Koki Horikoshi Alkaliphiles -Genetic Properties and Applications of Enzymes- Koki Horikoshi Alkaliphiles Genetic Properties and Applications of Enzymes With 108 Figures and 60 Tables fe\ Kodansha ^ Springer Koki Horikoshi Professor Emeritus, Tokyo Institute of Technology and RIKEN Institute, Japan Director General, Japan Agency for Marine-Earth Science and Technology(JAMSTEC) ISBN 4-06-211513-1 Kodansha Ltd., Tokyo ISBN-10 3-540-33372-x Springer Berlin Heidelberg New York ISBN-13 3-540-33372-2 Springer Berlin Heidelberg New York Library of Congress Control Number: 2006926727 All rights are reserved. No part of this book may be reproduced in any form, by photostat, microfilm, retrieval system, or any other means, without the written permission of Kodansha Ltd. (except in the case of brief quotation for criticism or review). This work is subject to copyright. 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Cover design: design & production GmbH, Heidelberg, Germany Cover image : B. halodurans, by courtesy of Dr. H. Takami. Printed on acid-free paper To my wife, Sachiko Preface "If I have seen a little further it is by standing on the shoulders of Giants." Letter to Robert Hooke, Isaac Newton, Februarys, 1676 In November 1956, I encountered an alkaliphilic bacterium, although not alkaliphilic in the true sense of the word. I was a graduate student in the Department of Agricultural Chemistry, University of Tokyo, working under the direction of Professor Kin-ichiro Sakaguchi. Autolysis of Aspergillus oryzae was the research theme for my doctoral thesis. The reason why Professor Sakaguchi asked me to study the autolysis of Asp. oryzae was a somewhat practical one. He thought the flavor and taste of Japanese sake came from an autolysate of Asp. oryzae. Every day, I cultured stock strains of Asp. oryzae. After one week of culture, all I had to do was taste the cultured fluid. (I used my bero-meter, bero meaning tongue in Japanese.) Naturally, I was entirely disappointed in microbiology itself. One day in November, I found one cultivation flask in which mycelia of Asp. oryzae had completely disappeared. The night before, when I looked at the flasks, the mold was flourishing in all culture flasks. I still remember vivid pictures of bacteria thriving and mo\ing. No mycelium could be seen under the microscope. The microorganism isolated from that flask was Bacillus circulans, and strong endo-l,3-P-glucanase activity was detected in the culture fluid. This enzyme lyzed Asp. oryzae. It was the first time that mold cells had been found to be lyzed by bacteria, and these results were published in Nature (Horikoshi et al., 1958). However, this bacterium showed very poor growth in the absence of mycelia of Asp. oryzae and production of endo-l,3-(i-glu- canase was very low. Therefore, purification of endo-l,3-P-glucanase could be done only in culture fluid in the presence of mycelia of Asp. oryzae. I did not realize at the time that the culture fluid had alkaline pH value. A few years later, I attempted production of endo-l,3-p-glucanase in conventional media. I tested many culture media containing various nutrients. Addition of 0.5% sodium bicarbonate to conventional nutrient culture broth gave good growth and production of the enzyme. Autolysis of Asp. oryzae changed the culture medium from weakly acidic to alkaline pH. In this way I discovered that such a change in pH value accelerated bacterial growth and production of the enzyme (see p. 41). viii Preface In 1968, I visited Florence, Italy, and saw Renaissance buildings, which are so very different from Japanese architecture. Centuries earlier no Japanese could have imagined this Renaissance culture. Then suddenly I heard a voice whispering in my ear, "There could be a whole new world of microorganisms in different unexplored cultures." Memories of experi- ments on B. circulans done years ago flashed into my mind. Could there be an entirely unknown domain of microorganisms existing at alkaline pH? The acidic environment was being studied, probably because most food is acidic. However, very little work had been done in the alkaline region. Upon my return to Japan I prepared an alkaline medium containing 1 % sodium carbonate, put small amounts of soil collected from various areas of the Institute of Physical and Chemical Research (RIKEN), Wako, Japan, into 30 test tubes and incubated them overnight at 37°C. To my surprise, microorganisms flourished in all test tubes. I isolated a great number of al- kaliphilic microorganisms and purified many alkaline enzymes. The first paper concerning an alkaline protease was published in 1971. Then, in 1972, I was talking with my father-in-law, Shigeo Hamada, about alkaliphilic microorganisms. He had been in London almost a centu- ry ago as a businessman and was curious about everything. He showed in- terest in alkaliphiles. These microorganisms were unique, required high al- kalinity, and they could produce alkaline enzymes such as alkaline proteas- es, alkaline amylases, etc. As I was speaking, he said, "Koki, wait a minute, I have an interesting present for you." He brought out a sheet of old newspa- per, Nikkei Shimbun dated June 11, 1958. A short column with one electron micrograph was like a punch to my head. I had not known this! The article stated: In Japan, since ancient times, indigo has been naturally reduced in the presence of sodium carbonate. Indigo from indigo leaves can be reduced by bacteria that grow under high alkaline conditions. Indigo reduction was controlled only by the skill of the craftsman. Takahara and his colleagues isolated the indigo reducing bacterium from a ball of indigo. I then carefully checked scientific papers from Chemical Abstracts in the library of RIKEN. Only 16 scientific papers on alkaliphiles were discovered. Alkaliphiles remained little more than interesting biological curiosities. No industrial application was attempted at all before 1968. I named these mi- croorganisms that grow well in alkaline environments "alkaliphiles" and conducted systematic microbial physiological studies on them. It was very Johnson, 1928; Downie and Cruickshank, 1928; Vedder, 1934; Jenkin, 1936; Bornside and Kallio, 1956; Chesbro and Evans, 1959; Kushner and Lisson, 1959; Takahara and Tanabe 1960; Chisleu and Kushner, 1961; Shislett and Kushner, 1961b; Takahara et al., 1961 ; Takahara and Tanabe, 1962; Wiley and Stokes, 1962; Wiley and Stokes, 1963; Barghoorn and Tyler, 1965; Siegel and Giumarro, 1966. Preface ix surprising that these microorganisms, which are completely different from any previously reported, were widely distributed throughout the globe (even at the deepest point of the Mariana Trench in the Pacific Ocean) produc- ing heretofore unknown substances. Here was a new alkaline world that was utterly different from the neutral world. Over the past three decades my coworkers and I have focused on the enzymology, physiology, ecology, taxonomy, molecular biology and genetics of alkaliphilic microorganisms to establish a new microbiology of alka- liphilic microorganisms. A big question arises, "WTiy do alkaliphiles require alkaline environments?" The cell surface of alkaliphiles can keep the intra- cellular pH values about 7-8 in alkaline environments of pH 10-13. How the pH homeostasis is maintained is one of the most fascinating aspects of alkaliphiles. In order to understand this simple but difficult question, we carried out several basic experiments to establish gene recombination sys- tems. Finally, after almost two years, the whole genome sequence of alka- liphilic Bacillus halodurans C-125 was completed. This was the second whole genome sequence of spore-forming bacteria thus far reported. This se- quence work revealed interesting results. Many genes were horizontally transferred from different genera and different species as well. Small frag- ments of enzyme genes were inserted in opposite directions, or separated by insertion fragments. Therefore, these bacteria could not produce some en- zymes. However, we still have not found the crucial gene(s) responsible for alkaliphily in the true meaning. Our results indicate that many gene prod- ucts synergistically cooperate and exhibit alkaliphily (or adaptation to alka- line environments). Industrial applications of these microorganisms have also been investi- gated extensively and some enzymes, such as alkaline proteases, alkaline amylases, alkaline cellulases and alkaline xylanases have been put to use on an industrial scale. Subsequently, many microbiologists have published nu- merous papers on alkaliphilic microorganisms in various fields. At the be- ginning of our studies, very few papers were presented, but now thousands of scientific papers and patents have been published. It is not clear which field our study of alkaliphiles will focus on next, but the author is convinced that alkaliphiles wdll provide much important information. The author expresses sincere gratitude to his wife Sachiko for her in- valuable help over the past 40 years. Tokyo, Japan April 2006 Contents Preface vii Part I Alkaliphiles and Their Genetic Properties 1 Introduction 3 1.1 What Are Alkaliphiles? 3 1.2 History of Alkaliphiles 4 1.3 Why the Author Selected Alkaliphilic Bacillus Strains for Discussion in This Volume 5 2 Isolation, Distribution and Taxonomy of AlkaliphiHc Microorganisms 7 2.1 Isolation 7 2.2 Distribution 8 2.2.1 Soil Samples 8 2.2.2 Deep Sea Samples 9 2.2.3 Alkaline Lakes 11 2.2.4 Others 11 2.3 Taxonomy of Alkaliphiles 15 2.3,1 Phylogenetic Analysis of Alkaliphiles Based on 16S rDNA Analysis 15 3 Cell Structure 23 3.1 Flagella 23 3.1.1 Flagella Formation and Flagellin 23 3.1.2 Flagellar Motor 23 3.1.3 Inhibitors for Flagellar Motors 25 3.2 Cell Wall 28 3.2.1 Cell Wall of Neutrophilic Gram-positive Bacteria 28 3.2.2 Cell Wall of Alkaliphilic Bacillus Strains 30 3.3 Cell Membrane 36 4 Physiology 39 4.1 Growth Conditions 39 4.1.1 pH Values of Culture Media 39 xii Contents 4.1.2 Sodium Ion 42 4.1.3 Temperature and Nutrition 46 4.2 Mutants, Antiporters and Alkaliphily 47 4.2.1 Isolation and Properties of Alkali-sensitive Mutants 48 4.2.2 Antiporter (pALK) Mutants 48 4.2.3 Ubiquitous Distribution of Mrp Operons 55 4.2.4 Respiration-dependent ATP Synthesis 57 4.3 Intracellular Enzymes 57 4.3.1 a-Galactosidases 58 4.3.2 p-Galactosidases 58 4.3.3 RNA Polymerases 59 4.3.4 Protein Synthesizing System 59 5 Molecular Biology 63 5.1 Alkaliphilic Miroorganisms as DNA Sources 64 5.1.1 Secretion Vector 64 5.1.2 Promoters 73 5.2 Host-Vector Systems of Bacillus halodurans C-125 77 5.2.1 Selection of Host Strains 78 5.2.2 Stability of Antibiotics in Horikoshi-I Medium 78 5.2.3 Preparation of Mutants 79 5.2.4 Preparation of Stable Protoplasts, Transformation and Regeneration 80 5.2.5 Cell Fusion of Alkaliphiles 85 5.2.6 Selection of Vectors 87 5.2.7 Summary and Conclusion 88 5.3 Host Vector System of Bacillus pseudofirmus 0F4 88 5.3.1 Bacterial Strains and Plasmids 88 5.3.2 Preparation and Transformation of Protoplasts 89 5.3.3 Construction of «/iaC-deleted Bacillus pseudofirmus OF4(N13) 89 6 Whole Genome Sequences of Alkaliphilic Bacillus Strains 91 6.1 Introduction 91 6.2 Alkaliphilic Bacillus Species 91 6.2.1 Construction of the Physical Map of the Bacillus halodurans C-125 Chromosome 92 6.2.2 Sequencing and Assembly of the Whole Genome Shotgun Library 95 6.2.3 Annotation 95 6.2.4 General Feature of the Bacillus halodurans C-125 Genome 96 6.3 Further Works on Genome Analysis of Alkaliphilic Bacillus halodurans 100 6.3.1 Origin of Replication 100 6.3.2 Transcription and Translation 100