Structural Studies of the Lipopolysaccharide of Aeromonas veronii bv. sobria Strain K133 Which Represents New Provisional Serogroup PGO1 Prevailing among Mesophilic Aeromonads on Polish Fish Farms

In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes.


Introduction
The world production of farmed food fish relies increasingly on inland aquaculture, which is typically practiced in a freshwater environment in most countries. It is estimated that freshwater species, such as carp, rainbow trout, catfish, and tilapia, are expected to represent about 62% of total world aquaculture production in 2030, compared with 58 percent in 2016 [1,2]. However, a worrying phenomenon is the occurrence of health disorders and infectious diseases in freshwater fish species, which contribute to large economic losses exceeding $10 billion worldwide. Diseases in aquacultures can be caused by various factors, however the most important among bacterial infections are those caused by motile Aeromonas representatives [3,4].
Aeromonads are Gram-negative, rod-shaped bacteria occurring ubiquitously in freshwater and marine environments, including drinking-water distribution systems, even when the water supply is chlorinated [5][6][7], and wastewater systems [8,9]. Aeromonas bacteria are especially known as opportunistic pathogens of fish. Outbreaks in cultured fish are caused by stressful environmental conditions such as sudden water temperature changes, excessive stocking density, or poor water quality [10]. The infections observed in freshwater fish comprise a wide variety of clinical manifestations, from skin ulceration including gill and fin lesions known as MAI (motile aeromonad infection) to the acute form of the disease, which quickly leads to sepsis, called MAS (motile aeromonad septicemia). In both the MAI and MAS modes of the disease, a high mortality rate of approximately 80% of stock is observed [3].
The pathogenicity of Aeromonas is associated with the production and/or secretion of numerous virulence factors, such as aerolysin, haemolysin, enterotoxins, proteases, and hemagglutinins. Moreover, these proteins and enzymes help to distinguish between potentially pathogenic and non-pathogenic strains [11,12]. The mechanisms of iron acquisition known as metallostasis, have also been described as important factors in the development of aeromonad infections [13][14][15]. In addition, components of the Gram-negative bacterial cell envelope, i.e., the capsule, S-layer, lipopolysaccharide (LPS), outer membrane proteins, and structures involved in the colonization process, e.g., polar flagella and pili, play a significant role in the pathogenicity of Aeromonas [11,16].
The lipopolysaccharide molecule is a glycolipid and a key trigger of innate immune responses, ranging from local inflammation to disseminated sepsis [17,18]. LPS contains three structural regions: the hydrophobic lipid A (endotoxin), a phosphorylated and nonrepetitive core oligosaccharide (core OS), and an O-specific polysaccharide (O-polysaccharide, O-PS, O-antigen) [19]. The O-polysaccharide is a polymer with highly antigenically variable oligosaccharide repeating subunits (a heteropolymeric variant of the O-antigen) and, according to the structure and composition of this surface polysaccharide, bacteria can be classified into different serotypes and serovars [20], which is important for diagnostics and epidemiological monitoring.
Regarding the Aeromonas spp. serotyping, the original classification scheme proposed by Sakazaki and Shimada, which included 44 different O-serogroups (the NIH scheme; National Institute of Health, Japan), was later supplemented by addition of new provisional serogroups, and it currently consists of 97 O-serogroups [21,22]. Serotyping of pathogenic bacteria leads to recognition of the etiological agent associated with specific disease syndrome [23]. Since there are differences in the distribution of the dominant O-serogroups responsible for the onset of various diseases depending on geographical regions [24], as evidenced by epidemiological studies carried out in various areas, including Polish aquacultures indicating to the emergence of new O-antigen variants, there are reasons to extend further or modify the classical serogrouping scheme [25][26][27].
In the present work, we have shown the structural characterization of LPS (including the O-specific polysaccharide) isolated from A. veronii bv. sobria strain K133, which has been classified into the new provisional serogroup PGO1, i.e., the most commonly represented immunotype among isolates pathogenic to carp and trout in Polish aquacultures. We have demonstrated that the O-chain is composed of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of this new serogroup. To the best of our knowledge, this is the first paper describing the occurrence of bacillosamine as a component of Aeromonas O-antigens. Moreover, the composition and structure of the O-PS repeating unit elucidated in the paper is unique among known bacterial polysaccharides.

Bacterial Cultivation, Isolation of LPS, and SDS-PAGE Study
Aeromonas veronii bv. sobria K133 bacteria were cultivated in TSB (tryptic soy broth) at 28 • C for 72 h, and the cell biomass obtained was enzymatically treated, as described in Section 4, and then subjected to lipopolysaccharide extraction using the hot phenolwater method [28]. The HMW (high molecular weight) S-LPS species were recovered only from the phenol phase in a yield of 1.2% of the dry bacterial cell mass. After silver nitrate gel staining, the SDS-PAGE analysis of the phenol-soluble LPS revealed a profile characteristic for glycoforms isolated from smooth bacterial cells composed of both fastmigrating LMW (low molecular weight) species typical of rough R-and semi rough SR-LPS, and slow-migrating species representing S-LPS glycoforms with different chain lengths of the O-antigen polysaccharide (O-PS) (Figure 1a).

Serological Studies of the Aeromonas veronii bv. sobria Strain K133 O-PS
A. veronii bv. sobria strain K133 was serologically typed by agglutination tests using heat-inactivated bacteria and antisera for 44 defined Aeromonas spp. O-serogroups (NIH system) and 20 provisional serogroups (PGO1-PGO20) for selected Polish isolates. Based on these tests, strain K133 was classified into the serogroup PGO1 [27,29]. The affiliation to the serogroup was further confirmed by the reactivity of the PGO1 reference antiserum with the phenol-soluble LPS and with both the whole bacterial cells and the LPS of A. veronii bv. sobria strain K133 in the Western blotting and ELISA experiments, respectively.
The Western blot with the PGO1 antiserum revealed positive reaction in the region of S-LPS and SR-LPS species (Figure 1b). In addition, the stained bands corresponding to the fast-migrating R-LPS may also indicate the presence of immunoglobulins in the antiserum, which recognize structural determinants in the core oligosaccharide region.
Accordingly, in the ELISA experiment (Table 1), the rabbit polyclonal reference antiserum reacted with whole bacterial cells and the LPS of A. veronii bv. sobria strain K133 to the titer of 128,000 and 64,000, respectively. However, the reactions were weaker than that in the homologous system with the PGO1 cells. Adsorption of the PGO1 reference antiserum with the A. veronii bv. sobria K133 cells decreased its reactivity in the homologous system, while there was no reaction of the adsorbed PGO1 antiserum with both the whole bacterial cells and the LPS of A. veronii bv. sobria K133. The latter findings indicated that the adsorption process was complete and resulted in removal of anti-K133 antibodies from the PGO1 reference antiserum. Moreover, these data also demonstrated that the PGO1 antiserum contained additional immunoglobulins recognizing structural determinants that were not found in the A. veronii bv. sobria K133 O-antigen. Therefore, detailed chemical analyses were performed to establish the structure of the O-PS from A. veronii bv. sobria strain K133, which represents the PGO1 serogroup. Kdo (3-deoxy-D-manno-2-octulosonic acid)-the only acidic sugarwas found in the LPS after treatment of the LPS with 48% aqueous HF (hydrofluoric acid), which suggested its phosphorylation [30][31][32]. In addition, the GLC-MS analysis of fatty acids as methyl esters and O-TMS derivatives revealed 3-hydroxytetradecanoic C14:0(3-OH), 3-hydroxyisopentadecanoic Ci15:0(3-OH), dodecanoic C12:0, and tetradecanoic C14:0 acids as the major components in a ratio of 5.5:1.4:2.6:1.0. GlcN was identified as a constituent of the lipid A disaccharide.

Chemical and Mass Spectrometry Analyses of LPS
The negative ion (MALDI-TOF) mass spectrum of the A. veronii bv. sobria K133 lipopolysaccharide (Figure 2a) showed the most intensive signals in the m/z range 1600-2000, which were attributable to the lipid A and core oligosaccharide species (Y-and B-type fragment ions) arising from in-source fragmentation at the ketosidic bond between the core oligosaccharide and the lipid A [33]. The ions at m/z 1768. 19, 1796.23, and 1824.26 corresponded to hexaacylated lipid A species (Y-fragment ions) [25] composed of a bisphosphorylated glucosaminyl disaccharide and substituted by different chain-length fatty acids. The ion at m/z 1796.23 represented the lipid A species substituted by three 3hydroxytetradecanoic acids C14:0(3-OH), one 3-hydroxyisopentadecanoic acid Ci15:0(3-OH), and two tetradecanoic acids C14:0. In turn, the ion at m/z 1768.19 contained a sugar backbone acylated by four 3-hydroxytetradecanoic acids and two dodecanoic acids, instead of two tetradecanoic acids, compared with the ion at m/z 1824. 26. The composition of the ions is shown in Table 2.
The signals at m/z 2027. 6, 1947.6, and 1903.6 (B-fragment ions) were assigned to the core oligosaccharide with the following composition: HexNAc 1 HexN 1 Hex 2 Hep 5 Kdo anh P. The mass difference between the first two ions of 80 amu corresponded to bisphosphorylated and monophosphorylated core oligosaccharides, respectively. In turn, the most abundant ion at m/z 1903.6 represented a core decasaccharide with loss of the carboxyl group of Kdo.
The negative ion mass spectrum of the core oligosaccharide fraction (core OS), which was liberated from LPS after mild acid hydrolysis and separation by GPC, showed the major ion at m/z 1867.58 corresponding to the dephosphorylated core decasaccharide with the composition HexNAc 1 HexN 1 Hex 2 Hep 5 Kdo anh (calcd monoisotopic mass = 1868.629 amu, calcd mass of deprotonated molecule = 1867.621 amu) ( Figure 2b, Table 2). The proposed structure of the core oligosaccharide corresponded to the compositional analysis of the fraction performed by GLC-MS of alditol acetate derivatives.
In addition, the ion at m/z 2708.9, observed at a higher mass range of the MALDI-TOF mass spectrum, was attributed to the core oligosaccharide with one O-antigen repeating unit attached. The mass difference of 841. 31

Structural Studies of O-Polysaccharide (O-PS)
The O-PS was released from the phenol-soluble LPS by mild-acid degradation followed by centrifugation of the lipid A precipitate and isolated, in a void volume, by gel-permeation-chromatography (GPC) on a Sephadex G50 Fine column. The yield of the high-molecular-mass O-PS fraction was 22% of the LPS mass. The GLC-MS sugar analysis of alditol acetates obtained after full acid hydrolysis of the O-PS with 2 M CF 3 CO 2 H showed the presence of galactose (Gal) and galactosamine (GalN) as the major components, in a peak area ratio of 1.0:1.6. There were also smaller amounts of glucose, two heptose isomers, and a component with a longer retention time identified as 2,4-diamino-2,4,6trideoxyhexitol containing a 3-hydroxybutanoyl group (Hb) amido-linked at C-4. The electron impact EI mass spectrum (Figure 3a) of the latter compound as an alditol acetate derivative showed ions at m/z 145, 244, 374, and 387 for the (C-1 ÷ C-2), (C-4 ÷ C-6), (C-1 ÷ C-4) and (C-2 ÷ C-6) primary fragments, respectively, and the derived secondary fragment ions at m/z 85, 124, 254, and 207, which allowed to distinguish the location of the N-Hb group.
The O-polysaccharide structure of A. veronii bv. sobria strain K133 was then studied with the use of 1D and 2D NMR spectroscopy.   Table 3. NAc-N-acetyl groups.  A high-field region of the spectrum included signals for two CH 3 -CH groups: H-6 of QuiN4N and H-4 of Hb at δ 1.21-1.25, one CH 2 group of Hb at δ 2.35, and three signals for N-acetyl groups at δ 2.03, 2.05, and 2.07. Additionally, the 1 H NMR spectrum showed proton signals of CH 2 -OH of hexose and aminohexose residues and CH-OH of H-3 of the 3-hydroxybutanoyl group at δ 4.20, as well as the ring proton signals in the range of δ 3.54-4.21, some of which overlapped.
The analysis of the two dimensional homonuclear ( 1 H, 1 H DQF-COSY, TOCSY, and NOESY) and heteronuclear ( 1 H, 13 C HSQC, 1 H, 13 C H2BC, and 1 H, 13 C HMBC) NMR experiments resulted in the assignment of the 1 H and 13 C resonances to the O-PS of A. veronii bv. sobria strain K133. The 1 H and 13 C NMR chemical shifts are collected in Table 3.
The 1 H, 13 C HSQC spectrum ( Figure 5) contained four correlation signals at δ 4.12/50.6, 4.05/52.8, 3.87/56.2, and 3.87/58.0 of protons at the nitrogen-bearing carbons to the corresponding carbons and showed that the O-PS repeating unit contained N-acetamido sugars. Moreover, the absence of signals at the 13 C coordinate in the region of δ 83.0-88.0 characteristics of the furanose ring demonstrated that all the sugars were pyranoses [36].
The 1 H, 1 H TOCSY and DQF-COSY spectra revealed four spin systems for monosaccharide residues, which were labelled A-D in the order of the decreasing chemical shifts of their H1/H2 protons, and an additional non-sugar spin system corresponding to the N-3-hydroxybutanoyl group (Hb).
The correlations in the two dimentional homonuclear 1 H, 1 H TOCSY, DQF-COSY and NOESY (Supplementary Materials Figure S1) spectra revealed three spin systems for sugar residues having the galacto configuration and one monosaccharide with the gluco configuration (see below).
In the TOCSY spectrum, starting from the H-1 proton signal the correlations with H-2, H-3 and H-4 were visible for spin systems A, B, and D, indicating galacto-configured monosaccharides. The remaining resonances were assigned from the NOESY (Figure 6), DQF-COSY and heteronuclear experiments.
In the 1 H, 13 C HMBC spectrum, correlations of the anomeric proton with carbons C-3 and C-5 were found for the spin system A, and then the proton resonances were assigned from the 1 H, 13   Overlay of (a) the 1 H-detected HSQC spectrum with 13 C decoupling during acquisition, and (b) the anomeric region of the HSQC spectrum measured without decoupling presenting the 1 J C1,H1 coupling constant values of αor β-anomeric configurations of monosaccharides. 1 J C1,H1 for A (175 Hz), for B and C (163-167 Hz), and D (163 Hz). Capital letters and Arabic numerals refer to atoms in sugar residues denoted as shown in Table 3.
In the DQF-COSY and TOCSY spectra, correlations of H-1/H-2 up to H-6 typical of monosaccharide having the gluco configuration were found for spin system C. The corresponding carbons were inferred from the 1 H, 13 C HSQC spectrum. However, given the almost complete coincidence of the H-2,3,4 proton signals, the chemical shifts of C-2, C-3, and C-4 of spin system C were identified after consideration of the two-bond and long-range correlations in the H2BC and HMBC spectra, respectively, and the glycosylation effects on the 13 C NMR resonances [37].
The 1 H, 13 C HSQC spectrum at the 1 H coordinate (Figure 5a) showed two correlation signals at δ 3.87/56.2 and 3.87/58.0 of protons at the nitrogen-bearing carbons to the corresponding carbons, which after including the high-field positions of H-6/C-6 at δ 1.21/17.1 indicated that spin system C was a diamino-6-deoxysugar. The latter finding was confirmed in the HMBC spectrum (Figure 7) by the presence of a long-range correlation between H-6 and C-4 at δ 1.21/58.0, which allowed identifying C-4 as the second nitrogenbearing carbon. The high-field region of the HMBC spectrum at the 1 H coordinate of the methyl protons (δ 1.21) also showed a correlation to C-5 of residue C at δ 72.3, whose low field position indicated β-linked sugar [32,38]. Based on these data and after including a relatively large 3 J H1,H2 coupling constant value of~8 Hz, spin system C was assigned to β-QuipN4N.
The 13 C resonances of the two nitrogen-bearing carbons of QuiN4N were also verified by the two-bond correlations in the 1 H, 13 Table 3.
The 13 C resonance of the NAc carbonyl signals were inferred from the correlations between the H-2 protons of residues A and B (δ 4.12 and δ 4.05) and the corresponding carbons in the HMBC spectrum (δ 175. 5 and 176.4), and between the latter and the methyl proton signals at δ 2.05 and 2.07, respectively.  In the disaccharide fragment A-(1→3)-C, α-D-GalpNAc-(1→3)-β-QuipN4N, the small β-effect of glycosylation on C-4 of β-QuipN4N C (<0.5 ppm) indicated that the linked monosaccharides had the same D absolute configuration. In the case of different D-L absolute configurations, the higher negative β-effect of~1.4 ppm on C-4 would have been observed [41,42].
Based on the data obtained, it was concluded that the O-polysaccharide from the LPS of A. veronii bv. sobria strain K133 is composed of a tetrasaccharide repeating unit and has the structure presented below: To our knowledge, the O-PS from A. veronii bv. sobria strain K133 is unique among bacterial polysaccharide structures as indicated by the Bacterial Carbohydrate Structure Database (http://glyco.ac.ru/bcsdb, (accessed on 10 March 2021)) [43].

Discussion
The inland aquaculture in Poland is focused on two fish species, i.e., carp and rainbow trout [44], the farming of which is endangered due to the stressful environmental conditions and various diseases, with dominance of infections caused by motile Aeromonas bacteria [3,4]. Studies on the occurrence of mesophilic Aeromonas species associated with outbreaks of MAI/MAS in Polish culture facilities showed the A. veronii bv. sobria species as one of the dominant isolates on carp farms, while representatives of A. veronii bv. sobria, A. bestiarum, and A. salmonicida were pathogenic for both carp and trout. Moreover, the veterinary data indicated that, despite their large diversity, only some Aeromonas sp. serogroups seem to be associated with virulence for freshwater fish species [26]. As demonstrated recently, the majority of isolates pathogenic to carp and trout in Polish cultures were positively classified based on the somatic O-antigen variants when the 44 antisera of the NIH scheme were extended to include those for 20 new provisional serogroups of local origin. Moreover, it was evidenced that the highest number of Aeromonas strains isolated from carp and trout represented the serogroup PGO1 [27].
Here, we established the structure of the LPS and the O-specific polysaccharide from A. veronii bv. sobria strain K133, which was isolated from the kidney of carp during an outbreak of MAI/MAS on a Polish fish farm. Strain K133 was classified to the serogroup PGO1, i.e., an immunotype that is common among aeromonads with pathogenicity to fish in Polish aquacultures.
The sugar analysis of the core oligosaccharide revealed D,D-heptose and L,D-heptose residues indicating the type of the core OS shared by A. hydrophila [30,31,45] and A. bestiarum species [46]. This LPS core variant is different from those described for A. salmonicida subsp. salmonicida, in which there was only one heptose isomer (L,D-Hep) [47]. However, some differences were found in the structure of the core region studied here. The chemical and mass spectrometry experiments revealed the following composition of the core oligosaccharide: HexNAc 1 HexN 1 Hex 2 Hep 5 Kdo anh P 1 , whose structure slightly differs from those described for A. hydrophila and A. bestiarum species by the presence of five instead of six heptose residues and an additional N-acetamido hexose (compare with the composition of the core OS of A. hydrophila HexN 1 Hex 2 Hep 6 Kdo 1 P 1 ).
The SDS-PAGE analysis confirmed that the phenol-soluble LPS contained HMW S-LPS glycoforms and thus suggested a highly hydrophobic character of the O-polysaccharide chains. This finding was in agreement with structural analysis, which demonstrated the presence in the O-PS of deoxy-amino sugars and amino sugars with hydrophobic substituents i.e., N-acetyl and N-acyl groups.
The Western blotting and ELISA experiments supported the results of serotyping with the use of the agglutination test showing the classification of the strain K133 to the new provisional serogroup PGO1. As reported recently, using heat-inactivated bacteria and antisera for 44 defined Aeromonas O-serogroups of the NIH system and after complementing the classical scheme with 20 new antisera for provisional serogroups (PGO1-PGO20) of local origin, the positive classification of the Aeromonas sp. isolates based on appropriate somatic antigen increased from 53% (with the use of the NIH system) to nearly 90% after including the new antisera [27,29].
The structure of the O-PS of A. veronii bv. sobria strain K133 has been established and it has been found that the O-antigen of the strain is built up of linear tetrasaccharide repeating units. To the best of our knowledge, the composition of the O-PS is unique among O-chains of Aeromonas spp. and other bacterial polysaccharide structures (Bacterial Carbohydrate Structure Database: http://glyco.ac.ru/bcsdb, (accessed on 10 March 2021)) [43].
The ELISA experiment with the reference PGO1 antiserum, carried out in this study, revealed the positive recognition of surface antigenic elements within the whole bacterial cells and the isolated LPS molecules of A. veronii bv. sobria strain K133, and thus confirmed affiliation of the strain to the PGO1 serogroup. Moreover, the assay also demonstrated that the reference antiserum contained immunoglobulins recognizing a wider range of structural epitopes that were not found in the A. veronii bv. sobria K133 O-polysaccharide, suggesting both similarities and possible differences in the PGO1 and K133 O antigens. It is worth mentioning, that the O-PS structure of the Aeromonas sp. reference strain for the PGO1 serogroup remains unknown. The structural fragment of the O-PS of A. veronii bv. sobria strain K133, containing β1→3-linked 2-acetamido-4-[(S)-3-hydroxybutanoyl]amino-6-deoxyglucose, seems to be one of the key antigenic determinants of the immunospecificity of the PGO1 serotype.
In conclusion, the immunochemical studies of the LPS of Aeromonas spp. bacteria, which are pathogenic to freshwater fish species and represent the dominant serogroups, will contribute to advancement in research targeted at development of an effective vaccine based on the antigenic profile of emergent pathogens and dedicated to specific fish farms as an alternative to antibiotic therapy. In fisheries, where bacterial diseases appear systematically and may bring large losses, immunoprophylaxis consisting of the use of preparations that increase non-specific and/or specific immunity in fish should play a significant role in preventing bacterial diseases. Due to the emerging difficulties of effective prophylaxis caused by the lack of commercial vaccines, the interest in the use of autovaccinations that can offer protection against defined serotypes from a specific region or geographic area is increasing in veterinary fields, including ichthyopathology [4].

Bacterial Strain, Growth Conditions, and LPS Isolation
A. veronii bv. sobria strain K133, serogroup PGO1, was isolated from the kidney of a common carp during an outbreak of MAS/MAI on a Polish fish farm and classified to the species level by restriction analysis of 16S rDNA amplified by PCR [27]. For the LPS studies, strain K133 was obtained from the collection of the Department of Fish Diseases, National Veterinary Research Institute (Puławy, Poland). The bacterium was cultivated with shaking (120 rpm) on tryptic soy broth (TSB) for 72 h at 28 • C. The cells were harvested by low speed centrifugation (8000× g, 20 min). The recovered bacterial cell pellet was washed twice with 0.85% saline and once more with distilled water.
The bacterial cells (5 g dry mass) were suspended in 50 mM phosphate buffer (pH 7.0) containing 5 mM MgCl 2 and treated with lysozyme, RNAse, and DNAse (16 h, 0.6 mg/g) and then with Proteinase K (16 h, 0.6 mg/g). The enzymatically digested biomass was extracted three times with aqueous 45% phenol at 70 • C [35]. Layers separated by centrifugation (3000× g, 45 min, 4 • C) were dialyzed against tap and distilled water. LPS species recovered from the phenol and water layers were purified by ultracentrifugation at 105,000× g (4 h, 18 • C) and freeze-dried to give a yield of 3% of dry bacterial cell mass.

SDS-PAGE
The phenol-soluble LPS fraction (3 µg) of A. veronii bv. sobria strain K133 prepared in the sample buffer (2% SDS and 50 mM Tris/HCl (pH 6.8), 25% glycerol, 0.1% bromophenol blue) was separated in 12.5% SDS-Tricine polyacrylamide electrophoresis gel and the profile was visualized by staining with silver nitrate after oxidation with periodate according to the published method [61].

Serological Studies
Western blotting with the rabbit antiserum PGO1 was performed after transferring SDS-PAGE-separated phenol-soluble LPS of A. veronii bv. sobria strain K133 to Immobilon P (Millipore, St. Louis, MO, USA). The primary antibodies were detected using alkaline phosphatase-conjugated goat anti-rabbit antibodies (Sigma, St. Louis, MO, USA). The blot was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate toluidine (Sigma) for 5 min, as described elsewhere [25]. Polyclonal rabbit O-antiserum against the Aeromonas sp. reference strain for the provisional serogroup PGO1 was the kind gift from Professor Alicja Kozińska (the National Veterinary Research Institute, Puławy, Poland) [29].
The enzyme-linked immunosorbent assay (ELISA) was performed as described previously [62] with some modifications. In short: 1-2 µg of the A. veronii bv. sobria strain K133 LPS or 10-20 µg of whole cell biomasses of A. veronii bv. sobria strain K133 or the Aeromonas sp. PGO1 reference strain per well were coated on flat-bottom F96 Maxisorp Nunc-Immuno plates (Thermo Fisher Scientific, Roskilde, Denmark); polyclonal rabbit PGO1 antiserum and rabbit-IgG specific peroxidase-conjugated goat antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used. 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was used as a substrate for peroxidase; the absorbance (A 405 ) was measured with the help of a Multiskan Go microplate reader (Thermo Fisher Scientific USA, Vantaa, Finland). The reference PGO1 antiserum diluted 1:50 in PBS (phosphate-buffered saline) was adsorbed on K133 cells during 0.5 h incubation on ice, in the ratio of 100 µL of wet biomass to 1 mL of the serum. The cells were removed by centrifugation and the process was repeated two more times [63].

Degradation of LPS and Isolation of O-Polysaccharide
The phenol-soluble LPS sample (100 mg) was heated in aq 2% acetic acid at 100 • C for 3 h, and the lipid A precipitate was removed by centrifugation (13,000× g, 30 min). The supernatant was concentrated and then fractionated by GPC on a column (1.8 × 80 cm) of Sephadex G-50 Fine (Pharmacia, Sweden) using 1% acetic acid as the eluent and monitoring with a differential refractometer (Knauer, Berlin, Germany). The yield of the O-PS fraction was 22% of the LPS portion subjected to hydrolysis.

Chemical Analyses
For neutral and amino sugar analysis, the degraded polysaccharide (dgPS) fraction released from the phenol-soluble LPS after mild acid hydrolysis, the O-PS, and the core oligosaccharide samples were hydrolyzed with 2 M CF 3 CO 2 H (120 • C, 2 h), reduced with NaBD 4 , and peracetylated with a 1:1 (v/v) mixture of acetic anhydride and pyridine (85 • C, 0.5 h). The O-PS was also hydrolyzed with 4 M HCl for 16 h at 100 • C to release QuiN4N, N-acetylated, reduced with NaBD 4 , and peracetylated.
To release acidic sugar (Kdo), LPS was dephosphorylated with 48% aqueous HF (4 • C, 18 h) and dried under vacuum over sodium hydroxide [32]. Methanolysis was performed with 1 M MeOH/HCl (85 • C, 1 h), and the sample was extracted with hexane. The methanol layer was concentrated and the residue was dried and peracetylated.
The absolute configuration of the monosaccharides was determined by GLC of acetylated (S)-(+)-2-octyl glycosides using authentic sugars as standards according to a published method [34], except for QuiN4N, whose configuration was determined upon analysis of glycosylation effects on 13 C resonances in the O-PS.
The absolute configuration of 3-hydroxybutanoic acid (Hb) was determined according to the method of Kenne, et al. [35] with some modifications: after hydrolysis of the O-PS with 2 M CF 3 CO 2 H (120 • C, 4 h), the product was extracted three times with EtOAc, evaporated under nitrogen, and subjected to solvolysis with 2 M HCl in S(+)-2-octanol at 80 • C for 16 h. The sample was concentrated to dryness, and trimethylsilylated derivatives were analyzed by GLC-MS and compared with the retention time of O-TMS (S)-2-octyl esters of authentic (S)-and (R)-3-hydroxybutanoates as references.
Methylation of the O-PS (1.0 mg) was carried out with methyl iodide in dimethyl sulfoxide in the presence of powdered sodium hydroxide as described by Ciucanu and Kerek [64]. The products were recovered by extraction with chloroform/water (1:1, v/v), N-acetylated, hydrolyzed with 2 M CF 3 CO 2 H (120 • C, 2 h), N-acetylated, reduced with NaBD 4 and peracetylated. The partially methylated alditol acetates derivatives were analyzed by GLC-MS.
For fatty acid analysis, a lipid A sample (1 mg) was subjected to methanolysis in 2 M methanolic HCl (85 • C, 12 h). The resulting fatty acid methyl esters were extracted with hexane and converted to their O-trimethylsilyl (O-TMS) derivatives, as described elsewhere [53,65]. The methanol layer containing methyl glycosides was dried and peracetylated with a pyridine-acetic anhydride mixture. The fatty acid derivatives and acetylated methyl glycosides were analyzed by GLC-MS as above.
All the sample derivatives were analyzed on an Agilent Technologies 7890A gas chromatograph (Agilent Technologies, Wilmington, DE, USA) connected to a 5975C MSD detector (inert XL EI/CI, Agilent Technologies, Wilmington, DE, USA). The chromatograph was equipped with an HP-5MS capillary column (Agilent Technologies, 30 m × 0.25 mm, flow rate of 1 mL/min, He as carrier gas). The temperature program for all the derivatives was as follows: 150 • C for 5 min, then 150 to 310 • C at 5 • C/min, and the final temperature was maintained for 10 min.

NMR Spectroscopy
The O-PS sample (7 mg) was deuterium-exchanged by freeze-drying from a 99.95% D 2 O solution and examined in 99.98% D 2 O. 1D and 2D NMR spectra were recorded at 42 • C on a 500 MHz NMR Varian Unity Inova instrument and calibrated with external acetone (δ H 2.225, δ C 31.45). Additionally, for detection of nitrogen-linked protons, 1 13 C HMBC were conducted for signal assignments and determination of the sugar sequence in the repeating unit. The mixing time of 90 and 200 ms was used in the TOCSY and NOESY experiments, respectively. The 1 H, 13 C HSQC experiment (gHSQCAD) with CRISIS based multiplicity editing was optimized for a coupling constant of 146 Hz. The 1 H, 13 C HSQC spectrum (band-selective gHSQCAD) measured without 13 C decoupling was used to determine the 1 J C1,H1 coupling constants for the anomeric carbons. The heteronuclear multiple-bond correlation (HMBC) experiment was optimized for J C,H = 8 Hz, with 2-step low-pass filter 130 and 165 Hz to suppress one-bond correlations.

MALDI-TOF Mass Spectrometry (MS)
The LPS and oligosaccharide samples were analyzed with matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) using a Waters SYNAPT G2-Si HDMS instrument (Waters Corporation, Milford, MA, USA) equipped with a 1 kHz Nd:YAG laser system. Acquisition of the data was performed using MassLynx software version 4.1 SCN916 (Waters Corporation, Wilmslow, UK). Mass spectra were assigned with a multi-point external calibration using red phosphorous (Sigma) and recorded in the negative ion mode. Phenol-soluble LPS and oligosaccharide samples (both at a concentration of 15 µg/µL) were suspended in a water/methanol (1:1, v/v) solution (containing 2 mM EDTA for the LPS sample) and dissolved by ultrasonication. After desalting with the use of cation exchange beads (Dowex 50WX8-200; Sigma), one microliter of each sample was transferred onto a well plate covered with a thin matrix film and allowed to dry at room temperature. The matrix solution was prepared from 2 ,4 ,6 -trihydroxyacetophenone (THAP) (200 mg/mL in methanol) mixed with nitrocellulose (15 mg/mL) suspended in 2-propanol/acetone (1:1, v/v) in a proportion of 4:1 (v/v), according to the published method [65,66].