Patent application title: GLUCOSE DEHYDROGENASE AND PRODUCTION THEREOF
Inventors:
Ruth Meissner (Leverkusen, DE)
Ruth Meissner (Leverkusen, DE)
Walter Weichel (Odenthal, DE)
Rainhard Koch (Kleinmachnow, DE)
Irina Bachmatova (Vilnius, LT)
Liucija Marcinkeviciene (Vilnius, LT)
Rita Meskiene (Vilnius, LT)
Rasa Semenaite (Vilnius, LT)
Vida Casaite (Vilnius, LT)
Rolandas Meskys (Vilnius, LT)
Assignees:
BAYER TECHNOLOGY SERVICES GMBH
IPC8 Class: AC12Q132FI
USPC Class:
435 26
Class name: Measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving oxidoreductase involving dehydrogenase
Publication date: 2009-04-02
Patent application number: 20090087874
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Patent application title: GLUCOSE DEHYDROGENASE AND PRODUCTION THEREOF
Inventors:
Ruth MEISSNER
Walter WEICHEL
Rainhard KOCH
Irina BACHMATOVA
Liucija MARCINKEVICIENE
Rita MESKIENE
Rasa SEMENAITE
Vida CASAITE
Rolandas MESKYS
Agents:
NORRIS, MCLAUGHLIN & MARCUS, PA
Assignees:
BAYER TECHNOLOGY SERVICES GMBH
Origin: NEW YORK, NY US
IPC8 Class: AC12Q132FI
USPC Class:
435 26
Abstract:
The invention relates to novel PQQ-dependent soluble glucose
dehydrogenases (sPQQGDH) from Acinetobacter and to a process for their
preparation by overexpression in suitable microbial expression systems.Claims:
1.-8. (canceled)
9. A soluble pyrroloquinoline quinone glucose dehydrogenase (sPQQGDH) having the amino acid sequence of wild-type Acinetobacter LMD 79:41 (numbering as shown in SEQ ID NO: 2), wherein compared to the amino acid sequence of wild-type Acinetobacter LMD 79:41 (numbering as shown in SEQ ID NO: 2):a) the following amino acids are exchanged:i) position 21 has a N;ii) position 41 has an S;iii) position 47 has an L;iv) position 121 has a V or an A;v) position 149 has an A;vi) position 213 has an S;vii) position 244 has an I;viii) position 320 has a G;ix) position 391 has an S;x) position 452 has an S;xi) position 474 has an R; andxii) position 480 has a Q; andb) at least one of the following amino acids are also exchanged:i) position 16 has an H;ii) position 18 has an L;iii) position 20 has an F;iv) position 40 has a G;v) position 48 has an I;vi) position 61 has an A;vii) position 111 has a T;viii) position 154 has a D;ix) position 190 has an E;x) position 293 has an A;xi) position 311 has an S;xii) position 314 has an A;xiii) position 324 has an L;xiv) position 333 has an M;xv) position 339 has an S;xvi) position 355 has a G;xvii) position 366 has a D;xviii) position 417 has an N; orxix) position 418 has an A.
10. An isolated and purified DNA sequence encoding a sPQQGDH enzyme according to claim 9.
11. A vector comprising a DNA sequence according to claim 11.
12. A transformed microorganism expressing a DNA sequence according to claim 11.
13. Process for preparing a sPQQGDH enzyme, which comprises cultivating a microorganism according to claim 12, and then isolating the sPQQGDH enzyme.
14. Reagent for detecting glucose comprising one or more sPQQGDH enzymes according to claim 9.
15. Sensor for detecting glucose comprising one or more sPQQGDH enzymes according to claim 9.
16. A method for detecting glucose in a sample comprising contacting the sample with one or more sPQQGDH enzymes according to claim 9.
Description:
[0001]The invention relates to novel PQQ-dependent soluble glucose
dehydrogenases (sPQQGDH) from Acinetobacter and to a process for their
preparation by overexpression in suitable microbial expression systems.
[0002]Patients suffering from diabetes must measure their blood glucose regularly. Measurement of glucose likewise plays an important part in fermentation processes. In many cases, glucose is determined enzymatically. For this purpose, either glucose oxidase, or, in rare cases, also glucose-6-phosphate dehydrogenase are employed. The methods based on glucose oxidase have the disadvantage that the enzyme transfers the electrons not just to the added mediators but also to the oxygen present. The result of the measurement is therefore dependent on the oxygen partial pressure. Efforts have been made for some time to replace the present enzymes by sPQQ-dependent glucose dehydrogenases. PQQ stands for pyrroloquinoline quinone. PQQ is the prosthetic group of glucose dehydrogenase (GDH). It transfers redox equivalents. The advantage of these enzymes is that the measurement is independent of the oxygen partial pressure and a more accurate measurement is possible. In addition, measurement in smaller sample volumes is possible through use of these enzymes.
[0003]Two different PQQGDHs are to be found in the literature. One form is membrane-bound (mPQQGDH) and is unsuitable for use in glucose sensors. The other form is soluble (sPQQGDH) and has been found variously in strains of the genus Acinetobacter (Biosci. Biotech. Biochem. 59 (8), pp. 1548-1555, 1995). The enzyme is a homodimer and has a molecular weight of 50 kDa. The soluble and the membrane-bound PQQGDH have no sequence homology and are different immunologically and in terms of their kinetics (Cleton-Jansen et al., 172 (11), pp. 6308-6315, J. Bacteriol. 1990) (Matsushita et al., Biochemistry 28 (15), pp. 6276-6280, 1989).
[0004]The sPQQGDH from Acinetobacter has been known for some time. All authors use the strains LMD 79.41 (Kojima et al., Biotechnology Letters, 22, pp. 1343-1347, 2000), NCIMB 11517 or JCM 6841 (both: US 2001021523).
[0005]The DNA sequences of the sPQQGDH of these strains are deposited in the Genbank under the access numbers X15871 (LMD 79.41), E28183 (JCM 6841) and E28182 (NCIMB 11517).
[0006]Many attempts have been made to alter the properties of these sPQQGDH by modifying the gene sequences. (EP 1 167 519 A1, EP 1 176 202 A1, Sode und Kojima, Biotechn. Letters, Vol. 19, (11) pp. 1073-1077, 1997). The aim in these cases was to improve the substrate specificity and the thermal stability of the enzyme. EP 1 167 519 A 1 describes the replacement of individual amino acids of the sPQQGDH from Acinetobacter LMD 79.41 in order to obtain increased thermal stability. Alterations are made in amino acids 209, 210, 231, 420 and 421. WO 02/072839 A1 describes further alterations in the amino acid sequence in order to achieve increased thermal stabilities and improvements in the water solubility. Replacements were made in positions 167, 231, 340, 415 and 418. The numbering is based in both cases on that used in EP 1 167 519. When comparing the enzyme from Acinetobacter LMD 79.41 with the novel sPQQGDH according to the invention and that from Acinetobacter JCM 6841 it is necessary also to take account of the fact that the two last-mentioned enzymes are two amino acids longer than that from LMD 79.41. The mature enzyme and the signal peptide are considered in this connection.
[0007]WO 02/34919 A1 describes replacement of individual amino acids with the aim of reducing the affinity of the enzyme for maltose. The thermal stability of the enzyme is unaffected.
[0008]Enzymes are normally prepared by heterologous expression thereof in E. coli. The problem arising on expression of sPQQGDH is that although E. coli can synthesise the enzyme, it cannot synthesise cofactor PQQ. Although, according to Sode et al. (J. Biotechnology, 49, 239-243, 1996) expression of the apoenzyme mPQQGDH is possible in the case of the membrane-bound glucose dehydrogenase, it is unstable and is broken down again during culturing of the cells. Matsushita et al. (Biosci. Biotechnol. Biochem. 59, 1548-1555, 1995) postulate a conformational change in the enzyme occurring on binding of the cofactor and protecting the holoenzyme from tryptic digestion.
[0009]For this reason, all research groups and manufacturers have made efforts to prepare active and stable enzyme in such a way that either PQQ is added during culturing of the cells, or the expression takes place in strains themselves able to form PQQ. Sode et al. (J. Biotechnology, 49, 239-243, 1996) succeeded in coexpressing mPQQGDH together with the genes for PQQ synthesis in E. coli and achieved about 1500 U/l at an OD of 4. Addition of PQQ during culturing of the cells achieves 1100 U/l. However, the system has the problem that the cells do not reach high optical densities and die during enzyme production (Kojima et al., Biotechnology Letters, 22, 1343-1347, 2000). The authors in the previously cited publication describe the synthesis of active sPQQGDH in Klebsiella pneumoniae. This organism is genetically available and has the ability to synthesize PQQ. In this case, therefore, activities of about 14 000 U/l are reached, although additional PQQ feeding is necessary. Yoshida et al. (Enzym. Microb. Technol. 30, pp. 312-318, 2002) expressed soluble sPQQGDH in Pichia pastoris heterologously and thus achieved usable enzyme yields of up to 200 000 U/l. However, it is necessary for this purpose to reach very high cell densities. In addition, the enzyme is glycosylated and must additionally be purified by a precipitation and two ion exchanges. Cost-effective preparation is impeded in particular by the elaborate purification and the glycosylation.
[0010]The Toyobo Co. Ltd., Japan, describes in JP 09140378 the preparation of sPQQGDH using Acinetobacter calcoaceticus. 60 U/l are reached after three purification steps. In order to increase the productivity of the system, the enzyme was subsequently expressed heterologously in Pseudomonas putida, because this strain is able to synthesize PQQ.
[0011]Olsthoorn and Duine (Arch. Biochem. Biophys. 336 (1), 42-48, 1996) describe the batch culturing of an E coli clone which expresses sPQQGDH in a 100 l fermenter. After the culturing, the enzyme is purified by three column steps. The clone forms no PQQ; the cofactor is added only in the enzyme assay. The yields amount to 10 mg of pure protein per 1 of grown culture. The low cell density of the culture, the elaborate purification process and the yield, however, make the process appear uneconomic.
DESCRIPTION OF THE INVENTION
[0012]During a screening, 12 strains of the species Acinetobacter calcoaceticus which form an sPQQGDH were found. Typing of the novel strains by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) revealed 99.8% homology of the partial 16S rDNA sequence to the Acinetobacter calcoaceticus type strain. However, the observed utilization of L-malate and the inability to break down L-phenylacetate is unusual for the type strain. This property is shown by all 12 strains found.
[0013]The sPQQGDHs formed by these strains differ from the previously known enzymes in substantial properties. On the one hand, they have a thermal stability which is improved by comparison with the enzyme from the type strain (see Example 3). The nucleotide sequences and the sequence of the amino acids are likewise different.
[0014]Sequence analysis of the 12 genes which code for the novel sPQQGDHs revealed the following results (see FIG. 1):
[0015]The nucleotide sequences of all the newly found sPQQGDHs differ distinctly from those of the enzymes from Acinetobacter LMD 79.41 and Acinetobacter JCM 6841 and NCIMB 11517.
[0016]The amino acid sequences which can be derived from the respective nucleotide sequences likewise differ from the previously known sequences. The sPQQGDH from Acinetobacter LMD 79.41 has 478 amino acids, including the signal peptide, while those from the two other known strains Acinetobacter JCM 6841 and Acinetobacter NCIMB 11517, and those from the sequences found here each have 480 amino acids, including signal peptide.
[0017]The amino acid sequences of the newly found sPQQGDHs differ in numerous positions from those previously known. Surprisingly, an amino acid different from that in all previously described enzymes was found at 12 positions with all the genes newly described herein. In addition, at least 75% of the genes newly described herein have an amino acid different from that in the previously described genes at four positions. Moreover, in the enzymes newly described herein there are further exchanges, which occur singly or in some of the enzymes.
[0018]The specific replacements in the newly found sPQQGDH according to the invention are of the following amino acids. The numbering of amino acids is based on the numbering in the enzyme from the strain Acinetobacter JCM 6841, which comprises 480 amino acids, including signal peptide (see FIG. 1):
[0019]All the sPQQGDHs which have at positions 21 an N and 41 an S and 47 an L and 121 a V or an A and 149 an A and 213 an S and 244 an 1 and 320 a G and 391 an S and 452 an S and 474 an R and 480 a Q as amino acid are according to the invention.
[0020]Further according to the invention are all the sPQQGDHs in which the following exchanges may occur in addition to the exchanges described above:
[0021]There may be at positions 16 an H, at 18 an L, at 20 an F, at 40 a G, at 48 an, at 61 an A, at 111 a T, at 154 a D, at 190 a D, at 293 an A, at 311 an S, at 314 an A, at 324 an L, at 333 an M, at 339 an S, at 355 a G, at 366 a D, at 417 an N and at 418 an A.
[0022]It has surprisingly been found that the newly found enzymes are all more thermally stable than the wild-type enzyme. The thermal stability is an essential criterion for successful use of sPQQGDH as glucose sensor. Thus, the remaining activities found on incubation at 60° C. and 70° C. for one hour were all higher than with the enzyme from Acinetobacter LMD 79.41. The remaining activity found at 60° C. with the sPQQGDHs according to the invention were between 51.5% and 12.5%. The wild type showed only 1.8% of its original activity in an identical approach. Between 26.7% and 8.8% of the initial activity were observed at 70° C., whereas the wild-type enzyme had only 6.4% of the original activity.
[0023]Various attempts have been made in the past to increase the thermal stability of sPQQGDH by targeted and random exchange of amino acids.
[0024]However, EP 1 167 519 A1 claims the exchange of amino acids at positions markedly different from those in the sPQQGDHs according to the invention. The same applies to the exchanges described in WO 02/072839. This likewise applies to the exchanges described in WO 02/34919 A1. The increased thermal stability of the sPQQGDHs according to the invention thus correlates with the exchange of a whole group of amino acids whose influence on this property was not previously known.
[0025]The enzymes according to the invention can be prepared by cultivating the relevant strain in a suitable medium. It is possible to use for this purpose the media described in the literature, such as nutrient broth (Difco 0003). After the cells have grown, they are harvested and disrupted. The enzyme is purified as described below.
[0026]The sPQQGDHs from the wild type are preferably cloned into a suitable host. Possibilities therefor are the usual genetically readily available prokaryotes such as members of the genus Bacillus, Klebsiella, Pseudomonas, and E. coli. The enyzmes may furthermore also be expressed in eukaryotes such as members of the genera Pichia, Saccharomyces, Hansenula, Aspergillus or Kluyveromyces. The enzymes may also be expressed in plants or animal cell lines.
[0027]It is possible to employ for the cloning standard methods such as PCR with degenerate primers or hybridization of genomic libraries with suitable probes. Expression is preferably carried out in E. coli. The expression is normally carried out in the strains BL21, DH5, HB101, JM101, RV308, TOPP, TOP10, XL-1 and derivatives thereof. The strains W3110 and DH5 are preferably used. Conventional expression vectors can be employed for this purpose. The vectors typically comprise an origin of replication, an antibiotic resistance and a promoter sequence. Examples of vectors which can be employed are the following: pUC18/19, pBluescript, pTZ, pGEX, pPROEx, pcDNA3.1, YEp24, pBAC, pPICZ. Vectors of the pMAL, pET, pTrx, pCAL, pQE and pPROTet series are preferred. Expression vectors of the pASK-IBA 2 to pASK-IBA 7 series are particularly preferably employed. Usual antibiotics for resistance selection are, for example, ampicillin, kanamycin, tetracycline and chloramphenicol.
[0028]It is also possible to use expression systems which lead to the protein being secreted into the medium.
[0029]The vectors can be transferred to the host cell by conventional methods. Examples employed for this purpose are: electroporation, protoplast fusion, chemical transformation.
[0030]Synthesis of the cloned sPQQGDHs is induced by adding an inducer. The inducers employed for this purpose are those suitable for the chosen expression system, such as, for example, IPTG, tryptophan, glucose and lactose. The inducer anhydrotetracycline is particularly preferably used.
[0031]The recombinant cell lines are cultured in media suitable therefor. Conventional processes used for culturing prokaryotes and eukaryotes are suitable for this purpose. The culturing can be carried out in suitable fermenters. The organisms are preferably cultivated in such a way that very high cell densities are reached. To do this it is necessary for the feeding with a C source and an N source to be suitably controlled by a strategy such that no toxic metabolic products result (for overview: Schugerl et al. (editors) in: Bioreaction Engineering, pp. 374-390, Springer-Verlag, Berlin, 2000; Yee and Blanch, Bio/Technology, 10 (2), pp. 1550-1556, 1992).
[0032]A process suitable for the purposes of this invention is for example that of Riesenberg et al., Appl. Microbiol. Biotechnol, 34, pp. 77-82, 1990.
[0033]Whereas the cultivation of the cells ideally takes place at 28-37° C., the temperature is lowered to 10-28° C. for expression of the protein. It is preferably at 15-25° C. and particularly preferably at 20-22° C.
[0034]It has surprisingly been found that on use of the process according to the invention distinctly higher yields of active enzyme are attained than previously described in the literature. When an E. coli clone is cultured in a batch culture it is possible in this way to obtain up to 48 mg of pure protein from one litre of culture supernatant (see Examples 5 and 6). Yoshida et al. (Enzym. Microbiol. Technol., 30, pp. 312-318, 2002) attained 43 mg/l. However, elaborate purification of the enzyme is necessary, and it is glycosylated. Olsthoorne and Duine (Arch. Biochem. Biophys. 336 (1), pp. 42-48, 1996, report a yield of 10 mg/l.
[0035]If the cells having the enzymes according to the invention are cultured in fermentation with high cell density, it is in fact possible to reach more than 220 mg of pure enzyme per 1 of culture liquid (Example 8).
[0036]Culturing of the cells is followed by harvesting and disruption thereof by suitable methods such as, for example, French press, addition of detergents or ultrasound. The protein solution is buffered and brought to a slightly alkaline pH. The buffer is adjusted to a pH between 7 and 9, and is preferably between 7.8 and 8.2.
[0037]Buffer substances which can be employed are the buffers customary in biochemistry, such as Tris, MOPS or PIPES buffers, potassium phosphate buffer; the concentration ought to be 5-100 mM, and is preferably in a range of 20-70 mM and particularly preferably 50 mM.
[0038]The sPQQGDH can now be purified very easily from the protein solution. To do this, the protein solution is either purified by a conventional ion exchange chromatography, or the ion exchange material is directly added to the protein solution and then separated from the remainder of the liquid on a suction funnel. Suitable ion exchangers are cation exchangers such as, for example, Lewatit resins CM-, S-, SM-Sepharose, CM-, SP-Sephadex, Amberlyst 15, Amberlite CG-50, Amberlite IR-120, carboxymethyl-, sulphoxyethyl-, oxycellulose, cellulose phosphate and CM-Toyopearl. CM-Toyopearl is preferably employed.
[0039]The protein is then eluted from the ion exchange material by conventional methods. An increasing NaCl gradient is employed for this, and the buffer used is that also employed previously for binding the protein to the ion exchange material. The enzyme is normally eluted at a concentration of about 200 mM NaCl.
[0040]The enzyme can then be purified further, and it is likewise possible to reduce the salt content by conventional methods such as dialysis and ultrafiltration.
[0041]The preparation and purification of the sPQQGDH according to the invention preferably takes place as apoenzyme, and the cofactor PQQ is added only when the enzyme has been completely purified. Addition of the PQQ ideally takes place in conjunction with changing the buffer of the enzyme after purification on the ion exchanger.
[0042]The PQQ can be added before the buffer is changed or thereafter. It is preferably added beforehand. The amount depends on the content of the protein solution. From 0.1 to 5 mol of PQQ can be added per mole of active enzyme. It is ideal to add from 0.5 to 2 mol, particularly preferably 2 mol, of PQQ per mole of active protein. However, it is also possible for the enzyme to be prepared and purified as holoenzyme. For this purpose the PQQ can be added during cell culturing, during cell disruption or prior to purification. A further possibility is for the sPQQGDHs according to the invention also be prepared as holoenzymes by heterologous expression thereof in organisms able to synthesise PQQ. These may be for example organisms from the genus Klebsiella or Pseudomonas.
[0043]The novel sPQQGDHs are employed according to the invention for glucose measurement. They are particularly preferably employed in instruments which can be used to measure blood glucose. It is also possible in addition to employ the enzymes for glucose measurement for example in fermentation processes.
[0044]If the sPQQGDHs according to the invention are employed for diagnostic purposes, such a test kit typically includes a buffer, a mediator and some units of enzymic activity. 0.5-10 U are typically employed, and 1-5 U are preferably employed. Various formulations of the enzyme are possible for this purpose. It can for example be freeze- or spray-dried and formulated as solution. Suitable electrodes may be carbon, gold or platinum electrodes. The enzyme is normally immobilized on the electrode. Crosslinking agents are normally used for this purpose, but the enzyme can also be encapsulated. Further possibilities for immobilizing it are by means of a dialysis membrane, by photocrosslinking, electrically conducting or redox polymers. Combinations of the abovementioned methods are also possible.
[0045]The enzymes are preferably applied as holoenzymes but they can also be employed as apoenzymes, in which case the necessary PQQ is supplied in a second layer. The novel sPQQGDHs are preferably immobilized on a carbon electrode with glutaraldehyde and then treated with a reagent containing amines for complete reaction of excess glutaraldehyde.
EXAMPLES
Example 1
Search for Novel Strains Which Produce sPQQGDH
[0046]Soil samples were suspended in saline (0.9% NaCl), and aliquots were streaked onto agar plates which contained a nutrient medium with gluconate as sole carbon source. The medium had the following composition:
TABLE-US-00001 NaCl 5 g MgSO4 0.2 g NH4H2PO4 1 g K2HPO4 1 g Sodium gluconates 2 g Yeast extract 0.5 g Agar 15 g Dist. water 1000 ml pH 7.0
[0047]The plates were incubated at 30° C. for 24-48 hours.
[0048]Alternatively, Pseudomonas-Agar from Oxoid was also used. The incubation conditions were identical.
[0049]Selected grown colonies were isolated on glucose-eosin-methylene blue agar. This had the following composition:
TABLE-US-00002 K2HPO4 1 g Glucose 18 g Peptone 10 g Eosins 0.4 g Methylene blue 0.06 g Agar 16 g Dist. water. 1000 ml pH 7.6
[0050]The plates were incubated at 30° C. for 24 hours. Positive clones can be identified by being dark red and/or having a green lustre. These colonies are again streaked on nutrient agar (Oxoid) in order to check their purity. The composition thereof was as follows:
TABLE-US-00003 Meat extract 1 g Yeast extract 2 g Peptone 5 g NaCl 5 g Agar 15 g Dist. water 1000 ml pH 7.4
[0051]The plates were incubated at 30° C. for 48 hours and again isolated. The purified strains were subsequently cultured in 100 ml of liquid nutrient broth (Oxoid) at 30° C. for 20 h; the medium had the following composition:
TABLE-US-00004 Meat extract 3 g Bakto peptone 5 g Glucose 1 g Dist. water 1000 ml
[0052]Culturing was followed by harvesting of the cultures (4500×g, 40 min, 4° C.) and washing with saline (0.9% NaCl). The pellets were resuspended in 5 ml of 50 mM KP buffer of pH 7.2 and disrupted with ultrasound. The extract was again centrifuged at 10 000×g at 4° C. for 30 min, and the GDH activity in the cell-free supernatant was measured. It was possible to isolate 26 strains with GDH activity on the screening medium with gluconate as sole carbon source. It was possible to isolate four strains after culturing on the Pseudomonas medium.
Example 2
Purification of the Enzymes
[0053]The strains whose sPQQGDHs were to be investigated were cultured in 8 l of NB medium (Oxoid), which contained 0.1% glucose at 30° C. for 20 h. The cells were harvested (4500×g, 40 min, 4° C.), washed with 0.9% saline and taken up in 150-200 ml of 10 mM MOPS of pH 8.0. The cells were disrupted with ultrasound. The cell-free extract was loaded onto 300 ml of TSK gel CM-Toyopearl 650M (Tosoh Corp.) and washed with 3-4 column volumes of 10 mM MOPS of pH 8.0. Elution took place with a 0-0.3 M NaCl gradient with a 3-4-fold column volume. Active fractions were pooled, transferred into a dialysis tube and concentrated by addition of high-viscosity carboxylmethylcellulose as water absorber. The concentrated sample was resuspended with 3.5 volume of 10 mM K-MOPS of pH 6.8 and again purified on the CM-Toyopearl column. Equilibration of the column and washing took place with 10 mM K-MOPS of pH 6.8, and elution was carried out with a 0-0.3 M NaCl gradient. The active fractions were pooled and concentrated as described above. They served as starting material for determining the thermal stability.
Example 3
Test of Thermal Stability
[0054]The thermal stability was determined by incubating the purified enzymes at 50, 60 and 70° C. for 60 minutes. The incubation took place in 50 mM Pipes at pH 6.5 in the presence of 1 mM CaCl2, 0.1% Triton X-100, 0.1% BSA and 5 μM PQQ. After the incubation, the samples were cooled on ice and the remaining activity was determined. It was expressed as a percentage of the original activity.
TABLE-US-00005 TABLE 1 Thermal stability of novel sPQQGDH from various new isolates Strain 50° C. 60° C. 70° C. KGN25 100 14.3 13.1 KGN34 104.2 19.8 14.6 KGN80 94.3 22.6 18.9 KGN100 95.9 12.5 13.5 KG106 102.6 33.4 13.9 KG140 105.5 20.5 8.8 KOZ62 107.7 42.8 14.7 KOZ65 103.8 34.6 19.2 PT15 100.0 25.4 16.3 PT16 85.0 51.5 14.9 PTN26 120.7 27.5 22.4 PTN69 93.2 36.4 26.7 LMD79.41 wild type 95.6 1.8 6.4
Example 4
Cloning and Analysis of the Novel sPQQGDH Genes
[0055]For cloning the sPQQGDH genes from the novel strains described here it was initially attempted to amplify the complete coding sequence on genomic DNA with synthetic oligonucleotides in a PCR. The primers which were used for this and were derived from the published sPQQGDH sequence (X15871; LMD 79.41) did not, however, lead to PCR products, because the sequences of the sPQQGDHs according to the invention differ particularly greatly from the wild type in the region of the signal peptide and close to the stop codon. For this reason, various primers from both strands of the wild-type sequence which were intended to lead on use in a PCR to amplification of fragments of the coding sequence were used. It was possible with some of these primer combinations to isolate such fragments from the strains described in Example 3. Commercial kits were used for all the PCR reactions, usually the PCR master kit from Roche, in accordance with the manufacturer's instructions. It was possible with the primers
GDH-fwd P1 (5'-CCA GAT AAT CAA ATT TGG TTA AC-3') and
[0056]GDH-rev P7 (5'-CAT CAC GAT AAC GGT TYT TGC-3') to isolate fragments about 1200 bp in size. The resulting DNA pieces were then sequenced. An inverse PCR was carried out in order to obtain the complete sequence of the individual genes (Sambrook and Russell: Molecluar Cloning--A Laboratory Manual. CSHL Press (2001), p. 8.81). The primers employed for this purpose in the present case were each positioned on the margin of the fragment in such a way that the still unknown part of the GDH gene is synthesized in a further PCR. Such a PCR reaction was carried out on genomic DNA of the respective strain, which had been cut with the restriction endonucleases EcoRI and BglII and then recircularized by T4 ligase. The primers used were
GDH-3Mid (5'-GGGATATGACCTACATTTGCTGGC-3') and
[0057]GDH-5Mid (5'-TGTCCATCAGCRTCATTTACAAYCTCAG-3'), which initiated directed DNA synthesis respectively in the direction of the 3' end (GDH-3Mid) and 5' end (GDH-5Mid) of the GDH gene. The amplicons obtained in this way contained the as yet missing portions of the coding sequence, as emerged by cloning into the vector pCR2.1 and subsequent sequencing. The DNA sequences which were now completely available were used anew to develop primers which made it possible for the coding sequence from start codon to stop codon to be cloned directly. The primers were chosen in this case so that cloning into the vector pASK-IBA3 is possible in accordance with IBA's instructions. For this purpose, a BsaI cleavage site is positioned directly in front of the coding sequence (in the 5'-binding primer) and one was positioned directly thereafter (in the 3'-binding primer) so that directed ligation of the BsaI-cut PCR product into the BsaI open vector pASK-IBA3 is possible. The 5' primer used wasGDH-U3(5'-TGGTAGGTCTCAAATGAATAAACATTTATTGGCTAAAATTAC-3'), and the 3' primers used were GDH-L3(5'-ATGGTAGGTCTCAGCGCTCTGAGCTTTATATGTAAACCTAATCAAAG-3'; for the GDH from clone PT15) and GDH-L4
(5'-ATGGTAGGTCTCAGCGCTCTGAGCTTTATATGTAAATCTAATCAGAG-3';
[0058]for all other clones). The resulting plasmidas were referred to as pA13-X. Instead of "X", the strain number of the clone from which the genomic DNA was isolated is inserted. For the purposes of comparison, the sPQQGDH gene from the wild-type strain LMD79.41 was cloned in the same way. However, the primers used for this were derived from the published sequence. This plasmid was referred to as pAI3-wt.
[0059]The described plasmids were transferred into the E. coli strain DH5α (from Invitrogen) by chemical transformation. The bacterial strains obtained in this way were referred to as DH5α::pAI3-X. The plasmids were transferred in a similar way into a further E. coli strain, W3110 (ATCC 27325). These strains were then referred to as W3110::pAI3-X.
Example 5
Recombinant Preparation of sPQQGDH with E. coli
[0060]The preculture was prepared as follows. 0.1 ml of a glycerol stock of DH5α::pAI3-KOZ65 cells was added to 2 ml of LB medium (50 μg/ml ampicillin) and shaken at 37° C. and 225 rpm overnight. For the main culture, 11 of TB medium (50 μg/ml ampicillin) was inoculated with the fully grown preculture. The culture was shaken at 37° C. and 225 rpm until an OD of about 1 was reached. The main culture was then induced with an anhydrotetracycline (AHT) stock solution. The inducer was dissolved in DMF for this purpose. The final concentration of AHT in the culture was 0.2 μg/ml, and induction took place at 27° C. and 225 rpm for 24 hours. The OD of the main culture was then 4.2.
[0061]The cells were harvested by centrifugation of the main culture at 3220×g and 4° C. for 15 min. The cell pellets were resuspended in 40 ml (= 1/25 of the total volume) in 75 mM Tris-HCl of pH 8.0, and disrupted using a French press. This is done by treating the complete cell suspension with the French press twice. The lysate, which may be cloudy due to inclusion bodies and cell detritus, was centrifuged at 48 745×g and 4° C. for 10 min. The protein content and the activity of the supernatant were assayed. The protein content was 9.25 mg/ml, and the activity was 1.4 kU/ml. Based on the original culture, 56 kU were obtained per 1 of culture.
Example 6
Purification of the Recombinant sPQQGDH
[0062]11.2 ml of the supernatant from Example 5 were separated by chromatography on a cation exchanger (Toyopearl CM-650M, from TOSOH BIOSEP GmbH) at 4° C. An XK 50/20 column (from Amersham Pharmacia Biotech) with a column bed of about 130 ml is used for this purpose; the flow rate was 8 ml/min. The column was equilibrated with 10 column volume of 10 mM K-MOPS of pH 8.0+1 mM CaCl2, after which the sample is loaded. The column was washed with 4 column volume of 10 mM K-MOPS of pH 8.0+1 mM CaCl2, and it was then eluted with a linear salt gradient from 0 to 0.4 N NaCl in 10 mM K-MOPS of pH 8.0 (in each case including 1 mM CaCl2), collecting the eluate in fractions. Regeneration of the column takes place with 3 column volume of 1 N NaCl+1 mM CaCl2.
[0063]The eluate fractions were assayed for activity in 96-well microtitre plates with flat base in order to find the active fractions. The colour solution had the following composition:
0.2 mM PMS (phenazine methosulphate in H2O)+0.22 mM NTB (nitrotetrazolium blue in H2O)+3 μM PQQ Na salt (in DMSO)in 20 mM Tris/HCl of pH 7.5 with 2% glucose
[0064]The assay was carried out as follows: in each case 90 μl of sample were introduced into a microtitre plate, and 110 μl of colour solution were added to each; the colour reaction can usually be observed after only one minute. On the basis of the results in the online UV chromatogram and in the activity assay, the active fractions are combined and the pool is concentrated where appropriate by ultrafiltration (30 000 MWCO). A protein determination and a quantitative activity assay follow (see Example 9).
TABLE-US-00006 TABLE 2 Purification of the sPQQGDH from E. coli DH5α::pAI3-KOZ65 Total Total Specific Vol. protein activity activity [ml] [mg] [U] [U/mg] Yield [%] Sample loaded onto 11.2 103.6 15 680 151 100 column Active fractions 4.45 13.50 15 085.5 1117 96
[0065]Only one active band was detected in an SDS gel and in a native gel. The protein was thus pure. Based on the original culture, 48.2 mg of pure protein were obtained per 1 of culture.
Example 7
Preparation of Recombinant sPQQGDH from W3110 Strains and Testing of the Thermal Stability
[0066]For comparison of the thermal stability of recombinant sPQQGDH from LMD79.41 (plasmid pAI3-wt) and the sPQQGDH from KOZ65 (plasmid pAI3-KOZ65), the strains W3110::pAI3-wt and W3110::pAI3-KOZ65 were cultured in 200 ml of TB medium with 100 μg/ml ampicillin. After the cultures had reached an OD600 of 3, the bacteria were centrifuged at 4600 rpm for 10', and the pellets were taken up in each case in 25 ml of fresh TB medium with 100 μg/ml ampicillin. AHT was added to a concentration of 2 μg/ml for induction, and the cells were shaken at 22° C. for 6 h. The cells were then pelleted anew, and the pellets were stored at -80° C. until processed further. The frozen cells were resuspended in each case in 25 ml of MOPS buffer (10 mM MOPS pH 8, 2.5 mM CaCl2, 0.05% Triton X-100) and disrupted by ultrasound treatment until the suspension became distinctly clearer. Cell residues were removed at 20 000 rpm and 4° C. for 30 min, and the supernatant was purified on a Toyopearl CM-650 M column (20 ml bed volume). For this purpose, after the sample loading the column was washed with 50 ml of MOPS buffer (see above) and then bound protein was eluted with a gradient from 0 to 0.6 mM NaCl in MOPS buffer. Fractions with GDH activity were pooled, and the activity of the pool was determined (see Example 9).
[0067]The thermal stability of both enzyme preparations was determined by dilution with 50 mM Pipes, pH 6.5 with 1 mM CaCl2, 0.1% Triton X-100, 0.1% BSA and 5 μM PQQ to adjust to a solution of 20 U/ml. Aliquots of this solution were incubated in parallel at 4° C., 50° C., 57° C. and 64° C. for 60 min. The remaining activity is then found, based on the value at 4° C. as 100%, as follows (as triplicates; see Example 9):
TABLE-US-00007 Strain 50° C. 57° C. 64° C. W3110::pAI3-wt 95.1 ± 2.9 20.7 ± 0.8 3.4 ± 0.0 W3110::pAI3-KOZ65 95.1 ± 1.6 56.8 ± 3.6 8.7 ± 0.3
Example 8
Preparation by High Cell-Density Fermentation
[0068]The fermentation was carried out in a 10 litre steel fermenter (BIOSTAT C) from Braun.
[0069]5 litres of (modified) Riesenberg medium were employed for this purpose
TABLE-US-00008 KH2PO4 13.3 g (NH4)2HPO4 4.0 g Citric acid 1.7 g Magnesium 1.2 g sulphate × 7H2O Thiamine 0.5 g Tryptone 1.2 g Yeast extract 2.4 g Trace element solution 50 ml Dist. water ad 880 ml Glucose 5 g in 100 ml of dist. water (sterilized separately) Ampicillin 100 mg (separately dissolved in 20 ml of dist. water and sterilized by filtration)
[0070]Trace Element Solution
TABLE-US-00009 Titriplex III 0.84 g Fe(III) citrate 6.00 g MnCl2 × 4H2O 1.50 g ZnCl2 × 2H2O 0.80 g H3BO3 0.30 g Na2MoO4 × 2H2O 0.25 g CoCl2 × 6H2O 0.25 g CuCl2 × 2H2O 0.15 g Dist. water ad 1000 ml
[0071]The pH was adjusted after addition of all the medium ingredients to 6.80 with 5 N NaOH.
[0072]The nutrient solution was inoculated with 100 ml of a preculture (LB medium with 100 mg/l ampicillin) grown at 37° C. overnight.
[0073]The OD after the inoculation was 0.066. The aeration rate was adjusted to 2 l/min, the stirrer speed to 500 rpm and the temperature to 37° C. The pH was adjusted to pH 7.25 with 5 N NH3 and kept constant throughout the fermentation.
[0074]After growth for 12 hours, the PO2 had fallen below 10% and most of the glucose had been consumed. After growth for 15 hours, the OD600 was 14.2 and the dry matter was 6.2 μl. Feeding was started at this time. The feed solution consisted of:
TABLE-US-00010 Glucose 700 g Magnesium sulphate × 7H2O 28 g Thiamine 0.5 g Tryptone 1.2 g Yeast extract 2.4 g Dist. water ad 1000 ml
[0075]The solution was sterilized by filtration. It was fed to the fermenter by a tubing pump. The pump delivery was controlled via the target pO2, which was set at 20%. Control took place in such a way that the pump is switched on at a pO2 of >20%, and new glucose is fed in. The oxygen consumption which starts up then causes the pO2 to fall again. The pump is switched off if the pO2 falls below 20%. The stirrer speed and aeration rate were not changed. The OD600 after growth for 64 hours was 50.2. 5 litres of double concentrated TB medium were then fed into the fermenter, the temperature was reduced to 22° C., and the aeration rate was set at 4 l/min.
[0076]The stirrer speed was raised to 700 rpm. The culture was then induced with 10 ml of anhydrotetracycline solution (2 mg/ml in DMF) for 6 hours. A sample was then taken to determine the activity. The activity assay revealed a value of 223 U/ml of culture liquid. Over 220 mg of pure enzyme were obtained per ml of culture.
Example 9
Procedure for the Activity Assay
[0077]The measurements were carried out in an "Ultraspec 2000" photometer. The following solutions are employed for this purpose:
TABLE-US-00011 PIPES: 50 mM pH 6.5 incl. 2.2% Triton X-100 Glucose: 1 M in H2O PMS: 3 mM phenazine methosulphate in H2O NTB: 6.6 mM nitrotetrazolium blue in H2O CaCl2: 1 M in H2O PQQ: 3 mM in DMSO EDB *: 50 mM PIPES pH 6.5 incl. 0.1% Triton X-100, 1 mM CaCl2, 0.1% BSA, 6 μM PQQ. EDB = enzyme dilution buffer
[0078]The working reagent consists of the following components:
25.5 ml of PIPES incl. 2.2% Triton X-100
0.9 ml of Glucose
2.0 ml of PMS
1.0 ml of NTB
[0079]The EDB and the working reagent should if possible be made up freshly.
[0080]Procedure for the Activity Determination:
[0081]Introduce 20 μl of the chosen sample dilution into microcuvettes (20 μl of EDB instead of the sample solution are added as zero value),
add 600 μl of working reagent and immediately start the measurement at 570 nm for 3 minutes.
[0082]Calculation of the Activity:
[0083]The change in absorption is measured as change in extinction/min; one unit of GDH generates 0.5 μmol of formazan per minute. The following formula applies:
U/ml=change in extinction/min×1.54×dilution factor ε=40 200 M-1 cm-1
Sequence CWU
1
2811437DNAAcinetobacter sp. 1atgaataaac atttattggc taaaattgct ttattaagcg
ctgttcagct agttacactc 60tcagcatttg ctgatgttcc tctaactcca tctcaatttg
ctaaagcgaa atcagagaac 120tttgacaaga aagttattct atctaatcta aataagccgc
atgctttgtt atggggacca 180gataatcaaa tttggttaac tgagcgagca acaggtaaga
ttctaagagt taatccagag 240tcgggtagtg taaaaacagt ttttcaggta ccagagattg
tcaatgatgc tgatgggcag 300aatggtttat taggttttgc cttccatcct gattttaaaa
ataatcctta tatctatatt 360tcaggtacat ttaaaaatcc gaaatctaca gataaagaat
taccgaacca aacgattatt 420cgtcgttata cctataataa atcaacagat acgctcgaga
agccagtcga tttattagca 480ggattacctt catcaaaaga ccatcagtca ggtcgtcttg
tcattgggcc agatcaaaag 540atttattata cgattggtga ccaagggcgt aaccagcttg
cttatttgtt cttgccaaat 600caagcacaac atacgccaac tcaacaagaa ctgaatggta
aagactatca cacctatatg 660ggtaaagtac tacgcttaaa tcttgatgga agtattccaa
aggataatcc aagttttaac 720ggggtggtta gccatattta tacacttgga catcgtaatc
cgcagggctt agcattcact 780ccaaatggta aattattgca gtctgaacaa ggcccaaact
ctgacgatga aattaacctc 840attgtcaaag gtggcaatta tggttggccg aatgtagcag
gttataaaga tgatagtggc 900tatgcttatg caaattattc agcagcagcc aataagtcaa
ttaaggattt agctcaaaat 960ggagtaaaag tagccgcagg ggtccctgtg acgaaagaat
ctgaatggac tggtaaaaac 1020tttgtcccac cattaaaaac tttatatacc gttcaagata
cctacaacta taacgatcca 1080acttgtggag agatgaccta catttgctgg ccaacagttg
caccgtcatc tgcctatgtc 1140tataagggcg gtaaaaaagc aattactggt tgggaaaata
cattattggt tccatcttta 1200aaacgtggtg tcattttccg tattaagtta gatccaactt
atagcactac ttatgatgac 1260gctgtaccga tgtttaagag caacaaccgt tatcgtgatg
tgattgcaag tccagatggg 1320aatgtcttat atgtattaac tgatactgcc ggaaatgtcc
aaaaagatga tggctcagta 1380acaaatacat tagaaaaccc aggatctctc attaagttca
cctataaggc taagtaa 14372478PRTAcinetobacter sp. 2Met Asn Lys His Leu
Leu Ala Lys Ile Ala Leu Leu Ser Ala Val Gln1 5
10 15Leu Val Thr Leu Ser Ala Phe Ala Asp Val Pro
Leu Thr Pro Ser Gln 20 25
30Phe Ala Lys Ala Lys Ser Glu Asn Phe Asp Lys Lys Val Ile Leu Ser
35 40 45Asn Leu Asn Lys Pro His Ala Leu
Leu Trp Gly Pro Asp Asn Gln Ile 50 55
60Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro Glu65
70 75 80Ser Gly Ser Val Lys
Thr Val Phe Gln Val Pro Glu Ile Val Asn Asp 85
90 95Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala
Phe His Pro Asp Phe 100 105
110Lys Asn Asn Pro Tyr Ile Tyr Ile Ser Gly Thr Phe Lys Asn Pro Lys
115 120 125Ser Thr Asp Lys Glu Leu Pro
Asn Gln Thr Ile Ile Arg Arg Tyr Thr 130 135
140Tyr Asn Lys Ser Thr Asp Thr Leu Glu Lys Pro Val Asp Leu Leu
Ala145 150 155 160Gly Leu
Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile Gly
165 170 175Pro Asp Gln Lys Ile Tyr Tyr
Thr Ile Gly Asp Gln Gly Arg Asn Gln 180 185
190Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro
Thr Gln 195 200 205Gln Glu Leu Asn
Gly Lys Asp Tyr His Thr Tyr Met Gly Lys Val Leu 210
215 220Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn
Pro Ser Phe Asn225 230 235
240Gly Val Val Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln Gly
245 250 255Leu Ala Phe Thr Pro
Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly Pro 260
265 270Asn Ser Asp Asp Glu Ile Asn Leu Ile Val Lys Gly
Gly Asn Tyr Gly 275 280 285Trp Pro
Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr Ala 290
295 300Asn Tyr Ser Ala Ala Ala Asn Lys Ser Ile Lys
Asp Leu Ala Gln Asn305 310 315
320Gly Val Lys Val Ala Ala Gly Val Pro Val Thr Lys Glu Ser Glu Trp
325 330 335Thr Gly Lys Asn
Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr Val Gln 340
345 350Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly
Glu Met Thr Tyr Ile 355 360 365Cys
Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys Gly Gly 370
375 380Lys Lys Ala Ile Thr Gly Trp Glu Asn Thr
Leu Leu Val Pro Ser Leu385 390 395
400Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr Ser
Thr 405 410 415Thr Tyr Asp
Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg Tyr Arg 420
425 430Asp Val Ile Ala Ser Pro Asp Gly Asn Val
Leu Tyr Val Leu Thr Asp 435 440
445Thr Ala Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr Asn Thr Leu 450
455 460Glu Asn Pro Gly Ser Leu Ile Lys
Phe Thr Tyr Lys Ala Lys465 470
47531443DNAAcinetobacter sp. 3atgaataaac atttattagc aaaaatcact cttttaggtg
ctgcacaact atttacgttt 60catacggcat ttgcagatat acctctgaca cctgctcagt
tcgcaaaagc gaaaacagaa 120aattttgata aaaaagtgat tctgtccaat ttaaataaac
cacatgcttt gttatggggg 180ccagataatc aaatttggtt aaccgaacgt gcaactggca
aaattttaag agtaaatcct 240gtatctggta gcgcgaaaac agtatttcag gttcctgaaa
ttgtgagtga tgctgatggg 300caaaatggtt tgttaggttt tgcttttcat cctgacttta
aacataaccc ttatatctat 360atttcaggca cttttaaaaa tccaaaatct acagataaag
agttacctaa tcagacaatt 420attcgtagat atacctataa taaaactaca gatacatttg
aaaagcctat tgatttgatt 480gcaggtttac cgtcatcaaa agatcatcag tctggtcgtc
tcgttattgg tccagaccaa 540aaaatctact atacgattgg tgaccaaggt cgtaatcagt
tagcttatct attcttatcg 600aatcaggcac agcatactcc gactcagcaa gagctcaata
gtaaagacta ccatacatat 660atgggtaaag tattacgctt aaatctggac ggcagtatac
ctaaagacaa cccaagcttt 720aacggcgtag tgagtcatat ctacacttta gggcaccgta
atccacaagg tttagcattt 780gccccaaatg gaaagctttt acaatctgag caagggccaa
attctgatga tgaaattaac 840cttgtattaa aaggtggtaa ctatggctgg ccaaatgtag
ctggttataa agatgacagt 900ggttatgcct atgcaaacta ttcggcagca accaataaat
cacaaattaa agatttagct 960caaaacggga taaaagtagc aacaggtgtt cctgtgacta
aagagtctga atggactggt 1020aaaaactttg tgccaccttt gaaaacttta tatacggtac
aagataccta taactataat 1080gaccctactt gtggtgagat ggcatatatt tgctggccaa
cggttgcacc gtcatcggca 1140tatgtatata cgggaggcaa aaaagcgatt ccagggtggg
aaaatacatt attggtccca 1200tctttaaaac gtggggtgat tttccgtatt aaattggacc
cgacatatag cacgactttg 1260gatgatgcta tcccaatgtt taaaagcaat aaccgttatc
gtgatgtcat cgctagtcca 1320gaaggtaata ccttatatgt gctgactgat acagcgggaa
atgtacaaaa agatgatggt 1380tcagtcactc atactttaga gaatcccggt tctctcatta
aatttacata taacggtaag 1440taa
14434480PRTAcinetobacter sp. 4Met Asn Lys His Leu
Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln1 5
10 15Leu Phe Thr Phe His Thr Ala Phe Ala Asp Ile
Pro Leu Thr Pro Ala 20 25
30Gln Phe Ala Lys Ala Lys Thr Glu Asn Phe Asp Lys Lys Val Ile Leu
35 40 45Ser Asn Leu Asn Lys Pro His Ala
Leu Leu Trp Gly Pro Asp Asn Gln 50 55
60Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro65
70 75 80Val Ser Gly Ser Ala
Lys Thr Val Phe Gln Val Pro Glu Ile Val Ser 85
90 95Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe
Ala Phe His Pro Asp 100 105
110Phe Lys His Asn Pro Tyr Ile Tyr Ile Ser Gly Thr Phe Lys Asn Pro
115 120 125Lys Ser Thr Asp Lys Glu Leu
Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130 135
140Thr Tyr Asn Lys Thr Thr Asp Thr Phe Glu Lys Pro Ile Asp Leu
Ile145 150 155 160Ala Gly
Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile
165 170 175Gly Pro Asp Gln Lys Ile Tyr
Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180 185
190Gln Leu Ala Tyr Leu Phe Leu Ser Asn Gln Ala Gln His Thr
Pro Thr 195 200 205Gln Gln Glu Leu
Asn Ser Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210
215 220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp
Asn Pro Ser Phe225 230 235
240Asn Gly Val Val Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln
245 250 255Gly Leu Ala Phe Ala
Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260
265 270Pro Asn Ser Asp Asp Glu Ile Asn Leu Val Leu Lys
Gly Gly Asn Tyr 275 280 285Gly Trp
Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290
295 300Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser Gln
Ile Lys Asp Leu Ala305 310 315
320Gln Asn Gly Ile Lys Val Ala Thr Gly Val Pro Val Thr Lys Glu Ser
325 330 335Glu Trp Thr Gly
Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340
345 350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr
Cys Gly Glu Met Ala 355 360 365Tyr
Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Thr 370
375 380Gly Gly Lys Lys Ala Ile Pro Gly Trp Glu
Asn Thr Leu Leu Val Pro385 390 395
400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr
Tyr 405 410 415Ser Thr Thr
Leu Asp Asp Ala Ile Pro Met Phe Lys Ser Asn Asn Arg 420
425 430Tyr Arg Asp Val Ile Ala Ser Pro Glu Gly
Asn Thr Leu Tyr Val Leu 435 440
445Thr Asp Thr Ala Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr His 450
455 460Thr Leu Glu Asn Pro Gly Ser Leu
Ile Lys Phe Thr Tyr Asn Gly Lys465 470
475 48051443DNAAcinetobacter sp. 5atgaataaac atttattggc
taaaattact ttattaggtg ctgctcagct acttacgctc 60aattcagcat ttgctgatgt
ccctcttact ccatctcaat ttgctaaagc gaaaacagaa 120agctttgata agaaagttct
tctatctaat ttaaataagc cacatgcttt gttgtggggg 180cctgataatc aaatttggtt
aacggagcgg gcaacaggta agattctaag agtgaatcca 240gagtcgggca gtgtgaaaac
agtttttcag gttcctgaga ttgtaaatga tgctgatgga 300caaaacggtt tattaggttt
tgcttttcat cctgacttta aacataatcc ttatatctat 360gtttcaggta catttaaaaa
tccgaaatct acagataaag aattaccgaa tcaaactatt 420attcgtcgat atacctataa
caaagcaaca gatactcttg agaaaccagt agatttatta 480gcaggattac cttcatcgaa
agaccatcag tcgggtcgcc ttgttattgg tccagaccaa 540aagatttact atacgattgg
tgatcaggga cgtaaccagc tggcttattt attcttacca 600aatcaagcac agcatacgcc
gactcaacag gaactgagcg gcaaagacta tcatacttat 660atgggtaaag tattacgctt
aaatctggat ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa ttagccatat
ttatacgctc ggtcatcgta acccacaggg cttggcattt 780actccaaatg gtaaactgtt
gcaatctgaa caaggtccaa actctgatga tgaaattaat 840ctcattgtta aaggtggtaa
ctatggctgg ccaaatgtag ctggttataa agatgacagt 900ggttatgcct atgcaaatta
ttcggcagca accaataagt cacaaattaa agatttaggg 960caaaatggag taaaagtagc
ggcaggtgta cctgtgatga aagagtctga atggagtggt 1020aaaaactttg taccgccgtt
aaaaacttta tataccgtcc aagataccta taactataat 1080gacccaactt gtggggatat
gacctacatt tgctggccaa cagttgcgcc atcatctgct 1140tatgtctata aggggggcaa
aaaagcaatt tctggttggg aaaatacatt attggttcca 1200tctttaaagc gtggtgttat
tttccgtatt aagttagatc caacttacag tgctacttat 1260gatgatgctg taccgatgtt
taagagcaat aaccgttatc gtgacgtgat tgcaagtcca 1320gatgggaatg ttttatatgt
attgactgat acttccggaa atgtccaaaa ggatgatggc 1380tctgtaacga atacattaga
aaatccagga tctctgatta gatttacata taaagctcag 1440taa
14436480PRTAcinetobacter sp.
6Met Asn Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln1
5 10 15Leu Leu Thr Leu Asn Ser
Ala Phe Ala Asp Val Pro Leu Thr Pro Ser 20 25
30Gln Phe Ala Lys Ala Lys Thr Glu Ser Phe Asp Lys Lys
Val Leu Leu 35 40 45Ser Asn Leu
Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50
55 60Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu
Arg Val Asn Pro65 70 75
80Glu Ser Gly Ser Val Lys Thr Val Phe Gln Val Pro Glu Ile Val Asn
85 90 95Asp Ala Asp Gly Gln Asn
Gly Leu Leu Gly Phe Ala Phe His Pro Asp 100
105 110Phe Lys His Asn Pro Tyr Ile Tyr Val Ser Gly Thr
Phe Lys Asn Pro 115 120 125Lys Ser
Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130
135 140Thr Tyr Asn Lys Ala Thr Asp Thr Leu Glu Lys
Pro Val Asp Leu Leu145 150 155
160Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile
165 170 175Gly Pro Asp Gln
Lys Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180
185 190Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala
Gln His Thr Pro Thr 195 200 205Gln
Gln Glu Leu Ser Gly Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210
215 220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro
Lys Asp Asn Pro Ser Phe225 230 235
240Asn Gly Val Ile Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro
Gln 245 250 255Gly Leu Ala
Phe Thr Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260
265 270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile
Val Lys Gly Gly Asn Tyr 275 280
285Gly Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290
295 300Ala Asn Tyr Ser Ala Ala Thr Asn
Lys Ser Gln Ile Lys Asp Leu Gly305 310
315 320Gln Asn Gly Val Lys Val Ala Ala Gly Val Pro Val
Met Lys Glu Ser 325 330
335Glu Trp Ser Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr
340 345 350Val Gln Asp Thr Tyr Asn
Tyr Asn Asp Pro Thr Cys Gly Asp Met Thr 355 360
365Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val
Tyr Lys 370 375 380Gly Gly Lys Lys Ala
Ile Ser Gly Trp Glu Asn Thr Leu Leu Val Pro385 390
395 400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile
Lys Leu Asp Pro Thr Tyr 405 410
415Ser Ala Thr Tyr Asp Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg
420 425 430Tyr Arg Asp Val Ile
Ala Ser Pro Asp Gly Asn Val Leu Tyr Val Leu 435
440 445Thr Asp Thr Ser Gly Asn Val Gln Lys Asp Asp Gly
Ser Val Thr Asn 450 455 460Thr Leu Glu
Asn Pro Gly Ser Leu Ile Arg Phe Thr Tyr Lys Ala Gln465
470 475 48071443DNAAcinetobacter sp.
7atgaataaac atttattggc taaaattact ttattaggtg ctgctcagct acttacgctc
60aattcagcat ttgctgatgt ccctcttaca ccatctcaat ttgctaaagc gaaaacagaa
120agctttgata agaaagttct tctatctaat ttaaataagc cacatgcttt gttgtgggga
180cctgataatc aaatttggtt aacggagcgg gcaacaggta agattctaag agttaatcca
240gagtcgggca gtgtaaaaac agtttttcag gttcctgaga ttgtaaatga tgctgatgga
300caaaacggtt tattgggttt tgcctttcat cctgacttta aaaataatcc ttatatctat
360gtttcaggta catttaaaaa tccgaaatct acagataaag aattaccgaa tcaaactatt
420atccgtcgat atacctataa caaggcaaca gatacccttg agaaaccagt agatttattg
480gcaggattac cttcatcgaa agaccatcag tcgggtcgtc ttgtgattgg tccagaccaa
540aagatttact atacgattgg tgatcaggga cgtaaccagc tggcttattt attcttacca
600aatcaagcac agcatacgcc gactcaacag gaactgagcg gcaaagacta tcatacctat
660atgggtaaag tattgcgctt aaatctggat ggaagtattc caaaagataa tccaagcttt
720aacggtgtaa ttagccatat ttatacgctc ggtcatcgta acccacaggg cttggcattt
780actccaaatg gtaaactgtt gcaatctgaa cagggtccaa actctgatga tgaaattaac
840ctcattgtca aaggtggtaa ctatggctgg ccaaatgtag cgggttataa agatgatagt
900ggttatgcct atgcaaatta ttcggcagca agcaataaag cacaaattaa agatttagga
960caaaatggtt taaaagtggc ggcaggtgta cctgtgatga aagagtctga atggactggt
1020aaaaactttg taccgccgtt aaaaacttta tataccgtcc aagataccta taactataat
1080gacccaactt gtggggatat gacctacatt tgctggccaa cggttgcgcc gtcatctgct
1140tatgtctata agggaggcaa aaaagcaatt tctggttggg aaaatacatt attggttcca
1200tctttaaagc gcggtgttat tttccgtatt aagctagatc caacttacag tactacttat
1260gatgatgctg tgccgatgtt taagagcaac aatcgttatc gtgacgtgat tgcaagtcca
1320gatgggaatg ttttatatgt attgactgat acttccggaa atgtccaaaa agatgatggt
1380tctgtaacga atacattaga aaacccagga tctctgatta gatttacata taaagctcag
1440taa
14438480PRTAcinetobacter sp. 8Met Asn Lys His Leu Leu Ala Lys Ile Thr Leu
Leu Gly Ala Ala Gln1 5 10
15Leu Leu Thr Leu Asn Ser Ala Phe Ala Asp Val Pro Leu Thr Pro Ser
20 25 30Gln Phe Ala Lys Ala Lys Thr
Glu Ser Phe Asp Lys Lys Val Leu Leu 35 40
45Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp Asn
Gln 50 55 60Ile Trp Leu Thr Glu Arg
Ala Thr Gly Lys Ile Leu Arg Val Asn Pro65 70
75 80Glu Ser Gly Ser Val Lys Thr Val Phe Gln Val
Pro Glu Ile Val Asn 85 90
95Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala Phe His Pro Asp
100 105 110Phe Lys Asn Asn Pro Tyr
Ile Tyr Val Ser Gly Thr Phe Lys Asn Pro 115 120
125Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg
Arg Tyr 130 135 140Thr Tyr Asn Lys Ala
Thr Asp Thr Leu Glu Lys Pro Val Asp Leu Leu145 150
155 160Ala Gly Leu Pro Ser Ser Lys Asp His Gln
Ser Gly Arg Leu Val Ile 165 170
175Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn
180 185 190Gln Leu Ala Tyr Leu
Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr 195
200 205Gln Gln Glu Leu Ser Gly Lys Asp Tyr His Thr Tyr
Met Gly Lys Val 210 215 220Leu Arg Leu
Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe225
230 235 240Asn Gly Val Ile Ser His Ile
Tyr Thr Leu Gly His Arg Asn Pro Gln 245
250 255Gly Leu Ala Phe Thr Pro Asn Gly Lys Leu Leu Gln
Ser Glu Gln Gly 260 265 270Pro
Asn Ser Asp Asp Glu Ile Asn Leu Ile Val Lys Gly Gly Asn Tyr 275
280 285Gly Trp Pro Asn Val Ala Gly Tyr Lys
Asp Asp Ser Gly Tyr Ala Tyr 290 295
300Ala Asn Tyr Ser Ala Ala Ser Asn Lys Ala Gln Ile Lys Asp Leu Gly305
310 315 320Gln Asn Gly Leu
Lys Val Ala Ala Gly Val Pro Val Met Lys Glu Ser 325
330 335Glu Trp Thr Gly Lys Asn Phe Val Pro Pro
Leu Lys Thr Leu Tyr Thr 340 345
350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Asp Met Thr
355 360 365Tyr Ile Cys Trp Pro Thr Val
Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370 375
380Gly Gly Lys Lys Ala Ile Ser Gly Trp Glu Asn Thr Leu Leu Val
Pro385 390 395 400Ser Leu
Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr
405 410 415Ser Thr Thr Tyr Asp Asp Ala
Val Pro Met Phe Lys Ser Asn Asn Arg 420 425
430Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly Asn Val Leu Tyr
Val Leu 435 440 445Thr Asp Thr Ser
Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr Asn 450
455 460Thr Leu Glu Asn Pro Gly Ser Leu Ile Arg Phe Thr
Tyr Lys Ala Gln465 470 475
48091443DNAAcinetobacter sp. 9atgaataaac atttattggc taaaattact
ttattaggtg ctgctcagct acttacactc 60aattcagcat ttgctgatgt ccctcttact
ccatctcaat ttgctaaagc gaaaacagaa 120agctttgata agaaagttct tctatctaat
ttaaataagc cgcatgcttt gttgtgggga 180cctgataatc aaatttggtt aacagagcgg
gcaacaggta agattctaag agttaaccct 240gaatcaggca gtgtaaaaac agtttttcag
gttcctgaga ttgtaaatga tgctgatgga 300caaaacgggt tattgggttt tgcctttcat
cctgacttta aaaataatcc ttatatctat 360gtttcaggta catttaaaaa tccgaaatct
acagataaag aattaccgaa tcaaactatt 420atccgtcgat atacctataa caaagcaaca
gatactcttg agaaaccagt agatttatta 480gcaggattac cttcatcgaa agaccatcag
tcgggtcgcc ttgtgattgg tccagaccaa 540aaaatttact atacgattgg tgatcagggg
cgtaaccagc ttgcttattt attcttacca 600aatcaggcac aacatacgcc gactcaacag
gaactgagcg gcaaagacta tcatacctat 660atgggtaaag tattacgctt aaatctggat
ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa ttagccatat ttatacgctc
ggtcatcgta acccacaggg cttggcattt 780actccaaatg gtaaactgtt gcaatctgaa
cagggtccaa actctgatga tgaaattaac 840ctcattgtta aaggtggtaa ctatggctgg
ccaaatgcgg cgggttataa agatgacagt 900ggttatgcct atgcaaatta ttcggcagca
accaataagt cacaaattaa agatttaggg 960caaaatggag taaaagtagc agctggcgtt
ccagtgacta aagagtctga atggactggt 1020aaaaactttg taccgccgtt aaaaacttta
tataccgtcc aagataccta taactataat 1080gacccaacct gtggggagat gacctacatt
tgctggccaa cagttgcgcc atcatctgct 1140tatgtctata agggaggcaa aaaagcaatt
tctggttggg aaaatacctt attggttcca 1200tctttaaagc gtggtgttat ttttcgtatt
aagctagatc caacttacag tgctacttat 1260gatgatgctg tgccgatgtt taagagcaac
aatcgttatc gtgacgtgat tgcaagtcca 1320gatggaaatg ttttatatgt attgactgat
acttccggaa atgtccaaaa agatgatggt 1380tctgtaacga atacattaga aaacccagga
tctctgatta gatttacata taaagctcag 1440taa
144310480PRTAcinetobacter sp. 10Met Asn
Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln1 5
10 15Leu Leu Thr Leu Asn Ser Ala Phe
Ala Asp Val Pro Leu Thr Pro Ser 20 25
30Gln Phe Ala Lys Ala Lys Thr Glu Ser Phe Asp Lys Lys Val Leu
Leu 35 40 45Ser Asn Leu Asn Lys
Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50 55
60Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val
Asn Pro65 70 75 80Glu
Ser Gly Ser Val Lys Thr Val Phe Gln Val Pro Glu Ile Val Asn
85 90 95Asp Ala Asp Gly Gln Asn Gly
Leu Leu Gly Phe Ala Phe His Pro Asp 100 105
110Phe Lys Asn Asn Pro Tyr Ile Tyr Val Ser Gly Thr Phe Lys
Asn Pro 115 120 125Lys Ser Thr Asp
Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130
135 140Thr Tyr Asn Lys Ala Thr Asp Thr Leu Glu Lys Pro
Val Asp Leu Leu145 150 155
160Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile
165 170 175Gly Pro Asp Gln Lys
Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180
185 190Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln
His Thr Pro Thr 195 200 205Gln Gln
Glu Leu Ser Gly Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210
215 220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys
Asp Asn Pro Ser Phe225 230 235
240Asn Gly Val Ile Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln
245 250 255Gly Leu Ala Phe
Thr Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260
265 270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val
Lys Gly Gly Asn Tyr 275 280 285Gly
Trp Pro Asn Ala Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290
295 300Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser
Gln Ile Lys Asp Leu Gly305 310 315
320Gln Asn Gly Val Lys Val Ala Ala Gly Val Pro Val Thr Lys Glu
Ser 325 330 335Glu Trp Thr
Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340
345 350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro
Thr Cys Gly Glu Met Thr 355 360
365Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly
Trp Glu Asn Thr Leu Leu Val Pro385 390
395 400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu
Asp Pro Thr Tyr 405 410
415Ser Ala Thr Tyr Asp Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg
420 425 430Tyr Arg Asp Val Ile Ala
Ser Pro Asp Gly Asn Val Leu Tyr Val Leu 435 440
445Thr Asp Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val
Thr Asn 450 455 460Thr Leu Glu Asn Pro
Gly Ser Leu Ile Arg Phe Thr Tyr Lys Ala Gln465 470
475 480111443DNAAcinetobacter sp. 11atgaataaac
atttattggc taaaattact ttattaggtg ctgctcagct acttacgctc 60aattcagcat
ttgctgatgt ccctcttaca ccatctcaat ttgctaaagc gaaaacagaa 120agctttgata
agaaagttct tctatctaat ttaaataagc cacatgcttt gttgtgggga 180cctgataatc
aaatttggtt aacggagcgg gcaacaggta agattctaag agttaatcca 240gagtcgggca
gtgtaaaaac agtttttcag gttcctgaga ttgtaaatga tgctgatgga 300caaaacggtt
tattgggttt tgcctttcat cctgacttta aaaataatcc ttatatctat 360gtttcaggta
catttaaaaa tccgaaatct acagataaag aattaccgaa tcaaactatt 420atccgtcgat
atacctataa caaggcaaca gatacccttg agaaaccagt agatttattg 480gcaggattac
cttcatcgaa agaccatcag tcgggtcgtc ttgtgattgg tccagaccaa 540aagatttact
atacgattgg tgatcaggga cgtaaccagc tggcttattt attcttacca 600aatcaagcac
agcatacgcc gactcaacag gaactgagcg gcaaagacta tcatacctat 660atgggtaaag
tattgcgctt aaatctggat ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa
ttagccatat ttatacgctc ggtcatcgta acccacaggg cttggcattt 780actccaaatg
gtaaactgtt gcaatctgaa cagggtccaa actctgatga tgaaattaac 840ctcattgtca
aaggtggtaa ctatggctgg ccaaatgtag cgggttataa agatgatagt 900ggttatgcct
atgcaaatta ttcggcagca agcaataaag cacaaattaa agatttagga 960caaaatggtt
taaaagtggc ggcaggtgta cctgtgatga aagagtctga atggactggt 1020aaaaactttg
taccgccgtt aaaaacttta tataccgtcc aagataccta taactataat 1080gacccaactt
gtggggatat gacctacatt tgctggccaa cggttgcgcc gtcatctgct 1140tatgtctata
agggaggcaa aaaagcaatt tctggttggg aaaatacatt attggttcca 1200tctttaaagc
gcggtgttat tttccgtatt aagctagatc caacttacag tactacttat 1260gatgatgctg
tgccgatgtt taagagcaac aatcgttatc gtgacgtgat tgcaagtcca 1320gatgggaatg
ttttatatgt attgactgat acttccggaa atgtccaaaa agatgatggt 1380tctgtaacga
atacattaga aaacccagga tctctgatta gatttacata taaagctcag 1440taa
144312480PRTAcinetobacter sp. 12Met Asn Lys His Leu Leu Ala Lys Ile Thr
Leu Leu Gly Ala Ala Gln1 5 10
15Leu Leu Thr Leu Asn Ser Ala Phe Ala Asp Val Pro Leu Thr Pro Ser
20 25 30Gln Phe Ala Lys Ala Lys
Thr Glu Ser Phe Asp Lys Lys Val Leu Leu 35 40
45Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp
Asn Gln 50 55 60Ile Trp Leu Thr Glu
Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro65 70
75 80Glu Ser Gly Ser Val Lys Thr Val Phe Gln
Val Pro Glu Ile Val Asn 85 90
95Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala Phe His Pro Asp
100 105 110Phe Lys Asn Asn Pro
Tyr Ile Tyr Val Ser Gly Thr Phe Lys Asn Pro 115
120 125Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile
Ile Arg Arg Tyr 130 135 140Thr Tyr Asn
Lys Ala Thr Asp Thr Leu Glu Lys Pro Val Asp Leu Leu145
150 155 160Ala Gly Leu Pro Ser Ser Lys
Asp His Gln Ser Gly Arg Leu Val Ile 165
170 175Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp
Gln Gly Arg Asn 180 185 190Gln
Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr 195
200 205Gln Gln Glu Leu Ser Gly Lys Asp Tyr
His Thr Tyr Met Gly Lys Val 210 215
220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe225
230 235 240Asn Gly Val Ile
Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln 245
250 255Gly Leu Ala Phe Thr Pro Asn Gly Lys Leu
Leu Gln Ser Glu Gln Gly 260 265
270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val Lys Gly Gly Asn Tyr
275 280 285Gly Trp Pro Asn Val Ala Gly
Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290 295
300Ala Asn Tyr Ser Ala Ala Ser Asn Lys Ala Gln Ile Lys Asp Leu
Gly305 310 315 320Gln Asn
Gly Leu Lys Val Ala Ala Gly Val Pro Val Met Lys Glu Ser
325 330 335Glu Trp Thr Gly Lys Asn Phe
Val Pro Pro Leu Lys Thr Leu Tyr Thr 340 345
350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Asp
Met Thr 355 360 365Tyr Ile Cys Trp
Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly Trp Glu Asn Thr
Leu Leu Val Pro385 390 395
400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr
405 410 415Ser Thr Thr Tyr Asp
Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg 420
425 430Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly Asn Val
Leu Tyr Val Leu 435 440 445Thr Asp
Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr Asn 450
455 460Thr Leu Glu Asn Pro Gly Ser Leu Ile Arg Phe
Thr Tyr Lys Ala Gln465 470 475
480131443DNAAcinetobacter sp. 13atgaataaac atttattggc taaaattact
ttattaggtg ctgctcagct agttacgctc 60aattcagcat ttgctgatgt ccctcttact
ccatctcaat ttgctaaagc gaaaacagga 120agctttgaca agaaagttct tatatctaat
ttaaataagc cacatgcttt gttgtggggg 180cctgataatc aaatttggtt aacggagcgg
gcaacaggta agattctaag agttaatcca 240gagtcgggca gtgtaaaaac agtttttcag
gttcctgaga ttgtaaatga tgctgatgga 300caaaacggtt tattgggttt tgcctttcat
cctgacttta aaaataatcc ttatatctat 360gtttcaggta catttaaaaa tccgaaatct
acagataaag aattaccgaa tcaaactatt 420atccgtcgat atacctataa caaagcaaca
gatactcttg agaaaccagt agatttattg 480gcaggattac cttcatcgaa agaccatcag
tcgggtcgcc ttgtcattgg tccagaccaa 540aagatttact atacgattgg tgatcaggga
cgtaaccagc ttgcttattt attcttacca 600aatcaggcac aacatacgcc gactcaacag
gaactgagcg gaaaagacta tcatacctat 660atgggtaaag tattacgctt aaatctggat
ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa ttagtcatat ttatacgctc
ggtcatcgta atccacaggg cttggcattt 780actccaaatg gtaaactgtt gcaatctgaa
caaggtccaa actctgatga tgaaattaat 840ctcattgtta aaggtggcaa ctatggctgg
ccaaatgtag cgggttataa agatgacagt 900ggttatgcct atgcaaatta ttcggcagca
gccaataagt cacaaattaa agatttaggg 960caaaatggag taaaagtagc ggcaggtgta
cctgtgatga aagagtctga atggactggt 1020aaaaactttg taccgccgtt aaaaacttta
tataccgtcc aagataccta taactataat 1080gacccaacct gtggggatat gacctacatt
tgctggccaa cagttgcacc gtcatctgct 1140tatgtctata agggcggcaa aaaagcaatt
tctggttggg aaaatacatt attggttcca 1200tctttaaagc gtggtgttat tttccgtatt
aagctagatc caacttacag tgctacttat 1260gatgatgctg tgccgatgtt taagagcaac
aatcgttatc gtgacgtgat tgcaagtcca 1320gatgggaatg ttttatatgt attgactgat
acttccggaa atgtccaaaa agatgatggt 1380tctgtaacga atacattaga aaatccagga
tctctgatta gatttacata taaagctcag 1440taa
144314480PRTAcinetobacter sp. 14Met Asn
Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln1 5
10 15Leu Val Thr Leu Asn Ser Ala Phe
Ala Asp Val Pro Leu Thr Pro Ser 20 25
30Gln Phe Ala Lys Ala Lys Thr Gly Ser Phe Asp Lys Lys Val Leu
Ile 35 40 45Ser Asn Leu Asn Lys
Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50 55
60Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val
Asn Pro65 70 75 80Glu
Ser Gly Ser Val Lys Thr Val Phe Gln Val Pro Glu Ile Val Asn
85 90 95Asp Ala Asp Gly Gln Asn Gly
Leu Leu Gly Phe Ala Phe His Pro Asp 100 105
110Phe Lys Asn Asn Pro Tyr Ile Tyr Val Ser Gly Thr Phe Lys
Asn Pro 115 120 125Lys Ser Thr Asp
Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130
135 140Thr Tyr Asn Lys Ala Thr Asp Thr Leu Glu Lys Pro
Val Asp Leu Leu145 150 155
160Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile
165 170 175Gly Pro Asp Gln Lys
Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180
185 190Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln
His Thr Pro Thr 195 200 205Gln Gln
Glu Leu Ser Gly Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210
215 220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys
Asp Asn Pro Ser Phe225 230 235
240Asn Gly Val Ile Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln
245 250 255Gly Leu Ala Phe
Thr Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260
265 270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val
Lys Gly Gly Asn Tyr 275 280 285Gly
Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290
295 300Ala Asn Tyr Ser Ala Ala Ala Asn Lys Ser
Gln Ile Lys Asp Leu Gly305 310 315
320Gln Asn Gly Val Lys Val Ala Ala Gly Val Pro Val Met Lys Glu
Ser 325 330 335Glu Trp Thr
Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340
345 350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro
Thr Cys Gly Asp Met Thr 355 360
365Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly
Trp Glu Asn Thr Leu Leu Val Pro385 390
395 400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu
Asp Pro Thr Tyr 405 410
415Ser Ala Thr Tyr Asp Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg
420 425 430Tyr Arg Asp Val Ile Ala
Ser Pro Asp Gly Asn Val Leu Tyr Val Leu 435 440
445Thr Asp Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val
Thr Asn 450 455 460Thr Leu Glu Asn Pro
Gly Ser Leu Ile Arg Phe Thr Tyr Lys Ala Gln465 470
475 480151443DNAAcinetobacter sp. 15atgaataaac
atttattggc taaaattact ttattaggtg ctgctcagtt acttacgctc 60aattcagcat
ttgctgatgt ccctcttacc ccatctcaat ttgctaaagc gaaaacagaa 120agctttgaca
agaaagttct tctatctaat ttaaataagc cacatgcttt gttgtgggga 180cctgataatc
aaatttggtt aacggagcgg gcaacaggta agattctaag agttaatcca 240gagtcgggca
gtgtaaaaac agtttttcag gttcctgaga ttgtaagtga tgctgatgga 300caaaacggtt
tattgggttt tgcctttcat actgacttta aaaataatcc ttatatctat 360gtttcaggta
catttaaaaa tccgaaatct acagataaag aattaccgaa tcaaactatt 420atccgtcgat
atacctataa caaagcaaca gatactcttg agaaaccagt agatttatta 480gcaggattac
cttcatcgaa agaccatcag tcgggtcgtc ttgtcattgg tccagaccaa 540aagatttact
atacgattgg tgatcaggga cgtaaccagc ttgcttattt attcttacca 600aatcaggcac
aacatacgcc gactcaacag gaactgagcg gcaaagacta tcatacctat 660atgggtaaag
tattacgctt aaatctggat ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa
ttagccatat ttatacgctc ggtcatcgta acccacaggg cttggcattt 780actccaaatg
gtaaactgtt gcaatctgaa cagggtccaa actctgacga tgaaattaat 840ctcattgtta
aaggtggtaa ctatggctgg ccaaatgtag cgggttataa agatgacagt 900ggttatgcct
atgcaaatta ttcggcagca accaataagt cacaaattaa agatttaggg 960caaaatggag
taaaagtagc ggcaggtgta cctgtgatga aagagtctga atggactggt 1020aaaaactttg
taccgccgtt aaaaacttta tataccgtcc aagataccta taactataat 1080gacccaactt
gtggggatat gacctacatt tgctggccaa cggttgcgcc gtcatctgct 1140tatgtctata
agggaggcaa aaaagcaatt tctggttggg aaaatacatt attggttcca 1200tctttaaagc
gcggtgttat tttccgtatt aagctagatc caacttacag tgctacttat 1260gatgatgctg
tgccgatgtt taaaagcaac aatcgttatc gtgacgtgat tgcaagtcca 1320gatgggaatg
ttttatatgt attgactgat acttccggaa atgtccaaaa agatgatggt 1380tctgtaacga
atacattaga aaatccagga tccctgatta gatttacata taaagctcag 1440taa
144316480PRTAcinetobacter sp. 16Met Asn Lys His Leu Leu Ala Lys Ile Thr
Leu Leu Gly Ala Ala Gln1 5 10
15Leu Leu Thr Leu Asn Ser Ala Phe Ala Asp Val Pro Leu Thr Pro Ser
20 25 30Gln Phe Ala Lys Ala Lys
Thr Glu Ser Phe Asp Lys Lys Val Leu Leu 35 40
45Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp
Asn Gln 50 55 60Ile Trp Leu Thr Glu
Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro65 70
75 80Glu Ser Gly Ser Val Lys Thr Val Phe Gln
Val Pro Glu Ile Val Ser 85 90
95Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala Phe His Thr Asp
100 105 110Phe Lys Asn Asn Pro
Tyr Ile Tyr Val Ser Gly Thr Phe Lys Asn Pro 115
120 125Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile
Ile Arg Arg Tyr 130 135 140Thr Tyr Asn
Lys Ala Thr Asp Thr Leu Glu Lys Pro Val Asp Leu Leu145
150 155 160Ala Gly Leu Pro Ser Ser Lys
Asp His Gln Ser Gly Arg Leu Val Ile 165
170 175Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp
Gln Gly Arg Asn 180 185 190Gln
Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr 195
200 205Gln Gln Glu Leu Ser Gly Lys Asp Tyr
His Thr Tyr Met Gly Lys Val 210 215
220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe225
230 235 240Asn Gly Val Ile
Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln 245
250 255Gly Leu Ala Phe Thr Pro Asn Gly Lys Leu
Leu Gln Ser Glu Gln Gly 260 265
270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val Lys Gly Gly Asn Tyr
275 280 285Gly Trp Pro Asn Val Ala Gly
Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290 295
300Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser Gln Ile Lys Asp Leu
Gly305 310 315 320Gln Asn
Gly Val Lys Val Ala Ala Gly Val Pro Val Met Lys Glu Ser
325 330 335Glu Trp Thr Gly Lys Asn Phe
Val Pro Pro Leu Lys Thr Leu Tyr Thr 340 345
350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Asp
Met Thr 355 360 365Tyr Ile Cys Trp
Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly Trp Glu Asn Thr
Leu Leu Val Pro385 390 395
400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr
405 410 415Ser Ala Thr Tyr Asp
Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg 420
425 430Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly Asn Val
Leu Tyr Val Leu 435 440 445Thr Asp
Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr Asn 450
455 460Thr Leu Glu Asn Pro Gly Ser Leu Ile Arg Phe
Thr Tyr Lys Ala Gln465 470 475
480171443DNAAcinetobacter sp. 17atgaataaac atttattggc taaaattact
ttattaggtg ctgctcagct acttacgctc 60aattcagcat ttgctgatgt ccctcttact
ccatctcaat ttgctaaagc gaaaacagaa 120agctttgaca agaaagttct tctatctaat
ttaaataagc cacatgcttt gttgtgggga 180cctgataatc aaatttggtt aacggagcgg
gcaacaggta agattctaag agttaaccct 240gaatcaggca gtgtaaaaac agtttttcag
gttcctgaga ttgtaaatga tgctgatgga 300caaaacggtt tattgggttt tgcctttcat
cctgacttta aacataatcc ttatatctat 360gtttcaggta catttaaaaa tccgaaatct
acagataaag aattaccgaa tcaaactatt 420atccgtcgat atacctataa caaggcaaca
gatacccttg agaaaccagt agatttatta 480gcaggattac cttcatcgaa agaccatcag
tcgggtcgtc ttgtgattgg tccagaccaa 540aagatttact atacgattgg tgatcagggg
cgtaaccagc tggcttattt attcttgcca 600aatcaagcac agcatacgcc gactcaacaa
gagctgagcg gtaaagacta tcacacctat 660atgggtaaag tattacgctt aaatctagat
ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa ttagccatat ttatacactc
ggtcatcgta atccacaggg cttggccttt 780actccaaatg gtaaactgtt gcaatctgaa
caaggtccaa actctgatga tgaaattaat 840ctgattgtta aaggtggtaa ctatggctgg
ccaaatgtag cgggttataa agatgacagt 900ggttatgcct atgcaaatta ttcggcagca
accaataagt cacaaattaa agatttaggg 960caaaatggag taaaagtagc ggcaggtgta
cctgtaatga aagagtctga atggactggt 1020aaaaactttg taccgccgtt aaaaacttta
tataccgtcc aagataccta taactataat 1080gacccaacct gtggggatat gacctacatt
tgctggccaa cggttgcgcc gtcatctgct 1140tatgtctata agggaggcaa aaaagcaatt
tctggttggg aaaatacctt attggttcca 1200tctttaaagc gcggtgttat tttccgtatt
aagctagatc caacttacag tgctacttat 1260gatgatgctg tgccgatgtt taagagcaac
aatcgttatc gtgacgtgat tgcaagtcca 1320gatggaaatg ttttatatgt attgactgat
acttccggaa atgtccaaaa agatgatggc 1380tctgtaacga atacattaga aaatccagga
tctttgatta ggtttacata taaagctcag 1440taa
144318480PRTAcinetobacter sp. 18Met Asn
Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln1 5
10 15Leu Leu Thr Leu Asn Ser Ala Phe
Ala Asp Val Pro Leu Thr Pro Ser 20 25
30Gln Phe Ala Lys Ala Lys Thr Glu Ser Phe Asp Lys Lys Val Leu
Leu 35 40 45Ser Asn Leu Asn Lys
Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50 55
60Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val
Asn Pro65 70 75 80Glu
Ser Gly Ser Val Lys Thr Val Phe Gln Val Pro Glu Ile Val Asn
85 90 95Asp Ala Asp Gly Gln Asn Gly
Leu Leu Gly Phe Ala Phe His Pro Asp 100 105
110Phe Lys His Asn Pro Tyr Ile Tyr Val Ser Gly Thr Phe Lys
Asn Pro 115 120 125Lys Ser Thr Asp
Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130
135 140Thr Tyr Asn Lys Ala Thr Asp Thr Leu Glu Lys Pro
Val Asp Leu Leu145 150 155
160Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile
165 170 175Gly Pro Asp Gln Lys
Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180
185 190Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln
His Thr Pro Thr 195 200 205Gln Gln
Glu Leu Ser Gly Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210
215 220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys
Asp Asn Pro Ser Phe225 230 235
240Asn Gly Val Ile Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln
245 250 255Gly Leu Ala Phe
Thr Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260
265 270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val
Lys Gly Gly Asn Tyr 275 280 285Gly
Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290
295 300Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser
Gln Ile Lys Asp Leu Gly305 310 315
320Gln Asn Gly Val Lys Val Ala Ala Gly Val Pro Val Met Lys Glu
Ser 325 330 335Glu Trp Thr
Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340
345 350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro
Thr Cys Gly Asp Met Thr 355 360
365Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly
Trp Glu Asn Thr Leu Leu Val Pro385 390
395 400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu
Asp Pro Thr Tyr 405 410
415Ser Ala Thr Tyr Asp Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg
420 425 430Tyr Arg Asp Val Ile Ala
Ser Pro Asp Gly Asn Val Leu Tyr Val Leu 435 440
445Thr Asp Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val
Thr Asn 450 455 460Thr Leu Glu Asn Pro
Gly Ser Leu Ile Arg Phe Thr Tyr Lys Ala Gln465 470
475 480191443DNAAcinetobacter sp. 19atgaataaac
atttattggc taaaattact ttattaggtg ctgctcacct acttacgctc 60aattcagcat
ttgctgatgt ccctcttact ccatctcaat ttgctaaagc gaaaacagaa 120agctttgaca
agaaagttct tctatctaat ttaaataagc cgcatgcttt gttgtgggga 180cctgataatc
aaatttggtt aacagagcgg gcaacaggta agattctaag agttaaccct 240gaatcaggca
gtgtaaaaac agtttttcag gttcctgaga ttgtaaatga tgctgatgga 300caaaacggtt
tattgggttt tgcctttcat cctgacttta aacataatcc ttatatctat 360gtttcaggta
catttaaaaa tccgaaatct acagataaag aattaccgaa tcaaactatt 420attcgtcgat
atacctataa caaagcaaca gatactcttg ataaaccagt agatttatta 480gcaggattac
cttcatcgaa agaccatcag tcgggtcgtc ttgtgattgg tccagaccaa 540aagatttact
atacgattgg tgatcagggg cgtaaccagc tggcttattt attcttacca 600aatcaggcac
aacatacgcc gactcaacag gaactgagcg gcaaagacta tcatacctat 660atgggtaaag
tattacgctt aaatctggat ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa
ttagccatat ttatacgctc ggtcatcgta acccacaggg cttggcattt 780actccaaatg
gtaaactgtt acaatctgaa cagggtccaa actctgacga tgaaattaat 840ctcattgtta
aaggtggtaa ctatggctgg ccaaatgtag cgggttataa agatgacagt 900ggttatgcgt
atgcaaatta ttcagcagca accaataagt cacaaattaa agatttaggg 960caaaatggag
taaaagtagc ggcaggtgta cctgtgatga aagagtctga atggactggt 1020aaaaactttg
taccgccgtt aaaaacttta tataccgtcc aagataccta taactataat 1080gacccaactt
gtggggatat gacctacatt tgctggccaa cagttgcgcc atcatctgct 1140tatgtctata
aggggggcaa aaaagcaatt tctggttggg aaaatacatt attggttcca 1200tctttaaagc
gtggtgttat ttttcgtatt aagctagatc caacttacag tactacttat 1260gatgatgctg
tgccgatgtt taagagcaac aatcgttatc gtgacgtgat tgcaagtcca 1320gatgggaatg
ttttatatgt attgactgat acttccggaa atgtccaaaa agatgatggt 1380tctgtaacga
atacattaga aaacccagga tctctgatta gatttacata taaagctcag 1440taa
144320480PRTAcinetobacter sp. 20Met Asn Lys His Leu Leu Ala Lys Ile Thr
Leu Leu Gly Ala Ala His1 5 10
15Leu Leu Thr Leu Asn Ser Ala Phe Ala Asp Val Pro Leu Thr Pro Ser
20 25 30Gln Phe Ala Lys Ala Lys
Thr Glu Ser Phe Asp Lys Lys Val Leu Leu 35 40
45Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp
Asn Gln 50 55 60Ile Trp Leu Thr Glu
Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro65 70
75 80Glu Ser Gly Ser Val Lys Thr Val Phe Gln
Val Pro Glu Ile Val Asn 85 90
95Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala Phe His Pro Asp
100 105 110Phe Lys His Asn Pro
Tyr Ile Tyr Val Ser Gly Thr Phe Lys Asn Pro 115
120 125Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile
Ile Arg Arg Tyr 130 135 140Thr Tyr Asn
Lys Ala Thr Asp Thr Leu Asp Lys Pro Val Asp Leu Leu145
150 155 160Ala Gly Leu Pro Ser Ser Lys
Asp His Gln Ser Gly Arg Leu Val Ile 165
170 175Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp
Gln Gly Arg Asn 180 185 190Gln
Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr 195
200 205Gln Gln Glu Leu Ser Gly Lys Asp Tyr
His Thr Tyr Met Gly Lys Val 210 215
220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe225
230 235 240Asn Gly Val Ile
Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln 245
250 255Gly Leu Ala Phe Thr Pro Asn Gly Lys Leu
Leu Gln Ser Glu Gln Gly 260 265
270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val Lys Gly Gly Asn Tyr
275 280 285Gly Trp Pro Asn Val Ala Gly
Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290 295
300Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser Gln Ile Lys Asp Leu
Gly305 310 315 320Gln Asn
Gly Val Lys Val Ala Ala Gly Val Pro Val Met Lys Glu Ser
325 330 335Glu Trp Thr Gly Lys Asn Phe
Val Pro Pro Leu Lys Thr Leu Tyr Thr 340 345
350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Asp
Met Thr 355 360 365Tyr Ile Cys Trp
Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly Trp Glu Asn Thr
Leu Leu Val Pro385 390 395
400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr
405 410 415Ser Thr Thr Tyr Asp
Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg 420
425 430Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly Asn Val
Leu Tyr Val Leu 435 440 445Thr Asp
Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr Asn 450
455 460Thr Leu Glu Asn Pro Gly Ser Leu Ile Arg Phe
Thr Tyr Lys Ala Gln465 470 475
480211443DNAAcinetobacter sp. 21atgaataaac atttattggc taaaattact
ttattaggtg ctgctcagct acttacgctc 60aattcagcat ttgctgatgt ccctcttaca
ccatctcaat ttgctaaagc gaaaacagaa 120agctttgaca agaaagttct tctatctaat
ttaaataagc cacatgcttt gttgtggggg 180cctgataatc aaatttggtt aacggagcgg
gcaacaggta agattctaag agtgaatcca 240gagtcgggca gtgtaaaaac agtttttcag
gttcctgaga ttgtaaatga tgctgatgga 300caaaacggtt tattgggttt tgcctttcat
cctgacttta aaaataatcc ttatatctat 360gtttcaggta cttttaaaaa tccgaaatct
acagataaag aattaccgaa tcaaactatt 420attcgtcgat atacctataa caaagcaaca
gatactcttg ataaaccagt agatttatta 480gcaggattac cttcatcgaa agaccatcag
tcgggtcgcc ttgtgattgg tccagaccaa 540aagatttact atacgattgg tgatcagggg
cgtaaccagc ttgcttattt attcttacca 600aatcaggcac aacatacgcc gactcaacag
gaactgagcg gcaaagacta tcatacctat 660atgggtaaag tattacgctt aaatctggat
ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa ttagccatat ttatacgctc
ggtcatcgta acccacaggg cttggcattt 780actccaaatg gtaaactgtt gcaatctgaa
cagggtccaa actctgatga tgaaattaac 840ctcattgtca aaggtggtaa ctatggctgg
ccaaatgtag cgggttataa agatgatagt 900ggttatgcct atgcaaatta ttcagcagca
agcaataaag cacaaattaa agatttagga 960caaaatggtt taaaagtggc agctggcgtt
ccagtgacta aagagtctga atggactggt 1020aaaaactttg taccgccgtt aaaaacttta
tataccgtcc aagataccta taactataat 1080gacccaacct gtggggatat gacctacatt
tgctggccaa cggttgcgcc gtcatctgct 1140tatgtctata agggaggcaa aaaagcaatt
tctggttggg aaaatacctt attggttcca 1200tctttaaagc gcggtgttat tttccgtatt
aagctagatc caacttacag tgctacttat 1260gatgatgctg tgccgatgtt taagagcaac
aatcgttatc gtgacgtgat tgcaagtcca 1320gatggaaatg ttttatatgt attgactgat
acttccggaa atgtccaaaa agatgatggt 1380tctgtaacga atacattaga aaacccagga
tctctgatta gatttacata taaagctcag 1440taa
144322480PRTAcinetobacter sp. 22Met Asn
Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln1 5
10 15Leu Leu Thr Leu Asn Ser Ala Phe
Ala Asp Val Pro Leu Thr Pro Ser 20 25
30Gln Phe Ala Lys Ala Lys Thr Glu Ser Phe Asp Lys Lys Val Leu
Leu 35 40 45Ser Asn Leu Asn Lys
Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50 55
60Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val
Asn Pro65 70 75 80Glu
Ser Gly Ser Val Lys Thr Val Phe Gln Val Pro Glu Ile Val Asn
85 90 95Asp Ala Asp Gly Gln Asn Gly
Leu Leu Gly Phe Ala Phe His Pro Asp 100 105
110Phe Lys Asn Asn Pro Tyr Ile Tyr Val Ser Gly Thr Phe Lys
Asn Pro 115 120 125Lys Ser Thr Asp
Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130
135 140Thr Tyr Asn Lys Ala Thr Asp Thr Leu Asp Lys Pro
Val Asp Leu Leu145 150 155
160Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile
165 170 175Gly Pro Asp Gln Lys
Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180
185 190Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln
His Thr Pro Thr 195 200 205Gln Gln
Glu Leu Ser Gly Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210
215 220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys
Asp Asn Pro Ser Phe225 230 235
240Asn Gly Val Ile Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln
245 250 255Gly Leu Ala Phe
Thr Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260
265 270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val
Lys Gly Gly Asn Tyr 275 280 285Gly
Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290
295 300Ala Asn Tyr Ser Ala Ala Ser Asn Lys Ala
Gln Ile Lys Asp Leu Gly305 310 315
320Gln Asn Gly Leu Lys Val Ala Ala Gly Val Pro Val Thr Lys Glu
Ser 325 330 335Glu Trp Thr
Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340
345 350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro
Thr Cys Gly Asp Met Thr 355 360
365Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly
Trp Glu Asn Thr Leu Leu Val Pro385 390
395 400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu
Asp Pro Thr Tyr 405 410
415Ser Ala Thr Tyr Asp Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg
420 425 430Tyr Arg Asp Val Ile Ala
Ser Pro Asp Gly Asn Val Leu Tyr Val Leu 435 440
445Thr Asp Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val
Thr Asn 450 455 460Thr Leu Glu Asn Pro
Gly Ser Leu Ile Arg Phe Thr Tyr Lys Ala Gln465 470
475 480231443DNAAcinetobacter sp. 23atgaataaac
atttattggc taaaattact ttattaggtg ctgctcagct acttacactc 60aattcagcat
ttgctgatgt ccctcttaca ccatctcaat ttgctaaagc gaaaacagaa 120agctttgaca
agaaagttct tctatctaat ttaaataagc cacatgcttt gttgtggggg 180cctgataatc
aaatttggtt aacggagcgg gcaacaggta agattctaag agtgaatcca 240gagtcgggca
gtgtaaaaac agtttttcag gttcctgaga ttgtaaatga tgctgatgga 300caaaacggtt
tattgggttt tgcctttcat cctgacttta aaaataatcc ttatatctat 360gtttcaggta
cttttaaaaa tccgaaatct acagataaag aattaccgaa tcaaactatt 420attcgtcgat
atacctataa caaagcaaca gatactcttg ataaaccagt agatttatta 480gcaggattac
cttcatcgaa agaccatcag tcgggtcgcc ttgtgattgg tccagaccaa 540aaaatttact
atacgattgg tgatcagggg cgtaaccagc ttgcttattt attcttacca 600aatcaggcac
aacatacgcc gactcaacag gaactgagcg gcaaagacta tcatacctat 660atgggtaaag
tattacgctt aaatctggat ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa
ttagtcatat ttatacgctc ggtcatcgta atccacaggg cttagcattc 780actccaaatg
gtaaactgtt gcaatctgaa caaggtccaa actctgatga tgaaattaat 840ctcattgtta
aaggtggtaa ctatggctgg ccaaatgtag ctggttataa agatgatagt 900ggttatgcct
atgcaaatta ttcagcagca agcaataaag cacaaattaa agatttagga 960caaaatggtt
taaaagtggc agctggcgtt ccagtgacta aagagtctga atggactggt 1020aaaaactttg
taccgccgtt aaaaacttta tataccgtcc aagataccta taactataat 1080gacccaacct
gtggggatat gacctacatt tgctggccaa cggttgcgcc gtcatctgct 1140tatgtctata
agggaggcaa aaaagcaatt tctggttggg aaaatacctt attggttcca 1200tctttaaagc
gcggtgttat tttccgtatt aagctagatc caacttacaa tactacttat 1260gatgatgctg
tgccgatgtt taagagcaac aatcgttatc gtgacgtgat tgcaagtcca 1320gatggaaatg
ttttatatgt attgactgat acttccggaa atgtccaaaa agatgatggc 1380tctgtaacga
atacattaga aaacccagga tctctgatta gatttacata taaagctcag 1440taa
144324480PRTAcinetobacter sp. 24Met Asn Lys His Leu Leu Ala Lys Ile Thr
Leu Leu Gly Ala Ala Gln1 5 10
15Leu Leu Thr Leu Asn Ser Ala Phe Ala Asp Val Pro Leu Thr Pro Ser
20 25 30Gln Phe Ala Lys Ala Lys
Thr Glu Ser Phe Asp Lys Lys Val Leu Leu 35 40
45Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Pro Asp
Asn Gln 50 55 60Ile Trp Leu Thr Glu
Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro65 70
75 80Glu Ser Gly Ser Val Lys Thr Val Phe Gln
Val Pro Glu Ile Val Asn 85 90
95Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala Phe His Pro Asp
100 105 110Phe Lys Asn Asn Pro
Tyr Ile Tyr Val Ser Gly Thr Phe Lys Asn Pro 115
120 125Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile
Ile Arg Arg Tyr 130 135 140Thr Tyr Asn
Lys Ala Thr Asp Thr Leu Asp Lys Pro Val Asp Leu Leu145
150 155 160Ala Gly Leu Pro Ser Ser Lys
Asp His Gln Ser Gly Arg Leu Val Ile 165
170 175Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp
Gln Gly Arg Asn 180 185 190Gln
Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr 195
200 205Gln Gln Glu Leu Ser Gly Lys Asp Tyr
His Thr Tyr Met Gly Lys Val 210 215
220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe225
230 235 240Asn Gly Val Ile
Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln 245
250 255Gly Leu Ala Phe Thr Pro Asn Gly Lys Leu
Leu Gln Ser Glu Gln Gly 260 265
270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val Lys Gly Gly Asn Tyr
275 280 285Gly Trp Pro Asn Val Ala Gly
Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290 295
300Ala Asn Tyr Ser Ala Ala Ser Asn Lys Ala Gln Ile Lys Asp Leu
Gly305 310 315 320Gln Asn
Gly Leu Lys Val Ala Ala Gly Val Pro Val Thr Lys Glu Ser
325 330 335Glu Trp Thr Gly Lys Asn Phe
Val Pro Pro Leu Lys Thr Leu Tyr Thr 340 345
350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Asp
Met Thr 355 360 365Tyr Ile Cys Trp
Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly Trp Glu Asn Thr
Leu Leu Val Pro385 390 395
400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr
405 410 415Asn Thr Thr Tyr Asp
Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg 420
425 430Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly Asn Val
Leu Tyr Val Leu 435 440 445Thr Asp
Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr Asn 450
455 460Thr Leu Glu Asn Pro Gly Ser Leu Ile Arg Phe
Thr Tyr Lys Ala Gln465 470 475
480251443DNAAcinetobacter sp. 25atgaataaac atttattggc taaaattact
ttattaggtg ctgctcagct agttacgctc 60aattcagcat ttgctgatgt ccctcttact
ccatctcaat ttgctaaagc gaaaacagaa 120agctttgaca agaaagttct tatatctaat
ttaaataagc cacatgcttt gttgtggggg 180cctgataatc aaatttggtt aacggagcgg
gcaacaggta agattctaag agttaatcca 240gagtcgggca gtgtaaaaac agtttttcag
gttcctgaga ttgtaaatga tgctgatgga 300caaaacggtt tattgggttt tgcctttcat
cctgacttta aaaataatcc ttatatctat 360gtttcaggta catttaaaaa tccgaaatct
acagataaag aattaccgaa tcaaactatt 420atccgtcgat atacctataa caaagcaaca
gatactcttg agaaaccagt agatttattg 480gcaggattac cttcatcgaa agaccatcag
tcgggtcgcc ttgtcattgg tccagaccaa 540aagatttact atacgattgg tgatcaggga
cgtaaccagc ttgcttattt attcttacca 600aatcaggcac aacatacgcc gactcaacag
gaactgagcg gaaaagacta tcatacctat 660atgggtaaag tattacgctt aaatctggat
ggaagtattc caaaagataa tccaagcttt 720aacggtgtaa ttagtcatat ttatacgctc
ggtcatcgta atccacaggg cttggcattt 780actccaaatg gtaaactgtt gcaatctgaa
caaggtccaa actctgatga tgaaattaat 840ctcattgtta aaggtggcaa ctatggctgg
ccaaatgtag cgggttataa agatgacagt 900ggttatgcct atgcaaatta ttcggcagca
accaataagt cacaaattaa agatttaggg 960caaaatggag taaaagtagc ggcaggtgta
cctgtgatga aagagtctga atggactggt 1020aaaaactttg taccgccgtt aaaaacttta
tataccgtcc aagataccta taactataat 1080gacccaacct gtggggatat gacctacatt
tgctggccaa cagttgcacc gtcatctgct 1140tatgtctata agggcggcaa aaaagcaatt
tctggttggg aaaatacatt attggttcca 1200tctttaaagc gtggtgttat tttccgtatt
aagctagatc caacttacag tgctacttat 1260gatgatgctg tgccgatgtt taagagcaac
aatcgttatc gtgacgtgat tgcaagtcca 1320gatgggaatg ttttatatgt attgactgat
acttccggaa atgtccaaaa agatgatggt 1380tctgtaacga atacattaga aaatccagga
tctctgatta gatttacata taaagctcag 1440taa
144326480PRTAcinetobacter sp. 26Met Asn
Lys His Leu Leu Ala Lys Ile Thr Leu Leu Gly Ala Ala Gln1 5
10 15Leu Val Thr Leu Asn Ser Ala Phe
Ala Asp Val Pro Leu Thr Pro Ser 20 25
30Gln Phe Ala Lys Ala Lys Thr Glu Ser Phe Asp Lys Lys Val Leu
Ile 35 40 45Ser Asn Leu Asn Lys
Pro His Ala Leu Leu Trp Gly Pro Asp Asn Gln 50 55
60Ile Trp Leu Thr Glu Arg Ala Thr Gly Lys Ile Leu Arg Val
Asn Pro65 70 75 80Glu
Ser Gly Ser Val Lys Thr Val Phe Gln Val Pro Glu Ile Val Asn
85 90 95Asp Ala Asp Gly Gln Asn Gly
Leu Leu Gly Phe Ala Phe His Pro Asp 100 105
110Phe Lys Asn Asn Pro Tyr Ile Tyr Val Ser Gly Thr Phe Lys
Asn Pro 115 120 125Lys Ser Thr Asp
Lys Glu Leu Pro Asn Gln Thr Ile Ile Arg Arg Tyr 130
135 140Thr Tyr Asn Lys Ala Thr Asp Thr Leu Glu Lys Pro
Val Asp Leu Leu145 150 155
160Ala Gly Leu Pro Ser Ser Lys Asp His Gln Ser Gly Arg Leu Val Ile
165 170 175Gly Pro Asp Gln Lys
Ile Tyr Tyr Thr Ile Gly Asp Gln Gly Arg Asn 180
185 190Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln
His Thr Pro Thr 195 200 205Gln Gln
Glu Leu Ser Gly Lys Asp Tyr His Thr Tyr Met Gly Lys Val 210
215 220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys
Asp Asn Pro Ser Phe225 230 235
240Asn Gly Val Ile Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln
245 250 255Gly Leu Ala Phe
Thr Pro Asn Gly Lys Leu Leu Gln Ser Glu Gln Gly 260
265 270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val
Lys Gly Gly Asn Tyr 275 280 285Gly
Trp Pro Asn Val Ala Gly Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290
295 300Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser
Gln Ile Lys Asp Leu Gly305 310 315
320Gln Asn Gly Val Lys Val Ala Ala Gly Val Pro Val Met Lys Glu
Ser 325 330 335Glu Trp Thr
Gly Lys Asn Phe Val Pro Pro Leu Lys Thr Leu Tyr Thr 340
345 350Val Gln Asp Thr Tyr Asn Tyr Asn Asp Pro
Thr Cys Gly Asp Met Thr 355 360
365Tyr Ile Cys Trp Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly
Trp Glu Asn Thr Leu Leu Val Pro385 390
395 400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu
Asp Pro Thr Tyr 405 410
415Ser Ala Thr Tyr Asp Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg
420 425 430Tyr Arg Asp Val Ile Ala
Ser Pro Asp Gly Asn Val Leu Tyr Val Leu 435 440
445Thr Asp Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val
Thr Asn 450 455 460Thr Leu Glu Asn Pro
Gly Ser Leu Ile Arg Phe Thr Tyr Lys Ala Gln465 470
475 480271443DNAAcinetobacter sp. 27atgaataaac
atttattggc taaaattact ttattaggtg ctgctcagct acttacgttc 60aattcagcat
ttgctgatgt ccctcttact ccatctcaat ttgctaaagc gaaaacggaa 120agctttgata
agaaagttct tctatctaat ttaaataagc cacatgcttt gttgtgggga 180gctgataatc
aaatttggtt aacggagcgg gcaacaggta agattctaag agtgaatcca 240gagtcgggca
gtgtaaaaac agtttttcag gttcctgaga ttgtaaatga tgctgatgga 300caaaacggtt
tattgggttt tgcctttcat cctgacttta aaaataatcc ttatatctat 360gtttcaggta
catttaaaaa tccgaaatct acagataaag aattaccgaa tcaaactatt 420atccgtcgat
atacctataa caaggcaaca gatacccttg agaaaccagt agatttattg 480gcaggattac
cttcatcgaa agaccatcag tcgggtcgcc ttgtcattgg tccagaccaa 540aaaatttact
atacgattgg tgatcagggg cgtaaccagc ttgcttattt attcttgcca 600aatcaagcac
agcatacgcc gactcaacag gaactgagcg gcaaagacta tcatacctat 660atgggtaaag
tattacgctt aaatctggat ggaagtattc caaaagataa tccaagcttt 720aacggcgtaa
ttagtcatat ttatacgctc ggtcatcgaa acccacaggg cttggcattt 780actccaaatg
gtaaactgtt gcaatctgaa cagggtccaa actctgacga tgaaattaat 840ctcattgtta
aaggtggtaa ctatggctgg ccaaatgtag cgggttataa agatgacagt 900ggttatgcct
atgcaaatta ttcggcagca accaataagt cacaaattaa agatttaggg 960caaaatggag
taaaagtagc ggcaggtgta cctgtgatga aagagtctga atggagtggt 1020aaaaactttg
taccgccgtt aaaaacttta tataccgtcc aaggtaccta taactataat 1080gacccaacct
gtggggatat gacctacatt tgctggccaa cggttgcgcc gtcatctgct 1140tatgtctata
agggaggcaa aaaagcaatt tctggttggg aaaatacctt actggttcca 1200tctttaaagc
gcggtgttat tttccgtatt aagctagatc caacttacag tgctacttat 1260gatgatgcgg
tgccgatgtt taagagcaac aatcgttatc gtgacgtgat tgcaagtcca 1320gatgggaatg
ttttatatgt attgactgat acttccggaa atgtccaaaa agatgatggt 1380tctgtaacga
atacattaga aaacccagga tctctgatta gatttacata taaagctcag 1440taa
144328480PRTAcinetobacter sp. 28Met Asn Lys His Leu Leu Ala Lys Ile Thr
Leu Leu Gly Ala Ala Gln1 5 10
15Leu Leu Thr Phe Asn Ser Ala Phe Ala Asp Val Pro Leu Thr Pro Ser
20 25 30Gln Phe Ala Lys Ala Lys
Thr Glu Ser Phe Asp Lys Lys Val Leu Leu 35 40
45Ser Asn Leu Asn Lys Pro His Ala Leu Leu Trp Gly Ala Asp
Asn Gln 50 55 60Ile Trp Leu Thr Glu
Arg Ala Thr Gly Lys Ile Leu Arg Val Asn Pro65 70
75 80Glu Ser Gly Ser Val Lys Thr Val Phe Gln
Val Pro Glu Ile Val Asn 85 90
95Asp Ala Asp Gly Gln Asn Gly Leu Leu Gly Phe Ala Phe His Pro Asp
100 105 110Phe Lys Asn Asn Pro
Tyr Ile Tyr Val Ser Gly Thr Phe Lys Asn Pro 115
120 125Lys Ser Thr Asp Lys Glu Leu Pro Asn Gln Thr Ile
Ile Arg Arg Tyr 130 135 140Thr Tyr Asn
Lys Ala Thr Asp Thr Leu Glu Lys Pro Val Asp Leu Leu145
150 155 160Ala Gly Leu Pro Ser Ser Lys
Asp His Gln Ser Gly Arg Leu Val Ile 165
170 175Gly Pro Asp Gln Lys Ile Tyr Tyr Thr Ile Gly Asp
Gln Gly Arg Asn 180 185 190Gln
Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr 195
200 205Gln Gln Glu Leu Ser Gly Lys Asp Tyr
His Thr Tyr Met Gly Lys Val 210 215
220Leu Arg Leu Asn Leu Asp Gly Ser Ile Pro Lys Asp Asn Pro Ser Phe225
230 235 240Asn Gly Val Ile
Ser His Ile Tyr Thr Leu Gly His Arg Asn Pro Gln 245
250 255Gly Leu Ala Phe Thr Pro Asn Gly Lys Leu
Leu Gln Ser Glu Gln Gly 260 265
270Pro Asn Ser Asp Asp Glu Ile Asn Leu Ile Val Lys Gly Gly Asn Tyr
275 280 285Gly Trp Pro Asn Val Ala Gly
Tyr Lys Asp Asp Ser Gly Tyr Ala Tyr 290 295
300Ala Asn Tyr Ser Ala Ala Thr Asn Lys Ser Gln Ile Lys Asp Leu
Gly305 310 315 320Gln Asn
Gly Val Lys Val Ala Ala Gly Val Pro Val Met Lys Glu Ser
325 330 335Glu Trp Ser Gly Lys Asn Phe
Val Pro Pro Leu Lys Thr Leu Tyr Thr 340 345
350Val Gln Gly Thr Tyr Asn Tyr Asn Asp Pro Thr Cys Gly Asp
Met Thr 355 360 365Tyr Ile Cys Trp
Pro Thr Val Ala Pro Ser Ser Ala Tyr Val Tyr Lys 370
375 380Gly Gly Lys Lys Ala Ile Ser Gly Trp Glu Asn Thr
Leu Leu Val Pro385 390 395
400Ser Leu Lys Arg Gly Val Ile Phe Arg Ile Lys Leu Asp Pro Thr Tyr
405 410 415Ser Ala Thr Tyr Asp
Asp Ala Val Pro Met Phe Lys Ser Asn Asn Arg 420
425 430Tyr Arg Asp Val Ile Ala Ser Pro Asp Gly Asn Val
Leu Tyr Val Leu 435 440 445Thr Asp
Thr Ser Gly Asn Val Gln Lys Asp Asp Gly Ser Val Thr Asn 450
455 460Thr Leu Glu Asn Pro Gly Ser Leu Ile Arg Phe
Thr Tyr Lys Ala Gln465 470 475
480
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