Halomonas sp. pBMC5 and Sutcliffiella sp. pBMC6 as candidate novel species
Our results, particularly those based on the ANI and DDH values, suggest that both pBMC5 and pBMC6 are novel species, and further characterization is planned. The systematic classification of the family Bacillaceae has undergone numerous modifications in recent years due to the implementation of new taxonomic polyphasic techniques 78,79. This has resulted in the creation of new genera from the previously classified Bacillus genus, such as the new genus Sutcliffiella 79, and justifies the presence of B. horikoshii strains in the phylogenomic tree of pBMC6—likely an older classification of the current S. horikoshii—and the dominance of Bacillus sp. genomes in this tree. The identification of three putative prophages from pBMC6 further corroborates these data because three different bacterial genera were predicted as their hosts, namely, Marinisporobacter, Sutcliffiella, and Bacillus.
Genome screening reveals previously proposed beneficial traits of pBMC
Following the classification of each pBMC for identification and phylogenomic analysis, we screened the pBMC genomes for genes encoding proteins that are potentially beneficial for corals. We screened for genes related to catalase, urease, and siderophore production; phosphate assimilation; and nitrogen cycle and DMSP degradation through biochemical tests and PCR assays 44, which are typically employed for BMC selection. We also detected genes involved in other potential beneficial traits (Table S3), including those related to oxidative stress, such as superoxide dismutases (all pBMC genomes), which exert an antioxidant effect by catalyzing the dismutation of superoxide (an ROS molecule that causes cell damage) 82; catalase KatE (all pBMC genomes) and catalase-peroxidase KatG (pBMC1, pBMC2, pBMC5, and pBMC6 genomes), both of which protect cells from the toxic effects of H2O2 and aerated growth conditions 83–87; manganese catalase (pBMC5 and pBMC6 genomes), which is also involved in the protection of cells from H2O2 88,89; and glutathione synthetase (all pBMC genomes except for pBMC6), which produces glutathione that can subsequently be used by glutathione peroxidase (all pBMC genomes except for pBMC3 and pBMC4) to scavenge ROS, such as H2O2 90,91. When seawater temperatures rise, the coral holobiont produce ROS, resulting in cell damage in both host and its symbionts 92–94. A direct correlation between bleaching and ROS production has been previously reported 3, and ROS-scavenging pBMC strains were hypothesized to mitigate coral bleaching 33, making this a crucial trait when selecting pBMC.
Several genes involved in vitamin B-complex biosynthesis were found in our pBMC genomes, such as riboflavin synthase (all pBMC), pyridoxine 5’-phosphate synthase (all pBMC except for pBMC6), biotin synthase (all pBMC), dihydrofolate synthase and thymidylate synthase (all pBMC), and cobalamin synthase (all pBMC except for pBMC3 and pBMC4) for the biosynthesis of vitamins B2, B6, B7, B9, and B12, respectively. Vitamin B2 is necessary for glutathione reductase activity, which is involved in stress reduction by increasing antioxidant potential, and B2 deficiency increases lipid peroxidation 95. Vitamin B6 catalyzes approximately 2% of all prokaryotic functions 96, but it has not been widely studied in the marine environment 97,98. It acts as an antioxidant during light exposure and against oxidative stress 99,100. Vitamin B7 is a cofactor in several metabolic pathways, such as fatty acid biosynthesis, amino acid metabolism, and gluconeogenesis 101. Vitamin B12 is involved in several metabolic pathways, including the production of the antioxidants glutathione and DMSP 102, which are important for neutralizing high concentrations of ROS generated from heat stress events 33,43. Bacteria that exist in association with corals possess genes encoding for proteins related to the biosynthesis of essential vitamins, such as B1, B2, and B7, whereas their coral host does not have the capacity to produce them 103. This suggests that the coral holobiont can only take up these essential vitamins through heterotrophic feeding and/or from its bacterial symbionts. Furthermore, coral symbionts from the family Symbiodiniaceae are auxotrophs for various B-complex vitamins, which they acquire from exogenous sources such as bacteria 81,102,104–106. This highlights the important role of bacterial symbionts in ensuring coral health. For these reasons and because of the close interaction between several B-complex vitamins, the presence of genes encoding proteins related to B-complex vitamin biosynthesis is suggested as a BMC trait.
We also screened for other genes related to metabolism. Siderophore synthase was present in pBMC6. This enzyme produces siderophores that can scavenge iron from the environment, a trait that is beneficial for corals 33,80 and other organisms 107. In general, the bioavailability of iron in oceans is extremely low; consequently, the growth and survival of organisms that use iron for essential physiological processes, such as photosynthesis and nitrogen fixation, cannot be guaranteed 108,109. Thus, bacteria that exist in association with other marine organisms, such as corals and microalgae, produce siderophores to capture and concentrate iron into a bioavailable form that can be used by other organisms 110,111 Apart from siderosphere production, we also found that some of our pBMC produced ectoine (pBMC3, pBMC4, and pBMC5) and betaine (pBMC1, pBMC2, pBMC3, pBMC4, and pBMC5), which have been previously described in BMC genomes and proposed as compounds that plays a role in beneficial mechanisms in corals 80. Ectoines and betaines are important for osmoregulation and act as protective agents under thermal stress and high irradiance 112–114. They also contribute to the nitrogen biomass of corals in reefs 115. In marine microalgae, the ectoine content was found to increase in the presence of bacteria, highlighting the crucial role of these microorganisms in host health 116. Betaine and ectoine production significantly improves environmental stress tolerance, including pH stress 117 and heat stress 118 in aquatic organisms 119, such as corals and their symbionts 114. Ectoines can help mitigate the harmful effects of heat stress, high salinity, ROS, and radiation 120. Pei et al. identified betaine lipids as leading metabolite drivers for differentiating heat-bleached corals from healthy ones, revealing new tools to screen for heat-resistant corals and their symbionts, such as BMC 114,121.
We also found genes involved in the nitrogen cycle, including nitrate reductase (pBMC3, pBMC4, and pBMC5) and cyanate hydratase (pBMC5). The presence of these genes was previously proposed as a BMC trait 33,43 because balancing this nutrient’s availability contributes to maintaining desirable levels of bioavailable nitrogen, limiting algal growth and leading to an accumulation of photosynthates in algal cells that, when released, feed the coral host and promote its growth. Additionally, increased coral catabolic activity due to an environmental stressor leads to host starvation and increased nitrogen availability to Symbiodiniaceae members of the holobiont, potentially causing destabilization of the host’s nutrient cycle and of the Symbiodiniaceae–coral interaction 10,122.
Screening for genes related to DMSP degradation and sulfur metabolism revealed the presence of DMSP CoA transferase/lyase DddD (pBMC3 and pBMC4) and acryloyl-CoA reductase AcuI/YhdH (BMC1 and pBMC2). DMSP is found in several marine organisms, including Symbiodiniaceae 105,123, and is a ROS scavenger in marine algae 124, an attribute that has been proposed as a BMC trait due to its antioxidant activity 33. Mechanisms of DMSP breakdown have also been hypothesized as BMC traits because a high DMSP concentration can lead to dysbiosis and signal the location of more vulnerable coral to pathogens through chemotaxis 18,33,125.
Discovery of the presence and putative wide distribution of BMC-associated prophages
Prophages are DNA from bacteriophages (or bacteria-infecting viruses) that are integrated into the genomes of their bacterial host upon infection. They are mainly studied as contributors to the virulence of pathogenic reef bacteria, such as V. coralliilyticus, because they encode virulence factors 126. The detection of putative prophages in four of our six pBMC strains revealed the potentially widespread presence of prophages in marine bacteria, suggesting the importance of prophages associated with beneficial bacteria. Prophages also protect the bacterial host against virulent phage infections via superinfection exclusion 127. Thus, prophages might both expand the metabolic capabilities and protection of beneficial bacteria, and further research is warranted.
Pangenomes exhibit novel pBMC beneficial traits and other applicable functions
We conducted pangenomic analyses that included our pBMC strains and strains closely related to them in the phylogenomic trees to screen for genes unique to our pBMC strains. In the Pseudoalteromonas pangenome, 110 genes with known functions were only present in the pBMC genomes (i.e., they were present in one or both of our P. galatheae pBMC strains). This included a protein involved in chemotaxis and some proteins involved in iron/sulfur metabolism. Chemotaxis may be an important BMC trait because it was previously suggested to play a crucial role in defining patterns of microbial diversity, coral metabolism, coral infection dynamics, and chemical cycling processes, thereby influencing coral holobiont health 128. Despite the absence of DMSP-related genes in the genomes of the Pseudoalteromonas sp. pBMC strains compared with the other Pseudoalteromonas sp. strains, the metabolism and production of sulfur compounds has been proposed as a BMC trait because they inhibit the growth of coral pathogens and also play a role in the structure of bacterial communities of the coral holobiont 33.
In the Cobetia pangenome, we found five genes with known functions that were only present in the pBMC genomes, but none of them showed a clear benefit to the host. In the Halomonas pangenome, we found 204 genes with known functions that were only present in the pBMC5 genome, inclunding genes involved in carotenoid biosynthesis, antibiotic biosynthesis, and sulfur metabolism. Although not directly related to host health but more to the environment as a whole, the presence of mercury resistance-related genes in the pBMC5 genome is of crucial interest. Heavy metals are known to be environmental toxins due to their bioaccumulation in the food chain, becoming increasingly hazardous for the higher trophic levels 129,130 The use of bioremediation for the removal of toxic metals has been studied, but only a few studies were performed in a marine environment 131. The trend is similar for mercury-resistant marine bacteria, and the use of mercury-resistant marine bacteria for bioremediation of mercury contamination has received little attention 132. However, their use is associated with certain advantages, such as simple process, lower amount of secondary metabolites, and lower cost than commonly used chemical technologies as well as better adaptability and higher resistance to adverse environmental conditions compared with terrestrial bacteria 132,133. Some studies have reported that mercury-resistant marine bacteria show a higher capability for mercury bioremediation and can reduce the toxic effects of mercury in contaminated environments 132,134.
We found that the Sutcliffiella genome had the highest amount of singleton genes of all the pangenomes generated in this study, with 1333 genes with known functions present only in the pBMC6 genome, including genes encoding proteins related to the biosynthesis of siderophores, carotenoids, antibiotics, vitamins B1, B2 and B12; nitrogen, iron, and sulfur metabolism; chemotaxis; and oxidative stress resistance. Other than the abovementioned genes, we identified a gene that was involved in the detoxification of reactive aldehydes, which are highly reactive organic chemical compounds that mostly arise due to oxidative stress 135. We also identified proteins involved in the degradation of aromatic compounds, such as phenols, cresols, catechols, and diphenyl phosphate (DPHP). Phenols and cresols are harmful to the environment 136, and catechol bioaccumulation negatively affects the entire ecosystem 137. DPHP is used as a chemical additive in numerous industrial products, and because it does not bind with other chemicals, it is easily spread to the environment, where it has been widely detected 138–140. When in the environment, DPHP has a long half-life and is immunotoxic and neurotoxic to other organisms 141. Despite not being comprehensively studied in marine organisms, recent studies have reported its negative effects on fish growth, energy metabolism, and reproduction 142–145. Further investigations are necessary to understand the toxic and negative effects of DPHP on corals, and it may be beneficial to possess the necessary genetic machinery to degrade such harmful compounds. Lastly, we identified a regulatory system that functions under specific stress conditions, such as hypoxia and starvation, and appears to be beneficial to host health and resilience.
pBMC and the importance of their secondary metabolites
Secondary metabolites are important for defense against microorganisms, toxic compounds, and UV radiation as well as essential for symbiotic relationships 146; they are abundantly found within the coral holobiont 147. Accordingly, we assessed the BGCs and Pfams of our six pBMC strains using the antiSMASH platform, revealing 11 secondary metabolite production clusters: ectoine, beta-lactone, terpene, aryl polyene, lasso peptide, NRPS, nonribosomal peptide metallophores, NI siderophores, opine-like zincophores, class I and IV lanthipeptides, and type I and III PKS. Ectoines are important because they reduce the effects of heat stress, high salinity, ROS, and radiation. Beta-lactone derivatives are extremely diverse, comprising 30 distinct families, many of which have antimicrobial activity 148, while others are important elements for antibiotic production 146. Terpenes are diverse organic compounds that play a role in defense mechanisms in plants and fungi 149,150, serving as antioxidants and protecting cells from oxidative stress 151, and their presence indicates production of diverse bioactive compounds 152. Some terpenes and carotenoids, such as squalenes, are pigments involved in photosynthesis and signaling. However, they also possess antioxidant activity and can neutralize oxidative stress 153–155. Aryl polyenes are pigments that are structurally and functionally related to carotenoids and confer protection against photo-oxidative damage and lipid peroxidation 156,157. Lasso peptides have antimicrobial and antiviral properties and also show thermal and chemical resistance 146,158. NRPSs are sources of newly discovered antibacterial agents that have also been widely studied for their antiviral and anti-inflammatory properties 146,159. Microorganisms scavenge metal ions from the environment via metallophores 160. Some of these metallophores, such as siderophores like desferrioxamine E, play important roles in biocontrol and bioremediation 161–163, and are considered beneficial when selecting for BMC 33. Zinc is also an essential nutrient for several cellular processes and is taken from the surrounding environment by bacteria 164. Similar to iron availability, zinc is also present at low concentrations in the environment and is captured by zincophores (also called opine-like zincophores in bacteria), such as bacillopaline, produced by microorganisms 165. Lanthipeptides that possess antimicrobial activity are known as lantibiotics 166, but their functions are not limited to this; they may also possess antifungal and antiviral properties 167,168. The presence of at least one PKS BGC in each pBMC genome suggests that these bacterial strains have the necessary genomic tools to synthesize various polyketides that are likely to have beneficial properties 159.
Red Sea pBMC and their Indo-Pacific Ocean counterparts
Rosado and colleagues26 successfully manipulated the coral microbiome of P. damicornis by adding BMC to coral fragments in a mesocosm setting, and the genomes of these BMC were screened to identify potential beneficial mechanisms and/or traits based on previous literature 80. Some of the BMC from the study by Rosado and colleagues26 share several gene functions related to putative beneficial traits for corals with some of our pBMC (e.g., superoxide dismutase, glutathione synthetase, catalase-peroxidase, adenosylcobinamide-phosphate synthase, adenosylcobinamide kinase, betaine aldehyde dehydrogenase, choline dehydrogenase, ectoine hydroxylase, L-ectoine synthase, nitrite reductase, and CoA transferase) (Table S3) 80. However, when examining the pangenomes of each genus, we noted that our pBMC had a unique set of genes that were absent in the genomes of the BMC from the study by Rosado and colleagues26. Moreover, some of these genes represent potential beneficial traits or mechanisms (Figs. 6–8, Table 1) 26.
Our study results revealed potential insights into how bacteria help corals during periods of stress. Although some of the abovementioned beneficial traits are hypothetical, others have already been validated in previous studies on the differential expression of genes during heat stress experiments in corals 18. Notably, all pBMC examined in this study, even those from the same genus, were distinct and contributed differently to the health and resilience of corals, highlighting the advantages of using microbial consortia 107,169 and the need for continued efforts to isolate 147,170, explore 80,171–173 and test 169,174 novel pBMC.