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PubMed1998

Inhibition effect of transgenic tobacco plants expressing snowdrop lectin on the population development of Myzus persicae.

The cDNAs of snowdrop lectin mature protein and its precursor protein, GNA12 and GNA34, were inserted downstream of a 35S promoter with a double enhancer and a "omega" fragment of TMV-RNA cDNA in the binary vector pBin438, or the phloem-specific CoYMV promoter in the vector pBcop1, respectively, resulting in the construction of four plant expression vectors pBGna12, pBGna34, pBCGna12, and pBCGna34. Leaf stripes of Nicotiana tabacum var. K326 were transformed with A. tumefaciens LBA4404 harboring the above expression vectors, respectively. PCR and Southern blot analysis showed that foreign GNA genes were inserted into the genome of transformed tobacco plants. Western blot analysis indicated that GNA could be expressed efficiently up to 0.08-0.15% of total soluble proteins in transgenic tobacco plants with pBGna34 or pBCGna34, while in those with pBGna12 and pBCGna12 GNA were hardly detected by immunoassay. The results from insect bioassay with a peach aphid (Myzus persicae) demonstrated that the transgenic plants of pBGna34 and pBCGna34 were aphid-resistant as shown by a 45-60% reduction in insect population density, with some individual transgenic lines being reduced by over 90%. In addition, it was evident that the 35S promoter with a double enhancer and CoYMV promoter had similar abilities to direct the GNA gene to express in transgenic tobacco plants, but because the CoYMV promoter drives the foreign gene in a phloem-specific expression manner, the transgenic plants of pBCGna34 showed higher aphid resistance.

Animals↗

PubMed2006

Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase.

An important industrial enzyme, Candida rugosa lipase (CRL) possesses several different isoforms encoded by the lip gene family (lip1-lip7), in which the recombinant LIP1 is the major form of the CRL multigene family. Previously, 19 of the nonuniversal serine codons (CTG) of the lip1 gene hav been successfully converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP1 in the yeast Pichia pastoris. To improve the expression efficiency of recombinant LIP1 in P. pastoris, a regional synthetic gene fragment of lip1 near the 5' end of a transcript has been constructed to match P. pastoris-preferred codon usage for simple scale-up fermentation. The present results show that the production level (152 mg/L) of coLIP1 (codon-optimized LIP1) has an overall improvement of 4.6-fold relative to that (33 mg/L) of non-codon-optimized LIP1 with only half the cultivation time of P. pastoris. This finding demonstrates that the regional codon optimization the lip1 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP1 in the P. pastoris system. More distinct biochemical properties of the purified recombinant LIP1 for further industrial applications are also determined and discussed in detail.

Base Sequence↗

PubMed1999

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae.

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end-products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast-based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1.0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA-1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA-1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA-1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1P-MFa1S-pedA-ADH1T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1P) and terminator (ADH1T). Secretion of the PED1-encoded pediocin PA-1 was directed by the yeast mating pheromone alpha-factor's secretion signal (MFa1S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4.6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria.

Bacteriocins↗

PubMed2001

Overexpression, rapid isolation, and biochemical characterization of Escherichia coli single-stranded DNA-binding protein.

Escherichia coli (E. coli) single-stranded binding protein (SSB) is a valuable protein for various biotechnical applications, such as PCR and DNA sequencing. Here we describe an efficient expression and purification scheme where the tendency of SSB to aggregate at low salt concentration and high protein concentration is avoided. The method contains fewer steps of purification and results in high protein yield, compared to previous published protocols. In our protocol, cells are harvested after cultivation overnight and SSB is isolated by ammonium sulfate precipitation followed by anion-exchange chromatography. The yield from a 2-liter fed-batch fermentor is 2 g protein, which is higher than all production methods for SSB earlier reported. Moreover, the two classical isolation steps combined in the purification scheme are robust, cost-efficient, and suitable for scaling up. The resulting SSB is pure and a correctly folded tetramer with an apparent binding to single-stranded DNA with a K(D) of 10(-8) M, as determined by surface plasmon resonance.

Amino Acid Sequence↗

PubMed2000

Dox-dependent SIVmac with tetracycline-inducible promoter in the U3 promoter region.

An attenuated live vaccine is a candidate in developing vaccines against human immunodeficiency virus type 1 (HIV-1). The study using macaques and simian immunodeficiency virus (SIVmac) showed an attenuated virus to be more effective than any other vaccine candidate. However, development of a safer vaccine is required for clinical application. In this study, we constructed pSIVmac Delta nef with tetracycline inducible promoter (pTet) and tried to control viral expression in a drug-dependent manner. Promoter/enhancer motifs in the U3 region of the long terminal repeats (LTRs) were serially deleted and replaced with pTet. In mutant LTRs, which lack NF-kappaB and Sp1 binding sites, TATA box motifs, and the 5' half of the U3 region, promoter activity was stringently controlled by doxycycline (Dox). Their activities were similar to or higher than that of wild-type LTR in the presence of Dox, based on the transient chloramphenicol acetyltransferase reporter assay. Three of these mutant LTRs were introduced into the pSIVmac239 Delta nef genome. Viral protein from these viruses was efficiently expressed in a Dox-dependent manner after transfection to a HeLa cell, which expresses reverse tetracycline transactivator (rtTA). The 2-LTR-form viral DNA of these viruses could be detected in M8166 cells that had been infected with supernatants from the transfected rtTA HeLa cell. These results suggest that pSIVmac Delta nef containing mutant LTRs can proceed through one viral replication cycle consisting of transcription, formation of viral particles, infection to cells, and reverse transcription. Although continuous replication of these Dox-dependent viruses requires a supply of rtTA as a constituent for the pTet-On viral genome, the successful replacement of the original promoter with a drug-dependent promoter suggests a new possibility for developing a safer attenuated live virus.

Doxycycline↗

PubMed1986

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

Base Sequence↗

PubMed1989

Tilapia prolactin: molecular cloning of two cDNAs and expression in Escherichia coli.

We have isolated cDNA clones encoding tilapia prolactin (tiPRL) from a cDNA library prepared from tilapia (Oreochromis niloticus) anterior pituitary glands. A trout PRL cDNA fragment was used as hybridization probe to select the recombinant plasmids carrying the tiPRL coding sequence. Two types of PRL cDNA were isolated and their complete nucleotide sequence determined. The larger cDNA (tiPRL-I) codes for a polypeptide of 212 amino acids, including a putative signal sequence of 24 amino acids, and contains a 3' untranslated region of 787 bp. The second prolactin cDNA (tiPRL-II) encodes a polypeptide of 200 amino acids, including a presumptive signal peptide of 23 amino acids, and contains a noncoding region of 512 bp. tiPRL-I and tiPRL-II cDNA sequences are 81% similar, whereas the encoded proteins share 69% amino acid identity at optimal alignment. Mature tiPRL-I was efficiently expressed in Escherichia coli carrying a plasmid in which the tiPRL-I cDNA was under the control of the phi 10 promoter of T7 bacteriophage. The new recombinant protein representing about 45% of the total cellular proteins was found in inclusion bodies and cross-reacted with salmon PRL antiserum.

Amino Acid Sequence↗

PubMed1992

Altered-function mutations of the transcriptional regulatory gene virG of Agrobacterium tumefaciens.

Three point mutations were isolated in the Agrobacterium tumefaciens virG gene by screening for vir gene expression in the absence of added phenolic inducing compounds. All three mutations were localized in the predicted amino-terminal phosphoryl receiver domain of the protein. One mutant (N54D) bypasses the requirement for VirA and phenolic inducers both for transcriptional activation of all tested vir promoters and for plant tumorigenesis. This mutant also activates vir gene expression efficiently at neutral pH, indicating that the step in induction that is normally stimulated by acid pH occurs before or during VirG phosphorylation. The other two mutants (M13T and H15R) require VirA for activity but are sensitized to low levels of inducing stimuli.

Acetophenones↗

PubMed1980

The ral gene of phage lambda. I. Identification of a non-essential gene that modulates restriction and modification in E. coli.

Host controlled restriction in Escherichia coli can be relieved by pre-infecting restricting cells with modified lambda helper phages. This process, in which intact unmodified phage genomes are allowed to escape restriction attack, is mediated by a newly identified lambda function called ral. The ral gene has been located by deletion mapping between cIII and N. Efficient expression of the ral gene requires the product of the regulator gene N. Polyacrylamide gel analysis of the lambda proteins specified by the cIII-N region failed to reveal the product of the ral gene, but demonstrated that protein Ea10 is encoded by a gene located immediately to the left of ral. From these results the map order cIII-Ea10-ral-TL1-N was deduced. Ral specifically alleviates restriction in E. coli K and E. coli B, but does not affect restriction systems EcoRI, EcoRII and EcoP1. In addition, ral enhances the modification activity of the EcoK and EcoB restriction enzymes: we observed that efficient modification of progeny phages obtained by propagating unmodified lambda phages in r-m+ hosts, is dependent upon the presence of ral. We thus conclude that the ral gene product acts by modulating the restriction and modification activities of the type I restriction systems in E. coli, and the possible mechanisms will be discussed.

Bacteriophage lambda↗

PubMed2000

[Expression, purification and identification of human IFN-alpha 2 b/HBV Pre S2 fusion protein].

OBJECTIVE: To express a fusion protein of human interferon-a2b and HBV Pre S2 in E.coli for the purpose of investigating anti-HBV immunomodulatory protein. METHODS: Human interferon-a2b and HBV Pre S2 encoding genes were amplified from plasmid templates through PCR, then fused and cloned into plasmid pBV220 through engineering technique to generate expression plasmid pBV-IFN-Pre S2. The plasmid was transfected into E. coli to produce fusion protein. RP-HPLC and ion-exchange chromatography were employed to purify fusion protein. RESULTS: 27 kDa fusion protein was expressed up to 15% of total bacterial protein in E. coli. After purification, the purity of fusion protein reached 95% of total protein. Anti-viral assay showed that IFN-Pre S2 protein induced a VSV-resistant activity of 1.25 x 10(8) lU/mg protein in Wish cell line, similar to the bioactivity of original recombinant human IFN-alpha 2b. ELISA data showed that IFN-Pre S2 protein had antigenicities of both human IFN-alpha 2b and HBV Pre S2. In addition, fusion protein showed the feature of binding to polymeric human serum albumin (PHSA). CONCLUSIONS: The bifunctional fusion protein was efficiently expressed in E. coli. The work provided initial evidence for studying PHSA-receptor-targeting IFN-alpha 2b,which might have potential application for the treatment of HBV infection.

Amino Acid Sequence↗

PubMed2006

Characterization of herpes simplex virus type 1 recombinants that express and incorporate high levels of HCV E2-gC chimeric proteins.

We report the construction of two HSV-1 recombinants encoding chimeric forms of the E2 glycoprotein of HCV-1a composed of the ectodomain of E2 (aa384-611 or 384-711) fused to different parts of the transmembrane and cytoplasmic domain of the HSV-1 gC glycoprotein (gC). The parental HSV-1, known as KgBpK(-)gC(-), is deleted for gC and the main heparan sulphate (HS) binding domain of gB, and it exhibits impaired binding (ca. 80%) to HS compared to the wild type virus KOS [Laquerre, S., Argnani, R., Anderson, D.B., Zucchini, S., Manservigi, R., Glorioso, J.C., 1998. Heparan sulphate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J. Virol. 72, 6119-6130]. We show that gC:E2 proteins are efficiently expressed and transported to the cell surface. We also demonstrate that HSV-1 can incorporate both gC:E2 chimeric proteins into particles and show that incorporation of both chimeric molecules in the viral envelope partially restored binding (ca. 20%) of the HSV-1 recombinants to heparan sulphate. Finally, we showed that the gC:E2ScaI chimeric glycoprotein was able to bind a recombinant form of hCD81 and virion-expressed gC:E2ScaI permitted the binding of the HSV-1 recombinant virus to the hCD81 molecule.

Animals↗

PubMedDate not supplied

Application of inducible and targeted gene strategies to produce transgenic fish: a review.

Compared to mammals, fishes offer easier transgenic technology because each female produces hundreds of eggs, the manipulated embryos do not need to be incubated inside the mother, and the probability of their harboring human-related pathogens is lower. In the last 15 years, traditional methods using injections of fertilized fish eggs and strong viral promoters have resulted in the generation of many transgenic fish species; however, they showed random genome integration with some mosaicism and episomic expression. The use of inducible gene systems that control temporal and tissue expression and of gene-targeting methodologies based on homologous recombination is desirable to control the expression, efficiency of insertion, and locus of incorporation of transgenes into fish genomes. A variety of systems developed for mammals are now available to be tested in fishes. The use of such systems would require further development of stem cell or nuclear transplant technologies in fish. Most of that work remains to be explored.

Animals↗

PubMed1997

Enhancement of extracellular production of a Cellulomonas fimi exoglucanase in Escherichia coli by the reduction of promoter strength.

The enzymatic approach to the treatment of cellulosic wastes depends on the availability of cost-effective means for the production of cellulases. We have engineered an excretion construct, tacIQpar8cex, to investigate the extracellular production of a Cellulomonas fimi exoglucanase (Exg) in Escherichia coli. The overall yield of Exg expressed by the culture of JM101 (tacIQpar8cex) was 2-11 times higher than that obtained using other systems. Over 20% of the activity was detected in the medium. When the culture was induced with IPTG, the overall production of Exg dropped dramatically. The lower yield was found to be caused by both rapid cell death and plasmid curing. A derivative of tacIQpar8cex containing the weaker lacUV5 promoter, designated lacUV5par8cex, was constructed to enhance excretion of Exg from strain JM101. Even with IPTG induction, the JM101 (lacUV5par8cex) culture was found to show a high level of cell viability and plasmid stability as well as the ability to provide efficient expression and excretion of Exg. Upon IPTG induction for 12 h, the activity and specific activity of the excreted Exg obtained from the lacUV5par8cex construct were 143 U ml-1 and 793 U mg-1 protein, respectively, which are 2-5 times higher than that detected from the tacIQpar8cex construct and from the best construct expressing the same gene reported previously.

DNA Primers↗

PubMed1982

The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells.

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.

Amino Acid Sequence↗

PubMed2005

The contribution of RING and B-box 2 domains to retroviral restriction mediated by monkey TRIM5alpha.

TRIM5alpha is a cytoplasmic protein that mediates a post-entry block to infection by some retroviruses. TRIM5alpha contains a tripartite motif (TRIM), which includes RING, B-box 2, and coiled-coil domains, and a C-terminal B30.2 (SPRY) domain. We investigated the contribution of the RING and B-box 2 domains to the antiretroviral activity of rhesus monkey TRIM5alpha (TRIM5alpharh), which potently restricts infection by human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus of African green monkeys (SIVagm). Disruption of the RING domain caused mislocalization of TRIM5alpharh so that the cytoplasmic level of the protein was decreased compared with that of the wild-type protein. Nonetheless, partial ability to restrict HIV-1 and SIVagm was retained by the RING domain mutants. By contrast, although TRIM5alpharh mutants with disrupted B-box 2 domains were efficiently expressed and correctly localized to the cytoplasm, antiretroviral activity was absent. The B-box 2 mutants colocalized and associated with wild-type TRIM5alpharh and exerted dominant-negative effects on the antiretroviral activity of the wild-type protein. Taken together with other data, these results indicate that functionally defective TRIM5alpharh molecules that retain a coiled coil can act as dominant-negative inhibitors of wild-type TRIM5alpharh function. The RING domain of TRIM5alpharh is not absolutely required for retrovirus restriction but can influence cytoplasmic levels of the protein and thus indirectly alter function. The B-box 2 domain, by contrast, appears to be essential for efficient retrovirus restriction.

Animals↗

PubMed1993

A new Salmonella tester strain expressing a hamster acetyltransferase shows high sensitivity for arylamines.

A hamster acetyltransferase, AT-I, has high activities for N-acetylation of arylamines, O-acetylation of N-hydroxyarylamines and N,O-acetyltransfer of N-hydroxyarylacetamides. In the present study, the cDNA was expressed in Salmonella typhimurium TA1538. The new SAT138 strain expressing high levels of AT-I showed remarkably high sensitivity (> 10,000 fold) for a carcinogenic intermediate, N-hydroxy-2-acetylaminofluorene, in an Ames mutagenesis test as compared to the parental TA1538 strain. SAT138 had 650-1,600-fold higher sensitivities for mutagenesis induced by 2-acetylaminofluorene and benzidine in the presence of S9 mix. Higher sensitivities (32-560-fold) were also observed with N-hydroxy-2-aminofluorene, N-hydroxy-4-aminobiphenyl, N-hydroxy-4-acetylaminobiphenyl, N-hydroxy-4-propionylaminobiphenyl and N-hydroxy-phenacetin in the absence of S9 mix. These high sensitivities to arylamines and the related chemicals are accounted for by the efficient expression of AT-I in the cytosol of this bacterium. The unique characteristics of SAT138 having high N-hydroxyarylacetamide N,O-transacetylating activity, which is defective in Salmonella acetyltransferase, provide broadened and high sensitivities for the detection of mutagenic N-substituted chemicals in the Salmonella mutagenesis test.

Acetyltransferases↗

PubMed1990

Clustered point mutation analysis of the rat prolactin promoter.

To identify DNA regions important for basal and hormone-stimulated transcription of the rat PRL gene, a series of clustered point mutations were prepared within the immediate 5' flanking region. DNA fragments representing the wild-type and 19 different linker-scanner mutations of the PRL gene were each linked to a luciferase marker gene, and the DNA constructs were transferred into GH3 pituitary tumor cells by electroporation. Luciferase activity was determined 24 h after transfection in extracts from control cells or cells treated with 0.5 mM chlorophenylthio-cAMP, 100 nM TRH, or 100 nM phorbol myristate acetate. The individual clustered point mutations covered a region from just up-stream of the TATA box (position -30) to a position 193 basepairs up-stream from the start of transcription. Five regions in which mutations produced substantial decreases in both basal and cAMP-, TRH-, or phorbol ester-stimulated expression of the marker gene were detected. Three of these regions (positions -41 to -58, -113 to -124, and -149 to -156) correspond to previously identified binding sites for the pituitary-specific, homeobox protein, Pit-1/GHF-1. The fourth and fifth regions do not correspond to Pit-1/GHF-1-binding sites and presumably represent sites for an unidentified factor. Within these regions, sequences with some similarity to a consensus cAMP response element and an AP-2-binding site have been detected. These data confirm the importance of Pit-1/GHF-1 as a key factor in PRL gene transcription. In addition, the results suggest that additional transcription factors are probably required for efficient expression of the PRL gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1.

A plasmid that included both an 8.9 kb chromosomal DNA insert containing genes from the rfb cluster of Shigella dysenteriae 1 and a smaller insert containing the rfp gene from a S. dysenteriae 1 multicopy plasmid resulted in efficient expression of O antigen in an rfb-deleted strain of Escherichia coli K-12. Eight genes were identified in the rfb fragment: the rfbB-CAD cluster which encodes dTDP-rhamnose synthesis, rfbX which encodes a hydrophobic protein involved in assembly of the O antigen, rfc which encodes the O antigen polymerase, and two sugar transferase genes. The production of an O antigen also required the E. coli K-12 rfe gene, which is known to encode a transferase which adds N-acetylglucosamine phosphate to the carrier lipid undecaprenol phosphate. Thus Rfe protein appears to function as an analogue of the Salmonella RfbP protein to provide the first sugar of the O unit. Functional analysis of the other genes was facilitated by the fact that partial O units of one, two or three sugars were efficiently transferred to the lipopolysaccharide core. This analysis indicated that the plasmid-encoded Rfp protein is the transferase that adds the second sugar of the O unit while the two rfb transferases add the distal sugars to make an O antigen whose structure is (Rha-Rha-Gal-GlcNAc)n. The use of the rfe gene product as the transferase that adds the first sugar of an O unit is a novel mechanism which may be used for the synthesis of other enteric O antigens.

Carbohydrate Metabolism↗