Functional Genomics of the Aeromonas salmonicida Lipopolysaccharide O-Antigen and A-Layer from Typical and Atypical Strains

The A. salmonicida A450 LPS O-antigen, encoded by the wbsalmo gene cluster, is exported through an ABC-2 transporter-dependent pathway. It represents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Until now, only repeating units with one or two different monosaccharides have been described. Functional genomic analysis of this wbsalmo region is mostly in agreement with the LPS O-antigen structure of acetylated l-rhamnose (Rha), d-glucose (Glc), and 2-amino-2-deoxy-d-mannose (ManN). Between genes of the wbsalmo we found the genes responsible for the biosynthesis and assembly of the S-layer (named A-layer in these strains). Through comparative genomic analysis and in-frame deletions of some of the genes, we concluded that all the A. salmonicida typical and atypical strains, other than A. salmonicida subsp. pectinolytica strains, shared the same wbsalmo and presence of A-layer. A. salmonicida subsp. pectinolytica strains lack wbsalmo and A-layer, two major virulence factors, and this could be the reason they are the only ones not found as fish pathogens.


Introduction
Currently, there are five accepted subspecies of Aeromonas salmonicida: A. salmonicida subsp. salmonicida, masoucida, achromogenes, pectinolytica and smithia [1]. Aeromonas salmonicida subsp. salmonicida is the etiological agent producing the systemic disease named furunculosis, being then an important fish pathogen [2]. This pathogen has been subjected to considerable investigation due to its importance in the farmed fish industry. Its major virulence factor is an S-layer (named A-layer), which principally consists of a unique two-dimensional crystalline tetragonal protein (A-protein with a molecular weight of 49 kDa) array [3], which is tethered to the cell by the lipopolysaccharide (LPS) [4].
Immunolabeling studies have been able to show that the A-layer appears to cover most of the surface of the virulent A. salmonicida [5], nevertheless some LPS molecules are still surface exposed [6]. Both LPS O-antigen and the A-layer are required to fully protect this bacterium from serum killing [7]. Although the A-layer is not completely necessary for the bacterium's resistance to serum killing, it is an important barrier against opsonophagocytosis [8].
In Gram-negative bacteria, the LPS are large amphiphilic molecules consisting of a hydrophilic polysaccharide part, and a covalently bound hydrophobic and highly conserved lipid component, termed lipid A (the bioactive endotoxin subunit). The polysaccharide part can be conceptually divided into two sub-domains: 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. These three regions have been differentiated and formally classified by their chemical structure, degree of conservation, biosynthetic pathways and genetic determination (see general review, [9]).
Some studies have chemically characterized structures of the O-antigen polysaccharide and the core oligosaccharide regions of the LPS from A. salmonicida strain SJ-15 [10,11]. More recent studies describe the structural elucidation of the O-antigen LPS of the A. salmonicida subsp. salmonicida from strains A449 and 80204-1 [12], and their core oligosaccharide region [13]. We studied the functional genetics of the O-antigen of the LPS from A. salmonicida subsp. salmonicida strain A450, whose chemical structure is similar to the previously described for other strains [12]. Furthermore, we found genes encoding for the production and export/assembly of the A-layer characteristic from A. salmonicida subsp. salmonicida strains, between the biosynthetic genes for the LPS O-antigen production (cluster named wbsalmo). We also studied the wbsalmo and genes encoding for the production and export/assembly of the A-layer in different strains of subspecies masoucida, achromogenes, pectinolytica and smithia.

Results
LPS was extracted from enzymatically digested A. salmonicida A450 cells by the Westphal procedure [14] and the O-polysaccharide isolated after mild acid degradation. Sugar analysis by gas-liquid chromatography (GLC) of resultant monosaccharides as alditol acetates and (S)-octyl glycosides revealed that it was composed of L-rhamnose (Rha), D-glucose (Glc), and 2-amino-2-deoxy-D-mannose (ManN) in the approximate molar ratio of 1:0.95:0.9. The methylation analysis of the O-polysaccharide revealed the presence of 2,3-di-O-methylrhamnose, 2-O-methylrhamnose, 2-deoxy-4,6-di-O-methyl-2-(N-methylacetamido) mannose and 2,3,4,6-tetra-O-methylglucose (Table 1), suggesting that the O-antigen polysaccharide contained 3,4-substituted Rha, 3-substituted ManNAc and terminal Glc. The high-resolution electrospray ionization mass spectrum results obtained were consistent with those of compositional and methylation analyses. We found the presence of the fragment ion at m/z 392.5 suggesting that HexNAc was attached to RhaOAc, and a fragment ion at m/z 554.5 was consistent with the consecutive addition of Hex (+162). Additional ions (m/z 757.5, 946.5 and 1108.5) corresponded to the consecutive addition of sugar residues HexNAc, RhaOAc and Hex, respectively. From these initial studies, we concluded that the A. salmonicida A450 LPS O-antigen seems to be identical to the one described for strains A449 and 80204 [12], which can be depicted as follows:

LPS O-Antigen Biosynthesis Gene Cluster (wbsalmo)
An A. salmonicida A450 cosmid-based genomic library was constructed and introduced into E. coli DH5α as previously described for other Aeromonas strains [15]. As we and others reported, A. salmonicida LPS O-antigen contains rhamnose. Thus, we used previously constructed DNA probes from A. hydrophila strain AH-3 rmlA and B genes (two biosynthetic rhamnose genes, [16]) due to their high DNA sequence conservation among Aeromonas strains, and screened the A. salmonicida A450 genomic library by colony Southern blot. Several tetracycline-resistant clones able to cross react with both probes were isolated and sequences flanking the DNA inserted were determined by using oligonucleotides complementary to the pLA2917 [15] cosmid. To complete the nucleotide sequence (GenBank KR704893) other sequence-derived oligonucleotides were designed by us, purchased (Sigma-Aldrich) and used. Analysis of the sequenced regions showed 26 complete putative open reading frames (ORFs) transcribed in the same direction, being 13 of them (ORF1 to 5 and ORF18 to 25) genes involved in the LPS O-antigen biosynthesis (wbsalmo cluster) as indicated in Figure 1   does not belong to any of both clusters. The orf named tnp is encoding a putative transposase protein with a DDE domain from superfamily endonuclease. The oprM gene is incompletely sequenced and it should be noticed that it is also adjacent to the wb O34-antigen LPS from strain AH-3 previously characterized [16]. Triangles indicate the mutants obtained.
The putative transposase protein showed a DDE domain from a superfamily of endonucleases. This domain contains 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.
We observed several ORFs (6 to 17), indicated in blue (Figure 1), between the genes involved in the LPS biosynthesis that belong to a type II secretion system (T2SS) plus the structural gene vapA (ORF17) which encodes the surface A-layer protein. It seems logical that these ORFs are genes that encode for the production and export/assembly of the A. salmonicida A-layer characteristic from these strains. Interestingly, the insertion point of the A-layer genes is immediately downstream of a gene encoding for a Wzm putative protein. Downstream of the A-layer genes, a complete ORF encoding a Wzt putative protein was observed. Wzm and Wzt proteins are characteristics of an ABC-2 type transporter. Finally, we found a gene (ORF26), labeled in red (Figure 1), that encodes for a NAD-dependent dehydratase or UDP-sugar epimerase (named accordingly UDP-ep). This gene does not initially belong to the wbsalmo cluster, because its mutation does not abolish the LPS O-antigen (see next section). Sequence analysis of the wbsalmo gene cluster revealed a conserved JUMPstart sequence with the 8 bp ops (operon polarity suppressor) sequence (GGCGGTAG) 119 bp upstream of the ORF1. The ops sequence is recognized by the bacterial antiterminator RfaH, which can be recruited by the transcription elongation complex to reduce pausing and termination at intergenic sites of polycistronic operons, allowing RNA polymerase to finalise transcription of the distal genes in large operons [17,18].
Analysis of ORFs from wbsalmo and S-layer clusters with their predicted function based on their homology to proteins of known function is shown in Table 2.

Mutant Isolation and Characterization
As described in Materials and Methods section, we obtained in-frame mutants in ORFs 1, 17, 18, 20, 22 and 26 named A450ΔrmlB, A450ΔvapA, A450Δwzt, A450ΔwbbB, A450Δgtf-2 and A450ΔUDP-ep, respectively. As shown in Figure 2, mutants A450ΔrmlB, A450Δwzt, A450ΔwbbB and A450Δgtf-2 were unable to produce LPS O-antigen when analyzed in a SDS-PAGE gel. However, A450ΔvapA and A450ΔUDP-ep mutants showed in the same gels an identical LPS profile as the wild-type strain with O-antigen ladder repetitions. We obtained outer-membrane proteins (OMp) from A450ΔvapA mutant as described in Materials and Methods. Analysis by SDS-PAGE gels showed the lack of the major protein band of approximately 49 kDa compared with the wild type ( Figure 3A). This band reacts with specific serum anti-VapA protein in Western blot analysis ( Figure 3B). Mutant A450ΔUDP-ep showed no changes in OMp. The reintroduction of the corresponding wild-type genes in A450ΔrmlB, A450Δwzt, A450ΔwbbB, and A450Δgtf-2 fully restored the LPS profile of the wild-type strain in silver-stained SDS gels ( Figure 2). A similar situation was observed when wild-type vapA was reintroduced in mutant A450ΔvapA, the presence in the OMp profile of the 49 kDa protein reacting with specific serum anti-A protein was restored ( Figure 3).

Different A. salmonicida Subspecies Strains
When we analyzed the LPS by SDS-PAGE gels from different A. salmonicida subsp. salmonicida strains, we found an identical O-antigen LPS profile in all of them. However, when we analyzed the LPS profile in gels from different A. salmonicida subspecies strains, we found that the strains belonging to A. salmonicida subsp. pectinolytica lack the characteristic LPS O-antigen homogeneous band, while the rest of the subspecies showed it ( Figure 4A).
The analysis of the OMp from different A. salmonicida subspecies strains by Western blot using specific anti-A protein antiserum is shown in Figure 4B. With the exception of two strains identified as A. salmonicida subsp. pectinolytica, all other strains produced a band of approximately 49 kDa which reacted with antiserum against A-protein ( Figure 4B).

Analysis of Fully Sequenced Genomes
When we inspected all the currently available Aeromonas salmonicida genomes, we found two for A. salmonicida subsp. salmonicida strains A449 and 01-B526, one for A. salmonicida subsp. achromogenes strain AS03, one for A. salmonicida subsp. masoucida strain NBRC 13784, one for A. salmonicida subsp. pectinolytica strain 34melT, and none for A. salmonicida subsp. smithia. Despite the different genome annotations (in part because these DNA regions had not been properly studied) with a more accurate comparative genomic analysis, we can conclude that in all the A. salmonicida genomes, besides the one for A. salmonicida subsp. pectinolytica strain 34melT, the wbsalmo is nearly identical. Furthermore, the chromosomal location between the oprM and UDP-ep genes is conserved among them. However, in the A. salmonicida subsp. pectinolytica strain 34melT genome, we found a putative wb cluster completely different. Briefly, the rml genes for rhamnose biosynthesis are not together, wzm-wzt are not present, and besides finding an oprM gene upstream, downstream of the rmlC seems to be a region with partial genes and encoded bacteriophage proteins (see Figure 5 and Table 3 ).

Discussion
The A. salmonicida A450 LPS O-antigen gene cluster (wbsalmo) showed a G + C percentage of approximately 49.1%, which is significantly lower than the expected (59%-63%) for this species. This is characteristic of the wb clusters from different bacteria that can be 10% lower in G + C than the species average. Initially the encoded proteins showed some consistency with the chemical structure of the O-antigen LPS. ORF1 to 4 (in order RmlB, D, A and C, respectively) are the biosynthetic proteins for dTDP-rhamnose production, ORF21 (WecB) is the enzyme that converts UDP-GlcNAc into UDP-ManAc [19], and rhamnose, ManAc, and Glc are the monosaccharide components of the LPS O-antigen. There is no need for UDP-Glc specific genes. Three glycosyl transferases seem to be involved in the biosynthesis of the O-antigen repeating unit (ORF20, named WbbB; ORF22, named Gtf-2 and ORF25, named WbbL). WbbL is a presumptive rhamnosyl transferase, while the WbbB showed a HexNAc transferase domain (here probably acetyl-N-mannosamine), and Gtf-2 could be the transferase for the Glc incorporation to the LPS O-antigen repeating unit. There is also an acetyl transferase (ORF24 named Atf-3) which is in agreement with the acetylated rhamnose of the LPS O-antigen.
ORF5 and 18 were similar to an ABC-2 type transport system integral membrane and an ATP-binding protein, respectively ( Table 2). The ORF5 protein hydrophobicity analysis and identification of the putative transmembrane domains [20] indicate that it is an integral membrane protein. The ORF18 protein contains the sequence GHNGAGKS (amino acid residues 57 to 64) which corresponds to the Walker box A, a motif present in ATP-binding proteins, as well as the ABC transporter family signature YSSGMYVRLAFAVQA (amino acid residues 162-175). Thus, ORF5 and 18 were named wzm and wzt respectively, despite the fact that they are usually found adjacent. Two main known pathways for O-antigen export have been established [9], the Wzy-dependent pathway for heteropolysaccharide structures and the ABC-2 transporter-dependent pathway mainly for homopolysaccharides or disaccharides. The presence of complete Wzm (ORF5) and Wzt (ORF18) showed that this LPS O-antigen belongs to the second pathway and represents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Between the Wzm (ORF5) and Wzt (ORF18), we found the genes responsible for the biosynthesis and assembly of the S-layer (named A-layer), (Figure 1 and Table 2). The S-layer-encoding genes included a large group of T2SS genes plus some encoding hypothetical proteins related to this specific S-layer T2SS system (ORF6 to 16), and the last gene encoding the unique protein that forms the A-layer (vapA, ORF17) characteristic of A. salmonicida strains. The insertion point of this genomic cluster seems to be in the intergenic region between the genes encoding for a Wzm protein, located just upstream of the S-layer genes, and the gene encoding a Wzt protein (ORF18, as previously indicated). Accordingly to this, the A450ΔvapA in frame mutant showed identical LPS profile as the wild-type strain with the same O-antigen profile in SDS-PAGE gels, but lacked the VapA protein being then unable to produce the A-layer. Mutants A450ΔrmlB, A450Δwzt, A450ΔwbbB, and A450Δgtf-2, when analyzed in an SDS-PAGE gel, were unable to produce LPS O-antigen. However, the A450ΔUDP-ep mutant showed an identical LPS profile as the wild-type strain with the presence of O-antigen in the LPS. Then, we concluded that ORF22 (UDP-ep) does not belong to the wbsalmo, because it seems not to be need for LPS O-antigen biosynthesis.
It is characteristic of the LPS O-antigen disaccharides assembled and exported thought an ABC-2 type transporter to contain a large protein (WbbB), like in K. pneumoniae O12 [21,22], that shows two clear domains. The first domain, in K. pneumoniae WbbB consisted of a condensation or capsule polysaccharide biosynthesis domain from amino acid residues 125 to 300; and a second domain with glycosyltransferase activity from GT1 family (625 to 825 amino acid residues) related to HexNAc transferases [21]. The A. salmonicida WbbB (ORF20) is larger than the one in Klebsiella, and showed three domains: capsule polysaccharide biosynthesis (130 to 290 amino acid residues), a glycosyltransferase activity from GT1family 1 (500 to 825 amino acid residues), and an additional glycosyltransferase activity domain from GT2 family (850 to 1050 amino acid residues). The fact there is an additional glycosyltransferase domain in the A. salmonicida WbbB seems to be in accordance with the O-antigen being a trisaccharide instead of a disaccharide.
At this moment, not much can be said about the hypothetical protein with a sulfotransferase domain (ORF 19, named Hyp), no such residue seems to be found in the chemical structure of the LPS O-antigen in these strains [10,12], and also about the hypothetical protein (ORF23 named ArnT) with a 4-amino-4-deoxy-L-arabinose transferase domain. However, it is true that in Aeromonas has been described the presence of a 4-amino-4-deoxy-L-arabinose in the lipid A [23]. Both genes encoding these proteins are clearly conserved among the different A. salmonicida wbsalmo. The biosynthesis of O-antigens starts with the assembly of monomers on an undecaprenol phosphate (lipid carrier), before their incorporation into the LPS molecules, by enzymes that can be present or not in the wb gene cluster [9]. In wbsalmo we could not find such enzyme.
When we inspected and deeply studied all the currently available A. salmonicida genomes, we found that the wbsalmo was conserved in all of the different subspecies with identical gene location besides in A. salmonicida subsp. pectinolytica. This fact is in full agreement with the LPS profiles in SDS-PAGE obtained for the different A. salmonicida strains from several subspecies. Sequence analysis of the A. salmonicida subsp. pectinolytica wb gene cluster from strain 34melT revealed that the LPS O-antigen seems to be exported through a Wzx-Wzy pathway and not an ABC-2 transporter-dependent pathway. However, besides the presence of the typical genes encoding the Wzx and Wzz proteins, no gene encoding a Wzy protein could be found in this cluster. Wzy mutants showed only a single O-antigen repeating unit [9] and, in the case of the LPS profile of A. salmonicida subsp. pectinolytica strains, it seems that a single O-antigen unit could be observed. A. salmonicida subsp. pectinolytica strains clearly show a different O-antigen LPS compared to the other subspecies and lack the A-layer according to our results. No such A-layer protein encoded by vapA gene could be detected by blot hybridization or PCR using appropriate DNA primers ( Figure 6). This gene could not be found in the complete genome of A. salmonicida subsp pectinolytica strain 34melT.
These two features, lack of wbsalmo and A-layer, are in agreement with the fact that atypical A. salmonicida subsp. pectinolytica strains are the only ones not found as pathogens in a wide variety of fish species [24]. Besides, no genome is available for A. salmonicida subsp. smithia. Our results suggest that the wbsalmo should also be conserved in these strains, according to the LPS profile in SDS-PAGE gels.

General DNA Methods
General DNA manipulations were done essentially as previously described [30]. DNA restriction endonucleases, T4 DNA ligase, E. coli DNA polymerase (Klenow fragment), and alkaline phosphatase were used as recommended by the Sigma-Aldrich (St Louis, MO, USA).

DNA Sequencing and Computer Analysis of Sequence Data
Double-stranded DNA sequencing was performed by using the dideoxy-chain termination method [31] from PCR amplified DNA fragments with the ABI Prism dye terminator cycle sequencing kit (PerkinElmer, Barcelona, Spain). Oligonucleotides used for genomic DNA amplifications and DNA sequencing were purchased from Sigma-Aldrich (St Louis, MO, USA). Deduced amino acid sequences were compared with those of DNA translated in all six frames from non-redundant GenBank and EMBL databases by using the BLAST [32] network service at the National Center for Biotechnology Information and the European Biotechnology Information. ClustalW was used for multiple-sequence alignments [33].

Mutant and Plasmid Constructions, Mutant Complementation Studies
The chromosomal in-frame A450ΔrmlB, A450ΔvapA, A450Δwzt, A450ΔwbbB, A450Δgtf-2 and A450ΔUDP-ep deletion mutants were constructed by allelic exchange as described by Milton et al. [26]. Plasmids were transferred to A. salmonicida strains as previously indicated [27]. To complete the allelic exchange, the integrated suicide plasmid was forced to recombine out of the chromosome by growing on agar plates containing 10% sucrose. Mutants were selected based on their survival on plates containing 10% sucrose and loss of the chloramphenicol resistant marker of vector pDM4. The mutations were confirmed by sequencing of the whole constructs in amplified PCR products. The primers used are shown in Table 5.  For complementation studies, the A. salmonicida A450 rmlB, vapA, wzt, wbbB, and gtf-2 genes were PCR amplified using appropriate oligonucleotides obtained from the sequenced clones and chromosomal A450 DNA as template, ligated to plasmid pGEMT (Promega), and transformed into E. coli DH5α. After checked, the corresponding genes were subcloned on plasmid pBAD33-Gm (Table 5) with an arabinose-inducible and glucose-repressible promoter [27,29]. Induction was obtained by adding L-arabinose to a final concentration of 0.2% (w/v). Plasmids were transferred to A. salmonicida strains as previously indicated [27].

LPS Characterization and SDS-PAGE
LPS was obtained after proteinase K digestion of whole cells [34] for screening purposes. LPS samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by silver staining as previously described [34].

LPS Isolation and O-Deacetylation
Cells (3 g dried weight) were digested with DNase, RNase (24 h, 3 mg each) and Proteinase K (36 h, 3 mg) in 25 mM Tris-HCl buffer pH 7.63 containing 2 mM CaCl2 (30 mL), the suspension was dialysed against distilled water and freeze-dried. Digested cells were extracted with aqueous 45% phenol at 68 C [14], the extract was dialysed against tap water without separation of the layers, residual cells were removed by centrifugation, and the supernatant was freeze-dried to give lipopolysaccharide sample. An aliquot (150 mg) was degraded with 0.1 M sodium acetate buffer pH 4.2 for 4 h at 100 C, the lipid precipitate was removed by centrifugation (13,000× g, 20 min), and high-molecular-mass O-polysaccharide (40 mg) was isolated from the supernatant by gel-permeation chromatography on a column (50 × 2.5 cm) of Sephadex G-50 Superfine in pyridinium acetate buffer (4 mL pyridine and 10 mL HOAc in 1 L water) using a Knauer differential refractometer (Knauer, Germany) for monitoring. The