Role of ribosome recycling factor in natural termination and translational coupling as a ribosome releasing factor.

The role of ribosome recycling factor (RRF) of E. coli was studied in vivo and in vitro. We used the translational coupling without the Shine-Dalgarno sequence of downstream ORF (d-ORF) as a model system of the RRF action in natural termination of protein synthesis. For the in vivo studies we used the translational coupling by the adjacent coat and lysis genes of RNA phage GA sharing the termination and initiation (UAAUG) and temperature sensitive RRF. The d-ORF translation was measured by the expression of the reporter lacZ gene connected to the 5'-terminal part of the lysis gene. The results showed that more ribosomes which finished upstream ORF (u-ORF) reading were used for downstream reading when RRF was inactivated. The in vitro translational coupling studies with 027mRNA having the junction sequence UAAUG with wild-type RRF were carried out with measuring amino acids incorporation. The results showed that ribosomes released by RRF read downstream from AUG of UAAUG. In the absence of RRF, ribosomes read downstream in frame with UAA. These in vivo and in vitro studies indicate that RRF releases ribosomes from mRNA at the termination codon of u-ORF. Furthermore, the non-dissociable ribosomes read downstream from AUG of UAAUG with RRF in vitro. This suggests that complete ribosomal splitting is not required for ribosome release by RRF in translational coupling. The data are consistent with the interpretation that RRF functions mostly as a ribosome releasing factor rather than ribosome splitting factor. Additionally, the in vivo studies showed that short (less than 5 codons) u-ORF inhibited d-ORF reading by ribosomes finishing u-ORF reading, suggesting that the termination process in short ORF is not similar to that in normal ORF. This means that all the preexisting studies on RRF with short mRNA may not represent what goes on in natural termination step.

A Lytic Bacteriophage for Controlling Pseudomonas lactis in Raw Cow's Milk.

The control of bacterial growth during milk processing is crucial for the quality maintenance of commercial milk and milk products. During a period of cold storage prior to heat treatments, some psychrotrophic bacteria grow and produce extracellular heat-resistant lipases and proteases that cause product defects. The use of lytic bacteriophages (phages) that infect and kill bacteria could be a useful tool for suppressing bacterial growth during this cold storage phase. In this study, we isolated a Pseudomonas lactis strain and a phage from raw cow's milk. Quantitative characterization of the phage was used to elucidate whether this phage was active under low temperatures and neutral pH and whether it was inactivated during pasteurization. Phage titer determination was possible under conditions ranging from pH 4 to 9 and from 3°C to 25°C; the phage was inactivated under pasteurization conditions at 63°C for 30 min. Furthermore, we showed that this phage reduced viable bacterial cell counts in both skim and whole milk. The results of this study represent the potential uses of phages for controlling psychrotrophic bacterial growth in raw cow's milk during cold storage.IMPORTANCE Suppression of bacterial growth in raw milk under cold storage is crucial for the quality control of commercially supplied milk. The use of lytic phages as low-cost microbicides is an attractive prospect. Due to strict host specificities, phages must be isolated from the raw milk where the host bacteria are growing. We first isolated the P. lactis bacterial strain and then the phage infecting that strain. Partial phage genomic analysis showed that this is a newly isolated phage, different from any previously reported. This study reports a lytic phage for P. lactis, and we have presented evidence here that this phage reduced viable bacterial cell counts not only in rich medium but also in skim and whole milk. As a result, we have concluded that the phage reported in this study would be useful in milk processing.

Quantitative analysis of the bacteriophage Qbeta infection cycle.

In this study, the infection cycle of bacteriophage Qbeta was investigated. Adsorption of bacteriophage Qbeta to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4x10(-10) ml/cells/min. In infected cells, approximately 130 molecules of beta-subunit and 2x10(5) molecules of coat protein were translated in 15 min. Replication of Qbeta RNA proceeded in 2 steps-an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5x10(5) molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.