an R Factor on Escherichia coli Chromosome

Transcription

1 Japan. J. Microbiol. Vol. 13 (3), , 1969 Integration of Chloramphenicol Resistance Gene of an R Factor on Escherichia coli Chromosome Shizuko IYOBE, Hajime HASHIMOTO, and Susumu MITSUHASHI Department of Microbiology, School of Medicine, Gunma University, Maebashi (Received for publication, December 6, 1968) ABSTRACT An unstable mutant R factor conferring only chloramphenicol (CM) resistance was obtained by spontaneous segregation. After storage in broth culture, a stable CM-resistant mutant was obtained and its CM-resistance could not be cured by treatment with acriflavine or transduced to a recombination-deficient strain of Escherichia coli K12. Recombinational analysis indicated that the cml gene governing CM resistance had been integrated into the E. coli chromosome and closely linked with met B locus. The cml gene was co-transduced with both met and arg markers by phage P1, and the linkage order was considered to be mtl-cml-met-arg-thi. When the strain carrying this chromosomal CMresistance was infected with a transferable R (TC) factor capable of conferring tetracycline (TC) resistance, the CM-resistance became transferable by conjugation. This mechanism is considered to account for the formation of the recombinant R (TC.CM) factor. The R factors in bacteria have many common properties with those genetic elements known as episomes [1, 11, 18]. They may exist in the autonomous state and replicate independently of the bacterial chromosome, are conjugally transmissible and can also transfer the host chromosome but with low efficiency. However, different from F, no evidence of the integration of the whole genome of an R factor onto host chromosome has as yet been presented. It was reported from this laboratory that part of the R genome, the gene including the tet gene governing tetracycline (TC) resistance, was segregated by transduction of the R factor with bacteriophage epsilon in group E Salmonella. After transfer to E. coli K12 strain by the mediation of F factor, the tet gene was found to be located on the E. coli K12 chromosome between the lac and pro, loci, near the lac [6]. Integration of a tet-r or sul-r str-r (sulphonamide resistance, streptomycin resistance) determinant into a similar area of the host chromosome has also been reported from another laboratory using transduction of the R factor with bacteriophage P22 in S. typhimurium [3]. In these cases, the possibility that integration might be mediated. by a part of phage genome cannot be excluded. This paper deals with the integration of cml gene of R factor into the E. coli K12 chromosome without participation of any other genetic elements such as bacteriophage or other episomal entities. MATERIALS AND METHODS Bacterial strains. Strains used were all derivatives of E. coli K12 and are listed in 225

2 226 S. IYOBE, H. HASHIMOTO AND S. MITSUHASHI Table 1. Characteristics of the bacterial strains useda a Abbreviations and symbols: met, methionine; trp, tryptophane; par, purine; thr, threonine; leu, leucine; thi, thiamine; his, histidine; arg, arginine;, dependence; -F, independence. Fermentation markers include: lac, lactose ; ara, arabinose; gal, galactose; xyl, xylose; mtl, mannitol; mal, maltose; -, nonfermentation; +, fermentation. Drug-resistance markers include: str, streptomycin; cml, chloramphenicol; tet, tetracycline; sal, sulphanilamide; nal, nalidixic acid; r, resistant; s, sensitive. Episomal markers of R factors include: rmt, R-mating; if m, inhibition of F-mating; stb, stable inheritance; tra, transfer; irs, inhibition of R superinfection;, wild type; -, defective. sfa+, having sex factor affinity locus; rec-, recombination deficient. b Symbols in parenthesis indicate the markers on the R factor. Table 1. Strain W3630 is a female variant of a Richter type [15]. This strain transfers chromosome at higher frequency than normal female strain when infected with R factor. W3623 rec+ and rec- (recombination-deficient) strains were kindly supplied by J. Tomizawa, National Institute of Health of Japan. R factors. All of the R factors used were derivatives of the R-100 factor, ori- ginally isolated by Nakaya and resistant to 4 drugs; tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulphonamide (SA) [13]. Resistance genes responsible for TC, CM, SM, and SA were abbreviated as tet, cml, str, and sul, respectively. Cm/ is the renamed resistance gene of chi previously reported [8]. If m- is the renamed genetic locus of i- reported by Hirota et al. [4, 16]. Ifm indicates the gene on the R factor governing the inhibition of F-function, such as F-mediated chromosome transfer or F-phage sensitivity. Naturally occurring ifm+ or if in R factors have been grouped as fi+ or fi R factors by Watanabe [21]. Transfer frequency of R factor to the recipient carrying another R factor was found to be decreased, and referred to as inhibition by R factor. The gene governing this function was called irs. R is an ifm- mutant of R 100 [4]. R and R are the segregants obtained by transduction of R 100 with bacteriophage P1. R was obtained by penicillin screening methods and recombination of R factors [7]. R 9-4 has been previously reported [12].

3 INTEGRATION OF CM GENE OF R FACTOR ON CHROMOSOME 227 Media. Eosin-methylene blue (EMB)- lactose, EMB-maltose [10], and Brain Heart Infusion (BHI, Difco) were used routinely. For the selective media for Met+ or to determine various nutritionary-deficient markers, A-maltose or A-glucose agar was used. The minimal agar consisted of medium A [2] without citrate but containing 40 ml of 0.2% bromthymol blue, 1.5% agar and 10 g of sugar per liter. Amino acid supplements, when necessary, were added to a concentration of 20 mg per liter. For the selection of R+ cells, CM (25 g/m1) or TC (50 p.g/m1) was added to the selective plates. Transfer of R factor and transduction of R factor with phage P1. The methods were described in a previous paper [7]. RESULTS Mutation from Unstable to Stable CM Resistance As reported previously [12], unstable (stb-) mutants of an R factor were isolated Table 2. Test of stability of CM-resistance The strain harboring each R factor was inoculated in BHI broth containing 25 pg of CM/ml. After incubation at 37 C for 18 hr, a loopful of the culture was inoculated into 2 ml of fresh BHI broth with or without 5 pg of acriflavine per ml and incubated at 37 C. After overnight incubation, the penicillin screening method was applied to the culture (except W3630 R-1-9-4) to test of loss of CM-resistance. This method has been reported by Hashimoto et al. (7). Frequency of CMs cells is expressed as the ratio of CMs cells after the PC-screening to the total number of cells before the screening. from TC sensitive derivatives of an R factor. They were lost at the high frequency of over 10% from their host bacteria during overnight growth. One of these mutants was referred to as R 9-4 (stb-1) and capable of conferring CM resistance. E. coli W3630 R± 9-4 (stb-1) was inoculated in nutrient broth at 37 C. Stock cultures were kept at room temperature in this laboratory. After several months' storage, cultures were streaked out on agar plates and the resultant colonies were tested for their CM resistance. Among 100 colonies resistant to CM, CM-resistance of one colony was found to be stable and not cured by treatment with acriflavine, but the remaining colonies were still unstable in their CM-resistance. This mutant of R factor derived from R 9-4 (stb-1) was called R (Table 2). Transduction of Cml Gene to Rec+ and Rec- Strains CM-resistance of R was stable and the cml gene was assumed to have been integrated onto the host chromosome. Chromosomal markers are known to be scarcely transduced to a recombination deficient mutant by phage P1 [17]. The cml gene of R was then tested for its transducibility with phage P1 to W3623 rec+ and rec- strains. As shown in Table 3, cytoplasmic R factor, R or R 9-4, was easily transduced to rec- strains as well as to rec+ strains. Genetic instability of an R 9-4 factor was not changed after the transduction, when 10 colonies from each selective plate were tested. However, transduction frequency of cml gene from W3630 R was decreased markedly when the recipient was rec. Trp gene of E. coli chromosome was not transduced to a recrecipient. These results strongly suggest that R (crnl-r) was located on the host chromosome by integration of the unstable

4 228 S. IYOBE, H. HASHIMOTO AND S. MITSUHASHI Table 3. Transduction of chloramphenicol resistance to rec+ and rec- strains Input titer of 3 kinds of phage lysate was uniformly 1>< 108. Recipient was W3623 rec+ or rec-, and the number of bacteria was 1.5X 109 or 6x 108, respectively. Selective plates were EMB-maltose supplemented with 25 pg of CM/ ml for cml marker, and A-glucose for trp marker. Frequency of transduction is expressed as the ratio of the number of transductants per input phage. R 9-4 factor during strage. Location of the Cml Gene of R on the Host Chromosome Recombinational analysis was undertaken to know the location of cml gene of R To obtain suitable sets of reciprocal crossing, the CM-resistance of E. coli W3630 R was transduced with phage P1 to Hfr C and to JE346 F- strain of E. coli K12. As shown in Table 4, the cml gene proved to be linked closely to met gene. Transduction analysis with phage P1 was conducted. As shown in Table 5, co-transduction of the cml and met loci was seen at the high frequency of over 80 per cent, regardless of the selective markers of either cml or met. Table 4. Frequency of occurrencece of unselected male markers in gal+ met+ recombinants in reciprocal crosses W1895 HfrC R met- gal+ x je346 F- R- met+ gal- (1), and W1895 HfrC R- met- gai+ x JE346 F- R met gal- (2) Equal volumes of donor and recipient culture (Ca. 3x 108/ml) were mixed. After 2 hr at 37 C, the mixture was plated on A-galactose agar supplemented with 20 pg of adenosine. The met+ gal+ recombinant colonies formed were purified 3 times and scored for unselected markers by replica plating. Table 5. Linked transduction of cml and met loci with phage P1 Input phage, 2x 108; Input bacteria, 5X 109; multiplicity of infection, EMB-maltose-CM and A-maltose were used for the selection of CM, and Met+ transductants, respectively. Transduction frequency was about 10-6 in both cases. In each selection, 100 colonies were picked and the frequency of cotransduction of both met and cml loci was tested after 3 successive single colony isolations. a: Transductant obtained in Exp. 1 in this table.

5 INTEGRATION OF CM GENE OF R FACTOR ON CHROMOSOME 229 When PA200 (met+ arg-) was used as a recipient of transduction from metarg+ R (crnl-r) which was obtained in Exp. 2 described in Table 5, frequency of cotransduction of met- or arg+ with cml-s was 71% or 23% respectively and cml-r or met- with arg+ was 30% or 35% respectively. All of the cml-r arg+ transductants were met-. Linkage order is considered as m tl-cml- met-arg-thi- Loss of an Ability of Mutual Exclusion from R 9-4 by Integration on the Host Chromosome R 9-217, which had been derived from R 9-4, did not inhibit the superinfection of R (tet-r irs+) as well as R 9-4 (Table 6). R 9-4 showed mutual exclusion with the tct-r irs+ R factor, and CMS or TCS progenies were easily obtained after both R factors were doubly infected in a same bacterium. This mutual exclusion was not found between R and R In R the gene concerning mutual exclusion might have been lost or suppressed at the time of integration. Conversion to Cytoplasmic State of R by Recombination with R (TC) Factor A rec- strain, when it is used as a recipient, is known not to decrease the transduction frequency of cytoplasmic R factor from other rec+ strains [17]. As shown in Table 3, when cml was transduced from W3630 R to a rec- strain, very few CM-resistant transductants were obtained. The results could be due to a change from chromosomal state to cytoplasmic state, an unstable state of R 9-4, but CMresistance of all the 100 transductants was stable. Genes concerning autonomous replication in cytoplasm were considered to have been lost totally in R Ultraviolet irradiation of R± cells was also undertaken, but induction of cytoplasmic state could not be obtained. The crnl gene of R was transferable by R-mating when the host was infected with R (tet-r rmt+). Most exconjuo-ant selected for CMr had received the chromosomal met gene and cytoplasmic TCr R factor simultaneously (Table 7). These strains and 2 of 14 CMr, TCr, Metexconjugants could transfer cml gene with or without met gene to a second recipient, ML1410, by R-mating. The 12 CMr, TCr, Met- strains could transfer both CM- and TC-resistance at high frequency to ML1410. In these cases, cml gene was considered to be picked up from chromosome onto the introduced R factor. Three exconjugants were TC-sensitive. Among these strains, 2 Table 6. Absence of interfering ability in R Overnight culture of donor, R (ter-r) and recipient, W3630 R+(cnil-r) were mixed in equal volumes and the mixture incubated for 2 hr. W3630 R+(tet-r) R.+(crnl-r) was selected as Malcolonies grown on EMB-maltose supplemented with 25 jug/m1 of both CM and TC. Frequency of superinfection is expressed as the ratio of the number of CMr TCr exconjugants to that of input donor cells. One hundred Mal- colonies selected on CM plus TC plates were picked and restreaked on the same kind of drug plate, purified once on a plate containing no drugs, and then tested for the segregation of drug-resistance.

6 230 S. IYOBE, H. HASHIMOTO AND S. MITSUHASHI Table 7. Incorporation of chromosomal cml gene of R9-217 onto cytoplasmic R9-127(tet-r) factor Donor, W3630 mal-met+ R+9-217(cna-r) R (tet-r), Recipient, mal+met-. Equal parts of both donor and recipient cultures were mixed and incubated at 37C. After 2hr incubation, the mixture was spread on the selective plate. Selection was made by EMB-maltose-CM. Transfer frequency of CM-resistance counted as black (Mal+) colonies was 1.1 x Ninety-one colonies grown on the selective plate were picked and after 3 times purification, the characters responsible for TCr, CMr and Met+, and their conjugal transferability were examined. As the second recipient, ML1410 met-nal-r was used. Transfer frequency : high, ; low, 10-10; no, less than Met+ strains could not transfer their CMresistance again, but one Met- strain could transfer the CM-resistance at high frequency. Cm/ gene of the latter was assumed to have aquired transferability by recombination with R Among 12 transferable TCr, CMr clones, one strain was selected and transferred to W3630. From W3630 met+ R+ (tet-r cml-r), thus obtained, TCr or CMr was transduced to metby phage P1 (Table 8). The determinants governing both TC and CM resistance were transduced jointly, as well as the case from W3630 met+ R (tet-r cml-r str-r sul-r), and from these strains met + was not cotransduced with TCr or CMr. From W3630 met+ R (cml-r) R± (tet-r), met+ was cotransduced with chromosomal cml-r of R These results indicated that the cml gene of R was recombined with R (tet-r) and the recombinant R (TC.CM) factor was in a cytoplasmic state and became transmissible at high frequency. Table 8. Recombination of R (cinl-r) with R (tet-r) and formation of R (TC.CM) factor Input bacteria, 6X 108/ml; input phage, P1V4, 3x 107; M.O.I., EMB-maltose containing either CM(25 pg/m1) or TC(50 /m1) was used for the selective plate. In each selection, 100 colonies were picked and streaked on the same plate as the selective plate, and the donor markers of transductants were examined by replica plating. a R(TC CM) was transfered to W3630 from one of the 12 TCrCMr clones indicated in the last row in the previous table. b This strain had cml gene on the chromosome and TC resistant R factor in the cytoplasmic state. c This strain had an TC and CM resistant R factor in the cytoplasmic state. d S indicated selection for this phenotype.

7 INTEGRATION OF CM GENE OF R FACTOR ON CHROMOSOME 231 somal site. Pearce and Meynell [14] demonstrated evidence of unstable integration of R factor onto host chromosome near trp marker, but this variant R factor, R1, seems to be an exceptional case. R 100 was not reported to transfer any specific chromosomal markers with high frequency. Harada et al. [6] demonstrated the integration of TC-resistance gene derived from an R factor onto host chromosome near lac locus, but the origin of this fragment of an R factor is a segregant obtained by transduction with phage s (epsilon) in a strain of group E Salmonella [5]. Phage s cannot transduce a whole genome of ordinary R factor and most transduced fragments of resistance genes are assumed to lack replication loci and become abortive. Therefore, transduced resistance genes are considered to multiply in the recipient cell only after their integration onto the host chromosome. In the Salmonella typhimurium and P22 system, the R factor can only be integrated onto the host chromosome by mediation of a part of the phage genome [3]. The HFT character of a TC-resistant transductant in group E Salmonella strains also proves the existence of a phage genome and its mediation in the integration of tet gene [9]. Different from these cases, the integration of the cml gene described in this paper is the first demonstration of integration of an R genome onto the host chromosome without any participation of any other episome. This locus is not the only one for the cml gene of R factor, because other chromosomal locations for the crnl gene were found from R 9-4, and their further results will be published elsewhere. ACKNOWLEDGEMENT This work was supported in part by a research grant AI , Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland, U.S.A. We have used the following criteria for a gene which exists extrachromosomally. 1) Spontaneous or artificial loss from its host should occur and the loss should be irreversible, 2) There should be no linkage to known chromosomal markers, 3) There should be no decrease in transduction frequency to rec- recipient comparing that to?tr' one. From the data described in this article, it was concluded that the R (crnl-r), derived from R 9-4 (cml-r stbl, became stable because of integration into the host chromosome, near the met locus. The R 9-217, as well as its parent R 9-4, was lacking in many determinants on R factor such as the resistance determinants (tet, str, sill), the determinants governing. transferability (tra), R-mating (rmt), inhibition of F-mating (ifm), and inhibition of R-superinfection (irs). However different from R 9-4, R did not exclude superinfected ifm+ R factor and could not return to cytoplasmic state by transduction to rec- strain. Thus it may be assumed that a part of an R genome including loci concerning mutual exclusion and autonomous replication might have been lost at the time of integration. R is likely to be a piece of R 9-4. Watanabe et al. [20] examined cotransferability of an R factor with either of several chromosome markers from an Hfr strain and deduced the attachment site of a 4 drug-resistant R factor between thi and ma locus. In later studies [19], however, he found a fluctuation in the frequency at which R factors are transfered with other certain host chromosome markers, and could not isolate clones with integrated R factors, or clones having affinity to specific chromo-

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