Acinetobacter baumannii isolate LUH5553 carries the KL90 capsule gene cluster, which includes genes for three glycosyltransferases (Gtrs) and the ItrA3 initiating transferase, as well as a set of genes for synthesis of a higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic (di-N-acetylpseudaminic) acid (Pse5Ac7Ac). The K90 capsular polysaccharide (CPS) has a tetrasaccharide repeat (K90 unit), which begins with d-GlcpNAc and contains Pse5Ac7Ac. The higher sugar was cleaved by mild acid hydrolysis of the CPS, and structures of the initial and modified polysaccharides were established by 1D and 2D 1H and 13C NMR spectroscopy. K90 contains α-d-Galp-(1-6)-d-GlcpNAc and α-d-GlcpNAc-(1-3)-d-GlcpNAc fragments, and formation of these glycosidic linkages is catalysed respectively by Gtr14 and Gtr15. The gtr14 and gtr15 genes occur in several A. baumannii KL gene clusters, including KL5 and KL7 that carry itrA2 rather than itrA3. As ItrA2 introduces d-GalpNAc rather than d-GlcpNAc as the first monosaccharide, Gtr15 can transfer d-GlcpNAc to either of these amino sugars, suggesting that this enzyme has relaxed specificity. Consequently, the third, novel glycosyltransferase, Gtr163, forms the β-(2-3) linkage between Pse5Ac7Ac and d-Galp. Wzy polymerases encoded by KL90 and KL7 are 54% identical and form the same linkage between the K units to give branched polysaccharides with the same main chain but different disaccharide side chains, β-Pse5Ac7Ac-(2-3)-d-Galp in K90 and α-Leg5Ac7Ac-(2-6)-d-Galp in K7.

capsular polysaccharide, Acinetobacter baumannii, Di-N-Acetylpseudaminic acid, KL90 gene cluster

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@@H]1[C@@H](O)[C@@H](OC[C@H]2O[C@H](O[C@@H]3[C@@H](NC(C)=O)[C@H]([*])O[C@H](CO)[C@@H]3O)[C@H](NC(C)=O)[C@@H](O)[C@@H]2O)O[C@H](CO[C@]2(C(=O)O)C[C@H](OC(C)=O)[C@@H](NC(C)=O)[C@H]([C@H](NC(C)=O)[C@@H](C)O)O2)[C@@H]1O

926.876 g/mol (C₃₇H₅₈R₂N₄O₂₃, R = next and previous repeats, not counted in mol weight)

The K5 capsular polysaccharide (CPS) was isolated from the bacterium Acinetobacter baumannii (A. baumannii) SDF and studied by 1D and 2D 1H and 13C NMR spectroscopy before and after O-deacetylation and partial acid hydrolysis. The CPS was found to be composed of branched tetrasaccharide repeats (K units) containing 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic acid (Leg5Ac7Ac). The KL5 capsule biosynthesis gene cluster at the K locus is consistent with the composition and structure of the CPS. KL5 is almost identical to the KL7 gene ulosonic acid (Leg5Ac7Ac A. baumannii LUH5533 that has been characterized earlier, and differs only in the gene for Wzy polymerase that is responsible for the formation of the linkage between the K units. The K5 CPS from strain SDF and the K7 CPS from strain LUH5533 have the same K-unit structure but a different linkage between the K units, which is formed by distinct Wzy polymerases. As opposite to Leg5Ac7Ac in the K7 CPS, this monosaccharide is O-acetylated at position 4 in the K5 CPS. No acetyltransferase for this modification of the K5 CPS is present in the KL5 gene cluster, and hence it is encoded by a gene located elsewhere in the genome.

capsular polysaccharide, Acinetobacter baumannii, legionaminic acid, Wzy polymerase, gene polymorphism, KL gene locus

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
3,6,6,4	Ac	174.3	21.5
3,6,6,5	Ac	174.8-175.9	23.3-23.7
3,6,6,7	Ac	174.8-175.9	23.3-23.7
3,6,6	aXLegp	?	100.9	38.3	71.9	51.1	72.8	55.0	68.2	19.8
3,6	aDGalp	99.6	68.7	80.5	70.5	70.5	65.2
3,2	Ac	174.8-175.9	23.3-23.7
3	aDGlcpN	95.5	54.6	72.5	72.5	70.5	66.1
2	Ac	174.8-175.9	23.3-23.7
	bDGalpN	103.7	52.3	77.0	65.5	76.0	62.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
3,6,6,4	Ac	-	2.03
3,6,6,5	Ac	-	1.89-2.05
3,6,6,7	Ac	-	1.89-2.05
3,6,6	aXLegp	-	-	1.79-2.71	4.88	3.83	4.09	3.87	3.98	1.16
3,6	aDGalp	4.96	3.91	3.95	4.21	4.03	3.62-3.92
3,2	Ac	-	1.89-2.05
3	aDGlcpN	5.05	3.96	3.73	3.65	3.74	3.64-4.14
2	Ac	-	1.89-2.05
	bDGalpN	4.71	4.07	3.82	4.07	3.62	3.74-3.78


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
3,6,6,4	Ac		21.5/2.03
3,6,6,5	Ac		23.3-23.7/1.89-2.05
3,6,6,7	Ac		23.3-23.7/1.89-2.05
3,6,6	aXLegp			38.3/1.79-2.71	71.9/4.88	51.1/3.83	72.8/4.09	55.0/3.87	68.2/3.98	19.8/1.16
3,6	aDGalp	99.6/4.96	68.7/3.91	80.5/3.95	70.5/4.21	70.5/4.03	65.2/3.62-3.92
3,2	Ac		23.3-23.7/1.89-2.05
3	aDGlcpN	95.5/5.05	54.6/3.96	72.5/3.73	72.5/3.65	70.5/3.74	66.1/3.64-4.14
2	Ac		23.3-23.7/1.89-2.05
	bDGalpN	103.7/4.71	52.3/4.07	77.0/3.82	65.5/4.07	76.0/3.62	62.3/3.74-3.78

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@@H]1[C@@H](O)[C@@H](OC[C@H]2O[C@H](O[C@@H]3[C@@H](NC(C)=O)[C@H]([*])O[C@H](CO)[C@@H]3O)[C@H](NC(C)=O)[C@@H](O)[C@@H]2O)O[C@H](CO[C@]2(C(=O)O)C[C@H](OC(C)=O)[C@@H](NC(C)=O)[C@H]([C@H](NC(C)=O)[C@@H](C)O)O2)[C@@H]1O

926.876 g/mol (C₃₇H₅₈R₂N₄O₂₃, R = next and previous repeats, not counted in mol weight)

Acinetobacter baumannii isolate NIPH 329 carries a novel capsular polysaccharide (CPS) gene cluster, designated KL46, that is closely related to the KL5 locus in A. baumannii isolate SDF but includes genes for synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic (di-N-acetylpseudaminic) acid (Pse5Ac7Ac) instead of the corresponding D-glycero-D-galacto isomer (di-N-acetyllegionaminic acid) (Leg5Ac7Ac). In agreement with the genetic content of KL46, chemical studies of the K46 CPS produced by NIPH 329 revealed a branched tetrasaccharide repeat (K unit) with an overall structure the same as K5 from SDF but with β-Pse5Ac7Ac replacing α-Leg5Ac7Ac. As for K5, the K46 unit begins with d-GalpNAc and includes α-d-GlcpNAc-(1→3)-d-GalpNAc and α-d-Galp-(1→6)-d-GlcpNAc linkages, formed by Gtr14 and Gtr15 glycosyltransferases, respectively. The Gtr94K46 glycosyltransferase, which is related to Gtr13K5, links Pse5Ac7Ac to d-Galp in the growing K unit via a β-(2→6) linkage. Nearly identical Wzy enzymes connect the K46 and K5 units via a α-D-GalpNAc-(1→3)-α-D-Galp linkage to form closely related CPSs. Both Pse5Ac7Ac in K46 and Leg5Ac7Ac in K5 are acetylated at O4 but no acetyltransferase gene is present in KL46 or KL5. Related acetyltransferases were found encoded in the NIPH 329 and SDF genomes, but not in other strains carrying an unacetylated Pse or Leg derivative in the CPS. The genes encoding the acetyltransferases were in different putative phage genomes. However, related acetyltransferases were rare among the >3000 publically available genome sequences.

biosynthesis, capsular polysaccharide, locus, Acinetobacter baumannii, gene cluster, nonulosonic acid, glycosyltransferase

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@@H]1[C@@H](O)[C@@H](OC[C@H]2O[C@H](O[C@@H]3[C@@H](NC(C)=O)[C@H]([*])O[C@H](CO)[C@@H]3O)[C@H](NC(C)=O)[C@@H](O)[C@@H]2O)O[C@H](CO[C@@]2(C(=O)O)C[C@H](OC(C)=O)[C@@H](NC(C)=O)[C@H]([C@H](NC(C)=O)[C@@H](C)O)O2)[C@@H]1O

926.876 g/mol (C₃₇H₅₈R₂N₄O₂₃, R = next and previous repeats, not counted in mol weight)