Strains of Corynebacterium glutamicum with Different Lysine ... - NCBI

Vol. 59, No. 1

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1993, p. 316-321

0099-2240/93/010316-06$02.00/0

Strains of Corynebacterium glutamicum with Different Lysine Productivities May Have Different Lysine Excretion Systems STEFAN BROER, LOTHAR EGGELING, AND REINHARD KRAMER* Institut fiur Biotechnologie I, Forschungszentrum Julich, Postfach 1913, D-5170 Julich, Germany Received 2 July 1992/Accepted 20 October 1992

The lysine excretion systems of three different lysine-producing strains of Corynebacterium glutamicum were characterized in intact cells. Two strains (DG 52-5 and MH 20-22B) are lysine producers of different efficiency. They were bred by classical mutagenesis and have a feedback-resistant aspartate kinase. The third strain (KK 25) was constructed from the wild type by introducing the feedback-resistant aspartate kinase gene of strain MH 20-22B into its genome. The three strains were shown to possess different excretion systems. Export in strain KK 25 is much slower than in the two mutants. The differences between the two lysine-producing strains are more subtle. Km and Vm. are similar, but pH dependence and membrane potential dependence reveal differences in the intrinsic properties of the carrier system. kinetic properties. Most results concerning strain DG 52-5 have been published before (4), but are included in the figures for comparison.

Corynebacterium glutamicum and related organisms from the group of coryneform bacteria are widely used for the industrial production of amino acids, especially glutamate and lysine. Lysine biosynthesis in the wild type of C. glutamicum is mainly controlled by concerted feedback inhibition of lysine plus threonine on aspartate kinase (16). Effective lysine excretion is possible only if this regulation is impaired. Such mutants can be obtained by screening for resistance against the lysine analog S-(2-aminoethyl)-L-cysteine (15). It was shown that many of these mutants are lysine producers and that the aspartate kinase could not be inhibited by high concentrations of lysine plus threonine (19). In contrast to the knowledge about basic metabolic events leading to increased lysine biosynthesis, the mechanism of transport across the membrane was elucidated only recently (3, 4). Excretion proved to be an active process, since lysine can be accumulated up to concentrations of several hundred millimolar in the medium. In addition, lysine is a cation and export must proceed against the membrane potential (outside is positive). We showed that efflux of lysine is carrier mediated; the maximum velocity was determined to be 12 nmol min-1 mg (dry weight)-1 and the (internal) Km to be 20 mM. By detailed kinetic analysis, we found that lysine is excreted by a 20H-lysine symporter (4). Thereby, the positive charge of lysine is compensated and the membrane potential is actually used as the driving force for excretion. The rate of transport is modulated by the membrane potential, the internal and external pH, and the lysine gradient. It was assumed that the production strain DG 52-5, obtained by classical mutagenesis, differs from the wild-type strain ATCC 13032 only by a feedback-resistant aspartate kinase (14). This defect leads to an elevated internal lysine concentration (3). Because lysine export follows MichaelisMenten kinetics, a high excretion rate is the consequence of this alteration. It should be mentioned that, in addition to the lysine export permease, C. glutamicum also possesses export systems for isoleucine (7) and glutamate (10, 11). In the present investigation, we chose two additional strains in order to elucidate whether the lysine transport systems of different C. glutamicum strains differ in their *

MATERIALS AND METHODS Growth of organism. Cells were grown under aerobic conditions on a rotary shaker (150 rpm) in mineral medium (pH 7) containing (per liter): (NH4)2SO4, 5 g; urea, 5 g; KH2PO4, 0.5 g; K2HPO4, 0.5 g; MgSO4- 7H20, 0.25 g; FeSO4 7H20, 0.01 g; MnSO4- H20, 0.01 g; CaC12 2H20, 0.01 g; ZnSO4 7H20, 1 mg; CuSO4, 0.2 mg; NiCl2, 0.02 mg; biotin, 200 jig; glucose, 50 g. For growth of strain MH 20-22B, this medium was supplemented with 0.5 g of leucine per liter. For growth of strain KK 25, 10 mg of kanamycin per liter was added. Bacterial strains. Strain KK 25 was constructed by integration of a feedback-resistant aspartate kinase into the chromosome of the wild-type strain ATCC 13032 (this study). Strains DG 52-5 and MH 20-22B are lysine producers bred by classical mutagenesis. They are characterized by a feedback-resistant aspartate kinase (14, this study). Strain MH 20-22B is in addition a leucine auxotroph. Chemicals. Radiochemicals were purchased from Amersham International (Little Chalfont, Buckinghamshire, United Kin dom). The following labelled compounds were used: (U-1 C]taurine, [14C]methylamine, [U-14C]benzoic acid, 3H20, and 86Rb. Biochemicals were from Boehringer GmbH (Mannheim, Germany); all other chemicals were of analytical grade and obtained from E. Merck AG (Darmstadt, Germany) and Sigma Chemical Co. (St. Louis, Mo.). Genetic engineering. Plasmids were isolated by the alkaline lysis method (1), and enzymes necessary for in vitro recombination were used as recommended by the manufacturer (Boehringer). To introduce the feedback-resistant kinase gene lysC2(Fbr) into the wild-type chromosome, we constructed the Eschenchia coli vector plasmid pKK6. For this purpose, lysC2(Fbr) cloned from MH 20-22B was excised from pJC33 (5) as a 3.2-kb DraI fragment which was purified from a gel slice with Geneclean (Bio 101, Inc., La Jolla, Calif.). It was ligated with pSUP301 (21) which previously was cleaved with ScaI and treated with alkaline phosphatase. The ligation mixture was used to transform E. coli DH5 to kanamycin resistance (Kmr) (50 ,ug/ml) on Luria-

Corresponding author. 316

VOL. 59, 1993

Bertani plates (18). From several Km' Aps clones, plasmids analyzed by restriction analysis, and all clones found to contain the lysC2(Fbr)-containing DraI fragment in the mobilizable vector. One plasmid was designated pKK6 and introduced via transformation into E. coli S-17-1, providing the additional functions necessary to transfer pKK6 to C. glutamicum. The mating was performed with C. glutamicum ATCC 13032 as described previously (20) and Kmr colonies were obtained, indicating that pKK6 had been integrated by a single crossover into the C. glutamicum lysC' gene. Determination of lysine excretion. C. glutamicum strains were grown to the late exponential phase, at which maximal lysine production was achieved. Cells were harvested by centrifugation (5 min, 3,000 x g), washed twice with ice-cold Tris-buffer (50 mM Tris-HCl [pH 7.5], 10 mM NaCl, 10 mM KCl), and suspended in the same buffer, yielding a concentration of about 40 mg (dry weight) per ml. These cells can be stored on ice for 2 h without substantial loss of internal lysine. Excretion was initiated by 12-fold dilution of the cell suspension into prewarmed (25°C) buffer and terminated by silicone oil centrifugation (13). For this, an aliquot (100 RI) of the reaction mixture was transferred at different time intervals to a 400-,u microcentrifuge tube containing a layer of silicone oil (PN 200; Roth GmbH, Karlsruhe, Germany) floating on a layer of 20% perchloric acid. The tube was immediately centrifuged in a Beckman Microfuge E for 30 s. If external lysine concentrations were higher than 10 mM, an additional rapid washing procedure was used to minimize errors in the determination of the internal lysine. In this case, an aliquot of cells was centrifuged, the supernatant was aspirated, and the cell pellet was resuspended in ice-cold buffer. This suspension was then immediately used for silicone oil centrifugation as described above. The washing procedure was completed within 1.5 min. Losses of internal lysine were neglible. Lysine production in batch cultures was determined as the concentration of lysine in the supernatant after 48 h. Determination of aspartate kinase activity. Aspartate kinase activity was determined as described elsewhere (6). Determination of cytoplasmic volume and components of the proton motive force. The cytoplasmic volume was determined by the method of Rottenberg (17), using [14C]taurine as a nonpenetrating marker for the extracellular space by subtracting the taurine space from the total 3H20-permeable volume. The taurine space was found to be 3.0 RI/mg (dry weight) and the total water-permeable space was determined to be 4.9 RI/mg (dry weight), thus yielding a cytoplasmic volume of 1.9 ,ul/mg (dry weight). The membrane potential was determined by the distribution of 86Rb in the presence of 10 ,uM valinomycin and potassium concentrations below 100 p.M. By using higher potassium concentrations, the membrane potential was varied according to the Nernst equation: BA = 59 mV log [K+in]l[K out] Extraction and determination of intracellular amino acids. Cells in a 100-,ul culture aliquot were separated from the medium by silicone oil centrifugation. The sedimented cells in the acid layer were extracted by sonication, and the extracts were neutralized by adding 25 p.l of 5 M KOH-1 M triethanolamine. The extracts were centrifuged in the cold, and the supernatants were used for amino acid determination. Lysine was detected fluorimetrically after precolumn derivatization with o-phthaldialdehyde reagent (Pierce Europe B.V., Oud-Beijerland, The Netherlands) and separation by a reversed-phase column (Hypersil ODS 5,u, 125 by 4 mm) with a high-pressure liquid chromatography system (HP 1090 M; Hewlett-Packard, Avondale, Pa.). A standard pro-

C. GLUTAMICUM LYSINE EXCRETION SYSTEMS

317

0.04 -

were

0

0.03 0

I

0

E

l19

_0.02

-

0

I

0.01 I -

0.00

0.05

0.10

0.15

ki (min-1) FIG. 1. Correction curve for export rate constants in strain MH 20-22B. Differences between rate constants determined by internal concentrations (ki) and external concentrations (k0) are plotted against the rate constants (kg). The values were taken from an experiment on the pH dependence of this mutant.

tocol for the separation of amino acids was used (12). Using the internal cell volume, true internal concentrations could be calculated. Calculations. When using internal lysine concentrations for determination of the efflux activity, efflux curves could be linearized by plotting the logarithm of the internal lysine concentration against time. Assuming a single exponential decay, the slope then represents the (first-order) rate constant of efflux (k) and was used for comparison of efflux activity. Rates were determined by the law for first-order kinetics (V = k. [Lysi.]) or by direct derivation. Deviations from first-order rate kinetics at high internal lysine concentrations are due to substrate saturation of the carrier on the inside and were used for determination of the apparent Ki,m as described in detail elsewhere (4). If possible, efflux was determined both by the decrease of the internal concentration and by the increase of the external concentration. Because no metabolizable substrate was present in the assay, both movements should be well balanced. This is true for strain DG 52-5 (3). For strains MH 20-22B and KK 25, the amount of lysine appearing at the outside was higher than that disappearing at the inside, because of refilling of the internal lysine pool by anabolism from internal sources. This caused an underestimation of the efflux rate when determined by using internal concentrations; thus, generally external concentrations were used. In experiments in which only internal concentrations could be measured, i.e., at high external lysine concentrations, the corresponding values had to be corrected. The pH dependence of export in strain MH 20-22B gave a set of rate constants determined by external and internal concentrations. The difference between these rate constants plotted against the true rate constant, i.e., the rate constant determined by external concentrations, was used as a correction curve (Fig. 1). RESULTS To compare the lysine export systems of different lysine producers, we chose three strains, one defined strain with feedback-resistant aspartate kinase (in the wild-type background) and two lysine producers obtained from the wild type by classical mutagenesis. A comparison of lysine secretion of the wild type with that of the lysine producers was not possible. The reason for this was the low internal lysine

318

BROER ET AL.

APPL. ENVIRON. MICROBIOL.

TABLE 1. Aspartate kinase activity and lysine productivity

7.5

Sp act of aspartate

(nmol mg [dry kinasea Strain wt]-1 min-')

6.0

External lysine

concn (mM) E 4.5

+LT

-LT

0

0.028 0.015 0.012

KK 25 DG 52-5 MH 20-22B

E ,, 3.0

29 40 70

0.015 0.012 0.014

0

>

a Enzyme activity was determined in the absence (-LT) and presence (+LT) of the allosteric effectors L-lysine and L-threonine, 10 mM each.

1.5

0

w

0.0

4 0

concentration and hence the lack of lysine export, as observed during growth in mineral medium (3). The release of feedback inhibition of the aspartate kinase is the only mutation so far known within the sequence from aspartate to lysine that is common to most lysine producers and also to mutants obtained by classical breeding. To obtain a defined producer from the wild type, we therefore subcloned the feedback-resistant aspartate kinase gene, lysC2(Fbr), into a mobilizable nonreplicative vector to integrate it into the chromosome via homologous recombination with the wildtype aspartate kinase gene as a target. The resulting lysC2(Fbr)-containing vector was transferred via conjugation into the wild-type C. glutamicum ATCC 13032. Fiftytwo transconjugants selected for vector-borne kanamycin resistance were obtained, and in four of them kinase activities were determined. Strain KK 25 exhibited the expected twofold kinase activity in the in vitro assay (Table 1, -LT), due to the additional presence of lysC2 in its chromosome, resulting in the lysC+(Fbs) lysC2(Fbr) merodiploid. The activity attributable to lysC2 is feedback resistant (Table 1, +LT) and is responsible for the same uncontrolled flux to lysine as is observed in the production strains in the presence of lysine and threonine (5). Strain KK 25 excreted up to 29 mM lysine during growth in minimal medium, whereas the wild type excreted less than 2 mM under identical conditions. The classically obtained strain with feedback-resistant aspartate kinase (DG 52-5) produced 40 mM lysine. Strain MH20-22B, which resulted from two rounds of undirected mutagenesis and was selected for high lysine yields, produced about 70 mM lysine under comparable conditions. To characterize the lysine export in more detail, we used

i 0 x

.'A J-j

50

-i

40

20

10

30

40

Internal Lysine [mMl

FIG. 3. Dependence of the export velocity on internal lysine concentration. Symbols: *, MH 20-22B; A, DG 52-5; V, KK 25.

the efflux test (3) in the following experiments. For this, cells harvested in the late exponential phase and washed twice with cold buffer (50 mM Tris-HCl [pH 7.5], 10 mM NaCl, 10 mM KCI). When the temperature was increased to 250C, excretion of lysine was observed, as demonstrated both by a decrease of the internal and by a simultaneous increase of the external lysine concentration (Fig. 2). Kinetic constants of the excretion systems. The excretion systems of strains DG 52-5 and MH 20-22B showed Michaelis-Menten-type behavior (Fig. 3, Table 2). In strain KK 25, no excretion occurred below an internal lysine concentration of 15 mM. The maximum velocity and the Km of MH 20-22B and DG 52-5 were more or less identical between the strains, but the Vm. of both strains was about five to six times higher than for KK 25. Influence of proton concentration on export activity. We recently characterized lysine export in strain DG 52-5 as a 2OH-/lysine symport mechanism (4). In batch fermentations without pH control, we measured excretion in a range of pH values from 8.5 to 5. The data shown in Fig. 4 demonstrate that pH dependence is an important feature of these export systems. Although there was 20 to 30% variation in the absolute values (cf. Table 2), the relative differences among the three strains in the observed pH dependence were very similar in repeated experiments. Again, strain KK 25 had a much lower lysine export activity than did DG 52-5 and MH 20-22B. The latter two strains were very similar in secretion at alkaline pHs. Yet, export in strain MH 20-22B was considerably faster below pH 7.4. The pH optimum was 7.4 for KK 25 and MH 20-22B and 7.8 for strain DG 52-5. Influence of external lysine concentration on export activity. During fermentations, lysine can be accumulated up to concentrations of several hundred millimolar outside the cell (15). The external lysine concentration is an important parameter influencing export activity. Lysine showed significant trans effects (the cis side being the side from which the

were

v

30

TABLE 2. Kinetic parameters of lysine export in three different strains

v

20 10 0

0

6

12 18 24 Time [min]

Strain

Km (mM)

KK 25 DG 52-5 MH 20-22B

19 + 4 17 ± 6

30

FIG. 2. Export of lysine as determined by the increase of external (upper panel) and the decrease of internal (lower panel) lysine KK 25. concentration. Symbols: *, MH 20-22B; A, DG 52-5; 7,

,

a

no Michaelis-Menten-type kinetics.

nmol mi nVm,, mg (dry wt)-'

1.8 + 0.7 10 ± 2 11 ± 3

VOL. 59, 1993

C. GLUTAMICUM LYSINE EXCRETION SYSTEMS -

10

319

-

cm

8-

E

6-

I

E

-a 0I

E

E E

4 'D

E

jv

2-

cr: x

lU

0

6

7 pH

8

9

FIG. 4. Dependence of the export rate on external pH at low external lysine concentration. Symbols: *, MH 20-22B; A, DG 52-5; V, KK 25.

transport starts, hence the inside) on the export rate (Fig. 5). When the external lysine concentration was increased, excretion was considerably reduced. The relative trans effect was similar in the different strains tested. Again, lysine export in the wild-type background (KK 25) was slow compared with that of the two production strains. The data for strain MH 20-22B were corrected by using the correction curve of Fig. 1. Membrane potential and export. We recently showed (4) that the membrane potential is the predominant driving force for lysine export. As observed in bacteria in general and also in C. glutamicum, the membrane potential varies with pH (8). In all strains, we correspondingly measured a strong influence of this driving force (Fig. 6; note the logarithmic scale of the ordinate). As for pH dependence, differences between strains MH 20-22B and DG 52-5 became obvious. Altogether, the dependence of lysine secretion on membrane potential in the wild-type background was similar to that in strain DG 52-5; however, at much lower activity. Influence of pH at high external lysine concentration. The kinetic analysis of lysine export led to the conclusion that symport of lysine and OH- proceeds in the same step (4). Important experimental evidence for this model was the observation that variation of external lysine and H+ concentration showed mutual influence on lysine secretion. At high external lysine concentration, export was much more sensitive to changes in the external pH (compare Fig. 4 and 7). Strain MH 20-22B was less sensitive to alkaline pH at elevated external lysine concentrations than was strain DG

Membrane potential [mVl

FIG. 6. Dependence of the export velocity on membrane potential. Symbols: U, MH 20-22B; A, DG 52-5; V, KK 25.

52-5. Strain KK 25 could not be tested with respect to this effect because its export activity is too low. In Fig. 7, the first-order rate constants for lysine efflux are also given. This allows direct comparison with the results presented recently (4). The export rate (V = k [Lys]j.) was calculated by using an internal concentration of 42 mM in all cases. The data for strain MH 20-22B were corrected in accordance with Fig. 1.

DISCUSSION In this study, we analyzed the differences in lysine secretion among two different lysine producers bred by classical mutagenesis and the defined strain KK 25. The latter is the wild-type strain containing the feedback-resistant aspartate kinase activity of strain MH 20-22B. The presence of the lysC' copy in this strain is unlikely to change the properties of lysine export, since lysC+ when present on a multicopy plasmid had no detectable effect on export activity (5). The main difference between strain KK 25 and the production strains was the export rate. Strain KK 25 showed low lysine secretion activity in the medium used for these experiments (Vma = 1.8 nmol min-1 mg [dry weight] -1). The membrane potential dependence was similar to that of strain DG 52-5, and the pH dependence resembles that of strain MH 20-22B. Strains DG 52-5 and MH 20-22B are similar with respect to activity and affinity of the lysine export system. However, there are significant differences in dependence on external 0.10

12 m

0 10 E

0.08

8

0.06

Ic

9

l.

E

E .

E

6

0.04

3

4

0 -

j

a

0.02 2

0.00

0

6.8 0

50 100 150 200 250 300 350

External Lysine rmMI

FIG. 5. trans effect of lysine. Dependence of the export rate on external lysine concentration. Symbols: *, MH 20-22B; A, DG 52-5; V, KK 25.

7.2

7.6

8.0

8.4

8.8

9.2

pH

FIG. 7. Dependence of the export velocity on pH at high external lysine concentrations. Symbols: * and A, experiments at an external lysine concentration of 50 mM; O and A, experiments at an external lysine concentration of at 90 mM; * and El, MH 20-22B; A and A, DG 52-5.

320

BR6ER ET AL.

pH and on membrane potential. These differences are the reason for a twofold-higher export activity of strain MH 20-22B in comparison with strain DG 52-5 at low pH. Also, at alkaline pHs, differences were observed. The reduction of the transport rate at elevated external lysine concentrations was less pronounced for strain MH 20-22B. Recently, we have characterized the export system of strain DG 52-5 to be a 20H-/lysine symporter (4). The experimental results could be perfectly explained by a kinetic model, the essential properties of which are (i) transport of both substrates in one step and (ii) separation of this step from the membrane potential-dependent step. All results could be explained by different rate limitation due to one of these steps. The three strains proved to be similar with regard to membrane potential, lysine gradient, and pH gradient. We therefore conclude that the mechanism of lysine export (20H-/lysine symport) is conserved in all three strains and that the observed differences are caused by other, more subtle changes. The most obvious property of strain KK 25 is its low export activity. A possible explanation would be an increased expression of the export carrier in the mutants. The absence of export activity in strain KK 25 below 15 mM internal lysine cannot be explained conclusively. However, uptake of lysine may be able to balance export at this low rate to a significant extent. We recently determined the maximum velocity of the uptake system to 0.2 to 0.3 nmol min-' mg (dry weight)-1 (2). In contrast to these results, the observed differences between the two production strains DG 52-5 and MH 20-22B cannot be explained by a change in the overall activity. Since some mechanistic properties of the lysine carrier are changed, a mutation of the transport protein seems to be likely. Three lines of argument support this idea. (i) According to generally accepted models for carrier transport, the membrane potential dependence corresponds to the location of the energy barrier within the transport pathway (9). Recently, we have derived for the lysine export system that extrapolation of the membrane potential dependence to 0 mV is a good measure for the permeability coefficient (PO) of the electrogenic step, i.e., the reorientation of the unloaded carrier. In strain MH 20-22B, P0 is significantly higher than in DG 52-5 (k = 16 x 10-3 and 2 x 10-3, respectively). (ii) Lysine and OH- are transported in one step. Consequently, both substrates influence each other. This is shown in Fig. 7. The higher the lysine concentration, the higher is the trans effect of OH- and vice versa. The difference between strains DG 52-5 and MH 20-22B is expressed in a shift between these curves. This can be explained by our kinetic model (4) when changing the pK for the OH- binding site by about 0.2 U (pK = 7.4 instead pK = 7.2) and using a P, of 16 x 10-'. (iii) Lysine export in strain MH 20-22B is less sensitive to inhibition by low external pH. This corresponds well to the observed change in the dependence on membrane potential, since bT decreases with decreasing external pH (8). The results presented here support the idea that carriermediated lysine secretion occurs not only in mutants, where it was first observed, but also in the wild-type strain, provided the internal lysine concentration is elevated. We recently showed that lysine export can also reach significant activity in the wild type under conditions of peptide uptake (3). It is thus possible that in the course of mutations and selections for high productivity, an altered export permease was also selected. We therefore propose that the expression of the lysine export carrier in both production strains is higher than in the wild type and that at least strain MH 20-22B carries a mutated form of the export permease. It

APPL. ENVIRON. MICROBIOL.

must be taken into consideration, however, that alternative explanations for these results are, in principle, possible. Except for modulation by availability of substrates and influence of gradients (3, 4), the regulation properties of the lysine export permease on the levels of both synthesis and activity have not been studied so far. Changes in these properties may additionally account for the observed alterations in lysine export activity. ACKNOWLEDGMENTS We are indebted to H. Sahm for continuous support and for valuable discussions and suggestions. The work is part of a joint project with the DEGUSSA AG and is supported by grant 038409 from the Bundesministerium fur Forschung und Technologie and by the Fonds der Chemischen Industrie. Strain DG 52-5 was put at our disposal by the DEGUSSA AG.

REFERENCES 1. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 2. Broer, S., and R. Kramer. 1990. Uptake and exchange of lysine in Corynebacterium glutamicum. J. Bacteriol. 172:7241-7248. 3. Broer, S., and R. Kramer. 1991. Lysine excretion by Corynebacterium glutamicum. I. Identification of a specific carrier system. Eur. J. Biochem. 202:131-135. 4. Broer, S., and R. Kramer. 1991. Lysine excretion by Corynebacterium glutamicum. II. Energetics and mechanism of the transport system. Eur. J. Biochem. 202:136-142. 5. Cremer, J., L. Eggeling, and H. Sahm. 1991. Control of the lysine biosynthetic sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes. Appl. Environ. Microbiol. 57:1746-1752. 6. Cremer, J., C. Treptow, L. Eggeling, and H. Sahm. 1988. Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum. J. Gen. Microbiol. 134:3221-3229. 7. Ebbighausen, H., B. Weil, and R. Krimer. 1989. Isoleucine excretion in Corynebacterium glutamicum: evidence for a specific efflux carrier system. Appl. Microbiol. Biotechnol. 31:184190. 8. Ebbighausen, H., B. Weil, and R. Krimer. 1989. Transport of branched-chain amino acids in Corynebacterium glutamicum. Arch. Microbiol. 151:238-244. 9. Hall, J. E., C. A. Mead, and G. Szabo. 1973. A barrier model for current flow in lipid bilayer membranes. J. Membr. Biol. 11:7597. 10. Hoischen, C., and R. Kramer. 1989. Evidence for an efflux carrier system involved in the secretion of glutamate by Corynebacterium glutamicum. Arch. Microbiol. 151:342-347. 11. Hoischen, C., and R. Kriimer. 1990. Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum. J. Bacteriol. 172:3409-3416. 12. Jones, B. N., and J. P. Gilligan. 1983. o-Phthaldialdehyde precolumn derivatization and reversed phase high performance liquid chromatography of polypeptide hydrolysates and physiological fluids. J. Chromatogr. 266:471-482. 13. Klingenberg, M., and E. Pfaff. 1967. Means of terminating reactions. Methods Enzymol. 10:680-684. 14. Menkel, E., G. Thierbach, L. Eggeling, and H. Sahm. 1989. Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate. Appl. Environ. Microbiol. 55:684-688. 15. Nakayama, K. 1985. Lysine, p. 607-620. In M. Moo-Young (ed.), Comprehensive biotechnology, vol. 3. Pergamon Press, Inc., Oxford. 16. Nakayama, K., H. Tanaka, H. Hagino, and S. Kinoshita. 1966. Studies on lysine fermentation. Part V. Concerted feedback inhibition of aspartokinase and the absence of lysine inhibition on aspartic semialdehyde-pyruvate condensation in Micrococcus glutamicus. Agric. Biol. Chem. 30:611-616. 17. Rottenberg, H. 1979. The measurement of membrane potential

VOL. 59, 1993

and pH in cells, organelles, and vesicles. Methods Enzymol. 55:547-569. 18. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 19. Sano, K., and I. Shiio. 1970. Microbial production of L-lysine. III. Production by mutants resistant to S-(2-aminoethyl)-Lcysteine. J. Gen. Appl. Microbiol. 16:373-391.

C. GLUTAMICUM LYSINE EXCRETION SYSTEMS

321

20. Schifer, A., J. Kalinowski, R. Simon, A. Seep-Feldhaus, and A. Pfihler. 1990. High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria. J. Bacteriol. 172:1663-1666. 21. Simon, R., U. Priefer, and A. Pfihler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1:784791.