UNIVERSITI PUTRA MALAYSIA

SPECIES CLASSIFICATION AND MOLECULAR STUDIES OF BILE SALT HYDROLASE (bsh) GENE IN

BIFIDOBACTERIUM SPP

SHUHAIMI BIN MUSTAFA

FSMB 2003 2

SPECIES CLASSIFICATION AND MOLECULAR STUDIES OF BILE SALT HYDROLASE (bsh) GENE IN

BIFIDOBACTERIUM SPP

SHUHAIMI BIN MUSTAFA

DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

2003

SPECIES CLASSIFICATION AND MOLECULAR STUDIES OF BILE SALT HYDROLASE (bsh) GENE IN BIFIDOBACTERIUMSPP

By

SHUHAIMI BIN MUSTAFA

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in FuliJlment of the Requirements for the Degree of

Doctor of Philosophy

January 2003

Dedicated to .............................. .

teachers who dedicate the best moments of their life to teach us for a better tomorrow

ii

Abstract of the thesis presented to the Senate of Universiti Putra

Malaysia in fulfilment of the requirements for the degree of Doctor of

Philosophy

SPECIES CLASSIFICATION AND MOLECULAR STUDIES OF BILE SALT HYDROLASE (bsh) GENE IN BIFIDOBACTERIUM SPP

By

SHUHAIMI BIN MUSTAFA

January 2003

Chairperson : Associate Professor Mohd Yazid Manap, Ph.D

Faculty : Food Science and Biotechnology

Molecular methods were used to identify and characterise

Bifidobactenum isolated from the faeces of breast-fed infants. A

Bifidobactenum genus-specific primers based on the V9 variable region

of the 168 rDNA was used to identify Bifidobactenum isolates and to

distinguish them from other genera. All the Bifidobactenum species

tested generated peR product with the size of approximately 1.35 kb,

whereas other genera showed no band with these primers.

Furthermore, a sequence analysis of 168 rRNA gene of

Bifidobactenum isolates revealed high homology (98% and above) with

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BijidobacteriumpseudocatenulatumJCM1200. This result indicates that

the 16S rRNA gene sequence analysis is a useful and accurate tool for

the identification of the genus Bifidobacterium In addition, a set of B.

pseudocatenulatum species-specific primer was used as an alternative to

identify the species of B. psedocatenulatum using PCR technique. It was

found that this set of primer was able to generate PCR product with the

size of approximately 278 bp in all the B. pseudocatenulatum isolates as

well as the type strain of B. pseudocatenulatumJCM1200. Other species

of bifidobacteria gave no band.

A protocol for the rapid fingerprinting technique of Bifidobacterium

strains and other pro biotic microorganisms based on randomly

amplified polymorphic DNA (RAPD) has been developed. Three 10-mer

primers (GEN 1-60-03, GEN 1-60-06 and GEN 1-60-07) used generated

RAPD patterns with DNA fragments ranging from approximately 0.3 kb

to 10.0 kb in size. Examination of the DNA fingerprints using cluster

analysis showed a significant genetic diversity among the strains tested.

Another fingerprinting technique based on the distribution of dispersed

repetitive DNA [enterobacterial repetitive intergenic consensus (ERIC)

and repetitive extragenic palindromic (REP)] segments in the genome of

Bifidobacterium and other pro biotic bacteria was also examined for the

first time using primers derived from the REP and ERIC sequences and

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PCR. The patterns of the resulting PCR products were analysed on

agarose gel and were found to be highly specific for each species. All the

B. pseudocatenulatum isolates and B. pseudocatenulatum JCM 1200 type

strain presented an approximately 1.5 kb fragment by ERIC and a 800

bp fragment by REP. These two fragments were not detectable in other

species of bifidobacteria. This study demonstrates the presence of ERIC

and REP-like elements in the genome of bifidobacteria and other

probiotic bacteria.

PCR technique was also used to detect the presence of bsh gene in

Bifidobacterium longum B8536. In this regards, a pair of PCR primers

for the rapid detection of bsh gene from B. longum have been

synthesised and have revealed the bsh gene of appoximately 970 bp .

The bsh gene was cloned and sequenced showing a high homology to

bsh gene previously published. The resulting nucleotide sequence

encodes a predicted protein of 317 amino acids with a molecular weight

of approximately 35 kDa. The bsh gene from B. longum was also cloned

and expressed in E. coli BL21-S1 using pRSET -A expression vector. The

expressed protein was detected using immunoblot assay.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi syarat keperluan untuk ijazah Doktor

Falsafah

PENGENALPASTIAN SECARA MOLEKULAR SPESIS DAN GEN BILE SALT HYDROLASE (bsh) DAR! BIFIDOBACTERIUMSPP

Oleh

SHUHAIMI BIN MUSTAFA

Januari 2003

Pengerusi : Profesor Madya Mohd Yazid Manap, Ph.D

Fakulti : Sains Makanan dan Bioteknologi

Kaedah biologi molekul telah digunakan didalam kajian ini untuk

mengenalpasti dan mencirikan isolat bifidobakteria dari najis bayi yang

menyusu ibu. Primer genus-spesifik untuk bifidobakteria yang

berdasarkan kepada kawasan bolehubah V9 dari jujukan nukleotid 1 68

rDNA telah digunakan untuk mengenalpasti isolat bifidobakteria dan

untuk membezakan mereka dari genera bakteria yang lain. 8emua

bifidobakteria yang diuji menghasilkan produk peR bersaiz lebih

kurang 1 .35 kb, manakala genera yang lain tidak menghasilkan

sebarang jalur dengan primer inL

Kemudian, analisa jujukan gen 1 6S rRNA dari isolat

bifidobakteria menunjukkan persamaan yang tinggi (98% dan keatas)

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dengan Bifidobacterium pseudocatenulatum JCM 1200. Keputusan ini

menunjukkan bahawa analisis jujukan gen 168 rRNA adalah satu

kaedah yang berguna dan tepat untuk mengenalpasti genera

bifidobakteria. Berikutnya, satu set primer spesis-spesifik telah

digunakan sebagai alternatif untuk pengenalpastian B.

psedocatenulatum menggunakan kaedah PCR. Keputusan yang

diperolehi menunjukkan bahawa primer ini berupaya menghasilkan

jalur PCR bersaiz kira-kira 278 bp untuk semua isolat B.

pseudocatenulatum dan B. pseudocatenulatum JCM 1 200. Spesis lain

tidak memberikan sebarang jalur PCR.

Protokol untuk pengecap jarian secara cepat keatas strain

bifidobakteria dan mikroorganisma probiotik yang lain telah dilakukan

dengan menggunakan kaedah 'randomly amplified polymorphic DNA'

(RAPD). Tiga jenis primer 1 0-mer (GEN 1 -60-03, GEN 1 -60-06 dan GEN

1-60-007) yang telah digunakan menghasilkan corak RAPD dengan

jalur DNA bersaiz dari 0.3 hingga 1 0.0 kb. Ujian keatas corak cap jari

DNA yang diperolehi dengan menggunakan kaedah perkumpulan

menunjukkan kepelbagaian genetik yang ketara diantara strain-strain

yang dikaji. Kaedah pengecap jarian lain yang berasaskan kepada

taburan DNA berulang ['enterobacterial repetitive intergenic consensus'

(ERIC) dan 'repetitive extragenic palindromic' (REP)] didalam genom

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bifidobakteria dan bakteria probiotik yang lain telah dikaji untuk kali

pertama menggunakan primer yang diterbitkan dari jujukan REP dan

ERIC. Corak produk PCR yang dihasilkan diatas gel agaros adalah

spesifik untuk setiap spesis. Semua isolat B. pseudocatenulatum dan B.

pseudocatenulatum JCM 1 200 yang dikaji menghasilkan satu jalur

bersaiz 1 . 5 kb dengan kaedah ERIC dan satu jalur 800 bp dengan

kaedah REP. Kedua-dua jalur ini tidak terdapat pada lain-lain spesis

bifidobakteria yang dikaji. Kajian ini menunjukkan kehadiran un sur­

un sur ERIC dan REP didalam genom bifidobakteria dan bakteria

probiotik yang lain.

Kaedah PCR juga telah digunakan untuk mengesan kehadiran

gen bsh didalam Bifidobacterium longum BB536. Untuk tujuan ini,

sepasang primer PCR untuk pengesanan secara cepat gen bsh telah

dihasilkan dan dapat mengesan gen bsh bersaiz 970 bp. Gen bsh ini

telah diklonkan dan jujukan nukleotid yang diperolehi menunjukkan

persamaan yang tinggi dengan jujukan nukleotid gen bsh yang telah

diterbitkan. Jujukan nukleotid yang dihasilkan mengkodkan protin

yang mengandungi 3 1 7 asid amino yang mempunyai berat molekul

kira-kira 35 kDa. Gen bsh dari B. longum ini juga telah diklonkan dan

dizahirkan didalam E. coli BL21 -SI menggunakan vector pRSET -A.

Pro tin yang dihasilkan telah dibuktikan melalui kaedah immunoblot.

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ACKNOWLEDGEMENTS

In the name of Allah, Most gracious, Most merciful. Alhamdulillah,

with His blessing, I have completed the preparation of this manuscript.

I would like to express my sincere gratitude and respect to my

kind advisor, Associate Professor Dr Mohd Yazid Abd Manap for his

invaluable contribution, constructive criticisms and support throughout

my study.

Furthermore, I would like to extend my appreciation to my

advisory committee Professor Dr Abd Manaf Ali and Associate Professor

Dr. Norihan Mohd Saleh for their invaluable advice and support. My

sincere thank is extended to the Faculty of Food science and

Biotechnology, Universiti Putra Malaysia and to the Ministry of Science,

Technology and Environment for financial supports provided through

National Science Fellowship scheme and Intensified Research in Priority

Area funding for this research.

Paramount gratitude is also extended to my beloved wife Rossida

Mamat and my daughter Nadiah Syahirah for their love, understanding,

patience and support throughout my study at the university.

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Last but not least, I would like to expres s my deepest gratitude to

my colleagues Lim, Anis, Santhi and Wendy at the Probiotic Laboratory

for their kindness and friendship and to Dr Majid, Samuel, Tony, Yang

Moi, Boon Keat, Madihah, Nor Rizan, Safdi, Ruhaya and others for their

help in making my stay at UPM an enjoyable and rewarding experience.

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I certify that an Examination Committee met on 6th January 2003 to conduct the final examination of Shuhaimi Bin Mustafa on his Doctor of Philosophy thesis entitled "Species Classification and Molecular Studies of Bile Salt Hydrolase (bsh) gene in Bifidobacterium spp" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 198 1 . The committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

Hasanah Mohd Ghazali, Ph.D.

Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia ( Chairperson)

Mohd Yazid Abd Manap, Ph.D.

Associate Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

Abd Manaf Ali, Ph.D. Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

Norihan Mohd Saleh, Ph.D.

Associate Professor Faculty of Food Science and Biotechnology U niversiti Putra Malaysia (Member)

Glen. R. Gibson, Ph.D. Professor Department of Microbiology University of Reading, UK. (Independent Examiner)

.....:::SHAMSHER MOHAMAD RAMADILI, Ph.D. Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date' "1 7 ' ,\ �l ,/,,1'1')

xi

• ...J " ''4 '- 'J'..:\)

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of Supervisory Committee are as follows:

Mohd Yazid Abd Manap, Ph.D.

Associate Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Chairman)

Abd Manaf Ali, Ph.D.

Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

Norihan Mohd Saleh, Ph.D.

Associate Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

xii

AINI IDERIS, Ph.D.

Professor / Dean School of Graduate Studies Universiti Putra Malaysia Date: '1 3 MAI\ 200J

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

xiii

Shuhaimi Bin Mustafa Date: \s {\ (fJ-Of) l

TABLE OF CONTENTS

Page

DEDICATION ................. . . . . . ...... . . .................................................. ii ABSTRACT .......................................... . ... . ........................... . . ..... . .. iii ABSTRAK ......................................... . . . .......................................... vi ACKNOWLEDGEMENTS ..................... . .. . . .. . . ... . .. . ... . . .... . . . . . . . . ....... . .. ix APPROVAL SHEETS ...................... . . . . . ........... . ... . .. . . ............... . ... . .. xi DECLARATION FORM ................... . ... . ... . ....... . .. . . . . . . ........... . ...... .... xiii LIST OF TABLES .................... . ...... . .. . . ... . ........... . ....... . . . . ..... .......... xvii LIST OF FIGURES .................. . ....... . ... . ....... . ........... . ...................... xviii LIST OF ABBREVIATIONS ............ . . . . . . .. . ... . . .... . .. . .......................... xxii

CHAPTER

1 INTRODUCTION . . ... . ... . ...................................................... 1

2 LITERATURE REVIEW ................... .................... . .............. 6 Pro biotic .......... . ... . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 6

Definition of Probiotic ....................... . .... . ............ . ....... 6 Microorganisms Used as Probiotics ................. . ........... 7 Genus Bifidobactenum ............................................... 9

Characterisation of Bifidobactenum Species by Conventional Carbohydrate Fermentation Patterns .............. 10 Molecular Classification of Probiotic Microorganisms .......... 13

Ribosomal RNA (rRNA) Gene Sequence Analysis .... . ... . . 14 Random Amplification of Polymorphic DNA (RAPD) ...... 18 Short, Interspersed Repetitive DNA Sequences . . . ... . .. . . . 21

Role of Recombinant DNA Technology in Improvement Of Probiotic Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .... 28 Scientific Basis of Probiotic Functionality .. . . ...................... . . 35

Technological Aspects of Probiotic Starter Cultures In Food Fermentation ..... ..... . . .... . ..................... . ........... 38

Lactic and Acetic Acids ...... . . ............................. 43 Bacteriocins .... ................ . . . ........... . .................. 44 Technological Characteristics of Pro biotic Starter Cultures ............................. . . . ............... 46

Improvement of Human Health and Diseases Prevention by Probiotics .............................................. 49

Reduction of Serum Cholesterol LeveL .............. 50 Potential Antitumor Activity ............................. 52

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Restoration of Intestinal Tract Function ............ 53 Improvement of Constipation ............................ 55 Prevention of Diarrhoea .................................... 56

Pro biotic as Live Vaccines ............................................ 56 Enterohepatic Circulation of Bile Salts ................................. 61

Deconjugation of Bile Salts by Intestinal Microorganisms ........................................................... 62

3 CLASSIFICATION OF BIFIDOBACTERIUM ISOLATES FROM INFANT FAECES USING CONVENTIONAL CARBOHYDRATE FERMENTATION PATTERNS AND

PCR-BASED METHODS ..................... ........... . . .................... 66 Introduction ........................................................................ 66 Materials and Methods .......................................................... 68

Bacterial Isolates and Microbiological Media ................. 68 Analysis of the Fermentative Characteristics of Bifidobacterium Isolates ................................................ 70 DNA Extraction ............................................................ 71 Genus-Specific PCR Amplification and Purification of Products .................................................................. 72 1 6S rDNA Sequencing .................................................. 73 Analysis of Sequence Data ........................................... 73 Species-Specific PCR .................................................... 74

Results ... ................. . ....................... . . . ........ . . . . . . . ..... . ..... . .. . ..... 74 Phenotypic Analysis of Bifidobacterium Isolates ............ 74 Genus-Specific PCR ..................................................... 75 Sequence Analysis of 1 6SrDNA of Bifidobacterium Isolates ........................................................................ 75 Species-Specific Detection of B. pseudocatenulatum .... . . 80

Discussion ........................................................................... 80

4 DIFFERENTIATION OF BIFIDOBACTERIUM PSEUDOCA TENULA TUM ISOLATES FROM FAECES OF INFANT BY RAPD- AND REPETITIVE ELEMENT-PCR ....... . 85 Introduction .......................................................................... 85 Materials and Methods .......................................................... 87

Bacterial Strains .......................................................... 87 DNA Extraction ................................... . . . ...................... 88 RAPD-PCR Amplification .............................................. 89 rep-PCR Amplification .................................................. 90 Gel Electrophoresis .......... . .. . ...... .................................. 91 Analysis of RAPD- and rep-PCR Patterns ............. . ........ 91

Results ................. . . ................... . .......... .... ................. . . . ......... 92 RAPD-PCR Profiles of Bifidobacteria and Other

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Probiotic Bacteria ........................................................ 92 Eric-PeR Profiles of Bifidobacteria and Other Pro biotic Bacteria ..................... .. .. . . . . . . .. . .. . . ..... . .. . . ..... ... ....... .. ..... 95 REP-PCR Profiles of Bifidobacteria and Other Probiotic Bacteria . . . . . . . . . . .... . . . .. ... . . . . . .. . . . . . . . . . . . .... . . . . . . . . ... .. . . . . ........ . 1 06

Discussion .......... ... .. ............... .. .... . .. ..... .. .............. ............ .. . 107

5 CLONING, SEQUENCING AND EXPRESSION OF BILE SALT HYDROLASE (bsh) GENE FROM BIFIDOBACTERIUM

LONGUM INTO E. COLI ... .... .. .... .. .. ... .. .. ....... ... .. . .. . ..... .... . ...... 1 14 Introduction ..... ....... ..... ..... .. .. . .. .. .. ... .. ... ...... ..... .. ... . . .. .. . .. ..... .. 1 14 Materials and Methods ....... ............ .. .. ..... ........... . . ................ 1 1 6

Bacterial Strains and Plasmid ............... .......... .. . . .. .. .. .. 1 1 6 DNA Extraction .... ......... .. . . . .. .. .. . .. ....... .... ........... .... ...... 1 1 6 Rapid Detection of Bile Salt Hydrolase Gene From B. longum BB536 Using PCR ... . . .. .. . . ... . . .. .. ....... . . .. .. . ..... 1 1 7 Purification of PCR Product from Agarose Gel Using The 'Wizard PCR Preps DNA Purification System' (Promega, Madison, USA) . ........ .. .. ....... .. . ........... .......... 1 1 8 Preparation of Competent E. coli BL2 1 -SI Cells ....... .. . .. 1 1 9 pGEM-T Easy Cloning ........ ........ ........... .... . ................. 1 20 Plasmid DNA Isolation and Purification .............. . . .. ..... 1 20 Restriction Enzyme Analysis .... . . ................................. 1 2 1 DNA Sequencing of the Insert ... . . .. .. ..... .... ............ ..... ... 1 22 Analysis of Sequence Data . .. .. .. .. .... . .. .. .. ...... ...... ... ....... 1 22 pRSET A Cloning .. .. . . . .............. ...... .... ... .... .......... .. .... .. . 124 Detection of Expressed Protein Using Direct Plate Method .. .. .. .. .. . ... . .. .. .. .. . .. .. .... . . . . . . . . ......... .. .. .. .. .... ... . . ... . . 1 24 Preparation of Protein for Electrophoresis.. . .... ...... .... .. . 1 24 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) ..... . ... . . .... .. .. ................... .. 1 25 Staining of Proteins with Coomasie Blue .......... .. .... . ..... 1 26 Western Blotting ....................... .... .... ........ .. .. . . . .. . . . . . .. . . 1 26

Result . ..... ................. .. ...... ................ .. .. . .. .. . ... . . . . . . .. .... .. .. ... .... 1 28 Discussion . .. .. .. . . . . . .. . . ........... .. .... . .. .. .. .. . . . . .... .. ............ ...... .... . 1 36

6 GENERAL DISCUSSION AND CONCLUSION ............ . . . . . . . . . . .. 1 39

REFERENCES . .. ... . ... .. .. .. ... . .. .. .. . . .. .. .. ........ ..... . .. . ..... ... .. . . .. .... .... .. .. . . 1 46

APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 63

BIOGRAPHICAL SKETCH .. .. .. .... . . . .. ... .. .... .. ...... ..................... .. .. . . .. 1 7 1

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LIST OF TABLES

Table Page

1 Lactic acid bacteria and other microorganisms used as pro biotics . . . . ... . . .. .. .. ....... . . ... ... . . . . ... . . .. .. . .. . . . . . .. . . . . .. . .. . . .. ... . . . . . . 8

2 List of species in the genera of Bifidobacterium. . . . . . . . . . . . . . . . . . . . . . . 1 1

3 Analysis of the fermentation characteristics of Bifidobacterium sp (aKok et ai., 1996; bMatsuki et ai., 1998) . . . 13

4 Studied isolates/strains and their code . .. . . . ... . . .... .. . . . .. . .. . .. . . .. . 69

5 The sequences of oligonucleotides for peR and 1 6S rDNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

6 Sugar fermentation profiles of the genus Bifidobacterium isolated from infant faeces ..... . . . . . . . .. . . . . . . . . .. . . . . . . . .. .. . .... . .. ..... .. . . . 77

7 Percentage of homology of the 1 6S rDNA partial sequences of Bifidobacterium isolates. Sequences were compared with the sequences deposited in Genbank ... . . . . . ... . . .. . .. . . ......... .... . .. . 79

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Figure

1

LIST OF FIGURES

Agarose gel electrophoresis of PCR products using primers lm26/lm3 . (A) Lane: 2 , C83; 3, F34; 4, D36; 5, F1 12; 6, G41 ; 7, F1 1 7; 8, F91 ; 9, F69; 10, 245; 1 1 , G4; 12, C1 5; 13, C25; 14, D22; 1 5, negative control; M, 1 kb DNA ladder. (B) Lane: 1 , F66 ; 2, 286; 3, C1 7; 4, F81; 5, B. indicum ATCC 25913; 6, B. longum 8B536; 7, B. animalis ATCC 27672; 8, B. pseudocatenulatum JCM 1200; 9, S. faecalis T-I1 0; 10, B. mesentricus TO-A; 1 1 , L. rhamnosus GG; 1 2 , negative control; M , 1 kb DNA

Page

ladder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . .. . . . . . . . . . 78

2 Agarose gel electrophoresis of PCR products using primers PseudoF/PseudoR. (A) Lane: 1 ,C83; 2 , F34; 3, D36; 4, F1 12; 5, G41; 6, F1 1 7; 7, F91 ; 8, F69; 9, 245; 10, G4; 1 1 , C1 5; 12 , C25; 13, D22; 14, F66; 15, 286; 16, F81 ; M, I kb DNA ladder. (B) Lane: M, 1 kb DNA ladder; 1 , B. pseudocatenulatum JCM 1200; 2 , B. indicum ATCC 2591 3; 3, B . .zongum BB 536; 4, B. animalis ATCC 27672; 5, S. faecalis T-I10; 6, B. mesentricus TO-A; 7, L. rhamnosus GG; 8 , negative control. . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . 82

3 RAPD of B. pseudocatenulatum and references strains with primer GEN 1-60-03 . (A) Lane: 1 , lkb DNA ladder; 2, C83;3 , F34;4, D36; 5, Fl1 2;6, G41 ;7, FI1 7;8, F91 ; 9, F69; 10, 245; 1 1 , G4; 12, C15; 13, C25; 14, D22; 15, F66. (B) Lane: 1 , 1 kb DNA ladder; 2 , B. indicum ATCC 2591 3; 3, B. longum 88536; 4, B. animalis ATCC 27672; 5, Streptococcus faecalis T -1 1 0; 6, Bacillus mesentricus TO-A; 7, F81 ; 8, C1 7; 9, L.·rhamnosus GG; 10, 286; 1 1 , negative control. (C) Lane: I, 1 kb DNA ladder; 2 , F81 ; 3, C1 7; 4, Z86; 5, JCM1200; 6, negative controL . . . . . . . . .. .. . . .. . ,. . . . . . . . . . . . . . . . . .. .. .. . . . . . . . . . . .. .. .. . . . .... . . . . . . . . 96

4 RAPD of B. pseudocatenulatum and references strains with primer GEN 1 -60-03. (A) Lane: 1 , 1kb DNA ladder; 2, C83;3, F34;4, D36; 5, Fl1 2 ;6, G41 ; 7, F1 1 7;8, F91; 9, F69; 1 0, 245; 1 1 , G4; 12, C1 5; 1 3, C25; 1 4, D22; 1 5, F66. (8) Lane: 1 , 1 kb DNA ladder; 2 , B. indicum ATCC 2591 3; 3, B. longum BB536; 4, B. animalis ATCC 27672; 5, Streptococcus faecalis T -11 0; 6, Bacillus mesentricus

xviii

TO-A; 7, F8I; 8, C17; 9, L. rhamnosus GG; 10, Z86; 11, negative control. (C) Lane: 1, 1 kb DNA ladder; 2, F81; 3, C17; 4, Z86; 5, JCM1200; 6, negative controL........................................................................ 97

5 RAPD of B. pseudocatenulatum and references strains with primer GEN 1-60-03. (A) Lane: 1, lkb DNA ladder; 2,C83;3,F34;4,D36; 5, F112;6,G41;7, Fl17;8,F91; 9, F69; 10, Z45; 11, G4; 12, C15; 13, C25; 14, D22; 15, F66. (B) Lane: 1, 1 kb DNA ladder; 2, B. indicum ATCC 25913; 3, B. longum BB536; 4, B. animalis ATCC 27672; 5, Streptococcus faecalis T -110; 6, Bacillus mesentricus TO -A; 7, F81; 8, C17; 9, L. rhamnosus GG; 10, Z86; 11, negative control. (C) Lane: 1, 1 kb DNA ladder; 2, F81; 3, C17; 4, Z86; 5, JCM1200; 6, negative control......................................................................... 98

6 Dendrogram obtained by using RAPD primer GEN 1-60-03 on purified chromosomal DNA from Bifidobacterium species followed by evaluation using UPGMA clustering method........................................................................ 99

7 Dendrogram obtained by using RAPD using primer GEN 1-60-06 on purified chromosomal DNA from Bifidobacterium species followed by evaluation using UPGMA clustering method............................................. 100

8 Dendrogram obtained by using RAPD using primer GEN 1-60-07 on purified chromosomal DNA from Bifidobacterium species followed by evaluation using UPGMA clustering method............................................. 101

9 Dendrogram obtained by using RAPD using combination of primers GEN 1-60-03, GEN 1-60-06 and GEN 1-60-07 on purified chromosomal DNA from Bifidobacterium species followed by evaluation using UPGMA clustering method ......................................................................... 102

10 ERIC-PCR fmgerprint patterns,.of B. pseudocatenulatum and references strains. (A) Lane: 1, F34; 2, D36; 3, Fl12; 4, G41; 5, Fl17; 6, F91; 7, F6�;·8, Z45; 9, G4; 10, C15; 11, C25; 12, D22; 13, F66; M, 1kb DNA ladder. (8) Lane: 1, C83; 2, Z86; 3, C17; 4, F81; 5, B. indicumATCC 25913; 6, B. longum 88536; 7, B. animalis ATCC 27672;

xix

8, Streptococcus faecalis T - 1 1 0; 9 , Bacillus mesentricus TOA; 10, L. rhamnosus GG; 1 1 , negative control; M, 1 kb DNA ladder. (C) Lane: 1 , D36; 2 , JCM 1 200; 3, negative control; 4, lkb DNA ladder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 04

1 1 Dendrogram obtained by using ERIC-PCR on purified chromosomal DNA from Bifidobacterium species followed by evaluation using UPGMA clustering method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . 1 05

1 2 REP-PCR fingerprint patterns of B. pseudocatenulatum and references strains. (A) Lane: 1 , 1 kb DNA ladder; 2 , F34;3, F66; 4, D36; 5 , FI 1 2;6, G4 1 ; 7, FI 1 7; 8 , F9 1 ; 9 , F69; 1 0, G48; 1 1 , G4; 1 2 , C 1 5; 1 3 , C25; 1 4 , D22; 1 5, D 1 9; 16, JCM 1 200. (B) Lane: 1 , 1 kb DNA ladder; 2 , F8 1; 3, Z45; 4, Z86; 5, B. longum B B 536. (C) Lane: 1 , 1 kb DNA ladder; 2 , B animalis ATCC 27672; 3, S. faecalis T - 1 1 0; 4, B mesentricus TO-A; 5, L. rhamnosus GG; 6, B. indicumATCC 259 1 3; 7, negative control... . . ... . . . . . . . . . . . . . . 1 08

1 3 Dendrogram obtained by using REP-PCR on purified chromosomal DNA from Bifidobacterium species followed by evaluation using UPGMA clustering method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . .. 1 09

14 pGEM-T Easy vector circle map and sequence reference points. . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . .. . . . . . . . . . . . . .. . . . .. . . . . . . .. . . 1 23

1 5 PCR amplification of bsh gene from Bifidobacterium longum BB536. Lanes: 1 , 1 kb DNA ladder; 2 , Bifidobacterium longum B B 536; 3, B. pseudocatenulatum JCM 1 200; 4 , B. pseudocatenulatum F34; 5, B. infantis ATCC 25962; 6, B. infantis ATCC 1 5697; 7, B. animalis ATCC 27672; 8, Streptococcus faecalis T-II0; 9 , Bacillus mesentricus TO-A; 10, Lactobacillus rhamnosus GG ATCC 53 1 03; 1 1 , negative control (without template DNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 29

1 6 Plasmids digestion of white colonies cultures with EcoRl at 37°C for 4 hours. Lanes: 1 , 1 kb DNA ladder; 2 , colony 1 ; 3 , colony 2 ; 4, colony 3 ; 5, colony 4 ; 6, colony 5; 7, colony 6; and 8, 1 kb DNA ladder. All the 6 colonies isolated harboured inserted DNA with the size of approximately 970 bp. . . . .. . . . ... . . . . . . . . . . . . ..... . . . . . . . . . . . . .. . . . . . . . . 1 30

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17 Alignment of bsh genes from B. longum BB 536, B. longum SBT 2928, C. perfringens and L. acidophilus. Asterisks indicate identical nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . 1 33

18 The alignment of amino acid sequences of BSH from B. longum BB 536, B. longum SBT 2928, L. acidophilus and C. perJringens. Asterisks, identical amino acids; dots, similar amino acids; underlines, amino acids of the putative active site. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 33

19 Restriction enzyme analysis of the transformant showed that the bsh gene from B. longum was successfully cloned into pRSET A expression vector and transformed into E. coli BL 2 1-S1. (Double digestion with EcoRI and BamH l, Lane 1: 1 kb DNA ladder; lane 2: vector plus insert; lane 3: vector without insert) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 34

20 Manifestation of BSH activity by E. coli transformants on solid medium containing 0 . 5% (wjv) TDCA. The E. coli colony in the centre is the strain without bsh gene. The four colonies surrounded with halos of precipitated DCA are transformants E. coli carrying bsh gene. . . . . . . . . . . . . . . . . . . . . 1 34

2 1 SDS-PAGE analysis of whole cell protein from E. coli transformants . Lane: 1 , low range protein marker; 2, E. coli carrying bsh gene; 3, E. coli without bsh gene. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 1 35

22 Western blot analysis of BSH enzyme. Lane: M, low range proteins marker; 1 , whole cells protein from E. coli carrying bsh gene; 2, whole cells protein from E. coli without bsh gene (negative control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 35

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ATCC

Bp

BSH

CaCh

CBAH

cfu

C02

DCA

DNA

dNTPs

E. coli

EDTA

ERIC

g

h

HCI

19A

IPTG

IRU

JCM

K

kb

kDa

L

LAB

Lb

mM

LIST OF ABBREVIATIONS

American Type Culture Collection

base pair

bile salt hydrolase

calcium chloride

conjugated bile acid hydrolase

colony forming unit

carbon dioxide

deoxycholic acid

deoxyribonucleic acid

deoxynucleotide triphosphate

Escherichia coli

ethylenediamine tetraacetic acid

enterobacterial repetitive intergenic consensus

gram

hour

hydrochloric acid

immunoglobulin A

isopropyl-p-D-thiogalactopyranoside

intergenic repetitive unit

Japan collection of microorganisms

kalium

kilobase

kilodalton

litre

lactic acid bacteria

luria bertani

millimolar

xxii

mln

ml

Na

NaCl

Ng

NTSYS

nm

N

PBS

PCR

PFGE

PU

PVDF

RNA

RAPD

rONA

RE

REP

Rep

rRNA

s

SAHN

SDS

Subsp

spp

TOeA

molarity

magnesium chloride

magnesium sulphate

minute

millilitre

natrium

natrium chloride

nanogram

numerical taxonomy system

nanometre

normality

phosphate buffered saline

polymerase �hain reaction

pulse field gel electrophoresis

palindromic unit

polyvinylidene fluoride

ribonucleic acid

random amplification of polymorphic DNA

ribosomal DNA

restriction enzyme

repetitive extragenic palindromic

repetitive

ribosomal RNA

second

'sequential, agglomerative, hierarchical, nested

sodium dodecyl sulphate

subspecies

species

taurochenodeoxycholic acid

xxiii