Deformability-Based Microfluidic Microdroplet Screening to Obtain Agarolytic Bacterial Cells.

Environmental microorganisms possess enzymes that can digest macromolecules such as agarose into smaller molecules that can be utilized for growth. These enzymes could be valuable for the effective utilization of global resources. However, since most of the microorganisms on Earth remain uncultured, there is significant untapped enzymatic potential in nature. Therefore, it is necessary to develop innovative tools and strategies for exploring these enzymatic resources. To address this, we developed a method for screening microbial cells that secrete hydrogel-degrading enzymes using deformability-based microfluidic microdroplet sorting. In this method, microbial cells are encapsulated as single cells in water-in-oil (W/O) microdroplets with a hydrogel whose shape becomes deformable as the hydrogel is progressively degraded into smaller molecules. Screening is achieved using a microfluidic device that passively sorts the deformed W/O microdroplets. Using this method, we successfully sorted agarose-containing microdroplets, encapsulating single bacterial cells that hydrolyzed agarose. This method can be used to screen various hydrogel-degrading microbial cells.

Complete Genomic Sequence of an Agarolytic Pseudoalteromonas Species Isolated from Deep Seawater.

We report the complete genomic sequence of the agarolytic bacterium Pseudoalteromonas sp. strain MM1, recovered from deep seawater. The genome has two circular chromosomes with sizes of 3,686,652 bp and 802,570 bp and GC contents of 40.8 and 40.0%, and it carries 3,967 protein-coding sequences, 24 rRNA genes, and 103 tRNA genes.

Complete Genomic Sequences of Two Agarolytic Vibrio Species Isolates from the Red Algae Gracilaria.

We report the complete genomic sequences of two agarolytic Vibrio species strains, STUT-A11 and STUT-A16, isolated from the red algae Gracilaria. Genomic annotations revealed that both strains harbor four ß-agarases, α-neoagarooligosaccharide hydrolase, and agarolytic ß-galactosidase, which support efficient agarose catabolism.

Nascent SecM chain interacts with outer ribosomal surface to stabilize translation arrest.

SecM, a bacterial secretion monitor protein, posttranscriptionally regulates downstream gene expression via translation elongation arrest. SecM contains a characteristic amino acid sequence called the arrest sequence at its C-terminus, and this sequence acts within the ribosomal exit tunnel to stop translation. It has been widely assumed that the arrest sequence within the ribosome tunnel is sufficient for translation arrest. We have previously shown that the nascent SecM chain outside the ribosomal exit tunnel stabilizes translation arrest, but the molecular mechanism is unknown. In this study, we found that residues 57-98 of the nascent SecM chain are responsible for stabilizing translation arrest. We performed alanine/serine-scanning mutagenesis of residues 57-98 to identify D79, Y80, W81, H84, R87, I90, R91, and F95 as the key residues responsible for stabilization. The residues were predicted to be located on and near an α-helix-forming segment. A striking feature of the α-helix is the presence of an arginine patch, which interacts with the negatively charged ribosomal surface. A photocross-linking experiment showed that Y80 is adjacent to the ribosomal protein L23, which is located next to the ribosomal exit tunnel when translation is arrested. Thus, the folded nascent SecM chain that emerges from the ribosome exit tunnel interacts with the outer surface of the ribosome to stabilize translation arrest.