Yoshinori Hatakeyama

Development of Innovative Pediocin PA-1 by DNA Shuffling among Class IIa Bacteriocins

ABSTRACT Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products.

Determination of Essential and Variable Residues in Pediocin PA-1 by NNK Scanning

ABSTRACT Pediocin PA-1 is an antimicrobial peptide (called bacteriocin) that shows inhibitory activity against the food-borne pathogen Listeria monocytogenes. To elucidate which residue(s) is responsible for this function, the antimicrobial activities of pediocin PA-1 mutants were evaluated and compared. Each of the 44 native codons was replaced with the NNK triplet oligonucleotide in a technique termed NNK scanning, and 35 mutations at each position were examined for antimicrobial activities using a modified colony overlay screening method. As a consequence, the functional responsibility of each residue was estimated by counting the number of active mutants, allowing us to identify candidate essential/variable residues. Activity was abrogated by many of the mutations at residues Y2, G6, C9, C14, C24, W33, G37, and C44, indicating that these residues may be essential. In contrast, activity was retained by almost all versions harboring mutations at K1, T8, G10, S13, G19, N28, and N41, indicating that these are functionally redundant residues. Sequence analysis revealed that only the wild type was active and 14 and 11 substitutions were inactive at G6 and C14, respectively, while 12 and 11 substitutions were active and 2 and 0 substitutions were inactive at T8 and K1, respectively. These findings suggest that NNK scanning is effective for determining essential and variable residues in pediocin PA-1, leading to an elucidation of structure-function relationships and to improvements in the antimicrobial function efficiently by peptide engineering.

A new method of pebrine inspection of silkworm egg using multiprimer PCR

Using a mixture of several PCR primers, we evaluated whether multiprimer PCR is practically useful for the early and simultaneous detection of several kinds of microsporidia that cause silkworm pebrine. When genomic DNA extracted from silkworm eggs infected with Nosema bombycis was used as the DNA template, the specific DNA sequences were amplified by multiprimer PCR. In addition, similar results were obtained even when genomic DNA extracted from silkworms infected with N. bombycis was used as the DNA template. These findings suggest that multiprimer PCR using several primers designed for this study is practically useful for pebrine inspection of silkworm eggs.

Phylogenetic relationships among Bacillus thuringiensis (Bacillaceae: Bacillales) strains based on a comparison of SSU rRNA sequences and genome profiling

Bacillus thuringiensis Berliner has previously been classified via the serological identification of flagellar antigens. However, the phylogenetic relationships among strains of B. thuringiensis cannot be investigated by serotyping. Furthermore, high levels of homology have been found in gene sequences among various strains, complicating the determination of their evolutionary relationships. In order to elucidate the phylogenetic relationships within B. thuringiensis, we analyzed 40 strains belonging to typical serotypes using two approaches: an analysis of small subunit (SSU) rRNA sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The SSU rRNA analysis resulted in all 40 strains forming a single cluster with Bacillus cereus Frankland & Frankland. The distances among the subclusters were too small to further classify the strains. On the other hand, the phylogenetic analysis based on GP resulted in three clusters of B. thuringiensis strains. These results suggest that GP is a better method for the determination of phylogenetic relationships within B. thuringiensis.

Phylogenetic relationships among strains of the entomopathogenic fungus Beauveria bassiana (Hypocreales: Clavicipitaceae) isolated from Japan

The fungus Beauveria bassiana (Balsamo) Vuillemin has previously been classified using morphological characteristics, but morphology cannot reveal the phylogenetic relationships among conventionally classified strains. High levels of homology have been found in gene sequences among various B. bassiana strains, complicating the determination of their evolutionary relationships. To elucidate phylogenetic relationships among conventionally known Beauveria species, we analyzed 57 major strains of B. bassiana and 3 strains of B. brongniartii (Saccardo) Petch isolated from Japan by analysis of internal transcribed spacer (ITS) sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The ITS sequence analysis placed the 57 conventional B. bassiana strains into two clusters, B. bassiana and Beauveria pseudobassiana Rehner et Humber. In contrast, GP analysis produced five clusters of B. bassiana strains that included B. pseudobassiana clusters. These results suggested that GP was more accurate than ITS sequence analysis for determining phylogenetic relationships within B. bassiana. In addition, our findings suggested that conventional strains of B. bassiana isolated from Japan include both B. bassiana and B. pseudobassiana groups.