Interspecies bacterial interactions stabilize transcriptional responses of ancestral wild types and biofilm-optimised variants
Authors: Burmølle Mette , Olsen Nanna Mee Coops , Jacquiod Samuel , Røder Henriette Lyng 
Published: July 2020

&lt;p&gt;Most bacteria in natural environments live in multispecies biofilms, featuring high diversity and chemical heterogeneity. The cell-to-cell proximity found in these biofilms results in biotic interactions and niche-partitioning, facilitating co-existence of species that may otherwise out-compete each other. Additionally, due to the fast generation time of microbes and ceaseless biotic interactions, biofilms accelerate adaptation through the emergence of more fit genetic variants, most probably in response to niche-partitioning and local constraints. We have previously isolated and characterized biofilm-optimised (wrinkled) variants of &lt;em&gt;Xanthomonas retroflexus&lt;/em&gt;. These variants emerged in biofilm co-cultures with &lt;em&gt;Paenibacillus amylolyticus&lt;/em&gt; and reinforced the original interspecific mutualistic interaction, due to altered c-di-GMP regulation and spatial organisation.&lt;/p&gt;
&lt;p&gt;The aim of the present study was to examine the impact on gene expression profiles of either co-cultivation of the wild type (WT) or the wrinkled variant &lt;em&gt;X. retroflexus&lt;/em&gt; with &lt;em&gt;P. amylolyticus&lt;/em&gt; or its supernatant. We hypothesised that the gene expression of the two &lt;em&gt;X. retroflexus&lt;/em&gt; strains would differ significantly and that these differences would be even more pronounced when co-cultured with &lt;em&gt;P. amylolyticus&lt;/em&gt; or its supernatant.&lt;/p&gt;
&lt;p&gt;Mono- and dual species biofilms were grown in 24-well plates for 24 h. The liquid culture was removed, and the remaining biofilm from the sides of the wells and the air-liquid interface was sampled and processed for mRNA sequencing. After sequencing, &lt;em&gt;X. retroflexus&amp;#160;&lt;/em&gt;reads were mapped against its concatenated genome and genes of which expression differed by fold changes of log2 &lt;-1 and &gt;1 were considered differentially expressed.&lt;/p&gt;
&lt;p&gt;Unexpectedly, most marked differences in gene expression were observed when comparing mono-cultures of the WT and the wrinkled &lt;em&gt;X. retroflexus&lt;/em&gt;, as approximately 500 genes were differentially expressed in these biofilms. Of these, 30 genes were predicted to encode biofilm-associated functions. When exposed to either live &lt;em&gt;P. amylolyticus&lt;/em&gt; or its supernatant, expression profiles of the WT and the wrinkled variant were more similar, with the living partner &lt;em&gt;P. amylolyticus&lt;/em&gt; being the key factor of this stabilization. Specifically, the stabilisation was caused by opposite regulation of specific genes in the wrinkled &lt;em&gt;X. retroflexus&amp;#160;&lt;/em&gt;variant compared to the WT in mono- vs. co-culture conditions.&lt;/p&gt;
&lt;p&gt;In conclusion, our data indicates that differences in gene expression of &lt;em&gt;X. retroflexus&lt;/em&gt; WT and the biofilm-optimised variant were neutralised by co-cultivation with &lt;em&gt;P. amylolyticus&lt;/em&gt;. To our knowledge, such comparative analyses of ancestral and biofilm-optimised variants have not previously been presented, despite being instrumental in elucidating evolutionary trajectories of such variants in complex environments.&lt;/p&gt;