Structure of the K141 capsular polysaccharide produced by Acinetobacter baumannii isolate KZ1106 that carries KL141 at the chromosomal K locus.

The structure of the K141 type capsular polysaccharide (CPS) produced by Acinetobacter baumannii KZ1106, a clinical isolate recovered from Kazakhstan in 2016, was established by sugar analyses and one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS was shown to consist of branched tetrasaccharide repeating units (K-units) with the following structure: This structure was found to be consistent with the genetic content of the KL141 CPS biosynthesis gene cluster at the chromosomal K locus in the KZ1106 whole genome sequence. Assignment of the encoded enzymes allowed the first sugar of the K unit to be identified, which revealed that the ß-d-GlcpNAc-(1→3)-d-GlcpNAc bond is the linkage between K-units formed by the WzyKL141 polymerase.

The Acinetobacter baumannii K239 capsular polysaccharide includes heptasaccharide units that are structurally related to K86 but joined by different linkages formed by different Wzy polymerases.

The K239 type capsular polysaccharide (CPS) isolated from Acinetobacter baumannii isolate MAR19-4435 was studied by sugar analysis, one- and two-dimensional 1H and 13C NMR spectroscopy. K239 consists of branched heptasaccharide repeats (K-units) comprised of five residues of l-rhamnose (l-Rhap), and one residue each of d-glucuronic acid (d-GlcpA) and N-acetyl-d-glucosamine (d-GlcpNAc). The structure of K239 is closely related to that of the A. baumannii K86 CPS type, though the two differ in the 2,3-substitution patterns on the l-Rhap residue that is involved in the linkage between K-units in the CPS polymer. This structural difference was attributed to the presence of a gtr221 glycosyltransferase gene and a wzyKL239 polymerase gene in KL239 that replaces the gtr80 and wzyKL86 genes in the KL86 CPS biosynthesis gene cluster. Comparison of the two structures established the role of a novel WzyKL239 polymerase encoded by KL239 that forms the ß-d-GlcpNAc-(1→2)-l-Rhap linkage between K239 units. A. baumannii MAR19-4435 was found to be non-susceptible to infection by the APK86 bacteriophage, which encodes a depolymerase that specifically cleaves the linkage between K-units in the K86 CPS, indicating that the difference in 2,3-substitution of l-Rhap influences the susceptibility of this isolate to bacteriophage activity.

Revised structure of the polysaccharide from Acinetobacter baumannii LUH5551 assigned as the K63 type capsular polysaccharide.

K63 capsular polysaccharide produced by Acinetobacter baumannii isolate LUH5551 (previously designated isolate O24) was re-examined using sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. Though previously reported as O24 consisting of linear tetrasaccharide units that include a 7-acetamido-5-acylamino form of 8-epilegionaminic acid [8eLeg5R7Ac, acylated at C5 with (S)-3-hydroxybutanoyl or acetyl (1:1)], the elucidated structure of the K63 type capsule was found to include a derivative of 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid, Leg5Ac7R, where R is either (S)-3-hydroxybutanoyl or an acetyl group (∼1:1 ratio). This finding is consistent with the presence of the lgaABCHIFG gene module for Leg5Ac7R biosynthesis in the KL63 gene cluster at the capsular polysaccharide (CPS) biosynthesis K locus in the LUH5551 genome. The glycosyltransferases (Gtrs) and Wzy polymerase encoded by KL63 were assigned to linkages in the linear K63 tetrasaccharide unit and linkage of the K63 units.

Depolymerisation of the Klebsiella pneumoniae Capsular Polysaccharide K21 by Klebsiella Phage K5.

Klebsiella pneumoniae is a pathogen associated with various infection types, which often exhibits multiple antibiotic resistance. Phages, or bacterial viruses, have an ability to specifically target and destroy K. pneumoniae, offering a potential means of combatting multidrug-resistant infections. Phage enzymes are another promising therapeutic agent that can break down bacterial capsular polysaccharide, which shields K. pneumoniae from the immune response and external factors. In this study, Klebsiella phage K5 was isolated; this phage is active against Klebsiella pneumoniae with the capsular type K21. It was demonstrated that the phage can effectively lyse the host culture. The adsorption apparatus of the phage has revealed two receptor-binding proteins (RBPs) with predicted polysaccharide depolymerising activity. A recombinant form of both RBPs was obtained and experiments showed that one of them depolymerised the capsular polysaccharide K21. The structure of this polysaccharide and its degradation fragments were analysed. The second receptor-binding protein showed no activity on capsular polysaccharide of any of the 31 capsule types tested, so the substrate for this enzyme remains to be determined in the future. Klebsiella phage K5 may be considered a useful agent against Klebsiella infections.

The Acinetobacter baumannii K70 and K9 capsular polysaccharides consist of related K-units linked by the same Wzy polymerase and cleaved by the same phage depolymerases.

IMPORTANCE: Bacteriophage show promise for the treatment of Acinetobacter baumannii infections that resist all therapeutically suitable antibiotics. Many tail-spike depolymerases encoded by phage that are able to degrade A. baumannii capsular polysaccharide (CPS) exhibit specificity for the linkage present between K-units that make up CPS polymers. This linkage is formed by a specific Wzy polymerase, and the ability to predict this linkage using sequence-based methods that identify the Wzy at the K locus could assist with the selection of phage for therapy. However, little is known about the specificity of Wzy polymerase enzymes. Here, we describe a Wzy polymerase that can accommodate two different but similar sugars as one of the residues it links and phage depolymerases that can cleave both types of bond that Wzy forms.

Structure elucidation and gene cluster of the O-antigen of Shewanella xiamenensis strain DCB-2-1 containing an amide of d-glucuronic acid with d-alanine and its bonding with U, Cr and V.

The aim of this work was to examine the structure and gene cluster of O-OPS of S. xiamenensis strain DCB-2-1 and survey its conceivability for chelating uranyl, chromate and vanadate ions from solution. O-polysaccharide (OPS, O-antigen) was isolated from the lipopolysaccharide of Shewanella xiamenensis DCB-2-1 and studied by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and sugar analysis. The following structure of the brunched pentasaccharide was established: where d-ß-GlcpA(d-Ala) is d-glucuronic acid acylated with NH group of d-Ala. The OPS structure established is unique among known bacterial polysaccharide structures. Interestingly, that dN-(d-glucuronoyl)-d-alanine derivative is not found in bacterial polysaccharides early. The O-antigen gene cluster of Shewanella xiamenensis strain DCB-2-1 has been sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the OPS structure. Based on the analysis of the IR spectra of the isolated polysaccharide DCB-2-1 and the products of its interaction with UO2(NO3)2 ∗ 6H2O, NH4VO3 and K2Cr2O7, a method of binding them can be proposed. Laboratory experiments show that the use of polysaccharide can be effective in removing uranyl, chromate and vanadate from solution.

The structure of Klebsiella pneumoniae K108 capsular polysaccharide is similar to Escherichia coli colanic acid.

The clinical isolate of Klebsiella pneumoniae 1333/P225 was revealed as containing a KL108 K. pneumoniae K locus for capsule biosynthesis. The gene cluster demonstrated a high level of sequence and arrangement similarity with that of the E. coli colanic acid biosynthesis gene cluster. The KL108 gene cluster includes a gene of WcaD polymerase responsible for joining oligosaccharide K units into capsular polysaccharide (CPS), acetyltransferase, pyruvyltransferasefive and genes for glycosyltransferases (Gtrs), four of which have homologues in genetic units of the colanic acid synthesis. The fifth Gtr is specific to this cluster. The work involved the use of sugar analysis, Smith degradation and one- and two-dimensional 1H and 13C NMR spectroscopy to establish the structure of the K108 CPS. The CPS repetitive K unit is composed of branched pentasaccharide with three monosaccharides in the backbone and a disaccharide side chain. The main chain is the same as for colanic acid but the side chain differs. Two bacteriophages infecting K. pneumoniae strain 1333/P225 were isolated and structural depolymerase genes were determined; depolymerases Dep108.1 and Dep108.2 were cloned, expressed and purified. It was demonstrated that both depolymerases specifically cleave the ß-Glcp-(1→4)-α-Fucp linkage between K108 units in the CPS.

Loss of a Branch Sugar in the Acinetobacter baumannii K3-Type Capsular Polysaccharide Due To Frameshifts in the gtr6 Glycosyltransferase Gene Leads To Susceptibility To Phage APK37.1.

The type of capsular polysaccharide (CPS) on the cell surface of Acinetobacter baumannii can determine the specificity of lytic bacteriophage under consideration for therapeutic use. Here, we report the isolation of a phage on an extensively antibiotic resistant ST2 A. baumannii isolate AB5001 that carries the KL3 CPS biosynthesis gene cluster predicting a K3-type CPS. As the phage did not infect isolates carrying KL3 or KL22 and known to produce K3 CPS, the structure of the CPS isolated from A. baumannii AB5001 was determined. AB5001 produced a variant CPS form, K3-v1, that lacks the ß-d-GlсpNAc side chain attached to the d-Galp residue in the K3 structure. Inspection of the KL3 sequence in the genomes of AB5001 and other phage-susceptible isolates with a KL3 locus revealed single-base deletions in gtr6, causing loss of the Gtr6 glycosyltransferase that adds the missing d-GlсpNAc side chain to the K3 CPS. Hence, the presence of this sugar profoundly restricts the ability of the phage to digest the CPS. The 41-kb linear double-stranded DNA (dsDNA) phage genome was identical to the genome of a phage isolated on a K37-producing isolate and thus was named APK37.1. APK37.1 also infected isolates carrying KL116. Consistent with this, K3-v1 resembles the K37 and K116 structures. APK37.1 is a Friunavirus belonging to the Autographiviridae family. The phage-encoded tail spike depolymerase DpoAPK37.1 was not closely related to Dpo encoded by other sequenced Friunaviruses, including APK37 and APK116. IMPORTANCE Lytic bacteriophage have potential for the treatment of otherwise untreatable extensively antibiotic-resistant bacteria. For Acinetobacter baumannii, most phage exhibit specificity for the type of capsular polysaccharide (CPS) produced on the cell surface. However, resistance can arise via mutations in CPS genes that abolish this phage receptor. Here, we show that single-base deletions in a CPS gene result in alteration of the final structure rather than deletion of the capsule layer and hence affect the ability of a newly reported podophage to infect strains producing the K3 CPS.

Complete chemical structure of the K135 capsular polysaccharide produced by Acinetobacter baumannii RES-546 that contains 5,7-di-N-acetyl-8-epipseudaminic acid.

A structurally diverse capsular polysaccharide (CPS) in the outer cell envelope plays an important role in the virulence of the important bacterial pathogen, Acinetobacter baumannii. More than 75 different CPS structures have been determined for the species to date, and many CPSs include isomers of a higher sugar, namely 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acid. Recently, a novel isomer having the d-glycero-l-manno configuration (5,7-di-N-acetyl-8-epipseudaminic acid; 8ePse5Ac7Ac) has been identified in the CPS from A. baumannii clinical isolate RES-546 [Carbohydr. Res. 513 (2022) 108,531]. Here, the complete chemical structure of this CPS, designated K135, was elucidated. The CPS was found to have a branched tetrasaccharide K unit and to include the higher sugar as part of a 8ePse5Ac7Ac-(2 â†’ 6)-α-Gal disaccharide branching from a →3)-α-D-GlcpNAc-(1 â†’ 3)-ß-D-GlcpNAc-(1→ main chain. Assignment of glycosyltransferases encoded by the CPS biosynthesis gene cluster in the RES-546 genome enabled the first sugar of the K unit, and hence the topology of the K135 CPS, to be determined.

Structure elucidation and gene cluster annotation of the O-antigen of Halomonas titanicae TAT1 containing three residues of 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid.

A halotolerant hydrocarbon-oxidizing bacterium Halomonas titanicae strain TAT1 was isolated from a petroleum reservoir. The O-polysaccharide (O-antigen) was isolated from the lipopolysaccharide of H. titanicae TAT1 and studied by component analyses and 1D and 2D NMR spectroscopy. The following structure of the repeating linear pentasaccharide O-unit, containing only aminosugars, was established: →4)-ß-d-GlcpNAc3NAcA-(1 â†’ 4)-ß-d-GlcpNAc3NAcA-(1 â†’ 6)-α-d-GlcpNAc-(1 â†’ 4)-ß-d-GlcpNAc3NAcA-(1 â†’ 6)-α-d-GlcpNAc-(→, where d-GlcNAc3NAcA indicates 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid. The O-antigen gene cluster was identified in the genome of H. titanicae TAT1 and compared with available database sequences. The genes revealed in the O-antigen gene cluster and the assigned functions of putative proteins were consistent with the established polysaccharide structure.

The K218 capsular polysaccharide produced by Acinetobacter baumannii isolate 52-249 includes 5,7-di-N-acetylpseudaminic acid linked by a KpsS3 glycosyltransferase.

Two acylated forms of the higher sugar, 5,7-diamino-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid called pseudaminic acid, Pse5Ac7Ac and Pse5Ac7RHb where R indicates (R)-3-hydroxybutanoyl, have been found to occur in many capsular polysaccharide (CPS) types produced by isolates of an important human pathogen, Acinetobacter baumannii. The presence of either a psaABCEDF or psaABCGHF gene module at the K locus (KL) for CPS biosynthesis determines the type of the variant produced. Here, an A. baumannii clinical isolate 52-249, recovered in 2015 in Moscow, Russia, was found to include a novel psaABCIJF gene module in the KL218 sequence at the K locus. The CPS from 52-249 was extracted and studied by sugar analysis and partial acid hydrolysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. A branched tetrasaccharide repeating unit was identified, which included a →3)-α-d-Galp-(1→6)-α-d-GlcpNAc-(1→3)-ß-d-GalpNAc-(1→ main chain and Pse5Ac7Ac attached as a side branch, indicating that the psaABCIJF gene module is associated with synthesis of this variant. The K218 CPS was found to be structurally related to the K46 CPS of A. baumannii, and a comparison of the two structures enabled the assignment of glycosyltransferases. A KpsS3 protein for the α-(2→6) linkage of the Pse5Ac7Ac residue to D-Galp in K218 was identified.

Structure of the K98 capsular polysaccharide from Acinetobacter baumannii REV-1184 containing a cyclic pyruvic acid acetal.

The K98 capsular polysaccharide (CPS) from the Acinetobacter baumannii clinical isolate, REV-1184, was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. The CPS was found to consist of linear tetrasaccharide repeats (K-units) that include one residue each of d-GlcpNAc, d-GalpNAc, 2-acetamido-2-deoxy-d-galacturonic acid (d-GalpNAcA), and 2-acetamido-2,6-dideoxy-d-glucose (N-acetylquinovosamine, d-QuipNAc), with the GalpNAc residue decorated with a (R)-configurated 4,6-pyruvic acid acetal group. The CPS has a similar composition to that of A. baumannii K4 but the topology of the tetrasaccharide K-unit is different (linear in K98 versus branched in K4). This was due to a difference in sequence for the Wzy polymerases encoded by the CPS biosynthesis gene clusters KL98 and KL4, with the WzyK98 polymerase forming a ß-d-QuipNAc-(1→3)-d-GalpNAc linkage between the K98 units.

The K89 capsular polysaccharide produced by Acinetobacter baumannii LUH5552 consists of a pentameric repeat-unit that includes a 3-acetamido-3,6-dideoxy-d-galactose residue.

Acinetobacter baumannii isolate LUH5552 carries the KL89 capsule biosynthesis gene cluster. Capsular polysaccharide (CPS) isolated from LUH5552 was analyzed by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K89 CPS structure has not been seen before in A. baumannii CPS structures resolved to date and includes a 3-acetamido-3,6-dideoxy-d-galactose (d-Fucp3NAc) residue which is rare amongst A. baumannii CPS. The K89 CPS has a →3)-α-d-GalpNAc-(1→3)-ß-d-GlcpNAc-(1→ main chain with a ß-d-Glcp-(1→2)-ß-d-Fucp3NAc-(1→6)-d-Glcp side branch that is α-(1→4) linked to d-GalpNAc. The roles of the Wzy polymerase and the four glycosyltransferases encoded by the KL89 gene cluster in the biosynthesis of the K89 CPS were assigned. Two glycosyltransferases, Gtr121 and Gtr122, link the d-Fucp3NAc to its neighboring sugars.

Involvement of a Phage-Encoded Wzy Protein in the Polymerization of K127 Units To Form the Capsular Polysaccharide of Acinetobacter baumannii Isolate 36-1454.

A comprehensive understanding of capsular polysaccharide (CPS) diversity is critical to implementation of phage therapy to treat panresistant Acinetobacter baumannii infections. Predictions from genome sequences can assist identification of the CPS type but can be complicated if genes outside the K locus (CPS biosynthesis gene cluster) are involved. Here, the CPS produced by A. baumannii clinical isolate 36-1454 carrying a novel K locus, KL127, was determined and compared to other CPSs. KL127 differs from KL128 in only two of the glycosyltransferase (gtr) genes. The K127 unit in 36-1454 CPS was the pentasaccharide ß-d-Glcp-(1→6)-d-ß-GalpNAc-(1→6)-α-d-Galp-(1→6)-ß-d-Glсp-(1→3)-ß-d-GalpNAc in which d-Glcp at position 4 replaces d-Galp in K128, and the glycosyltransferases encoded by the different gtr genes form the surrounding linkages. However, although the KL127 and KL128 gene clusters encode nearly identical Wzy polymerases, the linkages between K units that form the CPS chains are different, i.e., ß-d-GalpNAc-(1→3)-d-Galp in 36-1454 (K127) and ß-d-GalpNAc-(1→4)-d-Galp in KZ-1093 (K128). The linkage between K127 units in 36-1454 is the same as the K-unit linkage in five known CPS structures, and a gene encoding a Wzy protein related to the Wzy of the corresponding K loci was found encoded in a prophage genome in the 36-1454 chromosome. Closely related Wzy proteins were encoded in unrelated phage in available KL127-carrying genomes. However, a clinical isolate, KZ-1257, carrying KL127 but not the prophage was found, and K127 units in the KZ-1257 CPS were ß-d-GalpNAc-(1→4)-d-Galp linked, confirming that WzyKL127 forms this linkage and thus that the phage-encoded WzyPh1 forms the ß-d-GalpNAc-(1→3)-d-Galp linkage in 36-1454. IMPORTANCE Bacteriophage therapy is an attractive innovative treatment for infections caused by extensively drug resistant Acinetobacter baumannii, for which there are few effective antibiotic treatments remaining. Capsular polysaccharide (CPS) is a primary receptor for many lytic bacteriophages, and thus knowledge of the chemical structures of CPS produced by the species will underpin the identification of suitable phages for therapeutic cocktails. However, recent research has shown that some isolates carry additional genes outside of the CPS biosynthesis K locus, which can modify the CPS structure. These changes can subsequently alter phage receptor sites and may be a method utilized for natural phage resistance. Hence, it is critical to understand the genetics that drive CPS synthesis and the extent to which genes outside of the K locus can affect the CPS structure.

The K139 capsular polysaccharide produced by Acinetobacter baumannii MAR17-1041 belongs to a group of related structures including K14, K37 and K116.

Capsular polysaccharide (CPS) is a key target for bacteriophage and vaccine therapies currently being developed for treatment of infections caused by the extensively antibiotic resistant bacterial species, Acinetobacter baumannii. Identification of new CPS structures and the genetics that drive their synthesis underpins tailored treatment strategies. A novel CPS biosynthesis gene cluster, designated KL139, was identified in the whole genome sequence of a multiply antibiotic resistant clinical isolate, A. baumannii MAR-17-1041, recovered in Russia in 2017. CPS material extracted from A. baumannii MAR-17-1041 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy, and the structure was found to include a branched pentasaccharide repeating unit containing neutral carbohydrates. This structure closely resembles the topology of the A. baumannii K14 CPS but differs in the presence of d-Glcp in place of a d-Galp sugar in the repeat-unit main chain. The difference was attributed to a change in the sequence for two glycosyltransferases. These two proteins are also encoded by the A. baumannii KL37 gene cluster, and a multiple sequence alignment of KL139 with KL14 and KL37 revealed a hybrid relationship. The global distribution of KL139 was also assessed by probing 9065 A. baumannii genomes available in the NCBI non-redundant and WGS databases for the KL139 gene cluster. KL139 was found in 16 genomes from four different countries. Eleven of these isolates belong to the multidrug resistant global lineage, ST25.

Correlation of Acinetobacter baumannii K144 and K86 capsular polysaccharide structures with genes at the K locus reveals the involvement of a novel multifunctional rhamnosyltransferase for structural synthesis.

Whole genome sequence from Acinetobacter baumannii isolate Ab-46-1632 reveals a novel KL144 capsular polysaccharide (CPS) biosynthesis gene cluster, which carries genes for d-glucuronic acid (D-GlcA) and l-rhamnose (l-Rha) synthesis. The CPS was extracted from Ab-46-1632 and studied by 1H and 13C NMR spectroscopy, including a two-dimensional 1H,13C HMBC experiment and Smith degradation. The CPS was found to have a hexasaccharide repeat unit composed of four l-Rhap residues and one residue each of d-GlcpA and N-acetyl-d-glucosamine (D-GlcpNAc) consistent with sugar synthesis genes present in KL144. The K144 CPS structure was established and found to be related to those of A. baumannii K55, K74, K85, and K86. A comparison of the corresponding gene clusters to KL144 revealed a number of shared glycosyltransferase genes correlating to shared glycosidic linkages in the structures. One from the enzymes, encoded by only KL144 and KL86, is proposed to be a novel multifunctional rhamnosyltransfaerase likely responsible for synthesis of a shared α-l-Rhap-(1 â†’ 2)-α-L-Rhap-(1 â†’ 3)-L-Rhap trisaccharide fragment in the K144 and K86 structures.

The K26 capsular polysaccharide from Acinetobacter baumannii KZ-1098: Structure and cleavage by a specific phage depolymerase.

The KL26 gene cluster responsible for the synthesis of the K26 capsular polysaccharide (CPS) of Acinetobacter baumannii includes rmlBDAC genes for l-rhamnose (l-Rhap) synthesis, tle to generate 6-deoxy-l-talose (l-6dTalp) from l-Rhap, and a manC gene for D-mannose (D-Manp) that is rare in Acinetobacter CPS. K26 CPS material was isolated from A. baumannii isolate KZ-1098, and studied by sugar analysis, Smith degradation, and one and two-dimensional 1H and 13C NMR spectroscopy before and after O-deacetylation with aqueous ammonia. The following structure of the branched hexasaccharide repeating unit of the CPS was established: →2)-ß-D-Manp-1→4-ß-D-Glcp-1→3-α-L-6dTalp-1→3-ß-D-GlcpNAc-(1→3↑14│Acα-L-Rhap-2←1-α-D-Glcp The structural depolymerase of phage vB_AbaP_APK26 cleaved selectively the ß-GlcpNAc-(1 → 2)-α-Manp linkage in the K26 CPS formed by WzyK26 to give monomer, dimer, and trimer of the CPS repeating unit, which were characterized by high-resolution electrospray ionization mass spectrometry as well as 1H and 13C NMR spectroscopy. The wzyK26 gene responsible for this linkage and the manC gene were only found in six A. baumannii genomes carrying KL26 and one carrying the novel KL148 gene cluster, indicating the rare occurrence of ß-GlcpNAc-(1 → 2)-α-Manp in A. baumannii CPS structures. However, K26 shares a ß-d-Glcp-(1 → 3)-α-l-6dTalp-(1 → 3)-ß-d-GlcpNAc trisaccharide fragment with a group of related A. baumannii CPSs that have varying patterns of acetylation of l-6dTalp.

Acinetobacter baumannii K106 and K112: Two Structurally and Genetically Related 6-Deoxy-l-talose-Containing Capsular Polysaccharides.

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated.

Structure of the K128 capsular polysaccharide produced by Acinetobacter baumannii KZ-1093 from Kazakhstan.

The structure of the K128 capsular polysaccharide (CPS) produced by Acinetobacter baumannii isolate KZ-1093 from Kazakhstan was established by sugar analysis and Smith degradation along with 1D and 2D 1H and 13C NMR spectroscopy. The CPS was found to consist of branched pentasaccharide repeating units containing only neutral sugars, and its composition and topology are closely related to those of the A. baumannii K116 CPS. The K128 and K116 oligosaccharide units differ in the linkage between the disaccharide side chain and the main chain, with a ß-(1 → 6) linkage in K128 replacing a ß-(1 → 4) linkage in K116. The linkages between the repeating units in the K128 and K116 CPSs are also different, with K128 units linked by ß-d-GalpNAc-(1 → 4)-d-Galp, and ß-d-GalpNAc-(1 → 3)-d-Galp linkages between K116 units. The KZ-1093 genome was sequenced and the CPS biosynthesis gene cluster at the chromosomal K locus was designated KL128. Consistent with the CPS structures, KL128 differs from KL116 in one glycosyltransferase gene and the gene for the Wzy polymerase. In KL128, the gtr200 glycosyltransferase gene replaces gtr76 in KL116, and Gtr200 was therefore assigned to the different ß-d-GalpNAc-(1 → 6)-d-Galp linkage in K128. Similarly, the WzyK128 polymerase could be assigned to the ß-d-GalpNAc-(1 → 4)-d-Galp linkage between the K128 units.

Acinetobacter baumannii K116 capsular polysaccharide structure is a hybrid of the K14 and revised K37 structures.

The genome of Acinetobacter baumannii clinical isolate, MAR-303, recovered in Russia was sequenced and found to contain a novel gene cluster at the A. baumannii K locus for capsule biosynthesis. The gene cluster, designated KL116, included four genes for glycosyltransferases (Gtrs) and a gene for a Wzy polymerase responsible for joining oligosaccharide K units into the capsular polysaccharide (CPS). The arrangement of KL116 was a hybrid of previously described A. baumannii gene clusters, with two gtr genes and the wzy gene shared by KL37 and the two other gtr genes found in KL14. The structure of the K116 CPS was established by sugar analysis and Smith degradation, along with one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS is composed of branched pentasaccharide K units containing only neutral sugars, with three monosaccharides in the main chain and a disaccharide side chain. The K116 unit shares internal sugar linkages with the K14 and K37 units, corresponding to the presence of shared gtr genes in the gene clusters. However, the specific linkage formed by Wzy was discrepant between K116 and the previously reported K37 CPS produced by A. baumannii isolate NIPH146. The K37 structure was therefore revised in this study, and the corrected Wzy linkage found to be identical to the Wzy linkage in K116. The KL116, KL14 and KL37 gene clusters were found in genomes of a variety of A. baumannii strain backgrounds, indicating their global distribution.