cloning of the determinants for microcin d93 production and analysis of three different d-type...

PLASMID 23,2 16-225 (1990)

Cloning of the Determinants for Microcin D93 Production and Analysis of Three Different D-Type Microcin Plasmids

Jo& L. MARTfNEZ’ AND Jo& c. %REZ-DfAZ2

Setvicio de Microbiologia, Hospital Ram& y Cajal, Ctra. Colmenar km. 4100, 28034 Madrid, Spain

Received February 7. 1990; revised May 8, 1990

Three different microcin plasmids coding for D-type microcins were analyzed. Two of the plasmids (pMccD93 and pCPl0 1) were small, multicopy plasmids and were closely related. The third plasmid (pCP106) was a conjugative, antibiotic multiresistance plasmid. Although plasmids pCPlO1 and pCP106 were previously classified as A-type microcin plasmids, we have determined that they are, in fact, D type. Furthermore, the determinants for microcin D93 production were cloned from plasmid pMccD93, and a DNA probe for the region implicated in the synthesis of microcin was obtained. This probe hybridized to plasmid C from Escherichia co/i strain V5 17, indicating that this plasmid might be involved in the synthesis of a D-type microcin. The characteristics of replication of plasmid pCP106 were analyzed and appeared to be similar to those of ColEl plasmids, although pCP106 is a conjugative single-copy plasmid. ~1 1990 Academic

Press, Inc.

The term microcins was proposed to define a group of low-molecular-weight antibacterial substances produced mainly by Enterobacte- riaceae (Asensio et al.) 1976). Microcins are hypothesized to play an ecological role in the interactions between microorganisms in the intestinal ecosystem (Asensio et al., 1976; Ba- quero and Asensio, 1979; De Lorenzo and Aguilar, 1984). These molecules differ from colicins in their low molecular weight and their relative insensitivity to proteases. They are synthesized constitutively, independently of SOS system inducing agents (Baquero and Moreno, 1984). However, as in the case of colicins, genetic determinants for the synthesis of microcins are located on plasmids (Baquero et al., 1978, Baquero and Moreno, 1984).

Microcins have been classified into five groups according to cross-immunity criteria (Baquero and Moreno, 1984). Among the microcinogenic strains so far studied, more than 50% encoded the synthesis of a microcin be-

’ Present address: Instituto de Investigaciones Biomt- dicas de1 CSIC Facultad de Medicina de la Universidad Autbnoma. Arzobispo Morcillo 4, 28029 Madrid, Spain.

’ To whom correspondence should be addressed.

longing to group D (F. Sanchez, Ph.D. Thesis, Autonoma University, Madrid, 1981). A pro- totypical microcin belonging to this group is microcin D93. This molecule is a small (ca. 500 Da), hydrophilic, basic peptide, is active against Escherichia coli, Proteus, Citrobacter, and Pseudomonas, and seems to produce its primary effect at the level of DNA biosynthesis (Martinez and Perez-Diaz, 1986). Both synthesis and immunity for microcin D93 are encoded by a 5.5-kb plasmid that was named pMccD93 (Baquero and Moreno, 1984).

Although the genetics of several microcins have been extensively studied (Baquero et al., 1978; Genilloud et al., 1989; Hemindez-Chico et al., 1986; Novoa et al., 1986; Perez-Diaz and Clowes, 1980; San Millin, et al., 1985a,b), there are no available studies on the genetics of D-type microcins, the major group. In the present work, we have characterized the plasmid pMccD93, which is responsible for the synthesis of microcin D93, and cloned the determinants for the synthesis of this microcin. We have also characterized two other D-type microcin plasmids that were previously described as A-type microcin plasmids (Perez- Diaz and Clowes, 1980).

0 147-6 19X/90 $3.00 216 Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

D-TYPE MICROCIN PLASMIDS 217

MATERIALS AND METHODS

Bacterial Strains and Plasmids

biotics for the selection of transformants: ampicillin, 100 &ml; tetracycline, 10 pg/ml; kanamycin, 40 pg/ml.

Bacterial strains used in this work are listed in Table 1. Plasmid pBR322 was from our laboratory collection. Plasmid R6K (Kon- lomichalou et al., 1970) was obtained from R. Clowes. Plasmids pUC8 and pUC-4K were obtained from Pharmacia (Madrid, Spain).

E. coli RC20 1 was grown in LB broth. Sub- sequent subcultures ( 1: 100) of the bacteria at 42°C were made until the desired number of generations was reached.

Culture Conditions and Microcin Analysis

Determination of the plasmid copy number was performed in M63-glucose supplemented with 0.1% casamino acids, 250 pg/ml 2’- deoxyadenosine, 5 pg/ml thymidine, and [3H]thymidine (to 0.5 pCi/ml).

The tests for microcin production and immunity were performed as described (San Millan et al., 1985a) in minimal medium M63 with 0.2% (wt/vol) glucose (Miller, 1972) using E. coli RYC8 19 as susceptible strain. E. coli strains BM21 and 93F were used respectively as negative and positive controls of microcin production. The tests to distinguish D- type microcin from A-type microcin were performed as described (Aguilar et al., 1983). Microcin quantification was performed and 1 activity unit (au) defined as previously described (Garcia-Bustos, et al., 1984).

Antibiotic Susceptibility Tests

Susceptibility testing was performed for streptomycin, kanamycin, gentamycin, amy- kacin, ampicillin, cefoxitin, cloxacillin, ce- phalotin, cefazolin, cefamandole, cefotaxime, ceftazidime, aztreonam sulfonamide, tri- methoprim, tetracycline, fosfomycin, and chloramphenicol, using standard susceptibility criteria (Barry and Thornsberry, 1980).

P-Lactamase Analysis

Selection of transformants was carried out Identification of P-lactamase from strains in LB-agar medium (Maniatis et al., 1982), carrying the plasmid pCP 106 was performed using the following concentrations of anti- by isoelectric focusing as previously described

strain Phenotype/genotype

TABLE 1

E. cob STRAINS USED

Ref. origin

BM21

HBlOl

v517

93F

RYC819

F-gyr A(l+)

Fhsd R-hsd MrecAsupE lac 21~~ Bpro Athr thi

Prototrophic

like BM2 1 (pMccD93)

Fara D139 Alac U169 Amal B 1 rps L relA thi A ret A56

RC20 1 his arg G met B leu rps L thy xyl lac Y pol A 0’s)

JCL16 like BM21(R6K)

Baquero et al. ( 1978)

Boyer and Roulland-Dussoix (1969)

Laboratory collection


Ayers and McCowen ( 1978)

Baquero and Moreno (1984)

San Millan et al. (1985b)


F. Baquero

F. Moreno

Perez-D& and Clowes (1980)

R. Clowes

This work Laboratory collection

218 MARTiNEZ AND PfiREZ-DiAZ

(Matthew et al., 1975). TEM-1 /3-lactamase was obtained from E. coli strain HBlO 1 (pUC8) and used as a standard. Quantification of &lactamase was performed, using sonicated cell extracts, by spectrophotometric methods as previously described (Broad and Smith, 1983). Protein concentration was determined by the method of Lowry et al. (195 1).

ally, the transformed strain was E. coli BM2 1. E. coli HB 10 1 was transformed for the direct selection of clones when the cloning vector was plasmid pUC8.

Restriction Endonucleases and DNAs

Restriction enzymes and DNA from phages X and 4X174, respectively cut with the enzymes HindIII and HaeIII, were obtained from New England Biolabs Inc. The enzyme T4 DNA ligase was obtained from Pharmacia (Madrid, Spain).

Preparation of DNA

Plasmid DNA was obtained and purified by ultracentrifugation in cesium chloride/ethidium bromide gradients as described (Maniatis et al., 1982). For the rapid analysis of clones, the niinilysis boiling method of Holmes and Quigley (198 1) was used.

Insertional mutations on plasmid pMccD93 were generated as follows: The plasmid was cut with the restriction endonuclease Suu3A 1 in the presence of ethidium bromide. The dye concentration was selected so that the enzyme cuts only once (Goppelt et al., 198 1). Plasmid pUC-4K was cut with BumHI, and the fragment encoding kanamycin resistance (Km)3 was purified as described above. After the elimination of the ethidium bromide, the Km fragment and the linearized plasmid pMccD93 were ligated, competent cells were transformed, and kanamycin-resistant colonies were selected. Insertional mutations on plasmid pCPl0 1 were obtained as described (Konlomichalou et al., 1970) using plasmid R6K as donor of transposon Tn3.

Hybridization Procedures

Purification of DNA fragments after electrophoresis was performed with DEAE-cellu- lose, as previously described (Lizardi et al., 1984).

Plasmid Manipulations

Restriction endonucleases were used according the manufacturer’s instructions. HindIII-cut X DNA and HaeIII-cut 4X174 DNA were used as molecular-weight markers.

Electrophoresis of DNA was performed in 0.8, 1 .O, or 1.5% horizontal agarose gels. Elec- trophoresis buffer was Tris-EDTA-borate (Maniatis et al., 1982).

DNA hybridization was carried out in ni- trocellullose filters by the method of Southern (1975). Conditions for hybridization were 6X SSC, 0.1% SDS, 5X Denhardt’s solution (Maniatis et al., 1982). The filters were washed twice in 2~ SSC, 0.1% SDS for 30 min each time and then twice more in 0.1 X SSC, 0.1% SDS for 30 min each time. Both hybridization and washing steps were carried on at 65°C. Probes were labeled using a nick-translation (Rigby et al., 1977) kit provided by BRL, with [cu-32P]dCTP.

RESULTS

Determination of plasmid copy number was performed as described (Nisioka et al., 1970). For the determinations of the plasmid copy number in the absence of protein synthesis, chloramphenicol was added at a concentration of 175 &ml.

Characterization of Plasmid pkfccD93

The physical map of plasmid pMccD93 was obtained by analyzing the molecular weights of the DNA fragments obtained after single or double digestions with the desired enzymes. This map, using eight different restriction endonucleases, is shown in Fig. 1. The enzymes

Genetic Methods

Transformation was carried out as previ- 3 Abbreviations used: Km, kanamycin; SDS, sodium ously described (Maniatis et al., 1982). Usu- dodecyl sulfate; SSC, standard sodium citrate.


pMccD93

pCPlOl

g I E

A B C 0 7 pCPIO6

4 5 Kb

FTG. 1. Restriction endonuclease maps of plasmids pMccD93, pCPl0 1, and pCP106. Insertions that did not inactivate microcin D93 synthesis are indicated by open circles. Insertions that inactivated the microcin production are showed as filled circles. The region of DNA used as a probe was the SmaI/AccI fragment that comprised the insertions inactivating microcin production.

XmaIII, &I, SphI, and &II did not cut this plasmid or plasmid pCPlO1 (see below).

In an attempt to identify the region of the plasmid involved in the synthesis of microcin D, we obtained mutants deficient in microcin production by inserting the fragment encoding kanamycin resistance from plasmid pUC-4K into the linearized plasmid pMccD93 (see Materials and Methods). We were not able to obtain mutations in the region implicated in the immunity to the microcin, probably because plasmids with such mutations were le- thal to the bacteria.

The location of the inserts into pMccD93 was determined by evaluation of the size of the different fragments of DNA obtained after the recombinant plasmids were cleaved with the restriction endonucleases SmaI, AccI, ClaJ, and PvuI. The positions of seven relevant inserts into plasmid pMccD93 are shown in Fig. 1. All of the insertions that inactivated the production of microcin (93K2, 93K4,93K5, 93KlO) were located in a small SmaI/AccI fragment of 0.6 kb, whereas insertions that did

not affect the synthesis of microcin (93K3, 93K9, 93Kll) were located outside the 2.3- kb PvuI/BamHI fragment. No other insertions that could further define the region involved in the synthesis of the microcin were obtained, probably because the determinants for micro- tin immunity are located in this region. At- tempts to obtain Ba131 deletion mutants (Maniatis et al., 1982) from the BumHI or PvuI points of pMccD93 were also unsuccessful (data not shown).

Cloning of Microcin 093 Production Determinants

Attempts to clone the determinants for microcin synthesis were unsuccessful, except when the entire plasmid was cloned. We have cloned the whole plasmid into the PvuI and BumHI sites of the vector pBR322 (Fig. 2). The Iirst chimera was named pPP 1, and strains carrying it produced the same quantity of microcin that E. coli 93F produced (ca. 2000 au/ mg of protein) (Martinez and Perez-D&,

220 MARTiNEZ AND Pl?REZ-DiAZ

FIG. 2. Strategy for cloning microcin D93 determinants. In all the plasmids: (-) DNA from plasmid pBR322; (0) DNA from plasmid pUC8; (m) DNA from plasmid pMccD93. The SmaI/Accl fragment shown is the same previously implicated in the synthesis of microcin D93 by insertion-mutation criteria (see Fig. 1 and text).

1986), while strains carrying the second one for bla in plasmid pBR322, and that this pro- (pPP2) produced 40 times more antibacterial moter is more potent than that of the original than the wild-type strain. microcin gene(s).

To determine whether this overproduction was due to a higher copy number of the recombinant plasmid, we determined the copy number of plasmids pMccD93 and pPP2. As Table 2 shows, pMccD93 was present as ca. 24 copies/chromosome, and pPP2 as ca. 22 copies/chromosome. Therefore, overproduction was not the consequence of a different copy number of the gene. A possible explanation is that the gene(s) encoding the synthesis of micro&r D93 is situated, in our con- struction, under the control of the promoter

The small BamHI fragment from plasmid pPP2 was cloned into pUC8 (Fig. 2). This new plasmid was named pPP3 and showed the same phenotype of microcin overproduction as plasmid pPP2. This result confirmed that the information for synthesis of micro& D93 is located in the 2.3-kb BamHI/fiuI fragment of plasmid pMccD93. Although the strains carrying plasmids pPP2 and pPP3 seemed to carry the determinants for microcin immunity by cross-streaking criteria, these plasmids were very unstable. This instability was more pro-

TABLE 2

Cow NUMBER OF DIFFERENT MICROCIN PLASMIDS

cpm in Plasmid Size (kb) plasmid peak

pMccD93 5.5 19,700 PPR 9.9 8,300 pCPlO6 30 4,500 pCP106 (Cm)’ 30 62,800

D Chloramphenicol was added at a concentration of 175 pg/ml.

cpm in COPY chromosome peak number

548,800 24 155,900 22 242,000 2 239,000 33


nounced when the bacterial host was a recA mutant, like E. coli HBlOl. Since microcin D93 exerts its effect at the level of DNA biosynthesis and is particularly active against recA mutant strains (Martinez and Perez-D&, 1986), it is conceivable that we have not cloned the whole region required for full microcin immunity.

Comparison between Plasmids pMccD93, pCPlO1, andpCP106

Plasmids pCP 10 1 and pCP 106 (Perez-Diaz and Clowes, 1980) were considered responsible for the synthesis of microcin Al5 (formerly 15m (Aguilar et al., 1982)). However, in our hands, and using cross-immunity criteria, these plasmids appeared to encode the production of D-type microcins. pCPl0 1 is a small (5.7 kb), nonconjugative plasmid, which is present in ca. 20 copies/chromosome (Perez- Diaz and Clowes, 1980). We have determined that the physical map of plasmid pCPl0 1 is very similar to that of pMccD93 (Fig. 1). The only difference is an insertion of ca. 150 bp which eliminates the BgZI site from pMccD93, introducing a new SmaI site into the sequence of the plasmid. This region did not appear to be involved in the synthesis of microcin. Fur- thermore, this plasmid showed positive hybridization with the 0.6-kb SmaI/AccI fragment from plasmid pMccD93, which contains at least part of the region implicated in the synthesis of microcin D93.

Insertions that did not inactivate the production of microcin were obtained with transposon Tn3. From one of these plasmids (pPPlO), a BamHI fragment was cloned into plasmid pBR322 (Fig. 3). This recombinant plasmid was named pPPl1, and strains carrying it were able to synthesize microcin. This BamHI fragment comprised the SmaI/AccI region involved in the synthesis of D93 microcin in plasmid pMccD93, confirming the similarity between pMccD93 and pCP 10 1.

Characterization of Plasmid pCP106

Plasmid pCP 106 is a conjugative plasmid with a molecular size of 30 kb and a copy

' BamHI

FIG. 3. Cloning of microcin determinants from plasmid pCP101.PlasmidpPPlOisaderivativeofplasmidpCPlOl obtained by the insertion of transposon Tn3. In all plasmids: (-) DNA from pBR322; (Cl) DNA from pCPlO1; (W) DNA from Tn3.

number of ca. 1 (Perez-Diaz and Clowes, 1980). This is the only microcin plasmid so far described that, besides microcin synthesis and immunity, also encodes antibiotic resistance (ampicillin and streptomycin (PCrez- Diaz and Clowes, 1980)). During the present work, we have detected that this plasmid also encodes for resistance to sulfonamides.

We have determined, by isoelectric focusing analysis, that the ampicillin resistance was due to the production of TEM-1 (Bush, 1989) p- lactamase. The quantity of P-lactamase produced by E. coli HB 10 1 transformed with the plasmid was 13 U/mg, which is a value higher than that previously described for other single- copy ampicillin-resistance plasmids (Reguera et al., 1988). Plasmid pCP106 was mapped with five different restriction enzymes (Fig. 1). When a BamHI digest of this plasmid was electrophoresed and hybridized with the whole pMccD93 plasmid under high stringency conditions, positive hybridization of BamHI fragments B and C, shown in Fig. 1, with molecular sizes of 5.1 and 7.6 kb, respectively, was detected. When the hybridization was performed using the 0.6-kb SmaI/AccI fragment from plasmid pMccD93, only the 5.1-kb band showed hybridization. Thus, this region ap-

222 MARTiNEZ AND PEREZ-DiAZ

pears to be that implicated in the synthesis of microcin in this plasmid.

Plasmid pV5 17C, from E. coli V5 17, used as standard for molecular weights of plasmid DNAs (Ayers and McCowen, 1978) also showed hybridization with this probe.

Characteristics of the Replication of Plasmid pCP106

The copy number of pCP 106 was evaluated in the presence and absence of chloramphenicol. Under “normal” conditions of growth, the plasmid was present in ca. 2 copies/chromosome, a value close to that previously described (Perez-Diaz and Clowes, 1980), and in the absence of protein synthesis, this value in- creased to 33 copies/chromosome.

The influence of DNA polymerase I on the replication of pCP106 was also evaluated by introducing the plasmid into a thermosensitive polA mutant. Derivatives of this strain carrying plasmid pCP106, plasmid pBR322, or plasmid Rl (as controls) were grown at restrictive temperature, with subsequent bacterial subcultures, at cellular densities between 10’ and 109. Samples were removed at different times, and the presence of the plasmids was determined by the ability to grow on ampicillin. As Table 3 shows, only 1O-6 of the host cells retained plasmid pCPlO6 after 16 generations, whereas plasmid Rl was fully maintained under the same conditions. It was then concluded that pCP106 needs an active polA gene for its replication.

DISCUSSION

The most detailed works on the genetics of microcin production that have been published are those concerning the synthesis of microcin B 17 (Genilloud et al., 1989; San Millan et al., 1985a,b) and microcin C7 (Novoa et al., 1986). In both cases, the determinants for the production of microcin are located in conjugative plasmids, and large regions of DNA (4- 5 kb) are involved in the synthesis of the antibiotics. These regions included four genes in the case of the synthesis of microcin B17 (Genilloud et al., 1989), and three at least in

TABLE 3

STABILITY OF pCP106 IN A polA MUTANT

Plasmid Total cfu”/ml Ap b cfu/ml Frequency

pCPlO6 1.8 X 10” 1.9 x lo4 10-f RI 2 x 1O’O 2 x 10’0 1 pBR322 1O’O 4x IO2 4 x 10-8

Note. The frequency of bacteria carrying the different plasmids was evaluated after growing 16 generations at restrictive temperature (see Materials and Methods).

a cfu, colony forming units. * Number of colonies resistant to ampicillin.

the case of the synthesis of microcin C7 (No- voa et al., 1986). These results indicated a somewhat high complexity in the genetics of the synthesis of these microcins.

In contrast, the synthesis of microcin D93 (Martinez and Perez-Diaz, 1986) is encoded by a small (5.5 kb) nonconjugative plasmid that resembles ColE 1 -type plasmids (Scott, 1984). The determinants for microcin D93 synthesis have been located on a 2.3-kb PvuI/ BamHI fragment. This size is the upper limit for the quantity of DNA necessary for the synthesis of microcin D93 and contrast with the 3.8 kb necessary for the synthesis of microcin B17 (Genilloud et al., 1989) and the 5 kb required to produce microcin C7 (Novoa et al., 1986), indicating that the genetics of production of microcin D93 are probably less com- plex than those of microcins B17 and C7.

The fragment encoding the synthesis of microcin D93 was cloned into plasmid pUC8. Strains carrying recombinant plasmid pPP3 appeared to be immune to the microcin by standard criteria. Indeed, the plasmid was quickly lost when these strains were grown without antibiotic selective pressure, particularly if the recipient strain was recA. We think that this phenomenon might be due to an im- balance between the immunity and the production of microcin, since microcin is over- produced by strains carrying plasmid pPP3. Alternatively, it can be thought that the fragment cloned did not include the whole system of immunity, as occurred in the case of the previous attempts for cloning Bl7 microcin


immunity (Garrido et al., 1988; San Mill&n et al., 1985a).

Microcin D93 is produced by E. coli LP93. This strain produced also another microcin which is antagonized by L-methionine (Mar- tinez and Perez-Diaz, 1986). Production of two microcins, one belonging to group A (antagonized by L-methionine (Aguilar et al., 1982)) and the other to group D (not antagonized by L-methionine (Aguilar et al., 1983)), is frequent among microcinogenic strains. The plasmids encoding production of microcins have been analyzed in different strains from this group (Perez-D& and Clowes, 1980). When this last work was published, production of a second activity by these strains had not yet been described, and hence it was thought that the microcin plasmids encoded the synthesis of A-type microcins (Perez-Diaz and Clowes, 1980). In the present work, we have determined that plasmids pCP 10 1 and pCP 106, formerly described as A-type micro- tin plasmids, are really D-type microcin plasmids. Plasmid pCP 10 1 is highly homologous to pMccD93, whereas plasmid pCP106 shared a 5.1 -kb region homologous to the region involved in the synthesis of microcin from plasmid pMccD93.

Plasmid pCP 106 also showed certain un- usual characteristics: (a) It is a conjugative plasmid that can replicate in the absence of protein synthesis and requires a functional DNA polymerase I, characteristics that are typical of ColEl-type plasmids (Scott, 1984). (b) It is the only microcin plasmid described so far that also determines antibiotic resistance. It has been suggested that the presence of antibiotic resistance and aerobactin (a system for bacterial iron uptake (De Lorenzo and Martinez, 1988)) in the same plasmid may en- hance the fitness of the bacterial host (Gonzalo et al., 1988). This suggestion was based on two facts. First, aerobactin production will contribute to the growth of the bacteria under low- iron conditions, which are the usual conditions in the vertebrate tissues (Weinberg, 1984). Second, antibiotic resistance is an important selective marker, as the consequence of the indiscriminate utilization of antibiotics in

therapeutics and animal production (Martinez and Baquero, 1988). Similar arguments can be made for microcin/antibiotic-resistance plasmids. It was suggested that microcins have a role in the competition between microorganisms in the intestinal tract (Asensio et al., 1976; Baquero and Asensio, 1979). In this ecosystem, strains carrying microcin/antibiotic-resistance plasmids have a better ability to survive both in their competition with other strains and in the situations of antibiotic selective pressure. Hence, plasmids like pCP 106 might contribute to the maintenance and dis- semination of both microcin production and antibiotic resistance.

Hybridization experiments have shown that E. coli strain V5 17, habitually used as source for molecular weight marker plasmids (Ayers and McCowen, 1978), also carried a plasmid homologous to D-type microcin plasmids. This fact is not surprising since it was previously mentioned that this strain produced A- and D-type microcins, and the presence of a A-type microcin plasmid in this strain has been recently described (Sanchez et al., 1986). Moreover, all of the microcinogenic strains so far described that produce A microcin also synthesize D microcin. It seems that the plasmid that hybridized with the probe for micro- tin D93 is pV5 17C, which has been previously shown to encode the synthesis of A-type microcin (S&rchez et al., 1986). Although it is possible that this plasmid carried the determinants for the synthesis of both microcins, an alternative explanation would be the presence of two different plasmids, pV5 1 7CA and pV5 17Cn, with the same molecular weight and encoding respectively for the synthesis of microcins A and D. We think that it is a suit- able hypothesis, because it has been described that the presence of two different plasmids with the same molecular weight and encoding for the synthesis of different microcins is a com- mon trait in E. coli strains producing both A and D microcins (F. Sanchez, Ph.D. Thesis, Autonoma University, Madrid, 1981).

Isolates producing D-type microcins con- stitute about 50% of all microcinogenic strains. All of these strains studied so far carried a

224 MARTiNEZ AND PtiREZ-DfAZ

small, nonconjugative microcin plasmid. The only D-type microcin conjugative plasmid so far described is plasmid pCP106. Location of the microcin determinants in a transposable element has been previously suggested (Perez- Diaz and Clowes, 1980), but there are no data supporting this hypothesis. Since this plasmid resembles in its replication ColE 1 -type plasmids, it is conceivable that integration of a microcin plasmid into an antibiotic-resistant conjugative monocopy plasmid may occur. This topic is currently under study in our laboratory.

ACKNOWLEDGMENT

BROAD, D. F., AND SMITH, J. T. (1983). Extraction and purification of antibiotic-destroying enzymes. In “An- tibiotics: Assessments of Antimicrobial Activity and Resistance” (E. D. Russell and L. B. Quesnel, Eds.), pp. 93-l 10. Academic Press, London.

BUSH, K. (1989). Classification of p-lactamases: Groups 1, 2a, 2b, and 2b’. Antimicrob. Agents Chemother. 33, 264-270.

DE LORENZO, V., AND AGUILAR, A. (1984). Antibiotics from gram-negative bacteria: Do they play a role in microbial ecology? Trends Biochem. Sci. 9, 266-269.

DE LORENZO, V., AND MARTINEZ, J. L. (1988). Aerobactin production as a virulence factor: A reevaluation. Eur. J. Clin. Microbial. Infect. Dis. I, 621-629.

GARCf A-BUSTOS, J. F., Pnzzr, N., AND MENDEZ, E. ( 1984). Microcin 7: Purification and properties. Biochem. Bio- phys. Res. Commun. 119, 779-785.

GARRIDO, M. C., HERRERO, M., KOLTER, R., AND Mo- J.L.M. was the recipient of a fellowship from the FISSS. RENO, F. (1988). The export of the DNA replication

inhibitor microcin B I7 provides immunity for the host

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Communicated by Stuart B. Levy

cloning of the determinants for microcin d93 production and analysis of three different d-type...

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