Biofilm Interaction Mapping and Analysis (BIMA) of Interspecific Interactions in Pseudomonas Co-culture Biofilms.

Suzanne M Kosina,Romy Chakraborty,Jennifer J Zulovich,Grant M Zane,Adam M Deutschbauer,Marcin Zemla,Benjamin P Bowen,Adam P Arkin,Kelly M Wetmore,Judy D Wall,Peter Rademacher,Trent R Northen,Markus De Raad,Manfred Auer

doi:10.3389/fmicb.2021.757856

Abstract

Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing Pseudomonas stutzeri have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of P. stutzeri biofilms, in combination with consortia-based approaches, may offer advantages for these processes. Understanding the interspecific interaction within biofilm co-cultures or consortia provides a means for improvement of current technologies. However, the investigation of biofilm-based consortia has been limited. We present an adaptable and scalable method for the analysis of macroscopic interactions (colony morphology, inhibition, and invasion) between colony-forming bacterial strains using an automated printing method followed by analysis of the genes and metabolites involved in the interactions. Using Biofilm Interaction Mapping and Analysis (BIMA), these interactions were investigated between P. stutzeri strain RCH2, a denitrifier isolated from chromium (VI) contaminated soil, and 13 other species of pseudomonas isolated from non-contaminated soil. One interaction partner, Pseudomonas fluorescens N1B4 was selected for mutant fitness profiling of a DNA-barcoded mutant library; with this approach four genes of importance were identified and the effects on interactions were evaluated with deletion mutants and mass spectrometry based metabolomics.

Highlights

Consortia based systems in biotechnologies are widespread, controlling them is challenging due to the genomic and metabolomic complexities of the interactions
As the first step of Biofilm Interaction Mapping and Analysis (BIMA), a colony printing method was developed on an automated liquid handling system for the purpose of scalability and transferability between labs
Biofilm formation and morphology in pseudomonas appears to be regulated by a combination of biotic factors including, but not limited to: phenazines, exopolysaccharide production, signaling molecules and flagellar activity (Merritt et al, 2010)

Summary

Introduction

Consortia based systems in biotechnologies are widespread, controlling them is challenging due to the genomic and metabolomic complexities of the interactions. Characterization of the genes and metabolites involved in the interactions opens up the possibility for improved consortia functionality by use of engineered strains and culture condition metabolite amendments. The ability to incorporate genomics into these types of approaches will allow for a better understanding of the interactions involved. Next-generation sequencing enables rapid profiling of the abundance of barcodes mapped to specific genes in transposon mutant libraries under a wide range of environmental conditions (Wetmore et al, 2015; Price et al, 2018) and when integrated with metabolomics, provides rapid functional assignment of transport and metabolic processes (Baran et al, 2009) important in microbial interactions

Methods

Results

Conclusion