The use of biomimetic surfaces to reduce single- and dual-species biofilms of Escherichia coli and Pseudomonas putida.

The ability of bacteria to adhere to and form biofilms on food contact surfaces poses serious challenges, as these may lead to the cross-contamination of food products. Biomimetic topographic surface modifications have been explored to enhance the antifouling performance of materials. In this study, the topography of two plant leaves, Brassica oleracea var. botrytis (cauliflower, CF) and Brassica oleracea capitate (white cabbage, WC), was replicated through wax moulding, and their antibiofilm potential was tested against single- and dual-species biofilms of Escherichia coli and Pseudomonas putida. Biomimetic surfaces exhibited higher roughness values (SaWC = 4.0 ± 1.0 µm and SaCF = 3.3 ± 1.0 µm) than the flat control (SaF = 0.6 ± 0.2 µm), whilst the CF surface demonstrated a lower interfacial free energy (ΔGiwi) than the WC surface (-100.08 mJ m-2 and -71.98 mJ m-2, respectively). The CF and WC surfaces had similar antibiofilm effects against single-species biofilms, achieving cell reductions of approximately 50% and 60% for E. coli and P. putida, respectively, compared to the control. Additionally, the biomimetic surfaces led to reductions of up to 60% in biovolume, 45% in thickness, and 60% in the surface coverage of single-species biofilms. For dual-species biofilms, only the E. coli strain growing on the WC surface exhibited a significant decrease in the cell count. However, confocal microscopy analysis revealed a 60% reduction in the total biovolume and surface coverage of mixed biofilms developed on both biomimetic surfaces. Furthermore, dual-species biofilms were mainly composed of P. putida, which reduced E. coli growth. Altogether, these results demonstrate that the surface properties of CF and WC biomimetic surfaces have the potential for reducing biofilm formation.

Flagellar interference with plasmid uptake in biofilms: a joint experimental and modeling study.

Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated fliM mutants of the X. retroflexus wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of Pseudomonas putida suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, Xanthomonas retroflexus and Pseudomonas putida. Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.

Draft genomes of seven isolates from Danish wastewater facilities belonging to Pseudomonas, Bacillus, Pseudochrobactrum, Brevundimonas, and Pandoraea.

We report here seven draft genomes of bacterial strains from two Danish wastewater facilities, two of which might be characterized as a new group within the Pseudomonas and Pseudochrobactrum genera, respectively. These genomes will provide useful references for understanding bacterial interactions and horizontal gene transfer within bacterial communities.

A Pseudomonas putida cbrB transposon insertion mutant displays a biofilm hyperproducing phenotype that is resistant to dispersal.

The CbrAB two-component system in the Pseudomonads controls a variety of metabolic and behavioural traits required for its adaptation to changing environmental conditions, including the uptake or assimilation of certain carbon sources, and processes such as chemotaxis or stress tolerance. In this work we characterize a miniTn5-luxAB-Km transposon insertion mutant in cbrB (MPO406) in Pseudomonas putida leading to a biofilm overproducing phenotype that is not dispersed when nutrients are depleted. Comparison with a cbrB deletion mutant revealed that all phenotypes previously attributed to CbrB in P. putida correlated in both strains, with the exception of biofilm overproduction and absence of dispersal. We show that in the insertion mutant, the expression of the downstream regulatory RNA CrcZ is upregulated, and also show the presence of a truncated form of CbrB. Also, two additional point mutations in lapG and lapD have been detected in MPO406 by whole genome sequencing. Combination of these effects provides a robust biofilm overproducing phenotype. We present the mutant strain MPO406 as a good candidate to perform bio-production of substances of biotechnological interest or other processes such as bioremediation, which take advantage of immobilized cells on solid surfaces.

Transcriptional activation of the CrcZ and CrcY regulatory RNAs by the CbrB response regulator in Pseudomonas putida.

The CbrAB two-component system has been described as a high-ranked element in the regulatory hierarchy of Pseudomonas putida that controls a variety of metabolic and behavioural traits required for adaptation to changing environmental conditions. We show that the response regulatory protein CbrB, an activator of σ(N) -dependent promoters, directly controls the expression of the small RNAs CrcZ and CrcY in P. putida. These two RNAs sequester the protein Crc, which is a translational repressor of multiple pathways linked to carbon catabolite repression. We characterized the in vivo and in vitro activation by CbrB at both crcZ and crcY promoters, and identified new DNA sequences where the protein binds. IHF, a co-activator at many σ(N) -dependent promoters, also binds to the promoter regions and contributes to the activation of the sRNAs. CbrB phosphorylation is necessary at physiological activation conditions, but a higher dose of the protein allows in vitro transcriptional activation in its non-phosphorylated form. We also show there is some production of CrcY coming from an upstream promoter independent of CbrB. Thus, CbrAB constitute a global signal transduction pathway integrated in a higher regulatory network that also controls catabolite repression through the expression of the two regulatory RNAs CrcZ and CrcY.

Lack of CbrB in Pseudomonas putida affects not only amino acids metabolism but also different stress responses and biofilm development.

The CbrAB two-component system has been described in certain species of Pseudomonads as a global regulatory system required for the assimilation of several amino acids (e.g. histidine, proline or arginine) as carbon or carbon and nitrogen sources. In this work, we used global gene expression and phenotypic analyses to characterize the roles of the CbrAB system in Pseudomonas putida. Our results show that CbrB is involved in coordination with the nitrogen control system activator, NtrC, in the uptake and assimilation of several amino acids. In addition, CbrB affects other carbon utilization pathways and a number of apparently unrelated functions, such as chemotaxis, stress tolerance and biofilm development. Based on these new findings, we propose that CbrB is a high-ranked element in the regulatory hierarchy of P. putida that directly or indirectly controls a variety of metabolic and behavioural traits required for adaptation to changing environmental conditions.