vvEPA
United States
Environmental Protection
Agency
Risk Reduction
Engineering Laboratory
Cincinnati, OH 45268
Research and Development EPA/600/M-89/011 June 1989
ENVIRONMENTAL
RESEARCH BRIEF
Genetic Engineering of Enhanced Microbial Nitrification
Michael Carsiotis and Sunil Khanna
Abstract
Experiments were conducted to introduce genetic
information in the form of antibiotic or mercuric ion
resistance genes into Nitrobacter hamburgensis strain X14.
The resistance genes were either stable components of
broad host range plasmids or transposable genes on
plasmids presumably unable to replicate in strain X14. Four
methods for plasmid transformation as well as conjugation
with various donor strains of Escherichia coli failed to
achieve this goal. We also undertook the cloning of an origin
of replication from strain X14; seven such experiments were
unproductive. Both the leuB and thrB gene, of strain X14
were successfully cloned by means of complementation of a
leuB thrB auxotroph of £ coli. The leuB gene containing
DNA was restriction-mapped and the 1.3 kilobase pair gene
was subcloned into a vector suitable for use in DNA
sequencing. To date, a tentative sequence comprising about
1300 bases has been obtained. There is extensive similarity
in three regions of the sequence with the amino acid
sequence of the leuB gene product of Thermus
thermophilus, Salmonella typhimurium, and Saccharomyces
cerevisiae.
Although the primary goal of developing a procedure for
introducing genetic material into a nitrifying organism has
not yet been achieved, the results achieved have produced
useful information on the genomic organization of
Nitrobacter as well as a plasmid-borne library of genes from
that organism. Future experiments can be made with this
library in order to provide additional basic information on
Nitrobacter's genomic organization.
The authors are with the University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267-0524. • , .,
Introduction
Nitrification, the conversion of ammonia to nitrite, is a
desirable process in wastewater treatment facilities.
Improvement of the nitrification process by genetic
engineering of nitrifiers would provide a variety of benefits,
e.g. nitrifiers needing less retention time, nitrifiers resistant
to pollutants, nitrifiers active in cold weather, nitrifiers that
grow more rapidly. The strains of nitrifiers required for such
improvement could, in theory, be derived by genetic
engineering.
Nitrification occurs in two steps: NH3-»NO2- + hT and NO2-
-»NO3-. It is accomplished by the sequential action of two
genera of autotrophic bacteria. Bacteria of the genera
designated by the prefix "Nitroso" oxidize ammonia,
liberated from organic matter by the action of heterotrophic
bacteria, to nitrite. Nitrite is then oxidized to nitrate by
autotrophs of the genera designated by the prefix "Nitro".
The best known species are Nitrosomonas europa and
Nitrobacter winogradsky. The term nitrifiers is used when
referring to both genera collectively. Most publications focus
primarily on the physiology, enzymology and structure-
function relationships of the nitrifiers. Despite the important
contribution of nitrifiers to wastewater treatment, as well as
to the global nitrogen cycle, virtually nothing is known of
their genomic organization.
Genetic engineering of bacteria usually involves either the
introduction of foreign genes or the alteration of the existent
bacterial genome. In either case, basic information on the
organization and regulation of the bacterial genome is
desirable. In this study, although such information on
nitrifiers was lacking, it seemed reasonable to introduce, by
conjugation or transformation, either an antibiotic or
mercuric ion resistance plasmid into a nitrifier. This decision
was based on the numerous successful introductions of
resistance plasmids into Enterobacteriaceae and the ease of
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positive selection for antibiotic and mercuric ion resistance.
The stable antibiotic kanamycin and the stable mercuric ion
were chosen as the selective agents since nitrifiers grow
extremely slowly.
We choose to use Nitrobacter hamburgensis strain X14,
henceforth simply strain X14 (4), in our studies because it
grew relatively rapidly. Furthermore, it and the related strain
Y (4), were the only Nitrobacter strains known to contain
plasmids (14). Both strains contain three large (110, 186,
and 273 kilobase) cryptic plasmids. This suggested that
strain X14 would be physiologically able to maintain a newly
introduced plasmid.
Procedures and Results
Introduction of Resistance Plasmids by
Conjugation
The bacterial strains used in this study are listed in Table 1.
A/, hamburgensis strain X14 was known to be resistant to
chloramphenicol (M. Pohl, unpublished observation).
Therefore, a concentration of 50 yg/ml in agar solidified
mixotrophic (21) or autotrophic (21) growth medium usually
was used to counterselect against the E. coli plasmid
donors. All matings between strain X14 recipients and E.
coli donors were performed by mixing together the two
organisms on a sterile 0.22n filter at a donor to recipient
ratio of 10 to 1. The filter was immediately placed on an
agar plate of mixotrophic medium and incubated (30 °C) for
either 2,5,8, or 24 h to allow conjugation. The cells on the
filter were harvested in either mixotrophic or autotrophic
modia, generally supplemented with chloramphenicol (50
ng/ml) and incubated for 24 h (30 °C) to allow phenotypic
expression of resistance. Cells were harvested by
centrifugation, resuspended in a small volume, and the
entire volume plated among several selective agar plates.
The plates were placed in plastic sleeves, which were
loosely sealed, and then incubated (30°C) for up to 30 days.
Control platings of strain X14 formed minute colonies in ca
21 days. Despite precautions, fungal contamination
occasionally occurred on some plates.
In only 1 of 13 instances, and then only in the form of a
single colony, was the E. coli donor able to emerge on the
selective plate.
The plasmids chosen for use (Table 2) were generally broad
host range plasmids that carried an antibiotic resistance
gene for a chemically stable antibiotic, e.g., kanamycin,
streptomycin. The stability of the antibiotic was necessary
because of the long incubation period. Other plasmids
carried the transposon Tn 501 (mercuric ion resistance),
which is useful since the mercuric ion in selective plates is
chemically stable. Three of the plasmids were incapable of
replicating except in £ coli and carried transposons that
could transpose to either the chromosome or the
endogenous plasmids of strain X14.
Although preliminary experiments with all donor stains
showed them to be fertile when conjugated with E. coli, in
no case were any resistant transconjugants of strain X14
isolated.
Introduction of Resistance Plasmids by
Transformation
The introduction by transformation of two broad host range
plasmids in strain X14 was attempted in two separate
experiments. In each experiment, four different protocols
were used (7,9,12,24). Three of the protocols (7,12,24) were
selected since they had been used with recipient organisms
resistant to the more traditional fourth procedure (9), which
we also employed. After exposure to the resistance plasmid,
the strain X14 cells were incubated in mixotrophic medium
to allow phenotypic expression before plating on selective
media. Although control experiments proved that the
plasmids were capable of transforming E. coli with high
efficiency, no resistant transconjugants of strain X14 were
isolated.
Isolation of Origin of Replication
Strain X14 contains four origins of replication (or/): one
chromosomal or/ and an or/ on each of the three cryptic
plasmids. Our inability to introduce broad host range
resistance plasmids into strain X14 may have been due to
the incompatibility of their or/ with the physiological
properties of strain X14. We, therefore, designed the
following experiments to clone an or/ of strain X14.
Chromosomal and plasmid DNA of strain X14 were digested
separately to completion with the restriction endonucleases,
BamHl, Pst\, and Sau3A. The resultant fragments were
ligated into digested vector pMK2004 to yield three gene
libraries. Each library was used to transform E. coli JZ294, a
polA strain. PolA strains are unable to use the ColEI-derived
or/ of the vector pMK2004 (Figure 1; ref. 13). They will,
however, replicate vectors that contain a non-ColE1-derived
on (10,11). Transformation of E. coli JZ294 with the three
gene libraries failed to yield any transformants. Because we
used three different gene libraries and because previously
identified or/'s of Gram-negative organisms have all been
relatively short sequences of ca 250 base pairs (27), our
negative results suggest very strongly that the or/
sequence(s) in Nitrobacter are non-functional in E. coli.
Construction of a Gene Library of Strain X14
The inability to introduce resistance plasmids into strain X14
and to isolate an origin of replication from strain X14 could
be interpreted as follows. The regulatory sequences on the
resistance plasmids that regulate transcription, translation,
and DNA replication are those commonly used by
Enterobacteriaceae. These regulatory signals may be
distinct from those used by Nitrobacter for these
physiological properties. To learn what regulatory
sequences strain X14 uses to regulate transcription and
translation, we undertook the isolation of the leuB gene from
strain X14. Genomic DNA was prepared from strain X14 as
follows. A suspension of 0.5 g cells in 6 ml TE (10 mM Tris,
1 mM EDTA; pH 8), 25 ml 2% sodium dodecyl sulfate, and
10 ml 0.35% proteinase K was incubated at 37°C for 2 h.
The resultant viscious lysate was extracted twice with an
equal volume of phenol and then twice with an equal volume
of chloroform-isoamyl alcohol (24:1, v/v). The aqueous
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Table 1. Bacterial Strains
Bacteria
Relevant bacterial genotype*
Source, reference or both
Nitrobacter hamburgensis
X14
XU-SmR
Nitrobacter agilis
E. co//
UW937 (pUW942)
HB101 (pUW964)
UB1636 (pMR5)
J53 (RP4)
MC1061 (pBR322 Tet:Tn5)
C
K12(RP4.8::7>?501)
JZ294
C600
CV438
Prototrophic autotroph; ,
3 cryptic plasmids
Spontaneous SmR mutant
Prototrophic autotroph
thr leu
pro
his trp lys
met pro
leu
ilv
Prototroph
polA1
leuBG thrB thi
\euB61 thi pro
E. Bock, Univ. of Hamburg, F.
This study
ATCC 14123
(25)
(26)
(19)
J. C. Loper
University of Cincinnati
J. Lodge
Washington University
St. Louis
J. C. Loper
University of Cincinnati
M. Davidson
University of Georgia
D. Smith
University of California at San
(15)
J. M. Calvo
Cornell University
R.G.; (4)
Diego
"Relevant genotype of plasmids shown in Table 2.
SmR, streptomycin resistant.
phase was mixed with 0.6 volume isopropanol and kept at
-20 °C for 2 h. The precipitated DNA was recovered by
centrifugation and dissolved in TE. After centrifugation in
CsCI-ethidium bromide, the chromosomal DNA band was
collected and dialyzed versus TE. The DNA was digested
partially with SauSA, and the resultant digestion mixture was
electrophoresed in 1% low melting agarose. The 4 to 6
kilobase (kb) portion of the gel was cut out, and the DNA
extracted. The DNA was ligated to BamHI-digested vector
pMK2004 (Figure 1) and the resultant ligation mixture was
used to transform JZ279, a recA derivative of LE 392 (15),
by the method of Hanahan (9). We calculated that a total of
3990 kanamycin resistant transformants had been obtained;
a unique copy of chromosomal DNA should be represented
Table 2. Plasmids Used or Derived in This Study
Plasmid
pUW942
pUW964
pMR5
RP4
RP4.8:: 7/7501
Relevant
genotype"
:7n501 (HgR) Col E1 rep
:Tn5 (KanR) Col E1 rep
:7n801 (AmpR) rep's
KanR IncP
KanR IncP
Source or reference
(25)
(26)
(19)
(11)
Michael Davidson
pBR322Tet::f/75 ::Tn5 (KanR) Col E1 rep
pCV57
pNBH1,6
pNBH3,8,10,11
PNBH601
pNBH602
* KanR
KanR
leuB+ KanR
* KanR
University of Georgia
Jennifer Lodge
Washington University
J. M. Calvo
Cornell University
This study
This study
This study
This study
* Genetic symbols: HgR, mercuric ion resistant; KanR, kanamycin resistant; AmpR,
ampicillin resistant; repts, replication temperature sensitive; IncP, incompatibility group
P; Col E1 rep, Colicin E1 replication.
-------
EcoRI
4.5
Sa/l
1.5
2.0
3.0
Figure 1. Map of relevant restriction sites in pMK2004.
Modified from reference 13. Coordinates are in
kilobaso pairs. Tet, tetracycline resistance gene;
Kanr, kanamycln resistance gene; Ampr, ampicillin
resistance gene; Rep, origin of replication.
at least ones in the library with 99% confidence (5). This
conclusion was based on the assumption that Jhe
chromosome of strain X14 and that of E. coli are of equal
sizo. Recombinant plasmid DNA, prepared from the
transformed cells by the method of Birnboim and Doly (3),
was purified by CsCI-ethidium bromide gradient
centrifugation.
Complementation of E. coll C600
Rocombinant plasmid DNA was mixed with competent (9)
cells of £ co// C600, a leuB6 thrB mutant. Part of the
mixture was plated on leucine-deficient and part on
threonine-deficient agar medium. Twelve Leu*
transformants and three Thr* transformants were so
isolated. Six of the leuB6 and one of the thrB
complementing plasrnids were stable; the reason for the
instability of the other plasrnids is unknown. No further work
was done with the //jrB-complementing plasmid.
Southern Analysis
It was necessary to prove that the complementing activity in
the recombinant plasrnids was due to Nitrobacter DNA
rather than contaminating E. coli DNA present in the
pMK2004 preparation. Nitrobacter genomic DNA and E. coli
C600 DNA were digested completely with EcoRI and Sa/l,
resolved on agarose gels, transferred to nitrocellulose, and
probed with nick translated pNBH6 and pMK2004. The
radiolabeled pNBH6 did not hybridize to £ coli DNA but did
hybridize to a Nitrobacter DNA restriction fragment of the
appropriate size, and to the vector pMK2004. As a
necessary negative control, we also showed that the vector
pMK2004 failed to hybridize to Nitrobacter DNA. This
Southern analysis confirmed our conclusion that the
complementing activity of pNBH6 was due to Nitrobacter
DNA.
Plasmid Coded Enzymatic Activity
The \euB gene of E. coli codes for p-isopropylmalate
dehydrogenase (6,20). This enzymatic activity should,
therefore, be present in soluble extracts made from E. coli
C600 strains which contain a /et/S-complementing plasmid.
As seen in Table 3, enzymatic activity was present in
recombinant plasmid bearing strains but absent in two E.
coli leuB strains. Since E. coli CV438 (feuS61) and C600
(\euB6) contain different \euB mutant alleles (18), the
expression of enzymatic activity when they bear the same
complementing plasmid argues against leuB
complementation by Nitrobacter DNA being due to a
Nitrobacter suppressor tRNA.
Table 3. B-lsopropylmalate Dehydrogenase Activity of E.
coli strains
Expt.
No.
1
2
Strain (plasmid)
CV438 (pMK2004)
CV438 (pCV57)
CV438 (pNBH1)
CV438 (pNBH3)
CV438 (pNBH6)
K12
C600 (pMK2004)
C600 (pMK2004)
C600 (pNBH6)
C600 (pNBH601)
C600 (pNBH602)
Leucine in
growth
medium
(US/ml)"
40
0
0
0
0
0
10
40
0
0
0
Specific
activity +
< 0.001
0.057
0.040
0.017
0.053
0.084
< 0.001
< 0.001
0.10
0.162
0.137
* Minimal salts medium (23) was supplemented with praline and
thiamine (Expt. 1) or with threonine and thiamine (Expt. 2).
* Micromoles a-ketoisocaproate formed min-1 mg protein-1.
To obtain additional evidence that the leucine-
complementing activity was not due to inadvertent cloning
of E. coli DNA, the soluble extracts were electrophoresed by
continuous polyacrylamide gel electrophoresis under
nondenaturing conditions, p-isopropylmalate dehydrogenase
activity was detected by a histochemical stain (22) in
extracts prepared from E. coli strains that bore
complementing plasrnids but not in those prepared from E.
coli C600 (pMK2004). The relative mobility of the cloned
Nitrobacter p-isopropylmalate dehydrogenase was slower
than that of E. coli.
Complementation of leuA, C, and D Mutants
The four leucine biosynthetic genes occur as a cluster of
four contiguous genes, i.e., as an operon, in both Salmonella
typhimurium (16) and E. coli (20). The amount of DNA
needed to code for the four S. typhimurium leu polypeptides
was estimated to be ca 4.1 kb (6).
Our DNA sequencing data (see below) indicated that the
leuB gene existed within the 1.4 kb SamHI-Smal fragment
of pNBH602 (Figure 2). Thus, there was sufficient DNA on
-------
either side of the comparable region in pNBHS (Figure 2) for
one or more of the other three leu genes. We, therefore,
transformed a leuA, a leuC, and a leuD mutant of E. coli with
pNBHS. Plating on kanamycin-supplemented agar indicated
that all three of the mutants were successfully transformed
by pNBHS. None of the three mutants had, however, been
transformed to leucine independence. We concluded that
the four leucine genes in Nitrobacter are not organized as
an operon.
Restriction Mapping and Subcloning
The size of the insert in the six stable /euS-complementing
plasmids was either ca 12.5 kb (pNBH1, pNBH6) or ca 6.7
kb (pNBH3, pNBHS, pNBHIO, pNBHH). We identified a
common 3.7 kb SamHI-EcoRl restriction fragment that
perforce contained the /euB-complementing activity (Figure
2). This fragment was isolated from a BamHI, EcoRI digest
of pNBH6 and ligated into similarly digested pMK2004. The
resultant plasmid, pNBH601 (Figure 2), complemented the
let/6 mutation in E. coli C600. The reverse orientation of the
3.7 kb BamHI-EcoRI fragment in pNBH6 and pNBH601
(footnote Figure 2) suggested strongly that transcription
originated at a promoter site within the cloned Nitrobacter
DNA. Further subcloning by means of SamHI-Sa/l digestion
of pNBH6 and subsequent ligation into pMK2004 yielded
the /eufl-complementing plasmid pNBH602 (Figure 2). The
resultant 2.4 kb SamHI-Sa/l fragment of pNBH602 was
isolated and a restriction map constructed (Figure 2).
Sequencing the leuB Gene of Strain X14
The leuB gene had been localized within the 2.4 kb SamHI-
Sa/l fragment of pNBH602 (Figure 2). We subcloned the ca
1.4 kb SamHI-Smal portion of that fragment in both
orientations into pAA 3.7X (1). The method described by
Ahmed (1) was used to generate sets of overlapping
deletions suitable for sequencing both strands of the DNA
fragment. To date, a total of 1311 bases have been
sequenced from both strands. At this point, two aspects of
the derived amino acid sequence are especially noteworthy.
First is the occurrence of three regions of homology among
the amino acid sequences of the {Hsopropylmalate
dehydrogenase of strain X14, S. typhimurium (J. Calvo,
pers.- comm.), Thermus thermophilus (17), and
Sacc/?aromyces cerevisiae (2) (Figure 3). Second is that the
cited correspondence between the strain X14 amino acid
sequence and the other amino acid sequences allows us to
predict that the SamHI-Smal fragment contains
approximately 120 bases of 5'-flanking and approximately
150 bases of 3'-flanking DNA. This amount of flanking DNA
should presumably encompass the sought for 5'-flanking
(promoter and ribosome-binding) and 3'-flanking
(transcription termination) sequences (8).
Conclusions
The original goal of introducing genetic information into
strain X14 has not been realized. One plausible
interpretation of the inability to transfer genetic information
by either transformation or conjugation is that the replication
and/or transcriptional and/or translational signals in the
various plasmids are not functional in strain X14. A second
possibility is that foreign DNA can not enter by the
biological transfer mechanisms of conjugation and
transformation. Efforts to clone the four replication origins
H B
P EJ
pNBH3
pNBH6
PNBH601
(1
0 22
1 1 "
1 1
74 6 6.7
B S P E •:
, 1 T „ i
12 12.5
P E
3.7
pNBH602
Sm
f-t-F
1 1.4 2 2.4
Figure 2. Relevant restriction sites in /euS-complementing
fragments derived from genomic DfJA of strain X14
or by subsequent subcloning. Each of the fragments
when cloned into appropriately digested vector
pMK2004 was shown to be /euB-complementing. The
coordinates are in kilobase (kb) pairs and are
approximate values. The leuB-complementing
fragments of pNBH1 and pNBH6 are assumed to be
identical, as are those of pNBHS, pNBHS, pNBHIO
and pNBH11. Note that the 3.7 kb Ba/nHI-EcoRI
fragment in pNBH6 and pNBH601 are in opposite
orientations within their respective plasmids. B,
BamHI; E, EcoRI; H, H/ndlll; J, BamHI/SauSA; P, Psfl;
S, Sa/l; Sm, Smal.
found in strain X14 were unsuccessful. The successful
cloning of the leuB gene of the strain X14, the first gene
cloned from a nitrifier, provided us with the opportunity to
examine the flanking regions of this gene for the presence
or absence of traditional transcriptional and translational
signals common in the enterobacteria. To date, a total of ca
1300 bases of the leuB gene of strain X14 have been
sequenced. Since these data are a composite of sequences
from both strands, contain ambiguous bases, and do not yet
include the two junction fragments where the subcloned
gene joins the vector, no definitive statement can be made
at this time about the sought-for transcriptional and
translational DNA sequences. However, three sequences of
amino acids derived from the DNA sequence contain,
respectively, 13, 9, and 18 amino acids that are either
identical or functionally equivalent to sequences reported in
the same enzyme from T. thermophilus, S. typhimurium,
and S. cerevisiae. No function(s) have been ascribed to any
of the amino acid sequences in the enzymes from the latter
three enzymes.
Recommendations
This research has provided the first basic information on the
genomic organization of a nitrifying organism, N.
hamburgensis strain X14. It is apparent that the leuB
biosynthetic enzyme of Nitrobacter shares considerable
-------
a ( 9}AlaValLeuPheGlyAlaValGlyGlyProLysTrpAsp(21)
b (64) AlaValLeuleuGlySerValGlyGlyProLysTrpAsp (76)
c {68) AlalleLeuPheGlySerValGlyGlyProLysMet (79)
d (71)AIaValLeuLeuGlyAlaValGlyGlyProLysTrp (82)
a (40) LeuTyrAlaAsnLeuArgProAla ( 47)
b (97) LeuPheAlaAsnLeuArgProAla(i04)
c (102) LeuPheSerAsnLeuArgProAla (109)
d (101) LeuTyrAlaAsnLeuArgPro (108)
a ( 67) ValAsplleMetlteValArgGluLeuThrXxxGlyValTyrPheGIyGluProLys ( 85)
b (124) ValAspValLeulleValArgGluLeuThrGlyGlylleTyrPheGlyGluProArg (142)
c (130) PheAsplleLeuCysValArgGIuLeuThrGlyGlylleTyrPheGlyGlnProLys (148)
d (130)ThrAspPheValValValArgGluLeuValG!yGlylleTyrPheGly (145)
Figure 3. Comparison of derived amino acid sequences of the leuB DNA sequences of (a)
strain X14, (b) T. thermophilus, (c) S. typhimurium, and (d) S. cerew's/'ae. Xxx,
ambiguous sequence; numbers in parenthesis indicate amino acid residue.
amino acid homology with the same enzyme in two other
Gram-negative bacteria and a yeast.
The partially completed sequencing of the leuB gene and its
Hanking regions should be completed. The resultant data
will establish whether the DNA sequences which serve as
signals in transcription and translation of Nitrobacter DNA,
aro similar to those found in other bacteria. Should the
completed analysis of the DNA flanking the leuB gene fail to
reveal any regulatory sequences common to other bacteria,
then other Nitrobacter genes should be isolated and
sequenced. The gene library of strain X14 constructed
during this study is available as a source of other
Nitrobacter genes. A comparison of the flanking DNA
sequences would reveal sequences common to Nitrobacter
gones; these would be prime candidates for the sought for
regulatory sequences. This information is critical for the
following reason. The inability to introduce resistance
plasmids into strain X14 may be because incompatible
regulatory sequences prevent expression of the resistance
gene. The identification of AMrooacter-specific regulatory
sequences would allow the modification of plasmid vectors
so (hat expression of an introduced resistance gene would
be assured.
Although resistance plasmids could not be introduced into
strain X14 by transformation or conjugation, their
introduction by electroporation should be attempted.
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x"
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-------
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