Radiation Studies on Azotobacter chroococcum

Zbl. Bakt. Abt. II, Bd. 129, S. 242-246 (1974)

[Division of Microbiology, Indian Agricultural Research Institute, New Delhi, India]

Radiation Studies on Azotobacter mroococcum. I. Photoreactivation and Dose Reduction Curve M. H. Ahmed and G. S. Venkataraman With one figure

Summary Lethal effects, produced by ultraviolet light (2537 A) in six strains of Azotobacter chroococcum, can be reversed by light of longer wavelengths, administered after ultraviolet irradiation. Except in one strain (P3), a constant dose-reduction factor (DRF) appears to be untypical in this organism, the theoretical considerations of which are given.

Lethal damage, produced in Azotobacter cells by ultraviolet light (2537 A), can be partially repaired by post-treatment with visible light (GOUCHER et al. 1956). The photoreactivation in this organism appears to be related to the porphyrin content (GOUCHER and KOCHOLATY 1957). The present report deals with the occurrence of the phenomenon of photoreactivation in si x strains of Azotobacter chroococcum and with theoretical considerations of the dose-reduction factor (DRF). Material and Methods Strains: Five strains of Azotobacter chroococcllm (A41, K, P 1, P 3, B 3) were isolated from the rhizosphere of crop plants; strain A 2 was an asparagine-requiring mutant, obtained from strain A41 (KHURANA and VENKATARAMAN 1972). All stocks were grown in Jensen medium (JENSEN 1951), except A2, for which the medium was supplied with 1.5 g. asparagine per liter. All the strains, except A2, were obtained from Dr. S. T. Shende. UV-irradiation: For irradiation, 10 ml. samples of 24-hrs.-old suspensions of 2 X 10 6 cells/ml. were placed in 10 cm. Petri dishes, one for each dose given. These were mixed on a magnetic stirrer. The UV irradiation source was a I-Ianovia mercury vapour germicidal lamp (2537 A) at a distance of 40 cm. To prevent photoreactivation, the UV source was shielded, and all operations were carried out in a darkened room, illuminated by either a dim yellow light from a shielded G E 60 W yellow bulb or in dim red light from aGE 40 W red bulb. Post- UV treatment: For photoreactivation, the irradiated suspensions were placed in a thermostated (30 ± 1°C) photoreactivating chamber, containing two G E 250 W lamps in a fixture 20 cm. from the tubes. A filter of 0.03 N aqueous CuCl 2 in a 3 cm. deep cell was routinely used to absorb a large part of the infrared. A 60 min. exposure caused maximum photoreactivation at this temperature in all UV exposures, and was used as the standard photoreactivation time. After treatment, all suspensions were suitably dilutcd for plate counts, and the plates were incubated in the dark for 48-72 hI'S. at 30 ± 1°C.

Results Strains A41, K, P 1, P3, and B3 were more scnsitive to UV than A2, and all the six strains tested showed photoreactivation (Fig. 1). Under these conditions, only in the case of strain P 3 the photoreactivation resulted in a constant dose-reduction factor

243

Radiation Studies on Azotobacter chroococcum. I.

(DRF) of 0.5 (that is, the photoreactivable sector was 0.5), indicating that in this strain the ratio of reversible to non-reversible damage was the same at all dose levels. However, the dose-reduction factor for the other fi ve strains was not constant. In the case of B 3 and A2 strains, only at survivals below 4 and 50 per cent, respectively, a given dose of visible light negated the lethal effect of ultraviolet irradiation by a constant factor. Thus, the dose-reduction principle is not applicable over the range of the ultraviolet doses used. The curves L(D) in Fig. 1 show the ratio of ultraviolet doses at a given dark survival (rig h t 0 rd ina te) to the ultraviolet dose, yielding the same survival after photoreactivation (abscissa). The curve L(D) is a straight line only for strain P3, whereas for others the curves are not straight (Fig. 1). Discussion A constant dose-reduction factor (KELNER 1949) seems to be ratler untypical for Azotobacter (GouOHER et al. 1956). For every UV dose D, which is followed by light reactivation and thus leads to a certain number of survivors B(D), there can be found 100

16'

...-11:"$:---------,

A2

244

M. H. Ahmed and G. S. Venkataraman

a lower UV dose, L, which in the absence of light reactivation would lead to the same number of survivors, i.e., for which we have B(D)

= A(L).

Plotting L as a function of D, we obtain for L(D) a set of broken straight lines for which we may write L(D) = qD-k (1) in an interval of UV dose, where q is a dose independent constant, smaller than 1, and k is also a dose independent constant in an interval of UV dose. This means that there is a simple relationship between the survival curves A and B which we may express by B(D)

= A(qD-k)

(2)

in an interval of UV dose, where q and k are constants. The first of the straight lines, given by equation (1), always passes through the origin, and hence in an interval, containing the origin, the value of k will be initially zero. As k takes some positive value in the next interval (since each of the subsequent straight lines have a point in common), the value of q will differ from that of the first. However, in particular, if If remains zero throughout, we get a single straight line L(D) = qD through the origin. The set of relationships of the form (2) was confirmed by a series of experiments. When the bacteria are irradiated with UV, a persistent change in the DNA results (dimer formation), which we may call damage, C, that is produced in an amount proportional to the UV dose. Let us assume another change, i.e., inactivation of the enzyme E, which gets re-activated in light to split or monomerize C. Let us further assume that C exists in two forms, Cx which is not sensitive to light reactivation (non-repairable damage) and which is produced in amount x o ' and a formC y which can be repaired (repairable damage), and is produced in an amount Yo. Let us also assume that E consists of two forms, form Ex which gets reactivated in light and repairs C in light and is formed in an amount Xi> and a form Ey which has no effect on C and is formed in an amount Yi. Let us suppose that, due to a UV dose, D, cx, Cy, Ex, and Ey are formed in amounts x o , Yo, Xi> and Yi, respectively. So Xo Yo Xi Yi is proportional to the UV dose D. After UV exposure D, the amount of damage present is Xo + Yo and after light reactivation, the amount of damage present is Xo - Xi. In the absence of light reactivation, E has no effect on C. We assume that the number of survivors after exposure to UV irradiation, with or without subsequent light reactivation, depends on the amount of damage that is present in the bacteria at the time they are incubated with nutrient medium and permitted to multiply. The proportionality of Xo Yo Xi Yi to the UV dose D is so delicately maintained that for shorter duration of UV exposure Cy and Ey are more predominant. and as the dose increases, Cx and Ex are more and more significant and predominant. It is worthwhile to note that both C and E depend solely on the UV dose. Also Ex depends only on the interval in which the UV dose lies. An UV exposure level is determined in such a way that for any UV dose, lying in that interval, the amount of E, activated by light reactivation, is a constant. The amount of E never exceeds the amount of cx, ie., always Xo - Xi ~ O. The production of Cx varies as the UV dose in any specified interval of UV dose, and we will call the constant of proportionality for that interval "q". This interval will be called the general interval, and we may suppose without loss of generality that D lies in this interval. These constants, q, for the successive intervals are in an ascending order, tending to unity. Thus, Xo = qD, where q < 1 and the value of q

+

+

+

+

+

+

245

Radiation Studies on Azotobacter chroococcum. 1.

increases in successive intervals of the UV dose. Let k be the constant amount of E, activated for the general interval of time corresponding to q. Thus Xi = k. Hence, due to light reactivation, the amount of C that remains is given by Xo -

Xi

= qD - k

in the general interval of UV dose. The amount of damage left in the bacteria after light reactivation for the general interval of the UV dose is then given by qD - k, and therefore the number of survivors after light reactivation B(D) can be taken from the survival curve, obtained in the absence of light reactivation A(D), by writing B(D)

= A(qD - k)

in the general interval of the UV dose. If there are many intervals of UV dose, in each of which q and k have constant values, then we obtain many broken lines, corresponding to these intervals. However, if " = 0, we obtain a single straight line through the origin, as in the case of strain P3 (Fig. 1). Thus, in this case, q has the same constant value throughout. At the beginning, for some intervals of UV exposure, we may expect that no Ex will be there, because only little damage will be caused initially. Ma t hematically, this fact can be explained, for at D = 0 (which means that at the time when the bacteria are not exposed to any UV dose) the number of survivors with or without light reactivation must be the same. Hence, from equation (1), we have" = 0, i.e., no Ex is inactivated initially. Alternately, qD - "?: 0 at D = 0 gives" = O. Thus, in all the cases, the first straight line of equation (2) will pass through the origin (Fig. 1; Table 1). Thus, in all cases for the first interval of the UV dose, the equation of the straight line will be Table 1 Mathematical equations for the curves L(D ) in Fig. 1 Strain P1

Strain A2

y=O Y = O.h: - 0.2 y = 0.2x - 0.6 y = O.h - 1.8 y= 0.6x - 3.4. y= x-7.2

0";;; 2";;; 4.";;; 6";;; 8";;; 9.6";;;

x";;; 2 x";;; 4. x";;; 6 x";;; 8 x";;; 9.6 x ,,;;; 12

Strain P3

0 ";;; x";;; 3 3";;; x";;; 9 9";;; x";;; 26

Strain B3

Y = 0.57x - 0.57

Strain K y = O.1x y = O.2x y = O.3x y = O.4. x y = 0.5x y = 0.55x

y=O Y = 0.27x - 0.7 y = 0.4.7x - 2.59

0 ";;; x";;; 11

y=O y = 0.15x - 0.6 y = 0.6x - 3.6 Y = 0.9x - 6.6 Strain A4.1

- 0.2 - 0.7 -1.4. - 2.3 - 2.85

0 ";;; x";;; 2 2 ";;; x ";;; 5

5
7";;; x";;; 9 9 ,,;;; x";;; 11 11 ,,;;; x ,,;;; 13

y=O y=0.1x y = 0.33x Y = 0.7x y = 0.8x

-0.1 - 0.97 - 3.0 - 3.6

0";;; 4.";;; 7 ";;; 10 ";;;

x";;; x";;; x";;; x";;;

o , ;;; x";;;

1.6 ";;; 3";;; 6";;; 7";;;

4. 7 10 12

1.6 x";;; 3 x";;; 6 x";;; 7 x";;; 12

246 M. H. Ahmed and G. S. V enka t ara man, Radiation Studies on Awtobacter chroococcum. I. of the form y = qx. Then after a while, in general, Ex is called into play, due to light reactivation. The constant terms in the subsequent equations are due to the presence of Ex only. The constant terms are negative, as should be expected, for Ex decreases the damage in the bacteria. Further, the absolute values of the constant term increases. Mathematically, in two strai.ght lines of the form y = Qlx - kl and y = q2X- k2' ql = q2 only if kl = k2' since they have a common point of intersection. Thus, the Ex which contributes the constant term alters also the value of q. Initially, i.e. at D = 0, the value of k = 0 and so, if no Ex is present at all, the value of q does not change. That is, we get a straight line passing through the origin. However, in general, Ex is activated in light reactivation in a constant amount in an interval of UV exposure, and these constant values increase in successive UV exposure intervals. Thus, as k changes, q also changes. This explains why we get different broken straight lines in intervals of UV exposures (Fig. 1, strains A2, A41, K, PI, and B3). Also the values of q increase for successive intervals of UV exposures and tend to 1. This clearly shows that for higher UV doses more ex is formed than Cy . Since q approaches 1 for larger UV exposures, the curves tend to become parallel for higher UV doses. We may obtain the same number of survivors after an UV dose (x) in a photoreactivated culture, even at a smaller UV dose (y) in a non-photoreaetivated one. Hence, light reactivation contrihutes si~nifirantly to a hi~her survival. Zusammenfassung Letalc Einfliisse einel· UV- Bestrahlung (2537 A) auf sechs Stiimme von Azotobacter cliroococclLIn konnen dmch die danacll folgende Bestrahlung mit liingeren Lichtwellen zum Teil aufgehoben werden. Der Grad diesel' Schadensbehebung ist bei unterschiedlichen UV-Gaben unterschiedlich. Eine Ausnahme bildet der Stamm P3. Die thcoretischen Grundlagen dieser Erscheinung werden diskutiert. A c knowled gement s Our thanks are due to the Head of the Division of l\Iicrobiology for facilities, and to Mr. T. R . n a l a krishn an, Assistant Professor of Mathematics, Government r.olIege, Coimbatore, for helpful discussions. One of us (M. lI.A.) is grateful to the Indian Council of Agriculturnl Research for the award of a Fellowship.

Literature GOUCHER, C. R., KAMEl, I., and KOCHOLATY, W.: Ultraviolet inactivation and photoreactivation of Azotobacter. J. Baeter. 72 (1956), 184. - GOUCHER, C. R., and KOCHOLATY, W.: A comparison of the cytochrome structure and radiation effects in Azotobacter. Arch. Biochem. Biophys. 68 (1957),30. - JENSEN, H. L.: Notes on the biology of Azotobacter. Proc. Soc. App!. Bact. 14 (1951), 89. - KELNER, A.: Photorenctivation of ultrnviolet-irrndiated Escherichia coli with special reference to the dose reduction principle and to ultraviolet induced mutation. J. Bacter. 58 (1957), 511. - KHURANA, A. S., amI VENKATARAMAN, G. S.: Non-nitrogen fixing mutants of Azotobacter chroococcum. Indian J. exptl. BioI. 10 (1972), 136.

Authors' nddress: Dr. M. 1-1. Ahmed and Dr. G. S. Venkataraman, Division of Microbiology, Indian Agricultural Research Institute, New Delhi-110012 (India).