A three-step method for analysing bacterial biofilm formation under continuous medium flow

Abstract

For the investigation and comparison of microbial biofilms, a variety of analytical methods have been established, all focusing on different growth stages and application areas of biofilms. In this study, a novel quantitative assay for analysing biofilm maturation under the influence of continuous flow conditions was developed using the interesting biocatalyst Pseudomonas taiwanensis VLB120. In contrast to other tubular-based assay systems, this novel assay format delivers three readouts using a single setup in a total assay time of 40 h. It combines morphotype analysis of biofilm colonies with the direct quantification of biofilm biomass and pellicle formation on an air/liquid interphase. Applying the Tube-Assay, the impact of the second messenger cyclic diguanylate on biofilm formation of P. taiwanensis VLB120 was investigated. To this end, 41 deletions of genes encoding for protein homologues to diguanylate cyclase and phosphodiesterase were generated in the genome of P. taiwanensis VLB120. Subsequently, the biofilm formation of the resulting mutants was analysed using the Tube-Assay. In more than 60 % of the mutants, a significantly altered biofilm formation as compared to the parent strain was detected. Furthermore, the potential of the proposed Tube-Assay was validated by investigating the biofilms of several other bacterial species.

Trophic regulation of autoaggregation in Pseudomonas taiwanensis VLB120

“…1a, b). In P. taiwanensis VLB120ΔCeGFP Δ04710 and Δ05695, site-specific deletions affected genes which encode for proteins carrying a GGDEF and EAL domain (Schmutzler et al 2015). The scenario that a GGDEF domain that is responsible for c-di-GMP synthesis and an EAL domain that degrades c-di-GMP are arranged in tandem in the same enzyme is very common, but frequently, one of the two domains is catalytically inactive (Roemling et al 2013).…”

“…The five P. taiwanensis VLB120ΔCeGFP mutants showing an autoaggregative behaviour were constructed using two different methods. P. t a i w a n e n s i s V L B 1 2 0 Δ C e G F P Δ 0 4 7 1 0 a n d P. taiwanensis VLB120ΔCeGFP Δ05695 were generated in a previous study (Schmutzler et al 2015) using a markerless gene deletion protocol (Martinez-Garcia and de Lorenzo 2011) which produces site-specific in-frame deletions.…”

Continuous multistep synthesis of perillic acid from limonene by catalytic biofilms under segmented flow

“…End‐product toxification was overcome by solid‐liquid extraction of perillic acid using an anion exchange resin (Mirata et al, ). We developed a biofilm based process concept as an alternative strategy for overcoming toxicity limitations by exploiting the catalytic performance of the native strain P. putida GS1 and the likewise solvent‐tolerant recombinant P. taiwanensis VLB120 which has been extensively studied for biofilm formation and fine‐chemical synthesis (Gross et al, , ; Karande et al, ; Schmutzler et al, ). This optimized process stability during perillic acid production and resulted in the continuous production of perillic acid as the sole oxidation product.…”

Hyperadherence of Pseudomonas taiwanensis VLB120ΔC increases productivity of (S)‐styrene oxide formation

“…In this work, the problem of weak initial attachment forces was addressed by applying a hyperadherent mutant of the ( S )‐styrene oxide production strain P. taiwanensis VLB120ΔCeGFP. The variant P. taiwanensis VLB120ΔCeGFP Δ04710 possessing a genetically engineered cyclic diguanylate (c‐di‐GMP) metabolism (Schmutzler et al ., , ) carried a deletion of a phosphodiesterase highly similar to BifA of other pseudomonads (Kuchma et al ., , ; Jiménez‐Fernández et al ., ). This mutant developed macroscopic aggregates in dependency of the supplied carbon source.…”

“…This mutant developed macroscopic aggregates in dependency of the supplied carbon source. In minimal medium supplemented with carbohydrates or gluconate the variant showed heavy autoaggregation with enhanced adhesiveness of the cells due to excreted EPS material and/or enhanced cell surface hydrophobicity compared with the parent strain (Schmutzler et al ., , ). The attachment behaviour of the autoaggregates to silicone rubber and the biofilm formation of P. taiwanensis VLB120ΔCeGFP Δ04710 were studied in detail.…”