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Journal of Bacteriology, December 2005, p. 8332-8339, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8332-8339.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Maltodextrin System of Escherichia coli: Glycogen-Derived Endogenous Induction and Osmoregulation

Renate Dippel, Tobias Bergmiller, Alex Böhm,{dagger} and Winfried Boos*

Department of Biology, University of Konstanz, 78457 Konstanz, Germany

Received 27 May 2005/ Accepted 23 August 2005


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ABSTRACT
 
Strains of Escherichia coli lacking MalQ (maltodextrin glucanotransferase or amylomaltase) are endogenously induced for the maltose regulon by maltotriose that is derived from the degradation of glycogen (glycogen-dependent endogenous induction). A high level of induction was dependent on the presence of MalP, maltodextrin phosphorylase, while expression was counteracted by MalZ, maltodextrin glucosidase. Glycogen-derived endogenous induction was sensitive to high osmolarity. This osmodependence was caused by MalZ. malZ, the gene encoding this enzyme, was found to be induced by high osmolarity even in the absence of MalT, the central regulator of all mal genes. The osmodependent expression of malZ was neither RpoS nor OmpR dependent. In contrast, the malPQ operon, whose expression was also increased at a high osmolarity, was partially dependent on RpoS. In the absence of glycogen, residual endogenous induction of the mal genes that is sensitive to increasing osmolarity can still be observed. This glycogen-independent endogenous induction is not understood, and it is not affected by altering the expression of MalP, MalQ, and MalZ. In particular, its independence from MalZ suggests that the responsible inducer is not maltotriose.


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INTRODUCTION
 
The Escherichia coli maltodextrin system has become a paradigm for the understanding of a complex sugar-utilizing system in bacteria (3, 33). The regulon, controlled by MalT, the central activator of the system, consists of 10 coordinately regulated genes that are geared for the utilization of maltose and maltodextrins (for a detailed description of the different aspects of transport, enzymatic activity, and regulation, see the introduction of the accompanying publication [13]).

One of the less clear phenomena in maltose regulation is endogenous induction. The degradation of glycogen yields maltodextrins which are channeled back into metabolism by MalQ and MalP (Fig. 1). Among these dextrins is maltotriose. In the absence of MalQ, maltotriose is no longer channeled into metabolism and is therefore able to activate MalT (14). This is the reason why malQ strains appear constitutive (12). We will refer to this type of endogenous induction as glycogen-derived endogenous induction. However, even in strains lacking glycogen, the maltose system can be induced internally by growing the cells on carbon sources that yield internal glucose and {alpha}-glucose-1-phosphate (glucose-1-P) or glucose-6-P. For instance, the metabolism of trehalose is notorious for this production of internal inducer (11, 22). It remains unclear whether the active inducer formed under these conditions is in fact maltotriose. Glycogen-independent induction will not be dealt with in this publication.



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  		FIG. 1. Enzymatic function of MalQ, MalZ, and MalP. Numbers give the approximate stoichiometry in the MalQ-catalyzed formation of the different maltodextrins when derived from maltose under equilibrium conditions (see accompanying publication [13]). Circles indicate glucosyl residues, with the arrows showing the reducing end. The action of MalP is the sequential formation of glucose-1-P from maltodextrins larger than maltotriose; the action of MalZ is the sequential formation of glucose from maltodextrins larger than maltose. Note that the final product of MalZ on any maltodextrin is maltose, whereas the final product of MalP is maltotriose. Not shown is that glucose and glucose-1-P enter glycolysis after their transformation to glucose-6-P by glucokinase and phosphoglucomutase, respectively.

In the absence of MalK, which is the major inhibiting factor of MalT (and which is counteracted by maltotriose) (20, 29), endogenous induction can most easily be recognized since little endogenous inducer is necessary under these conditions. Thus, even cultures grown in glycerol, which is known to cause catabolite repression (15, 16), are constitutive for the maltose system (5). Constitutivity in strains lacking MalK is most easily followed when malK+ is replaced by a transcriptional malK-lacZ fusion. This fusion is extensively used in this publication as a reporter for mal gene expression in a malK genotype. It has been observed that endogenous induction in malK-lacZ mutants is strongly reduced at high osmolarity of the medium (5). Osmoregulation is not observed when wild-type cells are grown on maltose or other maltodextrins as a carbon source (5). This indicates that osmoregulation is exclusively connected to endogenous induction. The mechanism of glycogen-derived endogenous induction, as well as its dependence on osmolarity, is the subject of this publication. In particular, we characterize the role of the three maltodextrin-specific enzymes in this process: MalQ, MalP, and MalZ. Amylomaltase, MalQ (26, 38), an obligatory glucanotransferase, cleaves any linear maltodextrin, releasing the reducing-end glucose or dextrin, and transfers the nonreducing dextrinyl moiety onto glucose or another maltodextrin (28). In this way, any maltodextrin (including maltose) is transformed into a series of maltodextrins plus glucose, while the number of glucosidic linkages stays constant. Thus, MalQ can degrade as well as synthesize maltotriose, ensuring induction when the bacteria are grown on any maltodextrin. MalP, maltodextrin phosphorylase, cleaves glucosyl residues from the nonreducing end of maltodextrins under the formation of glucose-1-P (27, 34). Its smallest substrate is maltotetraose. Therefore, the end product of MalP action on any maltodextrin is the inducer maltotriose (see the accompanying publication [13]). For maltodextrin metabolism, the combined action of MalQ and MalP yields, from any maltodextrin, glucose and glucose-1-P, both of which enter glycolysis as glucose-6-P after the action of ATP-dependent glucokinase (24) and of phosphoglucomutase (23). The third maltodextrin-specific enzyme is MalZ. This has been discovered as hydrolyzing p-nitrophenyl-{alpha}-maltoside and has been recognized as a member of the maltose regulon, being MalT dependent (31). It hydrolyzes glucose sequentially from the reducing ends of maltodextrins, maltotriose being the smallest substrate (35).


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. We used three sets of isogenic E. coli K-12 strains, derivatives of MC4100. The first set carries an intact maltose/maltodextrin ABC transporter and is derived from strain HS3166, which carries an amber mutation in malQ (31) and therefore lacks amylomaltase. The second set consists of derivatives of strain Bre1162, which carries a transcriptional malK-lacZ fusion. This strain is MalK deficient, does not transport maltose, and, due to the polar effect of the fusion, lacks the {lambda} receptor. Its LacZ activity was taken as a measure for mal gene expression. The last set consists of derivatives of strain XY100, which harbors a malT(Con) mutation rendering MalT partially independent of the inducer maltotriose. XY100 carries a malP-lacZ fusion. The isogenic derivatives of these strains were constructed by P1vir transduction (25) using selectable antibiotic resistance insertions in the relevant genes. Some mutations (deletions) were constructed by the technique of Datsenko and Wanner (8) using the heat-inducible {lambda}RED recombination system of strain DY330 (39). The deletions constructed in this strain were then transduced into Bre1162. In some instances, the antibiotic resistance cassette was removed with plasmid pCP20 harboring the saccharomyces cerevisiae FLp recombinase (FLP) (8). All strains are listed in Table 1.


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  		TABLE 1. Strains of E. coli K-12 used in this study

For testing LacZ activity at different osmolarities, we first grew the strains in tryptone broth (10 g tryptone and 8 g NaCl per liter). Then the strains were transferred into minimal medium A (MMA) (25) with 0.4% glycerol and finally into the same medium containing, in addition, 50 to 300 mM NaCl. We used overnight cultures since their activities were most reproducible (±5%) of individually grown cultures.

ß-Galactosidase activity was determined according to the method of Miller (25) with alterations. We omitted mercaptoethanol from the Z buffer. Hydrolysis of ortho-nitrophenyl-ß-galactoside (ONPGal) was done at a constant temperature of 28°C. After stopping the reaction with sodium carbonate, we clarified the suspension by centrifugation before measuring the optical density at 405 nm (OD405). To calculate the specific activity, we used an extinction coefficient of 4,860/mol · cm for o-nitrophenol. Specific activity (U/mg protein) was given in µmol ONPGal hydrolyzed per min per mg of protein at 28°C. A specific activity of 1 corresponds to about 1,000 Miller units.

malZ assays in whole cells were done analogously to the assay for ß-galactosidase. Since MalZ activity is intrinsically rather low, we harvested 20-ml samples of the overnight cultures (grown in MMA and 0.4% glycerol with or without 250 and 350 mM NaCl). The cell pellet was resuspended in 1 ml Z buffer (25), and the OD578 was determined (OD of 1 was taken as 0.107 mg protein/ml) (25). Cultures (0.8 ml) were made permeable with 2 drops of 1% sodium dodecyl sulfate and chloroform and incubated at 28°C for 10 min. Next, 0.14 ml p-nitrophenyl-{alpha}-maltoside (1 mg/ml; Sigma) was added. The reaction was stopped with 0.4 ml of 1 M sodium carbonate and clarified by centrifugation before measuring the OD405. To calculate the specific activity, we used an extinction coefficient for p-nitrophenol of 14,000/mol · cm. Specific activity (U/mg protein) is given in µmol p-nitrophenyl-{alpha}-maltoside hydrolyzed per min per mg protein at 28°C.

Maltose transport activity was measured after growth overnight in MMA with 0.4% glycerol or with 0.4% Casamino Acids (CAA) as the carbon source. The cells were washed three times in MMA without a carbon source. For the transport assay, cells were resuspended to an OD578 of 0.1 and uptake was initiated by the addition of 50 nM [14C]maltose (600 mCi/mmol; Amersham) at room temperature. Five 0.5-ml samples were taken within 90 s, filtered, and counted in a scintillation counter. Initial rates of uptake were determined by extrapolation to time zero.

Staining for glycogen. Colonies were streaked on agar plates containing Kornberg medium (phosphate-buffered yeast extract plus 0.5% glucose) as described previously (32). The plate was positioned upside down over iodine crystals and incubated for 5 min at room temperature. A dark brown reaction of the colonies indicated a high level of glycogen. glgA mutants appeared yellowish in this test.


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RESULTS
 
Endogenous induction of the mal system in malQ mutants lacking additional maltodextrin-utilizing enzymes. Strains lacking MalQ (amylomaltase) were endogenously induced (Table 2, compare, e.g., MC4100 and HS3166). To test the effect of maltodextrin phosphorylase (MalP) and maltodextrin glucosidase (MalZ) on the level of a glycogen-derived endogenous inducer in a strain lacking MalQ, we assayed the initial rate of maltose transport as a measure of mal gene expression after growth on minimal medium with 0.2% glycerol or 0.4% Casamino Acids. For this set of experiments, we constructed isogenic strains derived from strain HS3166. They carried combinations of the following alleles: malQamber malP::Tn10 malZ::Spec and glgA::lacZ (Kan) fusion (lacking glycogen synthase). These strains grew well on 0.2% glycerol and encoded an intact maltose/maltodextrin ABC transporter whose activity (of outgrown overnight cultures) was taken as a measure for the level of mal gene expression. The results are summarized in Table 2. The first set of tests was done with strains synthesizing glycogen (glgA+). For endogenous induction to be optimal, MalP had to be present. Thus, MalP was instrumental in producing maltotriose from the degradation products of glycogen. In contrast, the lack of MalZ slightly increased induction, indicating that MalZ is able to remove maltotriose by hydrolyzing it to maltose. The second set of experiments was done in mutants lacking glycogen (glgA). Under these conditions, transport activity was low (reduced about 20-fold in comparison to that for HS3166, the glgA+ malP+ malZ+ strain). The residual transport activity (or remaining endogenous induction) in the glgA series did not respond further to mutations in malP or malZ. When these two sets of experiments were repeated after growth in MMA and 0.4% CAA as the carbon source, endogenous induction was higher than that with cells grown in glycerol, consistent with the effects of catabolite repression exerted by glycerol (15, 16). But endogenous induction was also dependent on the presence of glycogen under these conditions. The absence of MalP reduced induction, and the absence of MalZ increased it, most strongly when MalP was present (compare JH1 with HS3166). Yet, MalP appeared not to be essential for the glycogen-derived inducer formation since it occurs even in the absence of MalP and the presence of MalZ did affect this (reduced) induction (compare RD30 and RD31). In the absence of glycogen, cells grown in CAA showed a five- to sixfold-higher endogenous induction than cells grown on glycerol (catabolite effect of glycerol). But again, the presence or absence of MalP had no effect and the lack of MalZ showed only slightly increased expression.


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  		TABLE 2. Effect of maltodextrin enzymes MalP, MalZ, and GlgA on the endogenous induction of maltose transport activity

Endogenous induction of the mal system in malQ mutants at different osmolarities: the role of MalZ and MalP in the osmoregulation of glycogen-derived endogenous induction. Endogenous induction of the maltose system has been known for some time to be subjected to repression by high osmolarity (5). It was therefore of interest whether or not this osmodependency is caused by any of the maltodextrin enzymes. Since transport activity of the ABC transporter for maltose is highly sensitive to salt, we used a different test system. Mutants lacking MalK, the ATP-hydrolyzing subunit of the maltodextrin ABC transporter, exhibited high expression of the MalT-dependent genes. This was due to the lack of an interaction of MalK with MalT that results in reduced activity of MalT as a transcriptional activator (20). Mutants harboring a malK-lacZ fusion instead of a wild-type malK allele have been used in the past to analyze this effect. malK-lacZ fusion strains do not transport maltose or maltodextrins; they lack MalK activity, but measuring their ß-galactosidase activity allowed us to follow mal gene expression. The high level of ß-galactosidase of these mutants when grown on a nonmaltodextrin carbon source has been interpreted as being due to the presence of an endogenous inducer whose action on MalT is unrestrained by MalK. Typically, the constitutivity of malK-lacZ expression is sensitive to high osmolarity (5). Therefore, we analyzed malK-lacZ expression in various mutants lacking combinations of MalQ, MalP, MalZ, and GlgA. The data are summarized in Fig. 2.



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  		FIG. 2. Expression of malK-lacZ in isogenic strains differing in the presence or absence of MalQ (amylomaltase), MalP (maltodextrin phosphorylase), MalZ (maltodextrin glucosidase), and glycogen. Each strain was grown overnight in MMA with glycerol as the carbon source and, in addition, 0, 50, 100, 150, 200, 250, and 300 mM NaCl (from left to right for each of the 10 strains). The phenotype of each strain is shown underneath the groups of graphs for each strain and is given in a one-letter code, as follows: Q, MalQ; Z, MalZ; P, MalP; A, glycogen. Strains lacking glycogen are carrying a null mutation in glgA (encoding glycogen synthase).

The following conclusions could be drawn. (i) In the presence of glycogen and in the absence of MalQ, endogenous induction is increased about twofold over the activity seen in an equivalent mutant expressing MalQ (SI1 versus BRE1162). This is consistent with the effect of the malQ mutation in a malK+ strain when transport was measured (Table 2) even though the effect there was much less dramatic. Expression in the malQ mutant remained osmosensitive even though high osmolarity did not reduce it to the low values observed in the malQ+ strain (SI1 versus BRE1162). (ii) Lack of MalZ does not significantly alter the expression in a malQ+ strain (BRE1162 versus TB4) but dramatically alters it in a strain lacking MalQ (SI1 versus SI2). The absence of MalZ in a malQ mutant strongly increased expression, which became insensitive to increased osmolarity. Thus, in a malQ mutant, the glycogen-derived inducer maltotriose was hydrolyzed by MalZ to the noninducer maltose and the osmosensitivity seen in SI1 was due to the salt-induced expression of MalZ. In the presence of MalQ, the loss of MalZ had little or no effect. Thus, the endogenous inducer derived from glycogen (in the absence of MalQ) must clearly be maltotriose. The observation that the presence of MalZ had no effect on induction when MalQ was present (compare Bre1162 with TB4) is somewhat puzzling. The effect of MalZ in a MalQ+ strain (in degrading the inducer maltotriose) was nearly abolished. The reason for this curious phenomenon was the repolymerization by MalQ of maltose (the end product of the MalZ action) to maltotriose. MalQ is more active than MalZ, and thus, a substantial level of maltotriose is maintained in a MalQ+ MalZ+ strain. (iii) The presence of MalP increases glycogen-derived endogenous induction. This was most clearly seen when MalQ and MalZ were both absent (compare SI2 with TB41, the pair lacking MalZ and with or without MalP). It could also be seen when MalQ and MalZ were present (compare Bre1162 with TB3). When glycogen was absent, endogenous induction became low but was still sensitive to osmolarity. It did not respond to the presence or absence of MalZ in either a MalQ+ (compare RD38 with RD36) or a MalQ– (compare SI4 with SI3) background.

The reduction of glycogen-derived endogenous mal gene expression in malQ mutants at elevated salt concentrations is due to the differential expression of malZ. As seen in Fig. 2, the high malK-lacZ expression in SI1 (malQ malZ+ malP+ glgA+) decreased after growth at increasing NaCl concentrations. In contrast, strain SI2 (malQ malZ malP+ glgA+) showed elevated malK-lacZ expression that was no longer reduced after growth at increasing salt concentrations. This indicated that MalZ was responsible for the reduction of mal gene expression at increasing salt concentrations. We tested the role of MalZ by measuring its activity in HS3166 (MalZ+) and JH1 (MalZ–) and in a malT strain (lacking the general gene activator of the mal system) after growth at different salt concentrations (Table 3). The MalZ assays showed high activity in HS3166 (malZ+) and no reduction at increasing osmolarity. The background value in JH1 (lacking malZ) was low and not altered at various salt concentrations, reflecting the background hydrolysis of p-nitrophenyl-{alpha}-maltoside (the standard MalZ assay). Most importantly, in the malT mutant (MD11), MalZ activity that was induced at increasing salt concentrations could be detected. (It was surprising that malZ expression in MD11 grown in MMA without additional salt was relatively high. This was due to the 130 mM salt contained in MMA. Reducing MMA to 1/6 MMA reduced MalZ activity from 0.6 to 0.08, comparable to that for a malZ mutant.) This demonstrated that malZ can be transcribed independently of MalT and that this MalT-independent expression is increased at increasing salt concentrations. Thus, the elevated expression of MalZ at increasing salt concentrations must be responsible for the repression of the remaining mal genes. The role of MalZ then is to hydrolyze the glycogen-derived inducer maltotriose to maltose.


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  		TABLE 3. malZ expression in dependence of osmolarity during growth

The malT-independent expression of malZ is neither RpoS nor OmpR dependent. RpoS is known to be responsible for the increased expression of a number of genes in response to high salt concentrations (17). Thus, it was of interest whether or not the MalZ-dependent control of malK-lacZ expression was governed by RpoS. Table 4 compares malK-lacZ expression at increasing salt concentrations in malQ (SI1, normal amounts of RpoS), malQ rpoS (RD44, lacking RpoS), and malQ rssB (RD45, elevated levels of RpoS) strains (2). Strain SI2 lacking MalZ is shown for comparison. All three mutants showed the same activity at low osmolarity and decreased expression at high osmolarity. Thus, the lack of RpoS did not mimic a MalZ– phenotype, excluding the possibility that malZ is under the control of RpoS. Irrespective of the presence or absence of RpoS, the lack of MalZ dramatically increased malK-lacZ expression that became insensitive to salt. The rpoS mutant RD45 (in the malZ+ background) showed a much stronger decline in activity at increasing osmolarity than the other two rpoS+ strains. Therefore, if anything, RpoS positively controls a gene whose product is involved in the formation of a glycogen-derived inducer. As is shown below, this was most likely malP. We also tested MalZ activity in a mutant lacking RpoS and a mutant lacking OmpR, the response regulator involved in osmoregulation (21) (Table 3). In both cases, an increase of MalZ activity at increased osmolarity was observed, excluding the involvement of these known osmoregulators in the expression of malZ.


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  		TABLE 4. RpoS is not responsible for the osmoinduced expression of malZ

rpoS mutants are defective in glycogen synthesis (18). Thus, the formation of maltotriose derived from glycogen in rpoS malQ mutants could be altered just due to a reduced level of glycogen. Then, an rpoS mutant should equally be reduced in its malK-lacZ fusion activity as in a mutant lacking glycogen (for instance, strain RD36, glgA malQ). However, this is not the case. Testing the glycogen content by iodine staining of colonies revealed that the rpoS mutants RD45 and RD47 still showed a weak but clear glycogen response (data not shown). Apparently, the reduced amount of glycogen seen in rpoS mutants can still provide sufficient maltotriose for full glycogen-derived endogenous induction.

MalP is needed for the glycogen-dependent high expression of malK-lacZ in malQ mutants at elevated osmolarity and is partially dependent on RpoS. In a MalQ+ GlgA+ MalZ+ background, the absence of MalP reduced malK expression by a factor of 2 and the osmosensitivity of malK expression was maintained (Fig. 2, compare Bre1162 with TB3). In a MalQ– MalZ– GlgA+ background, the absence of MalP had a drastic effect on malK expression (Fig. 2, compare SI2 with TB41). Obviously, the malP-encoded maltodextrin phosphorylase played a prominent role in providing the internal inducer from glycogen, whereas MalZ broke it down. In particular, at a high osmolarity, even in the absence of MalZ, the lack of MalP reduced mal gene expression more than 100-fold. This indicates that malP (and possibly malQ) is osmoregulated and induced at a high osmolarity. Therefore, we tested the expression of malP in a mutant that carries a malP-lacZ fusion. Strain XY100 is constitutive for mal gene expression. It carries a malT(Con)1 mutation (7, 10) encoding a MalT protein that has become partially independent of the inducer, and it harbors a malP-lacZ fusion. Indeed, the activity of this malP-lacZ fusion increased at increasing osmolarity (Table 5). The introduction of an rpoS::Tn10 mutation into XY100 (strain RD54) reduced the expression of malP-lacZ and rendered it sensitive to increasing osmolarity. Thus, malP expression is partially dependent on RpoS and is increased at elevated osmolarity. In a malT::Tn10 derivative, malP-lacZ expression was very low and was not increased at elevated osmolarity. Thus, in contrast to malZ, whose expression was increased at high osmolarity even in a malT::Tn10 background, RpoS-dependent malP expression remained MalT dependent. The effect of an rpoS mutation on the expression of malP at high osmolarity is only partial. In the absence of MalZ, the MalP-dependent degradation of glycogen leading to maltotriose in the rpoS mutant was only marginally hampered at a high osmolarity (Table 4, strains RD46 and RD47).


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  		TABLE 5. RpoS-dependent expression of malP-lacZ


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DISCUSSION
 
Glycogen-derived endogenous induction for the maltose system has been observed for some time. It is particularly evident in mutants lacking malQ (12). The explanation for endogenous induction in malQ strains has been that turnover of glycogen results in the formation of maltodextrins, including maltotriose, the established inducer of the maltose system (30). In the malQ+ background, these degradation products of glycogen (including maltotriose) would be recycled essentially to glucose-6-P and eliminated by glycolysis. In this publication, we analyzed the role of the individual maltodextrin-specific enzymes in glycogen-derived endogenous induction observed in a malQ mutant. The first set of experiments was done with the genetic background of an intact maltose/maltodextrin transporter. MalK, the ATP-hydrolyzing ABC subunit of the transporter, was present and able to curb the activity of MalT, the general transcriptional mal gene activator. Therefore, with this background, the level of internal inducer needed to achieve the same mal gene expression will be higher than that in a strain lacking MalK. We tested two sets of carbon sources: 0.4% glycerol would be expected to exert catabolite repression, whereas 0.4% CAA would not interfere. As a parameter for mal gene expression, we measured maltose transport activity at sub-Km levels of substrate concentration. The data corroborated the conclusions obtained from the in vivo enzymatic activity of MalP and MalZ reported in the accompanying paper (13). Thus, in the absence of MalP but the presence of MalZ, glycogen-derived endogenous induction was lowest, and conversely, in the presence of MalP and the absence of MalZ, mal gene expression was highest, demonstrating that MalP degrades glycogen-derived maltodextrins to the inducer maltotriose but that MalZ hydrolyzes maltotriose to the noninducer maltose. The difference in expression with glycerol and CAA as carbon sources, i.e., low expression in glycerol and high expression in CAA, again can be explained by the catabolite repression exerted by glycerol, which leads to a decrease of malT and malK transcription (15, 16). From the results summarized in Table 2, it is clear that the elevated constitutive mal gene expression seen in malQ strains is indeed dependent on the presence of glycogen. However, we would like to emphasize that there was still significant remaining mal gene expression that was independent of glycogen. In strains lacking glycogen, there was no significant difference in expression whether or not MalZ or MalP was present (Table 2). Since MalZ is known to degrade the inducer maltotriose to the noninducer maltose and since the presence of MalZ has no effect on low-level expression in mutants lacking glycogen, we concluded that there has to be a small amount of an additional endogenous inducer that is not derived from glycogen and that is most likely not maltotriose. The present publication does not offer an explanation for the origin of this glycogen-independent endogenous induction of mal gene expression. It is conceivable that it is due to inducer-free MalT. Yet, the reducing effect of glucokinase on this type of endogenous induction points to an involvement of free internal glucose in the synthesis of an as-yet-unidentified inducer (24).

Sensitivity of endogenous induction to high osmolarity has been known for some time (5), even though the molecular mechanism has remained elusive. The availability of mutants defective in malP and malZ has enabled us to test the possible involvement of these enzymes in the osmoregulation of glycogen-derived endogenous induction. For this analysis, we switched to a second test for mal gene expression by analyzing the ß-galactosidase activity of a malK-lacZ reporter fusion that had lost MalK function. As MalK interferes with MalT, acting as a transcriptional activator, endogenous induction needs less inducer and can be measured more easily. In order to avoid interference with the accumulation of compatible solutes from the CAA medium at increasing salt concentrations, we used MMA and glycerol as the carbon source. The data shown in Fig. 2 define our understanding of glycogen-derived endogenous induction and osmoregulation. It is clear that even in the absence of glycogen, there is a relatively high residual mal gene expression characterized by a value of about 0.8 to 1.0 U/mg protein of the malK-lacZ fusion that was no longer influenced by the presence or absence of MalQ, MalZ, and MalP. This residual glycogen-independent mal gene expression is also sensitive to osmolarity during growth, and neither its origin nor its osmosensitivity is understood. This publication deals exclusively with glycogen-derived endogenous induction and its dependence on MalQ, MalP, and MalZ, as well as with its osmoregulation. The following conclusions were reached. (i) The absence of MalQ strongly increases endogenous induction. Thus, we conclude that among the dextrins derived from glycogen degradation, there is maltotriose, the established inducer of MalT. MalQ will reshuffle all maltodextrins, including maltotriose, to longer maltodextrins and glucose (Fig. 2B), thus effectively removing maltotriose. The conclusion that the inducer derived from glycogen must be maltotriose is borne out by the action of MalZ on endogenous induction. A malQ malZ malP+ glgA+ strain showed very high endogenous induction (Fig. 2, compare strain SI1 with strain SI2). Since MalZ is known to hydrolyze maltotriose to maltose, the glycogen-derived endogenous inducer must be maltotriose. The malQ malZ malP+ glgA+ strain also has lost its sensitivity to increased osmolarity, indicating that the osmosensitivity of glycogen-derived endogenous induction is due to the osmoinduction of MalZ. Indeed, increased MalZ activity was found after growth at increasing osmolarities, even in a mutant lacking MalT. This demonstrates that malZ has two overlapping modes of expression: one is MalT dependent; the other is osmodependent. The mechanism of its osmodependent expression remains unclear, but we found that it was independent of the general stress sigma factor RpoS.

(ii) The degradation of glycogen-derived maltodextrins leading to endogenous induction is controlled by MalP, the maltodextrin phosphorylase. This can be seen in Fig. 2. The high expression in a malQ malZ malP+ glgA+ strain became severely reduced after the introduction of a deletion in malP. Most significantly, malK-lacZ expression became very sensitive to high osmolarity (compare strain SI2 with strain TB41). From the comparison of mal gene expression levels in these two strains, one would conclude that malP expression should not be affected during growth at high osmolarity. Indeed, we could demonstrate that malP-lacZ expression was not only insensitive against high osmolarity but even increased by it. The data in Table 5 show that the osmoinduction of malP was partially dependent on RpoS, the global stress sigma factor. Yet, in contrast to malZ, whose osmoinduction is MalT independent, the RpoS-dependent increase of malP at elevated osmolarity still required MalT, the central mal gene activator. The visual inspection (R. Hengge, personal communication) of the MalT-dependent promoter in front of malP gave no indication of an RpoS recognition element (1, 36). Thus, the effect of RpoS on the malP promoter is probably indirect, possibly via an as-yet-unidentified RpoS-dependent transcriptional regulator (37). There was the possibility that the RpoS dependency may have been caused indirectly by the known RpoS-dependent control of the genes encoding the trehalose-synthesizing enzymes (otsBA) (19). Yet, the introduction of an otsA insertion mutation did not abolish the effect of RpoS on malP expression (data not shown).

Considering the degradation of glycogen, the current model (9) (Fig. 3) foresees, first, the consecutive phosphorolytic cleavage at the nonreducing ends of the linear branches by glycogen phosphorylase (GlgP) up to a point where only linear maltotetrayl or, in rare cases, maltotriosyl residues remain. These residues in the phosphorylase-limited glycogen are all linked via an {alpha}(1-6) linkage to the main chain. In the next step, maltotetraose is released by GlgX, the glycogen-debranching enzyme. Apparently, mainly maltotetrayl residues linked {alpha}(1-6)-glucosidically to the main glycogen chain are substrates of GlgX. Thus, it is maltotetraose that will become the substrate for MalP to produce maltotriose, the inducer of the system. Maltotetraose is also a substrate for MalZ. But in this case, hydrolysis continues, yielding, in the end, maltose that is no longer an inducer for MalT. Only when MalQ is present can maltose again be recycled to maltodextrins, including maltotriose, thus ensuring a low level of endogenous induction. This situation must prevail in a wild-type strain that grows on a nonmaltodextrin carbon source, explaining the relatively high uninduced level of the maltose/maltodextrin system.



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  		FIG. 3. Model of glycogen degradation and maltodextrin metabolism. The model of glycogen degradation as adapted from Dauvillée et al. (9). The distances between the branch points on one linear chain are not to scale but are much larger in vivo. The scheme includes the formation from glycogen of phosphorylase-limited glycogen by GlgP and the formation of maltotetraose by GlgX, the debranching enzyme. In the lower part, formation by MalP of maltotriose, the inducer of the maltose system (boxed), and the action of MalZ in removing maltotriose and the formation of maltose are shown. The glucosyl residues are indicated by circles, the {alpha}(1-4) linkage by the linear circle connector, and the reducing end by a straight arrow. The {alpha}(1-6) linkage is indicated by an angled arrow. The filled circles help to identify the origin of maltotriose.


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ACKNOWLEDGMENTS
 
We are grateful to Erika Oberer-Bley for her help in improving the manuscript.

This research was supported by grants from the Deutsche Forschungsgemeinschaft.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, University of Konstanz, 78457 Konstanz, Germany. Phone: 49-7531-882658. Fax: 49-7531-883356. E-mail: Winfried.Boos{at}uni-konstanz.de. Back

{dagger} Present address: Division of Molecular Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland. Back


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Journal of Bacteriology, December 2005, p. 8332-8339, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8332-8339.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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