1One hundred one antibacterial Pseudoalteromonas strains that inhibited growth of a Vibrio 2 anguillarum test strain were collected on a global research cruise (Galathea 3), and 51 of the strains 3 repeatedly demonstrated antibacterial activity. Here, we profile secondary metabolites of these 4 strains to determine if particular compounds serve as strain or species markers and to determine if 5 the secondary metabolite profile of one strain represents the bioactivity of the entire species. 16S 6 rRNA gene similarity divided the strains into two primary groups: One group (51 strains) consisted 7 of bacteria which retained antibacterial activity, 48 of which were pigmented, and another group (50 8 strains) of bacteria which lost antibacterial activity upon sub-culturing, two of which were 9pigmented. The group that retained antibacterial activity consisted of six clusters in which strains 10 were identified as Pseudoalteromonas luteoviolacea, Pseudoalteromonas aurantia, 11 Pseudoalteromonas phenolica, Pseudoalteromonas ruthenica, Pseudoalteromonas rubra and 12Pseudoalteromonas piscicida. HPLC-UV/VIS analyses identified key peaks, such as violacein in P. 13 luteoviolacea. Some compounds, such as a novel bromoalterochromide were detected in several 14 species. HPLC-UV/VIS detected systematic intra-species differences for some groups and testing 15 several strains of a species was required to determine these differences. The majority of non-16 antibacterial, non-pigmented strains were identified as Pseudoalteromonas agarivorans and HPLC-17 UV/VIS did not further differentiate this group. Pseudoalteromonas retaining antibacterial were 18 more likely to originate from biotic or abiotic surfaces in contrast to planktonic strains. Hence, the 19 pigmented, antibacterial Pseudoalteromonas have a niche specificity, and sampling from marine 20 biofilm environments is a strategy for isolating novel marine bacteria that produce antibacterial 21 compounds. 22 23Words: 252 24 25

We here combine chemical analysis and genomics to probe for new bioactive secondary metabolites based on their pattern of distribution within bacterial species. We demonstrate the usefulness of this combined approach in a group of marine Gram-negative bacteria closely related to Pseudoalteromonas luteoviolacea, which is a species known to produce a broad spectrum of chemicals. The approach allowed us to identify new antibiotics and their associated biosynthetic pathways. Combining chemical analysis and genetics is an efficient “mining” workflow for identifying diverse pharmaceutical candidates in a broad range of microorganisms and therefore of great use in bioprospecting.

Some microbial species are chemically homogenous, and the same secondary metabolites are found in all strains. In contrast, we previously found that five strains of P. luteoviolacea were closely related by 16S rRNA gene sequence but produced two different antibiotic profiles. The purpose of the present study was to determine whether such bioactivity differences could be linked to genotypes allowing methods from phylogenetic analysis to aid in selection of strains for biodiscovery. Thirteen P. luteoviolacea strains divided into three chemotypes based on production of known antibiotics and four antibacterial profiles based on inhibition assays against Vibrio anguillarum and Staphylococcus aureus. To determine whether chemotype and inhibition profile are reflected by phylogenetic clustering we sequenced 16S rRNA, gyrB and recA genes. Clustering based on 16S rRNA gene sequences alone showed little correlation to chemotypes and inhibition profiles, while clustering based on concatenated 16S rRNA, gyrB, and recA gene sequences resulted in three clusters, two of which uniformly consisted of strains of identical chemotype and inhibition profile. A major time sink in natural products discovery is the effort spent rediscovering known compounds, and this study indicates that phylogeny clustering of bioactive species has the potential to be a useful dereplication tool in biodiscovery efforts.

Many species of marine bacteria elicit a weak immune response. In this study, the aim was to assess the immunomodulatory properties of Gram-negative Pseudoalteromonas strains compared with other marine Gram-negative bacteria and to identify the molecular cause of the immunomodulation. Using murine bone-marrow derived dendritic cells (DCs), it was found that Pseudoalteromonas strains induced low cytokine production and modest up-regulation of surface markers CD40 and CD86 compared with other marine bacteria and Escherichia coli LPS. Two strains, Ps. luteoviolacea and Ps. ruthenica, were further investigated with respect to their immunomodulatory properties in DCs. Both inhibited IL-12 and increased IL-10 production induced by E. coli LPS. LPS isolated from the two Pseudoalteromonas strains had characteristic lipid A bands in SDS-PAGE. Stimulation of HEK293 TLR4/MD2 cells with the isolated LPS confirmed the involvement of LPS and TLR4 and established Pseudoalteromonas LPS as TLR4 antagonists. The isolated LPS was active in the endotoxin limulus amoebocyte lysate assay and capable of inducing increased endocytosis in DCs. This study highlights that antagonistic LPS from Pseudoalteromonas strains has potential as a new candidate of therapeutic agent capable of modulating immune responses.

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