Exploring the Potential of Micrococcus luteus Culture Supernatant With Resuscitation-Promoting Factor for Enhancing the Culturability of Soil Bacteria

Abstract

A bacterial species is best characterized after its isolation in a pure culture. This is an arduous endeavor for many soil microorganisms, but it can be simplified by several techniques for improving culturability: for example, by using growth-promoting factors. We investigated the potential of a Micrococcus luteus culture supernatant containing resuscitation-promoting factor (SRpf) to increase the number and diversity of cultured bacterial taxa from a nutrient-rich compost soil. Phosphate-buffered saline and inactivated SRpf were included as controls. After agitation with SRpf at 28°C for 1 day, the soil suspension was diluted and plated on two different solid, oligotrophic media: tenfold diluted Reasoner’s 2A agar (R2A) and soil extract-based agar (SA). Colonies were collected from the plates to assess the differences in diversity between different treatments and cultivation media. The diversity on both R2A and SA was higher in the SRpf-amended extracts than the controls, but the differences on R2A were higher. Importantly, 51 potentially novel bacterial species were isolated on R2A and SA after SRpf treatment. Diversity in the soil extracts was also determined by high-throughput 16S rRNA amplicon sequencing, which showed an increase in the abundance of specific taxa before their successful cultivation. Conclusively, SRpf can effectively enhance the growth of soil bacterial species, including those hitherto uncultured.

Pedomonas mirosovicensis gen. nov., sp. nov., a bacterium isolated from soil with the aid of Micrococcus luteus culture supernatant containing resuscitation-promoting factor

“…To conduct the phylogenetic analysis, DNA was extracted from the strain growing in 0.5×LB using a PureLink Genomic DNA Mini Kit (Invitrogen), following the manufacturer's instructions. A nearly complete 16S rRNA gene was amplified and sequenced commercially using the primers: 8 forward (5' -AGAGTTTGATCMTGGCTCAG-3′) and 1509 reverse (5′-GYTACCTTGT-TACGACTT-3′) [7], as described elsewhere [5]. The resulting 16S rRNA gene sequence was input into EzBioCloud's 16S rRNA gene database to obtain closely related species [8].…”

“…Strain A1X5R2 T was isolated thanks to an effort to increase the culturability of yet-uncultured soil bacteria using Micrococcus luteus NCTC 2665 T supernatant containing a resuscitation-promoting factor [5]. Before its isolation, the 16S rRNA gene sequence of strain A1X5R2 T was listed in the silva reference database as the unknown genus DSSF69 [5, 6].…”

“…Some other common features are the presence of Q-10 as the major ubiquinone; phosphatidylethanolamine, phosphatidylglycerol or diphosphatidylglycerol as the major polar lipids, and C 18 : 1 ω7c, C 16 : 0 and C 16 : 1 ω7c as the major fatty acids [1]. Strain A1X5R2 T was isolated thanks to an effort to increase the culturability of yet-uncultured soil bacteria using Micrococcus luteus NCTC 2665 T supernatant containing a resuscitation-promoting factor [5]. Before its isolation, the 16S rRNA gene sequence of strain A1X5R2 T was listed in the silva reference database as the unknown genus DSSF69 [5,6].…”

Culturomics of Bacteria from Radon-Saturated Water of the World’s Oldest Radium Mine

“…Due to unusual metabolic pathways, microorganisms isolated from extreme habitats are often used as biocatalysts or as a source of extremozymes and extremolytes ( 16 ). However, the isolation of such microorganisms is significantly complicated, for example, by the dormant state in which most of these microorganisms occur ( 17 ). Endeavoring to increase cultivation yields, several cultivation improvements have been implemented in methodical procedures, ranging from the 'provision of basic and specific needs' such as the addition of essential substances (nutrients, trace elements), growth factors such as Rpf ( 17 , 18 ), cAMP, and homoserine lactones ( 19 ); through the usage of high-tech isolating devices such as micromanipulation equipment ( 20 , 21 ), streaking pens ( 22 ) or ichips ( 23 ); to the application of knowledge gathered from metagenomic ( 24 ) and metatranscriptomic ( 25 ) analyses.…”

“…However, the isolation of such microorganisms is significantly complicated, for example, by the dormant state in which most of these microorganisms occur ( 17 ). Endeavoring to increase cultivation yields, several cultivation improvements have been implemented in methodical procedures, ranging from the 'provision of basic and specific needs' such as the addition of essential substances (nutrients, trace elements), growth factors such as Rpf ( 17 , 18 ), cAMP, and homoserine lactones ( 19 ); through the usage of high-tech isolating devices such as micromanipulation equipment ( 20 , 21 ), streaking pens ( 22 ) or ichips ( 23 ); to the application of knowledge gathered from metagenomic ( 24 ) and metatranscriptomic ( 25 ) analyses. It is not only sophisticated cultivation approaches that have borne fruit, but also simple cultivation strategies such as the application of oligotrophic media, possibly based directly on the environmental matrix and/or in combination with a culture temperature relevant to the environment from which the microbe is isolated ( 26 – 28 ).…”