Most of the microbes from natural habitats cannot be cultivated with standard cultivation in laboratory, and sponge-associated microbes are no exception. We used two advanced methods based on a continuous-flow bioreactor (CF) and in situ cultivation (I-tip) to isolate previously uncultivated marine sponge-associated bacteria. We also characterized the physiological properties of the isolates from each method and attempted to clarify the mechanisms operating in each cultivation method. A greater number of novel bacteria were isolated using CF and in situ cultivation compared to standard direct plating (SDP) cultivation. Most isolates from CF cultivation were poor growers (with lower specific growth rates and saturated cell densities than those of isolates from SDP cultivation), demonstrating that it is effective to carry out pre-enrichment cultivation targeting bacteria that are less competitive on conventional cultivation, especially K-strategists and bacterial types inhibited by their own growth. Isolates from in situ cultivation showed a positive influence on cell recovery stimulated by chemical compounds in the extract of sponge tissue, indicating that some of the bacteria require a “growth initiation factor” that is present in the natural environment. Each advanced cultivation method has its own distinct key mechanisms allowing cultivation of physiologically and phylogenetically different fastidious bacteria for cultivation compared with conventional methods.

Nitrifying chemoautotrophs support the growth of diverse concomitant heterotrophs in natural or engineered environments by supplying organic compounds. In this study, we aimed to investigate this microbial association, especially (i) to distinguish whether the relationship between nitrifying chemoautotrophs and heterotrophs is commensal or mutualistic, and (ii) to clarify how heterotrophs promote the growth of autotrophic nitrite-oxidizing bacteria (Nitrospira). Pure cultured Nitrospira (Nitrospira sp. ND1) was employed in this study. Heterotrophs growing with metabolic by-products of Nitrospira as a sole carbon source were isolated from several environmental samples and used to test the growth-promoting activity of Nitrospira. Furthermore, liquid chromatography-mass spectrometry analysis was conducted to evaluate how heterotrophs consumed chemical compounds produced by Nitrospira and newly produced during co-cultivation. Notably, Nitrospira growth was stimulated by co-cultivation with some heterotrophs and the addition of spent media of some strains, suggesting that not only heterotrophs but also Nitrospira received benefits from their mutual co-existence. Furthermore, the data suggested that some of the growth-promoting heterotrophs provided as-yet-unidentified growth-promoting factors to Nitrospira. Overall, Nitrospira and heterotrophs thus appear to exhibit a mutualistic relationship. Such mutualistic relationships between autotrophs and heterotrophs would contribute to the stability and diversity of microbial ecosystems.

Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, cultivating microbes in a membrane-bound device incubated in nature (in situ cultivation) can be an effective approach to overcome this limitation. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this method's efficiencies with those of the conventional method. Then, we attempted to investigate the key and previously unidentified mechanism of growing uncultivated microorganisms by in situ cultivation focusing on growth triggering via growth initiation factor. Significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). We hypothesized that some of environmental microorganisms which resist cultivation are in a non-growing state and require growth initiation factors for the recovery and that these can be provided from the environment (in this study from marine sponge). According to the hypothesis, the effect of the sponge extract on recovery on agar medium was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or saturated cell densities of all tested strains were not positively affected. These results indicate that, (1) the sponge extract contains chemical compounds that facilitate recovery of non-growing microbes, (2) these substances worked on the in situ strains, and (3) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from non-growing state).

Here, we report peptide probes with either single or cyclic double stranded collagen-like sequences that spontaneously acquire collagen-hybridizing ability at physiological pH. These peptides have ester bonds derived from O-acyl isopeptide units that are converted to amide bonds via intramolecular O-to-N acyl migration by a pH shift. The peptides that do not require pre-treatment for disassembly will be useful as prodrugs in theranostic treatments targeting unfolded collagen.

Sameuramide A (1), a new cyclic depsipeptide encompassing one each of alanine, N-methyl alanine, N-methyl dehydroalanine, N,O-dimethyl threonine, phenyllactic acid, three β-hydroxy leucines, and two propionates, was isolated from a didemnid ascidian collected at the northern part of Japan. The planar structure was established based on the interpretation of MS and NMR data. The absolute configuration of the subunits was determined by the advanced Marfey's method and the chiral LC-MS analysis. Compound 1 exhibited the activity of maintaining colony formation of murine embryonic stem (mES) cells without leukemia inhibitory factor (LIF). Down regulation of the gene expression of Krüppel-like transcription factor 4 (Klf4) indicated that 1 itself was not able to maintain the undifferentiated state of the mES cells. However, the expression levels of the marker genes (Nestin, T, Sox17) for three germ layers were upregulated in embryoid bodies (EBs) after treatment of 1 together with LIF, suggesting that 1 plays a supportive role for LIF in maintaining the multipotency of mES cells.