Chemical and Enzymatic Synthesis of Sialylated Glycoforms of Human Erythropoietin.

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life. EPO carries three N-glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline-assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor.

Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction.

Enzymes activity in a cell is determined by many factors, among which viscosity of the microenvironment plays a significant role. Various cosolvents can imitate intracellular conditions in vitro, allowing to reduce a combination of different regulatory effects. The aim of the study was to analyze the media viscosity effects on the rate constants of the separate stages of the bacterial bioluminescent reaction. Non-steady-state reaction kinetics in glycerol and sucrose solutions was measured by stopped-flow technique and analyzed with a mathematical model developed in accordance with the sequence of reaction stages. Molecular dynamics methods were applied to reveal the effects of cosolvents on luciferase structure. We observed both in glycerol and in sucrose media that the stages of luciferase binding with flavin and aldehyde, in contrast to oxygen, are diffusion-limited. Moreover, unlike glycerol, sucrose solutions enhanced the rate of an electronically excited intermediate formation. The MD simulations showed that, in comparison with sucrose, glycerol molecules could penetrate the active-site gorge, but sucrose solutions caused a conformational change of functionally important αGlu175 of luciferase. Therefore, both cosolvents induce diffusion limitation of substrates binding. However, in sucrose media, increasing enzyme catalytic constant neutralizes viscosity effects. The activating effect of sucrose can be attributed to its exclusion from the catalytic gorge of luciferase and promotion of the formation of the active site structure favorable for the catalysis.

The moonlighting protein fructose 1,6-bisphosphate aldolase as a potential vaccine candidate against Photobacterium damselae subsp. piscicida in Asian sea bass (Lates calcarifer).

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish.

A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. piscicida Cleaves the Peptidoglycan of Potentially Competing Bacteria.

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-containing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA ( PhotobacteriumNlpC-like protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-d-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and Vibrio vulnificus This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG.IMPORTANCE Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.

Identification and biochemical characterization of a novel chondroitin sulfate/dermantan sulfate lyase from Photobacterium sp.

Chondroitin sulfate (CS)/dermatan sulfate (DS) lyases play important roles in structural and functional studies of CS/DS. In this study, a novel CS/DS lyase (enCSase) was identified from the genome of the marine bacterium Photobacterium sp. QA16. This enzyme is easily heterologously expressed and purified as highly active form against various CS, DS and hyaluronic acid (HA). Under the optimal conditions, the specific activities of this enzyme towards CSA, CSC, CSD, CSE, DS and HA were 373, 474, 171, 172, 141 and 97 U/mg of proteins, respectively. As an endolytic enzyme, enCSase degrades HA to unsaturated hexa- and tetrasaccharides but CS/DS to unsaturated tetra- and disaccharides as the final products. Sequencing analysis showed that the structures of tetrasaccharides in the final products of CS variants were not unique but were highly variable, indicating the randomness of substrate degradation by this enzyme. Further studies showed that the smallest substrate of enCSase was octasaccharide for HA but hexasaccharide for CS/DS, which could explain why this enzyme cannot degrade HA hexa- and tetrasaccharides and CS/DS tetrasaccharides further. It is believed that enCSase may be a very useful tool for structural and functional studies and related applications of CS/DS and HA.

Characterization of a novel enzyme from Photobacterium phosphoreum with histidine decarboxylase activity.

Histamine or scombrotoxin fish poisoning is caused by ingestion of bacterially produced histamine in fish. Histamine-producing bacteria generally contain the histidine decarboxylase gene (hdc). However, some strains of Photobacterium phosphoreum are known to produce significant levels of histamine, although the hdc gene in these strains has not been recognized. The objective of this study was to investigate a previously unidentified mechanism of histamine production by P. phosphoreum. We identified a protein with histidine decarboxylase (HDC) activity comparable to activity of the pyridoxal-5-phosphate (PLP) dependent HDC from P. kishitanii and M. morganii. The newly identified protein (HDC2) in P. phosphoreum and P. kishitanii strains, was approximately 2× longer than the HDC protein from other Gram-negative bacteria and had 12% similarity to previously identified HDCs. In addition, the hdc2 gene cluster in P. phosphoreum was identical to the hdc gene cluster in P. kishitanii. HDC2 had optimal activity at 20-35 °C, at pH 4, and was not affected by 0-8% NaCl concentrations. Compared to the hdc gene from P. kishitanii, expression of the hdc2 gene was constitutive and not affected by pH or excess histidine. This newly identified protein explains possible mechanisms of histamine production in P. phosphoreum. Characterization of this protein will help in designing control measures to prevent or reduce histamine production in fish.

Viscous Media Slow Down the Decay of the Key Intermediate in Bacterial Bioluminescent Reaction.

The effects of medium viscosity on the decay rate of the 4a-hydroperoxyflavin intermediate of the bioluminescent reaction was investigated. It was found that at low concentrations of glycerol or sucrose (viscosity 1.1-1.3 cP) the decay rate rises, whereas a further increase in viscosity to 6.2 cP leads to a decrease in the decay rate following a power function with an exponent of 0.82-0.84. Using molecular dynamics methods, it was shown that the presence of glycerol and sucrose molecules causes a change in the mobility of the amino acid residues in the active center of luciferase, particularly those responsible for binding of flavin. The results obtained are indicative of two opposite effects of viscous media with glycerol and sucrose: (1) destabilization of 4a-hydroperoxyflavin due to a change in the structural and dynamic properties of the protein and (2) stabilization of this intermediate by the decrease in the diffusion rate of its decay products.

Engineering a bacterial sialyltransferase for di-sialylation of a therapeutic antibody.

Terminal α-2,6-sialylation of N-glycans is a humanized glycosylation that affects the properties and efficacy of therapeutic glycoproteins. Fc di-sialylation (a biantennary N-glycan with two α-2,6-linked sialic acids) of IgG antibodies imparts them with enhanced anti-inflammatory activity and other roles. However, the microheterogeneity of N-glycoforms presents a challenge for therapeutic development. Therefore, controlled sialylation has drawn considerable attention, but direct access to well-defined di-sialylated antibodies remains limited. Herein, a one-pot three-enzyme protocol was developed by engineering a bacterial sialyltransferase to facilitate the modification of therapeutic antibodies with N-acetylneuraminic acid or its derivatives towards optimized glycosylation. To overcome the low proficiency of bacterial sialyltransferase in antibody remodeling, the Photobacterium sp. JT-ISH-224 α-2,6-sialyltransferase (Psp2,6ST) was genetically engineered by terminal truncation and site-directed mutagenesis based on its protein crystal structure. With the optimized reaction conditions and using activity-based screening of various Psp2,6ST variants, a truncated mutant Psp2,6ST (111-511)-His6 A235M/A366G was shown to effectively improve the catalytic efficiency of antibody di-sialylation. Herceptin and the donor substrate promiscuity allow the introduction of bioorthogonal modifications of N-acetylneuraminic acid into antibodies for site-specific conjugation. 2-AB hydrophilic interaction chromatography analysis of the released N-glycans and intact mass characterization confirmed the high di-sialylation of Herceptin via the optimized one-pot three-enzyme reaction. This study established a versatile enzymatic approach for producing highly di-sialylated IgG antibodies. It provides new insights into engineering bacterial sialyltransferase for adaptation to the enzymatic glycoengineering of therapeutic antibodies and the glycosite-specific conjugation of antibodies.

Cloning and expression of the flavin reductase LuxG from Photobacterium leiognathi YL and its improvement for NADH detection.

In the present study, we aimed to purify and characterize LuxG obtained from Photobacterium leiognathi YL and examine its improvement for NADH detection. To this end, we cloned and expressed the putative luxG gene of P. leiognathi YL in the Escherichia coli BL21 strain. The product of luxG is a flavin reductase that consists of 206 amino acids, corresponding to a subunit molecular mass of ∼26 kDa. Phylogenetic analysis demonstrated that P. leiognathi YL LuxG has a rather distant evolutionary relationship with Frase I of Aliivibrio fischeri and Frp of Vibrio harveyi, but a close evolutionary relationship with Fre from Escherichia coli, which are all enzymes related to oxido-reductase. Further comparison shows that the changes in the functionally conserved sites may contribute to the functional divergence of LuxG and Fre. LuxG could supply reduced flavin mononucleotide (FMN) for bacterial luminescence by catalyzing the oxidation of nicotinamide adenine dinucleotide hydrogen (NADH). Based on this, a coupled pure enzyme bioluminescent system was established and used for NADH detection. The NADH samples with concentrations of 0.1-1 nM were used to validate the linear relationship, and it was found that the logarithmic deviations were less than 3%, which showed more sensitive and stable results than the NADH detection by recombinant E. coli including the exogenously expressed luciferase and intrinsic Fre. Investigation of P. leiognathi YL LuxG would provide a basic understanding of its evolution, and structural and functional properties, which might contribute to the development of a NADH detection kit in the future.

Exploiting the Disialyl Galactose Activity of α2,6-Sialyltransferase from Photobacterium damselae To Generate a Highly Sialylated Recombinant α-1-Antitrypsin.

Sialic acids are sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where a lack of sialylation can reduce bioactivity. Here, we describe how α-2,6-sialyltransferase from Photobacterium damselae can be used to markedly increase the level of sialylation of CHO-produced α-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second disialyl galactose motif Neu5Ac-α2,3(Neu5Ac-α2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the P. damselae in a multienzyme reaction to produce a highly sialylated α-1-antitrypsin. The influence of this unique disialylation on the in vitro activity of α-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.

Comparison of α2,6-sialyltransferases for sialylation of therapeutic proteins.

The development of therapeutic proteins for the treatment of numerous diseases is one of the fastest growing areas of biotechnology. Therapeutic efficacy and serum half-life are particularly important, and these properties rely heavily on the glycosylation state of the protein. Expression systems to produce authentically fully glycosylated therapeutic proteins with appropriate terminal sialic acids are not yet perfected. The in vitro modification of therapeutic proteins by recombinant sialyltransferases offers a promising and elegant strategy to overcome this problem. Thus, the detailed expression and characterization of sialyltransferases for completion of the glycan chains is of great interest to the community. We identified a novel α2,6-sialyltransferase from Helicobacter cetorum and compared it to the human ST6Gal1 and a Photobacterium sp. sialyltransferase using glycoprotein substrates in a 96-well microtiter-plate-based assay. We demonstrated that the recombinant α2,6-sialyltransferase from H. cetorum is an excellent catalyst for modification of N-linked glycans of different therapeutic proteins.

Photobacterium sp. NNA4, an efficient hydroxylamine-transforming heterotrophic nitrifier/aerobic denitrifier.

An efficient heterotrophic nitrifying/aerobic denitrifying strain, Photobacterium sp. NNA4 was isolated from a recirculating aquaculture system (RAS). NNA4 was capable of utilizing ammonia, nitrate or nitrite as sole N-source with maximal removal rates of 12.5 mg/L/h for NH4+N, 16.4 mg/L/h for NO3--N, and 4.5 mg/L/h for NO2--N, respectively. Optimal nitrification conditions were: sodium succinate as C-source, 30-37°C, NaCl 1-4%, pH 7.0-8.0, dissolved oxygen 5.89 mg/L, C/N > 10. Gas chromatography/mass spectrometry and gas chromatography/isotope ratio mass spectrometry analyses showed that N2 and N2O were aerobic denitrification products of nitrite and nitrate. NNA4 could tolerate high concentration of hydroxylamine and displayed efficient hydroxylamine-transforming capability. Hydroxylamine oxidoreductase activity using potassium ferricyanide as electron acceptor was 0.042 U. Results revealed that strain NNA4 could oxidize NH2OH directly to N2O at aerobic conditions. In view of its high removal ability of inorganic nitrogen pollutants and broad salinity tolerance range, NNA4 has great potential in denitrification treatment of types of wastewater with either low salinity (e.g., municipal facilities) or high salinity (e.g., aquaculture, seafood processing).

Development of a novel l-histidine assay method using histamine dehydrogenase and a stable mutant of histidine decarboxylase.

l-Histidine analysis is essential in physiological research and clinical applications because l-histidine concentrations in biofluids are associated with various diseases. However, an enzymatic method for l-histidine quantitation has not yet been established. Here, we describe a novel l-histidine quantitation assay using a combination of histidine decarboxylase (HDC) and histamine dehydrogenase (HDH) enzymes. Wild-type HDC is unstable and completely lost its activity within 50 days of storage at 4 °C in solution. We rationally designed a HDC C57S mutant with markedly improved stability (storage at 4 °C for over 200 days) without altering the enzyme's substrate specificity. Together with HDH, the HDC C57S mutant was applied to quantify l-histidine concentrations in human plasma. The assay showed high precision (<2.0% inter-assay variation) and high accuracy (<5.8% deviation from the results of LC/MS).

Crystallization and structure elucidation of GDSL esterase of Photobacterium sp. J15.

GDSL esterase J15 (EstJ15) is a member of Family II of lipolytic enzyme. The enzyme was further classified in subgroup SGNH hydrolase due to the presence of highly conserve motif, Ser-Gly-Asn-His in four conserved blocks I, II, III, and V, respectively. X-ray quality crystal of EstJ15 was obtained from optimized formulation containing 0.10 M ammonium sulphate, 0.15 M sodium cacodylate trihydrate pH 6.5, and 20% PEG 8000. The crystal structure of EstJ15 was solved at 1.38 Šwith one molecule per asymmetric unit. The structure exhibits α/ß hydrolase fold and shared low amino acid sequence identity of 23% with the passenger domain of the autotransporter EstA of Pseudomonas aeruginosa. The active site is located at the centre of the structure, formed a narrow tunnel that hinder long substrates to be catalysed which was proven by the protein-ligand docking analysis. This study facilitates the understanding of high substrate specificity of EstJ15 and provide insights on its catalytic mechanism.

Biogenic Amine Production by and Phylogenetic Analysis of 23 Photobacterium Species.

Photobacterium species are members of the bacterial communities typically associated with scombrotoxin-forming fish. Reclassification and discovery of new Photobacterium species has caused confusion as to which species are capable of biogenic amine production. We analyzed histamine, cadaverine, and putrescine production by 104 Photobacterium strains representing 23 species. The presence of the genes for histidine decarboxylase ( hdc), lysine decarboxylase ( ldc), and ornithine decarboxylase ( odc) was determined by real-time or conventional PCR and whole genome sequencing. Significant histamine production (>200 ppm) was detected in five Photobacterium species: P. angustum, P. aquimaris, P. kishitanii, P. damselae, and P. phosphoreum. The hdc gene was detected in all of these histamine-producing species except P. phosphoreum. Cadaverine was produced by eight Photobacterium species: P. angustum, P. aquimaris, P. damselae, P. iliopiscarium, P. kishitanii, P. leiognathi, P. mandapamensis, and P. phosphoreum. Putrescine was produced by six Photobacterium species: P. angustum, P. aquimaris, P. kishitanii, P. leiognathi, P. mandapamensis, and Photobacterium sp. Cadaverine production correlated closely with the presence of the ldc gene, but putrescine production did not correlate closely with the presence of the odc gene. Characterization of the biogenic amine production by Photobacterium species will allow identification of these marine bacteria and help ensure that current guidelines account for mitigation of these bacteria.

Disposable luciferase-based microfluidic chip for rapid assay of water pollution.

In the present study, we demonstrate the use of a disposable luciferase-based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3-dihydroxybenzene and 1,4-benzoquinone for the proposed microfluidic biosensor measured 3 µM, 15 mM, and 2 µM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers.

Expression of genes encoding the luciferase from Photobacterium leiognathi in Escherichia coli Rosetta (DE3) and its application in NADH detection.

Cloning of genes encoding the luciferase from Photobacterium leiognathi YL in Escherichia coli Rosetta (DE3) was performed successfully and the expressed forms of lux AB were purified to homogeneity. Experimental measurements revealed that luciferase from Photobacterium leiognathi YL has good thermal stability and a high residual activity at extreme pH values, which are extremely important for its various ecological, industrial and medical applications. Furthermore, we made a first attempt for quantitative detection of NADH by recombinant E. coli Rosetta (DE3) coupled enzyme system. A good linear relationship between luminescence intensity and NADH with low (1-12 nmol/L) and high (10-500 nmol/L) concentration was observed, whose standard curve was y = 772.97× + 4041.1, R2  = 0.9884 and y = 1710× + 4.99 × 105 , R2  = 0.9727, respectively. Our results demonstrate a high sensitivity of recombinant E. coli coupled enzyme system to NADH on the basis of high soluble expression of recombinant luciferase and continuous and stable expression of some NAD(P)H-dependent flavin mononucleotide (FMN) reductases.

α2-6-Neosialidase: A Sialyltransferase Mutant as a Sialyl Linkage-Specific Sialidase.

The lack of α2-6-linkage specific sialidases limits the structural and functional studies of sialic-acid-containing molecules. Photobacterium damselae α2-6-sialyltransferase (Pd2,6ST) was shown previously to have α2-6-specific, but weak, sialidase activity. Here, we develop a high-throughput blue-white colony screening method to identify Pd2,6ST mutants with improved α2-6-sialidase activity from mutant libraries generated by sequential saturation mutagenesis. A triple mutant (Pd2,6ST S232L/T356S/W361F) has been identified with 100-fold improved activity, high α2-6-sialyl linkage selectivity, and ability to cleave two common sialic acid forms, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). It is a valuable tool for sialoglycan structural analysis and functional characterization. The sequential saturation mutagenesis and screening strategy developed here can be explored to evolve other linkage-specific neoglycosidases from the corresponding glycosyltransferases.

Chemobacterial Synthesis of a Sialyl-Tn Cyclopeptide Vaccine Candidate.

A conjugatable form of the tumour-associated carbohydrate antigen sialyl-Tn (Neu5Ac-α-2,6-GalNAc) was efficiently produced in Escherichia coli. Metabolically engineered E. coli strains overexpressing the 6-sialyltransferase gene of Photobacterium sp. and CMP-Neu5Ac synthetase genes of Neisseria meningitidis were cultivated at high density in the presence of GalNAc-α-propargyl as the exogenous acceptor. The target disaccharides, which were produced on the scale of several hundreds of milligrams, were then conjugated by using copper(I)-catalysed azide-alkyne cycloaddition click chemistry to a fully synthetic and immunogenic scaffold with the aim to create a candidate anticancer vaccine. Four sialyl-Tn epitopes were introduced on the upper face of an azido-functionalised multivalent cyclopeptide scaffold, the lower face of which was previously modified by an immunogenic polypeptide, PADRE. The ability of the resulting glycoconjugate to interact with oncofoetal sialyl-Tn monoclonal antibodies was confirmed in ELISA assays.

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish.

Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacterized chromosomal loci encoding a phospholipase (PlpV) and a collagenase (ColP). PlpV was ubiquitous in the subspecies and exerted hemolytic activity against fish erythrocytes, which was enhanced in the presence of lecithin. ColP was restricted to a fraction of the isolates and was responsible for the collagen-degrading activity in this subspecies. Consistent with the presence of signal peptides in PlpV and ColP sequences, mutants for the type II secretion system (T2SS) genes epsL and pilD exhibited impairments in phospholipase and collagenase activities. Sea bass virulence experiments and cell culture assays demonstrated major contributions of PhlyC and PlpV to virulence and toxicity.IMPORTANCE This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, Photobacterium damselae subsp. damselae To date, studies on the genetic basis of virulence were restricted to the pPHDD1 plasmid-encoded toxins Dly and PhlyP. However, the vast majority of the recent isolates of this pathogen from fish farm outbreaks lack this plasmid. Here we demonstrate that the plasmidless strains produce two hitherto uncharacterized ubiquitous toxins encoded in chromosome I, namely, the hemolysin PhlyC and the phospholipase PlpV. We report the main roles of these two toxins in fish virulence and in cell toxicity. Our results constitute the basis for a better understanding of the virulence of a widespread marine pathogen.