Heterologous expression and characterization of the exopolysaccharide from Streptococcus thermophilus Sfi39.

The genes responsible for exopolysaccharide (EPS) synthesis in Streptococcus thermophilus Sfi39 were identified on a 20-kb genomic fragment. The two genes, epsE and epsG, were shown to be involved in EPS synthesis as their disruption lead to the loss of the ropy phenotype. Several naturally selected nonropy mutants were isolated, one acquired an insertion sequence (IS)-element (IS905) in the middle of the eps gene cluster. The eps gene cluster was cloned and transferred into a nonEPS-producing heterologous host, Lactococcus lactis MG1363. The EPS produced was shown by chemical analysis and NMR spectroscopy to be identical to the EPS produced by S. thermophilus Sfi39. This demonstrated first that all genes needed for EPS production and export were present in the S. thermophilus Sfi39 eps gene cluster, and second that the heterologous production of an EPS was possible by transfer of the complete eps gene cluster alone, provided that the heterologous host possessed all necessary genetic information for precursor synthesis.     

Functional Analysis of Glycosyltransferase Genes from Lactococcus lactis and Other Gram-Positive Cocci: Complementation, Expression, and Diversity

Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the eps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis or Streptococcus thermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS and Streptococcus pneumoniae capsule biosynthesis could completely restore or even increase EPS production in L. lactis.     

Exopolysaccharide biosynthesis genes required for social motility in Myxococcus xanthus

Social (S)-motility in Myxococcus xanthus is a flagellum-independent gliding motility system that allows bacteria to move in groups on solid surfaces. S-motility has been shown to require type IV pili (TFP), exopolysaccharide (EPS; a component of fibrils) and lipopolysaccharide (LPS). Previously, information concerning EPS biogenesis in M. xanthus was lacking. In this study, we screened 5000 randomly mutagenized colonies for defects in S-motility and EPS and identified two genetic regions essential for EPS biogenesis: the EPS synthesis (eps) region and the EPS-associated (eas) region. Mutants with insertions in the eps and eas regions were defective in S-motility and fruiting body formation. These mutants failed to bind the dye calcofluor white, indicating that they lacked EPS; however, they retained normal TFP and LPS. Analysis of the eps locus showed several open reading frames (ORFs) that encode homologues to glycosyltransferases, glucanases and EPS transporters as well as regulatory proteins; the eas locus contains two ORFs: one exhibits homology to hypothetical proteins with a conserved domain of unknown function and the other displays no apparent homology to other proteins in the database. Further genetic mutagenesis analysis indicates that the whole eps region is involved in the biosynthesis of fibrils and fibril EPS. The operon at the proximal end of the eps region was analysed by generating in-frame deletion mutations. These mutants showed varying degrees of defects in the bacterium's ability to produce EPS or perform EPS-related functions, confirming the involvement of these genes in M. xanthus EPS biogenesis.     

The constant gene orf14.9, which belongs to the variable eps (exopolysaccharide) cluster, is involved in the cell growth of Streptococcus thermophilus

In Streptococcus thermophilus, the eps clusters involved in exopolysaccharide (EPS) biosynthesis are very polymorphic, nevertheless they all contain a highly conserved sequence corresponding to that of orf14.9. This open reading frame (ORF) is transcribed in a reverse direction with respect to eps genes. Amino acid sequence analysis showed a possible transmembrane location of the putative Orf14.9 protein but did not permit a proposed function. Insertional mutants of orf14.9 were obtained in strains NST2280 and A054 of S. thermophilus. EPS yields of these mutants are similar to those of their respective wild strains, suggesting that orf14.9 does not modify the quantity of produced EPS. Growth parameter determination for wild strains and their respective mutants showed that orf14.9 is involved in the cell growth of S. thermophilus.     

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster.

Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -->2)-beta-D-Galf-(1-->6)-alpha-D-Glcp- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-GlcpNAc-(1-->, with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.     

Relationships between rfb gene clusters required for biosynthesis of identical D-galactose-containing O antigens in Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16.

The lipopolysaccharide O antigens of Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16 both contain a repeating unit disaccharide of [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]; the resulting polymer is known as D-galactan I. In K. pneumoniae serotype O1, the genes responsible for the synthesis of D-galactan I are found in the rfb gene cluster (rfbKpO1). We report here the cloning and analysis of the rfb cluster from S. marcescens serotype O16 (rfbSmO16). This is the first rfb gene cluster examined for the genus Serratia. Synthesis of D-galactan I is an rfe-dependent process for both K. pneumoniae serotype O1 and S. marcescens serotype O16. Hybridization experiments with probes derived from each of the six rfbKpO1 genes indicate that the cloned rfbSmO16 cluster contains homologous genes arranged in the same order. However, the degree of homology at the nucleotide sequence level was sufficiently low that hybridization was detected only under low-stringency conditions. rfbABSmO16 genes were subcloned and shown to encode an ABC-2 (ATP-binding cassette) transporter which is functionally identical to the one encoded by the corresponding rfb genes from K. pneumoniae serotype O1. The amino acid sequences of the predicted RfbA and RfbB homologs showed identities of 75.7% (87.9% total similarity) and 78.0% (86.5% total similarity), respectively. The last gene of the rfbKpO1 cluster, rfbFKpO1, encodes a bifunctional galactosyltransferase which initiates the formation of D-galactan I. RfbFKpO1 and RfbFSmO16 are 57.6% identical (with 71.1% total similarity), and both show similarity with RfpB, the galactosyltransferase involved in the synthesis of Shigella dysenteriae type I O-polysaccharide. The G+C contents of the rfbAB genes from each organism are quite similar, and values are lower than those typical for the species. However, the G+C content of rfbFSmO16 (47.6%) was much higher than that of rfbFKpO1 (37.3%), despite the fact that the average for each species (52 to 60%) falls within the same range.     

Role of Rfe and RfbF in the initiation of biosynthesis of D-galactan I, the lipopolysaccharide O antigen from Klebsiella pneumoniae serotype O1.

The 6.6-kb rfb gene cluster from Klebsiella pneumoniae serotype O1 (rfbKpO1) contains six genes whose products are required for the biosynthesis of a lipopolysaccharide O antigen with the following repeating unit structure: -->3-beta-D-Galf-1-->3-alpha-D-Galp-1-->(D-galactan I). rfbFKpO1 is the last gene in the cluster, and its gene product is required for the initiation of D-galactan I synthesis. Escherichia coli K-12 strains expressing the RfbFKpO1 polypeptide contain dual galactopyranosyl and galactofuranosyl transferase activity. This activity modifies the host lipopolysaccharide core by adding the disaccharide beta-D-Galf-1-->3-alpha-D-Galp, representing a single repeating unit of D-galactan I. The formation of the lipopolysaccharide substituted either with the disaccharide or with authentic polymeric D-galactan I is dependent on the activity of the Rfe enzyme. Rfe (UDP-GlcpNAc::undecaprenylphosphate GlcpNAc-1-phosphate transferase) catalyzes the formation of the lipid-linked biosynthetic intermediate to which galactosyl residues are transferred during the initial steps of D-galactan I synthesis. The rfbFKpO1 gene comprises 1,131 nucleotides, and the predicted polypeptide consists of 373 amino acid residues with a predicted M(r) of 42,600. A polypeptide with an M(r) of 42,000 was evident in sodium dodecyl sulfate-polyacrylamide gels when rfbKpO1 was expressed behind the T7 promoter. The carboxy-terminal region of RfbFKpO1 shares similarity with the carboxy terminus of RfpB, a galactopyranosyl transferase which is involved in the synthesis of the type 1 O antigen of Shigella dysenteriae.     

Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide

Streptococcus thermophilus Sfi6 produces an exopolysaccharide (EPS) composed of glucose, galactose and N-acetylgalactosamine in the molar ratio of 1:2:1. The genes responsible for the EPS biosynthesis have been isolated previously and found to be clustered in a 14.5 kb region encoding 13 genes. Transfer of this gene cluster into a non-EPS-producing heterologous host, Lactococcus lactis MG1363, yielded an EPS with a similar high molecular weight, but a different structure from the EPS from the native host. The structure of the recombinant EPS was determined by means of 1H homonuclear and 1H-13C heteronuclear two-dimensional nuclear magnetic resonance (NMR) spectra and was found to be --> 3)-beta-D-Glcp-(1 --> 3)-alpha-D-Galp-(1 --> 3)-beta-D-Galp-(1 --> as opposed to --> 3)[alpha-D-Galp-(1 --> 6)]-beta-D-Glcp-(1 --> 3)-alpha-D-GalpNAc-(1 --> 3)-beta-D-Galp-(1 --> for the wild-type S. thermophilus Sfi6. Furthermore, L. lactis MG1363 (pFS101) was also lacking a UDP-N-acetylglucosamine C4-epimerase activity, which would provide UDP-GalNAc for a GalNAc incorporation into the EPS and probably caused the substitution of GalNAc by Gal in the recombinant EPS. This modification implies that (i) bacterial glycosyltransferases could potentially have multiple specificities for the donor and the acceptor sugar molecule; and (ii) the repeating unit polymerase can recognize and polymerize a repeating unit that differs in the backbone as well as in the side-chain from its native substrate.     

Unraveling the function of glycosyltransferases in Streptococcus thermophilus Sfi6.

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (EPS) consisting of a -->3)[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->3)-alpha-D-GalpNAc-(1--> 3)-beta-D-Galp-(1--> repeating unit. We previously identified and analyzed a 14.5-kb gene cluster from S. thermophilus Sfi6 consisting of 13 genes responsible for its EPS production. Within this gene cluster, we found a central region of genes (epsE, epsF, epsG, and epsI) that showed similarity to glycosyltransferases. In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the biosynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose onto the lipophilic carrier. The second step is fulfilled by EpsG, which transfers an alpha-N-acetylgalactosamine onto the first beta-galactoside. The activity of EpsF was determined by characterizing the EPS produced by an S. thermophilus epsF deletion mutant. This EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approximately equimolar ratio, thus suggesting that epsF codes for the branching galactosyltransferase. epsI probably codes for the beta-1,3-glucosyltransferase, since it is the only glycosyltransferase to which no gene has been assigned and it exhibits similarity to other beta-glycosyltransferases. EpsE shows the conserved features of phosphoglycosyltransferases, whereas EpsF and EpsG exhibit the primary structure of alpha-glycosyltransferases, belonging to glycosyltransferase family 4, whose members are conserved in all major phylogenetic lineages, including the Archaea and Eukaryota.     

A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice.

The attenuated Sabin strain of poliovirus type 1 (PV-1) differs from the neurovirulent PV-1 Mahoney strain by 55 nucleotide mutations. Only one of these mutations (A-480-->G, in the 5' noncoding (5' NC) region of the genome, is well characterized, and it confers a strong attenuating effect. We attempted to identify genetic attenuation determinants in the 3'-terminal part of the Sabin 1 genome including the 3D polymerase (3Dpol) gene and the 3' NC region. Previous studies suggested that some of the 11 mutations in this region of the Sabin 1 genome, and in particular a mutation in the polymerase gene (U-6203-->C, Tyr-73-->His), are involved to some extent in the attenuation of PV-1. We analyzed the attenuating effect in the mouse model by using the mouse-adapted PV-1/PV-2 chimeric strain v510 (a Mahoney strain carrying nine amino acids of the VP1 capsid protein from the Lansing strain of PV-2). Mutagenesis of locus 6203 was performed on the original v510 (U-6203-->C) and also on a hybrid v510/Sabin 1 (C-6203-->U) carrying the downstream 1,840 nucleotides of the Sabin 1 genome including the 3Dpol and 3' NC regions. Statistical analysis of disease incidence and time to disease onset in numerous mice inoculated with these strains strongly suggested that nucleotide C-6203 is involved in the attenuation of the Sabin 1 strain. Results also suggested that, among the mutations located in the 3Dpol and 3' NC regions, nucleotide C-6203 may be the principal or the only one to be involved in attenuation in this mouse model. We also found that the effect of C-6203 was weaker than that of nucleotide G-480; the two nucleotides acted independently and may have a cumulative effect on attenuation. The U-6203-->C substitution also appeared to contribute to the thermosensitivity of the Sabin 1 strain.     

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.     

Beta-glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta-(1-->6) linkages.

Bradyrhizobium japonicum synthesizes periplasmic cyclic beta-(1-->3),beta-(1-->6)-D-glucans during growth in hypoosmotic environments, and evidence is growing that these molecules may have a specific function during plant-microbe interactions in addition to osmoregulation. Site-directed Tn5 mutagenesis of the DNA region upstream of ndvB resulted in identification of a new gene (ndvC) involved in beta-(1--> 3), beta-(1-->6)-glucan synthesis and in nodule development. The predicted translation product was a polypeptide (ca. 62 kDa) with several transmembrane domains. It contained a sequence characteristic of a conserved nucleoside-sugar-binding motif found in many bacterial enzymes and had 51% similarity with a beta-glucanosyltransferase from Candida albicans. B. japonicum carrying a Tn5 insertion in ndvC resulted in synthesis of altered cyclic beta-glucans composed almost entirely of beta-(1--> 3)-glycosyl linkages. The mutant strain was only slightly sensitive to hypoosmotic growth conditions compared with the ndvB mutant, but it was severely impaired in symbiotic interactions with soybean (Glycine max). Nodulation was delayed by 8 to 10 days, and many small nodule-like structures apparently devoid of viable bacteria were formed. This finding suggests that the structure of the beta-glucan molecule is important for a successful symbiotic interaction, and beta-glucans may have a specific function in addition to their role in hypoosmotic adaptation.     

SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis.

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.     

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.     

Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties?

Over the last years, important advances have been made in the study of the production of exopolysaccharides (EPS) by several lactic acid bacteria, including Lactococcus lactis. From different EPS-producing lactococcal strains the specific eps gene clusters have been characterised. They contain eps genes, which are involved in EPS repeating unit synthesis, export, polymerisation, and chain length determination. The function of the glycosyltransferase genes has been established and the availability of these genes opened the way to EPS engineering. In addition to the eps genes, biosynthesis of EPS requires a number of housekeeping genes that are involved in the metabolic pathways leading to the EPS-building blocks, the nucleotide sugars. The identification and characterisation of several of these housekeeping genes (galE, galU, rfbABCD) allows the design of metabolic engineering strategies that should lead to increased EPS production levels by L. lactis. Finally, model developme nt has been initiated in order to predict the physicochemical consequences of the addition of a EPS to a product.