Kornberg & Jones-Mortimer, 1975
). PtsG is, however, normally incapable of transporting the amino sugars so that in a wild-type E. coli strain GlcN is only transported by the Man PTS.
A spontaneous mutation was selected which allowed an E. coli manXYZ (ptsM) mutant strain to grow on GlcN (Jones-Mortimer & Kornberg, 1980 ). This mutation, called umgC, mapped to the ptsG loci (ptsG was previously called umg, uptake of
-methylglucoside). It was believed that this mutation rendered the expression of ptsG constitutive and thus capable of transporting GlcN which was not itself capable of inducing ptsG expression, unlike Glc (Jones-Mortimer & Kornberg, 1980
).
It has been shown that expression of ptsG is controlled by the mlc-encoded transcription factor. Growth on Glc induces ptsG expression (Erni & Zanolari, 1986 ; Kornberg & Reeves, 1972
), presumably by relieving Mlc repression (Kimata et al., 1998
; Plumbridge, 1998b
, 1999
). Mlc has also been shown to control the expression of the manXYZ transporter (Plumbridge, 1998a
) and the central genes of the PTS, ptsHI (Kim et al., 1999
; Plumbridge, 1999
; Tanaka et al., 1999
). These experiments suggested there was a correlation between the activity of PtsG, in particular the state of phosphorylation of IIBGlc, and the derepression of Mlc-controlled genes (Fig. 1
). In the light of this information, a possible explanation for the effect of the umgC mutation was that it rendered PtsG expression constitutive by affecting Mlc regulation. In this work the umgC mutation has been characterized and its effect on Mlc regulation of ptsG investigated.
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METHODS |
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RESULTS |
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The capacities of the manXYZ- or umgC-encoded transporters to allow growth on Man and GlcN were investigated by measuring the growth rates of strains using either of these systems in the presence or absence of the mlc mutation (Table 2). Mlc controls the expression of both manXYZ and ptsG. An mlc mutation increases their expression 4- and 20-fold respectively, in low catabolite repression media like glycerol (Plumbridge, 1998a
, b
, 1999
). The effect of the mlc mutation on manXYZ expression is detected by an increase in the growth rate on both GlcN and Man. The introduction of the mlc mutation into a wild-type strain decreases the doubling time (DT) on GlcN from about 150 to 100 min (30 °C) (Table 2
, line 2). A similar decrease in DT is seen with Man. This decrease is almost exclusively due to the effect of the mlc mutation on manXYZ expression since the PtsG-encoded transporter is effectively incapable of allowing growth on GlcN and allows only slow growth on Man. This is shown by the fact that introduction of a manXYZ mutation into a wild-type strain (ptsG+mlc+) eliminates measurable growth on GlcN and increases the DT on Man from about 140 to 300 min (Table 2
, line 3). Subsequent introduction of an mlc mutation into the manXYZ strain has only a very small effect on growth on Man or GlcN. The growth rate on Man is slightly improved and growth on GlcN can now be measured, but with a DT of about 600 min (Table 2
, line 4). This shows that GlcN can be transported, but very inefficiently, by the PtsG transporter when its expression is increased.
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These experiments strongly suggest that constitutive expression of wild-type ptsG, as produced by the mlc mutation, is not sufficient to allow PtsG to transport GlcN efficiently and so imply that the umgC mutation is acting in a different, specific way to permit GlcN and Man uptake and that the umgC mutation is not just enhancing ptsG expression as expected for a constitutive mutation.
The umgC mutation enhances ptsG expression
The effect of the umgC mutation on growth rates compared to that of the transcription factor mlc strongly suggests that the umgC mutation is altering PtsG sugar specificity and thus allowing it to transport GlcN. However, the umgC mutation does also increase ptsG expression as shown by an effect in trans of the umgC mutation on the ptsG-lacZ fusion. Growth of a wild-type strain on either GlcN or Man increases ptsG expression three- to fourfold but not so much as growth on Glc (eightfold; Table 2, line 1). These and other PTS sugars are capable of inducing ptsG expression presumably by partially relieving Mlc repression, although the molecular mechanism of the derepression has not been fully elucidated (Plumbridge, 1999
). The presence of the mlc mutation derepresses expression in all four media relative to mlc+, but the level of ptsG-lacZ expression observed is different in each medium (Table 2
, line 2). The P1 promoter of the ptsG gene is dependent upon cAMP/CAP and the level of expression in the different sugars is related to the level of catabolite repression generated by growth on the different sugars (Epstein et al., 1975
; Hogema et al., 1998
; Plumbridge, 1999
).
The effect of the umgC mutation on the level of expression of the ptsG-lacZ fusion was compared to that of the mlc mutation (Table 2, lines 2 and 5). Two effects are discernible. Even in a non-PTS medium like glycerol, the umgC mutation causes about a fourfold increase in ptsG-lacZ expression. Introduction of an mlc mutation to the umgC strain increased expression in glycerol to the same level as in the mlc strain. In GlcN or Man media the umgC mutation also increases ptsG-lacZ expression about five- to sixfold (i.e. at least 20-fold compared to the basal level of the wild-type strain in glycerol), producing levels of expression even higher than that produced by the mlc mutation in the same medium. Subsequent introduction of the mlc mutation into the umgC strain had no further effect on expression during growth of the PTS sugars (Table 2
, line 6). This can be correlated with the similar growth rates of strains carrying the umgC and umgC mlc mutations. Although the presence of the umgC mutation affects ptsG expression in the absence of any PTS substrate, expression is considerably increased in the presence of the UmgC substrates, Man or GlcN. It can also be noted that even in Glc medium the umgC mutation appears to increase ptsG expression somewhat, producing about a 30% increase in ptsG-lacZ expression.
The strong effect of the umgC mutation during growth on GlcN and Man in the manXYZ background on expression of the ptsG-lacZ reporter is reduced by the presence of a manXYZ+ allele. The presence of a second transporter for Man and GlcN reduces the level of expression of the ptsG-lacZ fusion, presumably by reducing the flux through the UmgC transporter. On the other hand the level of expression of ptsG-lacZ during growth on glycerol is not affected by the presence of the manXYZ + allele.
The umgC mutation enhances ptsG mRNA levels
The increase in ptsG-lacZ expression is paralleled by an increase in chromosomal ptsG mRNA levels as measured by an S1 protection experiment using the Glc5-Glc2 probe (Fig. 2). The umgC mutation produces a small increase during growth on glycerol (Fig. 3
, lane 2) but a considerable increase during growth on GlcN (Fig. 3
, lane 6). The mlc mutation in glycerol produces a comparable enhancement in ptsG mRNA (Fig. 3
, lanes 3 and 4,) but the mlc mutation in GlcN (Fig. 3
, lane 7) seems to produce a somewhat lower level of ptsG mRNA than the umgC mutation, with or without the mlc mutation (Fig. 3
, lanes 6 and 8). This correlates with a lower value for the ptsG-lacZ fusion in the mlc strain grown on GlcN than for the umgC strain in the same medium (Table 2
). The position of the two transcription start sites for ptsG, the major P1 transcript and the minor P2 transcript, are not changed by the umgC mutation and both mRNAs are similarly affected, which is the expected observation if umgC is altering Mlc regulation, since Mlc controls both promoters (Plumbridge, 1998b
). The S1 analysis was repeated using a probe specific for the ptsG-lacZ fusion. The pattern of expression was the same as for the chromosomal copy of ptsG (data not shown).
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DISCUSSION |
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Interestingly, the IIC mutation found in the umgC strain studied here is the same as one of the ribose-positive mutations, G176D (Oh et al., 1999 ). Moreover, recent experiments, which repeated the selection for a GlcN+ phenotype, yielded several different mutations, including changes in both V12 and G176 (Notley-McRobb & Ferenci, 2000
; Zeppenfeld et al., 2000
). As in the case of V12F, the umgC mutation also allows uptake of fructose (data not shown), implying that V12F and G176D allow a general reduction in sugar specificity and transportphosphorylation coupling. It should be noted that the umgC mutation does not eliminate all sugar specificity. It does not, for example, allow the uptake of GlcNAc; introduction of the umgC mutation to a manXYZ nagE (GlcNAc-) strain does not enable the strain to grow on GlcNAc.
The umgC mutation affects Mlc regulation
Two distinct, but interrelated, phenotypes can be assigned to the umgC mutation: an enhanced transport capacity for GlcN or Man and an increase in the expression of ptsG and other genes regulated by Mlc. Several pieces of evidence support the hypothesis that the umgC mutation affects regulation by Mlc: the umgC mutation produces parallel effects in cis on ptsG mRNA levels and in trans on ptsG-lacZ expression; three different Mlc-controlled operons, ptsG, manXYZ and ptsHI, are similarly affected; and the mlc mutation is epistatic to umgC mutations. During growth on GlcN and Man, when the umgC mutation has its maximum effect, there is no additional increase in expression when an mlc mutation is introduced, consistent with the idea that the mlc and umgC mutations are affecting the same regulatory pathway. However, it is clear that the increase in ptsG expression produced by the umgC mutation is not the only reason that growth on GlcN and Man improves since an mlc mutation, which produces increased ptsG expression, does not allow good growth on GlcN or Man. Notice that there is relatively little effect of the umgC mutation during growth on Glc. This is presumably because the level of catabolite repression generated by Glc dominates the expression level of ptsG and other Mlc-controlled genes, all of which are strongly cAMP/CAP-dependent.
The G176D mutation both causes the change in sugar specificity and the increase in ptsG expression
The analysis of the two mutations found in the umgC strain, G176D and E472K, individually on plasmids show that it is the G176D mutation which is responsible for both phenotypes, i.e. growth on GlcN and increased ptsG expression. This same mutation has already been shown to permit the uptake of ribose. Since several other mutations affecting PtsG sugar specificity map to the IIC domain, it is not surprising that the G176D mutation is responsible for the change in sugar specificity. It is perhaps less expected that the regulatory phenotype, increased expression of Mlc-controlled genes, is also associated with this same mutation. Previous results (Plumbridge, 1999 ) implicated the soluble IIB domain and the level of PtsG phosphorylation in the regulation of Mlc-controlled genes. Only in the presence of a ptsG+ allele were Mlc-regulated genes derepressed by growth on PTS sugars or by mutations which interrupted the flow of phosphates from PEP to the sugar.
Growth on Glc reasonably leads to induction by relieving Mlc repression but unlike other classical repressors no low-molecular-mass metabolite has been identified which is capable of displacing Mlc from its DNA targets. One hypothesis to explain the role of PtsG in Mlc induction is that transport of Glc permits an interaction between Mlc and membrane-bound PtsG, thus sequestering Mlc away from its DNA targets (Fig. 1; Plumbridge, 1999
). Recent experiments have indeed shown that such an interaction between membrane-bound PtsG and Mlc exists (S.-J. Lee, W. Boos and J. Plumbridge, unpublished). The predicted location of G176D in a periplasmic loop strongly suggests that there is no direct interaction between G176D and Mlc which is presumably confined to the cytoplasm or the cytoplasmic side of the inner membrane. Instead it would imply that the G176D mutation has a global effect on IICBGlc structure which is monitored by Mlc in the soluble part of the cell. Since the G176D mutation affects regulation in the absence of PTS sugar transport, the mutation might be mimicking an active transport conformation which is able to signal to the IIB domain. There is evidence for such a conformational coupling between the IIC and IIB domains of the mannitol-specific PTS permease (Meijberg et al., 1998
). The V12F mutation in the N-terminal cytoplasmic helix of the IIC domain, which shows similar characteristics to G176D (increased ptsG expression and the ability to transport fructose and GlcN), could, however, be in direct contact with Mlc.
The E472K mutation near the C terminus of the IIB domain appears to have no direct effect on ptsG expression levels. As the G176D mutation in the umgC strain is sufficient for growth on GlcN, one can wonder what is the origin of the E472K mutation. The presence of a plasmid carrying E472K appears to have a detrimental effect during growth on GlcN or Man compared to the wild-type ptsG allele (Table 4), suggesting that E472K might decrease PtsG activity. If the G176D mutation produces a very strong increase in PtsG transport activity, as has been shown for V12F (Kornberg et al., 2000
; Manché et al., 1999
), a second mutation reducing this hyperactivity might have arisen. This hypothesis is supported by the observation that when another example of strain JM2053, which had been stored independently for many years, was analysed, it was also found to carry two mutations: G176D and a UAG stop codon at position E472 (L. Notley-McRobb & T. Ferenci, personal communication) rather than the AAG lysine found in JM2053 obtained from the CGSC and analysed in this study.
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 9 May 2000;
revised 30 June 2000;
accepted 13 July 2000.