FAPA 2002 Seoul
19th Congress of Asian Pharmaceutical Sciences and Practice
Ishidate Awardee Paper
Genetic and Biochemical Studies on the Regulatory Mechanism of SelfResistance and Biosynthesis of Antibiotics
HO Bio Institute
33-9, Yushima-2, Bunkyo-ku, Tokyo 113-0034, Japan
Hiroshi Ogawara
Self-resistance
-Lactam antibiotics are distinct in that a -Lactam antibiotic, penicillin, was the first antibiotic discovered and
-Lactam antibiotics have been widely used for over 60 years since penicillin's rediscovery. However, as a natural
consequence of selection under the pressure of antibiotics, resistant bacteria are. becoming prevalent in natural
environment such as MRSA and VRSA. At the same time, most of the antibiotics are produced by bacteria,
especially Streptomyces. Therefore, Streptomyces must have mechanisms for protecting themselves from their own
antibiotics, self-resistance mechanisms. In addition, self-resistance is also related to antibiotic resistance in
pathogenic bacteria (1).
In general, bacteria and cells become resistant to drugs mainly through three mechanisms: that is, chemical
modification of drugs including degradation such as -Lactamase, changes of targets of drugs such as penicillinbinding proteins (PBP) and changes of permeability. So, as the first candidate of self-resistance mechanism, I
studied -Lactamase. For this purpose, I measured the -Lactamase activity of Streptomyces isolated from soil
newly and about 60 years ago, that means that the soil at that time has rarely been exposed to -Lactamse
antibiotics. As a result, about three fourths of Streptomyces isolated newly as well as about, 60 years ago were
found to produce -Lactamase constitutively (2). Furthermore, no difference of the physico-chemical properties and
substrate specificity was observed between -Lactamases from two groups. Therefore, it is suggested that unlike
pathogenic bacteria, Streptomyces has not been affected in the level of resistance against -Lactam antibiotics by the
introduction of various kinds of -Lactam antibiotics. It should be pointed out here that many Streptomyces species
produce -Lactam antibiotics. In contrast to Streptomcyes, rare actinomycetes are known for a long time to produce
-Lactam antibiotics very infrequently. Therefore, we determined -Lactamase productivity in rare actinomycetes
(3). Among 127 species in 36 genera, only 6 species in 4 genera produced -Lactamase constitutively. Again, there
is no relation between -Lactamase activity and minimum inhibitory concentration (MIC) of actinomycete species.
Moreover, there is no relationship between -Lactamase production and phylogenetic relatedness based on 16S
rRNA. From these results, it is suggested that even though there is some relationship between productivities of Lactamase and -Lactam antibiotics, there is no relation between -Lactamase production and resistance against Lactam antibiotics in actinomycetes, because actinomycetes produce -Lactamase irrespective of their MIC against
-Lactam antibiotics. That is, -Lactamase is a minor cause of -Lactam resistance in Streptomyces/actinomycetes.
According to the phylogenetic tree, -Lactamases from Streptomyces are divided into two groups (4). -Lactamases
in one group are related to those from Gram-negative bacteria such as Yersinia and Klebsiella and those in other
group are related to the enzymes from Gram-positive bacteria such as Bacillus. Thus, the regulatory mechanism of
-Lactamase genes was analyzed. Consequently, regulatory system in Streptomyces cacaoi is similar to that in both
Gram-positive and Gram-negative bacteria. In contrast, -Lactamase from Streptomyces fradiae has no regulatory
protein in the upstream region of the -Lactamase gene. Intriguingly, -Lactamases of this group bound bluedextran and NADP+. Such proteins generally show dehydrogenase activities. So it is interesting to know if Lactamases of this group have dehydrogenase activity. This topic remains to be clarified.
Next, I examined PBP, the second candidate. PBP in Streptomyces have following properties: first, PBP from
Streptomyces have very low affinity to benzylpenicillin and so Streptomyces in general have very high MIC values,
even though they are Gram-positive bacteria. Second, -Lactam-non-producing Streptomyces such as Streptomyces
cacaoi show 5 to 9 PBP, while -Lactam-producing Streptomyces such as Streptomyces clavuligerus show 2 to 5
PBP. PBP pattern in Streptomyces cacaoi is also similar to that of other bacteria such as Bacillus. Third, clavulanic
acid, a -Lactam antibiotic, binds to its specific PBP in Streptomyces cacaoi, but in Streptomyces clavuligerus it
does not bind to PBP. From these results, it is concluded that Streptomyces are resistant to -Lactam antibiotics by
possessing PBP with very low affinity for -Lactam antibiotics (5).
-Lactamase, PBP and -Lactam in Streptomyces are related to each other by forming a triangle. In addition, Lactamase and PBP n pathogenic bacteria are related to those in Streptomyces. Therefore, to prevent from bacterial
infection and resistance, it is important to investigate these relationships in more detail.
Protein kinase
Streptomyces are Gram-positive soil bacteria, possess 8 Mbp linear chromosome with proteins at 5' ends and show
characteristic morphological differentiation like aerial mycelium and spore formation and characteristic biochemical
differentiation like secondary metabolise formation such as antibiotics. These two differentiation systems are
interrelated with each other.
Protein phosphorylation is one of the major regulatory mechanisms in. signal transduction systems. In eukaryotes,
serine-, threonine- and tyrosine protein' kinases are involved in many regulatory systems. On the other hand, in
prokaryotes histidine- and aspartic acid kinases are the typical kinases known as two-component systems.
To know if eukaryotic-type protein kinases are involved in the regulation of morphological and biochemical
differentiation in Streptomyces, we screened eukaryotic-type protein kinases in Streptomyces coelicolor and found
seven kinase genes (6). At least two of them, pkaAand pkaB, were found to be involved in the regulation of
morphological and biochemical differentiation. Interestingly, these genes are located in various positions of the
chromosome.
At the present time, the whole nucleotide sequences of chromosomes in over 50 bacterial species have been
determined (http://www.tigr.org/tdb/mdb). So, it is interesting to know how prokaryotic-type as well as eukaryotictype protein kinases are distributed in these species. Accordingly, two-component systems are distributed
ubiquitously in almost all the bacteria, while eukaryotic-type protein kinases are detected in only one third of them.
Moreover, all the bacteria possessing more than three kinases show characteristic differentiation, such as
Myxococcus xanthus, Mycobacterium tuberculosis, Streptomyces coelicolor, and Synechocystis. Therefore, it is
suggested that eukaryotic-type protein kinases have been preserved only in these specially differentiated species
during the long history of evolution (7).
Next. I analyzed the evolutional relationships among these kinases by constructing a phylogenetic tree. Even
protein kinases from one species are located randomly in this tree, although most of the kinases from one species
form a cluster, indicating that some of the kinases from one species are interrelated far distantly. G-C contents of the
kinase genes are very similar to those of other genes of that species.
Summarizing these results, it is suggested that the eukaryotic-type protein kinases were already present before the
branching of eukaryotes from prokaryotes and that these kinases disappeared in some prokaryotes during the course
of evolution because they were dispensable in these species (7,8).
Genistein, a specific inhibitor for tyrosine protein kinases
In 1985, we discovered genistein as a specific inhibitor for tyrosine protein kinases from Pseudomonas (9, 10).
After discovery of genistein, it has been widely and popularly used as a reagent in a variety of signal transduction
systems, that is cancer including tumor cell invasion and metastasis, cell growth and differentiation, immune
response, neuroscience ,and brain, inflammation and rheumatoid arthritis, the five sensory systems and many others.
I am very much proud of the fact that I can contribute somewhat to the development of diverse fields through this
useful reagent not only as a scientist but also as a pharmacist. In this connection, I am very pleased to know that
Gleevec, an inhibitor for tyrosine protein kinase, was recently approved by the FDA in the United States for the
treatment of chronic myeloid leukemia (CML) and is used in clinic with great success to treat CML.