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The Roseobacter-group bacterium Phaeobacter as safe probiotic solution for aquaculture
Sonnenschein, Eva C.; Jimenez, Guillermo; Castex, Mathieu; Gram, Lone
Published in: Applied and Environmental Microbiology
Link to article, DOI: 10.1128/AEM.02581-20
Publication date: 2021
Document Version Peer reviewed version
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Citation (APA): Sonnenschein, E. C., Jimenez, G., Castex, M., & Gram, L. (2021). The Roseobacter-group bacterium Phaeobacter as safe probiotic solution for aquaculture. Applied and Environmental Microbiology, 87(5), [e02581- 20]. https://doi.org/10.1128/AEM.02581-20
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1 The Roseobacter-group bacterium Phaeobacter as safe probiotic solution for aquaculture
2 Eva C. Sonnenschein1*, Guillermo Jimenez2, Mathieu Castex2, Lone Gram1
3 1 Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts
4 Plads Bldg. 221, 2800 Kgs. Lyngby, Denmark.
5 2 Lallemand SAS, Blagnac Cedex, France.
6 * corresponding author e-mail: [email protected]
7 Abstract
8 Phaeobacter inhibens has been assessed as a probiotic bacterium for application in aquaculture.
9 Studies addressing the efficacy and safety indicate that P. inhibens maintains it antagonistic
10 activity against pathogenic vibrios in aquaculture live cultures (live feed and fish egg/larvae),
11 while having no or a positive effect on the host organisms, and a minor impact on the host
12 microbiomes. While producing antibacterial and algicidal compounds, no study has so far found
13 a virulent phenotype of P. inhibens cells against higher organisms. Additionally, an in silico
14 search for antibiotic resistance genes using published genomes of representative strains did not
15 raise concern regarding the risk for antimicrobial resistance. P. inhibens occurs naturally in
16 aquaculture systems supporting its safe usage in this environment. Concluding, at the current
17 state of knowledge, P. inhibens is a “safe-to-use” organism.
18
19 INTRODUCTION
20 Phaeobacter inhibens is a marine alphaproteobacterium with potential for application as a
21 probiotic bacterium in marine aquaculture systems. P. inhibens is a representative of the
22 Roseobacter group that is widespread and an environmentally important marine group of
1
23 bacteria. P. inhibens serves as a heterotrophic marine model organism to understand fundamental
24 processes such as adaptation to an attached lifestyle and interactions with higher organisms (1).
25 Its probiotic activity against fish pathogenic bacteria is primarily due to the production of the
26 antibacterial compound tropodithietic acid (TDA) (2). TDA is bacteriocidal to both fish and
27 human bacterial pathogens. The applicability of P. inhibens as a probiotic bacterium in marine
28 aquaculture has been assessed in several studies and has recently been compared to that of other
29 probiotics (3). The bacterium is naturally present in aquaculture systems and it is able to inhibit
30 fish pathogenic bacteria. Most studies have been performed on its activity against economically
31 important Vibrio spp. such as V. anguillarum and V. vulnificus, but it also inhibits other fish
32 pathogens such as Aeromonas and Tenacibaculum spp. (4–7). This is seen both in laboratory
33 based agar-assays, and in live feed used in marine larval rearing. The ability to inhibit fish
34 pathogenic Vibrio has been demonstrated in both axenic and non-axenic live feed systems (6, 8–
35 11). Also, in model challenge trials, P. inhibens can decrease the mortality of fish larvae
36 challenged with pathogenic Vibrio (10).
37 Farming of fish and shellfish is of great importance to supply protein for food and feed for the
38 growing world population. Catches from wild fish have stagnated (or even declined) since the
39 mid 1980s and the increase in fish production comes almost exclusively from aquaculture (12). In
40 intense animal rearing systems, infectious agents such as pathogenic bacteria spread rapidly and
41 one particularly sensitive stage is the larval development. Fish larvae do not have a developed
42 immune system and vaccination can therefore not be used as disease preventive measure (13).
43 During larval rearing, opportunistic and pathogenic bacteria are easily introduced via live feed
44 and infections can spread rapidly, eradicating the complete larval batch (14). Antibiotics have
45 been used to control these infections, however due to the risk of bacteria developing and
46 spreading antibiotic resistance, other measures must be found. One strategy for limiting the
2
47 proliferation of bacterial pathogens in live feed and fish larvae is the use of probiotic bacteria
48 (15). Probiotic microorganisms have been defined by FAO and WHO (16) as “live
49 microorganisms which when administered in adequate amounts confer a health benefit on the
50 host”. This approach presents an environmentally sustainable and economically viable solution to
51 counteract the economic loss caused by bacterial pathogens in aquaculture systems.
52
53 P. inhibens has been shown to antagonize many fish pathogenic bacteria such as Vibrio spp. (6,
54 7). In the laboratory, resistance to the effector molecule, TDA, could not be selected for in
55 pathogenic bacteria, likely because the molecule as an antiporter destabilizes the bacterial proton
56 motive force (17, 18). Interestingly, despite this broad range effect, addition of the bacterium to
57 live feed only causes minor changes in the microbiome of the live feed (19). A search in the
58 whole genome sequence of P. inhibens DSM 17395 for the presence of known antimicrobial
59 resistance (AMR) genes to antimicrobials relevant to their use in humans and animals was
60 performed, as indicated in the European Food Safety Authority (EFSA) Guidance on the
61 characterization of microorganisms used as feed additives or as production organisms (EFSA,
62 2018). For this purpose, a comparison against up-to-date databases (e.g. ARG-ANNOT, CARD
63 and ResFinder) was performed. This article provides an overview of the genetics and physiology
64 of P. inhibens and summarizes the current information on the safety of P. inhibens for its use as a
65 probiotic in aquaculture.
66
67 TAXONOMIC CLASSIFICATION, PHENOTYPE AND GENETIC DIVERSITY OF P.
68 INHIBENS
69 The obligate marine genus Phaeobacter belongs to the Roseobacter group, the marine subgroup
70 within the family Rhodobacteraceae (20). Due to the ease at which Rhodobacteraceae can be
3
71 cultured and the prevalence of this family in marine ecosystems, today, 181 different genera have
72 been reported (according to NCBI taxonomy); however, over the recent years, there has also been
73 several drastic reclassifications at genus and species level (21, 22). Currently, the Phaeobacter
74 genus comprises six species: P. gallaeciencis (21), P. inhibens (21), P. marinintestinus (23), P.
75 piscinae (24), P. porticola (25), and P. italicus (26) (Fig. 1). However the phylogenetic diversity
76 within the genus is low (22) and species level distinction based on 16S rRNA gene sequences is
77 difficult (27). In particular, differentiating between P. inhibens, P. gallaeciensis and P. piscinae
78 is challenging (24). When comparing the whole nucleotide data per genome using the average
79 nucleotide identity, P. piscinae (89.7%) and P. gallaeciensis (89.3%) are closest related to P.
80 inhibens (Fig. 1). P. porticola (84.5%) and P. italicus (78.6%) are less similar to P. inhibens.
81
82 The species Phaeobacter inhibens with the type strain DSM 16374T (also named LMG 22475T or
83 T5T) was described in 2006 as a reclassification of the species Roseobacter gallaeciensis (21).
84 Additional well-characterized strains include DSM 17395 (initially isolated as type strain of P.
85 gallaeciensis BS107; (28, 29)) and 2.10 (also named DSM 24588) (30). The strain DSM 17395
86 was considered identical with the strain CIP 105210 as both represented culture collection
87 deposits of the P. gallaeciensis type strain BS107T; however, further genomic analysis revealed
88 that DSM 17395 is more closely affiliated to P. inhibens T5T and represents a strain distinct from
89 CIP 105210 (29). CIP 105210T (= DSM 26640T = BS107T) is now the type strain of P.
90 gallaeciensis. In many studies, the descriptions of the source of strain BS107T is unclear and
91 accordingly, the biological identity of the strain described in these studies remains unknown.
92
93 Extensive physiological data on P. inhibens are available (21). P. inhibens clearly differentiates
94 morphologically from other marine bacteria due to the formation of brown colonies on nutrient
4
95 rich iron-containing medium due to precipitation of a brown TDA-iron complex (31). In liquid
96 medium, cells are motile, but tend to form star-shaped aggregates, also called rosettes (32). Based
97 on the currently available 22 genomes (Table 1), genomes of P. inhibens strains have an average
98 size of 4.36 Mb (4.02 – 4.84 Mb) and an average GC content of 59.8% (59.5 – 60.0%). The
99 nucleotide information is structured in one chromosome and several plasmids (3 to 10 plasmids)
100 including few very large (up to 262 kb) plasmids, or chromids. The genomes encode on average
101 4128 genes (3771 – 4609 genes). Chromosomes are largely syntenic and tetranucleotide
102 frequency distribution is highly homogenous (27). The pangenome of 22 P. inhibens strains
103 comprises 9,683 genes and the core genome consists of 2,980 genes (Fig. 2). The pangenome did
104 not reach saturation.
105
106 OCCURRENCE OF P. INHIBENS
107 P. inhibens DSM 16374T was isolated in 1999 from the German Wadden Sea (53° 42’ 20” N 07°
108 43’ 11” E) in marine broth (MB 2216, Difco) due to its strong antibacterial activity (2). Further
109 strains were isolated globally (Europe, Asia, North America, Australia) from coastal waters (27,
110 33) and marine aquaculture facilities (algae, zooplankton, clam, fish) (7, 34–36). At the genus
111 level, Phaeobacter represents approx. 0.03% of all sequences of bacteria in marine surface waters
112 as found in the global marine metagenomic sequence data (37). As experimentally assessed by
113 CARD-FISH, the Roseobacter group is of cosmopolitan distribution and represent 2-7% of the
114 bacterioplankton in surface waters (38). A recent genomic analysis of the Phaeobacter genus
115 (being represented by nearly 50% of genomes of P. inhibens) demonstrated that the genomic
116 clustering of the strains was independent of their geographic distribution (27).
117 Interestingly, Phaeobacter species have not been isolated from open ocean waters (39), but
118 occur in harbor areas or aquaculture tanks (7, 25, 33, 40, 41). Roseobacters could be isolated
5
119 from water samples of aquaculture larval rearing tanks (41), but most Phaeobacter isolates in
120 culture today were obtained from biofilm samples e.g. from walls of fish tanks or
121 macrobiological (algae, bryozoan, barnacles) or inert surfaces (wood, metal, plastic) submerged
122 in seawater (Table 1) (27, 28, 30, 34, 42). Overall, the knowledge pertaining to the natural
123 environmental habitat of the genus remains limited. However, laboratory experiments and
124 genomic analyses indicate an adaptation of P. inhibens to a biofilm lifestyle (1). In laboratory
125 cultures, P. inhibens forms rosettes that assemble into biofilms on the air-liquid interface and the
126 walls of the culture container when growing under agitation and even more so when growing
127 under stagnant conditions (43, 44). Also, P. inhibens was demonstrated to attach to microalgae
128 and their exudates in laboratory cultures (10, 45–47) indicating not only a preference to thrive in
129 a biofilm, but also an adaptation to an eukaryote-associated lifestyle.
130
131 EFFECT OF P. INHIBENS CELLS ON AQUACULTURE ORGANISMS
132 Antagonistic activity of P. inhibens against Vibrio
133 Pathogenic outbreaks in hatcheries can cause great economic damage such as the loss of 40% of
134 juvenile turbot in a Norwegian hatchery in 1988 due to vibriosis (48, 49). Vibriosis affecting
135 marine fish and shrimps still represents a major issue for the wealth of the aquaculture sector (50,
136 51). While vaccines can be used in adult fish, treatment of larvae and juveniles with a not- or
137 underdeveloped immune system must rely on other procedures. Traditionally, these are antibiotic
138 treatments, which also pose the risk of antibiotic resistance development (52, 53). For more than
139 two decades, scientists have evaluated probiotics for aquaculture (49, 54). In the 1990s,
140 Phaeobacter strains were first isolated and proposed as probiotics for aquaculture due to their
141 inhibitory activities against fish pathogenic bacteria such as Vibrio anguillarum (28, 54).
142 Subsequently, many more Phaeobacter isolates were obtained and their anti-Vibrio activity
6
143 confirmed (7, 34–36, 55). Over a seven day co-cultivation experiment, Phaeobacter inhibens
144 DSM 17395 (inoculum: ~106 CFU/mL) was capable of preventing growth of V. vulnificus
145 (inoculum: ~103 CFU/mL), while mono-cultures of V. vulnificus would reach a cell concentration
146 of ~106 CFU/mL (6). More recently, strong in vitro antagonistic effects of P. inhibens DSM
147 17395 toward various Vibrio species of aquaculture interest such as V. aestuarianus, V.
148 alginolyticus, V. harveyi, V. parahaemolyticus, V. splendidus, were also confirmed (Lallemand,
149 unpublished).
150 After having established the anti-Vibrio activity of Phaeobacter inhibens in laboratory dual-
151 cultivation setups, its activity against fish pathogenic Vibrio strains was also assessed in
152 aquaculture-related multi-species experiments (Table S1). Since pathogenic bacteria may enter
153 the larvae tanks via live feed (56, 57), challenge trials were conducted in cultures of rotifers,
154 brine shrimps, copepods and microalgae as well as in marine fish larvae. In algal cultures, the
155 reduction of Vibrio is typically 2 to 4 log10-fold (6, 8, 10, 58–60). The extent of the effect may
156 sometimes lead to the pathogen being undetectable by cultivation (10), while no adverse effect of
157 the probiotic bacterium on the algae was observed. The same level of activity was observed in
158 small zooplankton cultures (copepod, brine shrimp, rotifer, cod larvae) (8, 10, 58, 60), while no
159 reduction of Vibrio spp. by P. inhibens was observed in adult oysters (6, 42, 61, 62). Phaeobacter
160 inhibens was able to level off the mortality of cod-larvae challenged with Vibrio anguillarum
161 (accumulated control mortality reaching 100%) while substantially increasing the survival of
162 unchallenged larvae administrated with the probiotic (10).
163
164 Effect of P. inhibens on the host
165 In general, the probiotic bacterium P. inhibens appears to have no or a positive effect on higher
166 organisms (Table S1). In Vibrio-challenge trials as well as in dual co-cultivation with
7
167 microalgae, P. inhibens had no effect on the host (8, 10, 58, 59). In co-cultivation with the
168 microalgae Emiliania huxleyi, P. inhibens initially promoted algal growth, but subsequently
169 reduced algal cell numbers during senescence (63, 64).
170 Phaeobacter inhibens has a growth promoting effect on rotifers (10). It caused improved survival
171 of various host organisms (brine shrimp, cod larvae, fish cell line) (8, 10), while other studies
172 found no significant effect of P. inhibens on survival of brine shrimp nauplii or copepods (58,
173 65). P. inhibens had no adverse effect on the survival of the nematode Caenorhabditis elegans or
174 turbot larvae (35, 65). The strain LSS9 of the related species P. piscinae induced symptoms of
175 the bleaching disease in defense-deficient red algae Delisea pulchra, but it did not invade algal
176 cells (66). To the authors’ knowledge, there is only one report of the potential beneficial effect of
177 a Phaeobacter sp. strain on shrimp survival (67) even though the family Rhodobacteraceae, and
178 Phaeobacter spp. species are largely represented within shrimp aquaculture systems. The
179 Rhodobacteraceae family was found to comprise 10 to 30% of the microbial community (relative
180 abundance) in water of shrimp hatchery and nursery systems in Vietnam, while its level can reach
181 up to 70-80% within the shrimp-associated microbiome. A Phaeobacter daeponensis strain was
182 detected at a relative abundance of 3% in shrimp larvae (Lallemand, unpublished). Interestingly,
183 Zhao et al. (68) recently performed a feeding trial where the improving effect of a P. daeponensis
184 strain was determined on abalone health, V. harveyi resistance, performance parameters, and
185 modulation of the gut microbiota.
186
187 Effect of P. inhibens on the host microbiome
188 The microbiome associated with an organism significantly contributes to its health and
189 accordingly, maintaining a balanced microbiome on all trophic levels is key to sensitive and
190 intensive fish-rearing systems. Phaeobacter is endemic to aquaculture systems (7, 28, 34);
8
191 however, artificial addition of a high load of a given microorganism might cause imbalance in
192 aquaculture-related microbiomes. So far, high dose addition of P. inhibens to the microbiomes of
193 microalgae (Tetraselmis suecica, Thalassiosira rotula, Emiliania huxleyi), copepods (Acartia
194 tonsa), fish larvae (Scophthalmus maximus) and oysters (Ostrea edulis) has been investigated
195 using cultivation-dependent techniques and amplicon sequencing approaches (6, 19, 45, 69). In
196 natural biofilm samples, the relative abundance of P. inhibens is estimated to be 0.02–0.68% of
197 the bacterial community (33). In contrast, the dosages of P. inhibens administered in laboratory
198 experiments were higher, resulting in relative abundances of 5% of a natural seawater community
199 added to a microalgae, 0.9-90% of a native microalgal microbiome or 23% of a native oyster
200 microbiome (45, 69). The results of the microbiome studies indicate that the effect of P. inhibens
201 was dependent on the overall complexity of the native microbiome; i.e. low-complexity
202 microbiomes such as those of algae and copepods were structurally changed due to addition of P.
203 inhibens, while the more complex microbiomes of oysters and turbot fish larvae were less
204 affected. Addition of P. inhibens reduced or maintained the abundance of individual OTUs of
205 putative pathogenic groups such as vibrios and Pseudoalteromonas sp..
206 To test if the antibacterial activity of P. inhibens would affect the microbiome assembly of a
207 microalgae, Thalassiosira rotula was incubated with microbial communities from natural
208 seawater and either P. inhibens 2.10 or a variant strain lacking the antibacterial activity (70).
209 Only minor differences between the microbiomes in the two setups were identified (45)
210 suggesting that the antibacterial activity of P. inhibens did not have a major effect on bacterial
211 community assembly. Overall, the available data suggests that P. inhibens has minor impact on
212 the microbiomes of marine eukaryotes. The observed changes are highly specific i.e. partially
213 decreasing the abundance of putative pathogenic or opportunistic bacteria such as vibrios.
9
214 Available studies were conducted over a maximum time period of eight days and long-term
215 exposure of microbiomes to TDA-producing bacteria are required.
216
217 BIOACTIVE MOLECULES PRODUCED BY P. INHIBENS
218 Apart from TDA (2), several other small molecules of biological interest have been isolated from
219 P. inhibens: methyltroposulfenin (71), roseobacticides A-K (72), roseochelin A and B (73),
220 acylated homoserine lactones (AHLs), and siderophores (1) (Table 2). Small molecules are key
221 to the interaction between bacteria as well as between bacteria and their host and thus likely
222 involved in the putative probiotic effect of P. inhibens.
223
224 Tropodithietic acid
225 TDA is a disulfide-containing tropone derivative (2). The current knowledge on the molecule has
226 been recently reviewed and we therefore focus on the safety-related aspects (74). TDA was first
227 discovered from a Pseudomonas sp. from soil as its tautomer thiotropocin (75, 76). Besides its
228 biosynthesis by Phaeobacter spp. (2, 63), TDA is also produced by strains of the genera
229 Epibacterium (formerly Ruegeria or Silicibacter) (63, 77, 78), and Pseudovibrio (79, 80). An S-
230 methylated analogue of TDA, namely methyl troposulfenin, was recently characterized (71). Its
231 inhibitory effect against Vibrio anguillarum was 4-fold to 100-fold lower than that of TDA
232 indicating that methylation of TDA turns the potent compound inactive.
233 TDA is produced under iron-rich conditions and has a weak iron-chelating activity (31). Its
234 production is regulated by acyl homoserine lactone-quorum sensing, and auto induction (81), but
235 cell aggregation and biofilm formation are not physiological prerequisites for TDA production
236 (43, 62). Also, it was proposed that TDA biosynthesis is post-transcriptionally regulated and that
237 it requires a cross-regulation of carbon, nitrogen and sulfur metabolism (82, 83). It is bactericidal
10
238 against Gram-positive and Gram-negative bacteria including human pathogens such as Proteus
239 mirabilis, Mycoplasma spp., Staphylococcus aureus, and Klebsiella pneumoniae (MIC ≤ 3.13
240 μg/mL) (75) and fish pathogens such as Vibrio anguillarum (MIC of 90-11287 = 40-80 μg/mL,
241 MIC of NB10Sm = 1.25 μg/mL), V. parahaemolyticus (MIC = 3.31 μg/mL), and V. vulnificus
242 (MIC ≤ 0.83 μg/mL) (6, 17, 62) (see Table S2 for all details). Antibacterial activity is dependent
243 on pH and was found stronger in the acidic range (test range: pH 5 to 9); e.g. the MIC against E.
244 coli was 50 μg/mL at pH 9 decreasing to 0.1 μg/mL at pH 5 (75, 84). Stability of anti-Vibrio
245 activity (thus presumably TDA-containing) of P. piscinae 27-4 supernatant was assessed across
246 ranges of temperature (-80 to 37°C) and pH (1 to 9) (85). The antibacterial activity of the
247 supernatant was stable after 210 days of storage at -80°C, but decreased after approx. 30 days at -
248 20°C. Over a period of 3 days, the activity was stable after storage at 5°C, but decreased with
249 increasing temperature and time of storage (~50% loss at 37°C after one day of storage and
250 complete loss of activity after two days in comparison to 5°C). In this assessment of Phaeobacter
251 supernatant, exposure to different pH values for 1 to 1.5 h did not affect bioactivity. Stability of
252 anti-Vibrio activity of P. inhibens will have to be studied and potentially improved in order to
253 develop a robust product for use in aquaculture systems, which are exposed to a broad range of
254 temperatures and where storage conditions are not always optimal for biological products.
255 TDA is also active against pathogenic fungi including Rizoctonia solani and Pyricularia oryzae
256 (MIC = 3.13 μg/mL, Table S2) or Candida albicans (inhibition zone of 43 mm in agar diffusion
257 assay with 50 µL TDA on ∅ = 9 mm filter-paper disk, conc. = 700 μg/mL) (86). An agar
258 diffusion assay against three strains of microalgae (Chlorella, Scenedesmus) resulted in inhibition
259 zones of 17 – 24 (50 µL TDA on ∅ = 9 mm filter-paper disk, conc. = 700 μg/mL) (86). The
260 survival of the aquaculture feed algae Tetraselmis suecica was affected by TDA at a
261 concentration of 10.5 μg/mL, but no effect was observed at 0.2 μg/mL (10). 42.4 μg/mL of TDA
11
262 caused rapid loss of motility in the amoeba Dictyostelium discoideum followed by cell death (18).
263 It had no significant effect on survival of Artemia nauplii at a concentration of 3.2 μg/mL (65),
264 but 100% mortality was observed at 1 mg/mL (86). The IC50 of TDA against the nematode
265 Caenorhabditis elegans was determined at 25 µg/mL (86).
266 TDA was cytotoxic to mammalian neuronal and glial cell lines at concentrations > 0.1 μg/mL
267 (87) and to cancer cell lines (MCF7 breast carcinoma, HM02 gastric carcinoma, HEPG2
268 hepatocellular carcinoma) (GI50 = 5.0 – 6.7 µg/mL) (86). A screening against the NCI-60 cell
269 collection demonstrated the broad-spectrum lethal and growth-inhibitory activities of TDA
270 against cancer cells particularly against certain renal cancer cell lines with LC50s around 1.3
271 µg/mL (18). In contrast, IC50 of TDA against noncancerous MCF10A epithelial cells was 4.1
272 µg/mL. In neuronal cells, TDA may cause disruption of the mitochondrial membrane potential
273 and activation of oxidative stress response (87). Thus, the mechanism of TDA might be
274 dependent on the cell type and target organism. However, as mentioned above, no negative effect
275 of the live P. inhibens cells producing TDA was so far observed on eukaryotic host organisms
276 indicating that TDA is produced at concentrations unlikely to cause concerns for the host.
277 The usage in aquaculture (and elsewhere) of probiotic bacteria that produce antibacterial
278 molecules raises the concern of resistance development against these bioactive compounds; in
279 this case: TDA. P. inhibens carries genes conferring it self-resistance to TDA (18) and TDA-
280 resistant or -tolerant bacterial strains were isolated from eukaryote-associated microbiomes (79),
281 however, no tolerance or resistance of pathogenic vibrios to TDA could be induced in long-term
282 exposure experiments (17, 88). The genes conferring self-resistance to P. inhibens are co-located
283 to the TDA gene cluster on the largest plasmid (262 kbp), which has so far not been demonstrated
284 to be transmissible or to carry the necessary genes for transmission (89).
12
285 In E. coli, TDA was proposed to act as electroneutral proton-antiporter creating an acidic cytosol
286 by the import of hydrogen ions while exporting metal ions (18). Subsequently, TDA would cause
287 the disruption of the proton motive force. At sub-inhibitory concentrations, TDA caused cell
288 regeneration such as the biogenesis of the cell envelope and upregulation of defense mechanisms
289 (oxidative stress defense and iron uptake) proposing a more complex mode of action in the fish
290 pathogen Vibrio vulnificus (90). Also, TDA was shown to act as regulator of quorum sensing
291 similar to homoserine lactones and can affect ~10% of the producer’s gene expression (91).
292
293 Roseobacticides
294 Phaeboacter inhibens can produce the algicidal compounds, roseobacticides A-K, upon induction
295 by the algal breakdown products ferulic acid, sinapic acid, or p-coumaric acid (72). The
296 biosynthetic pathway and chemical structure of roseobacticides are similar to TDA (92). In
297 contrast to TDA production, the ability to produce roseobacticides is however limited to a narrow
298 phylogenetic subgroup within the Phaeobacter genus (63). The antibacterial and algicidal
299 activities of roseobacticides A and B, or roseobacticide-containing extract, has been assessed
300 against the bacteria P. gallaeciensis, Ruegeria sp., Bacillus subtilis, V. anguillarum and
301 Pseudoalteromonas tunicata and against the algae Emiliania huxleyi, Rhodomonas salina,
302 Chaetoceros muelleri, Thalassiosira pseudonana, Tetraselmis suecica and Isochrysis sp. (46, 63).
303 Roseobacticides did not inhibit any bacterial strains at the tested concentrations (≤ 160 μM).
304 Lowest IC50s (0.1-0.2 μM) were detected against the microalgae Emiliania huxleyi and
305 Rhodomonas salina. The aquaculture strains Tetraselmis suecica and Isochrysis sp. were not
306 affected by roseobacticides and also R. salina was able to recover from addition of
307 roseobacticide-containing extract. In the event that production of roseobacticides might cause
13
308 concern in usage of Phaeobacter as a probiotic in aquaculture systems, a TDA-positive
309 Phaeobacter strain has already been isolated that does not produce roseobacticides (63).
310
311 Siderophores and acylated homoserine lactones
312 Addition of sinapic acid also induces production of the small molecules roseochelin A and B in
313 P. inhibens (73). The molecules were chemically characterized, but the biosynthetic genes are
314 unknown. Roseochelin B has iron-chelating activity and is algicidal against the microalgae
315 Emiliana huxleyi, but no growth inhibition was detected for Chaetoceros muelleri and Dunaliella
316 tertiolecta. Antibacterial activity of roseochelin B was detected against Vibrio strains (IC50 of 75
317 – 891 μM), while other bacterial pathogens (Pseudomonas aeruginosa, Bacillus subtilis,
318 Staphylococcus aureus, Enterococcus faecalis) and Saccharomyces cerevisiae were not affected.
319
320 Iron-chelating activity of P. inhibens DSM 17395 was also described in a phenotypic chrome
321 azurol S assay (without induction by sinapic acid) and a putative biosynthetic gene cluster (BGC)
322 with homology to the petrobactin BGC of Bacillus cereus and Marinobacter aquaeolei was
323 identified (1, 93) (Table 3). However, the chemical structure of this siderophore and its broader
324 activity range remain to be determined. Siderophores are iron chelating compounds, but since
325 marine waters are already iron limited and the natural microbiome produces siderophores, the
326 addition of a single organism with siderophoric activity is unlikely to have any specific effects or
327 raise any concerns.
328
329 Production of the quorum sensing signals, acylated homoserine lactones (AHLs) by P. inhibens
330 was identified in an Agrobacterium tumefaciens bioassay (94, 95). This assay typically detects
331 regular, 3-hydroxy- and oxo-HLs and is sensitive to longer chain AHLs (>C4) (96). The AHLs
14
332 N-(3-hydroxydecanoyl)-L-HL and N-(octadecanoyl)-L-HL were confirmed chemically to be
333 produced by T5 (94) and N-(3-hydroxydecanoyl)-L-HL, N-(dodecanoyl-2,5-diene)-L-HL, and N-
334 (3-hydroxytetradecanoyl-7-ene)-L-HL by S45m. The S45m AHLs inhibited the protease activity
335 of V. coralliilyticus RE22 with IC50s of 0.26 µM, 3.7 µM, and 2.9 µM, respectively. N-(3-
336 hydroxydecanoyl)-L-HL is produced by the AHL synthase PgaI (BGC no. 6, Table 3) and partly
337 regulates the production of TDA (81). The supernatant of a 24-h culture of an AHL negative
338 mutant was not inhibitory against V. anguillarum as was the wildtype, however, after 3 days of
339 incubation, the inhibitory activity of the mutant was at the level of the wildtype (9).
340
341 OTHER FACTORS POTENTIALLY MEDIATING INTERACTIONS OF P. INHIBENS
342 According to the current state of knowledge, P. inhibens has shown no or a positive effect on
343 aquaculture host organisms. To identify potential functional traits of concern, the genome
344 annotation was screened for putative virulence factors. However, all genes identified can also be
345 associated with traits of basic cellular functions or communication. The genome of DSM 17395
346 encodes a complete Type IV secretion system (1, 89), a versatile system found in both Gram-
347 negative and Gram-positive bacteria and that secretes a wide range of substrates and compounds
348 (97). Furthermore, the genome annotations of P. inhibens DSM 16374T and DSM 17395 contain
349 genes associated with type I, II and IV secretion proteins, toxin-antitoxin systems, hemolysin and
350 up to 19 proteases (Table S3). Genomic analysis of DSM 17395 using the KEGG database (98)
351 indicates the presence of several complete ABC transporters such as ZnuABC (Zinc) or
352 YejABEF (microcin C) and two-component systems such as ChvGI. Phaeobacter inhibens
353 carries the necessary genes for the secretory pathways twin arginine translocation system
354 (TatABC) and the Sec-signal recognition particle (SRP) pathway (FtsY, Ffh, SecABDEFGY,
355 YajC, YidC). Many of these secretion pathways were also experimentally confirmed to be
15
356 produced (99). Furthermore, the genome indicates the ability of P. inhibens to perform
357 chemotaxis and as mentioned above, P. inhibens is capable of quorum sensing, biofilm
358 formation, and motility. No genes were found to other virulence factors such as adhesin and
359 invasin. Although, genetically encoded, the herein identified proteins and pathways have so far
360 not been tested for their possible virulence in P. inhibens.
361
362 Antimicrobial resistance
363 Antimicrobial susceptibility can be determined by phenotypic testing and search for AMR genes
364 through whole genome sequences analysis. In agar diffusion disc assays, P. inhibens DSM
365 16374T was found susceptible to many antibiotics and strongly susceptible (inhibition zones > 40
366 mm) to ampicillin (10 µg), cephalosporins (30 µg), chloramphenicol (30 µg), imipenem (10 µg),
367 linezolid (30 µg), penicillin G (10 IU), piperacillin/tazobactam (40 µg), and ticarcillin (75 µg)
368 (24). These susceptibilities were also observed for DSM 17395, P. gallaeciensis DSM 26640T
369 and two P. piscinae strains. Other studies described susceptibility of DSM 16374T to
370 streptomycin sulphate (6 µg), kanamycin (30 µg), gentamycin (30 µg), and neomycin (30 µg)
371 using disc assays, which also applied to the close relatives P. gallaeciensis, Leisingera
372 daeponensis and L. methylohalidivorans (21, 100).
373 P. inhibens DSM 16374T and DSM 17395 were resistant (disc assay, inhibition zone = 0 mm) to
374 nystatin (100 IU), clindamycin (10 µg), lincomycin (15 µg), teicoplanin (30 µg), fosfomycin (50
375 µg) and this was also observed for P. gallaeciensis DSM 26640T and four P. piscinae strains
376 (24), thus appears inherent to this phylogenetic group. Furthermore, resistance of the strain (and
377 DSM 26640T and the aforementioned Leisingera strains) to novobiocin (5 µg) was reported by
378 Yoon et al. (100). Otherwise, P. inhibens strains were found sensitive to copper (≥ 0.5 mM
379 CuCl2) (24).
16
380 In the genome of P. inhibens DSM 17395 (chromosome and three plasmids), no acquired
381 antimicrobial resistance genes for the available antibiotics (i.e. Aminoglycoside, Beta-lactam,
382 Colistin, Fluoroquinolone, Fosfomycin, Fusidic Acid, Glycopeptide, “MLS - Macrolide,
383 Lincosamide and Streptogramin B”, Nitroimidazole, Oxazolidinone, Phenicol, Rifampicin,
384 Sulphonamide, Tetracycline, and Trimethoprim were detected using ResFinder 4.0 (database
385 version 2020-07-28) (with default settings: 90% identity threshold, 60% minimum length and
386 with other less stringent settings: 70% threshold for ID and 60% coverage of the query sequence)
387 (101).
388 The blast results of the DSM 17395 genome against the latest ARG-ANNOT (102) database
389 (ARG-ANNOT_V6_NT_July2019) using the two available online tools (e.g. NCBI Blast tool
390 version +2.10.1 (103) with low stringency settings: 70% threshold for ID and 60% coverage of
391 the query sequence and the BLAST ARG-ANNOT NT tool) did not show the presence of any
392 known antimicrobial resistance gene to the antimicrobials listed in the EFSA Guidance.
393 Furthermore, the DSM17395 genome sequence was screened with CARD’s Resistance Gene
394 Identifier (RGI) version 5.1.1 (104) and the CARD database version 3.1.0. “Perfect”, “Strict”,
395 and “Loose” hits were evaluated, and for complete and partial prodigal gene predictions with the
396 default filter parameters (e.g. threshold percentages for identity and minimum sequence length)
397 were applied by the RGI tool (RGI tool does not provide information on such default filter
398 parameters nor does it offer the user the possibility of selecting filter parameters). The CARD
399 analysis (RGI criteria) resulted in 0 “Perfect hits”, 0 “Strict hits” and 251 “Loose hits” in the P.
400 inhibens DSM 17395 whole genome sequence. After filtering out the 251 loose hits (with less
401 than 70% identity against their matched CARD references and hits matching less than 60% of the
402 length of their CARD reference sequences, only 2 AMR genes loose hits remained,
403 corresponding both to elfamycin antibiotic, which is not included in the EFSA list of
17
404 antimicrobials of human and veterinary importance in the EFSA Guidance. However with the
405 CARD database, in addition to detecting an AMR gene or a variant of an AMR gene (100%
406 identical sequence), very distant homologs of AMR genes can also be detected: so called “loose”
407 hits. Thus, homologous sequences and very partial hits not playing a role in antibiotic resistance
408 are always detected.
409 Concluding, under the new EFSA guidance about characterization of microorganisms used as
410 feed additives (105), even though phenotypic resistance was detected for clindamycin and
411 fosfomycin, no known AMR gene were identified that can be linked to these phenotypes,
412 therefore no further studies would be required. Interestingly, resistance to clindamycin was also
413 detected for closely related P. gallaeciensis and piscinae strains (24), hence suggesting an
414 intrinsic resistance mechanism.
415
416 CONCLUSIONS AND PERSPECTIVE
417 In conclusion, P. inhibens is a promising probiotic candidate for marine aquaculture systems due
418 to efficient inhibition of fish pathogenic vibrios. P. inhibens maintains its antagonistic effect in
419 presence of aquaculture-relevant live feed organisms (algae, rotifers, crustaceans) and fish eggs
420 and larvae, while there is none or a positive effect on the eukaryotic host. Also, the effect on the
421 host-associated microbiomes is minor. Importantly, Phaeobacter spp. have repeatedly been
422 isolated from aquaculture systems and thus are intrinsic to these environments. P. inhibens
423 produces few natural products and its bioactive genomic potential is low in comparison to other
424 marine bacteria such as Pseudoalteromonadaceae and Vibrionaceae (93, 106, 107), but among
425 those molecules is the potent, antibacterial agent TDA. TDA is the major driver of Vibrio
426 inhibition. Algicidal molecules produced by P. inhibens did not show an inhibitory effect on
427 aquaculture-relevant algae. P. inhibens demonstrated resistance to several antimicrobial
18
428 compounds, however, this phenotype was observed across several strains and Phaeobacter
429 species and no dedicated resistance genes were identified in P. inhibens genomes.
430
431 While the results of the herein comprised studies are promising for the probiotic application of P.
432 inibens, a number of tests remain to be conducted to assess its safe use. The probiotic has been
433 tested in fish eggs and larvae (turbot), which are the trophic levels in need of treatment against
434 vibrios. However, carryover of the probiotic to juvenile and adult fish cannot be excluded and
435 thus, its effect on the higher trophic levels and their microbiomes needs to be evaluated. Even
436 though the ubiquitous and natural high abundance of the family Rhodobacteraceae, among which
437 Phaeobacter sp., in aquatics-associated microbiota including those of healthy eukaryotes further
438 support the safe use of such organisms as potential probiotics. Current tests have been limited to
439 feed cultures and turbot larvae on laboratory scale. However, aquaculture systems as well as the
440 fish species produced are very diverse. Also, the production units comprise different ecological
441 niches (such as tank water, tank walls, biofilters) with their own unique microbiota. Accordingly,
442 the effect of the probiotic in these environments would require further assessment.
443
444 Addressing these challenges will not only confirm the applicability and safe usage of P. inhibens
445 in aquaculture systems, but will drive the sustainable development of the industry for the
446 production of high-quality protein according to the UN sustainable development goals.
447
448 ACKNOWLEDGEMENTS
19
449 This work was financially supported by the Danish National Research Foundation to the Center
450 for Microbial Secondary Metabolites (CeMiSt), (DNRF137). Thanks to Karen K. Dittmann for
451 helpful discussion. The work was commissioned and co-funded by Lallemand.
452
453 REFERENCES
454 1. Thole S, Kalhoefer D, Voget S, Berger M, Engelhardt T, Liesegang H, Wollherr A,
455 Kjelleberg S, Daniel R, Simon M, Thomas T, Brinkhoff T. 2012. Phaeobacter
456 gallaeciensis genomes from globally opposite locations reveal high similarity of
457 adaptation to surface life. ISME J 6:2229–2244.
458 2. Brinkhoff T, Bach G, Heidorn T, Liang L, Schlingloff A, Simon M. 2004. Antibiotic
459 Production by a Roseobacter Clade-Affiliated Species from the German Wadden Sea and
460 Its Antagonistic Effects on Indigenous Isolates. Appl Environ Microbiol 70:2560–2565.
461 3. Ringø E. 2020. Probiotics in shellfish aquaculture. Aquac Fish 5:1–27.
462 4. Tesdorpf JE, Geers AU, Strube ML, Gram L, Bentzon‐Tilia M. Differential inhibition of
463 the emerging fish pathogenic Tenacibaculum genus by probiotic roseobacters. Prep.
464 5. D’Alvise P. 2008. Inhibition of fish-pathogenic bacteria by Phaebacter and Ruegeria
465 isolates. MSc thesis.
466 6. Porsby CH, Gram L. 2016. Phaeobacter inhibens as biocontrol agent against Vibrio
467 vulnificus in oyster models. Food Microbiol 57:63–70.
468 7. Grotkjær T, Bentzon-Tilia M, D’Alvise P, Dourala N, Nielsen KF, Gram L. 2016.
469 Isolation of TDA-producing Phaeobacter strains from sea bass larval rearing units and
20
470 their probiotic effect against pathogenic Vibrio spp. in Artemia cultures. Syst Appl
471 Microbiol 39:180–188.
472 8. Rasmussen BB, Kalatzis PG, Middelboe M, Gram L. 2019. Combining probiotic
473 Phaeobacter inhibens DSM17395 and broad-host-range vibriophage KVP40 against fish
474 pathogenic vibrios. Aquaculture 513:734415.
475 9. Prol García MJ, D’Alvise PW, Gram L. 2013. Disruption of cell-to-cell signaling does not
476 abolish the antagonism of Phaeobacter gallaeciensis toward the Fish pathogen Vibrio
477 anguillarum in algal systems. Appl Environ Microbiol 79:5414–7.
478 10. D’Alvise PW, Lillebø S, Prol-Garcia MJ, Wergeland HI, Nielsen KF, Bergh Ø, Gram L.
479 2012. Phaeobacter gallaeciensis reduces Vibrio anguillarum in cultures of microalgae and
480 rotifers, and prevents vibriosis in cod larvae. PLoS One 7:e43996.
481 11. Grotkjær T, Bentzon-Tilia M, D’Alvise P, Dierckens K, Bossier P, Gram L. 2016.
482 Phaeobacter inhibens as probiotic bacteria in non-axenic Artemia and algae cultures.
483 Aquaculture 462:64–69.
484 12. FAO. 2018. The state of world fisheries and aquaculture.
485 13. Ringø E, Olsen RE, Jensen I, Romero J, Lauzon HL. 2014. Application of vaccines and
486 dietary supplements in aquaculture: Possibilities and challenges. Rev Fish Biol Fish
487 24:1005–1032.
488 14. Reid HI, Treasurer JW, Adam B, Birkbeck TH. 2009. Analysis of bacterial populations in
489 the gut of developing cod larvae and identification of Vibrio logei, Vibrio anguillarum and
490 Vibrio splendidus as pathogens of cod larvae. Aquaculture 288:36–43.
21
491 15. Dittmann KK, Rasmussen BB, Castex M, Gram L, Bentzon-Tilia M. 2017. The
492 aquaculture microbiome at the centre of business creation. Microb Biotechnol 10:1279–
493 1282.
494 16. FAO and WHO. 2001. Probiotics in food - Health and nutrional properties ad guidelines
495 for evaluation. Food Nutr Pap 85.
496 17. Porsby CH, Webber M a, Nielsen KF, Piddock LJ V, Gram L. 2011. Resistance and
497 tolerance to tropodithietic acid, an antimicrobial in aquaculture, is hard to select.
498 Antimicrob Agents Chemother 55:1332–7.
499 18. Wilson MZ, Wang R, Gitai Z, Seyedsayamdost MR. 2016. Mode of action and resistance
500 studies unveil new roles for tropodithietic acid as an anticancer agent and the γ-glutamyl
501 cycle as a proton sink. Proc Natl Acad Sci 113:1630–5.
502 19. Dittmann KK, Rasmussen BB, Melchiorsen J, Sonnenschein EC, Gram L, Bentzon‐Tilia
503 M, Bentzon-Tilia M. 2020. Changes in the microbiome of mariculture feed organisms after
504 treatment with a potentially probiotic strain of Phaeobacter inhibens. Appl Environ
505 Microbiol https://doi.org/10.1128/AEM.00499-20.
506 20. Simon M, Scheuner C, Meier-Kolthoff JP, Brinkhoff T, Wagner-Döbler I, Ulbrich M,
507 Klenk H-P, Schomburg D, Petersen J, Göker M. 2017. Phylogenomics of
508 Rhodobacteraceae reveals evolutionary adaptation to marine and non-marine habitats.
509 ISME J 1–17.
510 21. Martens T, Heidorn T, Pukall R, Simon M, Tindall BJ, Brinkhoff T. 2006. Reclassification
511 of Roseobacter gallaeciensis Ruiz-Ponte et al. 1998 as Phaeobacter gallaeciensis gen.
22
512 nov., comb. nov., description of Phaeobacter inhibens sp. nov., reclassification of
513 Ruegeria algicola (Lafay et al. Int J Syst Evol Microbiol 56:1293–1304.
514 22. Breider S, Scheuner C, Schumann P, Fiebig A, Petersen JJ, Pradella S, Klenk H-PP,
515 Brinkhoff T, Göker M, G??ker M. 2014. Genome-scale data suggest reclassifications in
516 the Leisingera-Phaeobacter cluster including proposals for Sedimentitalea gen. nov. and
517 Pseudophaeobacter gen. nov. Front Microbiol 5:416.
518 23. Lee M-H, Song E-J, Seo M-J, Hyun D-W, Bae J-W, Lee S-Y, Roh SW, Nam Y-D. 2015.
519 Phaeobacter marinintestinus sp. nov., isolated from the intestine of a sea cucumber
520 (Apostichopus japonicus). Antonie Van Leeuwenhoek 107:209–216.
521 24. Sonnenschein EC, Phippen CBW, Nielsen KF, Mateiu RV, Melchiorsen J, Gram L,
522 Overmann J, Freese HM. 2017. Phaeobacter piscinae sp. nov., a species of the
523 Roseobacter group and potential aquaculture probiont. Int J Syst Evol Microbiol 67:4559–
524 4564.
525 25. Breider S, Freese HM, Spröer C, Simon M, Overmann J, Brinkhoff T. 2017. Phaeobacter
526 porticola sp. nov., an antibiotic-producing bacterium isolated from a sea harbour. Int J
527 Syst Evol Microbiol 67:2153–2159.
528 26. Wirth JS, Whitman WB. 2018. Phylogenomic analyses of a clade within the roseobacter
529 group suggest taxonomic reassignments of species of the genera Aestuariivita, Citreicella,
530 Loktanella, Nautella, Pelagibaca, Ruegeria, Thalassobius, Thiobacimonas and
531 Tropicibacter, and. Int J Syst Evol Microbiol 68:2393–2411.
532 27. Freese HM, Sikorski J, Bunk B, Scheuner C, Meier-Kolthoff JP, Spröer C, Gram L,
23
533 Overmann J. 2017. Trajectories and Drivers of Genome Evolution in Surface-Associated
534 Marine Phaeobacter. Genome Biol Evol 9:3297–3311.
535 28. Ruiz-Ponte C, Cilia V. 1998. Roseobacter gallaeciensis sp. nov., a new marine bacterium
536 isolated from rearings and collectors of the scallop Pecten maximus. Int J Syst Bacteriol
537 48:537–542.
538 29. Buddruhs N, Pradella S, Göker M, Päuker O, Pukall R, Spröer C, Schumann P, Petersen J,
539 Brinkhoff T. 2013. Molecular and phenotypic analyses reveal the non-identity of the
540 Phaeobacter gallaeciensis type strain deposits CIP 105210T and DSM 17395. Int J Syst
541 Evol Microbiol 63:4340–9.
542 30. Rao D, Webb JS, Kjelleberg S. 2005. Competitive interactions in mixed-species biofilms
543 containing the marine bacterium Pseudoalteromonas tunicata. Appl Environ Microbiol
544 71:1729–1736.
545 31. D’Alvise PW, Phippen CBW, Nielsen KF, Gram L. 2016. Influence of Iron on Production
546 of the Antibacterial Compound Tropodithietic Acid and Its Noninhibitory Analog in
547 Phaeobacter inhibens. Appl Environ Microbiol 82:502–509.
548 32. Segev E, Tellez A, Vlamakis H, Kolter R. 2015. Morphological Heterogeneity and
549 Attachment of Phaeobacter inhibens. PLoS One 10:e0141300.
550 33. Gram L, Rasmussen BB, Wemheuer B, Bernbom N, Ng YY, Porsby CH, Breider S,
551 Brinkhoff T. 2015. Phaeobacter inhibens from the Roseobacter clade has an
552 environmental niche as a surface colonizer in harbors. Syst Appl Microbiol 38:483–493.
553 34. Porsby C, Nielsen K, Gram L. 2008. Phaeobacter and Ruegeria species of the
24
554 Roseobacter clade colonize separate niches in a Danish Turbot (Scophthalmus maximus)-
555 rearing farm and antagonize Vibrio anguillarum under different growth conditions. Appl
556 Environ Microbiol 74:7356–7364.
557 35. Hjelm M, Bergh O, Riaza A, Nielsen J, Melchiorsen J, Jensen S, Duncan H, Ahrens P,
558 Birkbeck H, Gram L. 2004. Selection and identification of autochthonous potential
559 probiotic bacteria from turbot larvae (Scophthalmus maximus) rearing units. Syst Appl
560 Microbiol 27:360–371.
561 36. Prado S, Montes J, Romalde JL, Barja JL. 2009. Inhibitory activity of Phaeobacter strains
562 against aquaculture pathogenic bacteria. Int Microbiol 12:107–114.
563 37. Sunagawa S, Coelho LP, Chaffron S, Kultima JR, Labadie K, Salazar G, Djahanschiri B,
564 Zeller G, Mende DR, Alberti A, Cornejo-Castillo FM, Costea PI, Cruaud C, D’Ovidio F,
565 Engelen S, Ferrera I, Gasol JM, Guidi L, Hildebrand F, Kokoszka F, Lepoivre C, Lima-
566 Mendez G, Poulain J, Poulos BT, Royo-Llonch M, Sarmento H, Vieira-Silva S, Dimier C,
567 Picheral M, Searson S, Kandels-Lewis S, Bowler C, de Vargas C, Gorsky G, Grimsley N,
568 Hingamp P, Iudicone D, Jaillon O, Not F, Ogata H, Pesant S, Speich S, Stemmann L,
569 Sullivan MB, Weissenbach J, Wincker P, Karsenti E, Raes J, Acinas SG, Bork P, Boss E,
570 Bowler C, Follows M, Karp-Boss L, Krzic U, Reynaud EG, Sardet C, Sieracki M,
571 Velayoudon D. 2015. Structure and function of the global ocean microbiome. Science (80-
572 ) 348:1261359–1261359.
573 38. Wietz M, Gram L, Jørgensen B, Schramm a. 2010. Latitudinal patterns in the abundance
574 of major marine bacterioplankton groups. Aquat Microb Ecol 61:179–189.
575 39. Gram L, Melchiorsen J, Bruhn JB. 2010. Antibacterial Activity of Marine Culturable
25
576 Bacteria Collected from a Global Sampling of Ocean Surface Waters and Surface Swabs
577 of Marine Organisms. Mar Biotechnol 12:439–451.
578 40. Bernbom N, Ng YY, Kjelleberg S, Harder T, Gram L. 2011. Marine Bacteria from Danish
579 Coastal Waters Show Antifouling Activity against the Marine Fouling Bacterium
580 Pseudoalteromonas sp. Strain S91 and Zoospores of the Green Alga Ulva australis
581 Independent of Bacteriocidal Activity. Appl Environ Microbiol 77:8557–8567.
582 41. Hjelm M, Riaza A, Formoso F, Melchiorsen J, Gram L. 2004. Seasonal incidence of
583 autochthonous antagonistic Roseobacter spp. and Vibrionaceae strains in a turbot larva
584 (Scophthalmus maximus) rearing system. Appl Environ Microbiol 70:7288–7294.
585 42. Karim M, Zhao W, Rowley D, Nelson D, Gomez-Chiarri M. 2013. Probiotic strains for
586 shellfish aquaculture: Protection of eastern oyster, crassostrea virginica, larvae and
587 juveniles againsl bacterial challenge. J Shellfish Res 32:401–408.
588 43. Prol Garcia MJ, D’Alvise PW, Rygaard a M, Gram L. 2014. Biofilm formation is not a
589 prerequisite for production of the antibacterial compound tropodithietic acid in
590 Phaeobacter inhibens DSM17395. J Appl Microbiol https://doi.org/10.1111/jam.12659.
591 44. Bruhn JB, Gram L, Belas R. 2007. Production of antibacterial compounds and biofilm
592 formation by Roseobacter species are influenced by culture conditions. Appl Environ
593 Microbiol 73:442–50.
594 45. Majzoub ME, Beyersmann PG, Simon M, Thomas T, Brinkhoff T, Egan S. 2019.
595 Phaeobacter inhibens controls bacterial community assembly on a marine diatom. FEMS
596 Microbiol Ecol 95:1–12.
26
597 46. Seyedsayamdost MR, Case RJ, Kolter R, Clardy J. 2011. The Jekyll-and-Hyde chemistry
598 of Phaeobacter gallaeciensis. Nat Chem 3:331–335.
599 47. Segev E, Wyche TP, Kim KH, Petersen J, Ellebrandt C, Vlamakis H, Barteneva N,
600 Paulson JN, Chai L, Clardy J, Kolter R. 2016. Dynamic metabolic exchange governs a
601 marine algal-bacterial interaction. Elife 5:e17473.
602 48. Ina-Salwany MY, Al-saari N, Mohamad A, Mursidi FA, Mohd-Aris A, Amal MNA, Kasai
603 H, Mino S, Sawabe T, Zamri-Saad M. 2019. Vibriosis in Fish: A Review on Disease
604 Development and Prevention. J Aquat Anim Health 31:3–22.
605 49. Westerdahl A, Olsson JC, Kjelleberg S, Conway PL. 1991. Isolation and characterization
606 of turbot (Scophtalmus maximus)-associated bacteria with inhibitory effects against Vibrio
607 anguillarum. Appl Environ Microbiol 57:2223–2228.
608 50. Mohamad N, Amal MNA, Yasin ISM, Zamri Saad M, Nasruddin NS, Al-saari N, Mino S,
609 Sawabe T. 2019. Vibriosis in cultured marine fishes: a review. Aquaculture 512.
610 51. Tran L, Nunan L, Redman RM, Mohney LL, Pantoja CR, Fitzsimmons K, Lightner D V.
611 2013. Determination of the infectious nature of the agent of acute hepatopancreatic
612 necrosis syndrome affecting penaeid shrimp. Dis Aquat Organ 105:45–55.
613 52. Aoki T, Egusa S, Kimura T, Watanabe T. 1971. Detection of R factors in naturally
614 occurring Aeromonas salmonicida strains. Appl Microbiol 22:716–7.
615 53. Hjeltnes B, Andersen K, Egigius E. 1987. Multiple antibiotic resistance in Vibrio
616 salmonicida. Bull Eur Ass Fish Pathol 7:85.
617 54. Ruiz-Ponte C, Samain JF, Sánchez JL, Nicolas JL. 1999. The Benefit of a Roseobacter
27
618 Species on the Survival of Scallop Larvae. Mar Biotechnol 1:52–59.
619 55. Hjelm M, Riaza A, Formoso F, Melchiorsen J, Gram L. 2004. Seasonal incidence of
620 autochthonous antagonistic Roseobacter spp. and Vibrionaceae strains in a turbot larva
621 (Scophthalmus maximus) rearing system. Appl Environ Microbiol 70:7288–7294.
622 56. Eddy SD, Jones SH. 2002. Microbiology of summer flounder Paralichthys dentatus
623 fingerling production at a marine fish hatchery. Aquaculture 211:9–28.
624 57. Douillet PA, Pickering PL. 1999. Seawater treatment for larval culture of the fish
625 Sciaenops ocellatus Linnaeus (red drum). Aquaculture 170:113–126.
626 58. Rasmussen BB, Erner KE, Bentzon-Tilia M, Gram L. 2018. Effect of TDA-producing
627 Phaeobacter inhibens on the fish pathogen Vibrio anguillarum in non-axenic algae and
628 copepod systems. Microb Biotechnol https://doi.org/10.1111/1751-7915.13275.
629 59. Prol García MJ, D’Alvise PW, Gram L. 2013. Disruption of cell-to-cell signaling does not
630 abolish the antagonism of Phaeobacter gallaeciensis toward the Fish pathogen Vibrio
631 anguillarum in algal systems. Appl Environ Microbiol 79:5414–7.
632 60. Grotkjær T, Bentzon-Tilia M, D’Alvise PW, Dierckens K, Bossier P, Gram L. 2016.
633 Phaeobacter inhibens as probiotic bacteria in non-axenic Artemia and algae cultures.
634 Aquaculture 462:64–69.
635 61. Modak TH, Gomez-Chiarri M. 2020. Contrasting immunomodulatory effects of probiotic
636 and pathogenic bacteria on eastern oyster, crassostrea virginica, larvae. Vaccines 8:1–23.
637 62. Zhao W, Dao C, Karim M, Gomez-Chiarri M, Rowley D, Nelson DR. 2016. Contributions
638 of tropodithietic acid and biofilm formation to the probiotic activity of Phaeobacter
28
639 inhibens. BMC Microbiol 16:1.
640 63. Sonnenschein EC, Phippen CBW, Bentzon-Tilia M, Rasmussen SA, Nielsen KF, Gram L.
641 2018. Phylogenetic distribution of roseobacticides in the Roseobacter group and their
642 effect on microalgae. Environ Microbiol Rep 10:383–393.
643 64. Segev E, Wyche TP, Kim KH, Petersen J, Ellebrandt C, Vlamakis H, Barteneva N,
644 Paulson JN, Chai L, Clardy J, Kolter R. 2016. Dynamic metabolic exchange governs a
645 marine algal-bacterial interaction. Elife 5:e17473.
646 65. Neu AK, Månsson M, Gram L, Prol-García MJ. 2014. Toxicity of bioactive and probiotic
647 marine bacteria and their secondary metabolites in Artemia sp. and Caenorhabditis elegans
648 as eukaryotic model organisms. Appl Environ Microbiol 80:146–153.
649 66. Fernandes N, Case R, Longford S, Seyedsayamdost M, Steinberg P, Kjelleberg S, Thomas
650 T. 2011. Genomes and virulence factors of novel bacterial pathogens causing bleaching
651 disease in the marine red alga Delisea pulchra. PLoS One 6:e27387.
652 67. Xue M, Liang H, He Y, Wen C. 2016. Characterization and in-vivo evaluation of potential
653 probiotics of the bacterial flora within the water column of a healthy shrimp larviculture
654 system. Chinese J Oceanol Limnol 34:484–491.
655 68. Zhao J, Ling Y, Zhang R, Ke C, Hong G. 2018. Effects of dietary supplementation of
656 probiotics on growth, immune responses, and gut microbiome of the abalone Haliotis
657 diversicolor. Aquaculture 493:289–295.
658 69. Dittmann KK, Sonnenschein EC, Egan S, Gram L, Bentzon-Tilia M. 2019. Impact of
659 Phaeobacter inhibens on marine eukaryote-associated microbial communities. Environ
29
660 Microbiol Rep 11:401–413.
661 70. Majzoub ME, McElroy K, Maczka M, Thomas T, Egan S. 2018. Causes and
662 Consequences of a Variant Strain of Phaeobacter inhibens With Reduced Competition.
663 Front Microbiol 9:1–10.
664 71. Phippen CBW, Jørgensen CM, Bentzon-Tilia M, Gotfredsen CH, Larsen TO, Gram L,
665 Sonnenschein EC. 2019. Isolation of Methyl Troposulfenin from Phaeobacter inhibens. J
666 Nat Prod acs.jnatprod.8b01097.
667 72. Seyedsayamdost MR, Carr G, Kolter R, Clardy J. 2011. Roseobacticides: small molecule
668 modulators of an algal-bacterial symbiosis. J Am Chem Soc 133:18343–18349.
669 73. Wang R, Seyedsayamdost MR. 2017. Roseochelin B, an Algaecidal Natural Product
670 Synthesized by the Roseobacter Phaeobacter inhibens in Response to Algal Sinapic Acid.
671 Org Lett acs.orglett.7b02424.
672 74. Duan Y, Petzold M, Saleem-Batcha R, Teufel R. 2020. Bacterial tropone natural products
673 and derivatives: Overview on the biosynthesis, bioactivities, ecological role and
674 biotechnological potential. ChemBioChem https://doi.org/10.1002/cbic.201900786.
675 75. Kintaka K, Ono H, Tsubotani S, Harada S, Okazaki H. 1984. Thiotropocin, a new sulfur-
676 containing 7-membered-ring antibiotic produced by Pseudomonas sp. J Antibiot (Tokyo).
677 76. Greer E, Aebisher D, Greer A, Bentley R. 2008. Computational studies of the tropone
678 natural products, thiotropocin, tropodithietic acid, and troposulfenin. Significance of
679 thiocarbonyl-enol tautomerism. J Org Chem 73:280–283.
680 77. Geng H, Bruhn J, Nielsen K, Gram L, Belas R. 2008. Genetic dissection of tropodithietic
30
681 acid biosynthesis by marine roseobacters. Appl Environ Microbiol 74:1535–1545.
682 78. Sonnenschein EC, Nielsen KF, D’Alvise P, Porsby CH, Melchiorsen J, Heilmann J,
683 Kalatzis PG, López-Pérez M, Bunk B, Spröer C, Middelboe M, Gram L. 2017. Global
684 occurrence and heterogeneity of the Roseobacter-clade species Ruegeria mobilis. ISME J
685 11:569–583.
686 79. Harrington C, Reen FJ, Mooij MJ, Stewart FA, Chabot J-B, Guerra AF, Glöckner FO,
687 Nielsen KF, Gram L, Dobson ADW, Adams C, O’Gara F. 2014. Characterisation of Non-
688 Autoinducing Tropodithietic Acid (TDA) Production from Marine Sponge Pseudovibrio
689 Species. Mar Drugs 12:5960–5978.
690 80. Penesyan A, Tebben J, Lee M, Thomas T, Kjelleberg S, Harder T, Egan S. 2011.
691 Identification of the antibacterial compound produced by the marine epiphytic bacterium
692 Pseudovibrio sp. D323 and related sponge-associated bacteria. Mar Drugs 9:1391–402.
693 81. Berger M, Neumann A, Schulz S, Simon M, Brinkhoff T. 2011. Tropodithietic acid
694 production in Phaeobacter gallaeciensis is regulated by N-acyl homoserine lactone-
695 mediated quorum sensing. J Bacteriol 193:6576–85.
696 82. Wünsch D, Strijkstra A, Wöhlbrand L, Freese HM, Scheve S, Hinrichs C, Trautwein K,
697 Maczka M, Petersen J, Schulz S, Overmann J, Rabus R. 2020. Global Response of
698 Phaeobacter inhibens DSM 17395 to Deletion of Its 262-kb Chromid Encoding Antibiotic
699 Synthesis. Microb Physiol 1–16.
700 83. Trautwein K, Hensler M, Wiegmann K, Skorubskaya E, Wöhlbrand L, Wünsch D,
701 Hinrichs C, Feenders C, Müller C, Schell K, Ruppersberg H, Vagts J, Koßmehl S,
31
702 Steinbüchel A, Schmidt-Kopplin P, Wilkes H, Hillebrand H, Blasius B, Schomburg D,
703 Rabus R. 2018. The marine bacterium Phaeobacter inhibens secures external ammonium
704 by rapid buildup of intracellular nitrogen stocks. FEMS Microbiol Ecol 94:1–14.
705 84. Tsubotani S, Wada Y, Kamiya K, Okazaki H, Harada S. 1984. Structure of thiotropocin, a
706 new sulfur-containing 7-membered antibiotic. Tetrahedron Lett 25:419–422.
707 85. Bruhn J, Nielsen K, Hjelm M, Hansen M, Bresciani J, Schulz S, Gram L. 2005. Ecology,
708 inhibitory activity, and morphogenesis of a marine antagonistic bacterium belonging to the
709 Roseobacter clade. Appl Environ Microbiol 71:7263–7270.
710 86. Lanfang Liang. 2003. Investigation of Secondary Metabolites of North Sea Bacteria :
711 Fermentation , Isolation , Structure Elucidation and Bioactivity. Thesis.
712 87. Wichmann H, Vocke F, Brinkhoff T, Simon M, Richter-Landsberg C. 2015. Cytotoxic
713 Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial
714 Origin. Mar Drugs 13:7113–7123.
715 88. Rasmussen BB, Grotkjær T, D’Alvise PW, Yin G, Zhang F, Bunk B, Spröer C, Bentzon-
716 Tilia M, Gram L. 2016. Vibrio anguillarum is genetically and phenotypically unaffected
717 by long-term continuous exposure to the antibacterial compound tropodithietic acid. Appl
718 Environ Microbiol https://doi.org/10.1128/AEM.01047-16.
719 89. Petersen J, Frank O, Göker M, Pradella S. 2013. Extrachromosomal, extraordinary and
720 essential--the plasmids of the Roseobacter clade. Appl Microbiol Biotechnol 97:2805–15.
721 90. Dittmann KK, Porsby CH, Goncalves P, Mateiu RV, Sonnenschein EC, Bentzon‐Tilia M,
722 Egan S, Gram L. 2019. Tropodithietic acid induces oxidative stress response, cell envelope
32
723 biogenesis and iron uptake in Vibrio vulnificus. Environ Microbiol Rep 1758-2229.12771.
724 91. Beyersmann PG, Tomasch J, Son K, Stocker R, Göker M, Wagner-Döbler I, Simon M,
725 Brinkhoff T. 2017. Dual function of tropodithietic acid as antibiotic and signaling
726 molecule in global gene regulation of the probiotic bacterium Phaeobacter inhibens. Sci
727 Rep 7:730.
728 92. Wang R, Gallant É, Seyedsayamdost MR. 2016. Investigation of the Genetics and
729 Biochemistry of Roseobacticide Production in the Roseobacter Clade Bacterium
730 Phaeobacter inhibens. MBio 7:e02118-15.
731 93. Machado H, Sonnenschein EC, Melchiorsen J, Gram L. 2015. Genome mining reveals
732 unlocked bioactive potential of marine Gram-negative bacteria. BMC Genomics 16:1365.
733 94. Wagner-Döbler I, Thiel V, Eberl L, Allgaier M, Bodor A, Meyer S, Ebner S, Hennig A,
734 Pukall R, Schulz S. 2005. Discovery of complex mixtures of novel long-chain quorum
735 sensing signals in free-living and host-associated marine alphaproteobacteria.
736 Chembiochem 6:2195–206.
737 95. Martens T, Gram L, Grossart H-P, Kessler D, Müller R, Simon M, Wenzel SC, Brinkhoff
738 T. 2007. Bacteria of the Roseobacter clade show potential for secondary metabolite
739 production. Microb Ecol 54:31–42.
740 96. Cha C, Gao P, Chen YC, Shaw PD, Farrand SK. 1998. Production of acyl-homoserine
741 lactone quorum-sensing signals by gram-negative plant-associated bacteria. Mol Plant
742 Microbe Interact 11:1119–1129.
743 97. Fronzes R, Christie PJ, Waksman G. 2009. The structural biology of type IV secretion
33
744 systems. Nat Rev Microbiol 7:703–714.
745 98. Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. 2015. KEGG as a reference
746 resource for gene and protein annotation. Nucleic Acids Res 44:D457-62.
747 99. Koßmehl S, Wöhlbrand L, Drüppel K, Feenders C, Blasius B, Rabus R. 2013. Subcellular
748 protein localization (cell envelope) in Phaeobacter inhibens DSM 17395. Proteomics
749 13:2743–2760.
750 100. Yoon J-H, Kang S-J, Lee S-Y, Oh T-K. 2007. Phaeobacter daeponensis sp. nov., isolated
751 from a tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol 57:856–861.
752 101. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM,
753 Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob
754 Chemother 67:2640–2644.
755 102. Gupta SK, Padmanabhan BR, Diene SM, Lopez-Rojas R, Kempf M, Landraud L, Rolain
756 JM. 2014. ARG-annot, a new bioinformatic tool to discover antibiotic resistance genes in
757 bacterial genomes. Antimicrob Agents Chemother 58:212–220.
758 103. Zhang Z, Schwartz S, Wagner L, Miller W. 2000. A greedy algorithm for aligning DNA
759 sequences. J Comput Biol 7:203–214.
760 104. Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, Lago BA, Dave BM,
761 Pereira S, Sharma AN, Doshi S, Courtot M, Lo R, Williams LE, Frye JG, Elsayegh T,
762 Sardar D, Westman EL, Pawlowski AC, Johnson TA, Brinkman FSL, Wright GD,
763 McArthur AG. 2017. CARD 2017: Expansion and model-centric curation of the
764 comprehensive antibiotic resistance database. Nucleic Acids Res 45:D566–D573.
34
765 105. Rychen G, Aquilina G, Azimonti G, Bampidis V, Bastos M de L, Bories G, Chesson A,
766 Cocconcelli PS, Flachowsky G, Gropp J, Kolar B, Kouba M, López-Alonso M, López
767 Puente S, Mantovani A, Mayo B, Ramos F, Saarela M, Villa RE, Wallace RJ, Wester P,
768 Glandorf B, Herman L, Kärenlampi S, Aguilera J, Anguita M, Brozzi R, Galobart J. 2018.
769 Guidance on the characterisation of microorganisms used as feed additives or as
770 production organisms. EFSA J 16:1–24.
771 106. Paulsen SS, Strube ML, Bech PK, Gram L, Sonnenschein EC. 2019. Marine Chitinolytic
772 Pseudoalteromonas Represents an Untapped Reservoir of Bioactive Potential. mSystems
773 4:1–12.
774 107. Cimermancic P, Medema MH, Claesen J, Kurita K, Wieland Brown LC, Mavrommatis K,
775 Pati A, Godfrey PA, Koehrsen M, Clardy J, Birren BW, Takano E, Sali A, Linington RG,
776 Fischbach MA. 2014. Insights into Secondary Metabolism from a Global Analysis of
777 Prokaryotic Biosynthetic Gene Clusters. Cell 158:412–421.
778 108. Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. 2016. JSpeciesWS: a web
779 server for prokaryotic species circumscription based on pairwise genome comparison.
780 Bioinformatics 32:929–931.
781 109. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–
782 9.
783 110. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MTG, Fookes M, Falush D,
784 Keane JA, Parkhill J. 2015. Roary: Rapid large-scale prokaryote pan genome analysis.
785 Bioinformatics 31:3691–3693.
35
786 111. Berger M, Brock N, Liesegang H, Dogs M, Preuth I, Simon M, Dickschat J, Brinkhoff T.
787 2012. Genetic analysis of the upper phenylacetate catabolic pathway in the production of
788 tropodithietic acid by Phaeobacter gallaeciensis. Appl Environ Microbiol 78:3539–3551.
789 112. Zhao W, Yuan T, Piva C, Spinard EJ, Schuttert CW, Rowley DC, Nelson DR. 2019. The
790 Probiotic Bacterium Phaeobacter inhibens Downregulates Virulence Factor Transcription
791 in the Shellfish Pathogen Vibrio coralliilyticus by N-Acyl Homoserine Lactone
792 Production. Appl Environ Microbiol 85:1–14.
793
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794 Authors’ bio
795 Eva Sonnenschein studied at Kiel University, Germany, and received her PhD in Marine
796 Microbiology from Jacobs University Bremen and the Max Planck Institute for Marine
797 Microbiology in 2011. After exploring the secondary metabolism in microalgae for two years as
798 a postdoctoral researcher at UC San Diego, she worked first as postdoctoral researcher, now as
799 Senior Scientist at the Technical University of Denmark. Here, she investigates the molecular
800 interactions of bacteria and microalgae and exploits them for biotech applications.
801
802 Lone Gram received her MSc in 1985 and her PhD in 1989 from the Royal Veterinary and
803 Agricultural University in Copenhagen. She has since 2000 been a professor in bacteriology at
804 the Technical University of Denmark. Lone studies bacterial ecophysiology and biotechnology
805 and is especially interested in marine bacteria. One key interest, as featured in the paper, is the
806 use of marine bacteria as probiotics in aquaculture. She spent research visits at University of New
807 South Wales and at Harvard Medical School. She received the Villum Annual Award in 2016 and
808 has since January 2018 been leading a Centre of Excellence on Microbial Secondary Metabolites.
809 She is decorated with the Order of Dannebrog and is a member of the Royal Danish Academy of
810 Sciences and Letters. Lone has published more than 240 scientific papers that are cited more than
811 12,000 times.
812
813 Mathieu Castex studied at Paris Institute of Technology for Life, Food and Environmental
814 Sciences (AgroParisTech) and obtained his Phd from the same Institute in 2009. He has since
815 then occupied various roles, from research to business development, at Lallemand, a global
37
816 leader in the development, production and marketing of yeast, bacteria and specialty ingredients.
817 Since 2013 he acts as the RnD Director of Lallemand Animal Nutrition, managing a team of
818 microbiologists, physiologists, nutritionists and animal husbandry experts dedicated to the
819 development of microorganisms intended for use in animal nutrition and health.
820
821 Guillermo Jimenez studied at the Faculty of Veterinary Medicine, Autonomous University of
822 Barcelona (UAB), and received his degree in Veterinary Medicine (DVM) in 1989 and his MSc
823 in Animal Production in 2002. In 1990 he started his professional career at Rubinum, S.A.
824 (former Asahi Vet, S.A.) in the research, development and registration of microorganisms
825 intended for use in animal nutrition, thus gaining experience in the fields of taxonomy, genomics,
826 bioinformatics, safety and efficacy of probiotics. He has participated in some scientific papers
827 either as author or co-author. As from 2016 he is R&D Project Manager at Lallemand Animal
828 Nutrition.
829
830
38
831 List of Figures and Tables
832 Figure 1 Comparison by average nucleotide identity of the genomes of P. gallaeciencis DSM
833 26640T, P. inhibens DSM 16374T and DSM 17395, P. piscinae 27-4T, P. porticola P97T, and P.
834 italicus CECT 7645T using JSpeciesWS (108).
835
836 Figure 2. Gene saturation curve for the core genome (white boxes) and the pan genome (blue
837 boxes) of 22 Phaeobacter inhibens genomes. Nucleotide data annotated with Prokka (109) and
838 pan genome calculated with Roary (110). Visualization in R with ggplot2.
839
840 Table 1. Phaeobacter inhibens genome sequences used in this study.
841 Table 2. Small molecules produced by Phaeobacter inhibens. ND = not defined. See Table S2
842 for extended data.
843 Table 3. Bioinformatic analysis for biosynthetic gene clusters (BGC) of Phaeobacter inhibens
844 DSM 16374T (NCBI accession no.s NZ_KI421498.1, NZ_AXBB01000007.0,
845 NZ_AXBB01000008.0) extracted from the antiSMASH database (in fast mode) (Blin 2018
846 NAR). HSL = homoserine lactone
847
848 Supplementary Material
849 Table S1. Bioactivity of Phaeobacter inhibens in host systems.nd = not determined.
850 Table S2. Bioactivity of TDA with minimal inhibitory concentration (MIC). Fish pathogenic
851 species marked with an asterisk.
39
852 Table S3. Genes annotated in the genomes of P. inhibens DSM 16374T and DSM 17395
853 associated with type I, II and IV secretion proteins, toxin-antitoxin systems, hemolysin or
854 proteases
855
856
40
857
858
859 Figure 1 Comparison by average nucleotide identity of the genomes of P. gallaeciencis DSM
860 26640T, P. inhibens DSM 16374T and DSM 17395, P. piscinae 27-4T, P. porticola P97T, and P.
861 italicus CECT 7645T using JSpeciesWS (108).
862
41
863
864 Figure 2. Gene saturation curve for the core genome (white boxes) and the pan genome (blue
865 boxes) of 22 Phaeobacter inhibens genomes. Nucleotide data annotated with Prokka (109) and
866 pan genome calculated with Roary (110). Visualization in R with ggplot2.
867
42
868 Table 1. Phaeobacter inhibens genome sequences (August 2019) with genome features and strain information.
Size GC Scaffol Gen Protei Strain Country Coordinates Environment Habitat Assembly (Mb) % ds es ns
GCA_0034435 2.10 Australia NA Ulva australis algal surface 4.16 59.8 4 3913 3802 55.1
37° 14' 24.0"N 131° 52' GCA_0019693 DOK1-1 South Korea seawater surface water 4.29 59.6 5 4071 3855 12.0" E 45.1
DSM 53° 42' 20"N 07°43'11'' GCA_0004731 Germany natural water_sediment 4.13 60.0 6 3916 3820 16374 E 05.1
DSM 43° 23' 0.128"N 8° 24' clam_larvae_aquaculture, Pecten GCA_0001547 Spain aquaculture 4.23 59.8 4 3992 3880 17395 31.23"W maximus 65.2
48° 21' 16"N 04° 33' GCA_0028886 P10 France natural biofilm_boat 4.24 59.8 4 3981 3854 31"W 85.1
56° 49' 02"N 08° 31' GCA_0028916 P24 Denmark aquaculture system_aquaculture 4.40 59.9 9 4198 4084 47"E 65.1
56° 49' 02"N 08° 31' GCA_0028922 P30 Denmark aquaculture system_aquaculture 4.32 59.9 6 4089 3962 47"E 05.1
56° 49' 02"N 08° 31' fish_larvae_aquaculture, Scophthalmus GCA_0028922 P48 Denmark aquaculture 4.11 60.0 4 3898 3792 47"E maximus 65.1
56° 49' 02"N 08° 31' fish_larvae_aquaculture, Scophthalmus GCA_0028919 P51 Denmark aquaculture 4.12 60.0 6 3926 3816 47"E maximus 85.1
56° 49' 02"N 08° 31' GCA_0028921 P54 Denmark aquaculture zooplankton_aquaculture 4.25 59.8 6 4010 3903 47"E 85.1
56° 49' 02"N 08° 31' GCA_0028920 P57 Denmark aquaculture zooplankton_aquaculture 4.02 60.0 5 3771 3662 47"E 05.1
43
56° 49' 02"N 08° 31' GCA_0028921 P59 Denmark aquaculture zooplankton_aquaculture 4.10 59.9 4 3845 3737 47"E 65.1
43° 23' 0.128"N 8° 24' algae_aquaculture, phytoplankton GCA_0028920 P66 Spain aquaculture 4.78 59.7 10 4593 4457 31.23"W mixture 65.1
43° 23' 0.128"N 8° 24' algae_aquaculture, phytoplankton GCA_0028921 P70 Spain aquaculture 4.48 59.9 7 4275 4153 31.23"W mixture 25.1
43° 23' 0.128"N 8° 24' GCA_0028919 P72 Spain aquaculture clam_larvae_aquaculture, Ostrea edulis 4.43 59.8 6 4209 4093 31.23"W 45.1
43° 23' 0.128"N 8° 24' clam_larvae_aquaculture, Venerupis GCA_0028920 P74 Spain aquaculture 4.48 59.9 7 4276 4152 31.23"W philippinarum 45.1
43° 23' 0.128"N 8° 24' clam_larvae_aquaculture, Venerupis GCA_0028919 P78 Spain aquaculture 4.27 60.0 7 4045 3921 31.23"W philippinarum 65.1
43° 23' 0.128"N 8° 24' clam_larvae_aquaculture, Venerupis GCA_0028921 P80 Spain aquaculture 4.70 59.6 11 4478 4342 31.23"W philippinarum 45.1
43° 23' 0.128"N 8° 24' clam_larvae_aquaculture, Venerupis GCA_0028922 P83 Spain aquaculture 4.65 59.6 11 4419 4283 31.23"W philippinarum 25.1
43° 23' 0.128"N 8° 24' clam_larvae_aquaculture, Venerupis GCA_0028920 P88 Spain aquaculture 4.84 59.5 10 4609 4466 31.23"W philippinarum 85.1
43° 23' 0.128"N 8° 24' clam_larvae_aquaculture, Donax GCA_0028920 P92 Spain aquaculture 4.48 59.9 7 4275 4153 31.23"W trunculus 25.1
USA: Rhode Crassostrea GCA_0015597 S4Sm NA Inner shell 4.40 59.8 80 4221 3972 Island virginica 85.1
869
44
870 Table 2. Small molecules produced by Phaeobacter inhibens. ND = not defined. See Table S2 for extended data.
Compound(s) Bioactivity Concentration Biosafety Reference Tropodithietic acid (TDA) Antibacterial, iron MIC of Mycoplasma Potential effect on (2, 18, 75) chelating, anticancer gallisepticum = 0.39 μg/mL; MIC aquaculture microbiome of Vibrio anguillarum = 40 μg/mL;0.39 LC50 of U251 CNS renal cancer = 1.2 μg/mL
Methyltroposulfenin Antibacterial IC50 of Vibrio anguillarum = 0.14 unknown (71) mM Roseobacticides A-K Algicidal IC50 of Emiliania huxleyi = 0.2 Potential effect on feed (46) μM algae Roseochelin B Algicidal, antibacterial IC50 of Emiliania huxleyi = 64 Potential effect on feed (73) μM; IC50 of Vibrio orientalis = 75 algae or aquaculture μM microbiome Acylated homoserine Quorum sensing ND Potential effect on (1, 94, 111, lactones aquaculture microbiome 112) Siderophore Iron-chelating ND Potential effect on (1) aquaculture microbiome 871
45
872 Table 3. Bioinformatic analysis for biosynthetic gene clusters (BGC) of Phaeobacter inhibens DSM 16374T (NCBI accession no.s
873 NZ_KI421498.1, NZ_AXBB01000007.0, NZ_AXBB01000008.0) extracted from the antiSMASH database (in fast mode) (Blin 2018
874 NAR). HSL = homoserine lactone
875 Cluster 5 Type I PKS with identified resistance model according to ARTS
NCBI On BGC Most similar MIBiG BGC type accession From To contig Results URL no. known cluster BGC-ID of contig edge NZ_KI421 https://antismash- 1 bacteriocin 368979 379828 - None None 498.1 db.secondarymetabolites.org/go/GCF_000473105/1 NZ_KI421 https://antismash- 2 HSL 490450 511158 - None None 498.1 db.secondarymetabolites.org/go/GCF_000473105/2 NZ_KI421 https://antismash- 3 HSL 1725917 1746526 - None None 498.1 db.secondarymetabolites.org/go/GCF_000473105/3 NZ_KI421 https://antismash- 4 HSL 1856118 1876691 - None None 498.1 db.secondarymetabolites.org/go/GCF_000473105/4 NZ_KI421 https://antismash- 5 type I PKS 2000683 2052870 - None None 498.1 db.secondarymetabolites.org/go/GCF_000473105/5 NZ_KI421 https://antismash- 6 HSL 2179961 2200603 - None None 498.1 db.secondarymetabolites.org/go/GCF_000473105/6 NZ_AXBB https://antismash- 7 siderophore 53610 67178 - None None 01000007.0 db.secondarymetabolites.org/go/GCF_000473105/7 NZ_AXBB Polysaccharide BGC000 https://antismash- 8 NRPS 35116 68567 + 01000008.0 B; 6% similarity 1411_c1 db.secondarymetabolites.org/go/GCF_000473105/8 876
46