The polysialic acid synthesis pathway in E. coli consists of three steps: (i) formation of the precursor, CMP-NeuNAc, (ii) polymerization of sialic acid, and (iii) export of the polymer to the cell surface. The polysaccharide is polymerized by the transfer of sialic acid from CMP-NeuNAc to the nonreducing end of the growing chain in a linkage-specific fashion. Polymerization is believed to be initiated by transfer to an endogenous acceptor molecule and would therefore imply two types of sialyltransferase reactions, (i) one in which NeuNAc is transferred from CMP-NeuNAc to a growing chain of polysialic acid and (ii) a second in which NeuNAc is transferred from CMP-NeuNAc to an endogenous acceptor. The nature of this acceptor remains unknown.
Although there are 14 genes in the E. coli
K1/K92 gene cluster, only one gene, neuS
, is known to encode a sialyltransferase. The neuS
-encoded polysialyltransferase can only catalyze the elongation reaction in the absence of the other proteins encoded by the gene cluster (16
). In this study we identified the minimum combination of gene products required to observe the overall synthesis of polysialic acid de novo from CMP-sialic acid in vitro. Our results show that there are three genes necessary to observe de novo polysialic acid synthesis in E. coli
K1 in vitro, the neuES
of region 2 and kpsC
of region 1. KpsS increases polysaccharide production by several fold (about 10 to 15 times) over that observed with neuES
. We think that it is unlikely the difference is due to the level of KpsC expression, since the constructs only differ by the presence or absence of a complete kpsS
gene. Nevertheless, it is conceivable that KpsS could influence the stability of expressed KpsC protein. The size and repeating structure of the polysialic acid produced in membranes or cells lacking kpsS
do not appear to be affected by the absence of this gene. Gel filtration and PAGE experiments showed that in the presence of [14
C]CMP-NeuNAc three proteins (NeuES-KpsC) were able to assemble high-molecular-weight polymer in vitro. Intracellular polysaccharide isolated from a kpsS
deletion mutant was structurally similar to wild-type K1 polysaccharide as determined by 1
H and 13
C NMR. We conclude that kpsC
plays some role in de novo polysaccharide synthesis since region 2 gene products were not observed to polymerize sialic acid in the absence of kpsC
Strains carrying neuES
also produce polysialic acid in vivo when supplied with a source of CMP-NeuNAc. We coexpressed NeuES-KpsCS proteins together with CMP-NeuNAc synthetase NeuA (31
) and supplied free sialic acid in the medium. We purified polysaccharides produced in vivo during equivalent time periods by NeuES-KpsC and NeuES-KpsCS under these growth conditions. Polysaccharides isolated from these constructs were similar to each other in size and very similar to K1 wild-type extracellular polysaccharide. The only difference observed between NeuES-KpsC and NeuES-KpsCS samples was the decreased amount of polysaccharide produced in the absence of KpsS. Based on these experiments, the minimum combination of K1 capsule cluster genes required for de novo synthesis of polysialic acid from CMP-NeuNAc in vivo and in vitro is the same.
One of the interesting aspects of this study was the determination of the size of the neuE
ORF and the conclusion that the NeuE protein plays some role in the initiation of polysialic acid synthesis. An acapsular phenotype has been reported for a neuE
deletion mutant (35
), implying that the product of this gene participates in capsule production. Since the original report of the neuE
ORF sequence by Steenbergen et al. (27
), other neuE
sequences have been submitted to the data bank, suggesting that this gene might encode a larger protein than was presumed initially. Our results showed that the shorter version neuE
did not support a polysialic acid synthesis initiation in vitro in a region 1- and 3-positive background. To determine the size of the neuE
ORF we sequenced the region between and overlapping neuC
. A large ORF was observed which overlaps both neuC
genes. The fact that the plasmid containing the longer ORF could support de novo polysialic acid synthesis in the appropriate background suggested not only that the neuE
ORF is longer than it was assumed before, but also that the product of this gene plays a role in de novo polysialic acid synthesis. Our results indicate that NeuE is the only region 2-encoded protein other than NeuS needed for synthesis of polysialic acid from CMP-NeuNAc. Our results established the minimal effective size of the neuE
gene. We demonstrated that translation of an active protein could begin at ATG 88; however, the ATG 61 of the neuE
ORF is most likely the native translation start in vivo.
In earlier reports KpsC and KpsS were suggested to a play role in phosphatidyl-KDO substitution of group II E. coli
capsular polysaccharide (10
mutants accumulate intracellular polysaccharide (23
), which in K5 lacks a phosphatidyl-KDO moiety at the reducing end of the chain (23
). Because kpsC
mutations did not prevent production of K5 polysaccharide in vivo, it was proposed that phosphatidyl-KDO substitution occurs after initiation of synthesis (10
). In the absence of the exogenous acceptor we were unable to detect [14
C]NeuNAc incorporation by the membranes harboring K1 NeuES KpsS in vitro, but a low level of synthesis by the membranes harboring K1 NeuES KpsC was detected. Thus, in our hands KpsC appears to be essential for de novo synthesis of polysialic acid.
In E. coli
K5, KpsC and KpsS proteins have been shown to be associated with the inner membrane (21
). Deletion of kpsS
in E. coli
K5 results in failure of membrane targeting of both K5 glycosyltransferases KfiA and KfiC (21
). We do not know how far we can draw parallels between K1 and K5 mechanisms of polysaccharide synthesis. The membrane localization of K1 and K92 polysialyltransferases does not depend on the presence of the other capsule proteins. However, the fact that K1 proteins NeuES were able to initiate polysialic acid synthesis without exogenous acceptor in the MS108 (K5) background suggests that the K5 and K1 KpsCS have similar functions in both strains.
The major focus of this report was to describe the genetic requirement for the polymerization reaction catalyzed by the polysialyltransferase. We have clearly demonstrated that membranes isolated from E. coli carrying neuES and kpsC are sufficient to observe synthesis of polysaccharide from the polysialyltransferase substrate CMP-NeuNAc. Exactly what reaction occurs prior to polymerization will not be known until an endogenous acceptor can be isolated. In preliminary experiments we have extracted a hydrophobic substance that can act as an acceptor for TOP10 membranes harboring NeuS. This hydrophobic substance has been extracted from a neuS neuB-negative K1 strain and is currently being characterized (J. Vionnet and W. F. Vann, unpublished results). Nevertheless, we have learned from the present experiments that NeuE and KpsC function along with the polysialyltransferase to form a polymer. Whether these two proteins function in the formation of endogenous acceptor or support the transferase reaction as part of a complex is not clear at this point. These experiments lead one to ask whether NeuE, KpsC, and KpsS interact with the polysialyltransferase or whether either of these participates directly in the catalytic reaction.
One cannot deduce the function of these proteins from sequence homology since homologs do not have established functions. Interestingly, the translated Neisseria meningitidis
gene (NMB0065) shares homologous amino acid sequences with the NeuE gene (5
) (Table ). This gene or its homologs are present in sialic acid-producing N. meningitidis
(serogroups B, C, Y, and W135) but are not present in serogroup A, which does not produce a sialic acid-containing polysaccharide (6
). Transposon insertion into the N. meningitidis
serogroup C 0065 gene resulted in an unencapsulated phenotype (12
). It would be interesting to know if NMB0065 and NeuE play similar roles in polysaccharide production. While there are not many genes with significant homology to neuE
, there is a larger group of the gram-negative bacteria (encapsulated and acapsular) possessing “kpsCS
tandem,” a conserved pair of genes with significant homology to kpsC
and in the same direction of the transcription (Table ). No definitive function has been assigned to any of the homologs of neuE
, and kpsS
. In the future it would be interesting to investigate if the kpsCS
of other organisms play a similar role in capsule polysaccharide initiation as in K1/K92 E.coli.
Homologs of NeuE, KpsC, and KpsS proteins found in gram-negative bacterial strains
KpsC and KpsS were analyzed using the NCBI BLAST software and shown to share homologous sequences with two families of glycosyltransferases. It is conceivable that these proteins may be involved in the formation of acceptor molecules. Both proteins have been suggested (22
) to play a role in the addition of phosphatidyl KDO to the reducing end of E. coli
K5 polysaccharide. Thus, one could speculate that transfer of KDO to a phospholipid by KpsC would yield a putative acceptor molecule. This would in turn allow the polysialyltransferase to begin de novo synthesis. If the interaction of the polysialyltransferase with this phospholipid acceptor were less than ideal, a low level of polymer synthesis would be expected. The role of KpsS could then be to improve this interaction and facilitate normal de novo polysialic acid synthesis. Whether this scenario actually occurs awaits further experimentation.