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Journal of Bacteriology, August 1999, p. 4598-4604, Vol. 181, No. 15
Sector of Organic Chemistry and Biochemistry,
Department of Chemistry, University of Ioannina, 45110 Ioannina,
Greece
Received 8 April 1999/Accepted 19 May 1999
Exponentially growing cells of Zymomonas mobilis
normally exhibit a lag period of up to 3 h when transferred from
0.11 M (2%) to 0.55 M (10%) glucose liquid medium. A mutant of
Z. mobilis (CU1Rif2), fortuitously isolated, showed more
than a 20-h lag period when grown under the same conditions, whereas on
0.55 M glucose solid medium, it failed to grow. The growth of CU1Rif2 on elevated concentrations of other fermentable (0.55 M sucrose or
fructose) or nonfermentable (0.11 M glucose plus 0.44 M maltose or
xylose) sugars appeared to be normal. Surprisingly, CU1Rif2 cells grew
without any delay on 0.55 M glucose on which wild-type cells had been
incubated for 3 h and removed at the beginning of their
exponential phase. This apparent preconditioning was not observed with
medium obtained from wild-type cells grown on 0.11 M glucose and
supplemented to 0.55 M after removal of the wild-type cells. Undelayed
growth of CU1Rif2 on 0.55 M glucose previously conditioned by the wild
type was impaired by heating or protease treatment. It is suggested
that in Z. mobilis, a diffusible proteinaceous heat-labile
factor, transitionally not present in 0.55 M glucose CU1Rif2 cultures,
triggers growth on 0.55 M glucose. Biochemical analysis of glucose
uptake and glycolytic enzymes implied that glucose assimilation was not
directly involved in the phenomenon. By use of a wild-type Z. mobilis genomic library, a 4.5-kb DNA fragment which complemented
in low copy number the glucose-defective phenotype as well as
glucokinase and glucose uptake of CU1Rif2 was isolated. This fragment
carries a gene cluster consisting of four putative coding regions,
encoding 167, 167, 145, and 220 amino acids with typical Z. mobilis codon usage, Zymomonas mobilis, a
strictly fermentative gram-negative ethanologenic bacterium, obtains
its metabolic energy anaerobically via the Entner-Doudoroff pathway
(9, 20, 31, 44, 50, 51). It is an ideal organism for
studying unclarified aspects of glycolytic flux in conjunction with
sugar tolerance mechanisms. Its carbohydrate range is limited to
glucose, fructose, and sucrose, with the last being hydrolyzed to its
component hexoses via two extracellular hydrolases (37, 56).
Both glucose and fructose are taken up by a low-affinity-,
high-velocity-facilitated diffusion system (11, 34, 53)
encoded by glf (2). However, Z. mobilis definitely prefers the former, as indicated by the much
higher affinity of the transport system for glucose as well as by the inhibition of fructose kinase by glucose (35). Z. mobilis as a typical saccharophilic organism may thrive on
exceptionally high concentrations of sugars (45, 50). The
ability of Z. mobilis to counteract detrimental osmotic
effects when grown on sucrose or mixtures of glucose and fructose has
been attributed to the formation of sorbitol (25, 27) as a
result of the activity of glucose-fructose oxidoreductase (GFOR)
(57). However, sorbitol or any other compatible solute is
not formed by Z. mobilis when grown on glucose as a sole
carbon source, at least not to amounts sufficient to account for
osmotic protection (27). On the other hand, all strains of
Z. mobilis tested so far could grow on 1.11 M (20%) glucose
within 34 h, whereas some strains were able to grow on up to 2.22 M (40%) glucose after a long lag phase of 4 to 20 days
(50). It appears that Z. mobilis cells can be
adjusted to grow on glucose following a lag period, the length of which depends upon the glucose concentration. For instance, strain ATCC 10988 proliferates on 0.55 and 1.11 M glucose media after lag periods of 3 and 40 h, respectively (12). The ability of Z. mobilis to grow on high glucose concentrations was originally explained by a rapid equilibration of the external and internal glucose
concentrations achieved by the glucose facilitator system (11,
48). However, later findings showed that the internal concentration of glucose in growing Z. mobilis cells
remained low (19), whereas after analysis with
13C nuclear magnetic resonance spectroscopy, no other major
compatible solutes were found (27). The basis of this
phenomenon has not been studied before for Z. mobilis. For
the clarification of this puzzle, the availability of Z. mobilis mutants with impaired growth on high glucose
concentrations is indispensable. In the present report, the ability of
Z. mobilis to grow on elevated glucose concentrations is
investigated by use of a derivative of strain ATCC 10988 with delayed
growth on high glucose concentrations (1, 12).
Strains, plasmids, and growth conditions.
Z. mobilis
wild-type ATCC 10988 (50) and mutants CU1 (12)
and CU1Rif2 (1) were grown semianaerobically at 30°C in
complete liquid or solid medium as described before (1). To
avoid caramelization, carbohydrate solutions were sterilized separately
as concentrated stock solutions and then added to liquid medium at the
desired concentrations. Exponentially growing cells were used as
inocula to yield a starting liquid culture of approximately
107 cells per ml. Growth was monitored turbidimetrically at
a wavelength of 600 nm. An optical density at 600 nm
(OD600) of 0.9 corresponds to 0.35 mg of dry cell
weight · ml Estimation of glucose concentrations.
The amount of glucose
consumed during inoculation was calculated by subtracting the amount of
glucose remaining in the culture broth at the time of assay from the
initial amount of glucose. The amount of glucose was estimated with a
hexokinase Olympus System Reagent Kit (Olympus Diagnostics GmbH,
Hamburg, Germany).
Lipid analysis.
For phospholipid and fatty acid analysis,
cells were harvested in the late exponential phase by centrifugation
(6,000 × g, 10 min, 4°C), washed with distilled
water, lyophilized, and extracted by the method of Bligh and Dyer
(5). The amounts of phospholipids and fatty acids were
determined as described previously (23). Hopanoids were
analyzed by gas-liquid chromatography following three extractions (1 h
each) of freeze-dried cells under reflux with chloroform-methanol (2:1
[vol/vol] and treatment with
H5IO6-NaBH4 as described by Rohmer
et al. (38).
Enzyme assays.
Cells from 200 ml of liquid culture were
harvested at the mid-exponential phase by centrifugation
(6,000 × g, 10 min), washed with enzyme assay buffer
containing mercaptoethanol (14 mM), resuspended in 1 ml of the same
buffer, and disrupted in a Mini Bead Beater (Biospec Products,
Bartlesville, Okla.) essentially as previously reported
(22). The homogenate was centrifuged (10,000 × g, 5 min), and the supernatant was used as the crude cell extract. Glucokinase (GLK) and glucose-6-phosphate dehydrogenase were assayed as
described by Scopes et al. (42). GFOR was assayed
comparatively for wild-type and mutant cells essentially as described
by Zachariou and Scopes (57) by coupling the reaction with
indigenous gluconolactonase activity. Pyruvate decarboxylase (PDC) was
assayed by coupling to alcohol dehydrogenase (ADH) and measuring the
oxidation of NADH at 340 nm as described by Neale et al.
(32). ADH was assayed by determining the production of
ethanol as described by Conway et al. (10). All enzyme
reactions were initiated by adding the cell extract, and enzyme
activities were expressed in micromoles per minute per milligram of
protein. Protein concentration was determined by the method of Lowry et
al. (28).
Glucose uptake assays. (i) Z. mobilis.
Cells were
harvested at the mid-exponential phase, washed with phosphate buffer
(100 mM, pH 6.5), and resuspended in the same buffer essentially as
described by Walsh et al. (52). Glucose uptake was measured
with D-[U-14C]glucose (291 mCi/mmol;
Amersham, Buckinghamshire, England) at concentrations ranging from 0.25 to 50 mM. Z. mobilis cells (50 µl) and
fivefold-concentrated radiolabelled glucose (12.5 µl) were
preincubated separately at 20°C, mixed together to yield the
appropriate glucose concentration, and vortexed immediately. Uptake was
stopped by the addition of 10 ml of cold ( (ii) E. coli.
Glucose uptake was assayed with
2-deoxy-D-[U-14C]glucose (308 mCi/mmol;
Amersham) at a final concentration of 5 mM at 10°C essentially as
described by Weisser et al. (53) with the following
modifications. Briefly, E. coli was grown in M9 minimal
medium (40) supplemented with 0.5% gluconate, thiamine (1 µg/ml), and chloramphenicol (25 µg/ml).
Isopropyl- Bacterial conjugation.
Conjugal transfer of recombinant
plasmids in Z. mobilis CU1Rif2 was performed as described
previously (1) with double-donor filter matings and pRK2013
(14) (Kmr; 50 µg/ml) as a helper plasmid.
DNA methods.
Preparation of plasmids from E. coli, restriction enzyme digestions, ligations, DNA
electrophoresis, and Southern blot analysis were performed by standard
protocols (40). Plasmid DNA was isolated from Z. mobilis as previously described (43). Transformations of E. coli were done by chemical treatment (24).
DNA was isolated from agarose gels by use of GeneClean II (Bio 101, Inc., La Jolla, Calif.). DNA labelling and hybridization were performed
by the digoxigenin nonradioactive labelling method (Boehringer).
Genomic libraries of Z. mobilis CP4 (G. A. Sprenger,
Forschungszentrum, Jülich, Germany) and ATCC 10988 (C. Drainas)
were prepared by digestion of Z. mobilis genomic DNA with
Sau3A (~25-kb fragments). The digested fragments were
ligated into the BamHI-ScaI sites of the cosmid
vector pLAFR5 (21) and in vitro packaged by use of a
Stratagene Gigapack II packaging extract according to the instructions
of the manufacturer. Following transfection in E. coli
DH5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Zymomonas mobilis Mutant with Delayed
Growth on High Glucose Concentrations
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
35 and 10 promoter elements, and
individual Shine-Dalgarno consensus sites. However, strong homologies
were not detected in a BLAST2 (EMBL-Heidelberg) computer search with
known protein sequences.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
1. Dry cell weight was determined as
described by Loos et al. (27). For minimal medium cultures,
a chemically defined solution was used as described by Galani et al.
(17). When needed, complete or minimal medium with 0.55 M
glucose was conditioned with ATCC 10988 prior to inoculation with
CU1Rif2 cells. In these cases, ATCC 10988 inoculum was removed by
centrifugation (6,000 × g, 10 min), and the medium
supernatant was filtered (0.2-µm-pore-diameter Millipore filter) to
remove unsedimented cells. The filtrate was used untreated, heated, or
incubated with hydrolytic enzymes for 1 h before being inoculated.
Escherichia coli DH5
(18) was grown at 37°C
in Luria broth (29). The low-copy-number cosmid pLAFR5
(21) (Tcr; 20 µg/ml) was used for expression
in Z. mobilis. Plasmid pZY507 (53)
(Cmr; 25 µg/ml) was used for expression in E. coli ZSC112L
pts (53), and pUC18 (Boehringer Mannheim
Biochemicals) (Apr; 100 µg/ml) was used for subcloning
and sequencing. Transconjugants of Z. mobilis CU1Rif2 were
selected with tetracycline (40 µg/ml) and rifampin (20 µg/ml).
2.5°C) phosphate buffer
(100 mM, pH 7.5) containing 500 mM unlabelled glucose. Cells were
immediately filtered and washed with 10 ml of the same buffer. The
uptake rate was expressed as nanomoles of glucose taken up per minute
per milligram of total protein.
-D-thiogalactopyranoside (IPTG) (1 mM) was added at an OD600 of 0.2 to induce the tac
promoter. Cells were harvested in the late logarithmic phase
(OD600, 0.7) and assayed for uptake. The uptake reaction
was stopped at different times by the addition of 0.1 M phosphate
buffer (pH 7.5) containing 500 mM glucose and rapid filtration.
, two libraries of approximately 500 cell clones each were
produced. All transfected E. coli cells tested had
recombinant cosmids with inserts ranging from 24 to 35 kb.
TABLE 1.
Plasmids
DNA sequencing. Sequencing of the 4.5-kb Z. mobilis DNA fragment of pUC1845 was done by the dideoxy termination method (41) on an automated DNA sequencer (Applied Biosystems ABI Prism model 211) at the UCLA DNA Sequencing Facility (Erik Avaniss-Aghajani).
Computer analysis. A computer search for homologies to known nucleotide or protein sequences was performed with the BLAST2 program at the EMBL-Heidelberg website (13a). Analysis of the Z. mobilis sequences was aided by use of IntelliGenetics PC/Gene software (Oxford Molecular).
Nucleotide sequence accession number. The complete nucleotide sequence of the 4.5-kb Z. mobilis DNA fragment has been submitted to the EMBL database under accession no. AJ009974.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Growth of wild-type Z. mobilis and derivative CU1Rif2 on media with high sugar concentrations. Mutant CU1 was fortuitously isolated by mild acridine orange treatments in the course of screening for plasmid-cured isolates of Z. mobilis ATCC 10988 (12). This strain, appearing to have lost one of the smaller cryptic plasmids, was checked for growth on various concentrations of glucose and found to exhibit a pronounced lag period when grown in complex or minimal liquid medium with 0.55 M glucose, whereas it failed to grow at all on either solid medium. Mutant CU1Rif2 is a rifampin-resistant derivative of CU1 isolated to facilitate conjugal transfers from E. coli to Z. mobilis (1) and has been used in our laboratory since isolation as a routine experimental strain. CU1 and CU1Rif2 have identical phenotypes for growth on various sugar concentrations, plasmid content, lipid content, glucose uptake, and glycolytic enzyme activities. Therefore, only CU1Rif2 is discussed here.
We checked the growth of CU1Rif2 on high concentrations of various carbon sources. As shown in Fig. 1B, a pronounced extension of the lag phase (up to 20 h) was observed for the mutant when grown in liquid media with 0.55 M glucose, whereas it failed to grow at all on similar solid media. Its growth rate was unaffected by high concentrations of other fermentable sugars, such as fructose (up to 0.55 M), sucrose (up to 0.55 M), or glucose plus fructose (0.11 and 0.44 M, respectively). Similarly, the presence of high concentrations (up to 0.44 M) of nonfermentable sugars, such as maltose (not taken up) or xylose (taken up but not metabolized) (48, 54), in 0.11 M glucose media did not affect the growth of CU1Rif2 (data not shown). On the contrary, a similar extension of the lag phase (20 h) was observed in cultures containing 0.11 M glucose and a 0.44 M concentration of the glucose analog 2-deoxyglucose (DOG), which does not support growth (Fig. 1B). The addition of sorbitol at 50 mM did not reduce the lag period on 0.55 M glucose but expedited the exponential growth of CU1Rif2 in the presence of higher glucose concentrations (1.38 M), as in the wild-type strain (Fig. 1A and B). The growth of CU1Rif2 was the same in complete or minimal medium.
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) or 0.55 M glucose or 0.11 M glucose plus 0.44 M
DOG (
). After being precultured in the presence of 0.55 M glucose or
after 20 h of incubation with 0.55 M DOG, cells were inoculated
into liquid complete medium containing 0.55 M glucose (
), or 1.38 M glucose plus 50 mM sorbitol (
). Each
experiment was repeated six times with a standard error of less than
5%.
Detection of a diffusible factor affecting growth on high glucose concentrations. Once the growth of the mutant was established on 0.55 M glucose, or after 20 h of incubation in medium with 0.55 M DOG, growth occurred as for the wild type upon transfer to fresh solid or liquid medium with 0.55 M glucose (Fig. 1B). This result was observed even when the transferred mutant cells were washed with fresh liquid medium prior to inoculation, implying that they had at least phenotypically changed. However, subculturing of such cells through a growth cycle on 0.11 M glucose medium once again revealed the mutant phenotype of a long lag in the high-glucose liquid medium and no growth on the high-glucose solid medium. These results argue against reversion or suppression occurring during the lag phase. Instead, it appeared that a preconditioning of the medium was involved. Thus, when the wild-type strain was incubated to the beginning of exponential growth (3 h) in 0.55 M glucose complete or minimal medium and the cells were then removed (see Materials and Methods; the glucose concentration at this point was 0.54 M), mutant cells grew without delay. When the wild-type strain was preincubated in 0.11 M glucose medium which was supplemented to 0.55 M glucose after removal of the cells, a normal delay again occurred with mutant cells, as if the preconditioning required the high glucose concentration. The same phenomenon of preconditioning of the medium also occurred with the mutant culture itself; incubation of the mutant in 0.55 M glucose medium to the end of the 20-h lag phase (instead of the 2 h required for the wild type), followed by removal of the cells, allowed growth without delay of fresh mutant cells not exposed to high glucose. The putative growth lag factor was lost after treatment at 50°C for 30 min, at 75°C for 5 min, or with proteinase K (150 mU/ml) for 1 h but was stable after 1 h of treatment with phospholipase D (50 mU/ml), phospholipase A2 (500 mU/ml), DNase (150 U/ml), or RNase (75 U/ml). None of these treatments affected the growth of the wild type.
Lipid composition of Z. mobilis CU1Rif2. Due to the unusual lipid composition and the reported correlation with high sugar tolerance (6, 7), we examined the lipid composition of the Z. mobilis strains used in this work. No significant differences in the major phospholipid, fatty acid, and hopanoid contents of the wild type, CU1, and CU1Rif2 were observed.
Glucose uptake and enzyme activities.
Glucose transport was
measured in cells taken from 0.11 M glucose medium and subcultured for
3 h in medium with 0.11 or 0.55 M glucose. A decrease in transport
activity was found in the mutant (see fig. 3B) but not in the wild type
(see fig. 3A). In both cases, analysis of uptake kinetics (data not
shown) did not reveal clear differences in Km
values (for wild type versus mutant or for 0.11 versus 0.55 M glucose).
The apparent Km values were between 5.55 and
15.7 mM, in agreement with earlier reports (11, 35, 48),
although the Vmax values of 15 to 25.4 nmol
· min1 · mg of protein1 were low.
Enzyme activities measured for similarly treated cells (Table
2) showed no differences (for wild type
versus mutant or for glucose concentrations) for glucose-6-phosphate
dehydrogenase, PDC, or ADH. GLK activity, on the other hand, was
approximately doubled in the wild type exposed to the higher glucose
concentration but not in the mutant; no differences were seen in
Km values with glucose (Table 2). Although the
differences in transport and GLK activity between the wild type and the
mutant are likely to be related to the lag in mutant growth on high
glucose, they offer no clear explanation for the phenomenon.
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Isolation of a DNA fragment which complements the phenotype of
CU1Rif2.
A genomic library from Z. mobilis CP4 was
transferred to strain CU1Rif2 by bacterial conjugation, and 600 transconjugant colonies were isolated. Each colony was tested for
sensitivity to media containing 0.55 M glucose, and two clones
resistant to 0.55 M glucose were selected. Restriction analysis of the
DNA fragments (~25 kb each) isolated from both transconjugants
indicated that they contained overlapping regions. One of them was
further subcloned in pLAFR5 (Fig. 2). The
resulting pLAFR5 recombinants were transformed in DH5 and
transferred by conjugation to CU1Rif2, and transconjugants were tested
again for growth on 0.55 M glucose medium (Fig. 1C). Initially, a
9.5-kb fragment complementing CU1Rif2 was isolated. Further subcloning
of this fragment led to the isolation of functional subfragments of
8.9, 5.1, and 4.5 kb, whereas the HindIII and ApaI-SalI fragments were not functional (Fig. 2).
The 9.5-kb fragment hybridized strongly with chromosomal DNAs from both
Z. mobilis CP4 and Z. mobilis ATCC 10988. Furthermore, a similar clone was isolated from the ATCC 10988 genomic
library by hybridization under high-stringency conditions with the
4.5-kb fragment as a probe. Analysis of the ATCC 10988 clone revealed
the same restriction pattern and complementing function as for the CP4
4.5-kb fragment.
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Nucleotide sequence of the 4.5-kb Z. mobilis fragment in pUC1845. Both strands were sequenced throughout the 4.5-kb region, and the sequence revealed the existence of four putative coding regions (open reading frames [ORFs]) (Fig. 2). ORF 1 consists of 501 bp (nucleotides 376 to 876), ORF 2 has 501 bp (nucleotides 1032 to 1532), ORF 3 has 435 bp (nucleotides 1648 to 2082), and ORF 4 contains 660 bp (nucleotides 2115 to 2774). Each of the four ORFs is preceded by potential ribosome-binding sites (Shine-Dalgarno consensus sequences). All four predicted ORFs begin with an ATG start codon, which is typical for Z. mobilis protein-coding sequences, and use a TAA stop codon (except for ORF 1, which uses TGA), which appears to be the most commonly used in Z. mobilis. Furthermore, each of the four ORFs displays synonymous codon usage statistics, a finding which is typical for protein-coding sequences of Z. mobilis (data not shown).
The overall organization of the cluster of the four ORFs suggests that they may be cotranscribed as an autonomous operon, as in the cases of glf-zwf-edd-glk for glucose metabolism (3) and gluEMP for glutamate transport (36). Putative35 and 10 Z. mobilis promoter elements (47)
are located at bases 261 to 273 and 298 to 306, respectively, i.e., 70 bp upstream of the start codon of ORF 1, whereas a putative
transcription terminator sequence is found immediately downstream of
the stop codon of ORF 4 (bases 2819 to 2851).
The 3.3-kb BamHI-HindIII segment of pUC1845
containing all four putative coding regions along with the promoter and
terminator sequences was transferred to pLAFR5 (pLAFR533; Table 1),
expressed in CU1Rif2, and found to be sufficient for complementation of the glucose-sensitive phenotype. On the other hand, complementation was
not achieved with pLAFR531 (Fig. 2), in which the cluster sequence is
disrupted at the ApaI site of ORF 4 (nucleotide 2582). Removal of the 260-bp 5'-terminal sequence of the 4.5-kb fragment by
partial HindIII digestion resulted in abrogation of the
complementing phenotype, probably due to disruption of the 35
promoter sequence. The possibility that products of the cloned DNA
fragment could be involved with the expression of secondary glucose
uptake in Z. mobilis was ruled out because the functional
3.3-kb fragment subcloned in the pZY507 vector did not complement
E. coli ZCL11Lpts for glucose uptake as it did strain
ZCL11Lpts/glf (53; data not shown).
Additionally, plasmid pLAFR5glf (Table 1) was transferred to
Z. mobilis CU1Rif2 and found to be negative in functional
tests, indicating that glf does not restore the phenotype of CU1Rif2.
A computer search for similar sequences deposited in the database at
the EMBL-Heidelberg website (13a) yielded no information on
the functions of the proteins. No sequence similarities between any of
the four ORFs and cloned DNA fragments encoding glucose transporters
(33) or other outer membrane proteins associated with
glucose uptake (39, 55) were identified. However, the search
revealed a set of proteins from other species highly homologous to the
products of ORF 1, ORF 2, and ORF 4. ORF 1 and ORF 2 show the strongest
homology to a Rhodobacter capsulatus ORF upstream of the
nifR3 nitrogen regulatory gene (15). The ORF 1 product shows 40% sequence identity over 137 residues (positions 10 to 146 of the R. capsulatus ORF), and the ORF 2 product shows
59% sequence identity over 160 residues (positions 226 to 376 of the R. capsulatus ORF). In addition, ORF 2 has 50 to 54%
sequence identity with a set of sequences homologous to the C-terminal half (positions 226 to 376) of the R. capsulatus ORF,
including a 158-amino-acid ORF immediately upstream of the
gltX glutamyl-tRNA synthetase gene in Bacillus
subtilis (16) and a 159-amino-acid ORF in the region
upstream of a cluster of four genes involved in stationary-phase
survival in E. coli (26). No significant homology
to known sequences was detected for the ORF 3 product. Finally, the ORF
4 product displays 40 to 46% sequence identity over its last 150 to
170 residues with an ORF of 160 to 165 nucleotides identified upstream
of the homologous recA DNA recombination gene in E. coli (13), Enterobacter agglomerans, and
Pseudomonas putida. A similar degree of homology is found
between ORF 2 and an ORF of 177 amino acids located upstream of the
cheA, cheW, and cheY chemotactic
factor genes in Thermotoga maritima (49);
interestingly, cheY is often encountered upstream of a
recA gene (8). The same region of ORF 2 has 33 to
35% sequence identity with the C-terminal half of an ORF
(cinA) encoding a putative competence-damage protein in
Streptococcus pneumoniae or B. subtilis;
cinA is also located upstream of the recA gene in
these gram-positive bacteria (30).
In conclusion, a new phenomenon has been observed: the impaired
adaptation of a mutant to growth on a high glucose concentration appears to be related, surprisingly, to the delayed production of a
presumably proteinaceous and diffusible factor produced in response to
the high glucose concentration. The factor may be related to the
as-yet-unexplained mechanism of adaptation of the wild-type strain to
growth on high glucose. In keeping with the knowledge that sorbitol
does not accumulate during growth on high glucose (in contrast to
growth on sucrose), the mutant has been shown not to be defective in
GFOR (data not shown), and changes in other functions, such as glucose
transport and GLK activity, are no more than suggested. A cluster of
genes of unknown function, with similarity only to unassigned sequences
in other organisms, complements in low copy numbers the various
phenotypes of the mutant. Hence, analysis of these genes may reveal the
mutant lesion and mechanism of adaptation.
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ACKNOWLEDGMENTS |
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We thank Georg A. Sprenger (Forschungszentrum, Jülich,
Germany) for critical reading of the manuscript as well as for
providing the genomic library of Z. mobilis CP4, the
ZCL11Lpts E. coli strains, and the plasmids
pUC18glf, pZY507, and pZY507glf.
This study was supported financially by the Greek General Secretariat of Research and Technology (program PENED 1996; contract 95ED39) and by the Greek and French governments (French-Hellenic 1997 and 1998 PLATON programs).
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FOOTNOTES |
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* Corresponding author. Mailing address: Sector of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece. Phone: 30-651-98372. Fax: 30-651-47832. E-mail: cdrainas{at}cc.uoi.gr.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Journal of Bacteriology, August 1999, p. 4598-4604, Vol. 181, No. 15
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