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1-Zeitschrift fur Allg. Mikrobiologie I 2 4 19G2 320-323

(Grigore Alexandrescu Hospital, Central Laboratory Bucharest, Roumania Ilie Pintilie Boulevard 32)

Influence of some medium conditions on the manifestation of fermentative mutations

S. SCHAFLER

(Eingegangen am 12.3.1962)

The evidence of several fermentative mutations is to a greater extent subjected to the influence of different medium factors than that of drug resistance or auxotro- phic mutations. In this case may interfere complex interrelations with some sugars of the culture medium (MONOD 1942, SCHAFLER et a1 1954, LEMER 1958) or with other constituents of the medium (SCHAFLER et a1 1960). The lack of fermentation can depend on the absence of the corresponding enzyme, as well as on the imper- meability of the cell wall, the lack of the transport system, deficiences in inductibility, etc. All these factors can modify the required conditions for the evidence of a muta- tive fermentation. For that reason, the knowledge of the conditions for the mani- festation of the fermentative mutations may be useful for many genetic and taxono- mic purposes. Some of the influencing factors are described below.

Results I. The appearance of papillae

Several investigators believe the papillae are originating from individual mutations, the mutant cells giving rise to clones with selective advantages within the colonies - the papillae. Papillae were so used for the determination of the mutation rate (RYAN et a1 1955, GOETZE and SACHS 1960), and the estimation of the mutation frequency. HADOW (1937) and especially DEAN and HINSHELWOOD (1957) emphasize the im- portance of the culture age and of some micro-ecological conditions. One of the most important, but nearly not a t all studied influence is that of the composition of the culture medium, especially its contents in amino-acids, peptones, vitamines etc.

The appearance of lactose' papillae in E . coli, strain K-12 W 677 was studied in KOSER'S saline medium (NaNH4HP0,.5H20 1,5 g; KH,P04 1 g ; MgSO, . 7 H,O 0,2 g; H,O 1OOOml) with 2y0 agar, 1% lactose, 0,004~0 bromthymol blue and different concentrations of yeast extract (Difco). The papillae were tested in a confluent cultu- re film (Table 1). Table 1. The appearance of Lac+ papillae in function of the concentration of yeast extract - -

Concentration of yeast extract (yo)

0,2

1 2 5

.

0,5

- -

Time of appearance of papillae (days)

~~ ~~~

4-6 3 4 2-3 5-7

no papillae in 12 days

Number of papillae/ cma (mean number of

6 plates)

471 1 6 3 22,2 3 2 -

Prevalent fermentation type

slow fermentation slow fermentation slow fermentation fast fermentation

-

-

Manifestation of fermentative mutations 32 1

In 5% yeast extract, the appearance of papillae in the periphery of the growth area, was sometimes observed. The total cell count was 60-70"/0 greater in 2% yeast extract than in 1% (1,l-1,3 + lo9 cells/cm2) and it was 130-150% greater in 5%

tn log. nat. 2 1) yeast extract. The calculation of the mutation rate after the formula (RYAN et a1 1955), would give in this case very different results in function of t,he concentration in yeast extract.

In small isolated colonies on 1% yeast extract medium, the mean number of pa- pillae is smaller and sometimes the papillae are absent (see also DEAN and HINSHEL- WOOD 1957). In this case, a modification of the pH indicator by the whole colony can be observed occasionally.

I n the presence of 0,2 and 0,5% Bacto Peptone Difco, no Lac+ colonies during the 12 days period were observed. In the presence of 1% Bacto Peptone, 1-2 papillae/ ema were observed, in 2% Bacto Peptone, 4-8 papillae/cm2 appeared in 3-5 days, 5% gave a complete inhibition of the Lac' papillae. The total cell count in 2% Bacto Peptone is 70-80y0 of the number in 1% yeast extract. I n Bacto Casitone Difco similar results were obtained, but 5-7% Protese Peptone Difco had only a slow inhibitory effect,

I n nutritive agar (beef infusion, Witte Peptone 1%, NaCl 0,5y0, agar l,8y0, amino nitrogen 100-110 mg yo) the Lac+ papillae are nearly completely inhibited. In EMB lactose agar, the number of papillae was similar to that in KOSER'S medium with 0,5% yeast extract.

Addition of threonine, leucine and B, vitamin (growth factors of strain W 677) and increase of lactose concentration have a reducing influence on the appearence of the Lac+ papillae.

The formation of papillae in presence of salicin, maltose and mannitol is in a lesser extent inhibited by 2% yeast extract and nutritive agar than the lactose fermenta- tation, but is wholly inhibited with 5% yeast extract. In nutritive agar, or 2% yeast extract, the inhibition of the appearence of cellobiose + papillae by Salmonella Heidel- berg is only insignificant.

It seems that the presence of different nutritive components of the culture medium may have a decisive influence on the appearance of fermenting papillae. The ratio 1 papilla - 1 mutation appears only in special conditions. It seems that for the rapid appearance of papillae, the presence of an optimal concentration of some nutritive elements is required (see the delayed appearence of the Lac+ papillae in presence of 0,2--0,570 yeast extract and their absence in the first 12 days in 0,5% Baeto Peptone). Increased concentrations of some nutritive substances as peptones, amino acids, inter- mediates of the KREBS cyclus etc., can have a pronounced inhibitory effect ( S C ~ F L E R et al, 1960). This effect depends on the selective value of different mutants fermen- ting the same sugar (slow and fast fermenting Lac+ mutants), the kind of sugar and the strain used. 0,5-1y0 yeast extract and the EMB medium provide in many cases optimal conditions, but the concrete limiting conditions have to be determined for each instance.

N -~

2. Influence of the composition of tk liquid medium

Carrying on former investigations, concerning the influence of sugar (XCHAFLER and MINTZER 1959), and peptone concentration (SCHAFLER et al, 1960) on lactose fermentation by Sulmonellae, the influence of these factors on lactose fermentation by E. coli K-12 W 677 was tested. In peptone water (1 "/o Bacto Peptone, 0,5% NaCl ~ ~.

m = mean number of papillae, N = total number of cells.

322 S. SCHAPLER

and 0,004~0 bromthymol blue) the optimal concentration of lactose i s P 5 % , the fermentation time being in the average by 3,2 days shorter than in the presence of 0,5% lactose (50 tubes of seach concentration). In KOSER’S salt solution with 0,10//, yeast extract, the optimal concentration of lactose is 1,5-2yo. With the optimal concentration of lactose in peptone water there is a great difference between the fer- mentation times of the different tubes (from 3 to 12 days, with a mean value of 5,8 days in presence of 4% lactose). In KOSER’S medium + O,lyo yeast extract and 1,5% lac- tose, 94% of the tubes ferment lactose between the 6t1r and the 7t11 day (mean value 6,2 days).

The differences observed can be partially explained by the activation of the lactose fermentation by the Lac + mutants by increased concentrations of lactose and by the appearence of a greater ratio of slow fermenting-types (75-78y0), which are partially inhibited in peptone water (selective advantages in KOSER’S solution + O , l % yeast extract). The fast-fermenting types appear in a smaller number and have selective advantages in peptone water. Their smaller number explains the differences in fermentation time in parallel tubes with peptone water. I n fact, in both media, there is a mixture of slow and fast-fermenting types in different ratios. With the same strain, the mutative fermentation of salicin and mannitol is promoted in peptone water.

Investigations made with Salmonellae showed that, contrary to the fermentation of lactose, the d-arabinose fermentation by Salmonella Dublin is not influenced by the arabinose concentration.

We obtained very different results in function of the culture medium, with regard to the xylose fermentation by S. paratyphi A (18 strains) and the L-arabinose and trehalose fermentation by S. chlerae suis (21 strains). The lack of these fermentations is an important differential feature of these Salmonella species (KAUFFMANN, 1954).

In peptone water, none of the strains fermented xylose, arabinose and trehalose (except one strain). In KOSER’S saline medium + 0,l yo yeast extract all strains fer- mented in 9-14 days the above mentioned sugars (2 strains excepted), + and - co- lonies were isolated on the same medium with l,8% agar. Sucrose and inositol were fermented in neither medium. I n the culture media mentioned above, the fermen- tation of inositol and sucrose can be considered as negative and the fermentation of xylose, trehalose and arabinose as conditional positive. OLINICI~) found partially similar results with some &‘higella strains. Many negative fermentations seem to be such mutative conditional positive, slow fermentations.

Another feature which can influence the calculation of the mutation rate in defined media with the corresponding sugar as sole carbon source, is the various residual growth of the inoculum. In a preliminary study with salicin and lactose fermenting mutants it was found that the residual growth is due not only to the impurities of the culture medium, but in some instances to a slow utilization of the sugar, especially when the lack of fermentation is associated with other metabolic deficiencies than the absence of the corresponding enzyme.

Achnowledgement Thanks are due to Prof. C. N. HINSHELWOOD and Dr. A. C. R. DEAN for reading the manu-

script.

Summary The mutative fermentation is often a complex microecological processus in which the absence

of fermentation does not necessarly implicate the absence of cells ablc t o ferment the sugar.

’) unpublished data.

Manifestation of fermentative mutations 323

The formation of papillae is depending on the concentration of other elements of the nutritive medium. The ratio 1 papilla - 1 mutation-event is valid only in special optimal cases. The xylose fermentation by S. paratyphi A and the arabinose and trehalose fermentations by S. cliolerue suis may be positive or negative in function of other nutrients of the culture medium.

L i t e r a t u r e DEAN, A. C’. R.. and HINSHELWOOD, C., 1957. Tlic formation of papillae in bacterial colonies 11.

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