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KL55, KL74, and KL85 capsular polysaccharide (CPS) biosynthesis loci in Acinetobacter baumannii BAL_204, BAL_309, and LUH5543 genomes, respectively, are related and each contains genes for l -Rha p and d -Glc p A synthesis. The CPSs were isolated and studied by sugar analysis, Smith degradation, and 1H and 13C NMR spectroscopy. The K55 and K74 CPSs are built up of branched octasaccharide repeats (K units) containing one residue each of d -Glc p A and d -Glc p NAc and six residues of l -Rha p. The K55 unit differs from the K74 unit in the linkage between D-Glc p A and an l -Rha p residue in the K unit (1 → 3 versus 1 → 2) and linkage between K units. However, most K units in the isolated K74 CPS were modified by β-elimination of a side-chain α- l -Rha p -(1 → 3)-α- l -Rha p disaccharide from position 4 of GlcA to give 4-deoxy- l - threo -hex-4-enuronic acid (1:~3 ratio of intact and modified units). The K85 CPS has a branched heptasaccharide K unit similar to the K74 unit but with one fewer α- l -Rha p residue in the side chain. In contrast to previous findings on A. baumannii CPSs, each K locus includes fewer glycosyltransferase (Gtr) genes than the number required to form all linkages in the K units. Hence, one Gtr appears to be multifunctional catalysing formation of two 1 → 2 and one 1 → 3 linkages between the l -Rha residues. [ABSTRACT FROM AUTHOR]

The K17 capsular polysaccharide (CPS) produced by Acinetobacter baumannii G7, which carries the KL17 configuration at the capsule biosynthesis locus, was isolated and studied by chemical methods along with one- and two-dimensional 1H and 13C NMR spectroscopy. Selective cleavage of the glycosidic linkage of a 2,4-diacetamido-2,4,6-trideoxy- d -glucose (d -QuiNAc4NAc) residue by (i) trifluoroacetic acid solvolysis or (ii) alkaline β-elimination (NaOH-NaBH 4) of the 4-linked D-alanine amide of a 2-acetamido-2-deoxy- d -galacturonic acid residue (d -GalNAcA6DAla) yielded trisaccharides that were isolated by Fractogel TSK HW-40 gel-permeation chromatography and identified by using NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. The following structure was established for the trisaccharide repeat (K unit) of the CPS: →4)- α -d -Gal p NAcA6 d Ala-(1 → 4)- α -d -Gal p NAcA-(1 → 3)- β- d -Qui p NAc4NAc-(1 →. The presence of the itrA1 gene coding for the initial glycosylphosphotransferase in the KL17 gene cluster established the first sugar of the K unit as d -Qui p NAc4NAc. KL17 includes genes for three transferases that had been annotated previously as glycosyltransferases (Gtrs). As only two Gtrs are required for the K17 structure and one d -Gal p NAcA residue is modified by a d -alanine amide, these assignments were re-assessed. One transferase was found to belong to the ATPgrasp_TupA protein family that includes d -alanine- d -alanine ligases, and thus was renamed Alt1 (al anine t ransferase). Alt1 represents a novel family that amidate the carboxyl group of d -Gal p NAcA or d -Gal p A. [ABSTRACT FROM AUTHOR]

Infections caused by Acinetobacter baumannii isolates from the major global clones, GC1 and GC2, are difficult to treat with antibiotics, and phage therapy, which requires extensive knowledge of the variation in the surface polysaccharides, is an option under consideration. The gene clusters directing the synthesis of capsular polysaccharide (CPS) in A. baumannii GC1 isolate A388 and GC2 isolate G21 differ by a single glycosyltransferase (gtr) gene. They include genes encoding a novel UDP-glucose dehydrogenase (Ugd2) and a putative pyruvyl transferase (Ptr2). The composition and structures of the linear K20 and K21 tetrasaccharide repeats (K units) of the CPSs isolated from A338 and G21, respectively, were established by sugar analyses and Smith degradation along with 1D and 2D 1H and 13C NMR spectroscopy. The K20 and K21 CPSs are the first known to include Glc p A produced by Ugd2 and d -galactose with an (R)-configured 4,6-pyruvic acid acetal added by Prt2. The first sugar in the tetrasaccharide K units is 2-acetamido-4-amino-2,4,6-trideoxy- d- glucose (d -Qui p NAc4N) that carries a 4-N-[(S)-3-hydroxybutanoyl] group in some K units and a 4-N-acetyl group in the others. Accordingly, K unit polymerases WzyK20 and WzyK21 form a β- d -Qui p NAc4NR-(1→2)- d -Gal p bond. The K20 and K21 units differ only in the configuration of the glycosidic linkages of d -Glc p NAc allowing the unique inverting glycosyltransferases Gtr43 and the retaining glycosyltransferase Gtr45 to be assigned to the formation of the β- d -Glc p NAc-(1→4)- d -Glc p A and α- d -Glc p NAc-(1→4)- d -Glc p A linkages, respectively. [ABSTRACT FROM AUTHOR]

Capsular polysaccharides were recovered from four Acinetobacter baumannii isolates, and the following related structures of oligosaccharide repeating units were established by sugar analyses along with 1D and 2D 1 H and 13 C NMR spectroscopy: NIPH 60 and LUH5544 (K43) Image 2 NIPH 601 (K47) The K locus for capsule biosynthesis in the genome sequences available for NIPH 60 and LUH5544, designated KL43, was found to be related to gene clusters KL47 in NIPH 601 and KL88 in LUH5548. The three clusters share most gene content differing in only a small portion that includes an additional glycosyltransferase genes in KL47 and KL88, as well as genes encoding distinct Wzy polymerases that were found to form the same α- d -Glc p NAc-(1 → 6)-α- d -Glc p NAc linkage in K43 and K47. [ABSTRACT FROM AUTHOR]

Capsular polysaccharides (CPSs), from Acinetobacter baumannii isolates 1432, 4190 and NIPH 70, which have related gene content at the K locus, were examined, and the chemical structures established using 2D 1H and 13C NMR spectroscopy. The three isolates produce the same pentasaccharide repeat unit, which consists of 5-N-acetyl-7-N-[(S)-3-hydroxybutanoyl] (major) or 5,7-di-N-acetyl (minor) derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7R), D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. However, the linkage between repeat units in NIPH 70 was different to that in 1432 and 4190, and this significantly alters the CPS structure. The KL27 gene cluster in 4190 and KL44 gene cluster in NIPH 70 are organized identically and contain lga genes for Leg5Ac7R synthesis, genes for the synthesis of the common sugars, as well as an itrA2 initiating transferase and four glycosyltransferases genes. They share high-level nucleotide sequence identity for corresponding genes, but differ in the wzy gene encoding the Wzy polymerase. The Wzy proteins, which have different lengths and share no similarity, would form the unrelated linkages in the K27 and K44 structures. The linkages formed by the four shared glycosyltransferaseswere predicted by comparison with gene clusters that synthesize related structures. These findings unambiguously identify the linkages formed by WzyK27 andWzyK44, and show that the presence of different wzy genes in otherwise closely related K gene clusters changes the structure of the CPS. This may affect its capacity as a protective barrier for A. baumannii. [ABSTRACT FROM AUTHOR]

The structure of the capsular polysaccharide (CPS) recovered from D46, an extensively antibiotic resistant ST25 Acinetobacter baumannii clinical isolate, was elucidated. The structure was resolved on the basis of NMR spectroscopy and chemical analyses, and was found to contain a branched neutral pentasaccharide with a backbone composed of Gal p NAc and Gal p residues, all d configured, and a d -Glc p side group. The KL14 gene cluster found in the D46 genome includes genes for four glycosyltransferases but no modules for synthesis of complex sugars, and this is consistent with the structure of K14. The K14 structure and KL14 sequence clarify the relationship between the structure and K locus sequence for A. nosocomialis isolate LUH5541. The identity of the first sugar of the K14 repeat unit (K unit), and the functions of the four encoded glycosyltransferases and Wzy polymerase were predicted. [ABSTRACT FROM AUTHOR]

Neutral capsular polysaccharides (CPSs) were isolated from Acinetobacter baumannii NIPH190, NIPH201, and NIPH615. The CPSs were found to contain common monosaccharides only and to be branched with a side-chain 1→3-linked β- d -glucopyranose residue. Structures of the oligosaccharide repeat units (K units) of the CPSs were elucidated by 1D and 2D 1 H and 13 C NMR spectroscopy. Novel CPS biosynthesis gene clusters, designated KL30, KL45, and KL48, were found at the K locus in the genome sequences of NIPH190, NIPH201, and NIPH615, respectively. The genetic content of each gene cluster correlated with the structure of the CPS unit established, and therefore, the capsular types of the strains studied were designated as K30, K45, and K48, respectively. The initiating sugar of each K unit was predicted, and glycosyltransferases encoded by each gene cluster were assigned to the formation of the linkages between sugars in the corresponding K unit. [ABSTRACT FROM AUTHOR]

The repeat unit of the K12 capsular polysaccharide isolated from the Acinetobacter baumannii global clone 1 clinical isolate, D36, was elucidated by means of chemical and spectroscopical methods. The structure was shown to contain N-acetyl-D-galactosamine (D-GalpNAc), N-acetyl-D-fucosamine and N-acetyl-L-fucosamine linked together in the main chain, with the novel sugar, 5,7-diacetamido- 3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic acid (5,7-di-N-acetylacinetaminic acid or Aci5Ac7Ac), attached to D-GalpNAc as a side branch. This matched the sugar composition of the K12 capsule and the genetic content of the KL12 capsule gene cluster reported previously. D-FucpNAc was predicted to be the substrate for the initiating transferase, ItrB3, with the Wzy polymerase making a a-D-FucpNAc-(13)-D-GalpNAc linkage between the repeat units. The three glycosyltransferases encoded by KL12 are all retaining glycosyltransferases and were predicted to form specific linkages between the sugars in the K12 repeat unit. [ABSTRACT FROM AUTHOR]

The structure of the capsular polysaccharide (CPS) from an Acinetobacter baumannii global clone 2 (GC2) clinical isolate RBH4 that carries the KL6 gene cluster was elucidated by means of chemical and spectroscopical methods. The repeating unit of K6 CPS is linear and contains N-acetyl- d -galactosamine ( d -Gal p NAc), two d -galactose ( d -Gal p ) residues and 5,7-di-N-acetylpseudaminic acid (Pse5Ac7Ac). The synthesis of these sugars could be attributed to genes in the KL6 capsule biosynthesis gene cluster, and the formation of the linkages between the sugars were assigned to glycosyltransferases or the Wzy polymerase encoded in KL6. [ABSTRACT FROM AUTHOR]

The repeat unit structure of the K2 capsule from an extensively antibiotic-resistant Acinetobacter baumannii global clone 2 (GC2) strain was determined. The oligosaccharide contains three simple sugars, d-glucopyranose, d-galatopyranose and N-acetyl-d-galactosamine, and the complex sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (Pse5Ac7Ac or pseudaminic acid), which has not previously been reported in any A. baumannii capsule. The strain was found to carry all the genes required for the synthesis of the sugars and construction of the K2 structure. The linkages catalyzed by the initiating transferase, three glycosyltransferases and the Wzy polymerase were also predicted. Examination of publicly available A. baumannii genome sequences revealed that the same gene cluster, KL2, often occurs in extensively antibiotic-resistant GC2 isolates and in further strain types. The gene module responsible for the synthesis of pseudaminic acid was also detected in four other K loci. A related module including genes for an acylated relative of pseudaminic acid was also found in two new KL types. A polymerase chain reaction scheme was developed to detect all modules containing genes for sugars based on pseudaminic acid and to specifically detect KL2. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The K8 and K54 capsular polysaccharides were isolated from Acinetobacter baumannii BAL 097 and RCH52, respectively, and studied by sugar analysis, partial acid hydrolysis and selective solvolysis with CF 3 CO 2 H in the presence of 2-methyl-1-propanol, along with 1D and 2D 1H and 13C NMR spectroscopy. The following structures of related branched tetrasaccharide repeats (K units) of the polysaccharides were established: Image 2 where Leg indicates 5,7-diamino-3,5,7,9-tetradeoxy- d -glycero- d -galacto -non-2-ulosonic (legionaminic) acid and R indicates (R)-3-hydroxybutanoyl or acetyl in the ratio ~2.5:1. The sequences of the KL8 and KL54 capsule gene clusters were closely related. The difference in the acyl group at O-7 on the sidechain legionaminic acid is due to differences in two genes in the legionaminic acid biosynthesis cluster. The wzy genes encoding the K unit polymerases are also different and make different linkages between the K units, allowing the first sugar of both K units to be identified as d -Glc p NAc. The shared Gtr20 glycosyltransferase, also encoded in KL63, forms the α- l -Fuc p NAc-(1 → 3)- d -Glc p NAc linkage, and Gtr19 was predicted to form α- d -Gal p NAc-(1 → 3)- l -Fuc p NAc. Gtr18 from KL8 is 75% identical to Gtr108 from KL54 and both would link the Leg derivative to d -Gal p NAc. Hence the genes present at the K locus were consistent with the composition and structures of the K8 and K54 capsular polysaccharides. Image 1 • Capsule structures were obtained for Acinetobacter baumannii clinical isolates carrying KL8 and KL54. • Capsule structures are related, differing in the presence of a non-2-ulosonic acid and linkage between units. • The function of the glycosyltransferases, Wzy polymerases and initiating transferase were unambiguously assigned. [ABSTRACT FROM AUTHOR]

Capsular polysaccharides were isolated from A. baumannii LUH5535 (K35 CPS) and LUH5554 (K15 CPS) and studied by 1D and 2D 1 H and 13 C NMR spectroscopy. The CPSs were found to consist of linear tetrasaccharide repeats (K units) containing 2-acetamido-2-deoxy- d -galacturonic acid (K35) or 2,3-diacetamido-2,3-deoxy- d -glucuronic acid (K15) and 2,4-diacetamido-2,4,6-trideoxy- d -glucose (both CPSs). The K35 unit includes three O-acetyl groups on different GalNAcA residues. A. baumannii LUH5535 and LUH5554 carry the KL35 and KL15 gene clusters, respectively, and putatively assigned functions of genes in these clusters were consistent with the CPS structures established. [ABSTRACT FROM AUTHOR]

Capsular polysaccharide (CPS) is a heteroglycan that coats the cell surface of most isolates of the important Gram-negative bacterial pathogen, Acinetobacter baumannii. Strain MAR 15–4076, a clinical isolate recovered in Russia in 2015, was found to carry the KL129 sequence at the CPS biosynthesis K locus. The CPS was isolated from the strain and studied by sugar analysis, Smith degradation, one- and two-dimensional 1H and 13C NMR spectroscopy. It was composed of branched pentasaccharide units that include a →3)-α- l -Rha p -(1 → 3)-α- l -Rha p -(1 → 3)-β- d -Glc p NAc-(1→ mainchain and α- d -Man p NAc-(1 → 3)- l -Rha p side branch. Though the pentasaccharide units are identical to those that make up the K84 CPS produced by A. baumannii LUH5540, the units are linked differently via the substitution of an alternate l -Rha p residue, resulting in a difference in the overall topology of the CPS. This was due to the replacement of the Wzy polymerase gene encoded at the K locus. [Display omitted] • Structure of the Acinetobacter baumannii K129 CPS determined. • K129 and K84 CPS have the same units but differ in CPS branching patterns. • Difference between structures was explained by a replacement of the Wzy polymerase gene at the CPS biosynthesis K locus. [ABSTRACT FROM AUTHOR]

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 -Glc p NAc, d -Gal p NAc, 2-acetamido-2-deoxy- d -galacturonic acid (d -Gal p NAcA), and 2-acetamido-2,6-dideoxy- d -glucose (N -acetylquinovosamine, d -Qui p NAc), with the Gal p NAc 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 Wzy K98 polymerase forming a β- d -Qui p NAc-(1→3)- d -Gal p NAc linkage between the K98 units. [ABSTRACT FROM AUTHOR]

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 -Gal p -(1→6)-α- d -Glc p NAc-(1→3)-β- d -Gal p NAc-(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-Gal p in K218 was identified. [ABSTRACT FROM AUTHOR]

Capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii NIPH146, and the following structure of branched pentasaccharide repeating unit was established by sugar analyses along with 1D and 2D NMR spectroscopy: In comparison to most other known capsular polysaccharides of A. baumannii , the CPS studied is neutral and lacks any specific monosaccharide component. The synthesis, assembly and export of this structure could be attributed to genes in a novel capsule biosynthesis gene cluster, designated KL37, which was found in the NIPH146 genome. The CPS of A. baumannii NIPH146 shares the α- d -Gal p -(1→6)-β- d -Glc p -(1→3)- d -Gal p NAc-(1→ trisaccharide fragment with the CPS units of several A. baumannii strains, including ATCC 17978 and LUH 5537 that carry the KL3 and KL22 gene clusters, respectively. KL37 contains two genes for glycosyltransferases that are related to two glycosyltransferase genes present in both KL3 and KL22, and the encoded proteins could be tentatively assigned to linkages between sugars in the CPS repeat. [ABSTRACT FROM AUTHOR]

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 -Fuc p 3NAc) residue which is rare amongst A. baumannii CPS. The K89 CPS has a →3)-α- d -Gal p NAc-(1→3)-β- d -Glc p NAc-(1→ main chain with a β- d -Glc p -(1→2)-β- d -Fuc p 3NAc-(1→6)- d -Glc p side branch that is α-(1→4) linked to d -Gal p NAc. 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 -Fuc p 3NAc to its neighboring sugars. [ABSTRACT FROM AUTHOR]

Capsular polysaccharide (CPS), a heteropolymeric carbohydrate structure present on the cell surface of most isolates of the bacterial pathogen Acinetobacter baumannii , is a major virulence determinant. Here, the CPS produced by A. baumannii MRSN 31468, which carries the KL58 CPS biosynthesis locus, was studied by sugar analysis, one- and two-dimensional 1H and 13C NMR spectroscopy. The structure was found to consist of a repeating tetrasaccharide K-unit that includes glucose (d-Glc p), galactose (d-Gal p), N -acetyl-galactosamine (d-Gal p NAc), and 5,7-diacetamido-3,5,7,9-tetradeoxy- l - glycero - l - manno -non-2-ulosonic acid (5,7-di- N -acetylpseudaminic acid; Pse5Ac7Ac). The CPS has a branched repeating unit with the disaccharide →3)-β-d-Glc-(1→3)-β-d-GalNAc-(1→ as the mainchain and O-6 of the Glc unit substituted with the disaccharide β-Pse5Ac7Ac-(2→6)-α-d-Gal, and Pse5Ac7Ac is partially acetylated at O-4. The presence of Pse5Ac7Ac in the K58 structure is consistent with the presence of psaA-F genes in KL58, which are responsible for Pse5Ac7Ac synthesis. 4-O-acetylation of Pse5Ac7Ac was traced to an acetyltransferase, Atr44 , which was found to be closely related to Atr29 that similarly decorates Pse5Ac7Ac with 4OAc in the K46-type CPS. Atr44 like Atr29 is encoded by a gene found in a prophage. The K58 CPS produced by MRSN 31468 did not include the 8-epimer of Pse5Ac7Ac (5,7-di- N -acetyl-8-epipseudaminic acid; 8ePse5Ac7Ac) found in the closely related CPS from BAL062 that also carries KL58. Hence, the gene(s) for conversion of Pse5Ac7Ac to 8ePse5Ac7Ac must lie elsewhere. [Display omitted] • Structure of the CPS from Acinetobacter baumannii isolate MRSN31468 was determined. • Tetrasaccharide repeat units include D-Glc, D-Gal, D-GalNAc and Pse5Ac7Ac. • Structure was found to be consistent with genetic content of KL58 locus in the genome of MRSN31468. [ABSTRACT FROM AUTHOR]

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 -Glc p in place of a d -Gal p 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. [ABSTRACT FROM AUTHOR]

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: [Display omitted] 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 β-d-Glc p NAc-(1→3)-d-Glc p NAc bond is the linkage between K-units formed by the Wzy KL141 polymerase. [Display omitted] • Complete structure of Acinetobacter baumannii K141 CPS solved. • Annotations for the KL141 gene cluster reported. • Genetic content of KL141 is consistent with the structure reported. [ABSTRACT FROM AUTHOR]

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 -Rha p), and one residue each of d -glucuronic acid (d -Glc p A) and N -acetyl- d -glucosamine (d -Glc p NAc). 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 -Rha p 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 wzy KL239 polymerase gene in KL239 that replaces the gtr80 and wzy KL86 genes in the KL86 CPS biosynthesis gene cluster. Comparison of the two structures established the role of a novel Wzy KL239 polymerase encoded by KL239 that forms the β-d-GlcpNAc-(1→2)-l-Rha p 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 -Rha p influences the susceptibility of this isolate to bacteriophage activity. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • The structure of the A. baumannii K63 capsule has been determined. • This structure revises that reported for O24. • K63 includes 5,7-diamino-3,5,7,9-tetradeoxy- d - glycero - d - galacto -non-2-ulosonic acid. • Roles for the glycosyltransferases and Wzy polymerase encoded by KL63 are assigned. [ABSTRACT FROM AUTHOR]

K87 capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii isolate LUH5547 that carries the KL87 capsule biosynthesis gene cluster at the chromosomal K locus. Studies by sugar analysis, selective chemical cleavages, and 1D and 2D 1H and 13C NMR spectroscopy showed that the CPS has a branched heptasaccharide repeat (K unit) containing one residue each of d -glucose (d -Glс p), d -glucuronic acid (d -Glс p A), N -acetyl- d -glucosamine (d -Glс p NAc), 6-deoxy- l -talose (l -6dTal p), and three residues of l -rhamnose (l -Rha p). The following structure of the CPS was established: → 3) − α - L - R ha p − (1 → 2) - α - L - R h a p - (1 → 3) - α - L - 6 d T a l p - (1 → 3) - β - D - G l c p N A c - (1 → 2 ↑ 1 β - D - G l c p A - (4 ← 1) - α - D - G l c p (2 ← 1) - α - L - R h a p The position of a minor O-acetyl group present in the CPS was not determined. Functions of enzymes coded by genes in the KL87 gene cluster were tentatively assigned and found to be consistent with the CPS structure. [Display omitted] • Capsule structure from A. baumannii LUH5547 correlates with K locus genes. • Structure belongs to a group of 6-deoxy- l -talose containing capsules from A. baumannii. • The linkage cleaved by a bacteriophage depolymerase was identified. [ABSTRACT FROM AUTHOR]

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 β-(1 → 6) linkage in K128 replacing a β-(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 β- d -Gal p NAc-(1 → 4)- d -Gal p, and β- d -Gal p NAc-(1 → 3)- d -Gal p 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 β- d -Gal p NAc-(1 → 6)- d -Gal p linkage in K128. Similarly, the Wzy K128 polymerase could be assigned to the β- d -Gal p NAc-(1 → 4)- d -Gal p linkage between the K128 units. Image 1 • The Acinetobacter baumannii K128 capsule structure is closely related to the K116 capsule structure. • The function of the glycosyltransferases, Wzy polymerases and initiating transferase were unambiguously assigned. • The KL128 gene cluster includes genes for glycosyltransferases and simple sugar synthesis. [ABSTRACT FROM AUTHOR]

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. Image 1 • The Acinetobacter baumannii K116 capsule structure is established and is closely related to K14 and K37 capsules. • Correlation to capsule biosynthesis genes revealed an error in the K37 structure, which was revised in this study. • The function of the glycosyltransferases, Wzy polymerases and initiating transferase were unambiguously assigned. [ABSTRACT FROM AUTHOR]