Joining the bacterial conversation: increasing the cultivation efficiency of soil bacteria with acyl-homoserine lactones and cAMP

Abstract

ABSTRACT Bacteria need to be isolated in pure culture to gain access to the wide array of interesting functions they conceal. This is challenging because they do not live in isolation but instead in communities where they actively communicate and interact with each other. In this study, we aimed to increase the culturability of soil bacteria using three different signaling molecules: N-(3-oxohexanoyl)-L-homoserine lactone, N-octanoyl-L-homoserine lactone, and 3',5'-cyclic adenosine monophosphate (cAMP). The signals were added individually to soils suspended in PBS buffer to a final concentration of 5 µM. Soil suspensions were agitated for 24 hours, after which they were serially diluted and plated on tenfold-diluted Reasoner’s 2A agar that either contained or did not contain the same signaling molecule used during the extraction step. DNA was isolated from both soil suspensions and grown cultures and, after a high-throughput amplicon sequencing, differences in bacterial abundances and diversity were determined across treatments. To further explain the action of the signaling molecules on the treated soil communities, their metagenomic functions were predicted using the software PICRUSt2. N-octanoyl-L-homoserine lactone was found to increase the diversity observed on solid media, while N-(3-oxohexanoyl)-L-homoserine lactone and cAMP were not. Potentially novel isolates aided by the signaling molecules were affiliated with the genera Pseudomonas and Nocardioides. IMPORTANCE Microorganisms are a repository of interesting metabolites and functions. Therefore, accessing them is an important exercise for advancing not only basic questions about their physiology but also to advance technological applications. In this sense, increasing the culturability of environmental microorganisms remains an important endeavor for modern microbiology. Because microorganisms do not live in isolation in their environments, molecules can be added to the cultivation strategies to “inform them” that they are present in growth-permissive environmental conditions. Signaling molecules such as acyl-homoserine lactones and 3',5'-cyclic adenosine monophosphate belong to the plethora of molecules used by bacteria to communicate with each other in a phenomenon called quorum sensing. Therefore, including quorum sensing molecules can be an incentive for microorganisms, specifically soil bacteria, to increase their numbers on solid media.

Keywords

• signaling molecules
• acyl-homoserine lactones
• cAMP
• non-culturable bacteria
• increased culturability
• oligotrophic medium