Hyaluronan Biosynthesis Research Articles

Manipulation of Hyaluronan Synthase Expression in Prostate Adenocarcinoma Cells Alters Pericellular Matrix Retention and Adhesion to Bone Marrow Endothelial Cells

Prostate cancer metastasis to bone marrow involves initial adhesion of tumor cells to the bone marrow endothelium, followed by transmigration and proliferation within the marrow. Rapid, specific adhesion of highly metastatic prostate adenocarcinoma cells (PC3M-LN4) to bone marrow endothelial cell (BMEC) lines requires a pericellular hyaluronan (HA) matrix and correlates with dramatically up-regulated HA synthase (HAS) expression. Non-metastatic prostate tumor cells (LNCaP) do not assemble a HA matrix, adhere poorly to BMECs, and express normal levels of HAS. Preferential bone metastasis of prostate carcinoma cells may therefore be facilitated by tumor cell HA biosynthesis. In this report, HAS gene expression was manipulated to investigate the direct impact of prostate tumor cell HA production on adhesion to BMECs. PC3M-LN4 cells stably transfected with antisense HAS2 and HAS3 failed to form pericellular matrices. Adhesion of these transfectants to BMECs was significantly diminished, comparable to the low level exhibited by LNCaP cells. Upon transfection with full-length HAS2 or HAS3, the non-adherent LNCaP cells retained pericellular HA and adhered to BMECs. The results of this study are consistent with a model in which HA matrix formation, BMEC adhesion, and metastatic potential are mediated by HAS expression.

Epithelial repair: roles of extracellular matrix.

To review studies of the roles of extracellular matrix (ECM) metabolism in corneal epithelium during wound repair. Methods. 1) Alterations in the structure and composition of epithelial basement membrane during corneal epithelial healing were examined histologically and immunohistochemically. 2) The effects of procollagen and hyaluronan synthesis inhibitors on the spread of rabbit corneal epithelium were determined in organ culture. 3) Expression of keratan sulfate proteoglycan (KSPG) proteins in corneal epithelium was examined during repair after injury in wild-type and lumican-null mice. 1) Corneal epithelial basement membrane was transiently degraded and reassembled during tissue repair. Patterns of type IV collagen immunoreactivity were also transiently altered. The system of matrix metalloproteinase-tissue inhibitors of metalloproteinases may play an important role in the disassembly and reorganization of epithelial basement membrane. 2) Inhibitors of procollagen secretion and hyaluronan biosynthesis disrupted the spread of a corneal epithelial sheet in situ. 3) Among the corneal KSPG proteins examined, lumican was transiently expressed in migrating murine corneal epithelial cells. Anti-lumican antibody inhibited corneal epithelial resurfacing in organ culture. The absence of lumican was found to delay corneal epithelial wound healing in mice. Extracellular matrix metabolism by the injured corneal epithelium is important in the repair process.

EFFECT OF HYALURONAN ON MMP EXPRESSION AND ACTIVATION

Matrix metalloproteases (MMPs) play a crucial role in tissue remodelling in a variety of physiological and pathological processes. Hyaluronan is also involved in the same processes. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan and also of MMPs. The activity of MMPs has been shown to be regulated at the level of transcription and activation of the zymogen form.In order to explore the possible relationship between matrix components and especially hyaluronan, we studied the effect of hyaluronan on MMP expression (biosynthesis and activation) in the culture of human skin fibroblasts and corneal keratocytes (explant cultures and cell cultures). These cells were shown to exhibit distinct phenotypes as far as matrix biosynthesis is concerned. Using a synthetic substrate N-Suc(ala)3pNA, we measured elastase-type endopeptidase activity produced by fibroblasts and keratocytes and characterized the MMPs by zymography.Hyaluronan added to fibroblast cultures stimulated the membrane-bound elastase-type endopeptidase activity in a dose dependent fashion. Similar results were obtained with keratocyte cultures. In the presence of 1mg/ml hyaluronan there was an increase in MMP expression and also an activation of latent MMPs both by fibroblasts and keratocytes.

Stimulation of hyaluronan metabolism by interleukin‐1α in human articular cartilage

To determine the effects of interleukin-1alpha (IL-1alpha) on the expression of hyaluronan synthase (HAS), CD44, and aggrecan in human articular chondrocytes, and to assess the net result of these metabolic changes on the accumulation of hyaluronan within articular cartilage.Normal human articular cartilage slices, as well as isolated chondrocytes, were treated with IL-1alpha. Changes in the relative expression of messenger RNA (mRNA) for HAS-2, CD44, and aggrecan were determined by competitive, quantitative reverse transcriptase-polymerase chain reaction. Hyaluronan accumulation was characterized by staining with a hyaluronan-specific binding protein and by fluorophore-assisted carbohydrate electrophoresis, while proteoglycan content was determined by alcian blue and Safranin O staining, CD44 protein expression by immunohistochemistry, and aggrecan biosynthesis by 35S-sulfate incorporation. Changes in cell-associated matrix sizes were visualized by a particle exclusion assay.IL-1alpha stimulated the expression of HAS-2 and CD44 mRNA (3.5-fold and 3-fold, respectively), but inhibited the expression of aggrecan mRNA. In IL-1-treated chondrocytes, extracellular hyaluronan decreased, while intracellular accumulation of hyaluronan was enhanced. Together with the decrease in expression of aggrecan, a dramatic reduction in cell-associated matrix was observed. IL-1-treated cartilage slices displayed a prominent depletion of aggrecan as well as hyaluronan within the upper layers of the tissue. The regional loss of hyaluronan coincided with a regional up-regulation of CD44.These data demonstrate that IL-1alpha stimulates HAS-2 at the same time as it inhibits the expression of aggrecan. Although hyaluronan biosynthesis is up-regulated, so too is the expression of CD44 and the internalization/catabolism of hyaluronan. The net result is a loss of hyaluronan in areas of the articular cartilage where increases in CD44 expression are most prominent. This depletion of hyaluronan in the upper layers of the tissue likely facilitates the prominent loss of aggrecan from the tissue.

Stimulation of hyaluronan metabolism by interleukin-1alpha in human articular cartilage.

To determine the effects of interleukin-1alpha (IL-1alpha) on the expression of hyaluronan synthase (HAS), CD44, and aggrecan in human articular chondrocytes, and to assess the net result of these metabolic changes on the accumulation of hyaluronan within articular cartilage. Normal human articular cartilage slices, as well as isolated chondrocytes, were treated with IL-1alpha. Changes in the relative expression of messenger RNA (mRNA) for HAS-2, CD44, and aggrecan were determined by competitive, quantitative reverse transcriptase-polymerase chain reaction. Hyaluronan accumulation was characterized by staining with a hyaluronan-specific binding protein and by fluorophore-assisted carbohydrate electrophoresis, while proteoglycan content was determined by alcian blue and Safranin O staining, CD44 protein expression by immunohistochemistry, and aggrecan biosynthesis by 35S-sulfate incorporation. Changes in cell-associated matrix sizes were visualized by a particle exclusion assay. IL-1alpha stimulated the expression of HAS-2 and CD44 mRNA (3.5-fold and 3-fold, respectively), but inhibited the expression of aggrecan mRNA. In IL-1-treated chondrocytes, extracellular hyaluronan decreased, while intracellular accumulation of hyaluronan was enhanced. Together with the decrease in expression of aggrecan, a dramatic reduction in cell-associated matrix was observed. IL-1-treated cartilage slices displayed a prominent depletion of aggrecan as well as hyaluronan within the upper layers of the tissue. The regional loss of hyaluronan coincided with a regional up-regulation of CD44. These data demonstrate that IL-1alpha stimulates HAS-2 at the same time as it inhibits the expression of aggrecan. Although hyaluronan biosynthesis is up-regulated, so too is the expression of CD44 and the internalization/catabolism of hyaluronan. The net result is a loss of hyaluronan in areas of the articular cartilage where increases in CD44 expression are most prominent. This depletion of hyaluronan in the upper layers of the tissue likely facilitates the prominent loss of aggrecan from the tissue.

Three Isoforms of Mammalian Hyaluronan Synthases Have Distinct Enzymatic Properties

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.

Mammalian hyaluronan synthases: investigation of functional relationships in vivo.

Introduction Hyaluronan (HA) biosynthesis occurs at the inner face of the plasma membrane [1,2], in contrast to the biosynthesis of other glycosaminoglycans, which are synthesized within the Golgi network. Three HA synthase (ID1S) genes have been identified in vertebrates [3-81. Each gene encodes a predicted plasma membrane glycosyltransferase with multiple transmembrane domains. The mammalian HAS proteins share between 55 and 71% amino acid identity, and are encoded by distinct genes located on separate autosomes [9]. Expression of any one IfAS gene is sufficient to drive the biosynthesis of HA in mammalian cells ([S]; N. Itano, M. Yosida, P. Lenas, Y. Yamada, A. P. Spicer, J. A. McDonald and K. Kimata, unpublished work). We have previously demonstrated the differential expression of ID1S genes during embryonic development and in adult tissues [S]. Thus, promoter and enhancer sequences responsible for regulating the transcription of the respective €US genes have diverged substantially since this small gene family arose through gene duplication. However, it remains unclear as to whether or not the enzymic functions of the three HAS proteins have also diverged. Recent data obtained through cell culture studies suggest that although any one HAS protein is sufficient for HA biosynthesis,

ヒアルロン酸の生物学的活性を考える

Hyaluronan (HA) is an unbranched glycosaminoglycan (GAG) which is the only nonsulfated disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. HA is synthesized by most cells in various animal tissues and has by far the largest molecular size, with an average molecular weight of several million. HA plays important roles in tissue development, tissue hydration, cell surface-matrix interactions, cell migration, inflammation, and wound healing. The biosynthesis of HA is unique among the GAG. Rather than being made in the Golgi apparatus, HA is synthesized via HA synthase (HAS) which is located at the plasma membrane. Recently, it has been reported that 3 subtypes of HAS, named HAS 1, HAS 2 and HAS 3, have been cloned and that each of those plays a different role in HA production in vivo. Several lines of evidence demonstrated that expression of HAS and synthesis of HA are regulated by a variety of mechanisms other than cytokines and that the inducible HA production generates HA with relatively high molecular weight. Furthermore, recent observation demonstrated that HA with low molecular weight showed various biological activities such as induction of chemokines and iNOS. These data may suggest that HA plays crucial roles both in continuation of inflammatory responses and in restoration and maintenance of tissue homeostasis.

Stimulation of hyaluronan synthesis by tumor necrosis factor-α is mediated by the p50/p65 NF–κB complex in MRC-5 myofibroblasts

The lesions of fibrocontractive diseases result from an excessive myofibroproliferative response to numerous forms of inflammatory stimuli, which elicit the net deposition of extracellular matrix (ECM) in the interstitium of the affected tissue. Hyaluronan (HA), reported to be a key player supporting cellular migration and adherence, is a major component of ECM that undergoes dynamic regulation during inflammation. The molecular regulation of HA biosynthesis by inflammatory cytokines on myofibroblasts is not yet completely understood. Here we report the biochemical characteristics of the lung myofibroblast cell line MRC-5, and we demonstrate that the production of HA by this cell line is inducible by the proinflammatory cytokine, tumor necrosis factor-α (TNF-α), at the message level of HA synthase (HAS). In TNF-α-stimulated MRC-5 cells, DNA-binding and competition experiments indicated that the predominant NF–κB binding activity detected with nuclear extract-stimulated cells is mediated by the p50/p65 complex. Using antisense oligonucleotides, we confirmed that the TNF-α-stimulation of HA synthesis by MRC-5 cells is dependent on the activation of the p50/p65 NF–κB complex. These findings indicate that TNF-α production within inflamed tissues may enhance the HA synthesis via the transcriptional induction of HAS on myofibroblasts, thereby providing a provisional matrix for supporting cellular migration and adhesion, and that the p50/p65 NF–κB complex that plays an important role in the regulation of HA production by TNF-α might be an appropriate target for therapeutic compounds to treat tissue fibrosis accompanied by inflammation.

Differential Effects ofStaphylococcus aureusα-Hemolysin on the Synthesis of Hyaluronan and Chondroitin Sulfate by Rat Chondrosarcoma Chondrocytes

The plasma membranes of rat chondrosarcoma chondrocytes were permeabilized by treatment with α-hemolysin, the major toxin produced byStaphylococcus aureus,which forms small, stable, heptameric, transmembrane pores (1–2 nm in diameter) permitting influx/efflux of low-molecular-mass molecules (≤2000 Da). Treated chondrocytes were permeable to entry of trypan blue and exit of ATP. We describe the effects of α-hemolysin on the synthesis of hyaluronan (HA) and chondroitin sulfate (CS) by chondrocytes using the simple sugar [3H]glucosamine as a metabolic precursor. Chondrocytes permeabilized with α-hemolysin in serum-free media decreased intracellular ATP and synthesis of CS to ∼5% of control within 2–4 h, but synthesized HA (80% of control for 8 h; ∼65% of control at 24 h). Adding fresh medium (with or without serum) to permeabilized cells increased ATP significantly and increased HA synthesis to near initial control values. Under the same conditions, the recovery of CS synthesis approached initial levels in control but not permeabilized cells. Our model demonstrates that the biosynthesis of HA by these cellsin vitrois remarkably stable to cellular perturbations which drastically inhibit synthesis of CS on proteoglycans.

Characterization and Molecular Evolution of a Vertebrate Hyaluronan Synthase Gene Family

The three mammalian hyaluronan synthase (HAS) genes and the related Xenopus laevis gene, DG42, belong to a larger evolutionarily conserved vertebrate HAS gene family. We have characterized additional vertebrate HAS genes from chicken (chas2 and chas3) and Xenopus (xhas2, xhas3, and a unique Xenopus HAS-related sequence, xHAS-rs). Genomic structure analyses demonstrated that all vertebrate HAS genes share at least one exon-intron boundary, suggesting that they evolved from a common ancestral gene. Furthermore, the Has2 and Has3 genes are identical in structure, suggesting that they arose by a gene duplication event early in vertebrate evolution. Significantly, similarities in the genomic structures of the mouse Has1 and Xenopus DG42 genes strongly suggest that they are orthologues. Northern analyses revealed a similar temporal expression pattern of HAS genes in developing mouse and Xenopus embryos. Expression of mouse Has2, Has3, and Xenopus Has1 (DG42) led to hyaluronan biosynthesis in transfected mammalian cells. However, only mouse Has2 and Has3 expressing cells formed significant hyaluronan-dependent pericellular coats in culture, implying both functional similarities and differences among vertebrate HAS enzymes. We propose that vertebrate hyaluronan biosynthesis is regulated by a comparatively ancient gene family that has arisen by sequential gene duplication and divergence.

Chromosomal Localization of the Human and Mouse Hyaluronan Synthase Genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designatedHAS1, HAS2,andHAS3in humans andHas1, Has2,andHas3in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to theStreptococcus pyogenesHA synthase, HasA. Furthermore, expression of any oneHASgene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the threeHASgenes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes.HAS1was localized to the human chromosome 19q13.3–q13.4 boundary andHas1to mouse Chr 17.HAS2was localized to human chromosome 8q24.12 andHas2to mouse Chr 15.HAS3was localized to human chromosome 16q22.1 andHas3to mouse Chr 8. The map position forHAS1reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17.HAS2mapped outside the predicted critical region delineated for the Langer–Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.

Biosynthesis and degradation of hyaluronan by nonparenchymal liver cells during liver regeneration

Hepatic stellate cells (HSC) and endothelial cells of the liver sinusoids synthesize and degrade hyaluronan, respectively. The roles of these cell types in the biosynthesis and degradation of hyaluronan were studied during regeneration following partial hepatectomy. Pure cultures of HSC and liver endothelial cells (LEC) were obtained from regenerating liver at different stages using a Nycodenz gradient followed by discontinuous Percoll gradient. The HSC that established 3 or 4 days after partial hepatectomy synthesized large amounts of hyaluronan when cultured in the presence of fetal calf serum (FCS) or platelet-derived growth factor B-chain homodimer (PDGF)-BB. These cells, as well as LEC, expressed active PDGF beta-receptors. Furthermore, the ability of LEC to degrade hyaluronan was decreased at early stages of liver regeneration. The increased synthesis of hyaluronan by HSC and the failure of LEC to catabolize the polysaccharide resulted in elevated hyaluronan concentrations in the blood. (Hepatology 1996 Jun;23(6):1650-5)

Biosynthesis and degradation of hyaluronan by nonparenchymal liver cells during liver regeneration.

Hepatic stellate cells (HSC) and endothelial cells of the liver sinusoids synthesize and degrade hyaluronan, respectively. The roles of these cell types in the biosynthesis and degradation of hyaluronan were studied during regeneration following partial hepatectomy. Pure cultures of HSC and liver endothelial cells (LEC) were obtained from regenerating liver at different stages using a Nycodenz gradient followed by discontinuous Percoll gradient. The HSC that established 3 or 4 days after partial hepatectomy synthesized large amounts of hyaluronan when cultured in the presence of fetal calf serum (FCS) or platelet-derived growth factor B-chain homodimer (PDGF)-BB. These cells, as well as LEC, expressed active PDGF beta-receptors. Furthermore, the ability of LEC to degrade hyaluronan was decreased at early stages of liver regeneration. The increased synthesis of hyaluronan by HSC and the failure of LEC to catabolize the polysaccharide resulted in elevated hyaluronan concentrations in the blood.

Compression of Cartilage Results in Differential Effects on Biosynthetic Pathways for Aggrecan, Link Protein, and Hyaluronan

The differential effects of static compression and recovery from compression on biosynthesis and biosynthetic pathways of aggrecan, link protein, and hyaluronan were assessed. During compression, biosynthesis of aggrecan and link protein were inhibited to ∼25 and ∼40%, respectively, of free-swelling control levels. In marked contrast, hyaluronan synthesis was unaffected by static compression. After release from 12-h 50% static compression, aggrecan synthesis remained inhibited for up to 2.5 days; however, link protein synthesis completely recovered to free-swelling control levels within 8 h after release. Hyaluronan synthesis remained at control levels after release of compression. During compression, aggrecan core protein pool size was decreased, whereas the rate of processing into the proteoglycan form remained essentially the same as in free swelling control tissue. Four hours after release from compression, aggrecan core protein pool size remained small and the rate of intracellular processing of aggrecan had become slower than that of free swelling control tissue. Due to the altered core-protein processing kinetics, fewer but longer chondroitin sulfate chains were added to the core proteins. Sulfation was not markedly altered. The differential effects of static compression and release on the biosynthesis of aggrecan, link protein, and hyaluronan are similar to the changes in the biosynthetic pathways that are affected in response to IL-1 treatment, suggesting that the response to static compression is not a general inhibition of cellular activity, but appears to be a part of a specific transduction mechanism.

Regulation of hyaluronan synthesis and the interaction of hyaluronan with cells

Hyaluronan is a high molecular weight polysaccharide ubiquitously found in the extracellular matrix. It acts as a lubricant in joints and tissues and provides stability and elasticity to the extracellular matrix. In addition, hyaluronan is directly or indirectly implicated in cell growth, angiogenesis, differentiation and malignant tumor invasion (for a review see Laurent and Fraser 1992, Sherman et al. 1994). This review focuses on the regulation of the biosynthesis of hyaluronan and the formation and structure of the pericellular coat around hyaluronan producing cells.

Molecular mechanisms and genetics of hyaluronan biosynthesis

Hyaluronan is an extremely important polysaccharide from both the biological and commercial points of view. This review summarizes the present state of the art concerning the polymer and our understanding of the molecular mechanisms of its synthesis with emphasis on the implications of this understanding for polysaccharide engineering of hyaluronan.

The Streptococcus pyogenes Hyaluronan Synthase: Sequence Comparison and Conservation among Various Group A Strains

We recently cloned the hyaluronan synthase gene ( hasA) from Streptococcus pyogenes (DeAngelis et al., J. Biol. Chem. 268, 19181, 1993). Since this is the first glycosaminoglycan synthase gene to be cloned and these enzymes have also not been purified, nothing is yet known at the molecular level about the similarity or relatedness of hyaluronan synthase to other gene products. We found several proteins in the sequence database with substantial similarities to hyaluronan synthase (HasA), including NodC from Rhizobium, DG42 from Xenopus, and the three chitin synthases (Chs) from Saccharomyces. Like HasA, NodC and the Chs proteins all have at least an N-acetylglucosaminyl transferase activity in common and are membrane-associated. The polymerase chain reaction was also used to show that HasA, and probably the two gene operon for hyaluronan biosynthesis ( hasA/ hasB), is highly conserved among Group A streptococcal strains. In nine strains, isolated from 1917 through 1993, only five silent mutations were detected. The results show that hyaluronan synthase is highly conserved within Group A strains, although another virulence factor, the M protein, varies considerably in these same strains.

Molecular cloning, identification, and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes.

The hyaluronan (HA) synthase of Group A Streptococci has been identified by transposon mutagenesis and deletion analysis. The genes for the HA synthase and a recently identified UDP-Glc dehydrogenase (Dougherty, B. A., and van de Rijn, I. (1993) J. Biol. Chem. 268, 7118-7124) reside on a contiguous stretch of 3.2-kilobase pair DNA that can direct HA biosynthesis in Enterococcus faecalis and Escherichia coli as well as mutant Streptococcus (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 14568-14571). The synthase contains 395 residues (calculated Mr = 45,063) and migrates on SDS-PAGE with a molecular mass of 42 kDa. E. coli K5, which synthesizes UDP-glucuronic acid for production of its endogenous capsular polysaccharide, can make HA if it contains a plasmid encoding the intact 42-kDa protein. E. coli SURE or chi 1448 cells containing the same construct, however, cannot produce HA since these strains cannot make both required sugar nucleotide precursors. The HA synthase is predicted to be an integral membrane protein with four membrane-associated helices, which is consistent with the location of the enzyme activity in Streptococci. There is significant homology between the HA synthase and the Rhizobium nodC gene product, an enzyme that synthesizes chitin-like oligomers. This is the first description at the molecular level of an enzyme shown to synthesize a glycosaminoglycan.

Isolation of a Streptococcus pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria

A contiguous 3-kilobase pair region of DNA was isolated from Group A Streptococcus pyogenes (GAS) that can direct hyaluronic acid (HA) capsule biosynthesis in acapsular mutants as well as heterologous bacteria. The DNA was identified by transposon 916 insertional mutagenesis and subcloned into a plasmid shuttle vector. Mutant acapsular GAS or Enterococcus faecalis containing this plasmid, but not vector alone, displayed a mucoid phenotype on agar plates, possessed a capsule as seen by light microscopy, and produced HA in quantities comparable with wild-type GAS. The polysaccharide was shown to be authentic HA based on its recognition by a specific proteoglycan and its degradation by Streptomyces hyaluronate lyase. Escherichia coli with the complementing plasmid also produced HA but at only 10% of the level made by the above cells. E. coli minicell analysis showed that two proteins, 42 and 45 kDa, are expressed by the functional DNA insert. Deletion analysis of the insert in the minicells revealed that the 42-kDa protein is essential for HA production. This is the first demonstration of reconstitution of HA capsule biosynthesis in vivo.