The Aeromonas salmonicida Lipopolysaccharide Core from Different Subspecies: The Unusual subsp. pectinolytica

Initial hydridization tests using Aeromonas salmonicida typical and atypical strains showed the possibility of different lipopolysaccharide (LPS) outer cores among these strains. By chemical structural analysis, LPS-core SDS-PAGE gel migration, and functional and comparative genomics we demonstrated that typical A. salmonicida (subsp. salmonicida) strains and atypical subsp. masoucida and probably smithia strains showed the same LPS outer core. A. salmonicida subsp. achromogenes strains show a similar LPS outer core but lack one of the most external residues (a galactose linked α1-6 to heptose), not affecting the O-antigen LPS linkage. A. salmonicida subsp. pectinolytica strains show a rather changed LPS outer core, which is identical to the LPS outer core from the majority of the A. hydrophila strains studied by genomic analyses. The LPS inner core in all tested A. salmonicida strains, typical and atypical, is well-conserved. Furthermore, the LPS inner core seems to be conserved in all the Aeromonas (psychrophilic or mesophilic) strains studied by genomic analyses.


INTRODUCTION
The smooth lipopolysaccharide (LPS) in Gram-negative bacteria consists of large amphiphilic molecules with a hydrophilic polysaccharide and a hydrophobic highly conserved lipid component covalently bound. This lipid, named lipid A, is the bioactive endotoxin subunit. The polysaccharide section is mainly formed by two parts: one more internal and conserved, the core region, and one more external and highly variable, the O-specific chain, named also O-antigen for its immunogenic properties. Smooth LPS molecules show both polysaccharide parts, while rough LPS molecules only the completed or truncated LPS core. The lipid A, LPS-core, and O-antigen LPS have been differentiated and formally classified by their chemical structure, degree of conservation, biosynthetic pathways and genetic determination (see general review Aquilini and Tomás, 2015).
The LPS-core is also subdivided in two regions: inner and outer core. Within a genus or family, the structure of the inner core tends to be well-conserved, and typically consists of unusual sugars, particularly 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and heptoses (Hep; Holst, 2002). The outer core shows more structural diversity, is characterized by more common hexose sugars such as glucose (Glc), galactose (Gal), N-acetyl galactosamine (GalNAc) and N-acetyl glucosamine (GlcNAc), and is more variable than the inner core (Holst, 2007).

Genetic General Methodology
General DNA manipulations were done essentially as previously described, as well as the DNA sequencing and bioinformatics analysis of sequenced data (Aquilini et al., 2014).

Dot Blot Hybridizations
Total DNA was denatured after 5 min boiling, chilled on ice for 5 min. After, DNA samples were spotted onto prewetted in 2x SSC Hybond N1 (Amersham) nylon membrane and fixed by UV irradiation. Prehybridization was performed in a solution of 5x SSC, 0.1% N-lauroyl sarcosine, 0.02% SDS, 5% blocking reagent (Roche), and 50% formamide for 2 h at 42 • C. Hybridization with the correspondent labeled probe (20 ng/ml) with digoxigenin was performed for 18 h at 42 • C. The alkaline phosphatase detection system was finally carried out using the enhanced chemiluminescence detection system (Amersham) according to the manufacturer's instructions.

Plasmid Constructions for Gene Overexpression and Mutant Complementation Studies
For gene complementation studies of previously isolated A. salmonicida A450 and A. hydrophila AH-3 core mutants, the corresponding genes from chromosomal DNA of different A. salmonicida subspecies strains were PCR-amplified using specific WahD-Pect Rev: 5 -gcTCTAGAcgacaagatcatcgccaat-3 Primers contain SmaI(bold and capital letters) and XbaI(underlined and capital letters) restriction sites. The PCR amplified product was ligated to SmaI-XbaI digested pBAD33-Gm.
primer pairs ( Table 2) and ligated to pGEMTeasy plasmid. To generate pBAD33-Gm constructions pGEMT plasmids with the different genes were double digested with XbaI and SmaI and the DNA fragment obtained in each case ligated to pBAD33-Gm double digested with the same enzymes. Plasmid pBAD33-WaaL smi was generated using the primers designed for pBAD33-WaaL mas . pBAD33-Gm plasmids into E. coli MC1061 were then transferred into the different mutants by triparental mating using the mobilizing strain HB101/pRK2073 (Jimenez et al., 2009). Mutants were selected on plates containing gentamicin and nalidixic acid for the A450strain, and gentamicin and rifampicin in case of the AH-3 strain. Each gene was expressed from the arabinose-inducible and glucose-repressible pBAD33-Gm promoter.

LPS Isolation and SDS-PAGE
For screening purposes LPS was obtained after proteinase K digestion of whole cells and the LPS samples were separated by SDS-PAGE or SDS-Tricine-PAGE and visualized by silver staining as previously described (Aquilini et al., 2014). Cultures for analysis of LPS were grown in TSB at 20 • C. Dried bacterial cells of each strain in 25 mM Tris· HCl buffer containing 2 mM CaCl 2 pH 7.63 (10 ml g −1 ) were treated at 37 • C with RNAse, DNAse (24 h, 1 mg g −1 each), and then with proteinase K (36 h, 1 mg g −1 ). The suspension was dialyzed and lyophilized, and the LPS was extracted by the phenol-water procedure (Westphal and Jann, 1965). A portion of the LPS (∼50 mg) from each strain was heated with aqueous 2% acetic acid (6 ml) at 100 • C for 45 min. The precipitate was removed by centrifugation (13,000g × 20 min) and the supernatant fractionated on a column (56 cm × 2.6 cm) of Sephadex G-50 (S) in 0.05 M pyridinium acetate buffer pH 4.5 with monitoring using a differential refractometer. An oligosaccharide fraction was obtained in a yield 9-20 % depending on the strain.

Methylation Analysis and Mass Spectrometry
The methylation analyses were performed as previously described (Jimenez et al., 2009). Positive-ion reflectron time-of-flight mass spectra (MALDI-TOF) were acquired on a Voyager DE-PR instrument (Applied Biosystems) equipped with a delayed extraction ion source and used as previously described (Jimenez et al., 2009).

Comparative Genomics and Reannotation
For each analyzed genome we gathered all CDS and pseudo-CDS information by parsing NCBI GenBank records. When we obtained the UniProt Knowledge Base records for these loci using the cross-reference with Entrez GeneIDs and parsed them for gene names, functional annotations, and associated COG, PFAM, and TIGRFAM protein domains were studied. To annotate orthologs, we wrote custom scripts to analyze reference sequence alignments made to subject genomes with blastn and tblastn via NCBI's Web application programming interface. Briefly, we manually confirmed contextually accurate alignments, and then the script integrated coordinates and sequence information from both BLAST methods to locate the bounds of the reference gene in the subject genome; if an aligned start or stop codon was not located, we manually inspected the region. The script then analyzed alignments for insertions, deletions, premature stop codons, frameshifts, and changes to the start codon. An alignment in the same genomic context with >95% amino acid identity, excluding gaps and truncations, was our initial cutoff for orthology. The genomes of subsp. salmonicida A449, subsp. masoucida strain NBRC13784, subsp. achromogenes strain AS03 and subsp. pectinolytica strain 34melT are located at the GenBank accession numbers: CP000644, BAWQ00000000, AMQG00000000.2 and ARYZ00000000.2, respectively. The complete nucleotide sequences of the three A. salmonicida A450 chromosomal regions containing the LPS core biosynthetic genes described here have been assigned GenBank accession numbers FJ238464, FJ238465, and FJ238466, respectively. The complete nucleotide sequences of the three A. hydrophila AH-3 chromosomal regions containing LPS core biosynthesis genes described here have been assigned the following GenBank accession numbers: EU296246, EU296247, and EU296248.

RESULTS
We previously established the genomics and proteomics of the A. salmonicida subsp. salmonicida A450 strain waa (Jimenez et al., 2009; Figure 1). We studied by Colony Southern blot analysis, using several DNA probes, the waa region of A. salmonicida in subspecies masoucida, achromogenes, pectinolytica, and smithia. The initial selected DNA probes from strain A450 corresponded to complete wasC for chromosomal region 1, complete waaE for region 2, and complete waaC for region 3. WasC is the glycosyltransferase that links Gal to HepV in an α-1,6 linkage, WaaE the glycosyltransferase that links Glc to HepI in a β-1,4 linkage, and WaaC the heptosyltransferase that links HepI to Kdo in an α-1,5 linkage (Figure 1). A positive reaction was obtained with all the subspecies genomic DNA against probes from regions 2 and 3 (Table 3). However, either subspecies pectinolytica or achromogenes showed no reaction   Frontiers in Microbiology | www.frontiersin.org against wasC probe from region 1. Subspecies masoucida and smithia rendered a positive reaction against this probe. When we used two additional DNA probes from region 1, wahA and wasD (Figure 1), a positive reaction was obtained with either subspecies pectinolytica or achromogenes genomic DNA. These results prompted us to study the LPS-core of the different A. salmonicida subspecies masoucida, pectinolytica, and achromogenes.
A. salmonicida subsp. masoucida Composition analysis of the strain CECT896T core oligosaccharide from purified LPS by GLC showed the presence of Glc, Gal, GlcN (glucosamine), GalNAc, L,D-Hep, and Kdo in the ratios 1:0.9:0.9:0.8:4.7:0.9, respectively. The mass spectrum from this core oligosaccharide sample showed a major molecular ion peak at m/z 1.888,60 (Figure 2A), corresponding to the full core (calculated molecular mass, 1.887,60 atomic mass units). This molecular mass is essentially similar to those reported for both wild-type A. salmonicida subsp. salmonicida strains A449 and 80204-1 (Wang et al., 2006). Similar to other reported cases, some structural heterogeneity was observed, which was associated with the existence of Kdo in both normal and anhydro forms. The signal observed could be attributed to Kdo 1 , Hep 5 , Hex 2 , HexN 1 , HexNAc 1 . Methylation analysis showed that the core oligosaccharide was characterized by containing similar molar ratios of terminal Gal, GlcN, GalNAc, and L,D-Hep. In addition, 6-substituted Glc, 2-substituted Hep, 7-substituted Hep, 4,6-bisubstituted Hep, and 3,4,6-trisubstituted Hep were found. The complete presumptive structure of the LPS from A. salmonicida subsp. masoucida strain CECT896T is shown in Figure 2B.
The annotation of the waa region in A. salmonicida subsp. masoucida strain NBRC13784 was revised. Comparative genome analysis between the reannotated and the ortholog region in A. salmonicida subsp. salmonicida strain A450 (Jimenez et al., 2009), showed identical genes (Figure 3). The predicted functions encoded by the reannotated waa gene cluster of this A. salmonicida subsp. masoucida were in agreement with the chemical data obtained. Furthermore, the relative mobility of the LPS-core in a silver-stained SDS-PAGE gel from A. salmonicida subsp. masoucida strain NBRC13784 was identical to the mobility of the LPS-core from strain A. salmonicida subspecies salmonicida strain A450 (Figure 4).
A. salmonicida subsp. achromogenes GLC analysis of the strain CECT4238 core oligosaccharide from purified LPS showed the presence of Glc, GlcN, GalNAc, L,D-Hep, and Kdo in the ratios 1:1:0.9:4.5:0.9, respectively. The mass spectrum from this core oligosaccharide sample showed a major molecular ion peak at m/z 1.725,43 (Figure 5A), corresponding to the full core (calculated molecular mass, 1.726,10 atomic mass units). The signal observed was attributed to Kdo 1 , Hep 5 , Hex 1 ,   HexN 1 , HexNAc 1 . Similar to previous results some structural heterogeneity was observed due to Kdo in both normal and anhydro forms. Methylation analysis showed that the core oligosaccharide was characterized by containing similar molar ratios of terminal GlcN, GalNAc, and L,D-Hep. In addition, 6-substituted Glc, 2-substituted Hep, 7-substituted Hep, 4substituted Hep, and 3,4,6-trisubstituted Hep were found. This core fraction was found to be essentially similar to those reported for wild-type A. salmonicida subsp. salmonicida strains, with the lack of the Gal linked in a α1-6 linkage to L,D-HepV (Jimenez et al., 2009). The complete presumptive structure of the LPS from A. salmonicida achromogenes is shown in Figure 5B. Only one complete genome of A. salmonicida subsp. achromogenes is currently available from strain AS03 (Han et al., 2013). When we revised this region by comparative genomics data in other A. salmonicida, we found the genes indicated in Figure 3, with a completely lack of wasC and hldD and the presence of a putative transposase. WasC is the glycosyltransferase that links Gal in an α-1,6 linkage to L,D-HepV in the LPS core of A. salmonicida subsp. salmonicida A450 (Jimenez et al., 2009; Figure 1) and HldD is the epimerase for the L,D-Hep and D,D-Hep (Read et al., 2004). The predicted functions encoded by the genes in this region were in agreement with the chemical data. Furthermore, the relative mobility of the LPS-core from A. salmonicida subsp. achromogenes strain CECT4238 is in a silver-stained SDS-PAGE gel was higher than the mobility of the LPS-core from strain A. salmonicida subsp. salmonicida strain A450 (Figure 4), which was in agreement with the loss of a monosaccharide residue (Jimenez et al., 2009). A. salmonicida subsp. pectinolytica Composition analysis of the strain CECT5752T core oligosaccharide from purified LPS by GLC revealed the presence of Glc, Gal, GlcN, D-glycero-D-manno-heptose (D,D-Hep), L-glycero-D-manno-heptose (L,D-Hep), and Kdo in the ratios 1:0.7:0.9:2.1:4.3,respectively. The major molecular ion peak at m/z 1.857,63 in its mass spectrum ( Figure 6A) corresponded with calculated molecular mass 1.857,61 atomic mass units. The signal observed was attributed to Kdo 1 , Hep 6 , Hex 2 , HexN 1 . Methylation analysis resulted in identification of terminal Gal, 6-substituted Glc, terminal GlcN,terminal D,4, terminal L,D-Hep, 2-substituted L,D-Hep, 7-substituted L,D-Hep, and 3,4,6-trisubstituted L,D-Hep. The oligosaccharide sample from the A. salmonicida subsp. pectinolytica strain CECT5752T was found to be essentially identical to that of A. hydrophila AH-3 serogroup O34 (Jimenez et al., 2008), i.e., the same full core LPS. The complete presumptive structure of the LPS from A. salmonicida pectinolytica is shown in Figure 6B.
A comparative "in silico" analysis of the reannotated region 1 from the A. salmonicida subsp. pectinolytica strain 34melT showed identical genes to A. hydrophila AH-3 serotype O34 but not to any of the A. salmonicida strains. As can be observed in Figure 3, A. salmonicida subsp. pectinolytica strain 34melT shows wahB, wahC, and wahD genes from A. hydrophila AH-3 (in red) and lack the wasB, wasC, and wasD genes characteristic of A. salmonicida strains (in green). Also, Figure 4 shows that this strain lacks the characteristic A. salmonicida O-antigen LPS and present some bands probably from another kind of O-antigen LPS (Merino et al., 2015).

DISCUSSION
The bacterial species A. salmonicida comprises five subspecies. A. salmonicida subsp. salmonicida is known as typical A. salmonicida, causing furunculosis in salmonid fish (Bernoth, 1997). Atypical A. salmonicida include the other four subspecies: masoucida, achromogenes, smithia, and pectinolytica which, with the exception of A. salmonicida subsp. pectinolytica, are found as pathogens in a wide variety of fish species (Gudmundsdottir and Bjornsdottir, 2007). A. salmonicida subsp. pectinolytica strains are readily distinguished from the other psychrophilic aeromonads using the following phenotypic characteristics: growth at 35 • C, melanin production, growth on KCN broth, mannitol and sucrose fermentation with gas from glucose, and indole plus Voges Proskauer assays. Its ability to degrade polypectate is an unusual feature among Aeromonas species (Pavan et al., 2000).
Interestingly, the structure of the LPS core oligosaccharide from A. salmonicida subsp. pectinolytica is also consistent with the established core structure of A. hydrophila strain AH-3 serotype O34 (Jimenez et al., 2008). Both structures are identical with respect to its inner and outer core regions  from the published fully sequenced genome, the predicted gene functions were in agreement with the chemical structure. Either by gene analysis or by complementation studies the region 1 of waa from A. salmonicida subsp. pectinolytica corresponds to the A. hydrophila AH-3 waa determined. The genomic analyses of the A. salmonicida subsp. pectinolytica region 1 from strain 34melT versus the Aeromonas whole genomes from mesophilic strains found in Pubmed (http: //www.ncbi.nlm.nih.gov/genome/?term=Aeromonas) rendered that approximately 89% of the strains contain the same region 1. Nevertheless, from the 121 whole genomes inspected, 13 of them belonging to the species A. hydrophila, A. veronii, A. caviae, A. media, and Aeromonas sp. showed some different genes ( Table 4). Aeromonas salmonicida subsp. achromogenes showed a disaccharide in its LPS outer core of β-D-GalpNAc-(1→4)-L-α-D-Hepp-(1→) instead of the previously mentioned A. salmonicida subsp. salmonicida trisaccharide. When we inspected and deeply studied the unique A. salmonicida subsp. achromogenes fully sequenced genome, the analysis and reannotation of the region 1 was in agreement with the biosynthesis of this chemical structure. The wasC and hldD were absent from region 1 of A. salmonicida subsp. achromogenes waa and instead a transposase was present. The transposase DDE found in subsp. achromogenes strain AS03 contains two domains Pfam 13737 and 01609, which are members of the DDE superfamily, which contain three carboxylate residues that are believed to be responsible for coordinating metal ions needed for catalysis. The catalytic activity of this enzyme involves DNA cleavage at a specific site followed by a strand transfer reaction. This family contains transposases for mainly insertion sequence (IS) 4 or 421 (Klaer et al., 1981). WasC is the glycosyl FIGURE 8 | Alignment of the WaaL aminoacid sequence from A. salmonicida subsp. salmonicida A450, A. salmonicida subsp. masoucida NBRC13784, A. salmonicida subsp. achromogenes strain AS03, A. salmonicida subsp. pectinolytica strain 34melT, and A. salmonicida subsp. smithia CECT5179. Different aminoacids residues among the sequences are labeled in red and bold and inside a square box. transferase that links Gal in a α1-6 linkage to L,D-HepV in the LPS core (Figure 1) and this monosaccharide residue is missing in the outer core LPS. By genomic analyses we could confirm the complete absence of wasC over the genome and only 126bp are retained between the transposase and wahA genes (11,2% of total gene). No fragment of wasC, was found retained upstream of the transposase gene. Therefore, a complex rearrangement event is probably responsible of the loss of the hldD and wasC genes. HldD (the epimerase for D,D-Hep) is not needed in A. salmonicida subsp. achromogenes LPS-core because D,D-Hep is not found. No hldD gene could be found by genomic analyses in the subsp. achromogenes strain AS03 total genome. Accordingly, the A. salmonicida subsp. achromogenes strains LPS-core migration in SDS-PAGE is faster than the one observed for LPS-core of A. salmonicida subsp. salmonicida strains.
No changes in the outer core trisaccharide (α-D-Galp-(1→6)-β-D-GalpNAc-(1→4)-L-α-D-Hepp-1→) are found in A. salmonicida subsp. masoucida strains, being region 1 of A. salmonicida subsp. salmonicida waa identical to the subspecies masoucida according to chemical structure data, genomic information, LPS-core SDS-PAGE gel migration, and complementation studies. Besides that no full genome is still available for A. salmonicida subsp. smithia strains, the complementation studies and the LPS-core SDS-PAGE gel migration suggest that region 1 of A. salmonicida subsp. smithia is probably identical to the one of A. salmonicida subsp. salmonicida.
No changes were observed in regions 2 and 3 of waa from A. salmonicida subspecies. These data were obtained either by hybridization analysis or by genome study of the different public complete genomes of A. salmonicida strains independently of the subspecies. Furthermore, the genomic analyses of the Aeromonas whole genomes from mesophilic strains found in Pubmed (http://www.ncbi.nlm.nih.gov/genome/?term=Aeromo nas) indicate that these genomic regions were identical in all the Aeromonas strains studied, either psychrophilic or mesophilic.
WaaL is the ligase enzyme that links the O-antigen LPS to the lipidA-LPS core, and shows two clear features. The enzyme catalyzes the formation of a glycosidic bond but does not share any protein motif with usual glycosyltransferases, and second the specificity of the reaction is based on the requirement for a specific lipid A-core OS acceptor structure but not the O-antigen LPS or any other undecaprenol-P-linked substrate (Valvano, 2011). According to these features, the WaaL from subsp. salmonicida, subsp. masoucida, and subsp. smithia are identical in amino acid sequence (Figure 8). WaaL subsp. smithia sequence was obtained after sequencing pBAD33-WaaL smi . WaaL from subsp. achromogenes showed a large similarity (nearly identity only with a few amino acid residues changes) to the previous ones, while WaaL from subsp. pectinolytica showed a clearly decreased similarity versus the rest of the WaaL from other salmonicida subspecies (Figure 8). The A. salmonicida subsp. pectinolytica WaaL from strain 34melT showed more identity with many WaaL from several mesophilic Aeromonas strains belonging to different species than to WaaL from other A. salmonicida subspecies.
It can be observed that the LPS inner core in A. salmonicida strains is well-conserved; however, there is some structural diversity in the LPS outer core. From the different typical and atypical A. salmonicida strains we can conclude that subsp. salmonicida, subsp. masoucida, and probably subsp. smithia strains shared the same kind of LPS outer core. A. salmonicida subsp. achromogenes strains showed a similar LPS outer core but lacked one branched external residue not affecting the O-antigen LPS linkage. However, A. salmonicida subsp. pectinolytica strains showed a rather changed LPS outer core, identical to many mesophilic Aeromonas strains LPS outer core. However, these LPS-core genes those are structural non-variable genes could be among others of interest for specific phylogenetic analyses.