Modulation of fatty acid composition and growth in Sporosarcina species in response to temperatures and exogenous branched-chain amino acids.

Psychrotolerant endospore-forming Sporosarcina species have been predominantly isolated from minced fish meat (surimi), which is stored under refrigeration after heat treatment. To develop a better method for preserving surimi-based food products, we studied the growth and fatty acid compositions of the isolated strain S92h as well as Sporosarcina koreensis and Sporosarcina aquimarina at cold and moderate temperatures. The growth rates of strain S92h and S. koreensis were the fastest and slowest at cold temperatures, respectively, although these strains grew at a similar rate at moderate temperatures. In all three strains, the proportions of anteiso-C15:0 and unsaturated fatty acids (UFAs) were significantly higher at cold temperatures than at moderate temperatures. Furthermore, supplementation with valine, leucine, and isoleucine resulted in proportional increases in iso-C16:0, iso-C15:0, and anteiso-C15:0, respectively, among the fatty acid compositions of these strains. The proportions of the UFAs were also altered by the supplementation. At cold temperatures, the growth rates of strain S92h and S. koreensis, but not of S. aquimarina, were affected by supplementation with leucine. Supplementation with isoleucine enhanced the growth of S. koreensis at cold temperatures but not that of the other strains. Valine did not affect the growth of any strain. These results indicate that anteiso-C15:0 and UFAs both play important roles in the cold tolerance of the genus Sporosarcina and that these bacteria modulate their fatty acid compositions in response to the growth environment.

Putative intermediary stages for the molecular evolution from a ribozyme to a catalytic RNP.

A hypothetical evolutionary pathway from a ribozyme to a catalytic RNA-protein complex (RNP) is proposed and examined. In this hypothesis for an early phase of molecular evolution, one RNA-RNA interaction in the starting ribozyme is replaced with an RNA-protein interaction via two intermediary stages. At each stage, the original RNA-RNA interaction and a newly introduced RNA-protein interaction are designed to coexist. The catalytic RNPs corresponding to the intermediary stages were constructed by employing the Tetrahymena ribozyme together with molecular modeling. Analyses of the RNPs indicate that the protein can fully replace the original role of the RNA-RNA interaction in the starting ribozyme and that the association of a protein with a ribozyme might be beneficial for improving the ribozymatic activity.

Isolation and characterization of psychrotolerant endospore-forming Sporosarcina species associated with minced fish meat (surimi).

We studied the changes of resident microbiota in surimi-minced fish meat-during heat-treatment and subsequent cold-storage via the sequencing of partial 16S rRNA gene. Raw surimi made from Alaska pollock, pike conger, and white croaker was contaminated with 10(4) to 10(6)CFU/g of various non-endospore-forming bacteria. Immediately after heat-treatment, the bacterial counts were significantly reduced to less than 1CFU/g, and only endospore-forming bacteria, identified as Bacillus species were retrieved. Subsequently, the bacterial counts increased up to 10 to 10(5)CFU/g in the heated surimi after refrigerated storage at 5 °C for 2 weeks or at 10 °C for 1 week. Most of the isolates from the refrigerated surimi were identified as Sporosarcina species. The Sporosarcina isolates have an increased ability to grow at 10 °C than the isolates related to the other endospore-forming bacteria, such as Bacillus, Lysinibacillus, and Paenibacillus species. Endospores of the Sporosarcina isolates were able to germinate and proliferate in a fish-paste product model system stored at 10 °C within 8 days. In order to study the cold-adaptation mechanism of Sporosarcina species, the fatty acid composition of the isolates was analyzed. At the growth temperature of 10 °C, the proportions of unsaturated to saturated fatty acids and anteiso to iso fatty acids were higher than those at 28 °C. The alteration of the fatty acid composition suggests that Sporosarcina species adapt to cold by maintaining the fluidity of the cell membrane because unsaturated and anteiso fatty acids have lower melting points than saturated and iso fatty acids, respectively. We concluded that the endospores of Sporosarcina species are widely distributed in surimi, and that they can survive heat-treatment and proliferate during cold-storage in fish-paste products. Controlling Sporosarcina species would contribute to improving the quality of surimi product.

De novo synthesis and development of an RNA enzyme.

Arbitrary manipulation of molecular recognition at the atomic level has many applications. However, systematic design and de novo synthesis of an artificial enzyme based on such manipulation has been a long-standing challenge in the field of chemistry and biotechnology. In this report, we developed an artificial RNA ligase by implementing a synthetic strategy that fuses a series of 3D molecular modelings based on naturally occurring RNA-RNA recognition motifs with a small-scale combinatorial synthesis of a modular catalytic unit. The resulting ligase produces a 3'-5' linkage in a template-directed manner for any combinations of two nucleotides at the reaction site. The reaction rate is 10(6)-fold over that of the uncatalyzed reaction with a yield higher than those of previously reported ligase ribozymes. The strategy may be applicable to the synthesis and development of a variety of nonnatural functional RNAs with defined 3D structures.

Modeling of a possible evolutional process from a ribozyme to a catalytic RNP.

A model process for molecular evolution from an RNA enzyme to a catalytic RNA-protein complex (RNP) is proposed. In the model, one RNA-RNA interaction in the enzyme is replaced by an RNA-protein interaction via an intermediary state where the original RNA-RNA and newly introduced RNA-protein interaction co-exist. To test the model, a catalytic RNP was designed and examined by employing the Tetrahymena ribozyme.