(diploid) -- P6S (haploid) -* P6SP (diploid).

Transcription

1 EFFECT OF HAPLOID OR DIPLOID CONSTITUTION OF ESCHERICHIA COLI ON FORMIC HYDROGENLYASE ACTIVITY RONALD H. OLSEN AND JAMES E. OGG Department of Microbiology and Pathology, Colorado State University, Fort Collins, Colorado Received for publication 26 April 1963 ABSTRACT OLSEN, RONALD H. (Colorado State University, Fort Collins) AND JAMES E. OGG. Effect of haploid or diploid constitution of Escherichia coli on formic hydrogenlyase activity. J. Bacteriol. 86: Phenotypic differences occurring between haploid and presumed diploid strains of Escherichia coli containing homologous genetic material have been observed. In a growth medium containing glucose and Casamino Acids, formic hydrogenlyase (FHL) activity was induced in haploid strains but not in diploid cells. Other differences were noted when haploid and diploid resting-cell suspensions were assayed for their ability to induce maximal FHL activity. For FHL activity, diploid induction lags were significantly longer, and maximal inducible rates were lower than those observed for haploid strains. Ogg and Zelle (1957) and Zelle and Ogg (1957) reported the isolation of stable, presumed diploid strains of Escherichia coli after treatment of E. coli 82/r with camphor vapors. Diploids were found to be distinguished from haploid strains by their relatively higher resistance to the lethal effects of X rays, and by having twice the deoxyribonucleic acid (DNA) and twice the ribonucleic acid (RNA) content per cell. Ogg and Zelle (1957) also reported a delay in gas evolution by diploid strains when grown in a fermentation medium containing either pyruvate or formate. However, no definitive information has been reported on genotype-phenotype relationships for haploid or diploid cells as they may be affected by the level of ploidy. The present studies were designed to elucidate a change in the genotype-phenotype relationship occurring with the diploidization of cells containing genetic materials homologous to the parent haploids. To this end, the inducible formic hydrogenlyase (FHL) system as described by Lichstein and Boyd (1954) and others has been investigated as a phenotype parameter. This report is concerned with the apparent repression of FHL activity in diploid cells under proliferative conditions wherein haploid cells were induced to FHL activity. In addition, the induction to maximal FHL activity by resting-cell suspensions of haploid and diploid strains is described. MATERIALS AND METHODS Organisms. E. coli 82/r, an adenine-requiring mutant strain described by Anderson (1951), was used as the parent haploid strain. Treatments were applied to 82/r as described by Ogg and Humphrey (1963) to produce possibly diploid and subsequent haploid and diploid strains in the following sequence: 82/r (haploid) -* P6 (diploid) -- P6S (haploid) -* P6SP (diploid). These strains were maintained on nutrient agar slants, and were periodically streaked on nutrient agar plates to confirm their haploid or diploid identity. Haploid strains gave rise to smooth colonies, whereas diploid strains characteristically appeared as rough-granular colonies. Medium and culture. Diploid and haploid cell crops were obtained by the incubation of deepstanding cultures for 16.5 hr at 30 C. For this purpose, a modification of the medium described by Lichstein and Boyd (1952) was used. This growth medium was composed of the following: 0.4% (NH4)2SO4, 0.2% NaCl, 0.1% MgSO4' 7H20, 0.01% MnCl2, 0.001% Fe2(SO4)3, 0.3% K2HPO4, 0.1% KH2PO4, 0.2% trisodium citrate, 1.8% glucose, 1.0% vitamin-free Casamino Acids (Difco); and 1,ug of adenine per ml. The ph was adjusted to 7.0 with NaOH after sterilization. Glucose, Casamino Acids, and phosphate components in aqueous solutions were sterilized separately, and reconstituted to the given formulation immediately prior to use. The inoculum for the production of cell crops consisted of 0.2 ml of an aqueous cell suspension. A 1:10 dilution of the inoculum suspension gave an optical densitv of 494

2 VOL. 86, 1963 FORMIC HYDROGENLYASE ACTIVITY IN E. COLI when read at 425 m,u in a Bausch & Lomb Spectronic-20 colorimeter. Cells of this inoculum were derived from twice-subcultured cell suspensions produced under conditions analogous to those described for the production of cell crops. ilanometric procedures. Hydrogen gas l)roduction from 25,umoles of sodium formate, or the induction solution, was determined anaerobically by conventional manometric techniques (UImbreit, Burris, and Stauffer, 1957). The induction solution used contained, per 0.5 ml of solution: 25 Amoles of sodium formate; 25,umoles of glucose; vitamin-free Casamino A cids (Difco), 1.5 mg;(nh4)2so4, 1.2 mg; and adenine, 3.0,ug. Cell crops were harvested from the growth medium by centrifugation, washed once in distilled water, again sedimented by centrifugation, and resuspended in distilled water to approximately 0.1 mg of nitrogen per ml of cell suspension. Nitrogen was determined by modification of the procedure of Johnson (1941). Cell suspensions were assayed for FHL activity immediately after their preparation. Warburg flask contents included 1.0 ml of cell suspension and 1.5 ml of M l)otassium phosphate buffer (ph 6.0). The side arm contained 0.5 ml of either sodium formate or the induction solution, which was tipped in subsequent to gassing with nitrogen and temperature equilibration. RESULTS Under the specified conditions of culture, the haploid and presumed diploid strains showed approximately equivalent growth at 16.5 hr. Harvesting of cells after these incubations yielded cell populations representative of the late logarithmic or early stationary growth phase. Induction to FIIL activity by proliferating cultures. Haploid and diploid strains were initially tested for their ability to induce to FHL activity concomitant with proliferation of the culture. Since formate was not added to the growth medium, it is assumed that the appearance of FHL activity reflects the efficacy of induction by formate carbon derived from glucose or Casamino Acids. FHL activitv for cells as they were harvested from the growth medium is shown in Fig. 1. These data show that the original 82/r haploid strain and the recurring haploid derivative (P6S) produced FHL under the growth conditions cited. Furthermore, FHL activity was similar for both z LL -c w I 0 Cf) LU F- 100o 50[ 0 FIG. 1. A= 82/r (HAPLOID) B=P6 (DIPLOID) C = P6S (HAP LOID) / D = P6SP( DIPLOID)/ / /** Production of hydrogen gas from fornmate by anaerobically grown Escherichia coli. the haploid strains. The similar performance of the original haploid (82/r) and the recurring haploid (P6S) derived from a diploid indicates that genetic material directing FHL activity passed through the diploid state unchanged. On the other hand, the first diploid derivative (P6) and the recurring diploid derivative (P6SP) did not produce FHL activity under growth conditions identical to those used for growth of the haploid strains. Therefore, these data indicate a significant phenotypic difference occurring between haploid and diploid cells containing homologous chromosomal material but differing in ploidy. Induction to maximal FHL activity by resting cells. The failure of diploid strains to induce to FHL activity under conditions wherein haploid strains did induce then raised the question of whether diploid strains were completely repressed for FHL. Both haploid and diploid resting cells grown as (lescribed previously were assayed for their ability to induce to FHL activity in the presence of an induction milieu. Induction was followed in a Warburg vessel by the production of hydrogen gas (Fig. 2). The data in Fig. 2 show that the haploid strains (82/r and I'6S) respond

3 496 OLSEN AND OGG J.: BACTRoL. to a maximal rate of hydrogen gas production in approximately 10 min, subsequent to the addition of the induction solution to the cell suspensions. A similar induction response was noted for both haploid strains; this finding militates against a mutation-directed change in phenotype observed for the diploid derivatives. However, diploid strains showed a lag of approximately 30 min from the tip-in of the induction solution to the onset of hydrogen gas evolution. Also, diploid maximal rates of FHL activity were less than haploid rates. Since the haploids possessed some preformed FHL activity prior to contact with the induction solution in the resting-cell state, one might question whether the presence of preformed FHL activity by haploids facilitated subsequent induction to maximal activity with significantly shorter lags than the diploids. The diploids were in a repressed condition as they were harvested from the growth medium. To obtain cells without FHL activity, both haploid and diploid strains were grown aerobically by sparging with air the growth medium cited previously. Aerobiosis resulted in the production of both haploid and diploid cells repressed for FHL activity. When aerobically grown resting cells were examined, v FIG. 2. Induction of formic hydrogenlyase by anaerobically grown Escherichia coli. FIG. 3. Induction of formic hydrogenlyase by aerobically grown Escherichia coli. haploid strains induced more quickly to maxmal FHL activity than diploid strains. This relationship corresponds to that described in Fig. 2 for anaerobically grown cells. However, iiithis histance aerobically grown haploids lacked demoilstrable FHL activity as they were harvested from the growth medium. When induction responses for anaerobically grown cells (Fig. 2) and aerobically grown cells (Fig. 3) are compared, it is apparent that aerobiosis during the proliferation of the cell crop effected an extended lag in activity for diploid strains. Aerobically grown diploids lagged 100 min in FHL activity, whereas a lag of 30 min was observed for anaerqbically grown diploid resting cells. Consequently, aerobic growth of diploids accentuates the repreqson of their FHL activity when resting cells are subsequently assayed under anaerobic induct*iq conditions. In the case of haploid resting cellp, the lag in FHL induction is relatively unactd by prior growth of the cells under aerobic, or anaerobic conditions. DISCUSSION These studies have indicated that thenrelatiokship between genotype and phenotype may not be simple and direct. Although genetic material

4 VOL. 86, 1963 FORMIC HYDROGENLYASE ACTIVITY IN E. COLI497 possessed by the presumed diploids is homologous to the parent haploid (82/r), data presented here have shown an alteration in phenotype associated with the diploidization of haploid strains. Phenotype differences occurring between haploid and diploid strains may be summarized as follows. (i) Only haploid strains produced demonstrable FHL activity in proliferating cultures. (ii) Diploid strains induced more slowly to maximal FHL activity than haploid strains. (iii) Maximal FHL activity observed for diploids was less than that of haploid strains. In view of the noninduction of FHL occurring in proliferating diploid cultures, one might question whether other phenotypic properties could be associated with this observation. For example, the diploid cells are larger in size than the corresponding haploids (Ogg and Zelle, 1957). Hence, the lower surface area to volume ratio characteristic of diploid cells could impose limitations on the rate of entry of substrate carbon into the diploid cell, and indirectly regulate the availability of formate carbon derived from glucose for FHL induction. Evidence against this hypothesis comes from a report by Ogg and Zelle (1957), who found growth rates to be equivalent for haploids and diploids grown under aerobic conditions. Also, approximately equivalent growth was obtained for both haploid and diploid strains cultivated under the conditions described in this report. Additional support for the conclusion that cell size is not the rate-limiting factor in FHL induction in the diploids comes from the results of unpublished experiments by the authors on recombinants obtained from matings between a male haploid E. coli B derivative and female diploid P6 cells. The majority of recombinants from such crosses contained the same amount of DNA per cell, had the same size cells, and had the same radiation-resistant characteristics of the female diploid P6 parent; yet, a large number of these diploid recombinant strains showed derepression for FHL induction in growing cultures and reduced lags for FHL induction in resting cells. Since the difference in surface area to volume ratios between haploid and diploid strains does not account for dissimilar FHL induction kinetics, some other cell regulatory mechanism must be involved. It is possible that with the doubling of the DNA complement per cell, an increase in cytoplasmic repressors occurs, leading to greater enzyme repression in the diploid. To inactivate the additional repressors, a higher endogenous concentration of inducer would be required; this might account for the failure of proliferating diploid cells to induce to FHL and for the prolonged FHL induction lags observed for restingcell suspensions. This repressor inactivation hypothesis was proposed by Jacob and Monod (1961a, b) to account for the regulation of induced enzyme synthesis. It could also be proposed that doubling the cell size creates dissimilar ratios of other cytoplasmic constituents, e.g., the number of ribosomes per cell. The number of ribosomes per cell can influence the rate of protein synthesis (Schaechter, Maaloe, and Kjeldgaard, 1958; Neidhardt and Magasanik, 1960; Schaechter, 1961). Ogg and Zelle (1957) and Ogg and Humphrey (1963) found diploid cells in the stationary period of growth to have approximately twice as much RNA per cell as the haploids. Yet, these latter observations do not rule out the possibility that the ratio of ribosomal RNA to DNA in diploids and haploids is not the same during the period of active growth. Repression of FHL activity could also reflect the accumulation of a metabolite, and it might be proposed a priori that the large cell size of the diploids could allow greater amounts of repressor-metabolite to be accumulated intracellularly. One possible argument against this latter hypothesis is that with the double DNA and RNA content of the diploids one could expect greater enzymatic activity which would maintain a ratio of repressor-metabolite to genetic materials or enzymeforming sites similar to that of the haploid strains. This in effect would yield similar induction kinetics for both haploid and diploid strains, but data present in this report have shown that maximal inducible FHL activity for diploids is less than that observed for haploids. Therefore, considering the genetic homology existing between haploid and diploid strains, the differences in FHL activity may reflect an altered mechanism for the expression of genotype at the diploid level, or the cumulative effects of a doubled DNA content and possible altered ratios of cytoplasmic constituents for diploid cells. Comparative studies now in progress on the homozygous diploids, the diploid recombinant strains, and the parental haploid strains may yield information about genotype-phenotype relationships as expressed in enzymatic activity.

5 498 OLSEN AND OGG J. BACTERIOL. ACKNOWLEDGMENT This study was supported by research grant G from the National Science Foundation. LITERATURE CITED ANDERSON, E. H The effect of oxygen on mutation induction by X rays. Proc. Nat]. Acad. Sci. U.S. 37: JACOB, F., AND J. MONOD. 1961a. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3: JACOB, F., AND J. MONOD. 1961b. On the regulation of gene activity. Cold Spring Harbor Symp. Quant. Biol. 26: JOHNSON, M. J Isolation and properties of a pure yeast polypeptidase. J. Biol. Chem. 137: LICHSTEIN, H. C., AND R. B. BOYD A cofactor for the formic hydrogenlyase enzyme system. Proc. Soc. Exptl. Biol. Med. 79: LICHSTEIN, H. C., AND R. B. BOYD Studies on the formic hydrogenlyase system of bacteria. J. Bacteriol. 67: NEIDHARDT, F. C., AND B. MAGASANIK Studies on the role of ribonucleic acid in the growth of bacteria. Biochim. Biophys. Acta 42: OGG, J. E., AND R. D. HUMPHREY Small-cell segregants from a possibly homozygous diploid strain of Escherichia coli. J. Bacteriol. 85: OGG, J. E., AND M. R. ZELLE Isolation and characterization of a large cell possibly polyploid strain of Escherichia coli. J. Bacteriol. 74: SCHAECHTER, M Patterns of cellular control during unbalanced growth. Cold Spring Harbor Symp. Quant. Biol. 26: SCHAECHTER, M., 0. MAALOE, AND N. 0. KJELD- GAARD Dependency on medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium. J. Gen. Microbiol. 19: UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF- FER Manometric techniques. Burgess Publishing Co., Minneapolis. ZELLE, M. R., AND J. E. OGG Radiation resistance and genetic segregation in a large cell possibly polyploid strain of Escherichia coli. J. Bacteriol. 74: Downloaded from on November 10, 2018 by guest