Genetic Lesions Affecting Amino Acid Biosynthetic Pathways

JOURNAL OF BACTERIOLOGY, Oct. 1981, p. 64-71 0021-9193/81/100064-08$02.00/0

Vol. 148, No. 1

Multiple Nutritional Requirements of Lactobacilli: Genetic Lesions Affecting Amino Acid Biosynthetic Pathways TAKASHI MORISHITA,l* YORIKO DEGUCHI,' MASAKO YAJIMA,1 TOSHIZO SAKURAI,' AND TAKASHI YURA2 Yakult Institute for Microbiological Research, Kunitachi, Tokyo,' and Institute for Virus Research, Kyoto University, Kyoto,2 Japan Received 24 March 1981/Accepted 20 June 1981

Genetic lesions responsible for amino acid requirements in several species of multiple auxotrophic lactobacilli were investigated. Systematic attempts were made to isolate mutants that could grow in the absence of each of the amino acids required by the parental strains of Lactobacillus plantarum, L. casei, L. helveticus, and L. acidophilus. After treatment with appropriate mutagens, such mutants could be obtained with respect to many but not all required amino acids. Successful isolation of mutants for a given amino acid means that a minor genetic lesion reparable by single-step mutations affects its biosynthesis; a failure to isolate mutants suggests the involvement of more extensive lesions. Analysis of these results as well as the specific requirements exhibited by the parental strains revealed certain regularities; some of the biosynthetic pathways for individual amino acids were virtually unaffected or affected by minor lesions in all of the species tested, whereas others were affected by more extensive lesions in at least some species. Both the number and the kind of pathways affected by extensive lesions differed appreciably among different species. Furthermore, the growth response of the parental strains to some putative amino acid precursors revealed a clear correlation between the extent of genetic lesions and the occurrence and location of a genetic block(s) for a given pathway. These findings are discussed in relation to the phylogeny, ecology, and evolution of lactic acid bacteria. Most strains of lactobacilli exhibit characteristic requirements for a number of amino acids, vitamins, and other nutrients for growth in synthetic media. As one approach to understand the genetic and biochemical basis of multiple nutritional requirements in lactobacilli, we had previously undertaken systematic isolation and characterization of mutants of Lactobacillus casei strain S1 that had lost a requirement for specific nutrients (10). Such mutants could indeed be obtained for a majority of amino acids (7 of 12) and vitamins (3 of 4) tested. At least in one case studied in some detail, a single-step mutation was shown to cause the appearance of specific enzyme activity involved in biosynthesis of the particular amino acid. These results led us to a general conclusion that the nutritional requirements in L. casei are usually, if not always, due to minor defects in the bacterial genome rather than large deletions. In other words, all the genes essential for biosynthesis of most nutrients appeared to be present in the genome of L. casei S1, and many of them failed to produce functionally active proteins due to one or a few deleterious mutations. It seemed as

though L. casei had evolved by progressive loss of functions from an ancestral organism that once possessed perhaps all of the functionally intact biosynthetic pathways. Lactobacilli and other lactic acid bacteria are distributed widely in nature and have been isolated from various sources, including humans, lower animals, plants, dairy products, wines, and ensilages (11, 13). However, many individual species appear to have adapted to specific environments and are generally not found outside of their specialized habitats (7). It was thought, therefore, that the kind of experiments conducted with L. casei S1, if extended to other species, might reveal whether specialized habitats for each species influenced the nature and extent of genetic lesions affecting the biosynthetic pathways. The present paper reports on the results of experiments carried out with four species of Lactobacillus, L. plantarum, L. casei (ATCC 7469), L. helveticus, and L. acidophilus, chosen primarily on the basis of known natural habitats and taxonomic properties. L. casei ATCC 7469 was included in the present study because it 64

VOL. 148, 1981

AMINO ACID REQUIREMENTS IN LACTOBACILLI

exhibits nutritional requirements appreciably different from those of strain Si, which was used previously. Our results show that mutants that lost a specific requirement could be isolated for a number of amino acids required by each parental organism. The results indicate both similarities and differences among different species in the nature and extent of genetic lesions that have accumulated on the genome; similarities were found for some biosynthetic pathways, whereas distinctive differences were detected for others. The growth response of these organisms to some of the putative amino acid precursors indicates that the occurrence of a genetic block(s) for each pathway is correlated with the extent of genetic lesion on the genome.

65

(ii) Mutagenesis with EMS. Overnight cultures were diluted 50-fold in natural medium containing 2% (vol/vol) EMS and were incubated at 37°C for 45 min (L. plantarum and L. casei) or 30 min (L. helveticus and L. acidophilus). Cells were collected, washed in saline, suspended in the same volume of drug-free natural medium, and then incubated overnight at 370C. (iii) UV irradiation. UV irradiation was carried out by exposing washed cell suspensions (109 cells/ml in saline) to a Mitsubishi germicidal lamp (15 W) for 30 s (L. plantarum and L. casei) or 60 s (L. helveticus and L. acidophilus) at a distance of 50 cm. Irradiated cells were collected by centrifugation and incubated overnight in natural medium. To facilitate detection of rare mutants, the mutagenized cells prepared as above (109 cells/ml) were collected, washed twice in saline, suspended in appropriate selective liquid media (108 cells/ml), and incubated for about 40 h. The cells were collected, and appropriate dilutions were plated onto selective agar media. In some cases, cells were retransferred (one or two times) and incubated further in fresh selective liquid media. All agar plates were incubated for 3 to 4 days, and the colonies obtained were picked and purified on selective agar plates. Nutritional properties of these mutants were tested by streaking cell suspensions on a set of selective agar media from which a specific nutrient was omitted. All cultures were incubated at 37°C except where otherwise indicated.

MATERIALS AND METHODS Bacterial strains. Wild-type strains of lactobacilli used as the parental organisms were L. plantarum ATCC 8014, L. casei ATCC 7469, L. helveticus ATCC 15009, and L. acidophilus ATCC 11506. L. casei ATCC 7469 belongs to subspecies rhamnosus and differs taxonomically from L. casei Si (subspecies casei), which was used in the previous work (10). Media. Natural medium was essentially the same as Rogosa medium (2) and consisted of (per liter): Trypticase (BBL Microbiology Systems), 10 g; yeast extract (Difco Laboratories), 5 g; tryptose (Difco), 3 g; RESULTS D-glucose, 5 g; sodium acetate, 1.7 g; ammonium citother nutritional reAmino acid and 2 3 3 MgS04.7H20, rate, g; KH2PO4, g; K2HPO4, g; 0.575 g; FeSO4.7H20, 0.034 g; MnSO4.2H20, 0.12 g; L- quirements of parental strains. Nutritional cysteine hydrochloride, 0.5 g; and Tween 80, 1 g. Basal requirements of the four species of Lactobacil(synthetic) media were glucose-salts media supple- lus were first determined by examining growth mented with all of the nutrients required by each of cells in a series of liquid glucose-salts media strain for maximal growth (Table 1). The compositions with defined supplements (Table 1). All strains of the media were basically similar but differed from exhibited characteristic requirements for a numone another due to differences in specific growth requirements for each strain. Solid media contained 1.5% ber of amino acids and vitamins and, in some agar (Difco). The pH of all media was adjusted to 7.2. cases, purines and pyrimidines for maximal Chemicals. All chemicals used were commercial growth. The results were generally in agreement products. Amino acids were purchased from Wako with those reported in the literature (4, 6, 14, Pure Chemicals Co., and amino acid precursors were 16). The number of amino acids absolutely reobtained primarily from Sigma Chemical Co. quired by each strain was 7, 10, 14, and 15 for L. Growth experiments. Cells were grown overnight plantarum ATCC 8014, L. casei ATCC 7469, L. in natural medium, washed twice in saline, and used helveticus ATCC 15009, and L. acidophilus to inoculate a series of liquid basal media (about 106 cells/ml). Cultures were incubated by standing at 37°C ATCC 11506, respectively. A few additional for 72 h (unless otherwise indicated), and turbidity amino acids were also required for maximal was measured in a Klett-Summerson colorimeter (no. growth of these strains. Besides, some nutrients, including amino acids, stimulated growth on 66 filter). Isolation of mutants. Mutants that had lost a agar media significantly, though they were not requirement for a given nutrient were isolated after required in liquid media. The standard basal treatment of cells grown overnight in natural medium media contained all of these nutrients in addition (about 109 cells/mi) with N-methyl-N'-nitro-N-nitro- to those required for maximal growth in liquid soguanidine (NG), ethyl methane sulfonate (EMS), or media. UV light as follows. Mutations leading to loss of requirement (i) Mutagenesis with NG. Cells were washed in saline and treated with NG (100 tg/ml) in 0.05 M for individual amino acids. To determine phosphate buffer (pH 7.0) at 37°C for 30 min. After whether genetic lesions responsible for amino being washed twice in saline, cells were suspended and acid requirements could be rescued by singlestep mutations, a systematic attempt was made incubated in natural medium for 3 h.

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MORISHITA ET AL.

J. BACTERIOL.

TABLE 1. Composition of basal media useda Concn (mg/ml)

Concn (mg/ml)

Compound

Compound L.p.

D-Glucose .......... 10 6 Sodium acetate Sodium citrate 1 Ammonium citrate KH2PO4 ............ 3 K2HP04 ............ 3 0.5 MgSO4 7H20 MnSO4 7H20 ....... 0.05 0.02 FeSO4 7H20 Tween 80 .......... 1 Sodium thioglycolate Spermidine .......

........

L.c.

L.h.

10 6

10 6

1 3 3 0.5 0.05 0.02 1

1 3 3 0.5 0.05 0.02 1

phosphate

L-Alanine 0...........1" DL-Alanine

6.1b ......... L-Arginine . 0.2' L-Aspartic acid L-Cysteine .......... 0.2c

0.1 0.2 0.2c 0.2 L-Glutamic acid ..... 0.2 Glycine .L.............C 0.1 L-Histidine 0.1 0.1 0.1 L-Isoleucine ........ 0.1 0.1 L-Leucine .......... 0.1 0.1C 0.1 L-Lysine 0.1c 0.1 L-Methionine ....... 0.1 0.1 0.2 0.2 0.2

L.p.

L.a.

15 15 0.5 1

1 0.2 0.025 0.015 1 0.5

0.1

L-Tryptophan 0.1 .01b L-Tyrosine

0.1 0.1 0.1

L-Valine . 0.1 p-Aminobenzoic acid . 0.0002 Biotin .0.00001 Folic acid Nicotinic acid 0.001 0.005 Nicotinamide Pantothenic 0.2b acid . 0.001 0.1 Pyridoxal ..... 0.002

0.2 0.2 0.5

0.3b 0.1 0.1 0.1

0.1b

L.c.

L-Phenylalanine 0.1 L-Proline L-Serine O.1c L-Threonmne

Pyridoxol Riboflavin ..... Adenine Adenylic acid Cytidylic acid Deoxyguanosine

0.1b 0.lc

L.h

0.1 0.1

0.1b

0.1 0.1 0.1 0.1

0.0002c 0.0001 0.001

0.0001c 0.00016 0.001

0.001 0.001 0.002

0.001 0.002

0.001

0.001 0.01C

0.001 0.001C 0.001

0.016b 0.05b

Guanine .0.01 Uracil

L.a.

0.1 0.1 0.1 0.1 0.1 0.1 0.1

0.01

0.008 0.03c

0.01b

0.004b Thymine Xanthine 0.01C a Basal media for L. plantarum (L.p.), L. casei (L.c.), and L. helveticus (L.h.) were established by modifying the compositions of the synthetic medium used previously for L. casei S1 (10). For L. acidophilus (L.a.), the medium described by Soska (16) was used with minor modifications. The modifications were based on the growth requirements of each organism, which were determined as follows: washed cell suspensions were used to inoculate a set of liquid media lacking each of the nutrients listed, cultures were incubated at 37°C, and optical density was measured at 24-h intervals for 3 days. bNot required, but stimulates growth in both liquid and agar media. 'Not required for growth in liquid media, but stimulates growth on agar media. 0.1

to isolate mutants that no longer required each

of the amino acids required by the parental organisms. To facilitate detection of rare mutants as well as to permit full expression and segregation of the mutated genes, the mutagenized cells were first incubated in natural medium and then in selective medium before being plated onto a set of selective agar media at 37 or 300C (Table 2). In agreement with the previous results with L. casei S1 (10), mutants that had lost a specific requirement were obtained for many, but not all, amino acids tested. Treatment with NG generally proved most effective. A separate experiment using a higher concentration of NG gave essentially the same results for all of these strains (data not shown). Such mutants could not be obtained spontaneously for any of the amino acids or strains tested (