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Flavobacterium columnare . (A) SDS-PAGE profile of outer membrane proteins (OMPs) from F. columnare grown in FCGM with FeSO 4 (40 μM) (Lane 2); FCGM (Lane 3); and FCGM with 2, 2’-dipyridyl (100 μM) (Lane 4). Arrows indicate the iron-regulated OMPs. (B) 3D structure prediction of FhuA from F. columnare , SS: signal sequence
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Flavobacterium columnare, a fastidious Gram-negative pathogen and the causative agent of columnaris disease, is one of the most harmful pathogens in the freshwater fish-farming industry. Nevertheless the virulence mechanisms of F. columnare are not well understood. Bacterial iron uptake from the host during infection is an important mechanism of vi...
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... indicated that these 2 IROMPs corre- spond to the same protein, the TonB-dependent outer membrane ferrichrome-iron receptor precursor protein (FhuA) of F. psychro philum . The amino acid sequences of F. columnare FhuA were obtained through blasting the FhuA sequence of F. psychro philum with the non-redundant F. columnare protein database. Structural ana lysis showed that F. co lum nare FhuA presented a signal secretion peptide at the N-terminal region, indicating that the FhuA protein with the lower molecular weight observed in the IROMPs profile corresponded to the FhuA protein without the se cretion signal peptide, which was predicted to be cleaved between the 18th and 19th amino acid re sidues (data are not shown). The predicted 763 amino acids of the F. columnare FhuA protein sequence shared homologies with a variety of side ro phore receptors from many bacteria, such as the similarity to F. psychrophilum FhuA (55% identity; 70% similarity), Escherichia coli FhuA (23% identity; 42% similarity), Vibrio cholerae FhuA (21% identity; 39% similarity), Neisseria meningitidis FhuA (24% identity; 42% similarity), and Salmonella Ty phi murium FhuA (23% identity; 43% similarity). The 3D structure prediction also shows that F. co lum nare FhuA has similar domain architecture, with a 22-stranded transmembrane β -barrel that encloses a globular plug domain (Fig. 2B). Ligand-binding sites are formed from re sidues on the extracellular side of the plug domain, as well as from residues on the walls and extracellular loops of the β -barrel. The TonB box is found at the N-terminus of the plug domain, and in some structures it protrudes into the periplasm (Fig. 2B). The F. co lum nare fhuA gene (GeneID: 11477636) is highly homo logous with F. psychrophilum fhuA (GeneID: 5300370), with a similarity of 96.9%. Flavobacterium columnare belongs to the Bacteroi detes family, which have unique promoter elements with −33/−7 consensus sequences (TTG/TAnn TTTG) separated by a spacer of variable length (generally 17 to 23 nucleotides; Pérez-Pascual et al. 2011). The predicted promoter motif TTA-N18-TATATTTG was located upstream of the fhuA ORF (Fig. 3), which was the most highly conserved structure as TTG/ TAnnTTTG. The CAS assay was applied to test whether Flavo bacterium columnare could produce side ro phores. After 3 d of incubation on CAS agar, F. columnare produced a yellow halo around the colonies (Fig. 4), and a similar yellow halo also appeared around the positive control, Salmonella Ty phi murium fur mu tant, but not around the negative control, S. Ty phi murium wild type. These results indicate that F. columnare synthesizes and secrets siderophores on CAS me di um modified for F. columnare growth. The predicted promoter motif TTA-N18-TATCTTTG was located upstream of the fur ORF (Fig. 5). The F. columnare Fur protein had 152 amino acids, which shared similar size with common Fur from other bacteria such as E. coli (148 aa), Salmonella Ty phi murium (150 aa), and Vibrio cholerae (150 aa) (de Lorenzo et al. 1988, Litwin et al. 1992, Bjarnason et al. 2003). Sequence and structural alignment between functional representative bacterial Fur proteins revealed that 26 amino acid residues (~17%) are strictly conserved out of 153 residues in F. columnare Fur (Fig. 6). F. columnare Fur has 43, 43, 32, 42, and 90% amino acid similarity to the Fur of S. Ty phi murium, E . coli , Edwardsiella tarda , V. cholerae , and F. psychro philum , respectively, which means that F. columnare Fur may be functionally similar to Fur from other bacteria. To evaluate the functionality of the Flavobacterium columnare fur gene, a complementary CAS assay was performed. As shown in Fig. 7, after 12 h growth, on the CAS plate, the S . Typhimurium Δ fur- 44 mu tant, which contains the fur gene of F. columnare , showed a small orange halo around the colonies which is very close to the negative control (Salmonella Ty phi murium UK-1 wild type), but much smaller than the one produced by the fur mutant. This means that S . Ty phi murium UK-1 Δ fur- 44 was partially complemented by the F. columnare P fur - fur gene, showing intermediate se cretion of siderophores between the S. Ty phi murium Δ fur- 44 mutant and S . Ty phi murium UK-1 wild type. These results indicate that F. columnare Fur is functional and may play a re gulatory role in F. columnare iron homeostasis. Vertebrates sequester iron from invading patho gens as a means of nutritional immunity, using high- affinity iron-binding proteins to limit levels of free iron in biological fluids and tissues in order to de prive pathogens of this key nutritional component. In vading bacterial pathogens sense this iron depletion as a signal that they are within a host and induce the expression of genes that allow iron uptake in order to overcome the host defenses. Similar to other pathogens, Flavobacterium columnare can respond to iron- limited environments by upregulating the FhuA receptor protein (Fig. 2), indicating that F. co lumnare possesses at least one system for si dero phore uptake. Flavobacterium columnare belongs to the Bacte roidetes family (also known as the Cytophaga Flavobacterium - Bacteroides group; Gherna & Woese 1992). Bacteroidetes have strong differences in their mechanisms of gene regulation at either transcription or translation levels compared to proteobacteria (Vingadassalom et al. 2005). As mentioned previously, Bacteroidetes have unique promoter elements with −33/−7 consensus sequences (TTG/TAnnTTTG) separated by spacer or variable length nucleotides (generally 17 to 23 bases; Chen et al. 2010). The putative ribosomal binding site (RBS) consensus sequence has been described as TAAAA, typically found at 2 to 12 bases from the gene start codon (Staroscik et al. 2008). The F. columnare FhuA gene presents all of these elements at its promoter region as predicted (Fig. 3). However, the F. columnare fhuA gene is not part of an organized operon, and the surrounding genes have no similarities with the fhuBCD genes, which are required for siderophore transport. The rest of the machinery for siderophore transport across the F. columnare cell membrane still needs to be identified. As mentioned above, Flavobacterium co lum nare FhuA belongs to a siderophore receptor precursor protein family. Thus, the presence of sidero phores was evaluated. Siderophores of F. columnare were detected using CAS plates (Fig. 4). However, further studies are required to determine the gene regulation of siderophore synthesis and secretion for F. columnare . Usually in bacteria, iron acquisition is tightly con- trolled by the Fur protein (Crosa et al. 2004). A Fur- like regulation pattern may therefore exist during iron uptake in Flavobacterium columnare . We identified a putative fur gene in the chromosome of F. columnare . To evaluate its activity, Salmonella Typhimurium Δ fur- 44 mutant was complemented with the F. columnare putative fur gene. The F. columnare P fur - fur gene partially complements S . Typhimurium Δ fur- 44 mutant (Fig. 7). This partial complementation could be due to differences in the promoter regions between S. Typhimurium and Flavobacterium . Although the F. columnare Fur protein possesses a predicted functional 3D structure (data not shown), its amino acid sequence differed from the enteric Fur protein, which could lead to a partial repression activity. As mentioned, the promoter regions of the fur gene in S. Typhimurium are different from those in Flavobacterium , indicating that the F. columnare Fur binding box is also different. On the other hand, Bacteroides possesses an unusual primary sigma factor, called σ ABfr -like, that strongly differs from the σ 70 factor of proteobacteria (Chen et al. 2007). Thus, the genes in Bacteroides are not usually expressed when transferred into proteobacteria, and proteobacteria genetic elements do not function well in Bactero i detes (Chen et al. 2010). This correlates with the partial complementation of S. Typhimurium Δ fur- 44 by the F. columnare P fur - fur gene. Pathogenesis of Flavobacterium columnare is not clear, but it is known that the columnaris disease pro- cess involves bacterial invasion and external tissue damage, with no occurrence of systemic infection found (Tripathi et al. 2003). This may indicate that the extracellular capsule or proteases are more criti- cal than iron acquisition in establishing F. columnare infection. Therefore, more experiments are needed to identify whether iron acquisition of F. columnare can influence its virulence. In addition, only one strain, F. columnare ATCC 23463, was used in all of the experiments described here; thus our conclusions are not all-inclusive. The lack of feasible genetic means and techniques for mutant construction in F. columnare ATCC 23463 is also a major limitation in advancing in-depth investigation of the iron uptake mechanisms of F. columnare. In summary, we identified part of the principal iron uptake machinery of F. columnare, which contains a putative siderophore iron uptake system, FhuA, that is regulated in an iron-dependent fashion and likely regulated in a Fur- dependent fashion as well. Siderophores were detected in F. columnare . Further studies should be conducted at the genetic level to determine the regulatory relationship of these components of the iron uptake system, as well as their potential role in the pathogenesis of F. columnare ...
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... One remarkable difference between Proteobacteria and Bacteroidetes is their capability to synthesize siderophores. Proteobacteria have been shown to synthesize dozens of different siderophores (Vraspir & Butler, 2009), while almost no instances of siderophore synthesis are known among Bacteroidetes (Guan et al., 2013). However, Bacteroidetes are known to have receptors for iron-siderophore complexes (D'Onofrio et al., 2010;Zhu et al., 2020). ...
... Interestingly, the Proteobacteria MAGs examined in the same study lacked these genes. Moreover, some fish pathogens within the genus Flavobacterium have shown siderophore synthesis capabilities (Guan et al., 2013;Møller et al., 2005). ...
Microbial assemblages under the sea ice of the Dease Strait, Canadian Arctic, were sequenced for metagenomes of a small size fraction (0.2–3 μm). The community from early March was typical for this season, with Alpha‐ and Gammaproteobacteria as the dominant taxa, followed by Thaumarchaeota and Bacteroidetes. Toward summer, Bacteroidetes, and particularly the genus Polaribacter, became increasingly dominant, followed by the Gammaproteobacteria. Analysis of genes responsible for microbial acquisition of iron showed an abundance of ABC transporters for divalent cations and ferrous iron. The most abundant transporters, however, were the outer membrane TonB‐dependent transporters of iron‐siderophore complexes. The abundance of iron acquisition genes suggested this element was essential for the microbial assemblage. Interestingly, Gammaproteobacteria were responsible for most of the siderophore synthesis genes. On the contrary, Bacteroidetes did not synthesize siderophores but accounted for most of the transporters, suggesting a role as cheaters in the competition for siderophores as public goods. This cheating ability of the Bacteroidetes may have contributed to their dominance in the summer.
... Iron acquisition has been linked to virulence in many pathogens, including the fish pathogens Edwardsiella ictaluri (Abdelhamed et al., 2016), Vibrio anguillarum (Wolf and Crosa, 1986), Aeromonas salmonicida (Hirst et al., 1991), and Flavobacterium psychrophilum . Several studies explored iron acquisition by F. columnare and related fish pathogens (Avendano-Herrera et al., 2005;Moller et al., 2005;Guan et al., 2013;Bruce et al., 2021). Genes predicted to be involved in iron uptake have been identified by analyses of F. columnare genomes (Tekedar et al., 2017;Zhang et al., 2017). ...
... Siderophore-iron complexes (ferri-siderophores) are taken up by specific receptors on the bacterial cell surface (Sheldon et al., 2016). F. columnare produces siderophores under iron deprivation (Guan et al., 2013) and the expression of genes predicted to be involved in siderophore synthesis increases following exposure to fish mucus (Lange et al., 2018). F. columnare genes needed for siderophore production and secretion were recently identified and mutated Diagram of ferric iron uptake systems. ...
... Multiple TonB-dependent receptors have been described for ferric iron uptake, and expression of genes encoding these proteins is often upregulated in low-iron conditions (Braun, 2005;Bruce et al., 2021). Previous studies identified the F. columnare genes encoding the predicted TonB-dependent outer membrane siderophore receptors FhuA, IutA, and C6N29_04165 (Guan et al., 2013;Conrad et al., 2022), but their roles in iron utilization and virulence have not been explored further. The fate of ferrisiderophores and heme molecules that reach the periplasm has been studied in other Gram-negative bacteria (Davidson and Chen, 2004;Benson and Rivera, 2013;Sheldon et al., 2016). ...
Flavobacterium columnare, which causes columnaris disease, is one of the costliest pathogens in the freshwater fish-farming industry. The virulence mechanisms of F. columnare are not well understood and current methods to control columnaris outbreaks are inadequate. Iron is an essential nutrient needed for metabolic processes and is often required for bacterial virulence. F. columnare produces siderophores that bind ferric iron for transport into the cell. The genes needed for siderophore production have been identified, but other components involved in F. columnare iron uptake have not been studied in detail. We identified the genes encoding the predicted secreted heme-binding protein HmuY, the outer membrane iron receptors FhuA, FhuE, and FecA, and components of an ATP binding cassette (ABC) transporter predicted to transport ferric iron across the cytoplasmic membrane. Deletion mutants were constructed and examined for growth defects under iron-limited conditions and for virulence against zebrafish and rainbow trout. Mutants with deletions in genes encoding outer membrane receptors, and ABC transporter components exhibited growth defects under iron-limited conditions. Mutants lacking multiple outer membrane receptors, the ABC transporter, or HmuY retained virulence against zebrafish and rainbow trout mirroring that exhibited by the wild type. Some mutants predicted to be deficient in multiple steps of iron uptake exhibited decreased virulence. Survivors of exposure to such mutants were partially protected against later infection by wild-type F. columnare.
... Several studies of iron acquisition mechanisms of F. columnare and related fish pathogens have been reported (23)(24)(25)(26). Genome analyses revealed F. columnare genes predicted to be involved in iron acquisition (5,6). ...
... Studies of Flavobacterium psychrophilum revealed that iron deprivation increased expression of some antigenic proteins and increased the effectiveness of attenuated vaccine strains (28,29). F. columnare produces siderophores under iron-limited conditions (23). Exposure to fish mucus resulted in increased expression of genes predicted to be involved in siderophore production (30). ...
... A more complete understanding of the critical virulence factors involved in columnaris disease may result in improved strategies to control outbreaks. Iron-uptake systems have been linked to virulence in other fish pathogens (13,17), and F. columnare produces an iron-chelating siderophore (23). Here, we identified the critical F. columnare siderophore biosynthesis genes and examined the roles of F. columnare siderophores as possible virulence factors. ...
Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish. F. columnare virulence mechanisms are not well understood, and current methods to control columnaris disease are inadequate. Iron acquisition from the host is important for the pathogenicity and virulence of many bacterial pathogens. F. columnare iron acquisition has not been studied in detail. We identified genes predicted to function in siderophore production for ferric iron uptake. Genes predicted to encode the proteins needed for siderophore synthesis, export, uptake, and regulation were deleted from F. columnare strain MS-FC-4. The mutants were examined for defects in siderophore production, for growth defects in iron-limited conditions, and for virulence against zebrafish and rainbow trout. Mutants lacking all siderophore activity were obtained. These mutants displayed growth defects when cultured under iron-limited conditions, but they retained virulence against zebrafish and rainbow trout similar to that exhibited by the wild type, indicating that the F. columnare MS-FC-4 siderophores are not required for virulence under the conditions tested.
IMPORTANCE Columnaris disease, which is caused by Flavobacterium columnare, is a major problem for freshwater aquaculture. Little is known regarding F. columnare virulence factors, and control measures are limited. Iron acquisition mechanisms such as siderophores are important for virulence of other pathogens. We identified F. columnare siderophore biosynthesis, export, and uptake genes. Deletion of these genes eliminated siderophore production and resulted in growth defects under iron-limited conditions but did not alter virulence in rainbow trout or zebrafish. The results indicate that the F. columnare strain MS-FC-4 siderophores are not critical virulence factors under the conditions tested but may be important for survival under iron-limited conditions in natural aquatic environments or aquaculture systems.
... Invasins promote translocation of pathogens in host cell [(Meuskens et al., 2019), Yersinia spp.]. Once inside the host, the survival rate of pathogens is modulated by numerous structural and metabolic virulence factors such as capsules and iron acquisition systems, respectively (Lindler et al., 1998;Winkelmann et al., 2002;Hsieh et al., 2003;Møller et al., 2005;Guan et al., 2013;Balado et al., 2018). Encapsulated pathogens are resistant to phagocytosis due to the protective carbohydrate layer that blocks host immune response components from binding to immunogenic membrane proteins of the bacteria (Cress et al., 2014). ...
... Bacteroidetes harbor genes encoding superoxide dismutase, catalase-peroxidase, and thiol peroxidase to resist ROS-mediated killing Levipan et al., 2018). Iron acquisition systems have been identified in F. columnare (Guan et al., 2013;Zhang et al., 2017), F. psychrophilum (Møller et al., 2005), F. spartansii , F. branchiophilum, and T. maritimum (Avendaño-Herrera et al., 2005), whose function is to compete for the limited iron stock in host cells. The uptake of iron-siderophore complexes by bacteroidetes are not fully explored, however, several TonB-dependent outer membrane receptors such as, OmpA related proteins, ferrichrome-iron receptor precursor (FhuA) and ferric uptake receptor (Fur) protein are identified in the genomes of F. columnare (Dumpala et al., 2010;Guan et al., 2013), F. psychrophilum (Alvarez et al., 2008) T. maritimum (Avendaño-Herrera et al., 2005) that may function as iron importer across the outer membrane as is shown for several Gram-negative bacteria (Letain and Postle, 1997;Braun et al., 1998). ...
... Iron acquisition systems have been identified in F. columnare (Guan et al., 2013;Zhang et al., 2017), F. psychrophilum (Møller et al., 2005), F. spartansii , F. branchiophilum, and T. maritimum (Avendaño-Herrera et al., 2005), whose function is to compete for the limited iron stock in host cells. The uptake of iron-siderophore complexes by bacteroidetes are not fully explored, however, several TonB-dependent outer membrane receptors such as, OmpA related proteins, ferrichrome-iron receptor precursor (FhuA) and ferric uptake receptor (Fur) protein are identified in the genomes of F. columnare (Dumpala et al., 2010;Guan et al., 2013), F. psychrophilum (Alvarez et al., 2008) T. maritimum (Avendaño-Herrera et al., 2005) that may function as iron importer across the outer membrane as is shown for several Gram-negative bacteria (Letain and Postle, 1997;Braun et al., 1998). ...
Bacterial fish pathogens are one of the key challenges in the aquaculture industry, one of the fast-growing industries worldwide. These pathogens rely on arsenal of virulence factors such as toxins, adhesins, effectors and enzymes to promote colonization and infection. Translocation of virulence factors across the membrane to either the extracellular environment or directly into the host cells is performed by single or multiple dedicated secretion systems. These secretion systems are often key to the infection process. They can range from simple single-protein systems to complex injection needles made from dozens of subunits. Here, we review the different types of secretion systems in Gram-negative bacterial fish pathogens and describe their putative roles in pathogenicity. We find that the available information is fragmented and often descriptive, and hope that our overview will help researchers to more systematically learn from the similarities and differences between the virulence factors and secretion systems of the fish-pathogenic species described here.
... . For example, the host may utilize ferritin or transferrin to bind iron in order to evade bacterial acquisition along with neutral pH and aerobic environments within the host serum (Neilands, 1995;Skaar, 2010). Ferric uptake regulator (FUR) protein has been identified in the F. columnare genome and has been found to be conserved with other bacteria (Guan et al., 2013). In host fish species, hepcidin is also an important regulator in response to pathogen infection or increased circulating iron loads, and increased hepcidin levels will sequester iron from circulation (Rodrigues et al., 2006). ...
Iron is essential for growth and virulence in most pathogenic bacterial strains. In some cases, the hosts for these pathogenic bacteria develop specialized strategies to sequester iron and limit infectivity. This in turn may result in the invading pathogens utilizing high‐affinity iron transport mechanisms, such as the use of iron‐chelating siderophores, to extend beyond the host defences. Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD) in salmonids, relies on iron metabolism for infectivity, and the genome of the model CSF‐259‐93 strain has recently been made available. Further, this strain serves as a parent strain for a live‐attenuated vaccine strain, B.17, which has been shown to provide rainbow trout with protection against BCWD. To elucidate specific gene expression responses to iron metabolism and compare strain differences, both F. psychrophilum strains were grown under iron‐limiting conditions and 26 genes related to iron metabolism were mapped for 96 hr in culture via qPCR analyses. Results indicate increased production of the ferrous iron transport protein B (FITB; p =.008), and ferric receptor CfrA (FR 1; p =.012) in the wild‐type CSF‐259‐93 strain at 72 hr and 96 hr post‐exposure to iron‐limiting media. In the B.17 vaccine strain, siderophore synthase (SS) expression was found to be downregulated at 72 hr, in comparison with 0h (p =.018). When strains were compared, FITB (p =.021), FR1 (p =.009) and SS (p =.016) were also elevated in B.17 at 0 hr and TonB outer protein membrane receptor 1 (TBomr1; p =.005) had a lower expression at 96 hr. Overall, this study demonstrated strain‐related gene expression changes in only a fraction of the iron metabolism genes tested; however, results provide insight on potential virulence mechanisms and clarification on iron‐related gene expression for F. psychrophilum.
... Iron is a critical micronutrient, and availability of iron in biological fluids is tremendously limited. Iron acquisition in vivo is considered a significant factor in bacterial virulence [95][96][97]. Iron uptake mechanisms have been related to fish pathogens' virulence in Listonella anguillarum, Aeromonas salmonicida, Photobacterium damselae, and Tenacibaculum maritimum [98][99][100][101][102]. F. columnare, F. spartansii, F. tructae, F. hydatis, F. chilense, and F. araucananum have the most potential ironregulated and -acquisition related genes (Table 5). Thus, iron uptake mechanisms may have an important role in these Flavobacterium species' pathogenicity. ...
Flavobacterium species are considered important fish pathogens in wild and cultured fish throughout the world. They can cause acute, subacute, and chronic infections, which are mainly characterized by gill damage, skin lesions, and deep necrotic ulcerations. Primarily, three Flavobacterium species, F. branchiophilum, F. columnare, and F. psychrophilum, have been reported to cause substantial losses to freshwater fish. In this study, we evaluated genomes of 86 Flavobacterium species isolated from aquatic hosts (mainly fish) to identify their unique and shared genome features. Our results showed that F. columnare genomes cluster into four different genetic groups. In silico secretion system analysis identified that all genomes carry type I (T1SS) and type IX (T9SS) secretion systems, but the number of type I secretion system genes shows diversity between species. F. branchiophilum, F. araucananum, F. chilense, F. spartansii, and F. tructae genomes have full type VI secretion system (T6SS). F. columnare, F. hydatis, and F. plurextorum carry partial T6SS with some of the T6SS genes missing. F. columnare, F. araucananum, F. chilense, F. spartansii, F. araucananum, F. tructae, Flavobacterium sp., F. crassostreae, F. succinicans, F. hydatis, and F. plurextorum carry most of the type IV secretion system genes (T4SS). F. columnare genetic groups 1 and 2, Flavobacterium sp., and F. crassostreae encode the least number of antibiotic resistance elements. F. hydatis, F. chilense, and F. plurextorum encode the greatest number of antibiotic resistance genes. Additionally, F. spartansii, F. araucananum, and chilense encode the greatest number of virulence genes while Flavobacterium sp. and F. crassostreae encode the least number of virulence genes. In conclusion, comparative genomics of Flavobacterium species of aquatic origin will help our understanding of Flavobacterium pathogenesis.
... The synthesis of siderophores under iron-limiting conditions in vitro has been also demonstrated in the fish pathogens Yersinia ruckeri (Romalde et al. 1991;Fern andez et al. 2007), Lactococcus ruckeri (Schmidtke and Carson 2003), Tenacibaculum maritimum (Avendaño-Herrera et al. 2005), Flavobacterium psychrophilum (Moller et al. 2005), Streptococcus phocae (Retamales et al. 2012), Tenacibaculum mesophilum (Fujita et al. 2013), Flavobacterium columnare (Guan et al. 2013), Edwardsiella tarda (Castro et al. 2016), Edwardsiella ictaluri (Abdelhamed et al. 2016), Renibacterium salmoninarum (Bethke et al. 2016) and Piscirickettsia salmonis (Calquin et al. 2018). In none of these bacteria the siderophores produced have been chemically characterized, with the exception of T. mesophilum which produces the di-hydroxamate bisucaberin B (Fujita et al. 2013) and Edwardsiella tarda in which the production of vibrioferrin has been confirmed (Castro et al. 2016). ...
This review summarizes the current knowledge about iron uptake systems in bacterial fish pathogens and their involvement in the infective process. Like most animal pathogens, fish pathogens have evolved sophisticated iron uptake mechanisms some of which are key virulence factors for colonization of the host. Among these systems, siderophore production and heme uptake systems are the best studied in fish pathogenic bacteria. Siderophores like anguibactin or piscibactin, have been described in Vibrio and Photobacterium pathogens as key virulence factors to cause disease in fish. In many other bacterial fish pathogens production of siderophores was demonstrated but the compounds were not yet chemically characterised and their role in virulence was not determined. The role of heme uptake in virulence was not yet clearly elucidated in fish pathogens although there exist evidence that these systems are expressed in fish tissues during infection. The relationship of other systems, like Fe(II) transporters or the use of citrate as iron carrier, with virulence is also unclear. Future trends of research on all these iron uptake mechanisms in bacterial fish pathogens are also discussed.
... It is well known that most vertebrate hosts utilize different ironbinding proteins such as lactoferrin to limit the amount of free Fe + available to bacteria (Ángeles Esteban, 2012;Vogel, 2012); however, Gram-negative bacteria also employ different methods for retaining and acquiring Fe 2+ and Fe 3+ (Noinaj, Guillier, Barnard, & Buchanan, 2010;Porcheron, Garenaux, Proulx, Sabri, & Dozois, 2013). We and others have previously identified several F. columnare genes, which likely represent different iron acquisition pathways (Beck et al., 2015;Cai et al., 2019;Guan, Santander, Mellata, Zhang, & Curtiss, 2013;Lange et al., 2018). We utilized RT-qPCR to evaluate the expression of iron acquisition genes in mucus-stimulated planktonic and biofilm cells. ...
Columnaris disease is responsible for substantial losses throughout the production of many freshwater fish species. One of the ways in which the bacterium Flavobacterium columnare is so effective in initiating disease is through the formation of biofilms on fish skin and gills. To further explore the interaction between host factors and bacterial cells, we assayed the ability of vertebrate mucus to enhance F. columnare biofilm development. Different concentrations of catfish, tilapia and pig mucus (5–60 µg/ml) increased biofilm growth at varying degrees among F. columnare isolates. Our data suggest that vertebrate mucus acts as a signalling molecule for the development of F. columnare biofilms; however, there are clear disparities in how individual isolates respond to different mucus fractions to stimulate biofilms. The expression of iron acquisition genes among two genomovar II isolates showed that ferroxidase, TonB receptor and the siderophore synthetase gene were all significantly upregulated among F. columnare biofilms. Interestingly, the siderophore acetyltransferase gene was only shown to be significantly upregulated in one of the genomovar II isolates. This work provides insight into our understanding of the interaction between F. columnare and vertebrate mucus, which likely contributes to the growth of planktonic cells and the transition into biofilms.
... We found that Flavobacterium highly dominated the surface of blades and Paracoccus dominated the surface of vesicles, and both were prominent in iron transport. TonB-dependent OMP, which was crucial for transport of the siderophore-iron complex, has been recognized in several closely related strains of Flavobacterium (Møller et al., 2005;Guan et al., 2013) and several closely related Paracoccus strains had strong abilities to produce siderophores (Bergeron et al., 1985;Bergeron and Weimar, 1990;Sedlácek et al., 2009). These two dominant OTUs might play key roles in supplying iron to hosts under stress. ...
Golden tides dominated by Sargassum spp. are occurring at an accelerated rate worldwide. In China, Sargassum has started to bloom in the Yellow Sea and led to tremendous economic losses, but the underlying biological causes and mechanisms are still unclear. Although algae-associated bacteria were suggested to play crucial roles in algal blooms, the profiles of bacteria associated with drifting Sargassum remain unexplored. In this study, the community structures and functions of Sargassum associated bacteria were analyzed using the high-throughput sequencing data of the V5–V7 hypervariable region of the 16S rRNA gene. Molecular identification revealed that the golden tide analyzed in the Yellow Sea was dominated by a single species, Sargassum horneri. They were a healthy brown color nearshore but were yellow offshore with significantly decreased chlorophyll contents (P < 0.01), which indicates that yellow S. horneri was under physiological stress. The structural and functional analyses of bacterial communities indicated that the drifting S. horneri had an obvious selectivity on their associated bacteria against surrounding seawater. Although the bacterial communities phylogenetically differed between brown and yellow S. horneri (P < 0.01), their dominant functions were all nitrogen and iron transporters, which strongly indicates microbial contribution to blooming of the algal host. For the first time, potential epiphytic and endophytic bacteria associated with Sargassum were independently analyzed by a modified co-vortex method with silica sand. We showed that the composition of dominant endophytes, mainly Bacillus and Propionibacterium, was relatively consistent regardless of host status, whereas the epiphytic operational taxonomic units (OTUs) greatly varied in response to weakness of host status; however, dominant functions were consistent at elevated intensities, which might protect the host from stress related to nitrogen or iron deficiency. Thus, we propose that host physiological status at different intensities of functional demands, which were related to variable environmental conditions, may be a critical factor that influences the assembly of epiphytic bacterial communities. This study provided new insight into the structure and potential functions of associated bacteria with golden tide blooms.
... animal hosts, iron is typically present in transferrin or lactoferrin complexes, which are ironbinding glycoproteins that circulate through the bloodstream (Chart and Trust, 1983). Invading bacterial pathogens can sense this iron-depletion as a signal that they have successfully entered a host, and subsequently induce the expression of genes that facilitate iron uptake as a means to overcome host defenses (Chart and Trust, 1983;Ebanks et al., 2004;Guan et al., 2013;Hirst et al., 1991;Najimi et al., 2009;Santander, 2012). Therefore, the transcriptional stability of each reference genes was evaluated under iron-limiting and iron-rich conditions. ...
Aeromonas salmonicida subsp. salmonicida is a Gram-negative, facultative intracellular
pathogen of a wide range of freshwater and marine fish species. A. salmonicida is the causative
agent of furunculosis, an immunosuppressive disease that typically progresses to septicemia.
Several aspects of A. salmonicida pathogenesis has already been described, but fundamental
genetic aspects of the psychrophilic lifestyle of this bacterium remain unknown.
Reverse transcription quantitative real-time polymerase chain reaction (qPCR) is a
precise molecular technique used to detect very slight changes in gene expression. The
appropriate choice of reference genes is essential for accurate normalization of qPCR gene
expression data. Despite the available abundance of validated reference genes for mesophilic
pathogens, a broad list of validated reference genes for A. salmonicida is not available. Here, we
evaluated seven A. salmonicida reference genes under different culture conditions, including
different growth phases, iron-limited and iron-supplemented conditions, and thermal stress. We determined that hfq maintained the most stable expression, followed by era, recA, rpoB, 16S,
fabD, and gapA. The results of this study provided with an expanded list of reliable reference
genes for A. salmonicida gene expression studies using qPCR
