RESUMO
A large number of microbes are not able to form colonies using agar-plating methods, which is one of the reasons that cultivation based on solid media leaves the majority of microbial diversity in the environment inaccessible. We developed a new Non-Colony-Forming Liquid Cultivation method (NCFLC) that can selectively isolate non-colony-forming microbes that exclusively grow in liquid culture. The NCFLC method involves physically separating cells using dilution-to-extinction (DTE) cultivation and then selecting those that could not grow on a solid medium. The NCFLC was applied to marine samples from a coastal intertidal zone and soil samples from a forest area, and the results were compared with those from the standard direct plating method (SDP). The NCFLC yielded fastidious bacteria from marine samples such as Acidobacteriota, Epsilonproteobacteria, Oligoflexia, and Verrucomicrobiota. Furthermore, 62% of the isolated strains were potential new species, whereas only 10% were novel species from SDP. From soil samples, isolates belonging to Acidobacteriota and Armatimonadota (which are known as rare species among identified isolates) were exclusively isolated by NCFLC. Colony formation capabilities of isolates cultivated by NCFLC were tested using solid agar plates, among which approximately one-third of the isolates were non-colony-forming, approximately half-formed micro-colonies, and only a minority could form ordinary size colonies. This indicates that the majority of the strains cultivated by NCFLC were previously uncultured microbial species unavailable using the SDP method. The NCFCL method described here can serve as a new approach to accessing the hidden microbial dark matter.
RESUMO
Diol dehydratase-reactivase (DD-R) is a molecular chaperone that reactivates inactivated holodiol dehydratase (DD) by cofactor exchange. Its ADP-bound and ATP-bound forms are high-affinity and low-affinity forms for DD, respectively. Among DD-Rs mutated at the nucleotide-binding site, neither the Dα8N nor Dα413N mutant was effective as a reactivase. Although Dα413N showed ATPase activity, it did not mediate cyanocobalamin (CN-Cbl) release from the DD·CN-Cbl complex in the presence of ATP or ADP and formed a tight complex with apoDD even in the presence of ATP, suggesting the involvement of Aspα413 in the nucleotide switch. In contrast, Dα8N showed very low ATPase activity and did not mediate CN-Cbl release from the complex in the presence of ATP, but it did cause about 50% release in the presence of ADP. The complex formation of this mutant with DD was partially reversed by ATP, suggesting that Aspα8 is involved in the ATPase activity but only partially in the nucleotide switch. Among DD-Rs mutated at the Mg(2+)-binding site, only Eß31Q was about 30% as active as wild-type DD-R and formed a tight complex with apoDD, indicating that the DD-R ß subunit is not absolutely required for reactivation. If subunit swapping occurs between the DD-R ß and DD ß subunits, Gluß97 of DD would coordinate to Mg(2+). The complex of Eß97Q DD with CN-Cbl was not activated by wild-type DD-R. No complex was formed between this mutant and wild-type DD-R, indicating that the coordination of Gluß97 to Mg(2+) is essential for subunit swapping and therefore for (re)activation.