Determination of Glycosyltransferase Specificities for the Escherichia coli O111 O Antigen by a Generic Approach

We describe a bacterial strain developed to facilitate the determination of glycosyltransferase (GT) specificities for O antigens of known structure and gene cluster sequence. For proof of principle for the approach, the strain was used to determine the specificity of the Escherichia coli O111 O-antigen GT genes.     

Bacteriophage Types and O Antigen Groups of Escherichia coli from Urine

Urinary strains of Escherichia coli from seven geographical regions were typed serologically for O-specific antigens and with phages capable of lysing the majority of urinary isolated. The O antigen groups 4, 6, 75, 1, 50, 7, and 25 were the common ones found. Of the 454 cultures tested, 66.1% were phage typable and 65.2% were serotypable with the 48 antisera employed. Also, 71.6% of the cultures for which an O group could be determined were phage typable. Furthermore, of those seven O-antigen groups implicated in urinary tract infection, 80.2% exhibited a phage pattern. Various phage types were found within an O-antigen group, and, although one phage type associated a high percentage of the time with one O-antigen group, no correlation was observed between other O-antigen groups and phage types. Studies with bacteriuric patients by phage typing showed the presence of two strains of E. coli within an O-antigen group. Serogrouping and phage typing of fecal isolates of E. coli revealed the presence of some O-antigen groups and phage types also found as predominant types among urinary isolates. Phage typability correlated highly with hemolysis of human erythrocytes. Elevated temperatures of incubation and a chemical curing agent were used to enhance typability of cultures refractory to the typing phages. Phage typing, due to its rapidity, ease, and ability to distinguish strains of E. coli within an O-antigenic group, is suggested as a possible method by which a better insight into the epidemiology of urinary tract infections may be obtained.     

Escherichia coli O157:H7 Typing Phage V7 Is a T4-Like Virus

The complete genome sequence of the Escherichia coli O157:H7 typing phage V7 was determined. Its double-stranded DNA genome is 166,452 bp long, encoding 273 proteins and including 11 tRNAs. This virus belongs to the genus T4-like viruses within the subfamily Tevenvirinae, family Myoviridae.     

Bacteriophage Typing of Salmonella typhimurium by Use of a Mechanized Technique

A new mechanized technique for the application of drops of phages on agar plates is described. Drops of equal size are delivered by needles with the aid of filtered pressurized air. The part of the device which is in contact with phage is interchangeable as a whole.     

Virulence Genes and Molecular Typing of Different Groups of Escherichia coli O157 Strains in Cattle

Characterization of an Escherichia coli O157 strain collection (n = 42) derived from healthy Hungarian cattle revealed the existence of diverse pathotypes. Enteropathogenic E. coli (EPEC; eae positive) appeared to be the most frequent pathotype (n = 22 strains), 11 O157 strains were typical enterohemorrhagic E. coli (EHEC; stx and eae positive), and 9 O157 strains were atypical, with none of the key stx and eae virulence genes detected. EHEC and EPEC O157 strains all carried eae-gamma, tir-gamma, tccP, and paa. Other virulence genes located on the pO157 virulence plasmid and different O islands (O island 43 [OI-43] and OI-122), as well as espJ and espM, also characterized the EPEC and EHEC O157 strains with similar frequencies. However, none of these virulence genes were detected by PCR in atypical O157 strains. Interestingly, five of nine atypical O157 strains produced cytolethal distending toxin V (CDT-V) and carried genes encoding long polar fimbriae. Macro-restriction fragment enzyme analysis (pulsed-field gel electrophoresis) revealed that these E. coli O157 strains belong to four main clusters. Multilocus sequence typing analysis revealed that five housekeeping genes were identical in EHEC and EPEC O157 strains but were different in the atypical O157 strains. These results suggest that the Hungarian bovine E. coli O157 strains represent at least two main clones: EHEC/EPEC O157:H7/NM (nonmotile) and atypical CDT-V-producing O157 strains with H antigens different from H7. The CDT-V-producing O157 strains represent a novel genogroup. The pathogenic potential of these strains remains to be elucidated.Escherichia coli O157:H7 is a food- and waterborne zoonotic pathogen with serious effects on public health. E. coli O157:H7 causes diseases in humans ranging from uncomplicated diarrhea to hemorrhagic colitis and hemolytic-uremic syndrome (HUS) (30). Typically, enterohemorrhagic E. coli (EHEC) strains express two groups of important virulence factors: one or more Shiga toxins (Stx; also called verotoxins), encoded by lambda-like bacteriophages, and a pathogenicity island called the locus of enterocyte effacement (LEE) encoding all the proteins necessary for attaching and effacing lesions of epithelial cells (41). Comparative genomic studies of E. coli O157:H7 strains revealed extensive genomic diversity related to the structures, positions, and genetic contents of bacteriophages and the variability of putative virulence genes encoding non-LEE effector proteins (29, 43).Ruminants and, in particular, healthy cattle are the major reservoir of E. coli O157:H7, although the prevalence of O157:H7 strains in cattle may vary widely, as reviewed by Caprioli et al. (12). E. coli O157:H7 has been found to persist and remain infective in the environment for a long time, e.g., for at least 6 months in water trough sediments, which may be an important environmental niche.In Hungary, infections with E. coli O157 and other Shiga toxin-producing E. coli (STEC) strains in humans in cases of “enteritidis infectiosa” have been notifiable since 1998 on a case report basis. Up to now, the disease has been sporadic, and fewer than 100 (n = 83) cases of STEC infection among 2,700 suspect cases have been reported since 2001. However, until the present study, no systematic, representative survey of possible animal sources had been performed.In this study, our aim was to investigate healthy cattle in Hungary for the presence of strains of E. coli O157 and the genes encoding Shiga toxins (stx₁ and stx₂) and intimin (eae) and a wide range of putative virulence genes found in these strains. In addition, the phage type (PT) was determined, and pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to further compare the strains at the molecular level. Shiga toxin and cytolethal distending toxin (CDT) production was also examined, and phage induction experiments were conducted. The high incidence of enteropathogenic E. coli (EPEC; eae-positive) O157:H7 strains and atypical (eae- and stx-negative) O157 strains indicates that cattle are a major reservoir of not only EHEC O157 but also EPEC O157 and atypical E. coli O157 strains. These atypical, non-sorbitol-fermenting O157 strains frequently produced CDT-V and may represent a novel O157 clade as demonstrated by MLST and PFGE.     

Synthesis of Rat Casein Antigen in Escherichia coli

Fumes from phospholipids pyrolyzed at 500~700°C did not themselves show any mutagenicity on Salmonella strains, but when the pyrolyzates were treated with a sodium chloride precipitate, active carbon, or an anionic exchange resin, the filtrates were found to be mutagenic on Salmonella TA 100. Tests confirmed that the phospholipid pyrolyzates contained both mutagenic and inactivating substances of this mutagenicity. Low level mutagenicity was produced on Salmonella TA 98, but there was no such activity on the other strains. Preincubation of the pyrolyzates with S-9 mix had no activating effect on mutagenicity. The inactivating substances of the mutagenicity were isolated and identified as long chain fatty acids.     

Escherichia coli O157 Somatic Antigen Is Present in an Isolate of E. fergusonii

A bacterium that tested positive with antibodies specific for Escherichia coli O157 was isolated from beef during routine screening procedures. The bacterium was identified as E. fergusonii by biochemical testing and partial sequencing of 16S rRNA. The isolate was tested for the presence of genes encoding Shiga toxins, the E. coli attaching and effacing factor, enterohemolysin, and the O157 O antigen. The isolate tested negative for Shiga toxins and other E. coli O157 virulence markers but was found to harbor the genes encoding the O157 antigen. These results suggest genetic transfer of the O antigen gene cluster between E. coli O157:H7 and E. fergusonii.     

Domain Interactions Control Complex Formation and Polymerase Specificity in the Biosynthesis of the Escherichia coli O9a Antigen

The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for bacterial glycan synthesis and export by an ATP-binding cassette transporter-dependent pathway. The O9a O-PS possesses a tetrasaccharide repeat unit comprising two α-(1→2)- and two α-(1→3)-linked mannose residues and is extended on a polyisoprenoid lipid carrier by the action of a polymerase (WbdA) containing two glycosyltransferase active sites. The N-terminal domain of WbdA possesses α-(1→2)-mannosyltransferase activity, and we demonstrate in this study that the C-terminal domain is an α-(1→3)-mannosyltransferase. Previous studies established that the size of the O9a polysaccharide is determined by the chain-terminating dual kinase/methyltransferase (WbdD) that is tethered to the membrane and recruits WbdA into an active enzyme complex by protein-protein interactions. Here, we used bacterial two-hybrid analysis to identify a surface-exposed α-helix in the C-terminal mannosyltransferase domain of WbdA as the site of interaction with WbdD. However, the C-terminal domain was unable to interact with WbdD in the absence of its N-terminal partner. Through deletion analysis, we demonstrated that the α-(1→2)-mannosyltransferase activity of the N-terminal domain is regulated by the activity of the C-terminal α-(1→3)-mannosyltransferase. In mutants where the C-terminal catalytic site was deleted but the WbdD-interaction site remained, the N-terminal mannosyltransferase became an unrestricted polymerase, creating a novel polymer comprising only α-(1→2)-linked mannose residues. The WbdD protein therefore orchestrates critical localization and coordination of activities involved in chain extension and termination. Complex domain interactions are needed to position the polymerase components appropriately for assembly into a functional complex located at the cytoplasmic membrane.     

Bacteriophage Typing as an Epidemiological Tool for Urinary Escherichia coli

Phage typing was used to identify strains of Escherichia coli isolated from urinary and nonurinary sources. When eight phages isolated in Pennsylvania were used to type 717 cultures from Missouri, 50.3% of 624 urinary isolates and 34.4% of 93 nonurinary isolates were typable. Strains from nonurinary sources were not found commonly in urine. When five additional phages isolated in Missouri were added to the original set of eight phages, 80.4% of 331 urinary isolates were typable. When this set of phages was used to type 552 urinary cultures isolated in California, Minnesota, Ohio, Pennsylvania, Virginia, and West Virginia, 82.0% of the cultures were typable. Some common phage types were found in high incidence among cultures from the different regions. No correlation was found between phage type and the pattern of resistance to antibiotics. Phage typing data were presented also on the number of strains in individual urine specimens and the recurrences of strains in patients with chronic bacteriuria. Of 97 fecal isolates, 75.2% of the cultures were typable, and the most common phage type was observed in high incidence among the urinary isolates from this region. When 75 cultures from nine other genera of enteric bacteria were typed, only the shigellae were lysed. In view of the information obtained by phage typing and the ease and speed with which it can be done, it is suggested that phage typing be considered a new tool in epidemiological studies of urinary tract infections by E. coli.     

Waterborne Escherichia coli O157

The waterborne route of Vero cytotoxin-producing E. coli (VTEC) O157 infection was first suggested in two unconnected human cases in 1985. Since then, waterborne VTEC O157 has been identified in sporadic cases and in outbreaks of illness. Recreational waters, private and municipal supplies have been implicated from microbiological, environmental and epidemiological studies of cases. In addition, a research cohort study of farm workers identified exposure to private water supplies as a risk factor for having antibodies to E. coli O157. Sources of contamination are thought to be animal and human faeces or sewage. The presence of low numbers of target organisms in water makes microbiological confirmation difficult, therefore epidemiological evidence has been essential in outbreak investigations. Despite the potential for contamination of water with VTEC O157, waterborne infection is relatively rare largely due to the susceptibility of the organism to water treatment processes. This paper presents the evidence for waterborne VTEC O157 infection, considering current microbiological, environmental and particularly epidemiological information.     

rhs Genes Are Potential Markers for Multilocus Sequence Typing of Escherichia coli O157:H7 Strains

DNA sequence-based molecular subtyping methods such as multilocus sequence typing (MLST) are commonly used to generate phylogenetic inferences for monomorphic pathogens. The development of an effective MLST scheme for subtyping Escherichia coli O157:H7 has been hindered in the past due to the lack of sequence variation found within analyzed housekeeping and virulence genes. A recent study suggested that rhs genes are under strong positive selection pressure, and therefore in this study we analyzed these genes within a diverse collection of E. coli O157:H7 strains for sequence variability. Eighteen O157:H7 strains from lineages I and II and 15 O157:H7 strains from eight clades were included. Examination of these rhs genes revealed 44 polymorphic loci (PL) and 10 sequence types (STs) among the 18 lineage strains and 280 PL and 12 STs among the 15 clade strains. Phylogenetic analysis using rhs genes generally grouped strains according to their known lineage and clade classifications. These findings also suggested that O157:H7 strains from clades 6 and 8 fall into lineage I/II and that strains of clades 1, 2, 3, and 4 fall into lineage I. Additionally, unique markers were found in rhsA and rhsJ that might be used to define clade 8 and clade 6. Therefore, rhs genes may be useful markers for phylogenetic analysis of E. coli O157:H7.Escherichia coli O157:H7 was first described in 1983 as the causative agent of a food-borne outbreak attributed to contaminated ground beef patties (35), and it has subsequently emerged as a very important food-borne pathogen. Diseases caused by E. coli O157:H7, such as hemorrhagic colitis and hemolytic uremic syndrome, can be very severe or even life-threatening. Cattle are believed to be the main reservoir for E. coli O157:H7 (5, 15, 41), although other animals may also carry this organism (6, 21). Outbreaks are commonly associated with the consumption of beef and fresh produce that come into contact with bovine feces or feces-contaminated environments, such as food contact surfaces, animal hides, or irrigation water (12, 21, 30, 38).It is well-established that strains of E. coli O157:H7 vary in terms of virulence and transmissibility to humans and that strains differing in these characteristics can be distinguished using DNA-based methods (22, 29, 42). For example, octamer-based genome scanning, which is a PCR approach using 8-bp primers, provided the first evidence that there are at least two lineages of O157:H7, termed lineage I and lineage II (22). Strains classified as lineage I are more frequently isolated from humans than are lineage II strains (42). A later refinement of this classification system was coined the lineage-specific polymorphism assay (LSPA), which classified strains based upon the amplicon size obtained using PCRs targeting six chromosomal regions of E. coli O157:H7 and assigned a six-digit code based upon the pattern obtained (42). Most strains of lineage I grouped into LSPA type 111111, while the majority of lineage II strains fell into LSPA types 211111, 212111, and 222222. More recently, it was suggested that LSPA type 211111 strains comprise a separate group called lineage I/II (45).To gain greater insight into the recent evolution of E. coli O157:H7, a method that is more discriminatory than the LSPA method is desirable. Multilocus sequence typing (MLST) is a method that discriminates between strains of a bacterial species by identifying DNA sequence differences in six to eight targeted genes. Satisfactory MLST schemes exist for other bacterial pathogens (28, 43); however, due to the lack of sequence variations in previously targeted gene markers in E. coli O157:H7 (13, 33), MLST approaches for subtyping this pathogen have been more difficult to develop. More recently, high-throughput microarray and sequencing platforms have been used to identify hundreds of single nucleotide polymorphisms (SNPs) that are useful for discriminating between strains of E. coli O157:H7 during epidemiologic investigations and for drawing phylogenetic inferences (11, 20, 29, 44). Particularly noteworthy, Manning et al. (29) developed a subtyping scheme based upon the interrogation of 32 putative SNP loci. This method separated 528 strains into 39 distinct SNP genotypes, which were grouped into nine statistically supported phylogenetic groups called clade 1 through clade 9. By analyzing the rates of hemolytic uremic syndrome observed in patients infected with strains of clades 2, 7, and 8, it was also concluded that clade 8 strains are more virulent to humans than other strains (29).One drawback of current DNA sequence-based subtyping schemes for E. coli O157:H7 is that they require screening of at least 32 SNP loci. We were interested in asking whether a simpler approach that targets a few informative gene markers could be developed for rapid strain discrimination and phylogenetic determination. A recent analysis of E. coli genomes predicted that rearrangement hot spot (rhs) genes are under the strongest positive selection of all coding sequences analyzed (34). Therefore, we hypothesized that these genes would display significant sequence variations for subtyping O157:H7 strains. The rhs genes were first discovered as elements mediating tandem duplication of the glyS locus in E. coli K-12 (26); however, their function remains unknown. There are nine rhs genes within the genome of the prototypical E. coli O157:H7 strain Sakai, and these genes are designated rhsA, -C, -D, -E, -F, -G, -I, -J, and -K (see Table S1 in the supplemental material) (16). Three of these nine rhs genes, rhsF, -J, and -K, were previously studied by Zhang et al. (44), and a number of SNPs were identified among these genes. However, no studies have been conducted to comprehensively investigate rhs genes as markers in an MLST scheme for subtyping E. coli O157:H7.The primary purpose of the present study was to investigate whether there are sufficient DNA sequence variations among rhs genes to develop an MLST approach for subtyping E. coli O157:H7. In this study, a greater level of DNA sequence variation was observed among rhs genes than in gene markers targeted in previous studies (13, 33). Furthermore, phylogenetic analysis using these rhs genes generally agreed with the established lineage and clade classifications of O157:H7 strains defined previously. We also wanted to determine whether there is a correlation between the lineage classification of O157:H7 strains (42) and the recently proposed clade classification (29). The present study reports evidence that O157:H7 strains from clade 8 are classified as lineage I/II, which is a different lineage from well-studied E. coli O157:H7 outbreak strains, such as EDL933 and Sakai. Therefore, we suggest that outbreaks of O157:H7 are caused by two lineages of this pathogen, lineage I and lineage I/II.     

Plasmidome-Analysis of ESBL-Producing Escherichia coli Using Conventional Typing and High-Throughput Sequencing

Infections caused by Extended spectrum β-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used β-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements. We have characterized the plasmid content of ESBL-producing E. coli from human urinary tract infections. Ten diverse isolates were selected; they had unrelated pulsed-field gel electrophoresis (PFGE) types (<90% similarity), were from geographically dispersed locations and had diverging antibiotic resistance profiles. Three isolates belonged to the globally disseminated sequence type ST131. ESBL-genes of the CTX-M-1 and CTX-M-9 phylogroups were identified in all ten isolates. The plasmid content (plasmidome) of each strain was analyzed using a combination of molecular methods and high-throughput sequencing. Hidden Markov Model-based analysis of unassembled sequencing reads was used to analyze the genetic diversity of the plasmid samples and to detect resistance genes. Each isolate contained between two and eight distinct plasmids, and at least 22 large plasmids were identified overall. The plasmids were variants of pUTI89, pKF3-70, pEK499, pKF3-140, pKF3-70, p1ESCUM, pEK204, pHK17a, p083CORR, R64, pLF82, pSFO157, and R721. In addition, small cryptic high copy-number plasmids were frequent, containing one to seven open reading frames per plasmid. Three clustered groups of such small cryptic plasmids could be distinguished based on sequence similarity. Extrachromosomal prophages were found in three isolates. Two of them resembled the E. coli P1 phage and one was previously unknown. The present study confirms plasmid multiplicity in multi-resistant E. coli. We conclude that high-throughput sequencing successfully provides information on the extrachromosomal gene content and can be used to generate a genetic fingerprint of possible use in epidemiology. This could be a valuable tool for tracing plasmids in outbreaks.     

Genetic Typing of Shiga Toxin 2 Variants of Escherichia coli by PCR-Restriction Fragment Length Polymorphism Analysis

Shiga toxins Stx1 and Stx2 play a prominent role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) infections. Several variants of the stx₂ gene, encoding Stx2, have been described. In this study, we developed a PCR-restriction fragment length polymorphism system for typing stx₂ genes of STEC strains. The typing system discriminates eight described variants and allows the identification of new stx₂ variants and STEC isolates carrying multiple stx₂ genes. A phylogenetic tree, based on the nucleotide sequences of the toxin-encoding genes, demonstrates that stx₂ sequences with the same PvuII HaeIII HincII AccI type generally cluster together.     

Genetic Analysis for the Lack of Expression of the O157 Antigen in an O Rough:H7 Escherichia coli Strain

The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6.Shiga-toxigenic Escherichia coli (STEC) serotype O157:H7 carries O157 and H7 antigens, so these traits are extensively used in identification (1). Strain MA6, isolated from beef in Malaysia (8), carries the O157:H7 virulence factor genes, including the Shiga toxin 2 gene (stx₂), the γ intimin allele (γ-eae), the enterohemolysin gene (ehxA), and the +93 uidA single nucleotide polymorphism (SNP) found only in O157:H7 strains (1). Multilocus sequence typing also showed MA6 to have the most common sequence type (ST-66) for O157:H7 strains. However, and in spite the fact that MA6 had per gene sequences essential for O157 antigen synthesis (2), no O157 antigen is expressed (O rough), and therefore, it is undetectable with serological assays used in O157:H7 analysis.The biosynthesis and assembly of E. coli O antigen are highly complex (9). The rfb operon (12 genes) (16), along with 3 ancillary genes outside of the rfb, is required for the biosynthesis of the 4 sugar nucleotide precursors and the assembly of the O unit (11). This is then linked to the core antigen, comprising an inner and an outer component, which require 3 other operons for biosynthesis and assembly (9). As defects in any of these genes could produce the O-null phenotype (13), we systematically examined these genes (Table ​(Table1)1) to elucidate the cause of the absence of O157 expression in MA6.

TABLE 1.

rfb operon genes, ancillary genes, and waa cluster genes examined in this study

Typing of O26 enterohaemorrhagic and enteropathogenic Escherichia coli isolated from humans and cattle with IS621 multiplex PCR-based fingerprinting

Aims: This study evaluated a typing method of O26:H11 enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) based on the variation in genomic location and copy numbers of IS621. Methods and Results: Two multiplex PCRs, targeting either the left (5′) or right (3′) IS/chromosome junction of 12 IS621 insertion sites and one PCR specific of another truncated copy, were developed. Thirty‐eight amplification profiles were observed amongst a collection of 69 human and bovine O26:H11 EHEC and EPEC. Seventy‐one per cent of the 45 EHEC and EPEC with identical IS621 fingerprints within groups of two, three or four isolates had >85% pulsed field gel electrophoresis (PFGE) profile similarity, including four groups of epidemiologically related EHEC or EPEC, while most of the groups had <85% similarity between each others. Epidemiologically related EHEC from each of three independent outbreaks in Japan and Belgium also exhibited identical IS621 fingerprints and PFGE profiles. Conclusions: The IS621 fingerprinting and the PFGE are complementary typing assays of EHEC and EPEC; though, the former is less discriminatory. Significance and Impact of the Study: The IS621 printing method represents a rapid (24 h) first‐line surveillance and typing assay, to compare and trace back O26:H11 EHEC and EPEC during surveys in farms, multiple human cases and outbreaks.     

Genetic relationship of diarrheagenic Escherichia coli pathotypes among the enteropathogenic Escherichia coli O serogroup

The genetic relationship among the Escherichia coli pathotypes was investigated. We used random amplified polymorphic DNA (RAPD) data for constructing a dendrogram of 73 strains of diarrheagenic E. coli. A phylogenetic tree encompassing 15 serotypes from different pathotypes was constructed using multilocus sequence typing data. Phylogram clusters were used for validating RAPD data on the clonality of enteropathogenic E. coli (EPEC) O serogroup strains. Both analyses showed very similar topologies, characterized by the presence of two major groups: group A includes EPEC H6 and H34 strains and group B contains the other EPEC strains plus all serotypes belonging to atypical EPEC, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). These results confirm the existence of two evolutionary divergent groups in EPEC: one is genetically and serologically very homogeneous whereas the other harbors EPEC and non-EPEC serotypes. The same situation was found for EAEC and EHEC.     

Structure of a teichoic acid-like O-polysaccharide of Escherichia coli O29

A teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O29. The O-polysaccharide and an oligosaccharide obtained by dephosphorylation of the O-polysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy. The following structure of the branched oligosaccharide repeating unit, containing five monosaccharide residues and glycerol 1-phosphate (D-Gro-1-P), was established: [carbohydrate structure: see text].