S. Harayama - Academia.edu (original) (raw)

Papers by S. Harayama

Journal of Bacteriology, 1979

We determined the content of galactose-glucose-, maltose-, and ribose-binding proteins in cells o... more We determined the content of galactose-glucose-, maltose-, and ribose-binding proteins in cells of Escherichia coli K-12 grown in a variety of media and also measured the respective transport and chemotactic activities that depend on those binding proteins. Correlation of the level of induction of a particular binding protein with the extent of tactic activity mediated by that protein indicates that the magnitude of the tactic response to a particular stimulating compound is a direct function of the number of receptors per cell. In contrast, comparison of the magnitudes of response to substances recognized by independent receptors indicates that some stimulus-receptor complexes are more effective in eliciting tactic responses than are others. Thus, the magnitude of response to any particular stimulating compound is a function both of the number of receptors per cell and of the effectiveness of the stimulus-receptor complex. Considerations of available information about the tactic re...

Journal of Biological Chemistry, 1991

Earlier studies have shown that the major porin species in Pseudomonas aeruginosa outer membrane ... more Earlier studies have shown that the major porin species in Pseudomonas aeruginosa outer membrane is protein F (OprF), which produces channels wider than those produced by Escherichia coli porins. In contrast, Yoshihara and Nakae ((1989) J. Biol. Chen. 264, 6297-6301) reported that protein F has no pore-forming activity as measured by the flux of L-arabinose, and that the channels in P. aeruginosa outer membrane, being produced by proteins C, "D," and "E," are much narrower than E. coli porin channels. In this study, we followed the protein purification scheme of Yoshihara and Nakae as closely as possible, and found that protein F had a specific activity for pore formation similar to that of proteins D l , D2, and E2. Furthermore, proteoliposome reconstitution assays showed conclusively that the channels formed by protein F, as well as by unfractionated outer membranes, allowed the diffusion of a tetrasaccharide, stachyose, at a significant rate, indicating that these channels are much larger than E. coli porin channels. It appears likely that in the study of Yoshihara and Nakae protein F was inadvertently inactivated during purification. We further suggest a hypothesis that resolves the apparent conflict between the presence of large diameter channels and the low permeability of the outer membrane in P. aeruginosa. Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is becoming important especially in hospital-acquired infections because it is resistant to many of the commonly used antibiotics and chemotherapeutic agents (1). It has been shown that the permeability of the P. aeruginosa outer membrane to P-lactam antibiotics and also to some simple organic compounds is two to three orders of magnitude lower than the permeability of Escherichia coli outer membrane to the same or similar compounds (2, 3), and clearly this lower permeability of the outer membrane layer plays a major role in the general antibiotic resistance of this organism (4). Most of the antibiotics that are effective against enteric

Applied and Environmental Microbiology, 1999

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increa... more Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter −1 day −1 and then to 1.5 g liter −1 day −1 . After the loading rate was increased to 1.5 g liter −1 day −1 , nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter −1 day −1 . A total of 41 bacterial strains with different repetitive extragenic p...

Applied and Environmental Microbiology, 1998

A method for quantifying bacterial populations introduced into an activated-sludge microbial comm... more A method for quantifying bacterial populations introduced into an activated-sludge microbial community is described. The method involves extraction of DNA from activated sludge, appropriate dilution of the extracted DNA with DNA extracted from nonintroduced activated sludge, PCR amplification of a gyrB gene fragment from the introduced strain with a set of strain-specific primers, and quantification of the electrophoresed PCR product by densitometry. The adequacy of the method was examined by analyzing the population dynamics of two phenol-degrading bacteria, Pseudomonas putida BH and Comamonas sp. strain E6, that had been introduced into phenol-digesting activated sludge. The density of each of the two populations determined by the PCR method immediately after the introduction was consistent with the density estimated from a plate count of the inoculum. This quantitative PCR method revealed different population dynamics for the two strains in the activated sludge under different ph...

Molecular and General Genetics MGG, 1993

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidatio... more TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xyITEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xyITEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpGT, respectively. Comparison of the nucleotide sequences of the xyIXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xyITEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.

Artificial evolution by DNA shuffling

Trends in Biotechnology, 1998

Improvement of enzymes is one of the important objectives of biotechnology. In vitro evolution of... more Improvement of enzymes is one of the important objectives of biotechnology. In vitro evolution of enzymes using DNA shuffling involves the assembly of two or more DNA segments into a full-length gene by homologous, or site-specific, recombination. Before the assembly, the segments are often subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods. Many useful enzymes and peptides have been isolated following the artificial evolution.

Major Carotenoid Isolated from Paracoccus schoinia NBRC 100637T Is Adonixanthin Diglucoside

Journal of Natural Products, 2006

The structure of a novel major carotenoid glycoside (nearly 80% of total carotenoids) from a newl... more The structure of a novel major carotenoid glycoside (nearly 80% of total carotenoids) from a newly isolated bacterium, Paracoccus schoinia NBRC 100637T, was determined to be adonixanthin diglucoside using spectral data. By contrast, carotenoid diglycosides are rare and are usually minor carotenoids in nature. The minor carotenoids in this bacterium included astaxanthin diglucoside, zeaxanthin diglucoside, canthaxanthin, echinenone, zeaxanthin, beta-cryptoxanthin, and beta-carotene.

Journal of Bacteriology, 2008

The soil actinomycete Kocuria rhizophila belongs to the suborder Micrococcineae , a divergent bac... more The soil actinomycete Kocuria rhizophila belongs to the suborder Micrococcineae , a divergent bacterial group for which only a limited amount of genomic information is currently available. K. rhizophila is also important in industrial applications; e.g., it is commonly used as a standard quality control strain for antimicrobial susceptibility testing. Sequencing and annotation of the genome of K. rhizophila DC2201 (NBRC 103217) revealed a single circular chromosome (2,697,540 bp; G+C content of 71.16%) containing 2,357 predicted protein-coding genes. Most of the predicted proteins (87.7%) were orthologous to actinobacterial proteins, and the genome showed fairly good conservation of synteny with taxonomically related actinobacterial genomes. On the other hand, the genome seems to encode much smaller numbers of proteins necessary for secondary metabolism (one each of nonribosomal peptide synthetase and type III polyketide synthase), transcriptional regulation, and lateral gene transf...

Novel family shuffling methods for the in vitro evolution of enzymes

Gene, 1999

It has recently been shown that shuffling of the amino acid sequences of family enzymes allows th... more It has recently been shown that shuffling of the amino acid sequences of family enzymes allows the generation of improved enzymes. Family shuffling is generally achieved by a DNase I treatment and then by PCR. Shuffling of the xylE and nahH genes, both encoding catechol 2,3-dioxygenases, was carried out by the published method. However, nahH-xylE hybrids were only formed at a very low frequency (less than 1%). Therefore, we developed improved methods for family shuffling by which DNA was cleaved by restriction enzymes instead of by DNase I. With the first improved method, five nahH fragments and five xylE fragments that had been generated by restriction enzyme digestion were subjected to the PCR reactions in two steps, the first being without a primer and the second with a set of primers. This method enabled nahH-xylE hybrid genes to be formed at a high frequency (almost 100%). With the second improved method, nahH and xylE were cleaved by several sets of restriction enzymes, and these digests were then reassembled in two steps. The nahH and xylE DNAs were each cleaved by two (or three) sets of restriction enzymes, and one type of nahH digest and one type of xylE digest were mixed, thus making four (or nine) different mixtures of the nahH and xylE digests. These mixtures were used as templates to carry out PCR without a primer. After the first PCR reaction, all the mixtures were combined, and a second PCR reaction was carried out without a primer. Following these two PCR assembly steps, a third PCR reaction was carried out with two primers to amplify the full-length nahH-xylE hybrid genes. This second method also yielded nahH-xylE hybrids at a frequency of 100%. The degree of recombination of the products with the second method was higher than that with the first method. These methods were used to isolate catechol 2,3-dioxygenases exhibiting relatively high stability at high temperature, one of them being respectively 13- and 26-fold more thermostable than XylE and NahH at 50 degrees C.

Environmental Microbiology, 2001

In January 1997, the tanker Nakhodka sank in the Japan Sea, and more than 5000 tons of heavy oil ... more In January 1997, the tanker Nakhodka sank in the Japan Sea, and more than 5000 tons of heavy oil leaked. The released oil contaminated more than 500 km of the coastline, and some still remained even by June 1999. To investigate the long-term influence of the Nakhodka oil spill on marine bacterial populations, sea water and residual oil were sampled from the oil-contaminated zones 10, 18, 22 and 29 months after the accident, and the bacterial populations in these samples were analysed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments. The dominant DGGE bands were sequenced, and the sequences were compared with those in DNA sequence libraries. Most of the bacteria in the sea water samples were classified as the Cytophaga±Flavobacterium±Bacteroides phylum, a-Proteobacteria or cyanobacteria. The bacteria detected in the oil paste samples were different from those detected in the sea water samples; they were types related to hydrocarbon degraders, exemplified by strains closely related to Sphingomonas subarctica and Alcanivorax borkumensis. The sizes of the major bacterial populations in the oil paste samples ranged from 3.4 Â 10 5 to 1.6 Â 10 6 bacteria per gram of oil paste, these low numbers explaining the slow rate of natural attenuation.

Bioscience, Biotechnology, and Biochemistry, 2001

Bioconversion (biotransformation) experiments on arenes (arematic compounds), inc]uding yarious t... more Bioconversion (biotransformation) experiments on arenes (arematic compounds), inc]uding yarious tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the ce]ls of EScherichia coli transformants expressing seyeral arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phct4BCD) genes derived from the marine bacterium IVbcardioides sp. strain KP7 conyerted all of these tricyclic aromatic compounds, while E. coli carrying the Rseudomonas putidu Fl toluene dioxygenase (todCIC2B,tl) genes or the P. pseudoalcangenes KF707 biphenyl dioxygenase (ophAJA2A3A4 genes was not able to convert these substrates. Surprisingly, E. coti carrying hybrid dioxygenase (todCl::ophA2A3,44 genes with a subunit substitution between the toluene and biphenyl dioxygenases was ab]e to conyert fiuorene, dibenzofuran, and dibenzothiophene. The cel]s of a Streptomyces tiviclans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconyersion of various tricyc]ic fused aromatic compou"ds. The ability of this actinomycete in their conversion was similar to that of E coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cel]s were purified by column chromatography on silica gel, and identified by their MS and 'H and i3C NMR analyses.

Biochemical and Biophysical Research Communications, 1991

A mathematical model of the induction kinetics of RNAs and proteins in exponentially growing orga... more A mathematical model of the induction kinetics of RNAs and proteins in exponentially growing organisms is derived, and the cellular concentrations of the induced macromolecules at a given time after induction are related to three parameters: the fraction of the synthesis of these macromolecules in total synthesis, the half life of the inducible macromolecules, and the generation time of the organisms. The model predicts that the concentrations of the inducible macromolecules reach one half of the maximum induction level within one generation time after the onset of the induction. The model also predicts that induction curves of proteins are parabolic when their mRNAs are short-lived, but sigmoid when they are stable. Observed induction curves of B-galactosidase in Escherichia coli cells fit in the theoretical induction curves.

Applied and Environmental Microbiology, 2002

Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing d -glucos... more Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing d -glucose, d -galactose, d -mannose, d -glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the ba...

Environmental Microbiology, 2006

Bacterial transcriptional networks are built on a hierarchy of regulators, on top of which lie th... more Bacterial transcriptional networks are built on a hierarchy of regulators, on top of which lie the components of the RNA polymerase (in particular the sigma factors) and the global control elements, which play a pivotal role. We have designed a genome-wide oligonucleotide-based DNA microarray for Pseudomonas putida KT2440. In combination with real-time reverse transcription polymerase chain reaction (RT-PCR), we have used it to analyse the expression pattern of the genes encoding the RNA polymerase subunits (the core enzyme and the 24 sigma factors), and various proteins involved in global regulation (Crc,

Alonso-Gutierrez PCAP 2015 ME

Applied and Environmental Microbiology, 2005

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q25... more A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264: 6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61: 329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, whil...

Printed in Great Britain 1 73 Phototactic Response of Aerobically Cultivated Rhodospirillum rubrum

Motile cells of aerobically cultivated Rhodospirillum rubrum, containing no detectable bacterioch... more Motile cells of aerobically cultivated Rhodospirillum rubrum, containing no detectable bacteriochlorophyll, assembled at a spot of strong light projected through a dark-field condenser. Far-red light was not effective, indicating that bacteriochlorophyll and thus photosynthetic metabolism was not responsible for the phenomenon. Bacteria moving towards the centre of the light spot changed direction less frequently than those moving towards the margin. They also responded to temporal changes in the intensity of light, altering their swimming direction more frequently after a sudden decrease in light intensity than after an abrupt increase.

Journal of Biological Chemistry, 1989

PH/Hf 7a. Name of Monitoring Organization Naval Postgraduate School c. Address (city, state, and ... more PH/Hf 7a. Name of Monitoring Organization Naval Postgraduate School c. Address (city, state, and ZIP code) vlonterey, CA 93943-5000 7b. Address (city, state, and TIP code) Monterey, CA 93943-5000 a. Name of Funding/Sponsoring Organization 8b. Office Symbol (If Applicable) 9. Procurement Instrument Identification Number c. Address (city, state, and ZIP code) 10. Source of Funding Numbers Program Element Number Project No Task No Work Unit Accession No i. Tide (include Security Classification) Investigation of the Physical Characteristics of a Mass Element Resonator 2. Personal Author(s) Grant, Larry A. 3 a. Type of Report Master's Thesis 13b. Time Covered From Jul 89 To Mar 92 14. Date of Report (year, month.day)

Journal of Biological Chemistry, 1993

Journal of Bacteriology, 1984

Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mu... more Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mutagenized with transposon Tn1000 or Tn5. The resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in Escherichia coli. The physical locations of the insertions in each of 28 Tn1000 and 5 Tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis. This information permitted the construction of a precise physical and genetic map of the meta-cleavage pathway operon. The gene order xylD (toluate dioxygenase), L (dihydroxycyclohexidiene carboxylate dehydrogenase), E (catechol 2,3-dioxygenase), G (hydroxymuconic semialdehyde dehydrogenase), F (hydroxymuconic semialdehyde hydrolase), J (2-oxopent-4-enoate hydratase), I (4-oxalocrotonate decarboxylase), and H (4-oxalocrotonate tautomerase) was established, and gene sizes were estimated. Tn1000 insertions within catabolic genes exerted polar effects on distal stru...