Short peptide induces an "uncultivable" microorganism to grow in vitro - PubMed (original) (raw)
Short peptide induces an "uncultivable" microorganism to grow in vitro
D Nichols et al. Appl Environ Microbiol. 2008 Aug.
Abstract
Microorganisms comprise the bulk of biodiversity, but only a small fraction of this diversity grows on artificial media. This phenomenon was noticed almost a century ago, repeatedly confirmed, and termed the "great plate count anomaly." Advances in microbial cultivation improved microbial recovery but failed to explain why most microbial species do not grow in vitro. Here we show that at least some of such species can form domesticated variants capable of growth on artificial media. We also present evidence that small signaling molecules, such as short peptides, may be essential factors in initiating growth of nongrowing cells. We identified one 5-amino-acid peptide, LQPEV, that at 3.5 nM induces the otherwise "uncultivable" strain Psychrobacter sp. strain MSC33 to grow on standard media. This demonstrates that the restriction preventing microbial in vitro growth may be different from those offered to date to explain the "great plate count anomaly," such as deficiencies in nutrient composition and concentrations in standard media, medium toxicity, and inappropriate incubation time. Growth induction of MSC33 illustrates that some microorganisms do not grow in vitro because they are removed from their native communities and the signals produced therein. "Uncultivable" species represent the largest source of unexplored biodiversity, and provide remarkable opportunities for both basic and applied research. Access to cultures of some of these species should be possible through identification of the signaling compounds necessary for growth, their addition to standard medium formulations, and eventual domestication.
Figures
FIG. 1.
Growth induction of MSC33 by the helper strain MSC105. (A) General view of the two-compartment chamber for cocultivation of two microorganisms. A tissue culture insert with a porous (0.02-μm) bottom is placed on the surface of a petri dish, providing the means to grow two pure microbial cultures in chemical contact with each other. (B and C) Inoculation of the insert with the helper strain MSC105 leads to the induction of growth of MSC33 inoculated into the petri dish. (D and E) An empty insert produces no effect, and MSC33 remains in the form of single cells. In all cases, microorganisms were grown for 14 days on casein medium supplemented with Casamino Acids. (F) The microcolony counts of MSC33 in the presence of MSC105 are statistically significantly higher (P < 0.05) than the counts of MSC33 in the presence of the “helper” strain MSC109, which in separate experiments was found to be a “helper” strain to MSC101 (Table 1). Three replicate wells were observed for each treatment. Error bars indicate standard deviations. (Note that the growth of MSC33 with the insert containing no microbial culture and with no insert present [negative controls 1 and 2, respectively] is only apparent. CFU in the negative controls corresponded throughout the study to the number of small clumps of MSC33 cells present in the cells' stock suspension [data not shown]. In order to show the relative scale of such “noise” CFU, we chose not to subtract these counts from the counts of colonies that indeed grew during the experiments.)
FIG. 2.
MSC33 and growth properties of its cultivable variant MSC33c. (A) SEM view of MSC33c cells. (B) MSC33c shows substantial growth at 4°C after 23 days of incubation on LB medium. In parallel incubation, there was no proliferation of Escherichia coli. (C) MSC33 exhibits no growth after 23 h of incubation in LBSW with shaking at 240 rpm and 30°C. The cultivable variant MSC33c grew under the same conditions as E. coli (the latter was incubated at 37°C). For MSC33 and MSC33c, 1,000 cells were used as inocula. LBSW served as a sterile control. All the experiments were conducted in triplicate.
FIG. 3.
Growth induction of MSC33 by its cultivable variant MSC33c and peptides produced in the presence of MSC33c. (A) The microcolony counts of MSC33 in the presence of MSC33c are statistically significantly higher (P < 0.002) than the background counts (negative control 1, insert not inoculated; negative control 2, no insert present). (B) Inverted HPLC trace of the 75% methanol extract of MSC33c-conditioned medium (LBSW). Out of the 16 HPLC fractions tested, three fractions (5, 7, and 14) induced statistically significant (P < 0.05) MSC33 growth. LBSW medium, deionized water, and no amendment (negative controls 1, 2, and 3, respectively) had no effect on MSC33 growth. (C) MSC33 growth in media amended with 2 nM of 3- to 8-amino-acid fragments of the peptide isolated from HPLC fraction 14 (solid bars). The 5-amino-acid peptide was the strongest inducer of MSC33 growth. Individual amino acids produced no effect on MSC33 growth (open bars), as did deionized water and no amendment (negative controls 1 and 2, respectively). (D) Effect of the 5-amino-acid peptide concentration on MSC33 growth, as for panel C. Six replicate wells were observed for each treatment. Error bars indicate standard deviations. See the parenthetical material at the end of the Fig. 1 legend on the nature of CFU in negative controls.
FIG. 4.
Results of CAD/ECD experiment showing the characteristic fragmentation pattern of the peptide (bottom spectrum). The insets show an expanded view of the indicated ions illustrating the resolution of the mass spectrometric measurements. The sequence at the top is marked with observed cleavages, including the exact masses. For each pair of masses, the upper one represents the measured mass, while the lower is the mass of the fragment as calculated from the given sequence.
FIG. 5.
Growth induction of MSC33 in the absence of casein. (A) The microcolony counts of MSC33 in the presence of MSC33c are statistically significantly higher (P < 0.02) than the background counts in CF medium (negative control 1, insert not inoculated; negative control 2, no insert present). (B) The microcolony counts of MSC33 in CF medium amended with MSC33c-conditioned medium (artificial seawater-based yeast extract) are statistically significantly higher (P < 0.02) than control counts. Deionized water and unconditioned yeast extract medium (negative controls 1 and 2) had no effect on MSC33 growth. (C) The microcolony counts of MSC33 in CF medium amended with an extract of MSC33c-conditioned medium (artificial seawater-based yeast extract) are statistically significantly higher (P < 0.02) than control counts. Unconditioned medium extract, deionized water, and unconditioned yeast extract medium (negative controls 1, 2, and 3, respectively) had no effect on MSC33 growth. (D) CFU counts of MSC33 in CF medium amended with methanol extracts of MSC33c-conditioned medium (artificial seawater-based yeast extract) are statistically significantly higher (P < 0.05) than control counts. Negative controls are as for panel C. Six replicate wells were observed for each treatment. Error bars indicate standard deviations. See the parenthetical material at the end of the Fig. 1 legend on the nature of CFU in negative controls.
References
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