ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1991, 0066-4804/91/020242-10$02.00/0 Copyright © 1991, American Society for Microbiology



Vol. 35, No. 2

Sequence and Molecular Characterization of the ROB-1 3-Lactamase Gene from Pasteurella haemolytica VALERIE LIVRELLI,l* JEAN PEDUZZI,2 AND BERNARD JOLY' Laboratoire de Bacteriologie-Virologie, Faculte de Pharmacie, 28, place Henri-Dunant, 63001 Clermont-Ferrand Cedex,' and Unite' de Recherche Associe'e 401, Museum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, 75231 Paris Cedex 05,2 France Received 14 June 1990/Accepted 13 November 1990

The ROB-1 ,I-lactamase-encoding plasmids from eight Pasteurella and two Haemophilus strains were compared by restriction endonuclease and hybridization analyses. Two types of ROB-i-encoding plasmids, which differed in size, were detected. One (4.1 kb) was found only in Pasteurella strains. The other (4.4 kb) was found in both Haemophilus influenzae and in one of the eight Pasteurella strains examined. These two plasmids shared multiple homologous fragments, suggesting that one was derived from the other. The ROB-i-encoding gene from Pasteurella haemolytica LNPB 51 was cloned and sequenced. An open reading frame of 915 nucleotides was found; it encoded a 305-amino-acid protein. Analysis of this amino acid sequence confirmed that the enzyme is a class A (8-lactamase. It had 32 to 48% homology with other class A enzymes and exhibited several common features of the gram-positive ,I-lactamases. The ROB-1 mature protein, however, contained only one cysteine residue at position 123. These results suggest that ROB-1 is a link between I-lactamases of gram-negative and gram-positive bacteria. An internal 230-bp DraI fragment from ROB-1 hybridized only with plasmid DNA from ROB-i-producing strains. This specffic probe could be useful in epidemiological studies.

P-Lactamases are the major determinants of resistance to P-lactam antibiotics among microorganisms and are widely

mase gene from H. influenzae plasmid RROb was determined (26). We report here the molecular characterization of the

distributed in both gram-positive and gram-negative bacteria (11). These enzymes were first classified according to their substrate profiles, isoelectric points, and other properties such as molecular weight. A classification scheme based on amino acid composition and sequence homologies was later proposed by Ambler (1), who defined classes A and B. 3-Lactamases are now divided into four classes. Class A includes serine penicillinases such as chromosomally encoded 13-lactamases from Bacillus spp. (37, 42), Streptomyces spp. (17, 18), and Klebsiella spp. (3, 4); plasmid-mediated enzymes from Staphylococcus aureus PC1 (46); and TEM- and SHV-type enzymes from members of the family Enterobacteriaceae (2, 6, 43). All these class A P-lactamases show amino acid sequence homology, especially around the active serine site. Class B includes metalloenzymes (1), class C includes chromosomally encoded cephalosporinases (24), and class D includes oxacillinases (23). Most ,-lactamases have not yet been classified according to this scheme because their amino acid sequences are not available. The ROB-1 ,B-lactamase was first described in a human Haemophilus influenzae strain. The new enzyme had a TEM-like substrate profile, and its isoelectric point was estimated to be 8.1. In this strain ROB-1 was encoded by a plasmid of 4.4 kb, RROb (40). ROB-1 was later found in several H. influenzae strains and in porcine Actinobacillus pleuropneumoniae strains throughout the United States (34). More recently, in France we found (31) bovine and porcine Pasteurella multocida, Pasteurella haemolytica, and Pasteurella aerogenes strains that produce ROB-1. Furthermore, study of a ROB-1-encoding plasmid from an H. influenzae strain isolated in France revealed that it is identical to the 4.4-kb plasmid RROb (30). Recently, the nucleotide sequence of the ROB-1 13-lacta*

ROB-1-encoding plasmids from Pasteurella and Haemophilus strains. The nucleotide sequence of the ,-lactamase gene from P. haemolytica LNPB 51 was determined, and the deduced amino acid sequence was compared with those of other enzymes. Our data confirmed that ROB-1 is a class A 13-lactamase which shares several features with gram-positive bacterial 1-lactamases. MATERIALS AND METHODS

Bacterial strains and plasmids. Wild-type strains of P. multocida, P. haemolytica, and H. influenzae; reference strains; and the plasmids used for cloning and sequencing of the ROB-1 ,-lactamase gene and DNA hybridization are listed in Table 1. Media and reagents. Pasteurella and Haemophilus strains were grown in tryptic soy broth supplemented with 2% Fildes extract (Difco Laboratories, Detroit, Mich.). Restriction endonucleases, T4 DNA ligase, and bovine alkaline phosphatase were purchased from Boehringer Mannheim, Meylan, France. When needed, antibiotics were added, as follows: ampicillin, 20 jig/ml (Beecham, Paris, France); tetracycline, 15 ,ug/ml (Diamant, Paris, France); and chloramphenicol, 15 ,ug/ml (Hoechst-Roussel, Paris, France). Analytical isoelectric focusing. Crude enzyme extracts were obtained by sonication of bacterial suspensions at 4°C and were kept frozen at -20°C until use. Analytical isoelectric focusing was performed on 6% acrylamide gels containing ampholytes with a pH range from 3.5 to 9.5 (LKB products) at a constant power of 4 W with a maximum of 1,500 V for 6 h. ,-Lactamase activity was detected by pouring 10 ml of a 500-,uglml nitrocefin solution on the gel. The exact pl was determined by analytical isoelectric focusing in a sucrose density gradient with an LKB 8101 column, according to the recommendations of the manufacturer (LKB Instruments, Inc., Pharmacia LKB, France).

Corresponding author. 242



VOL. 35, 1991

TABLE 1. Bacterial strains and plasmids used in this study Characteristics

Bacterial strain or plasmid

Bacterial strains P. multocida LNPB 9 P. haemolytica LNPB 51 P. multocida LNPB 86 P. haemolytica LNPB 1721 P. haemolytica LNPB 1899 P. haemolytica LNPB 1928 P. haemolytica LNPB 1998 P. haemolytica LNPB 2308 H. influenzae 86 242 H. influenzae F990 E. coli p453 E. coli RGN238 P. aeruginosa Dalgleish P. aeruginosa Cilote E. coli HB101 E. coli JM103

Plasmids pACYC184 pUC18 pBR322


LNPBa LNPB LNPB LNPB LNPB LNPB LNPB LNPB Clinical isolate producing ROB-1 (plasmid pCFF242) Reference strain for ROB-1 3-lactamase (plasmid RROb) Reference strain for SHV-1 Reference strain for OXA-1 Reference strain for CARB-1 (PSE-4) Reference strain for CARB-3 F- leu proA2 recA13 thi ara-14 lacYl galK2 xyl-5 mtl-l rpsL20 supE44 hsdS20(rB- mB-) supE thi A(lac-proAB) (F' traD36 proAB lacIq lacZ AM15)

31 31 31 This This This This This 30 40 39 15 19 28 9

Cloning vector, Cmr Tcr Cloning and sequencing vector, Apr Cloning vector, Tcr Apr (TEM-1)

13 49 44

study study study study study


a LNPB, Strains were isolated from calf lung at the Laboratoire National de Pathologie Bovine (Lyon, France). The strains produced ROB-1, and the ampicillin MIC for these strains was .32 ,ug/ml.

Preparation of DNA. Plasmid DNA was prepared by the procedure of Bimboim and Doly (7) and purified by isopycnic centrifugation in a cesium chloride-ethidium bromide gradient (32). Restriction endonucleases were used as suggested by the manufacturers. After endonuclease digestion, DNA fragments were separated by electrophoresis in a 1% agarose gel in Tris borate-EDTA buffer. Fragments to be used as probes or in subcloning experiments were recovered by electroelution (32). Blotting and hybridization procedures. (i) Southern blot. Transfer of plasmid DNA from the agarose gel to nylon membranes (HYBOND N+; Amersham France, Les Ulis, France) was carried out as described by Maniatis et al. (32). (ii) Dot blot. Plasmid DNAs of the reference strains encoding TEM-1, SHV-1, OXA-1, CARB-1, CARB-3, and ROB-1 ,-lactamases were spotted onto nitrocellulose filters (Schleicher & Schuell). DNA was denatured by placing nitrocellulose filters on filter paper (Whatman, Inc., Clifton, N.J.) that was wetted with 0.5 M NaOH-1.5 M NaCl for 7 min. The nylon (Southern blot) or nitrocellulose (dot blot) membranes were then transferred twice onto filter papers that were wetted with 2 M NaCl-1 M Tris for 3 min, washed with 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and dried at room temperature before UV fixation for 2 min. Probes were prepared by labeling DNA fragments with [a-32P]dATP by using a randomly primed DNA labeling kit (Amersham). The 1.8-kb Sau3A and 230-bp DraI fragments from P. haemolytica LNPB 51 plasmid DNA were tested as specific ROB-1 probes. Filters were prehybridized at 42°C for 4 h in 6 ml of 5 x SSC-50% formamide-5 x Denhardt solution (ix Denhardt solution is 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and 0.02% Ficoll)-50 mM sodium phosphate buffer (pH 6.5)-0.2% sodium dodecyl sulfate-heat denatured

calf thymus DNA (10 ,ug/ml). Hybridization was carried out under the same conditions (50% formamide, 42°C) for 18 h with 106 dpm of specific probes per ml. Filters were then washed twice at 42°C in 2x SSC-0.1% sodium dodecyl sulfate for 15 min and in 0.5x SSC-0.1% sodium dodecyl sulfate for 30 min. The blots were then autoradiographed overnight at -80°C by using Amersham Hyperfilm MP. Cloning strategy. DNA fragments from pPH51, the ROB1-encoding plasmid of P. haemolytica LNPB 51, were cloned as follows. The 1.8-kb Sau3A fragment was cloned into both BamHI-digested pACYC184 and pUC18; the 1.6-kb AluI and the 1-kb AluI-DraI fragments were cloned into pACYC184 cut with EcoRV. The 1.6-kb AluI, 1-kb AluI-DraI, 230-bp DraI, and 350-bp DraI-Scal fragments were cloned into SmaI-digested pUC18 (see Fig. 3). Sau3A and BamHI sites were compatible; AluI, DraI, EcoRV, Scal, and SmaI sites were blunt. Transformation of Escherichia coli HB101 or E. coli JM103 was performed by the CaCl2 procedure (32). DNA sequencing. Double-stranded plasmid DNA was sequenced by the dideoxynucleotide chain-termination method of Sanger et al. (41). Sequencing was performed with Sequenase by following the protocol supplied with the Sequenase kit by United States Biochemical Corp, Cleveland, Ohio. The universal 17mer M13(pUC) sequencing primer or the 17mer M13(pUC) reverse primer was used to sequence the fragments cloned in pUC18. Six oligonucleotides were also synthesized (CNRS URA 487; Institut Pasteur, Paris, France) and were used as internal primers. Computer-assisted sequence analysis. Nucleotide sequence data analysis and amino acid sequence comparisons were performed on a VAX computer by using the software package provided by the BISANCE system at CITI-2 in Paris. Paired alignment of amino acid sequences were done by the method of Kanehisa (27) with a genetic code matrix. The multiple alignment of class A ,-lactamases was im-










FIG. 1. Analytical isoelectric focusing gel. Crude enzyme extracts were subjected to isoelectric focusing on a polyacrylamide gel

and revealed by the addition of a 500-p.gIml nitrocefin solution. P-Lactamases with known pis were included. Lane a, CARB-3 (p1 5.75); lane b, OXA-i (pl 7.4); lane e, SHV-i (pl 7.7); lane f, TEM-1 (pl 5.4). The ROB-i P-lactamases from H. influenzae 86 242 and P. haemolytica LNPB 51 were loaded in lanes c and d, respectively.

proved manually. Gaps were inserted to find alignment between the sequences.



Nucleotide sequence accession number. The i,482-bp ROB-i P3-lactamase-encoding region is EMBL accession number X52872.

RESULTS Determination of the ROB-i isoelectric point. Figure 1 compares the isoelectric patterns of reference 13-lactamases with those of the ROB-i P3-lactamase. The P3-lactamases produced by both P. haemolytica LNPB 51 and H. influenzae F990 (the ROB-i-producing reference strain) had identical isoelectric points. The exact pl determined by isoelectric focusing in a sucrose density gradient was 8.6. Analysis of plasmid DNA by digestion with restriction endonucleases and hybridization experiments. To compare ROB-i-encoding plasmids within Pasteurella strains, plasmid DNA was prepared from eight P. multocida and P. haemolytica wild-type strains isolated in France (strains LNPB 9, 5i, 86, 1721, 1899, 1928, 1998, 2308). All these

strains were ampicillin resistant (MIC, .32 p.g/ml) because of production of a ROB-1 3-lactamase. Because this enzyme was first described in H. influenzae F990 (40) and was then found in H. influenzae 86 242, which was isolated in France (40), plasmid DNAs were also extracted from these two strains. All Pasteurella strains tested harbored from one to three plasmids (molecular mass range, 4 to 10 kb). Only one plasmid of 4.1 kb was common to seven strains. Strain P. haemolytica LNPB 1899 contained only this 4.1-kb plasmid, it was chosen for further analysis. Its restriction map was determined. No sites were found for the following 6-bp recognition sequence endonucleases: AccI, BamHI, BglII, EcoRI, EcoRV, HinclI, HindlIl, HpaI, KpnI, NcoI, PvuII, Sacl, Sall, SmaI, SspI, and SstI. Only one AvaI, two Hinfl, two ScaI, and three DraI sites were found. Since restriction endonucleases with a 4-bp recognition sequence, such as Sau3A, AluI, or TaqI, cut more frequently, they were used to compare the plasmids. Figure 2B shows the Sau3A patterns obtained with Pasteurella and Haemophilus plasmid DNAs. Among H. influenzae strains, Sau3A digestion patterns of plasmid DNAs from strains F990 and 86 242 were identical. They showed five fragments of 1.4, 1.1, 0.9, 0.7, and 0.3 kb (Fig. 2B, lanes 1 and 2). The 0.3-kb fragment, although weak, is present in lanes I to 10 of Fig. 2B. Moreover, a 2-kb fragment corresponding to uncut DNA appears in some lanes. P. haemolytica LNPB 1899 (the Pasteurella strain that harbored only the 4.1-kb ROB-1encoding plasmid) yielded four fragments of 1.8, 1.1, 0.9, and 0.3 kb (Fig. 2B, lane 7). Other Pasteurella strains showed more complex patterns because of the presence of at least two plasmids. However, the same Sau3A fragments (1.8, 1.1, 0.9, and 0.3 kb) were detected in the following six strains, as shown in Fig. 2B: LNPB 9 (lane 3), LNPB 51 (lane 4), LNPB 86 (lane 5), LNPB 1721 (lane 6), LNPB 1928 (lane 8), and LNPB 1998 (lane 9). P. haemolytica LNPB 2308 showed a Sau3A pattern slightly different from those of other Pasteurella strains (Fig. 2B, lane 10). It lacked the 1.8-kb Sau3A fragment but presented 1.4- and 0.7-kb fragments identical to those found in Haemophilus strains. Therefore, three fragments (1.1, 0.9, and 0.3 kb) seemed to be common to all the ROB-1-producing plasmids from Pasteurella and Haemophilus strains, suggesting a close relationship. This hypothesis was confirmed by hybridization experiments after Southern transfer (Fig. 2C). The 4.4-kb plasmid RR.b digested with Sau3A was used as a probe. It hybridized with all the Sau3A fragments of pPH1899 (Fig. 2C, lane 7). Hybridization patterns were identical in all the Pasteurella strains (Fig. 2C, lanes 3 to 9), except P. haemolytica LNPB 2308 (Fig. 2C, lane 10), which gave a profile similar to those of pCFF242 and RRob (Fig. 2C, lanes 1 and 2). These results indicated that the plasmids specifying ROB-1 in Haemophilus and Pasteurella strains are very similar. Cloning experiments. To localize more precisely the ROB1-encoding gene, the following restriction fragments from P. haemolytica LNPB 51 were cloned: the 1.8-kb Sau3A fragment into BamHI-digested pACYC184 and the 1.6-kb AluI and 1-kb AluI-DraI fragments into EcoRV-digested pACYC184 (Fig. 3). When examined by isoelectric focusing, only recombinant plasmids harboring the 1.8-kb Sau3A fragment cloned into pACYC184 were found to produce ROB-1. The 1.6-kb AluI fragment led to unstable recombinant clones with a very low level of 1-lactamase expression. Other recombinant clones obtained with the 1-kb AluI-Dral fragment lacked 1-lactamase activity.


VOL. 35, 1991




FIG. 2. Comparison of Sau3A fragment patterns of plasmids encoding the ROB-1 ,-lactamase. (A) Schematic representation of plasmid DNAs from H. influenzae 86 242 (lane 1) and P. haemolytica 1899 (lane 2) digested with Sau3A. (B) Agarose gel electrophoresis of Sau3A-digested plasmid DNAs from H. influenzae F990 (lane 1), H. influenzae 86 242 (lane 2), P. multocida LNPB 9 (lane 3), P. haemolytica LNPB 51 (lane 4), P. multocida LNPB 86 (lane 5), P. haemolytica LNPB 1721 (lane 6), P. haemolytica LNPB 1899 (lane 7), P. haemolytica LNPB 1928 (lane 8), P. haemolytica LNPB 1998 (lane 9), P. haemolytica LNPB 2308 (lane 10), E. coli HB101(pBR322) (lane 11), and E. coli HB101(pACYC184) plus the 1.8-kb Sau3A fragment from pPH51 (lane 12). (C) Southern blot of the gel shown in panel B probed with Sau3A-digested RRob plasmid. Numbers on the left are molecular sizes (in kilobases).

DNA sequencing of the ROB-1 j8-lactamase. The strategy and fragments used for dideoxy sequencing are shown in Fig. 3. Approximately 80% of the sequence was determined from both strands. A 1,482-bp sequence and the deduced amino acid sequence of ROB-1 are shown in Fig. 4. There is an open reading frame of 915 nucleotides, starting with an




ATG codon (nucleotides 221 to 223) which codes for a protein of 305 amino acid residues and which terminates with TAA (nucleotides 1136 to 1138). The ATG codon is separated by 7 bp from the sequence AAGG, which is a putative ribosome-binding site. Further -upstream are sequences which could be the -10 region (TAGACT, nucleotides 92 to

i_ A i1 11 111I 1~~~~~~~

a am I 0cc 0 1144



Fzz1,f 6A




1 00 bp 1.8 Kb Sau3A


1.6 Kb Alul 1.0 Kb Alul-Dral

box FIG. 3. Restriction map and cloning and sequencing strategies. A 2-kb region from P. haemolytica LNPB 51 is shown. The hatched The represents the open reading frame. The 1.8-kb Sau3A, 1.6-kb AluI, and 1-kb AluI-DraI fragments cloned in pACYC184 are indicated. a vertical arrows below the map represent the individual DNA fragments that were sequenced and their polarities. An arrow beginning with Asterisks into pUC18. subcloned a and fragment reverse the or primer universal primer the using derived by a sequence bar represents represent sequences generated by using synthesized primers specific to a sequence within the clone.



97) and the -35 region (TTGAGA, nucleotides 70 to 75) of a mass of the pre-3lactamase is 33,830 Da. Analysis of the deduced amino acid sequence. The deduced amino acid sequence of the ROB-1 P-lactamase was compared with the sequences of related class A enzymes (Fig. 5). In the multiple alignment, gaps were introduced to maximize the homology. Amino acid sequences of putative or known mature f-lactamases were aligned in pairs. The number of identities between sequences was scored. Homology was expressed as a percentage of identical amino acids of the total number of amino acids in the longer sequence. From these alignments, a similarity matrix table was deduced (Table 2). A 32 to 47.5% homology was observed between the amino acid sequence of the P. haemolytica ROB-1 ,-lactamase and other class A ,-lactamases. With the exception of the S. aureus PC1 P-lactamase, the ROB-1 1-lactamase amino acid sequence presented a high degree of homology (>40%) with those of gram-positive bacterial enzymes. This observation was most obvious in the C-terminal region of amino acid sequences (residues 251 in the numbering of Ambler [1] to the C terminus), where ROB-1 and gram-positive enzymes shared 24 to 38% homology but only 7 to 5% homology with gram-negative bacterial P-lactamases (TEM-1, SHV-1, LEN-1). The insertion of two gaps in the region from nucleotides 80 to 90 (Fig. 5) was necessary to obtain an optimal matching. These gaps needed to be inserted only in gram-positive bacterial P-lactamases and ROB-1. Moreover, ROB-1 and gram-positive bacterial P-lactamases shared an asparagineaspartic acid doublet at positions 245 to 246. In the mature ROB-1 amino acid sequence, there was only a single cysteine residue (position 123 in the numbering of Ambler [1]), whereas other gram-negative enzymes except the Klebsiella oxytoca 3-lactamase all contained two cysteine residues (positions 77 and 123). Analysis of the multiple alignment also revealed the existence of several residues that were identical in all the sequences but that were not located in one of the areas of homology (boxes) defined by Joris et al. (25). Some of these residues (threonine-71, valine-103, serine-130, aspartic acid131, asparagine-132, leucine-169) were shown to be part of the active site of S. aureus PC1 (20). The codon usage in the synthesis of the ROB-1 ,-lactamase from P. haemolytica is identical to those of the 11 sequenced genes from the family Pasteurellaceae in GenBank. Hybridization with DNA probes. To verify that the ROB-1 determinant was not genetically related to other plasmid ,-lactamase genes and to develop a ROB-1-specific molecular probe, dot blot hybridization experiments were undertaken. The filters were probed under stringent conditions with either the 1.8-kb Sau3A fragment carrying the ROB-1 gene or an intragenic 230-bp DraI fragment of pPH51. Neither probes hybridized to plasmid DNAs from strains producing TEM-1, OXA-1, SHV-1, CARB-1, or CARB-3 and hybridized only with total DNA from ROB-1-producing strains (data not shown).

promoter. The calculated molecular

DISCUSSION The apparent pl of the P-lactamase produced by a P. multocida strain (LNPB 9) isolated in France was estimated to be 8.8 (38). The enzyme was later identified as the ROB-1 P-lactamase already described in H. influenzae strains, which has an estimated pl of 8.1 (40). In our hands, the two


enzymes focused at the same point and the pl, which was determined by analytical isoelectric focusing in a sucrose density gradient, was estimated to be 8.6. Such differences between pIs are not surprising and could be due to experimental conditions (5). Moreover, we lack reliable standards in the alkaline range of pIs. Wray' and Morrison (48) described a 1-lactamase that is produced by a P. haemolytica strain and estimated its pI to be 9.35. It would be of interest to compare the pI of this enzyme and that of ROB-1 on the same isoelectric focusing gel. In all eight Pasteurella strains tested, the ROB-1 1-lactamase was plasmid encoded. In seven of the eight strains (LNPB 9, 51, 86, 1721, 1899, 1928, 1998), the ROB-1encoding plasmid had a molecular mass of 4.1 kb. Thus, two types of ROB-1-specifying plasmids have been described. One (4.1 kb) was only found in Pasteurella strains; the other (4.4 kb) was found not only in H. influenzae strains (F990 and 86 242) (30, 40) but also in one P. haemolytica strain (LNPB 2308). These ROB-1-encoding plasmids from Pasteurella and Haemophilus strains were compared by restriction endonuclease analysis and hybridization experiments. Although a single clonal plasmid was not demonstrated, the two types of plasmids involved in ROB-1 production in members of the family Pasteurellaceae shared multiple homologous fragments, suggesting that one plasmid is derived from the other. F990, the original ROB-1-producing H. influenzae strain, was isolated in Maryland and harbors the 4.4-kb plasmid RRob. RRob and plasmid DNAs from porcine A. pleuropneumoniae or human H. influenzae ROB-1-producing strains showed identical restriction patterns (34). These strains were isolated in different states throughout the United States. H. influenzae 86 242 and LNPB Pasteurella strains were collected in France. Their ROB-1-encoding plasmids were shown to be very similar to RROb. These observations confirm the close relationship between ROB-1-specifying plasmids, regardless of the strains' geographical origin and the original host (16, 34). As reported by Daum et al. (16), the earliest ROB-1producing H. influenzae strain was isolated in 1979, P. multocida LNPB 9 was isolated in 1978, and Pasteurella aerogenes ATCC 27883 was isolated prior to 1973 (31). It is therefore likely that ROB-1 originated in Pasteurella strains. Only one plasmid was previously described in P. haemolytica LNPB 51 (31). Actually, this strain harbored two plasmids with identical molecular weights but with very different restriction patterns. The restriction map of the ROB-1-encoding plasmid from P. haemolytica LNPB 1899 revealed the low frequency of restriction endonuclease sites. Such a phenomenon has already been reported by Mayer and Robbins (33), who established a map of the 7.1-kb ,B-lactamase-encoding plasmid of Neisseria gonorrhoeae. With the set of enzymes that were used, there were only 12 sites per kilobase in the DNA portion of Haemophilus origin compared with 51 sites per kilobase in the TnA portions. This could be related to the G+C content, which is about 40% in members of the family Pasteurellaceae. Moreover, only three DraI sites were found by overnight digestion of plasmid DNA with DraI, although the DNA sequence presented two additional DraI sites (AAATTT, nucleotides 190 to 195 and 1165 to 1170). We previously showed that ROB-1 is chromosomally encoded in one P. aerogenes strain (31). In all the other strains studied, ROB-1 is encoded by small plasmids. Thus, the ROB-1-encoding gene could be part of a transposable element. A total of 22% of P. multocida isolates from

VOL. 35, 1991



acaagcaaattgcatacctagaggcgaacttacttgagaatatcaaagtgcatttcattgtagaaagcattgasLattaaaggctaaattgctagactttt 100 gcgcttaaattcgccaaaatctgtttttttgttctacaaacaaattatccgaatttccgcgcaatgtacgtttcaggctgcctgaaaagaaatttattta


gtaaaatcaaaggataattt ATG TTA MT MG TTA AAA ATC GGC ACA TTA TTA TTG CTG ACA TTA ACG GCT TGT TCG CCC 280 Met Leu Asn Lys Leu Lys lIe Gly Thr Leu Leu Leu Leu Thr Leu Thr Ala Cys Ser Pro 20

MT TCT GTT CAT TCG GTA ACG TCT MT CCG CAG CCT GCT AGT GCG CCT GTG CM CAA TCA GCC ACA CM GCC ACC Asn Ser Val His Ser Val Thr Ser Asn Pro Gin Pro Ala Ser Ala Pro Vai Gin Gin Ser Ala Thr Gin Aia Thr

355 45

TTT CM CAG ACT TTG GCG MT TTG GM CAG CAG TAT CAA GCC CGA ATT GGC GTT TAT GTA TGG GAT ACA GAA ACG Phe Gin Gin Thr Leu Ala Asn Leu Glu Gin Gin Tyr Gin Ala Arg Ile Gly Val Tyr Val Trp Asp Thr Giu Thr

430 70

GGA CAT TCT TTG TCT TAT CGT GCA GAT GMA CGC TTT GCT TAT GCG TCC ACT TTC MG GCG TTG TTG GCT GGG GCG Gly His Ser Leu Ser Tyr Arg Ala Asp Glu Arg Phe Ala Tyr Ala Ser Thr Phe Lys Aia Leu Leu Ala Gly Aia

505 95

GTG TTG CM TCG CTG CCT GM AAA GAT TTA MT CGT ACC ATT TCA TAT AGC CM AM GAT TTG GTT AGT TAT TCT Val Leu Gin Ser Leu Pro Glu Lys Asp Leu Asn Arg Thr Ile Ser Tyr Ser Gin Lys Asp Leu Val Ser Tyr Ser

580 120

CCC GAA ACC CAA MA TAC GTT GGC AAA GGC ATG ACG ATT GCC CAA TTA TGT GAA GCA GCC GTG CGG TTT AGC GAC Pro Glu Thr Gln Lys Tyr Val Gly Lys Gly Met Thr Ile Ala Gln Leu Cys Glu Ala Ala Val Arg Phe Ser Asp

655 145

MC AGC GCG ACC MT TTG CTG CTC AAA GAA TTG GGT GGC GTG GAA CAA TAT CAA CGT ATT TTG CGA CM TTA GGC Asn Ser Ala Thr Asn Leu Leu Leu Lys Glu Leu Gly Gly Val Glu Gln Tyr Gln Arg ILe Leu Arg Gln Leu Gly

730 170

GAT MC GTA ACC CAT ACC MT CGG CTA GM CCC GAT TTA MT CM GCC AM CCC MC GAT ATT CGT GAT ACG AGT Asp Asn Val Thr His Thr Asn Arg Leu Glu Pro Asp Leu Asn Gln Ala Lys Pro Asn Asp Ile Arg Asp Thr Ser

805 195

ACA CCC AA CM ATG GCG ATG MT TTA MT GCG TAT TTA TTG GGC MC ACA TTA ACC GM TCG CM MA ACG ATT Thr Pro Lys Gin Met Ala Met Asn Leu Asn Ala Tyr Leu Leu Gly Asn Thr Leu Thr Glu Ser Gin Lys Thr lie

880 220

TTG TGG MT TGG TTG GAC MT MC GCA ACA GGC MT CCA TTG ATT CGC GCT GCT ACG CCA ACA TCG TGG MA GTG Lou Trp Asn Trp Leu Asp Asn Asn Ala Thr Giy Asn Pro Leu Ile Arg Ala Ala Thr Pro Thr Ser Trp Lys Val

955 245

TAC GAT AA AGC GGG GCG GGT MA TAT GGT GTA CGC MT GAT ATT GCG GTG GTT CGC ATA CCA MT CGC AA CCG 1030 Tyr Asp Lys Ser Gly Ala Gly Lys Tyr Gly Val Arg Asn Asp lie Ala Val Val Arg Ile Pro Asn Arg Lys Pro 270

ATT GTG ATG GCA ATC ATG AGT ACG CM TTT ACC GM GM GCC MA TTC MC MT MA TTA GTA GM GAT GCA GCA 1105 Ile Val Met Ala lie Met Ser Thr Gln Phe Thr Glu Gtu Ala Lys Phe Asn Asn Lys Leu Val Gtu Asp Ala Ala 295

MG CM GTA TTT CAT ACT TTA CAG CTC MC TM caaattcattttgttagaaaaagtgaaaatttagggctgaatttacaatccaagtg 1194 305 Lys Gin Vai Phe His Thr Leu Gin Lou Asn ***

caacaaaaaagaagtactcatcaactagaatataattttgtttccacacaaaaaccacttccaaatgatgagtacttcctaccgacatcttacaataagc 1294 1394



FIG. 4. Nucleotide sequence of the 1,482-bp ROB-1 P-lactamase-encoding region. Bases underlined with dashes may constitute a ribosome-binding site. The putative -35 (nucleotides 70 to 75) and -10 (nucleotides 92 to 97) transcriptional initiation signals are underlined. The predicted amino acid sequence for ROB-1 13-lactamase (bp 221 to 1138) is shown beneath the DNA sequence. The stop codon is represented by three asterisks.









I *





** *







TEM-1 K.p LEN-1 SHV-1 PSE-3 PSE-4 RP.c 108





4Ha30 *


IV *




*** **

























1+k+++ 210 S.a PCI B.c 569/HI

111*+ .3



















VOL. 35, 1991


TABLE 2. Similarity matrix of class A P-lactamase protein sequences % Homology between amino acid sequencesa


PSE-4 PSE-3 SHV-1 K.p LEN-1 TEM-1 K.o.E23004 S.c 40057 Act R39 B.l 749/C B.c 569/HI S.a PC1 ROB-1

RP.c 108






K.o E23004

S.c 40057

Act. R39

B.l 749/C

B.c 569/HI

S.a PC1

38.5 39.1 40.6 39.1 40.8 37.4 32.5 35.5 30.8 33.3 23.2 31.7

44.2 44.8 42.0 42.0 36.2 33.0 29.7 28.4 35.3 33.7 34.3

47.1 45.5 46.6 38.3 33.7 41.2 34.6 39.5 30.0 36.6

86.4 67.5 38.0 36.4 32.5 32.1 33.8 27.7 36.3

64.6 37.5 36.7 31.8 34.0 34.2 27.2 36.7

38.6 35.9 31.4 35.1 35.7 30.2 37.1

40.1 40.3 39.6 39.2 30.9 40.6

54.6 46.3 45.7 34.3 42.8

49.6 51.7 34.1 44.5

56.2 41.0 41.7

37.5 47.5


a See the legend to Fig. 5 for

P-lactamase abbreviations.

turkeys contain small cryptic plasmids. These strains also harbor R plasmids encoding for resistance to tetracycline. The R plasmids appeared to be a result of the insertion of a tetracycline resistance-encoding transposon into small cryptic plasmids (21). Moreover, TEM-1 P-lactamase-encoding plasmids from N. gonorrhoeae, H. influenzae, Haemophilus parainfluenzae, and Haemophilus ducreyi arose by insertion of a transposon from the TnA family into small cryptic plasmids (10, 14). Such a phenomenon might have occurred for ROB-1. However, attempts to demonstrate ROB-1 transposition failed, possibly because of the tendency of Neisseria and Haemophilus species to select for deleted transposons that lack transposase and regulation functions (33). Thus, it would be interesting to determine the sequence of flanking regions of the chromosomal ROB-1 gene we described previously (31). To determine the relatedness of ROB-1 ,B-lactamase to other enzymes, the ROB-1-encoding gene was cloned and sequenced. An open reading frame of 915 nucleotides was found (nucleotides 221 to 1135). A sequence which could be a promoter was found 123 bp upstream from the starting codon (ATG, nucleotides 221 to 223). The sequence TTGAGA (nucleotides 70 to 75) matched five of the six nucleotides found in the consensus E. coli -35 box TTGACA. The sequence TAGACT (nucleotides 92 to 97) matched four of the six nucleotides of the consensus - 10 box TATAAT. Thus, the putative promoter of the P. haemolytica ROB-1 gene shares characteristics with the consensus E. coli promoter. This nucleotide sequence was compared with that of the ROB-1 ,-lactamase from H. influenzae plasmid RROb (26). Both sequences were identical from nucleotides 81 to 1179 of the Pasteurella gene (nucleotides 163 to 1261, respectively, of the Haemophilus gene) except for two mutations. One was located in the open reading frame at position 400 (nucleotide A in the Pasteurella gene versus C in the Haemophilus gene). This mutation occurred

on the third base of the arginine-60 codon and thus did not alter the amino acid sequence. The other mutation was located in the 3'-flanking region at position 1174 (nucleotide G in the Pasteurella gene versus nucleotide A in the Haemophilus gene). In contrast, the 5'-flanking sequences (upstream of nucleotide 81) and the 3'-flanking sequences (downstream of nucleotide 1179) were completely different in Pasteurella and Haemophilus ROB-1 genes. The 5' change occurred between the - 10 and -35 sequences of the Pasteurella gene promoter; thus, the ROB-1 ,-lactamases genes from Pasteurella and Haemophilus species have different promoters. This does not seem to affect the promoter's strength, because the MICs of ampicillin for Pasteurella and Haemophilus strains are comparable (30, 31). However, the finding of identical ROB-1 genes with different flanking sequences within very similar encoding plasmids brings up the question of the origin and spread of the ROB-1 ,-lactamase gene within the family Pasteurellaceae. 1-Lactamases are exported through membranes, and they are synthesized as a precursor with a typical signal peptide. The signal peptide of gram-negative bacterial P-lactamases consists of about 20 amino acids (24, 43). A 33-amino-acid signal peptide was arbitrarily chosen by Juteau and Levesque (26) for the ROB-1 P-lactamase of H. influenzae. By using the method of von Heijne (45) and analysis of amino acid alignment with other class A P-lactamases, a putative cleavage site for ROB-1 was located between alanine-44 and threonine-45, leading to an unusual 44-aminoacid signal peptide. However, several P-lactamases from gram-positive bacteria harbor a 33- to 48-amino-acid signal peptide (17, 22, 36). Moreover, ROB-1 was found to present the highest similarity with the P-lactamase of Bacillus cereus 569/H1, whose signal peptide is 43 amino acids in length (47). Determination of the NH2-terminal sequence of the mature ROB-1 1-lactamase should confirm this unusual feature. The amino acid sequence of the putative mature P-lacta-

FIG. 5. Amino acid alignment of class A P-lactamases. The numbering system for the residues was that of Ambler (1). One additional residue was observed in the PSE-3 amino acid sequence between positions 110 and 120. Gaps were introduced to optimize the alignment. Boxes were defined by conserved or homologous residues among the active-site-serine penicillin-recognizing enzymes (25). Secondary structures of the S. aureus PC1 P-lactamase are indicated by a (a helix) or P (p sheet) followed by a number described by Herzberg and Moult (20). The residues identical in all the sequences are marked by asterisks. Abbreviations for p-lactamases are as follows: S.a PC1, Staphylococcus aureus PC1 (46); B.c 569/HI, Bacillus cereus 569/HI (42); B.l 749/C, Bacillus licheniformis 749/C (37); Act. R39, Actinomadura R39 (22); S.c 40057, Streptomyces cacaoi DSM 40057 (18); ROB-1, P. haemolytica LNPB 51 (this study); K.o E23004, Klebsiella oxytoca E23004 (4); TEM-1, TEM-1 (43); K.p LEN-1, Klebsiella pneumoniae LEN-1 (3); SHV-1, SHV-1 (6); PSE-3, PSE-3 (12); PSE-4, PSE-4 (8); and RP.c 108, Rhodopseudomonas capsulata sp 108 (12).



mase was compared with the sequences of class A r-lactamases. ROB-1 showed about 40% homology (from 32 to 48%)

with all the known class A enzymes. Moreover, our data confirmed that ROB-1 is the enzyme from gram-negative bacteria which presents the highest similarity with the 13-lactamases from gram-positive bacteria. To get the best alignment, two gaps of one amino acid each were introduced (between amino acids 80 and 90) in the sequences of 1-lactamases from gram-positive bacteria. These gaps (positions 86 and 90) were also observed in the ROB-1 sequence. Around the consensus active site, ROB-1 contains a region identical to that of the S. aureus PCI enzyme (alanine-67, tyrosine-68, alanine 69) but shares the active-site STFK tetrad (serine-70, threonine-71, phenylalanine-72, lysine-73) with the gram-negative bacterial enzymes TEM-1, SHV-1, LEN-1, and PSE-4 and the gram-positive bacterial P-lactamase of Streptomyces cacaoi DSM 40057. Finally, ROB-1 contains in its amino acid sequence an asparagine-aspartic acid doublet (amino acids 245 to 246) which is only found in the 3-lactamases from gram-positive bacteria. Although ROB-1 is a gram-negative bacterial P-lactamase found only in members of the family Pasteurellaceae, it presented a high degree of homology with the enzymes from grampositive bacteria. Another striking feature of the ROB-1 protein is the presence of only one cysteine residue at position 123. All the 1-lactamases from gram-negative bacteria (except the K. oxytoca enzyme) contained two cysteine residues at positions 77 and 123. These residues are involved in the formation of a disulfide bond, which is important for enzyme stability. None of the gram-positive bacterial f-lactamases contained these cysteine residues; however, the Streptomyces cacaoi DSM 40057 enzyme had both of them. Thus, ROB-1 is the only 1-lactamase which has only one of the two cysteine residues. These features strongly indicate that ROB-1 could be a link between the 3-lactamases of grampositive and gram-negative bacteria. Whether or not the ROB-1-encoding gene has recently arisen in Pasteurella species from gram-positive bacteria remains to be elucidated. Epidemiological studies can be a useful way of elucidating the origin of an enzyme and how it spreads. Detection of the ROB-1 P-lactamase cannot be done by the analysis of antibiograms because its antimicrobial substrate profile is similar to that of TEM-1. A valuable alternative to pl determination is the use of DNA probes. A ROB-1 DNA probe was described previously (29), but it was later shown not to be intragenic from sequencing data (26). We tested two ROB-1 probes: the 1.8-kb Sau3A fragment and the 230-bp Dral fragment of the ROB-1-encoding plasmid of P. haemolytica LNPB 51. As expected from sequence findings, neither of these two probes hybridized with TEM-i-, SHV1-, OXA-i-, CARB-i-, or CARB-3-encoding genes. The 1.8-kb Sau3A fragment contains sequences outside of the structural gene, whereas the 230-bp DraI fragment is intragenic. This latter fragment is therefore suitable as a ROB-1specific probe for further molecular epidemiological studies. ACKNOWLEDGMENTS We thank Christiane Forestier and Kristin Swihart for critical reading of the manuscript. REFERENCES 1. Ambler, R. P. 1980. Structure of ,-lactamases. Phil. Trans. R. Soc. London Biol. Sci. 289:321-331. 2. Ambler, R. P., and G. K. Scott. 1978. Partial amino acid




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Sequence and molecular characterization of the ROB-1 beta-lactamase gene from Pasteurella haemolytica.

The ROB-1 beta-lactamase-encoding plasmids from eight Pasteurella and two Haemophilus strains were compared by restriction endonuclease and hybridizat...
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