Abstract: The research was conducted to investigate the culturability of the E. coli αDH5 (Wt)by using two different plasmids; Vitreoscilla hemoglobin gene (vgb), which was cloned into plasmid pUC8 and transformed into E. coli αDH5 (strain VHb) as well as the plasmid pUC8 that was similarly transformed into the same strain (strain pUC8) The culturability (expressed by the relative survival cells under all starvation conditions) was increased significantly in the order of pUC8, Wt and VHb. Similarly the total proteins of the strains were increased significantly in the order of pUC8, Wt and VHb. It seems that the first increase in culturability is related to the role of bacterial hemoglobin gene (vgb), irrespective of the plasmid size. However, the second increase in total proteins, could be more attributed to the function of bacterial hemoglobin gene (vgb), for enhancing the starvation-induced proteins rather than to the total cellular protein.
Introduction
The development of viable but nonculturable cells (VBNC) has been widely investigated in many organisms (Oliver et al., 1993 and Paludane-muller et al., 1996). Recently, a lot of information became available regarding the effect of individual nutrient starvation, on the maintenance of several bacterial cultures (Huisman and Kolter, 1994). In Vibrio vulnificus, the use of viable accounts at 24°C starvation of either one of the following elements: carbon(C), nitrogen(N), phosphorus(P) when present singly or simultaneously altogether (multiple effect) have shown similar patterns (Paludane-muller et al., 1996). Also in E. coli (Jenkins et al., 1988) a significant and overlapping responses to the individual starvation conditions were similarly found. Additionally, extracellular products of bacteria have been shown to regulate wide variety of microbial processes, fore example homoserine lactone has been proposed as a starvation signal in E. coli (Huisman and Kolter, 1994; Matin, 1991). Nevertheless, extracellular factors produced during starvation have no effect on the culturability of cells incubated at low temperature (Wolf and Oliver, 1992; Weichart et al., 1992).
In particular, some investigations have indicated that there is an inverse correlation between the size of a plasmid harbored by a bacterial strain and the growth of that strain ( et al., 1989; Liu et al., 1995). Also it is known that the presence of a recombinant plasmid can alter the levels of certain metabolites (Axe and Bailey, 1987) and that the presence of a plasmid vector can increase the oxygen demand of cells which harbor it and may result in general alteration of cell metabolism (Khosravi et al., 1990). Other studies have shown that, in the presence of a plasmid burden metabolism occurred on host bacteria, which is enhanced as the size of the plasmid increases.
It is well known that the hemoglobin of Gram-negative obligate aerobe, Vitreoscilla (VHb) is the best-characterized bacterial hemoglobin (Kallio et al., 1994). The VHb gene (vgb) has been isolated, sequenced and cloned in many species of bacteria, the new heterologous hosts of vgb were shown to enhance cellular growth, viability, ATP production and production of valuable proteins (Liu et al., 1994; Kallio et al., 1994). The hemoglobin gene enables cells to grow in a microaerophilic environment by serving as an oxygen-binding protein (Joshi and Dikshit, 1994). Also it was shown that the cell size in strains without the hemoglobin gene decrease during the late log phase in response to starvation (Khosravi et al., 1990). In contrast, strains that bearing vgb maintained a fairly constant cell size (White, 1995). It was realized that, the presence of VHb alleviate this stress and allows the size of the vgb-bearing cells to remain constant (Kallio et al., 1994).
The aim of this research work was to investigate the effect/s of vgb on the culturability alterations caused by a plasmid or in another meaning to assess the ability of bacterial hemoglobin gene (vgb) for compensating the heavy duty caused by a plasmid. To the best of my knowledge the effect of bacterial hemoglobin (vgb/VHb) on the culturability of E. coli has not been investigated before.
Materials and Methods
Bacterial strains and plasmids: The strains used were Escherichia coli
αDH5 as parental cell (denoted Wt). Second strain was the E. coli
αDH5 transformed with plasmid pUC8 denoted by pUC8 and was obtained from
the Illinois Institute of Technology, Chicago, IL, USA. Third strain was the
same, Wt transformed with Plasmid pUC8:16 (Liu et al., 1994) contains
Vitreoscilla fragment of l.4 kb which has been cloned into pUC8. (Dikshit
and Webster, 1988) and this fragment was engineered to have vgb. The
third strain was denoted by VHb.
Culture Conditions: Broth medium was used for growth (Mitra et al., 1975) and contained the following salts gram per liter: 1g NH4Cl, 1g NaCl, 1g KCl, 0.2g MgSO4 and 0.1g (NH4)2SO4 in K2HPO4 5 x10-5 M. Tris-HCl was added gradually with mixing up to a final concentration of 0.05M, pH was maintained at 7.2 and glucose was added to a final concentration of 0.5%. Medium for the transformed strains also contained ampicillin (Ap) at 100Fg ml-1 Cells of overnight cultures grown in a GFL Model 3032 shaker fisher model 129 at 37°C, 125 rpm in synthetic or synthetic-Ap of the untransformed and transformed strains, respectively, were inoculatated (1:50) into 50ml of starved-fresh synthetic or synthetic-Ap respectively. These media were modified to contain single defined inorganic N and P sources (9.25mM NH4Cl; 10.32mM K2HPO4) and 0.2% (w/v) glucose as a single carbon source as described by Paludane-muller et al. (1996). Transferred washed growing cells to the medium lacking carbon (SM-C) or nitrogen (SM-N) or phosphorus (SM-P) or C, N and P simultaneous (SM-CNP) attained starvation conditions. The experiments were performed without shaking at room temperatures (23±2 °C).
Plasmid stability: Cells were harvested at 2, 4, 6, 8, 16, 24, and 32 days of all starvation conditions and their Plasmid stability was monitored at each time point. 25 colonies from randomly selected viable cells were cultured into LB agar plates containing 100Fg ml-1 AP and left to grow overnight. The number of colonies growing indicated the stability of the Plasmid (Lenski et al., 1988).
Hemoglobin determination: The presence of Vitreoscilla hemoglobin and it’s concentration were determined by the CO-difference spectrum method according to Liu and Webster (1974). Cell cultures used were grown in synthetic medium to stationary phase.
Determination of c.f.u.: To test the culturability of E. coli αDH5 (Wt, pUC8 and VHb), samples were taken at the indicated times and diluted in the respective starvation medium. Plate counts (number of viable cells ml-1) were performed on LB agar and LB-Ap agar respectively. Plates were incubated for 24hr. at 37°C before the evaluation of c.f.u. Consequent to extended incubations for a total of 96 hr no longer colony development could be watched (Paludane- muller et al., 1996).
Prestarvation at different incubation temperatures: Cells of the three strains were grown at 37°C overnight in synthetic broth medium (SM) transferred to fresh medium at a dilution of 1:50 and grown overnight incubated at 20, 30, 37 and 45°C. The cells were grown to med-exponential phase OD=0.25-0.5. The cultures were divided into 4 samples, each was harvested and washed (8000 rpm, 15 °C , 15 min, Sorval RC5 B plus centrifuge, SS 34 rotor) in either SM-N, SM-P, SM-C or SM-CNP, and the cells resuspended in vol. 1 of the respective starvation medium. Subsamples of these suspensions were starved for 72hrs. at respective temperatures.
Protein determination: Protein was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard.
Results and Discussion
Culturability of the three strains (Wt, pUC8 and VHb) was followed up to 32 day (Table 1 and 2). To bear out that, the respective nutrients, limit the growth of these strains, the growth lost by starvation was restored by adding back the deficient nutritional source to a subsamples of the experiment after 72hrs. of starvation, and the cultures incubated at 37°C with shaking. After overnight incubations, the amended samples showed an increase in c.f.u. of 20, 16 and 28 folds as compared with the counts of C, N and P-starved cultures, respectively. However, the actual cultures showed 5-10% decrease in c.f.u., assuring that the starving cultures were growth-limited for the omitted substrate. Additionally, the simultaneous addition of the three nutrients resulted in growth, whereas, the addition of single and combination of two nutrients did not show any increase in c.f.u.
Also, the results showed that bacterial hemoglobin (VHb) is expressed in vgb-containing E. coli cells grown in the synthetic medium and under certain starvation conditions by using CO-difference spectra (Fig. 1), colony colors and AP resisting of the cells. Additionally the VHb strains showed more viability and culturability of their cells as compared with strains lacking vgb (parental cell and pUC8- containing cells) (Table 1). A few scientists reported that there is an inverse correlation between the size of a plasmid harbored by a bacterial strain and the growth of that strain (Zund and Lebek, 1980; Cheah et al., 1987; Ryan et al., 1989). While those have shown that, in the presence of a plasmid burden metabolism occurred on host bacteria, which elevated as the size of the plasmid increases. Strains harboring plasmids are more sensitive to lower oxygen levels than plasmid-free strains by the criterion of cell growth (Khosravi et al., 1990). This agreed with the results reported here, by which the survival of E. coli strain pUC8 under the starvation condition showed a little lower than that of the strain Wt. The strain VHb’s survival has been shown to be 1.5-2 folds as compared with the strains Wt and pUC8 after 32 days of incubations (Table 1).
The possible explanations why the strain VHb showed this advantage, are that, in strain VHb, the bacterial hemoglobin gene can compensate for the effects of plasmid burden. Joshi and Dikshit (1994) have shown that about 40% of the hemoglobin protein is in the periplasmic space, and this localization is suited it’s function to transfer oxygen to the terminal oxidase under hypoxic condition through the facilitated diffusion. The VHb may, also, carry oxygen directly to enhance viability by delivering oxygen to terminal oxidases and consequently increasing ATP production (Chen and Bailey 1994; Kallio et al., 1994). Another possible explanation, by enhancing oxygen delivery to enzymes which may expressed during starvation and keep cells culturable as compared with non-vgb strains. Additionally Liu et al. (1994) reported that as the vgb is transformed into heterologous bacterial host, it can enhance growth and production of valuable biochemicals. The results, may confirm that vgb seem to reflect the cell’s strategy to increase oxidative phosphorylation as energy requirement increase (Khosravi et al., 1990). This also emphasize that with the current limited amount of nutrition, the potential VHb effects should be evaluated on a case-by-case basis. Monitoring the survival of E. coli, non–vgb strains (Wt and pUC8) especially, their culturability after prestarvation may does not imply that they did not survive, but they may be viable but nonculturable.
Plasmid Stability: Plasmid stability was determined at each time point.
| Fig. 1: | Co-difference spectrum of whole cells (36 mg mL-1) of E. coli strain VHb grown in LB medium to late loge phase. The x-axis is nanometers intervals and the y-axis represents the OD measure. Bacterial hemoglobin (VHb) is determined by observing the peaks in this type of spectra at 419nm and a groove at 436 nm. BL means baseline. |
| Fig. 2: | Correlation of protein content of E. coli with two different size of plasmid including pUC8 and VHb (bacterial hemoglobin gene “vgb” containing pUC8 and the parental cells which have no plasmid (Wt). |
| Table 1: | Proportional survival of different E. coli strains at room temperature during starvation. |
| *: Starvation for Carbon(C), Nitrogen(N) and Phosphorus (P) simultaneously | |
At times 2, 4, 8, 16 and 24 days, the data revealed that pUC8 and VHb exhibited 100% plasmid stability; after 24 day, however pUC8 exhibited plasmid instability and at the 32 day time point only 55% of the colonies had plasmids.
| Table 2: | Effect of incubation temperatures (*) on the culturability of the prestarved E. coli strains Wt, pUC8 and VHb. |
| *10°C incubations was also tried several times but the incubation times used could not be enough for the cells to grow. | |
The instability in this strain was confirmed further to determine if it involved either or both strains, pUC8 and VHb. (Table 1)
Effect of incubation temperatures on the culturability of the E. coli strain Wt, PUC8 and VHb: The effect of incubation temperatures on the culturability of the three strains was studied by four temperature (20, 30, 37 and 45°C). The incubation times chosen was 72hr. In the three strains the results (Table 2) were paralleled with the results in Table 1, by which the strain VHb has been shown to has higher culturability as compared with the that of non-vgb strains on the c.f.u. basis. However, at incubation temperature (30°C), the viability of the VHb was the highest. This showed that, may be 30°C temperature is the optimal incubation temperature, for activity of starvation-induced proteins and the VHb at that temperature also enhances the production of these proteins that prolonged the cells culturability. The 20°C it may needs more incubation times because of the unsuitable temperature to the enzymes and proteins involved in bacterial culturability sustenance. However, 45°C mostly is an uncomfortably hot temperature that might inhibit the synthesis of proteins/enzymes involved in the prolongation of cell’s culturability.
Protein concentration: The total amount of protein was determined to see the effect of respective starvation and the vgb on the total protein. The starvation condition and the vgb dramatically affected the total protein content (Fig. 1 and 2). The total protein increase significantly in the order pUC8, Wt and VHb and the culturability expressed by the relative survival cells also increase significantly in the order pUC8, Wt and VHb irrespective to plasmid size. This increase, support the possible role of the vgb, by which it enhances the starvation–induced proteins but not the total proteins. Demodena et al. (1993), reported that modification of cell response by VHb give an evidence for association of it with the cell metabolism and it’s role in cell growth and increasing valuable products.
From this investigation, concluded that omitting any of these nutritional sources (C, P, N and multiple) generally showed the same pattern. However, irrespective to either starvation conditions or plasmid size, the results indicate that the effects of vgb/VHb on the culturability of cells appeared to be advantage. Additionally, this increase in culturability enhanced by VHb may also reflected in the response of physiological behavioral alterations that enabled transformed cells compensating for the heavy duty presented by plasmids.