Peptide-Mediated Gene Transfer into Marine Purple Photosynthetic Bacteria.

Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 °C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.

Method for the facile transformation of marine purple photosynthetic bacteria using chemically competent cells.

Marine purple photosynthetic bacteria are ideal organisms for the production of useful materials at reduced costs and contributing to a sustainable society because they can utilize sunlight, seawater, and components of air, including carbon dioxide and nitrogen gases, for their growth. However, conjugation is the only applicable method for the transformation of marine purple photosynthetic bacteria so far. Here, we examined a calcium chloride-mediated method for the transformation of marine purple photosynthetic bacteria. Plasmid DNAs containing the kanamycin resistance gene were successfully transferred into chemically competent cells of two strains of marine purple photosynthetic bacteria (Rhodovulum sulfidophilum and Roseospira marina). Heat shock treatment increased the transformation efficiency in R. sulfidophilum, whereas the addition of cell-penetrating peptide did not improve it. We also found that prolonged incubation in agar plates containing kanamycin led to spontaneous mutation of the 16S rRNA, resulting in kanamycin resistance in R. marina. Thus, we developed an efficient and facile transformation method using chemically competent cells of marine purple photosynthetic bacteria with calcium chloride.

A Screening Method for the Isolation of Polyhydroxyalkanoate-Producing Purple Non-sulfur Photosynthetic Bacteria from Natural Seawater.

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters accumulated by a variety of microorganisms as carbon and energy storage under starvation conditions. We focused on marine purple non-sulfur photosynthetic bacteria as host microorganisms for PHA production and developed a method for their isolation from natural seawater. To identify novel PHA-producing marine purple non-sulfur photosynthetic bacteria, natural seawaters were cultured in nutrient-rich medium for purple non-sulfur photosynthetic bacteria, and twelve pink- or red-pigmented colonies were picked up. Gas chromatography mass spectrometry analysis revealed that four isolates synthesized PHA at levels ranging from 0.5 to 24.4 wt% of cell dry weight. The 16S ribosomal RNA sequence analysis revealed that one isolate (HM2) showed 100% identity to marine purple non-sulfur photosynthetic bacteria. In conclusion, we have demonstrated in this study that PHA-producing marine purple non-sulfur photosynthetic bacteria can be isolated from natural seawater under nutrient-rich conditions.

Synthesis of High-Molecular-Weight Polyhydroxyalkanoates by Marine Photosynthetic Purple Bacteria.

Polyhydroxyalkanoate (PHA) is a biopolyester/bioplastic that is produced by a variety of microorganisms to store carbon and increase reducing redox potential. Photosynthetic bacteria convert carbon dioxide into organic compounds using light energy and are known to accumulate PHA. We analyzed PHAs synthesized by 3 purple sulfur bacteria and 9 purple non-sulfur bacteria strains. These 12 purple bacteria were cultured in nitrogen-limited medium containing acetate and/or sodium bicarbonate as carbon sources. PHA production in the purple sulfur bacteria was induced by nitrogen-limited conditions. Purple non-sulfur bacteria accumulated PHA even under normal growth conditions, and PHA production in 3 strains was enhanced by nitrogen-limited conditions. Gel permeation chromatography analysis revealed that 5 photosynthetic purple bacteria synthesized high-molecular-weight PHAs, which are useful for industrial applications. Quantitative reverse transcription polymerase chain reaction analysis revealed that mRNA levels of phaC and PhaZ genes were low under nitrogen-limited conditions, resulting in production of high-molecular-weight PHAs. We conclude that all 12 tested strains are able to synthesize PHA to some degree, and we identify 5 photosynthetic purple bacteria that accumulate high-molecular-weight PHA molecules. Furthermore, the photosynthetic purple bacteria synthesized PHA when they were cultured in seawater supplemented with acetate. The photosynthetic purple bacteria strains characterized in this study should be useful as host microorganisms for large-scale PHA production utilizing abundant marine resources and carbon dioxide.