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Copyright © 2016 Thøgersen, Delpin, Melchiorsen, Kilstrup, Månsson, Bunk, Spröer, Overmann, Nielsen and Gram. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level as the wild type strain. The mutant strain killed V. anguillarum in co-culture experiments as efficiently as the wild type. Using UHPLC-UV/Vis analyses, we quantified violacein and indolmycin, and the mutant strain only produced 7–10% the amount of violacein compared to the wild type strain. In contrast, the amount of indolmycin produced by the mutant strain was about 300% that of the wild type. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea.

Antifouling strategies to limit biofilms on submerged surfaces in the marine environment are of particular interest due to the economic and environmental impacts in industries such as shipping and aquaculture. Here, we investigate the influence of chemically modified hessian bag surfaces on the bacterial abundance and community composition of biofilm formation using flow cytometry and 16S rRNA pyrosequencing. Hessian bags were coated with 5% and 10% Propyl(trimethoxy) silane (PTMS) and half of the bags had their lignin and hemicellulose removed via NaOH mercerisation. Significantly lower bacterial abundance was observed on mercerised bags treated with 5% PTMS (p < 0.01). Significant shifts in bacterial taxa were also observed (p = 0.0004), whereby unmercerised bags exhibited higher relative abundances of the anaerobic family Desulfovibrionaceae (4.5 +/- 1.7%), while mercerised bags displayed higher relative abundances of the aerobic family Phyllobacteriaceae (3.6 +/- 1.7%). This suggests that the mercerisation process may lower colonization rates and subsequently produce a thinner biofilm. This hypothesis is strengthened by the lower abundance of bacteria on mercerised bags, particularly on the 5% PTMS coating. Our results show that modifying a hessian surface via non-toxic coating and mercerisation reduces biofilm formation and also shifts the dominant taxa, increasing our understanding of antifouling strategies in the marine environment. (C) 2015 Elsevier B.V. All rights reserved. Refereed/Peer-reviewed

A metagenomic analysis of two aquifer systems located under a dairy farming region was performed to examine to what extent the composition and function of microbial communities varies between confined and surface-influenced unconfined groundwater ecosystems. A fundamental shift in taxa was seen with an overrepresentation of Rhodospirillales, Rhodocyclales, Chlorobia and Circovirus in the unconfined aquifer, while Deltaproteobacteria and Clostridiales were overrepresented in the confined aquifer. A relative overrepresentation of metabolic processes including antibiotic resistance (beta-lactamase genes), lactose and glucose utilization and DNA replication were observed in the unconfined aquifer, while flagella production, phosphate metabolism and starch uptake pathways were all overrepresented in the confined aquifer. These differences were likely driven by differences in the nutrient status and extent of exposure to contaminants of the two groundwater systems. However, when compared with freshwater, ocean, sediment and animal gut metagenomes, the unconfined and confined aquifers were taxonomically and metabolically more similar to each other than to any other environment. This suggests that intrinsic features of groundwater ecosystems, including low oxygen levels and a lack of sunlight, have provided specific niches for evolution to create unique microbial communities. Obtaining a broader understanding of the structure and function of microbial communities inhabiting different groundwater systems is particularly important given the increased need for managing groundwater reserves of potable water. Refereed/Peer-reviewed

Ammonium concentration and nitrogen source regulate promoter activity and use for the transcription of chiA, the major chitinase gene of Pseudoalteromonas sp. S91 and S91CX, an S91 transposon lacZ fusion mutant. The activity of chiA was quantified by beta-galactosidase assay of S91CX cultures containing different ammonium concentrations (NH4+; 0, 9.5 or 191 mM) and with different nitrogen sources (N-acetylglucosamine (GlcNAc) or glutamate (glt)). S91 chiA expression was found to depend on both the NH4+ concentration and source of nitrogen in marine minimal medium (MMM). Pseudoalteromonas sp. S91 and S91CX can use either GlcNAc or glt as a sole source of carbon in MMM containing a standard concentration of 9.5 mM NH4+. Adding excess NH4+, 20 times the standard concentration, to MMM significantly reduced chiA activity below that found in the presence of either GlcNAc or glt. When no NH4+ was added to MMM, S91CX was also able to use either GlcNAc or glt as a source of nitrogen; under these conditions chiA activity was significantly increased. Under all conditions tested, GlcNAc induced chiA activity significantly more than glt. Regulation of bacterial chitinases by nitrogen has not been previously reported. Transcriptional start point analysis of S91 chiA, using 5'RACE (ligation-anchored PCR), showed that during growth in MMM supplemented with (1) maltose (solely a carbon source for S91), chiA transcription occurred from only one putative sigma(70)-dependent promoter; (2) the chitin monomer GlcNAc, transcription initiated from two putative sigma(54)-dependent promoters and (3) glt, transcription initiated from all three putative promoters. The ISME Journal (2009) 3, 1064-1069; doi:10.1038/ismej.2009.49; published online 14 May 2009 Refereed/Peer-reviewed