Previous Article | Next Article 
Applied and Environmental Microbiology, December 1998, p. 5016-5019, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Recombinant Klebsiella oxytoca Strains
with Improved Efficiency in Removal of High Nitrate Loads
Guadalupe
Piñar
and
Juan L.
Ramos*
Department of Biochemistry and Molecular and
Cellular Biology of Plants, Estación Experimental del
Zaidín
Consejo Superior de Investigaciones Científicas,
18008 Granada, Spain
Received 22 June 1998/Accepted 2 October 1998
 |
ABSTRACT |
Klebsiella oxytoca CECT 4460 removes high nitrate loads
from industrial wastewaters without accumulation of nitrite under optimal culture conditions; however, under nonoptimal conditions nitrite accumulates. This situation reflects an in vivo-limited functioning of nitrite reductase in this strain. As a way to overcome this limitation, an increase in the nitrite reductase gene dose in
K. oxytoca CECT 4460 was considered. To achieve this,
we cloned and transferred into this strain the Klebsiella
pneumoniae nasB gene, which encodes assimilatory nitrite
reductase (Lin et al., J. Bacteriol. 176:2551-2559, 1994). The
delivery vector was either the wide-host-range plasmid pUPE2, in
which the nasB gene is expressed from the Escherichia
coli Plac promoter, or a
mini-Tn5-Km vector, which upon random insertion
in the host chromosome allowed expression of the nasB gene
from an unidentified chromosomal host promoter. The effect of the
increase in the dose of the nasB gene in K. oxytoca CECT 4460 on the accumulation of nitrite in the culture medium was tested in two recombinant strains. The results obtained showed that K. oxytoca CECT 4460 bearing pUPE2
accumulated 88% less nitrite than the wild-type strain, while the
recombinant strain bearing the K. pneumoniae nasB gene
in the host chromosome showed a 25% lower level of nitrite
accumulation in the culture medium than that of the wild type.
| |
TEXT |
Biological removal of nitrogenous
compounds from industrial wastewaters with high nitrogen contents has
been the focus of many studies, mainly because of its potential
advantage and improved removal of these compounds over physical and/or
chemical processes (4, 9, 18). Effluents from industrial
facilities producing fertilizers, semiconductors, and munitions contain
high nitrate loads and are difficult to treat (3, 14, 15, 18, 20, 23). In fact, when these wastewaters reach conventional treatment plants a futile reduction of nitrate to nitrite takes place, which eventually leads to the full collapse of the treatment plants (10). In our efforts to provide microorganisms useful
for the biotreatment of wastewaters from the production of
dinitroethylene glycol, we isolated a strain of Klebsiella
oxytoca capable of tolerating high nitrate loads (up to at least 1 M NO3
) and capable of removing from a batch
nitrate at concentrations up to 150 mM if sufficient glycerol as a C
source was supplied in the culture medium (14). We found
that under continuous culture operation the strain could perform in a
zone of double nutrient limitation, so that at C/N ratios from 8 to 11 full removal of both C and N sources took place (16).
However, when conditions in batch or continuous cultivation were
suboptimal, e.g., nitrate load shock, when the C/N ratio was
inappropriate, and/or when sucrose was used in the batch instead of
glycerol, we observed that part of the nitrate (between 20 and 50%)
was reduced to nitrite, which accumulated in the culture medium and
eventually led to the arrest of cell growth. Since, during the
long-term operation of industrial wastewater treatment plants, it is
expected that sudden physicochemical alterations can lead to suboptimal
operational conditions, we have explored ways to avoid the accumulation
of nitrite. We came to the conclusion that for K. oxytoca CECT 4460 the limiting step in the removal of nitrate
under nonoptimal conditions might be the level of in vivo nitrite
reductase activity. We then reasoned that a way to bypass this
limitation might be to increase the dose of the gene for and expression
of the nitrite reductase in this strain. This hypothesis implies that
no limitation in the uptake and in the reduction of nitrate to nitrite
occurs under nonoptimal conditions.
Subcloning of the Klebsiella pneumoniae nasB gene and
its transfer to K. oxytoca CECT 4460.
The
assimilatory nitrate reductase (nasCA) and nitrite
reductase (nasB) genes of K. pneumoniae are
part of a cluster of genes involved in nitrate assimilation and have
previously been cloned (11, 12). Hybridization studies with
these genes revealed that in K. oxytoca the
nasCBA genes were present as a single copy on the host
chromosome and that these genes were clustered in a 17-kb
HindIII operon as in K. pneumoniae (Fig. 1). Given the strong homology of the nasB genes of these two strains, we
decided to increase the nasB gene dose in K. oxytoca CECT 4460 via the cloning of the K. pneumoniae
nasB gene in the former host strain.
View larger version (67K):
[in this window]
[in a new window]
|
FIG. 1.
Hybridization of total DNA of K. oxytoca
CECT 4460 and its recombinant derivative GP1 with the nasB
probe. Chromosomal DNA from K. oxytoca CECT 4460 (lane 2) and GP1 (lane 1) cells grown on minimal medium was prepared
(21), digested with HindIII, and hybridized
with the nasB gene of K. pneumoniae
labelled with digoxigenin as described before (19).
Immunodevelopment of the Southern blot was performed as recommended by
Boehringer Mannheim. The size marker was lambda DNA digested with
HindIII and labelled with digoxigenin (lane M); from top
to bottom, the sizes of the bands were 23.1, 9.4, 6.6, 4.4, 2.3, and
2.0 kb.
|
|
To this end two strategies were considered. In the first one, the
promoterless
K. pneumoniae nasB gene was excised from
plasmid
pVJS615 (
11) with
BamHI and
KpnI and ligated between the same
sites in plasmid pJB3Km
(
1). The ligation mixture was transformed
in
Escherichia coli JM109 (
23), and a clone bearing
the correct
plasmid was selected and called pUPE2 (Fig.
2). In this plasmid,
which encodes
kanamycin resistance, the
nasB gene is read from
the
P
lac promoter. The plasmid carrying
mob+ and
tra, pUPE2, was mobilized
from
E. coli JM109 to
K. oxytoca CECT 4460 by triparental mating in which
E. coli HB101
(pRK600)
was a helper strain (
2,
6-8). Kanamycin-resistant
(Km
r) transconjugants of
K. oxytoca were
selected on M8 minimal medium
with glycerol as the sole C
source, 50 µg of kanamycin per ml,
and 20 mM
NO
3. The frequency of transconjugants
was on the order of 10
5 per recipient cell. Because no
lacI gene encoding the LacI repressor
was present in
K. oxytoca CECT 4460, we concluded that the expression
of the cloned
nasB gene in pUPE2 was constitutive
(
17). In the
second strategy, the
nasB gene was
cloned into a mini-Tn
5Km in
plasmid pUTKm (
6),
whose replication depends on the presence
of the PIR protein
(
8). The cloning was designed so that reading
of the
nasB gene would require the expression of the gene from
a
host promoter upon the insertion of the mini-Tn
5 in the host
chromosome. Plasmid pUPE5 (Fig.
2) bears the gene within the borders
of
the mini-Tn
5-Km transposon. This plasmid was
propagated in
E. coli CC118
PIR, and it was
used as a delivery system of the
nasB gene into the
K. oxytoca chromosome. To this end a triparental
mating among
K. oxytoca CECT 4460 as a recipient,
E. coli CC118
PIR
(pUPE5), and
E. coli HB101 (pRK600) was performed (
7). Since
different
insertions could give rise to different levels of
nasB gene
expression, 50 random Km
r K. oxytoca
transconjugants were chosen for further studies and
grown in
liquid medium with 40 mM nitrate and 2% (wt/vol) sucrose.
Under
these conditions the wild-type strain accumulated a high
level of
nitrite in the culture medium (up to 30 mM). The transconjugants
were
grown on the same medium, and the transconjugant derivative
that
accumulated the lowest level of nitrite (about 10 mM in two
assays) was
selected for further studies (see also below). This
clone was called
K. oxytoca GP1. Southern blot analysis confirmed
that
K. oxytoca GP1 contained two copies of the
nasB gene, the
host
nasCBA operon,
which appeared in a 17-kb
HindIII fragment,
and another
gene acquired after the random insertion of the
mini-Tn
5/nasB gene in the host chromosome that was located
in a
HindIII fragment
of about 14 kb (Fig.
1).
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 2.
Maps of plasmids pUPE2 and pUPE5. Relevant restriction
sites, antibiotic markers, origin of transfer (oriT), and origin of
replication (oriV) are indicated.
|
|
To determine if the two different recombinant derivatives of
K. oxytoca,
K. oxytoca bearing pUPE2
and
K. oxytoca GP1, exhibited
higher nitrite
reductase activities than the parental strain,
the three strains were
grown on M8 minimal medium with 40 mM nitrate
and 2% (wt/vol) sucrose
or 2% (wt/vol) glycerol as the sole C
source (
15). Cells in
the mid-exponential growth phase were
harvested by centrifugation, and
cell extracts were prepared by
sonication. Cells resuspended in
potassium phosphate buffer (100
mM, pH 7.2) were disrupted by
sonication (Braun Biotech, Labsonic)
with 75-W 20-s pulses delivered
for 10 min. The unbroken cells
were removed by centrifugation
(10,000 ×
g for 10 min at 4°C),
and the supernatant
was used as a source of protein for enzymatic
assays. The levels of
nitrate reductase and nitrite reductase
activities in these
extracts were determined as described elsewhere
(
5).
The results obtained are shown in Table
1. As expected,
the levels of nitrate
reductase were equally high in the three
strains, although in the cells
grown on glycerol the levels (110
± 15 mU/mg of protein)
(mean ± standard error of the mean) were
nearly twice as high as
those in the cells grown on sucrose (40
to 60 mU/mg of protein). The
results presented are averages of
four independent determinations. The
levels of nitrite reductase
for the two recombinant strains were
significantly higher than
those determined for the wild-type strain.
For
K. oxytoca bearing
pUPE2 these levels were
three- to fourfold higher than for the
wild-type strain, while
for
K. oxytoca GP1 the level of activity
of this
enzyme was about threefold higher than for the wild-type
strain. These
results show that the recombinant strains exhibited
a higher dose of
nitrite reductase than the wild-type strain,
even though they had
similar nitrate reductase levels, as expected
from the lack of
alteration of the dose of the
nasCA genes.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Nitrate reductase and nitrite reductase activities in
cell extracts of wild-type K. oxytoca CECT 4460 cells
and recombinant derivatives bearing the K. pneumoniae
nasB gene
|
|
Effect of extra copies of the nasB gene in
K. oxytoca CECT 4460 on the accumulation of
nitrite and ammonium in the culture medium.
To determine the
effect of the extra nasB gene dose in K. oxytoca on nitrate removal and nitrite accumulation, cells of
K. oxytoca CECT 4460, K. oxytoca CECT
4460 (pUPE2), and K. oxytoca GP1 were grown in batch
cultures on minimal medium with 40 mM KNO3 and sucrose (3%
wt/vol). In the assays, growth measured as turbidity of the culture
(Fig. 3A) and the concentrations of
nitrate (not shown), nitrite, and ammonium in the culture medium were determined (Fig. 3C and D). We also determined nitrite reductase and
nitrate reductase activities during growth (13) (Fig. 3B). We observed a sequential pathway for the reduction of nitrate to
ammonium in the three strains in four independent assays. The results
of one assay for K. oxytoca CECT 4460 and K. oxytoca bearing pUPE2 are shown in Fig. 3. The results obtained
with K. oxytoca GP1 were similar to those obtained with
K. oxytoca bearing pUPE2 but with some differences
which are noted in the text. The wild-type K. oxytoca
and K. oxytoca bearing pUPE2 showed similar growth curves (Fig. 3A). The three strains consumed nitrate at similar rates
(0.7 ± 0.1 g of NO3
/g of protein per h), and the nitrate was fully consumed after 18 h (not shown). Nitrite accumulated after the reduction of nitrate (Fig. 3C) and was eventually reduced to ammonium, which again accumulated transiently in the culture
medium (Fig. 3D). The highest levels of nitrite in the culture media of
the wild-type and the recombinant strains were detected 18 h after
the cultures were set up. In the supernatants of the
culture of the wild-type strain up to about 18 mM
nitrite was found. In contrast, when K. oxytoca
CECT 4460 bore plasmid pUPE2 the level of nitrite in the medium was
lower (2 mM) (Fig. 3B). For strain GP1 the highest level of nitrite was
10 mM (not shown). The highest levels of ammonium in the culture medium
were detected 22 h after the cultures were set up. In
the supernatants of the culture of the wild-type strain up to about
5 mM ammonium was found. In contrast, when K. oxytoca
CECT 4460 bore plasmid pUPE2 the level of ammonium in the
medium was higher, 13 mM (Fig. 3D), and the level was 12 mM for
K. oxytoca GP1 (not shown). However, after some time
the ammonium was fully consumed (Fig. 3D).
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 3.
Effect of nasB gene copy number increase on
the accumulation of nitrite and ammonium in the culture medium.
K. oxytoca CECT 4460 ( ) and K. oxytoca CECT 4460 bearing UPE2 ( ) were grown on minimal medium,
with 40 mM KNO3 and 3% (wt/vol) sucrose at 30°C for
24 h. Growth (A), nitrite reductase activity (B), nitrite
concentration (C), and ammonium concentration (D) were determined
during the growth of both strains. Nitrite concentration was determined
by the method of Snell and Snell (22). Ammonium
concentration was determined enzymatically by using a commercial kit
from Boehringer Mannheim (Ref. 1112732). Nitrite reductase activity was
determined in permeabilized cells as described by Neubauer and
Götz (13).
|
|
The level of nitrite reductase in the three strains was determined at
various times. At the beginning of culture the activity
was highest,
and the activity had a tendency to decrease with
time, reaching about
30 to 10% of the maximum at the end of the
assay (Fig.
3B). For
K. oxytoca CECT 4460 bearing pUPE2 the level
of nitrite
reductase was always higher than that determined in
the wild-type
K. oxytoca CECT 4460 strain (Fig.
3B). Nitrite
reductase
activity showed a maximum level of 11 ± 0.1 mU/mg of
protein,
and the level was about threefold higher in the clone bearing
the pUPE2 plasmid than in the wild type. The nitrate reductase
activities of the three strains were also determined in permeabilized
cells and showed a maximum activity of 50 ± 7 mU/mg of
protein
(not
shown).
In summary, our results show that an increase in the dose of the
nasB gene in
K. oxytoca suffices to overcome
nitrite accumulation
under suboptimal growth conditions. The improved
strain represents
a more reliable one for the biotreatment of
wastewaters with high
nitrate
loads.
| |
ACKNOWLEDGMENTS |
This study was supported by grants from Unión Española
de Explosivos and PETRI from the Comisión Interministerial de
Ciencia y Tecnología. G. Piñar was a holder of a Mapfre
Foundation fellowship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: EEZ-CSIC,
Apdo. Correos 419, E-18008 Granada, Spain. Phone: 349-58-121011. Fax:
349-58-129600. E-mail: jlramos{at}eez.csic.es.
Present address: Department of Microbiology, University of
Vienna, Vienna, Austria.
| |
REFERENCES |
| 1.
|
Blatny, J. M.,
T. Brautaset,
H. C. Winther-Larsen,
K. Haugan, and S. Valla.
1997.
Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon.
Appl. Environ. Microbiol.
63:370-379[Abstract].
|
| 2.
|
Boyer, H. W., and D. Roulland-Dussoix.
1969.
A complementation analysis of the restriction and modification of DNA in Escherichia coli.
J. Mol. Biol.
41:459-472[Medline].
|
| 3.
|
Clarkson, W. W.,
B. J. B. Ross, and S. Krishnamachari.
1991.
Denitrification of high-strength industrial wastewaters, p. 347-357.
In
45th Purdue Industrial Waste Conference Proceedings. Lewis Publishers, Inc., Chelsea, Mich.
|
| 4.
|
Clifford, D., and X. Liu.
1993.
Biological denitrification of spent regenerant brine using a sequencing batch reactor.
Water Res.
27:1477-1484.
|
| 5.
|
Coleman, K. J.,
A. Cornish-Bowden, and J. A. Cole.
1978.
Purification and properties of nitrite reductase from Escherichia coli K12.
Biochem. J.
175:483-493[Medline].
|
| 6.
|
de Lorenzo, V.,
M. Herrero,
U. Jakubzik, and K. N. Timmis.
1990.
Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria.
J. Bacteriol.
172:6568-6572[Abstract/Free Full Text].
|
| 7.
|
de Lorenzo, V., and K. N. Timmis.
1994.
Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons.
Methods Enzymol.
235:386-405[Medline].
|
| 8.
|
Herrero, M.,
V. de Lorenzo, and K. N. Timmis.
1990.
Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.
J. Bacteriol.
172:6557-6567[Abstract/Free Full Text].
|
| 9.
|
Kamath, S.,
D. A. Sabatini, and L. W. Canter.
1992.
Treatment of high nitrogen (NaNO2) wastewater by biological nitrification/denitrification, p. 623-630.
In
46th Purdue Industrial Waste Conference Proceedings. Lewis Publishers, Inc., Chelsea, Mich.
|
| 10.
|
Krishnamachari, S., and W. W. Clarkson.
1993.
Nitrite accumulation in the effluents from high-strength denitrification of industrial wastewater, p. 383-392.
In
47th Purdue Industrial Waste Conference Proceedings. Lewis Publishers, Inc., Chelsea, Mich.
|
| 11.
|
Lin, J. T.,
B. S. Goldman, and V. Stewart.
1993.
Structure of genes nasA and nasB, encoding assimilatory nitrate and nitrite reductase in Klebsiella pneumoniae M5a1.
J. Bacteriol.
175:2370-2378[Abstract/Free Full Text].
|
| 12.
|
Lin, J. T.,
B. S. Goldman, and V. Stewart.
1994.
The nasFEDCBA operon for nitrate and nitrite assimilation in Klebsiella pneumoniae M5a1.
J. Bacteriol.
176:2551-2559[Abstract/Free Full Text].
|
| 13.
|
Neubauer, H., and F. Götz.
1996.
Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus.
J. Bacteriol.
178:2005-2009[Abstract/Free Full Text].
|
| 14.
|
Piñar, G.,
E. Duque,
A. Haïdour,
J. M. Oliva,
L. Sánchez-Barbero,
V. Calvo, and J. L. Ramos.
1997.
Removal of high concentration of nitrate from industrial wastewater by bacteria.
Appl. Environ. Microbiol.
63:2071-2073[Abstract].
|
| 15.
|
Piñar, G.,
J. M. Oliva,
L. Sánchez-Barbero,
V. Calvo, and J. L. Ramos.
1998.
Removal of nitrate from industrial wastewater in a pilot plant by nitrate-tolerant Klebsiella oxytoca clone 15 and Arthrobacter globiformis clone AH1.
Biotechnol. Bioeng.
58:510-514[Medline].
|
| 16.
|
Piñar, G.,
K. Ková óva,
T. Egli, and J. L. Ramos.
1998.
Influence of carbon source on nitrate removal by nitrate-tolerant Klebsiella oxytoca CECT 4460 in batch and chemostat cultures.
Appl. Environ. Microbiol.
64:2970-2976[Abstract/Free Full Text].
|
| 17.
| Piñar, G., and J. L. Ramos. 1998. Unpublished results.
|
| 18.
|
Pitt, W. W.,
C. W. Hancher, and B. D. Patton.
1981.
Biological reduction of nitrates in wastewater from nuclear processing using a fluidized-bed bioreactor.
Nucl. Chem. Waste Manag.
2:57-70.
|
| 19.
|
Ramos-González, M. I.,
E. Duque, and J. L. Ramos.
1991.
Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmid.
Appl. Environ. Microbiol.
57:3020-3027[Abstract/Free Full Text].
|
| 20.
|
Ramos, J. L.,
A. Haïdour,
E. Duque,
G. Piñar,
V. Calvo, and J. M. Oliva.
1996.
Metabolism of nitrate esters by a consortium of two bacteria.
Nature Biotechnol.
14:320-322[Medline].
|
| 21.
|
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.
|
| 22.
|
Snell, F. D., and T. Snell.
1949.
Colorimetric methods of analysis, vol. 2. , p. 802-807.
Van Nostrand Co., New York, N.Y.
|
| 23.
|
Walker, J. F., Jr.,
M. V. Helfrich, and T. L. Donaldson.
1989.
Biodenitrification of uranium refinery wastewaters.
Environ. Prog.
8:97-101.
|
Applied and Environmental Microbiology, December 1998, p. 5016-5019, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Senko, J. M., Dewers, T. A., Krumholz, L. R.
(2005). Effect of Oxidation Rate and Fe(II) State on Microbial Nitrate-Dependent Fe(III) Mineral Formation. Appl. Environ. Microbiol.
71: 7172-7177
[Abstract]
[Full Text]
Top
Abstract
Text
