BackgroundBioinformatic analysis of the genes coding for the chitinase in Pyrococcus furiosus and Thermococcus kodakarensis revealed that most likely a one nucleotide insertion in Pyrococcus caused a frame shift in the chitinase gene. This splits the enzyme into two separate genes, PF1233 and PF1234, in comparison to Thermococcus kodakarensis. Furthermore, our attempts to grow the wild type strain of Pyrococcus furiosus on chitin were negative. From these data we assume that Pyrococcus furiosus is most likely unable to use chitin as a carbon source. The aim of this study was to analyze in vivo if the one nucleotide insertion is responsible for the inability to grow on chitin, using a recently described genetic system for Pyrococcus furiosus.ResultsA marker-less genetic system for Pyrococcus furiosus was developed using simvastatin for positive selection and 6-methylpurine for negative selection. Resistance against simvastatin was achieved by overexpression of the hydroxymethylglutaryl coenzyme A reductase gene. For the resistance to 6-methylpurine the hypoxanthine-guanine phosphoribosyltransferase gene was deleted. This system was used to delete the additional nucleotide at position 1006 in PF1234. The resulting chitinase in the mutant strain was a single subunit enzyme and aligns perfectly to the enzyme from Thermococcus kodakarensis. A detailed analysis of the wild type and the mutant using counted cell numbers as well as ATP and acetate production as growth indicators revealed that only the mutant is able to use chitin as a carbon source. An additional mutant strain containing a reduced chitinase version containing just one catalytic and one chitin-binding domain showed diminished growth on chitin in comparison to the mutant containing the single large enzyme.ConclusionsWild type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. The overall high sequence identity of the two chitinases between P. furiosus and T. kodakarensis indicates that this mutation occurred very recently or there is still some kind of selection pressure for a functional enzyme using programmed +/−1 frameshifting.

Five mutants of Pseudomonas taiwanensis VLB120ΔCeGFP showed significant autoaggregation when growing on defined carbohydrates or gluconate, while they grew as suspended cells on complex medium and on organic acids like citrate and succinate. Surprisingly, the respective mutations affected very different genes, although all five strains exhibited the same behaviour of aggregate formation. To elucidate the mechanism of the aggregative behaviour, the microbial adhesion to hydrocarbons (MATH) assay and contact angle measurements were performed that pointed to an increased cell surface hydrophobicity. Moreover, investigations of the outer layer of the cell membrane revealed a reduced amount of O-specific polysaccharides in the lipopolysaccharide of the mutant cells. To determine the regulation of the aggregation, reverse transcription quantitative real-time PCR was performed and, irrespective of the mutation, the transcription of a gene encoding a putative phosphodiesterase, which is degrading the global second messenger cyclic diguanylate, was decreased or even deactivated in all mutants. In summary, it appears that the trophic autoaggregation was regulated via cyclic diguanylate and a link between the cellular cyclic diguanylate concentration and the lipopolysaccharide composition of P. taiwanensis VLB120ΔCeGFP is suggested.

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.

SummaryThe attachment strength of biofilm microbes is responsible for the adherence of the cells to surfaces and thus is a critical parameter in biofilm processes. In tubular microreactors, aqueous‐air segmented flow ensures an optimal oxygen supply and prevents excessive biofilm growth. However, organisms growing in these systems depend on an adaptation phase of several days, before mature and strong biofilms can develop. This is due to strong interfacial forces. In this study, a hyperadherent mutant of Pseudomonas taiwanensis VLB120ΔCeGFP possessing an engineered cyclic diguanylate metabolism, was applied to a continuous biofilm process for the production of (S)‐styrene oxide. Cells of the mutant P.  taiwanensis VLB120ΔCeGFP Δ04710, showing the same specific activity as the wild type, adhered substantially stronger to the substratum. Adaptation to the high interfacial forces was not necessary in these cases. Thereby, 40% higher final product concentrations were achieved and the maximal volumetric productivity of the parent strain was significantly surpassed by P. taiwanensis VLB120ΔCeGFP Δ04710. Applying mutants with strong adhesion in biofilm‐based catalysis opens the door to biological process control in future applications of catalytic biofilms using other industrially relevant strains.