Isolation and Characterization of Two Cryptic Plasmids in the ...

JOURNAL OF BACTERIOLOGY, June 1999, p. 3375–3381 0021-9193/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 181, No. 11

Isolation and Characterization of Two Cryptic Plasmids in the Ammonia-Oxidizing Bacterium Nitrosomonas sp. Strain ENI-11 AKIRA YAMAGATA, JUNICHI KATO, RYUICHI HIROTA, AKIO KURODA, TSUKASA IKEDA, NOBORU TAKIGUCHI, AND HISAO OHTAKE* Department of Fermentation Technology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8527, Japan Received 26 October 1998/Accepted 31 March 1999

Two plasmids were discovered in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11, which was isolated from activated sludge. The plasmids, designated pAYS and pAYL, were relatively small, being approximately 1.9 kb long. They were cryptic plasmids, having no detectable plasmid-linked antibiotic resistance or heavy metal resistance markers. The complete nucleotide sequences of pAYS and pAYL were determined, and their physical maps were constructed. There existed two major open reading frames, ORF1 in pAYS and ORF2 in pAYL, each of which was more than 500 bp long. The predicted product of ORF2 was 28% identical to part of the replication protein of a Bacillus plasmid, pBAA1. However, no significant similarity to any known protein sequences was detected with the predicted product of ORF1. pAYS and pAYL had a highly homologous region, designated HHR, of 262 bp. The overall identity was 98% between the two nucleotide sequences. Interestingly, HHR-homologous sequences were also detected in the genomes of ENI-11 and the plasmidless strain Nitrosomonas europaea IFO14298. Deletion analysis of pAYS and pAYL indicated that HHR, together with either ORF1 or ORF2, was essential for plasmid maintenance in ENI-11. To our knowledge, pAYS and pAYL are the first plasmids found in the ammonia-oxidizing autotrophic bacteria. 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.

The ammonia-oxidizing autotrophic bacteria are confined to the gram-negative b and l 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

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 mg per ml. Escherichia coli cells were grown at 37°C with shaking in 23 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-mF capacitance and 12 kV/cm. Kanamycinresistant (Kanr) transformants were selected around 14 days after being spread on MA solid medium. PCR was performed in a volume of 100 ml with respective sets of oligonucleotide primers (1 mM) 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).

* Corresponding author. Mailing address: Department of Fermentation Technology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8527, Japan. Phone: 81-824-24-7756. Fax: 81-824-22-3758. E-mail: [email protected]. 3375

3376

YAMAGATA ET AL.

J. BACTERIOL. TABLE 1. Bacterial strains and plasmids used in the present study

Strain or plasmid

E. coli MV1184 N. europaea IFO14298 Nitrosomonas sp. strain ENI-11 Plasmids pBR322 pCRII pHSG396 pUC119 pAYL pAYS pCL1 pCL2 pCS1 pCS2 pCSL1 pCSL2 pCSL3 pCSL4 pUHL pUHS pUL1 pUYL1 pUYS1 a

Description

Source or reference r

1

ara D(lac-proAB) rpsL thi (f80lacZDM15) D(srl recA)306::Tn10 (Tet ) F9[traD36 proAB lacI lacZDM15] Type strain, plasmidless Strain isolated from activated sludge

q

Cloning vector; Apr Tetr Cloning vector; Apr Kanr Cloning vector; Cmr Cloning vector; Apr Cryptic plasmid isolated from ENI-11; ORF2; pAYL HHR Cryptic plasmid isolated from ENI-11; ORF1; pAYS HHR pCRII containing StuI-digested pAYL; pAYL HHR pCRII containing KpnI-digested pAYL; ORF2 pCRII containing EcoRV-digested pAYS; pAYS HHR pCRII containing KpnI-digested pAYS; ORF1 Hybrid plasmid constructed by fusing pUL1 and pCS1; ORF2; pAYS HHR pCSL1 derivative; ORF2; pAYS HHR pCSL1 derivative; pAYS HHR pCSL1 derivative; ORF2 pUC119 containing 1.2-kb EcoRI-StuI fragment of pAYL and 1.1-kb HindIII-PstI fragment of ENI-11 genomic DNA including one copy of hao pUC119 containing 1.0-kb EcoRI-EcoRV fragment of pAYS and 1.1-kb HindIII-PstI fragment of ENI-11 genomic DNA including one copy of hao pUC119 containing KpnI-digested pAYL; ORF2 pUC119 containing 1.2-kb EcoRI-StuI fragment of pAYL pUC119 containing 1.0-kb EcoRI-EcoRV fragment of pAYS

24 IFOa 28

6 Invitrogen 23 24 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

IFO, Institute for Fermentation, Osaka, Japan.

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 StuIdigested 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.

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. 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

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)

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).

VOL. 181, 1999

PLASMIDS IN NITROSOMONAS SP. STRAIN ENI-11

3377

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.

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, 1029). 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. 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 (59-GAAACCCTCAGAATGCGATC) and AYS2 (59-TGCTGGAATTGCTCTCGATC) were designed to amplify the 570-bp sequence of pAYS (Fig. 2). AYL1 (59-AAAATCCCACCGGCTGAAAC) and AYL2 (59-CGTC GAGCTATTGATGATGC) were used for amplifying the 474bp 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). 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. 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 mg

3378

YAMAGATA ET AL.

J. BACTERIOL.

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.

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 59 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

PLASMIDS IN NITROSOMONAS SP. STRAIN ENI-11

VOL. 181, 1999

3379

FIG. 3—Continued.

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, TTA CNCNGTAA, 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

3380

YAMAGATA ET AL.

J. BACTERIOL.

FIG. 4. Nucleotide sequence similarities in pAYS HHR, ENI-11 HHR, and IFO14298 HHR. Shading indicates identical nucleotides.

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

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.

PLASMIDS IN NITROSOMONAS SP. STRAIN ENI-11

VOL. 181, 1999

maintenance in Nitrosomonas strains. It remains to be addressed whether additional regions are required for plasmid maintenance in these bacteria. ACKNOWLEDGMENTS This work was supported in part by Sumitomo Chemical Co. and Takeda Chemical Industries Co. REFERENCES 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. 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. 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. 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. 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. 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. 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. 11. Hyman, M. R., and P. M. Wood. 1985. Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene. Biochem. J. 227:719–725. 12. Iizumi, T., and K. Nakamura. 1997. Cloning, nucleotide sequence, and

13.

14. 15.

16.

17. 18.

19. 20. 21. 22. 23.

24. 25. 26. 27. 28.

3381

regulatory analysis of the Nitrosomonas europaea dnaK gene. Appl. Environ. Microbiol. 63:1777–1784. 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. Kornberg, A., and T. A. Baker. 1992. DNA replication, 2nd ed. W. H. Freeman and Company, New York, N.Y. 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. 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. 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. 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. 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. 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. 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. 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. Takeshita, S., M. Sato, M. Toba, W. Masahashi, and T. Hashimoto-Gotoh. 1987. High-copy-number and low-copy-number plasmid vectors for lacZacomplementation and chloramphenicol- or kanamycin-resistance selection. Gene 61:63–74. Vieira, J., and J. Messing. 1987. Production of single-stranded plasmid DNA. Methods Enzymol. 153:3–11. 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. 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. 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. Yamagata, A., J. Kato, and H. Ohtake. Unpublished data.