reeD41 mutation; Polsinelli et al. (1973). recE4. Friedman and Yasbin (1983). reeE45. reeE45 mutation; deVos and Venema. (1983). reeF15. reeF15 mutation ...
Mol Gen Genet (1987) 208:349-352 © Springer-Verlag 1987
Short communications Plasmid maintenance in Bacillus subtilis recombination-deficient mutants Juan C. Alonso, Jean-Franfois Viret, and Ravindra H. Tailor Max-Planck-Institut fiir molekulare Genetik, Ihnestrage 73, D-1000 Berlin 33, Federal Republic of Germany
Summary. An isogenic set of 11 recombination-deficient mutant strains of Bacillus subtilis has been constructed. Whereas plasmid p U B I I 0 is stably maintained in such R e c - cells, the high copy number plasmid pC194 is unstable. Instability in R e c - strains could be mostly attributed to the deleterious effect of the presence of the plasmid on the R e c - cells' growth capability. In part, instability of pC194 derivatives could also be correlated with the presence of an unusually high amount of multimeric D N A molecules. Key words: D N A repair deficient mutants - pC194 pUB110 - Plasmid replication - Plasmid stability
The stable maintenance of plasmids in bacterial cells requires functions that ensure duplication of replicons and ordered partitioning at cell division. Genes specifying plasmid copy distribution, termed par or seg, have been identifled on Escherichia eoli plasmids (Meacock and Cohen 1980; Nordstrom et al. 1980; Ogura and Hiraga 1983). Also, in the Bacillus subtilis plasmid pC194 a region adjacent to the minireplicon encodes a function necessary for stable inheritance at cell division (Alonso and Trautner 1985 a). The stable maintenance of high copy number plasmids during cell growth is in addition affected by the configuration of plasmids (Hakkaart et al. 1984; Summers and Sherrat 1984). In some recombination-deficient mutant strains of E. coli, plasmid oligomerization is drastically reduced (Fishel et al. 1981) and in all cases the stability of plasmids was directly correlated with the capability of the host to produce plasmid monomers, In B. subtilis, a systematic study of plasmid stability in R e c - mutants has not been performed. Previous comparisons of various R e c - strains were complicated by the fact that the rec mutations resided in different genetic backgrounds. To remedy this situation, a set of isogenic recombination-deficient mutants with the YB 886 genetic background, distinguished by the absence of prophage SPfl and by non-inducibility of PBSX (Yasbin et al. 1980), were constructed (Table 1). Some of the strains, namely recA1, recB2 and recE4, were already available in the YB 886 background (see Table 1). The reeF15 mutation was brought into YB 886 by allele transfer (" conversion"). The chromosomal allele of Rec + cells transformed with plasmid Offprint requests to: J.C. Alonso
precF15 was efficiently converted to the plasmid genotype
as reported by Iglesias and Trautner (1983). The remaining including addA5, were transfered into YB 886 by co-transformation using 0.5 gg/ml of both plasrec mutations,
Table 1. Bacillus subtilis strains and plasmids used Strain
Relevant Origin or reference of genotypea the rec Allele
YB 886 b YB 1260 BG 115
rec + fecal reeA73
YB 1290 BG 117 BG 121 YB 1015 BG 105
recB2 recB3 recD41 recE4 reeE45
BG BG BG BG BG BG
reeF15 reeG40 recL16 reeM13 rec-149 addA5
129 123 107 127 101 125
Yasbin et al. (1980) Love and Yasbin (1984) recA73 mutation; deVos and Venema (1983) Love and Yasbin (1984) recD3 mutation; Dubnau et al. (1973) reeD41 mutation; Polsinelli et al. (1973) Friedman and Yasbin (1983) reeE45 mutation; deVos and Venema (1983) reeF15 mutation; Harford et al. (1973) reeG40 mutation; Polsinelli et al. (1973) recF16 mutation; Dubnau et al. (1973) reeG13 mutation; Dubnau et al. (1973) rec-149 mutation; Chestukin et al. (1972) recE5 mutation; Doly et al. (1974); Anagnostopoulos, personal communication Spontaneous mutation of YB1015 background that enhances plasmid stability; J. Kupsch, personal communication
BG 113
recE4, stb-1
Plasmid
Genotype
Reference
pC194 pC1941 pBC30
Cm r, ine8 Cm r, inc8 Cm r, inc8, seg-
pUBll0 pSal
Nm r, inc13 Cm r, ine8, reeF Cm r, incS, reeF15
Iordanescu (1975) Alonso et al. (1986) Alonso and Trautner (1985a, 1985b) Gryczan et al. (1978) Lampe and Bott (1985) This work
PrecF15c
Cm, chloramphenicol; Nm, neomycin; inc, incompatibility grasp; seg, segregation
a Names of all rec mutations (except recE4, recE45 and addA5) are given in accordance to the classification of Mazza and Galizzi (1978) b The genotype of the parental strain YB 886 is trpC2, metB5, amyE, xin-I, attSP fl c The recF15 mutation was placed into the pSal plasmid by allele
transfer during propagation in the original strain (see text)
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Fig. 1. Stability of plasmid pC194 in different Bacillus subtilis recombination-deficient mutants. B. subtilis strains carrying pC194 were grown in chloramphenicol-containing TY medium. Overnight cultures were diluted (5 x 109 fold) and grown in antibiotic-free medium for approximately 100 generations; about every 12 generations the culture was diluted. The fraction of Cmr cells was determined each 25 generations, either directly or indirectly by replica plating onto chloramphenicol-containing plates. After t00 generations, pC194 curing in the reeD41 and rec-149 strains was identical to the wild type. The plasmid curing curves of strains recA1, recM13, recG40 and recA73 were nearly identical; only the latter one is presented. Plasmid segregation was similar in recE45 and reeF15. Strains: (.) tee+; (n) recA73; (0) recB3; (o) recE4; (o) recE4 stb-1; (u) recF15; (A) recH342; (z~)reeL16; (v) addA5
mid pBC30 (to select for competent cells) and chromosomal D N A purified from the original R e c - mutants. Chloramphenicol-resistant (Cm r) colonies were identified and scored for sensitivity to the D N A damaging agent methyl methane sulfonate (MMS). Of the Cm r colonies 1 % - 5 % were sensitive to at least 200 gg/ml final concentration of MMS, like the donor cells. Within the limit of detection of this analysis more than 50% of those differred only in the rec character from the recipient cell. In a second step, the derived transformants were cured of pBC30 by extensive growth in the absence of chloramphenicol. Using the above mutants, we compared the influence of various host recombination genes on plasmid stability. The segregational stability of plasmid pC194 was analysed during approximately 100 generations of growth in TY medium (Biswal et al. 1967) without selective pressure. From the results shown in Fig. 1, we conclude that pC194 is stably maintained in Rec + cells, whereas it is segregationally unstable in nearly all Rec- backgrounds. Various rec-deficient mutants gave different degrees of instability and the segregation kinetics are biphasic in most of them. In the addA5 mutant strain about 20% of the viable cells were plasmidfree at the beginning of non-selective growth and segrega-
tion was observed from the first generation without a detectable lag period. We have shown previously that plasmid pBC30 is segregationally unstable and that this could not be explained by variations in plasmid copy number (Alonso and Trautner 1985a). Unfractionated pBC30 D N A has a higher specific transforming activity than pC194 (5- to 7-fold) suggesting an enrichment in oligomers (see Canosi et al. 1978). The high degree of oligomerization, indicating defective plasmid functions determining stability, makes pBC30 a suitable candidate to study more specifically the influence of the host genetic background on plasmid segregational stability. As compared to pC194, the segregation instability of a Seg- plasmid such as pBC30, was drastically enhanced. Indeed, with the exception of the rec +, recH342 and recE4 stb-1 strains, more than 50% of the cells of all rec-deficient mutants were plasmid-free in less than 35 generations (data not shown). In the addA5 genetic background less than 5% of the viable cells contained plasmid under selective pressure (time zero of segregation experiments) indicating a stringent requirement of pBC30 for the ATP-dependent DNase or another function associated with these gene products (by analogy with the E. coli recBCD complex). As observed for pC194, the segregation kinetics for pBC30 were biphasic in some rec-deficient mutants, except the addA5 and recE4 mutant strains, with a slow rate of plasmid curing at very early stages and a faster curing at later stages. The recombination requirement is identical for both plasmids except for the recD function(s) which are required only for pBC30 (data not shown). In order to test whether the host recombination background has some influence on plasmid oligomerization, we took advantage of a mutation in which 2 bp have been added at the HpalI cleavage site of pC194 creating plasmid pC1941, leading to high oligomerization (see Fig. 2A). The multimeric pattern of pC1941 is nearly the same in all the strains examined but an additional band migrating between the tetramers and pentamers was found in recB3, recE4, recF15, recG40, and recH342 genetic backgrounds. Figure 2B shows that for plasmid pC194, purified in the late exponential growth phase, the major plasmid configuration in any genetic background is the monomeric form which is either covalently closed circular or open circular. The segregation kinetics of plasmid pC1941 in Rec + cells was indistinguishable from the pattern of plasmid pBC30 (data not shown). A direct association between plasmid oligomerization and instability may thus be possible but oligomerization alone cannot reconcile all our observations. Does plasmid replication and partitioning really require host recombination functions or are we dealing with an indirect effect, e.g. a rapid overgrowth of plasmid-cured cells? Should the second hypothesis be correct, we would predict that plasmid-containing cells would be under physiological stress. The doubling time of plasmid-free recE4 cells was 28 min in TY medium and increased to 38 rain and 35 min for cells growing in the same antibiotic-free medium when carrying pBC30 and pC194 respectively. When plasmidcarrying recE4 cells were observed in a light microscope, long chains, occasionally with ghost cells, were observed, but not filamentous forms (data not shown). In contrast, in those genetic backgrounds where the segregation curves
351 A
B
C D E
F G H
I
J
K L
A
C H
D
G
0
F
Fig. 2A, B. Plasmid multimerization in different Bacillus subtilis recombination-deficient mutants. Plasmid DNA was purified, according to Canosi et al. (1978), from different rec-deficient mutants containing either: A pC1941 or B pC194 and analysed on a 0.8% agarose gel. The position of supercoiled monomers, dimers etc. of plasmid can be counted from the bottom to the top (open circular monomers run at the same position as the dimer band). Lane A, rec + cells; lane B, recA73; lane C, recB3; lane D, recD41; lane E, recE4; lane F, reeF15; lane G, recG40; lane H, recH342; lane I, reeL16; lane J, recM13 lane K, rec-149; lane L, polA5; lane O, addA5. The arrow indicates the additional band observed in some mutants and (C) the chromosomal DNA were not biphasic, i.e. wild type or recH342, the doubling time was about 25 min, irrespective of whether or not the cells contained plasmids. A spontaneous host mutation, termed stb-1, which partially stabilizes plasmid pBC30 in the recE4 mutant strain, has been isolated (J. Kupsch, unpublished results) and plasmid curing was tested in the new mutant strain, B G l l 3 (recE4 stb-1). As shown in Fig. 1, plasmid stability was markedly increased as compared to the recE4 strain; the same was observed with pBC30 (data not shown). In BG113 the doubling time for plasmid-free cells was comparable to reeE4 (30 min) but the difference with plasmid-bearing cells is lower (35 min and 34 min for pBC30 and pC194, respectively). These results suggest that the first phase of plasmid curing observed in Fig. 1 reflects a specific need of some recombination functions for optimal maintenance. However, a strong interference with the cell's reproductive fitness could account for the second component of the curves. According to this hypothesis, any mutation like stb-1 that could reduce the observed physiological stress, may enhance plasmid stability. Recently teRiele et al. (1986) have found that circular single stranded D N A (ssDNA) molecules accumulate in pC194 as well as in pE194, pC221 and pT181 but very little in plasmid pUBll0. When the later plasmid was scored for segregational stability with the set of rec-deficient mutants of Table 1, we observed that more than 99% of the cells were plasmid-positive after 100 generations of growth in non-selective medium. In line with this, the presence of plasmid pUB110 did not affect cell physiology by any parameter tested (colony formation, doubling time, cell morphology).
Conclusions
The establishment, for the first time, of a set of isogenic recombination-deficient mutant strains lacking the bacteriophage SPfl and inducibility for PBSX will improve the understanding of B. subtilis D N A recombination and repair pathways (Alonso et al., to be published). Until now, among the 12 reported loci (Zeigler and Dean 1985; Piggot and Hoch 1985), functions have been associated only with the recE and addA loci (de Vos et al. 1983; Doly etal. 1974). We have shown previously that plasmid pC194 encodes a gene that controls segregation without affecting plasmid copy number (Alonso and Trautner 1985a). The results presented in this communication corroborate our previous observations that in rec + cells both a plasmid-encoded function, which is missing in plasmid pBC30, and some host rec functions are required for full stabilization. The latter functions are necessary for plasmid stability or avoidance of physiological stress. The nature of this requirement is not clear but to account for the present observations we must assume: (1) That segregation does not follow equipartition kinetics, since a very high degree of stability is predicted for a multicopy plasmid such as pBC30 (Alonso and Trautner 1985 a); (2) That the presence of pC194 or derivative plasmids severly affects some physiological parameter of the cells. The maximal instability was observed when the main recombination pathways were blocked. In the absence of either recE and/or recA (E. coli recA equivalent) addA and reeF (recBC and recF-like pathways) gene product(s) more than 50% of the cells had lost wild-type and pBC30 plasmids in less than 40 and 20 generations respectively.
352 The physiological stress linked to pC194 or derivative plasmids m a y be due to SOS induction. However, accumulation o f p C / 9 4 single-stranded D N A over the wild-type level was observed in none o f the strains presented in Table 1 (data not shown) and thus cannot account for the physiological stress. A l t h o u g h pC194 and p U B I 10 share an extensive h o m o l ogy at the replication level, plasmid p U B I 1 0 was stably m a i n t a i n e d in all the described recombination-deficient mutants. Plasmid p U B l l 0 has been r e p o r t e d to require the d n a B gene p r o d u c t that is essential for b o t h replication and m e m b r a n e a t t a c h m e n t (Wilson and Sueoka 1980; T a n a k a and Sueoka 1983). To account for our data, we assume that plasmid replication a n d m e m b r a n e a t t a c h m e n t could use the host replicosome system whereas plasmids replicating in a cytoplasmic form could use an alternative mechanism that requires rec functions. Interestingly, when some o f the p U B l l 0 m e m b r a n e binding sites are removed, the plasmid is destabilized and biphasic curing curves are also observed (see Bron and Luxen 1985), suggesting that membrane a t t a c h m e n t might account for plasmid stability. Experiments are in progress to u n d e r s t a n d the specific requirement o f plasmid for the corresponding function. Acknowledgements. We are grateful to T.A. Trautner for his contin-
uing interest in this project and many stimulating discussions. We thank R.E. Yasbin for providing bacterial strains, K.F. Bott and H. Yoshikawa for providing pSal and pSM1041 plasmids and C. Anagnostopoulos for communication of unpublished results and helpful discussions. J.-F. Viret was supported by a grant of the Fond national suisse de la Recherche scientifique. References
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