FEMS Microbiology Letters 115 (1994) 13-18 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

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FEMSLE 05738

Phylogeny of spore-forming lactic acid based on 16S rRNA gene sequences

bacteria

T o m o n o r i Suzuki * and K a z u h i d e Y a m a s a t o 1

Institute of Applied Microbiology/Molecular and Cellular Biosciences, The Unit,ersity of Tokyo, Bunkyo-ku, Tokyo 113, Japan

(Received 15 July 1993; revision received 4 October 1993; accepted 6 October 1993)

Abstract: The phylogeny of spore-forming lactic acid bacteria was investigated on the basis of 16S rRNA gene sequences. Sixteen strains were separated into three lines of descent; one consisted of 14 strains assigned to Sporolactobacillus spp. and Bacillus spp., and the other two each consisted of "Sporolactobacillus dextrus" and Bacillus coagulans. Strains of all the first lineage but one composed a cluster of similarity values of 97.2% and higher, and were represented by the type strain of S. inulinus. The cluster was further separated into five subclusters, four catalase negative and one positive. The definition of the genus Sporolactobacillus should be amended to accommodate catalase positive strains. Spore-forming lactic acid bacteria originated at different phyloge- netic positions, and would have evolved convergently in the area of Bacillus.

Key words: Spore-forming lactic acid bacteria; Sporolactobacillus; Bacillus; Phylogeny; 16S rNA gene

Introduction

Spore-forming lactic acid bacteria are an as- semblage of Gram-positive, motile, homo-lactic acid fermenting and spore-forming bacilli. Histor- ically, only Bacillus coagulans had been known to be such an organism until Sporolactobacillus in- ulinus was described [1]. S. mulinus is catalase negative, but shares chemotaxonomic characteris- tics with Bacillus, which suggests the organism is

* Corresponding author. (Present address) Department of Applied Biological Science, Science University of Tokyo, 2641, Yamazaki, Noda 278, Japan. Present address: Culture Collection Center, Tokyo Univer- sity of Agriculture, Setagaya-ku, Tokyo 156, Japan.

a possible member of the family Bacillaceae [2,3,4]. Sixteen S rRNA cataloging study also sug- gested its closeness to Bacillus [5], and 16S rRNA sequence analysis showed its phylogenetic posi- tion in the area of Bacillus [6].

In addition to B. coagulans and S. inulinus, several catalase negative and positive species were isolated by Nakayama et al. [7,8,9] as seen In Table 1, and their comparative studies have been reported for phenotypic features [7,8,10] and DNA relatedness [11]. The phylogenetic relation- ships among these species or to other bacteria have not yet been investigated, however. In the present study, 16S rRNA gene sequences were determined for 16 strains of those organisms and 10 strains of Bacillus spp. to elucidate the phy- logeny of spore-forming lactic acid bacteria.

SSDI 0378-1097(93)E0430-K

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Materials and Methods

Bacterial strains and cultivation The strains used in this study are listed in

Table 1. Spore-forming lactic acid bacteria except for

B. coagulans IAM 12463 T (T: type strain) were grown at 30°C In GYP medium containing 2% glucose, 2.5% 'Meast' (paste of yeast cell au- tolysate) (Asahi Beer Food Co. Ltd., Tokyo) or 1% yeast extract, 1% peptone (Kyokuto Seiyaku Co., Tokyo), 1% sodium acetate, and 0.5% (v/v) salts solution, pH 6.8. The salts solution con- tained 4% MgSO4.7H20 , 0.2% MnSO4.4H20, 0.2% FeSOa.7H20, and 0.2% NaC1. B. lentus IAM 12466 T was grown in Trypticase soy broth (BBL) at 26°C. Other strains of Bacillus were

grown in Nutrient broth (Kyokuto Seiyaku Co.) at 30°C, with the exception of B. psychrophilus IAM 12468 v which was grown at 15°C. All the catalase positive strains were grown with shaking.

DNA preparation Cells were suspended in Tris-EDTA buffer (33

mM Tris.HC1; pH 8.0 and 1 mM Na2EDTA) and treated with lysozyme (final concentration: 20 mg/ml) and sodium dodecyl sulfate (final con- centration: 0.5%) for lysis. Chromosomal DNA was purified according to standard procedures [121.

Amplification of 16S ribosomal RNA gene Amplification of 16S r R N A gene was per-

formed on a Thermal Reactor (Hybaid Ltd.) in

Table 1

Strains used for the determination of 16S rDNA sequences

Species Strain number Strain number Optical activity of of lactic acid O. Nakayama produced [7,8]

Bacillus coagulans Sporolactobacillus inulinus

"Sporolactobacillus dextrus" "Sporolactobacillus laevas"

"Sporolactobacillus laevas var. intermedius"

" Sporolactobacillus racemicus "

"Bacillus laevolacticus "

"Bacillus myxolacticus" "Bacillus racemilacticus"

Bacillus cereus Bacillus firm us Bacillus laterosporus Bacillus lentus Bacillus megaterium Bacillus pantothenticus Bacillus polymyxa Bacillus psychrophilus Bacillus thuringiensis

IAM12463 T JCM6014 T

NRIC 1134 IAM12380 IAM12388 IAM14263 IAM12384 IAMl2387 IAM12395 IAM12396 IAM14264 IAM12321 IAM12322 IAM12326 IAM12318 IAM12319 IAM12605 T IAM12464 T IAM12465 v IAM12466 x IAM13418 v IAM 11061T IAM 13419 v IAM 12468 T IAM12077 T

L(+ ) D ( - ) D( - )

M-15 L ( + ) * M-114 D ( - ) M-18 D( - ) M-86 D( - ) M-103 D( - ) M-16 DL M-17 DL M-116 DL M-8 D( - ) M-1 D ( - ) M-105 Not described M-14 DL M-5 DL

Abbreviations: x, type strain; IAM, IAM Culture Collection, Institute of Applied Microbiology, The University of Tokyo, Tokyo; JCM, Japan Collection of Microorganisms. RIKEN, Wako; NRIC, Culture Collection Center, Tokyo University of Agriculture, Tokyo. *, Determined by S. Okada.

100 /~1 reaction volume containing 100 ng of chromosomal DNA, 10 t~l of 10 X PCR reaction buffer (Takara Shuzo Co., Ltd., Kyoto), 200 /.~M of each dNTP, 1 /~M of each primer, and 5 units of AmpliTaq DNA polymerase (Perkin Elmer Cetus). The primers were 5' GAGTTFGATC- CTGGCTCAG (Escherichia coli numbering sys- tem: positions 9-27) and 5' AGAAAGGAGGT- GATCCAGCC (positions 1525-1544). Amplifica- tion conditions for the PCR included an initial denaturation step at 94°C for 2.5 min followed by 30 cycles (94°C for 1 rain, 58°C for 2.5 min, then 72°C for 2.5 min) and a final extension step at 72°C for 5 min.

Sequencing The amplified DNA fragments were purified

by gel electrophoresis on 1% Agarose S (Nippon Gene Co. Ltd., Tokyo), and recovered with glass powder using an EASYTRAP kit (Takara Shuzo Co., Ltd.). Template DNA was annealed to the respective sequencing primer [13] by snap cooling (heated at 95°C for 5 min, and then cooled at 0°C for 10 min). DNA sequencing was performed using [a-35S]dATP (Amersham Corp.) and the Sequenase version 2.0 kit (U.S. Biochemical Corp.).

Analysis of sequence data The sequences were aligned to B. subtilis 16S

rRNA sequence [14] taking account of the sec- ondary structure. Evolutionary distances (Knu o values) were calculated, and a phylogenetic tree was reconstructed by applying the algorithm of neighbor-joining method [15] to Knu c values.

Nucleotide sequence accession numbers The sequences reported here have been as-

signed (DDBJ) accession numbers D16266 to D16291.

Results and Discussion

We determined about 1,500 nucleotide se- quences of 16S rRNA gene (rDNA) of 16 strains of spore-forming lactic acid bacteria and 10 strains of Bacillus spp. which were used as reference.

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The phylogenetic tree was reconstructed from 1,092 nucleotide sequences which were unam- biguously alignable positions (Fig. 1). About 100 nucleotides of 3'-end of 16S rDNA could not be used, as reference sequences from the DNA data base lacked the sequences for this region. The variable regions corresponding to positions 70- 100 and 370-430 (E. coli numbering system) were also excluded from the calculation in order to achieve a more precise phylogenetic analysis.

The sixteen strains of spore-forming lactic acid bacteria used in this study were phylogenetically divided into three lines of descent. These were the branch represented by S. inulinus (B-1 branch) and branches consisting of 'S. dextrus' IAM 12380 (M-15) (B-2 branch) and B. coagulans IAM 12463 T (B-3 branch), respectively. The B-1 branch comprised a cluster (SI cluster) consisting of 9 strains assigned to Sporolactobacillus spp. (includ- ing the type strain of the type species, S. inulinus) and 4 strains assigned to Bacillus spp., and 'B. racemilacticus' IAM 12319 (M-5) (Fig. 1).

Ash et al. [6] indicated that the type strain of S. inulinus formed a separate line in the area of Bacillus. In the present report, we have shown that most of the spore-forming lactic acid bacte- ria were located on this line and clustered at its top.

Among the strains of the SI cluster, 16S rDNA similarity values calculated from 1,456 nu- cleotides were 97.2% and higher. These values and the topology of the tree indicate that those strains should be comprised in a single genus, Sporolactobacillus, even though they are not ho- mogeneous in catalase and lactic acid isomer.

The SI cluster branched into five twigs, and each of the five subclusters consisted of one to four strains. The inter-subcluster values of 16S rDNA similarity were 97.2 to 99.0% (14 to 42 nucleotides differences) and intra-subcluster ones were 99.7 to 100% (0 to 4 nucleotides differ- ences). The strains of the subclusters were lo- cated at the top of the twigs. Each subcluster was compact and composed a distinct phylogenetic unit at the infra-generic level. It might be as- sumed to correspond to a species, but its taxo- nomic appreciation will require further genetic and phenetic investigations.

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8. coagulans TAM 12463 ~

X :ac~lcus = M- 16 racewaY'as * II- 116

S ~hullhus Jell 6014 T "~ laevas ssp. in ter~dius ° M-86

inullnus NRIC 1134 " ~

"~ rnc~i lac tJcus" l-5

.8 l~a to t.g'ca ticus

"==/ I/ h'cbenifar#2/ R azotoforJans ,

racealcus" if- I 7 laevas " M-114 laevaa ssp. ia ter~d~k¢" M-f03

~ ~ laevaI.~ticu$'~=8 , . a :acexilacticus !-14

laevolac ta~s "li-1 #yxalacticus" M- 105

~ laevas" i-18

R stearatherat~Has

R ancar2holytl~as

c o s l ~ s l u s

, ~ latcrospotz~

R saithii~

t~ ~ g a ter~2~,

R polymxxa

R a#ylol:tlcus

sl~aen~us

~ dextr t~" I[- 15

0.01 K~o~

~,~ psycMol~ilus

cereus R ~dusa B. tharahgaensas

Fig . 1. Phylogenetic tree of spore-forming lactic acid bacteria and Bacil lus species. The name and number of a strain and its position in the SI cluster correspond in clockwise order.

The SI cluster includes catalase negative strains (Sporolactobacillus spp.) and positive strains (Bacillus spp.). Since these strains are to be as- signed together to Sporolactobacillus, the defini- tion of this genus should be amended to accom- modate both catalase negative and positive strains. The positive strains were exclusively col- lected in one subcluster of the five. The catalase activity is not a feature characterizing the genus but, rather, the subclusters.

Nakayama and Yanoshi [7] and Nakayama [9] defined species on the basis of optical isomers of lactic acid produced. The present results showed that this character could not be a criterion to define species for the bacteria of the cluster; strains of a species they described were scattered

among subclusters and each of subclusters in- cluded strains producing different isomers. Yanagida et al. [10,11] also reported an inconsis- tency between optical activity and their taxo- nomic grouping.

'B. racemilacticus' IAM 12319 (M-5) of the B-1 branch deviated on the way to the SI cluster. 'S. dextrus' IAM 12380 (M-15) of the B-2 branch was located in the B. cereus - B. megaterium group, though the strain is catalase negative. This bac- terium and B. coagulans of the B-3 branch pro- duced L( + )-lactic acid and was very remote from o(-)- and DE-acid producers of B-1 branch.

Spore-forming lactic acid bacteria are not monophyletic. They originated from at least three different phylogenetic positions in the area of

Bacillus, and would have evolved independently to create separate lines of descent, each converg- ing to live on homo-lactic acid fermentation.

Acknowledgments

We are indebted to those involved the IAM Culture Collection, Institute of Applied Microbi- ology, The University of Tokyo, the Japan Collec- tion of Microorganisms, RIKEN and the Culture Collection Center of the Tokyo University of Agriculture for providing bacterial strains. We thank Dr. Hiroshi Oyaizu (Department of Agri- cultural Chemistry, The University of Tokyo) for advice on the PCR-RI sequencing method and also Dr. Sanae Okada (Culture Collection Cen- ter, Tokyo University of Agriculture) for the de- termination of lactic acid isomers. We are grate- ful to Dr. Hiroshi Kuraishi and Dr. Yuzo Ya- mada (Department of Agricultural Chemistry, Shizuoka University) for helpful advice and en- couragement. This work was supported in part by a Grant-in-Aid for Cooperative Research (A) (No. 03304017) from the Ministry of Education, Sci- ence and Culture, Japan.

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