Department of Fermentation Technology,
Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8527, Japan
 |
INTRODUCTION |
The ammonia-oxidizing autotrophic
bacteria are confined to the gram-negative 
and 
subdivisions of
the class Proteobacteria (9, 26, 27). They obtain
all of their energy for growth from the oxidation of ammonia to nitrite
(25). In Nitrosomonas europaea, ammonia is
initially oxidized to hydroxylamine by the integral membrane enzyme
ammonia monooxygenase (11, 13, 17), and the subsequent
oxidation of hydroxylamine to nitrite is catalyzed by the multiheme
hydroxylamine oxidoreductase (HAO) (4, 21). Two of the four
electrons generated from hydroxylamine oxidation are used to support
the oxidation of additional ammonia molecules, while the other two
electrons enter the electron transfer chain and are used to support
CO2 reduction and ATP biosynthesis (5, 19).
Ammonia-oxidizing bacteria are found in a wide range of aerobic
environments ranging from soil and freshwater to seawater (5,
25). Interest in the ammonia-oxidizing bacteria stems from their
key role in the nitrogen cycle in nature (5) and also from
their importance in removing nitrogen from wastewaters (12).
However, relatively little is known about the genetics of
ammonia-oxidizing autotrophic bacteria, and improvements in the
efficiency of biological nitrogen removal are still difficult.
To date, native plasmids in the ammonia-oxidizing autotrophic bacteria
have not been described. In the present paper, we report two indigenous
plasmids in the ammonia-oxidizing bacterium Nitrosomonas sp.
strain ENI-11, which was originally isolated from activated sludge. The
plasmids, designated pAYS and pAYL, were relatively small (1.8 and 1.9 kbp) and cryptic, having no detectable plasmid-linked antibiotic
resistance or heavy metal resistance markers. Sequence analysis of pAYS
and pAYL revealed that they both contained a major open reading frame
(ORF) consisting of more than 500 bp and had a highly homologous
region, designated HHR, of 262 bp. HHR-homologous sequences were also
detected in the genomes of ENI-11 and the plasmidless strain N. europaea IFO14298. These plasmids are likely to be good candidates
for developing vectors for gene transfer in the ammonia-oxidizing
autotrophic bacteria.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
Bacterial strains and
plasmids used in the present study are shown in Table
1. Nitrosomonas sp. strain
ENI-11, which was originally isolated from activated sludge, was
obtained from the Process and Production Technology Center, Sumitomo
Chemical Co., Ehime, Japan. Nitrosomonas sp. strain ENI-11
and N. europaea IFO14298 were grown aerobically at 28°C in
modified Alexander (MA) medium [2 g of
(NH4)2SO4, 0.5 g of
K2HPO4, 0.5 g of NaHCO3, 50 mg
of MgSO4-7H2O, 5 mg of
CaCl2-2H2O, 2 mg of
MnSO4-4H2O, 5 mg of Fe-EDTA(III), 0.1 mg of
CuSO4-5H2O, 0.05 mg of
Na2MoO4-2H2O, 0.001 mg of
CoCl2-6H2O, 0.1 mg of
ZnSO4-7H2O, 50 mM HEPES (pH 7.8) per liter].
MA solid medium was prepared by adding 1% Gellan gum (Kanto Chemical
Co., Tokyo, Japan) to MA medium (22). To select
Nitrosomonas transformants, kanamycin was added to MA medium
at a final concentration of 25 µg per ml. Escherichia coli
cells were grown at 37°C with shaking in 2× YT medium
(20).
DNA manipulation.
Plasmid isolation from ENI-11 was
performed by the alkaline-lysis method (20). Genomic and
plasmid DNA preparations, DNA restriction digestions, and Southern
hybridizations were done as described previously (20).
Nitrosomonas cells were transformed by electroporation
(10). Cells grown to stationary phase were harvested by
centrifugation, washed three times with sterile distilled water, and
resuspended in sterile distilled water at an optical density at 600 nm
of about 5.0. The washed cells were kept on ice until use.
Electroporation was done in an Electro cell manipulator (BTX Inc., San
Diego, Calif.) in a 2-mm-gap cuvette at a 50-µF capacitance and 12 kV/cm. Kanamycin-resistant (Kanr) transformants were
selected around 14 days after being spread on MA solid medium. PCR was
performed in a volume of 100 µl with respective sets of
oligonucleotide primers (1 µM) and a Takara Ex Taq DNA
polymerase (Takara Shuzo Co., Shiga, Japan) on a DNA thermal cycler
(Perkin-Elmer). The reaction conditions were 96°C for 30 s,
55°C for 60 s, and 72°C for 90 s (25 cycles). DNA
sequence was determined by the dideoxy chain-termination method with an Auto Cycle kit (Pharmacia) and an ALFred DNA sequencer (Pharmacia).
Determination of plasmid copy number.
The average copy
numbers of pAYS and pAYL in ENI-11 were estimated based on molar ratios
of plasmid DNAs to the gene for HAO (hao). Like N. europaea, ENI-11 is known to have three copies of the
hao gene on the chromosome (28). A 1.0-kb
EcoRI-EcoRV fragment of pAYS and a 1.2-kb
EcoRI-StuI fragment of pAYL, both of which
contained no HHR sequence, were subcloned into pUC119 to make pUYS1 and
pUYL1, respectively. A 1.1-kb HindIII-PstI
fragment of ENI-11 genomic DNA, which contained one copy of
hao, was then cloned into pUYS1 and pUYL1 to construct pUHS
and pUHL. The total DNA of ENI-11 was digested with BamHI
and resolved by an agarose gel electrophoresis, blotted onto a nylon
membrane, and hybridized by either digoxigenin-labelled pUHS or pUHL.
The densities of the DNA-blotted regions of the membrane were
determined with a BioImage analyzer (BAS1000; Fuji Photo Film Co.,
Tokyo, Japan).
Construction of pAYS and pAYL derivatives.
The E. coli plasmid pCRII (Invitrogen), which carried a kanamycin
resistance gene and an ampicillin resistance gene, was digested with
EcoRV and ligated with EcoRV-digested pAYS to
construct pCS1. pCRII was also digested with EcoRV and
ligated with StuI-digested pAYL to construct pCL1. To make
pCS2 and pCL2, pAYS and pAYL were digested with KpnI and
ligated with KpnI-digested pCRII. Plasmid pCSL1 was made in
two steps. First, pAYL was digested with KpnI and ligated
with KpnI-digested pUC119 to make pUL1. pUL1 was then digested with SacI and BamHI and ligated with
pCS1 digested with SacI and BamHI, creating
pCSL1. Plasmids pCSL2 and pCSL3 were constructed by treating
BamHI-digested pCSL1 with exonuclease III and S1 nuclease
and self-ligation. To make pCSL4, pCSL2 was digested with
EcoRV, treated with exonuclease III and S1 nuclease, and
self-ligated.
Nucleotide sequence accession number.
The nucleotide
sequence data of plasmids pAYS and pAYL have been deposited in the
GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases under
accession numbers AB018480 and AB018481, respectively. The nucleotide
sequence data of the HHR-homologous regions of N. europaea
IFO14298 and Nitrosomonas sp. strain ENI-11 have also
appeared under accession numbers AB018482 and AB018483, respectively.
| |
RESULTS |
Characterization of Nitrosomonas plasmids.
Two
small plasmids, designated pAYS (1.8 kb) and pAYL (1.9 kb), were
detected in Nitrosomonas sp. strain ENI-11. Plasmid DNA purified from ENI-11 was observed to migrate as a single band when
electrophoresed in a 1.0% agarose gel (Fig.
1). The size of undigested plasmid was
estimated to be approximately 1.8 kb by using size reference control
plasmids pBR322 (4.3 kb) and pHSG396 (2.2 kb). However, when purified
plasmid DNA was digested with EcoRI, four bands were
observed, and the sum of the fragment sizes was approximately twofold
larger than the expected size, suggesting the presence of two different
plasmids.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 1.
Agarose gel electrophoresis of undigested plasmid DNAs.
Lanes: 1, undigested plasmids isolated from Nitrosomonas sp.
strain ENI-11; 2, undigested vector plasmid pHSG396 (2,239 bp); 3, undigested vector plasmid pBR322 (4,361 bp).
|
|
Plasmid DNA was partially digested with various restriction enzymes,
the randomly produced fragments were cloned by insertion into the
appropriate sites of pUC119, and the resulting clones were subjected to
sequencing. The nucleotide sequences of the clones were then assembled
into two separate sequences with the aid of a computer (Fig.
2). pAYS and pAYL were circular DNA
molecules of 1,823 and 1,910 bp, respectively. pAYS had one restriction site for AccI, BclI, and EcoRV; two
restriction sites for BalI, EcoRI, and
KpnI; and three restriction sites for ClaI and
PvuI. pAYL had one restriction site for BalI,
BclI, ClaI, KpnI, and StuI;
two restriction sites for DraI, EcoRI, and
SphI; and four restriction sites for HincII. The
copy numbers of pAYS and pAYL were estimated to be approximately 10 per
genome on the basis of the molar ratios of plasmid DNAs to the gene for
HAO (see Materials and Methods). There existed two major ORFs, ORF1 in
pAYS and ORF2 in pAYL, each of which was more than 500 bp long (Fig.
3). ORF1 and ORF2
predicted possible proteins of 297 and 319 amino acids, respectively.
The stretch of amino acid residues 54 to 226 of the predicted product
of ORF2 showed similarity to the replication protein RepA of a
Bacillus plasmid, pBAA1 (7). The positional identity with the similar region was 28% (e value,
10
9). The predicted products of ORF1 showed no
significant similarity to any known protein sequences.
Furthermore, there existed no similarity between the potential ORF1 and
ORF2 proteins.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 2.
Restriction maps of pAYS and pAYL. The thick solid lines
represent the regions of high homology (HHR) between pAYS and pAYL.
Arrows indicate PCR primers.
|
|
View larger version (137K):
[in this window]
[in a new window]
|
FIG. 3.
Complete nucleotide sequences of pAYS (A) and pAYL (B).
Nucleotide numbering starts at the EcoRI site of each
plasmid. Potential Shine-Dalgarno sequences are double underlined, and
asterisks indicate stop codons. Amino acids deduced from the nucleotide
sequences are specified by standard one-letter abbreviations. The HHR
sequences are shadowed. Horizontal arrows indicate direct repeats.
Related restriction enzyme sites are underlined.
|
|
To confirm that ENI-11 carries both pAYS and pAYL, PCR primers that had
a unique sequence for either pAYS or pAYL were designed. AYS1
(5'-GAAACCCTCAGAATGCGATC) and AYS2
(5'-TGCTGGAATTGCTCTCGATC) were designed to amplify the
570-bp sequence of pAYS (Fig. 2). AYL1 (5'-AAAATCCCACCGGCTGAAAC)
and AYL2 (5'-CGTCGAGCTATTGATGATGC) were used for
amplifying the 474-bp sequence of pAYL (Fig. 2). Three colonies of
ENI-11 were randomly chosen on MA solid medium, and the cells from each
colony were suspended in PCR buffer and lysed by incubating the mixture
at 95°C for 5 min. In each case, by using the four primers, PCR
generated DNA fragments of expected sizes (570 and 474 bp) (data not
shown). This result confirms that pAYS and pAYL coexist in ENI-11.
Identification of homologous sequence.
pAYS and pAYL had a
highly homologous region (designated HHR) of 262 bp (Fig. 3). The
overall identity of this region was 98% between the two nucleotide
sequences. Although the nucleotide sequence of HHR was subjected
to a computer-assisted homology search, no significant similarity
was detected either to structural genes or to the known origins of
replication listed in the DNA sequence databases. Interestingly,
however, the plasmidless strain N. europaea IFO14298
contained chromosomally an HHR-homologous sequence. Genomic DNA of
N. europaea IFO14298 was digested with several
restriction enzymes, and the resulting fragments were separated
by agarose gel electrophoresis. The presence of the HHR-homologous
sequence was detected by Southern hybridization with
digoxigenin-labelled pAYS DNA. A strong hybridization signal corresponding to the 3.4-kb EcoRI-HindIII
fragment of IFO14298 genomic DNA was detected (data not shown).
Nucleotide sequence analysis of the 3.4-kb
EcoRI-HindIII fragment revealed the presence of a sequence homologous to HHR (Fig. 4).
The IFO14298 HHR of 257 bp had 68% nucleotide identity with the pAYS
HHR. Particularly strong similarity was found in the last 140 nucleotides of the IFO14298 HHR (87% nucleotide identity).
View larger version (56K):
[in this window]
[in a new window]
|
FIG. 4.
Nucleotide sequence similarities in pAYS HHR, ENI-11
HHR, and IFO14298 HHR. Shading indicates identical nucleotides.
|
|
Southern blot analysis was also done to examine whether strain ENI-11
contains chromosomally HHR-homologous sequences. To construct a DNA
probe, the 3.4-kb EcoRI-HindIII fragment of
IFO14298 genomic DNA was digested with HincII and
PvuII, and the resulting 0.7-kb
HincII-PvuII fragment, which contained the
upstream region of the IFO14298 HHR, was labelled with digoxigenin. A
strong hybridization signal corresponding to the 3.0-kb
EcoRI-HindIII fragment of ENI-11 genomic DNA
was detected (data not shown), and this fragment was cloned into
pUC119. Nucleotide sequence analysis of the 3.0-kb EcoRI-HindIII fragment showed the presence of
a sequence homologous to HHR (Fig. 4). Sequence comparison of the
ENI-11 HHR revealed that it had 56 and 58% identity to the pAYS HHR
and the IFO14298 HHR, respectively. Strong similarity was also observed
in the last 140 nucleotides of the ENI-11 HHR and the pAYS HHR (Fig. 4).
Derivatives of pAYS and pAYL.
To investigate whether the major
ORFs, along with HHR, are essential for plasmid maintenance in
Nitrosomonas cells, various hybrid plasmids were
constructed in E. coli MV1184 (Fig.
5). Plasmids pCS1 and pCL1
contained pCRII (Invitrogen) in the internal EcoRV site of
ORF1 and the internal StuI site of ORF2, respectively (see
Materials and Methods). Plasmids pCS2 and pCL2 contained pCRII in the
KpnI site of the pAYS HHR and the pAYL HHR, respectively. Competent cells of E. coli MV1184 were transformed with
pCS1, pCL1, pCS2, or pCL2. Plasmids were then prepared from the
ampicillin-resistant E. coli transformants and used for
transforming ENI-11 cells by electroporation. However, no
Kanr transformants were obtained with these hybrid
plasmids.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 5.
Restriction maps of hybrid plasmids. Native plasmids
pAYS and pAYL are shown as linearized at the EcoRI site. The
thick lines represent pAYS and pAYL DNAs. The thin lines indicate
vector plasmid DNAs. The dashed lines show DNA fragments deleted from
pCSL1. ORF1 and ORF2 are shown by arrows below the restriction maps.
Open boxes indicate pAYS HHR and pAYL HHR. Restriction sites: B,
BamHI; E, EcoRI; EV, EcoRV; H,
HindIII; K, KpnI; Sa, SacI; S,
StuI; Xh, XhoI; Xb, XbaI. The
BamHI, HindIII, SacI,
XhoI, and XbaI sites of pCSL1 were derived from
pCRII and pUC119. Replicative abilities of these plasmids in IFO14298
and ENI-11 are shown on the right.
|
|
When pCSL1, which contained the entire ORF2 and the pAYS HHR, was
introduced into ENI-11, Kanr transformants were
obtained after 14 days of cultivation. To confirm that ENI-11
transformants harbored pCSL1, plasmid DNA was extracted from several
Kanr colonies and analysed by agarose gel
electrophoresis. pCSL1 was detected with all the Kanr
transformants (data not shown). Transformation experiments were also
performed with N. europaea IFO14298 by electroporation.
pCSL1 was able to express Kanr in IFO14298, while no
Kanr transformants were obtained with pCS1, pCS2, pCL1, or
pCL2. When pCSL1 was used as the transforming DNA, efficiencies of
about 102 Kanr transformants of ENI-11 per µg
of DNA were obtained. Similar frequencies were also observed with
N. europaea IFO14298.
To further investigate the essential regions for plasmid maintenance,
deletion-derivative plasmids of pCSL1 were constructed (Fig. 5), and
transformation experiments were performed with ENI-11. When pCSL2,
which carried the entire ORF2 and the pAYS HHR, was used in the
transformation experiments, it retained the ability to express
Kanr in ENI-11. Agarose gel electrophoresis analysis also
revealed the presence of pCSL2 in all the Kanr
transformants (data not shown). However, no Kanr
transformants were obtained with pCSL3, which contained a 16-bp deletion from the 5' end of ORF2. pCSL4, which was constructed by
removing the pAYS HHR from pCSL2, failed to express Kanr in
IFO14298. These results convincingly suggest that ORF2 and pAYS HHR
were essential for the maintenance of pCSL1.
| |
DISCUSSION |
Nitrosomonas sp. strain ENI-11 was originally isolated
from an activated sludge system designed for nitrogen removal
(28). This organism is obligately dependent on the oxidation
of ammonia to nitrite for energy. Biochemical and morphological
tests suggested that ENI-11 is most similar to N. europaea, differing only in that ENI-11 is nonmotile. Nucleotide
sequence analysis has revealed that ENI-11 is closely related to
N. europaea (28). The 16S ribosomal DNA of ENI-11
was identical to that of N. europaea, except for only 2 bp
in the 1,529-bp sequence (8, 9). Like N. europaea
(3, 16), the ammonia monooxygenase- and HAO-encoding genes
were present in multiple copies on the chromosomal DNA of ENI-11
(28). Furthermore, one of the HAO-encoding genes in ENI-11 has been cloned and sequenced, and the predicted protein has been shown
to have 99% amino acid identity with that of N. europaea (28). Growth of ENI-11 was approximately
twofold faster than that of N. europaea IFO14298 in MA
medium (data not shown). Consequently, ENI-11 could oxidize
ammonia to nitrite in MA medium at a rate higher than that of strain
IFO14298. This evidence is of practical importance, because the rate of
ammonia oxidation is critical to nitrogen removal from wastewaters.
To our knowledge, pAYS and pAYL are the first plasmids found in the
ammonia-oxidizing autotrophic bacteria. The plasmids appear to be
stable, since they were isolated from ENI-11, a strain that has been
maintained in the laboratory for over 3 years. However, the function of
pAYS and pAYL remains unknown. It has been shown that the IncQ plasmid
pKT240 can be used as a stable vector for the genetic study of
N. europaea (2, 12). pAYS and pAYL are also
potentially useful for constructing a shuttle vector for genetic
studies. The relatively small size of the plasmids makes them easy to
manipulate with a minimum amount of shearing. In fact, the chimeric
plasmids pCSL1 and pCSL2 could replicate in both E. coli and N. europaea. pCSL2 carries ampicillin and
kanamycin resistance determinants and contains unique restriction sites for EcoRV, HindIII, SacI,
XbaI, and XhoI. Insertions can be made into these
unique restriction sites without disrupting plasmid maintenance or
resistance functions.
pAYS and pAYL had a highly homologous region, designated HHR, of 262 bp. The overall identity was 98% between the two nucleotide sequences.
Nucleotide sequence analysis also revealed the presence of three 11-bp
direct repeats, TTACNCNGTAA, in both pAYS and pAYL sequences
(Fig. 3). Two of the three direct repeats existed in the HHR of each
plasmid. It is possible, therefore, that HHR may be an ori
for DNA replication of pAYS and pAYL, while ORF1 and ORF2 may represent
potential replicators that act on the same sequence. However, a highly
AT-rich region, which contains a sequence homologous to the E. coli oriC 13-bp repeats (14), was not identified in the
nucleotide sequences of pAYS and pAYL. Neither pAYS nor pAYL contained
a dnaA box sequence, TTATCCACA, that has been
found in E. coli plasmids (14). It is also
possible that HHR could be acted on by an unknown host protein, and
some other sequence might represent the target of each ORF. The fact
that the HHR-homologous sequence also existed in the ENI-11 chromosome
may lead to this hypothesis.
Interestingly, an HHR-homologous sequence existed even in the
plasmidless strain IFO14298, suggesting that this genetic information is of importance for these bacteria. Nucleotide sequence analysis of
the chromosomal HHR regions of ENI-11 and IFO14298 showed the presence
of a number of stop codons, suggesting that ENI-11 HHR and IFO14298 HHR
are not contained in a structural gene. It was also found that an ORF,
whose potential product had 45% amino acid identity with the E. coli elongation factor P (1), existed approximately 2.5 kb upstream of the IFO14298 HHR (data not shown). However, no
significant structural features or homologies were detected in the
chromosomal region between this ORF and the IFO14298 HHR. The
HHR-homologous sequence is likely unique for Nitrosomonas species, because no significant similarity was detected in the DNA
sequence databases.
pCSL1 and pCSL2 could transform ENI-11, which carried the homologous
resident plasmids pAYS and pAYL as well as the plasmidless strain
IFO14298. At this time it is difficult to understand how these
homologous plasmids are stably maintained in the same ENI-11 cell.
However, similar results have also been reported for
Acinetobacter calcoaceticus (18) and
Helicobacter pylori (15). Genetic studies of the
ammonia-oxidizing autotrophic bacteria are hampered by their
unfavorable physiological characteristics, namely, slow growth, small
biomass, and susceptibility of cultures to contamination (5). Although ENI-11 was relatively fast growing, it also
required at least 10 days to form visible colonies on MA solid medium. In the present study, therefore, we focused our attention on the essentiality of only two major ORFs and HHR for plasmid maintenance in
Nitrosomonas strains. It remains to be addressed whether
additional regions are required for plasmid maintenance in these bacteria.
This work was supported in part by Sumitomo Chemical Co. and
Takeda Chemical Industries Co.
| 1.
|
Aoki, H.,
S.-L. Adams,
D.-G. Chun,
M. Yaguchi,
S.-E. Chuang, and M. C. Ganoza.
1991.
Cloning, sequencing and overexpression of the gene for prokaryotic factor EF-P involved in peptide bond synthesis.
Nucleic Acids Res.
22:6215-6220.
|
| 2.
|
Bagdasarian, M. M.,
E. Amann,
R. Lurz,
B. Ruckert, and M. Bagdasarian.
1983.
Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors.
Gene
26:273-282[Medline].
|
| 3.
|
Bergmann, D. J., and A. B. Hooper.
1994.
Sequence of the gene amoB, for the 43-kDa polypeptide of ammonia monooxygenase of Nitrosomonas europaea.
Biochem. Biophys. Res. Commun.
204:759-762[Medline].
|
| 4.
|
Bergmann, D. J.,
D. M. Arciero, and A. B. Hooper.
1994.
Organization of the hao gene cluster of Nitrosomonas europaea: genes for tetraheme c cytochromes.
J. Bacteriol.
176:3148-3153[Abstract/Free Full Text].
|
| 5.
|
Bock, E.,
H.-P. Koops,
B. Ahlers, and H. Harms.
1992.
Oxidation of inorganic nitrogen compounds as energy source, p. 414-430.
In
A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes, 2nd ed. Springer-Verlag, New York, N.Y.
|
| 6.
|
Bolivar, F.,
R. L. Rodriguez,
P. J. Greene,
M. C. Betlach,
H. L. Heyneker, and H. W. Boyer.
1977.
Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.
Gene
2:95-113[Medline].
|
| 7.
|
Devine, K. M.,
S. T. Hogan,
D. G. Higgins, and D. J. McConnell.
1989.
Replication and segregational stability of Bacillus plasmid pBAA1.
J. Bacteriol.
171:1166-1172[Abstract/Free Full Text].
|
| 8.
|
Edwards, U.,
T. Rogall,
H. Blocker,
E. Monica, and E. C. Bottger.
1989.
Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.
Nucleic Acids Res.
17:7843-7853[Abstract/Free Full Text].
|
| 9.
|
Head, I. M.,
W. D. Hiorns,
T. Martin Embley,
A. J. McCarthy, and J. R. Saunders.
1993.
The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences.
J. Gen. Microbiol.
139:1147-1153.
|
| 10.
|
Hommes, N. G.,
L. A. Sayavedra-Soto, and D. J. Arp.
1996.
Mutagenesis of hydroxylamine oxidoreductase in Nitrosomonas europaea by transformation and recombination.
J. Bacteriol.
178:3710-3714[Abstract/Free Full Text].
|
| 11.
|
Hyman, M. R., and P. M. Wood.
1985.
Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene.
Biochem. J.
227:719-725[Medline].
|
| 12.
|
Iizumi, T., and K. Nakamura.
1997.
Cloning, nucleotide sequence, and regulatory analysis of the Nitrosomonas europaea dnaK gene.
Appl. Environ. Microbiol.
63:1777-1784[Abstract].
|
| 13.
|
Klots, M. G.,
J. Alzerreca, and J. M. Norton.
1997.
A gene encoding a membrane protein exists upstream of the amoA/amoB genes in ammonia oxidizing bacteria: a third member of the amo operon?
FEMS Microbiol. Lett.
150:65-73[Medline].
|
| 14.
|
Kornberg, A., and T. A. Baker.
1992.
DNA replication, 2nd ed.
W. H. Freeman and Company, New York, N.Y.
|
| 15.
|
Lee, W.-K.,
Y.-S. An,
K.-H. Kim,
S.-H. Kim,
J.-Y. Song,
B.-D. Ryu,
Y.-J. Choi,
Y.-H. Yoon,
S.-C. Baik,
K.-H. Rhee, and M.-J. Cho.
1997.
Construction of a Helicobacter pylori-Escherichia coli shuttle vector for gene transfer in Helicobacter pylori.
Appl. Environ. Microbiol.
63:4866-4871[Abstract].
|
| 16.
|
McTavish, H.,
F. LaQuier,
D. Arciero,
M. Logan,
G. Mundfrom,
J. A. Fuchs, and A. B. Hooper.
1993.
Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea.
J. Bacteriol.
175:2445-2447[Abstract/Free Full Text].
|
| 17.
|
McTavish, H.,
J. A. Fuchs, and A. B. Hooper.
1993.
Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea.
J. Bacteriol.
175:2436-2444[Abstract/Free Full Text].
|
| 18.
|
Minas, W., and D. L. Gutnick.
1993.
Isolation, characterization, and sequence analysis of cryptic plasmids from Acinetobacter calcoaceticus and their use in the construction of Escherichia coli shuttle plasmids.
Appl. Environ. Microbiol.
59:2807-2816[Abstract/Free Full Text].
|
| 19.
|
Olson, T. C., and A. B. Hooper.
1983.
Energy coupling in the bacterial oxidation of small molecules: an extracytoplasmic dehydrogenase in Nitrosomonas.
FEMS Microbiol. Lett.
19:47-50.
|
| 20.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 21.
|
Sayavedra-Soto, L. A.,
N. G. Hommes, and D. J. Arp.
1994.
Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonas europaea.
J. Bacteriol.
176:504-510[Abstract/Free Full Text].
|
| 22.
|
Takahashi, R.,
N. Kondo,
K. Usui,
T. Kanehira,
M. Shinohara, and T. Tokuyama.
1992.
Pure isolation of a new chemoautotrophic ammonia-oxidizing bacterium on gellan gum plate.
J. Ferment. Bioeng.
74:52-54.
|
| 23.
|
Takeshita, S.,
M. Sato,
M. Toba,
W. Masahashi, and T. Hashimoto-Gotoh.
1987.
High-copy-number and low-copy-number plasmid vectors for lacZ -complementation and chloramphenicol- or kanamycin-resistance selection.
Gene
61:63-74[Medline].
|
| 24.
|
Vieira, J., and J. Messing.
1987.
Production of single-stranded plasmid DNA.
Methods Enzymol.
153:3-11[Medline].
|
| 25.
|
Wood, P. M.
1986.
Nitrification as a bacterial energy source, p. 39-62.
In
J. I. Prosser (ed.), Nitrification. Society for General Microbiology and IRL Press, Oxford, United Kingdom.
|
| 26.
|
Worce, C. R.,
W. G. Weisburg,
B. J. Paster,
C. M. Hahn,
R. S. Tanner,
N. R. Krieg,
H. P. Koops,
H. Harms, and E. Stackebrandt.
1984.
The phylogeny of purple bacteria: the beta subdivision.
Syst. Appl. Microbiol.
5:327-336.
|
| 27.
|
Worce, C. R.,
W. G. Weisburg,
C. M. Hahn,
B. J. Paster,
L. B. Zablen,
B. J. Lewis,
T. J. Macke,
W. Ludwig, and E. Stackebrandt.
1985.
The phylogeny of purple bacteria: the gamma subdivision.
Syst. Appl. Microbiol.
6:25-33.
|
| 28.
| Yamagata, A., J. Kato, and H. Ohtake. Unpublished
data.
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
