Structure and gene cluster of the O-antigen of Enterobacter cloacae G2559.

The O-polysaccharide (OPS) was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G2559 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the branched pentasaccharide repeating unit was established. The O-antigen gene cluster of Enterobacter cloacae G2559 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the O-antigen structure.

Structure and gene cluster of the O-antigen of Enterobacter cloacae G3422.

The O-polysaccharide (OPS) was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G3422 and studied by chemical methods, including sugar analyses, Smith degradation, and solvolysis with anhydrous trifluoroacetic acid, along with 1H and 13C NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit was established: The O-antigen gene cluster of Enterobacter cloacae G3422 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in a good agreement with the O-antigen structure.

Revisiting Species Identification within the Enterobacter cloacae Complex by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is commonly used by clinical microbiology laboratories to identify pathogens, despite some limitations of the technique. The Enterobacter cloacae complex (ECC) taxonomy has recently been expanded, leading to uncertain identification of some species within the ECC when commercial MALDI-TOF MS is used. This technique is especially unsuited in the case of E. hormaechei, the main species responsible for infections and one of the most prone, within the ECC, to acquire antibiotic resistance. Hence, rapid and reliable identification at the species level could improve patient management. Here, we evaluated the performance of the Bruker Microflex MALDI-TOF MS instrument to identify ECC isolates using two databases and algorithms in comparison to the hsp60 gene sequencing reference method: the Bruker database included in the MALDI Biotyper software and an extensive online database coupled to an original Mass Spectrometric Identification (MSI) algorithm. Among a panel of 94 ECC isolates tested in triplicate, the online database coupled to MSI software allowed the highest rate of identification at the species level (92%) compared to the MALDI Biotyper database (25%), especially for the species E. hormaechei (97% versus 20%). We show that by creating a database of MALDI-TOF reference spectral profiles with a high number of representatives associated with the performant MSI software, we were able to substantially improve the identification of the E. cloacae complex members, with only 8% of isolates misidentified at the species level. This online database is available through a free online MSI application (https://msi.happy-dev.fr/). IMPORTANCE Creation of a database of MALDI-TOF reference spectral profiles with a high number of representatives associated with the performant MSI software enables substantial improvement in identification of E. cloacae complex members. Moreover, this online database is available through a free online MSI application (https://msi.happy-dev.fr/).

Protective effects of sulfated polysaccharide from Enterobacter cloacae Z0206 against DSS-induced intestinal injury via DNA methylation.

We previously obtained and characterized a novel sulfated derivative of the exopolysaccharides from Enterobacter cloacae Z0206 (SEPS). This study aimed at investigating the effects and mechanism of SEPS against dextran sulfate sodium (DSS) induced intestinal injury. The results showed that SEPS increased the proliferation and survival of intestinal epithelial cells during DSS stimulation. Furthermore, SEPS maintained the barrier function and inflammatory response via JAK2 and MAPK signaling to protect against DSS-induced intestinal injury. Mechanistically, SEPS elevated the DNA methylation in the promoter region to negatively regulate the JAK2 and MAPKs expression. Thus, the current study shows the potential effects and mechanism of SEPS on DSS-induced intestinal epithelial cell injury.

Crystal structure and functional implication of a bacterial cyclic AMP-AMP-GMP synthetase.

Mammalian cyclic GMP-AMP synthase (cGAS) and its homologue dinucleotide cyclase in Vibrio cholerae (VcDncV) produce cyclic dinucleotides (CDNs) that participate in the defense against viral infection. Recently, scores of new cGAS/DncV-like nucleotidyltransferases (CD-NTases) were discovered, which produce various CDNs and cyclic trinucleotides (CTNs) as second messengers. Here, we present the crystal structures of EcCdnD, a CD-NTase from Enterobacter cloacae that produces cyclic AMP-AMP-GMP, in its apo-form and in complex with ATP, ADP and AMPcPP, an ATP analogue. Despite the similar overall architecture, the protein shows significant structural variations from other CD-NTases. Adjacent to the donor substrate, another nucleotide is bound to the acceptor binding site by a non-productive mode. Isothermal titration calorimetry results also suggest the presence of two ATP binding sites. GTP alone does not bind to EcCdnD, which however binds to pppApG, a possible intermediate. The enzyme is active on ATP or a mixture of ATP and GTP, and the best metal cofactor is Mg2+. The conserved residues Asp69 and Asp71 are essential for catalysis, as indicated by the loss of activity in the mutants. Based on structural analysis and comparison with VcDncV and RNA polymerase, a tentative catalytic pathway for the CTN-producing EcCdnD is proposed.

An Unusually Rapid Protein Backbone Modification Stabilizes the Essential Bacterial Enzyme MurA.

Proteins are subject to spontaneous rearrangements of their backbones. Most prominently, asparagine and aspartate residues isomerize to their ß-linked isomer, isoaspartate (isoAsp), on time scales ranging from days to centuries. Such modifications are typically considered "molecular wear-and-tear", destroying protein function. However, the observation that some proteins, including the essential bacterial enzyme MurA, harbor stoichiometric amounts of isoAsp suggests that this modification can confer advantageous properties. Here, we demonstrate that nature exploits an isoAsp residue within a hairpin to stabilize MurA. We found that isoAsp formation in MurA is unusually rapid and critically dependent on folding status. Moreover, perturbation of the isoAsp-containing hairpin via site-directed mutagenesis causes aggregation of MurA variants. Structural mass spectrometry revealed that this effect is caused by local protein unfolding in MurA mutants. Our findings demonstrate that MurA evolved to "mature" via a spontaneous post-translational incorporation of a ß-amino acid, which raises the possibility that isoAsp-containing hairpins may serve as a structural motif of biological importance.

Ethylene glycol and glycolic acid production from xylonic acid by Enterobacter cloacae.

BACKGROUND: Biological routes for ethylene glycol production have been developed in recent years by constructing the synthesis pathways in different microorganisms. However, no microorganisms have been reported yet to produce ethylene glycol naturally. RESULTS: Xylonic acid utilizing microorganisms were screened from natural environments, and an Enterobacter cloacae strain was isolated. The major metabolites of this strain were ethylene glycol and glycolic acid. However, the metabolites were switched to 2,3-butanediol, acetoin or acetic acid when this strain was cultured with other carbon sources. The metabolic pathway of ethylene glycol synthesis from xylonic acid in this bacterium was identified. Xylonic acid was converted to 2-dehydro-3-deoxy-D-pentonate catalyzed by D-xylonic acid dehydratase. 2-Dehydro-3-deoxy-D-pentonate was converted to form pyruvate and glycolaldehyde, and this reaction was catalyzed by an aldolase. D-Xylonic acid dehydratase and 2-dehydro-3-deoxy-D-pentonate aldolase were encoded by yjhG and yjhH, respectively. The two genes are part of the same operon and are located adjacent on the chromosome. Besides yjhG and yjhH, this operon contains four other genes. However, individually inactivation of these four genes had no effect on either ethylene glycol or glycolic acid production; both formed from glycolaldehyde. YqhD exhibits ethylene glycol dehydrogenase activity in vitro. However, a low level of ethylene glycol was still synthesized by E. cloacae ΔyqhD. Fermentation parameters for ethylene glycol and glycolic acid production by the E. cloacae strain were optimized, and aerobic cultivation at neutral pH were found to be optimal. In fed batch culture, 34 g/L of ethylene glycol and 13 g/L of glycolic acid were produced in 46 h, with a total conversion ratio of 0.99 mol/mol xylonic acid. CONCLUSIONS: A novel route of xylose biorefinery via xylonic acid as an intermediate has been established.

Proteomic study of sulfated polysaccharide from Enterobacter cloacae Z0206 against H2O2-induced oxidative damage in murine macrophages.

The present study aimed to unveil potential protective mechanisms of SEPS-4, a novel sulfated derivative of exopolysaccharide produced by Enterobacter cloacae Z0206, against H2O2-induced oxidative damage in murine macrophages based on proteomic approaches. SEPS-4 pre-treatment was found to be capable of alleviating H2O2-induced reduction of RAW264.7 cell viability. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to evaluate proteins with significant expression alterations in H2O2-challenged RAW264.7 cells following pre-incubation with SEPS-4. Here 12 up-regulated proteins and 12 down-regulated proteins were successfully identified. Bio-informatic analysis was applied for further mechanistic studies. GO annotation and KEGG pathway enrichment analyses demonstrated that differentially expressed proteins were mainly clustered in stress-related biological processes, including metabolic process, stimulus to response, cell growth and death. Peroxiredoxin-2 and Eef1g were core modules of protein-protein interaction network. Collectively, our data indicated that SEPS-4 may exert protective effects against H2O2-induced oxidative damage via the regulation of these functional proteins and related biological processes.

Co-culture with Enterobacter cloacae does not Enhance Virus Resistance to Thermal and Chemical Treatments.

Human noroviruses (hNoV) are the primary cause of foodborne disease in the USA. Most studies on inactivation kinetics of hNoV and its surrogates are performed in monoculture, while the microbial ecosystem effect on virus inactivation remains limited. This study investigated the persistence of hNoV surrogates, murine norovirus (MNV) and Tulane virus (TuV), along with Aichi virus (AiV) under thermal and chemical inactivation in association with Gram-negative (Enterobacter cloacae) bacteria. Thermal inactivation of viruses in co-culture with E. cloacae revealed no protective effects of bacteria. At 56 °C, AiV with and without bacteria was completely inactivated by 10 min with decimal reduction values (D-values) of 41 and 43 s, respectively. Similar results were also observed for TuV. Conversely, MNV with bacteria was completely inactivated by 10 min while MNV alone remained stable up to 30 min at 56 °C. Both MNV and TuV were slightly more stable than AiV at 63 °C with TuV detection up to 2 min without bacteria. For chemical inactivation on stainless steel surfaces, viruses alone and in association with bacteria were treated with 1000 ppm sodium hypochlorite. Virus association with bacteria had no significant effect (p > 0.05) on virus resistance to bleach inactivation compared to virus alone. Specifically, exposure to 1000 ppm bleach for 5 min resulted in an average of 3.86, 2.14, and 0.94 log10 PFU/ml reductions for TuV, MNV, and AiV without bacteria, respectively. Reductions in TuV, MNV, and AiV were 3.50, 1.88, and 0.61 log10 PFU/ml when associated with E. cloacae, respectively.

A polysaccharide from Enterobacter cloacae induces apoptosis of human osteosarcoma cells through the activation of p53 and mitochondrial intrinsic pathway.

In the present study, a polysaccharide (ECP) from Enterobacter cloacae dose and time-dependently inhibited cell growth of human osteosarcoma U-2 OS cells via induction of apoptosis. ECP treatment was selectively toxic to U-2 OS cells whereas had no cytotoxic effect on normal human osteoblast cell line NHOst. ECP-induced apoptotic cell death was associated with collapse of mitochondrial membrane, cytochrome c release into the cytosol, activation of caspase-9 and-3, degradation of poly (ADP-ribose) polymerase (PARP), elevated the ratio of Bax/Bcl-2 protein and overexpression of p53, suggesting the involvement of the activation of p53 and mitochondrial intrinsic pathway in ECP-induced apoptosis. Likewise, ECP oral administration significantly inhibited the U-2 OS cancer growth in xenograft tumor model. All these first evidence indicated that ECP was a potential antitumor supplement for the treatment of human osteosarcoma.

Isolation and characterization of kurstakin and surfactin isoforms produced by Enterobacter cloacae C3 strain.

In this work, the extraction, structural analysis, and identification as well as antimicrobial, anti-adhesive, and antibiofilm activities of lipopeptides produced by Enterobacter cloacae C3 strain were studied. A combination of chromatographic and spectroscopic techniques offers opportunities for a better characterization of the biosurfactant structure. Thin layer chromatography (TLC) and HPLC for amino acid composition determination are used. Efficient spectroscopic techniques have been utilized for investigations on the biochemical structure of biosurfactants, such as Fourier transform infrared (FT-IR) spectroscopy and mass spectrometry analysis. This is the first work describing the production of different isoforms belonging to kurstakin and surfactin families by E cloacae strain. Three kurstakin homologues differing by the fatty acid chain length from C10 to C12 were detected. The spectrum of lipopeptides belonging to surfactin family contains various isoforms differing by the fatty acid chain length as well as the amino acids at positions four and seven. Lipopeptide C3 extract exhibited important antibacterial activity against Gram-positive and Gram-negative bacteria, antifungal activity, and interesting anti-adhesive and disruptive properties against biofilm formation by human pathogenic bacterial strains: Salmonella typhimurium, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus cereus, and Candida albicans.

Structural investigation of a polysaccharide from the mycelium of Enterobacter cloacae and its antibacterial activity against extensively drug-resistant E. cloacae producing SHV-12 extended-spectrum ß-lactamase.

In this study, a polysaccharide (ECP) was isolated from the mycelium of Enterobacter cloacae and was found to exhibit strong antibacterial activities against E. cloacae producing SHV-12 ESBL with the increase of the inhibition zone diameter. Its minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 12.5 mg/L and 25 mg/L, respectively. ECP at these concentrations immediately inhibited planktonic growth of the bacteria especially at the time from 2 to 10 h. Flow cytometry analysis further revealed that almost all the bacterial cells were damaged following ECP treatment. The permeability of the cytoplasmic membrane of E. cloacae was increased when ECP concentrations increasing, as evidenced by an influx of Na and an efflux of K, P or S, the leakage of intracellular ATP and the UV-absorbing substances, as well as the depolarization of the cytoplasmic membrane, indicating that bactericidal activity of ECP was achieved by inducing cell membrane damage.

A novel cold-adapted esterase from Enterobacter cloacae: Characterization and improvement of its activity and thermostability via the site of Tyr193Cys.

BACKGROUND: In industries lipolytic reactions occur in insensitive conditions such as high temperature thus novel stout esterases with unique properties are attracts to the industrial application. Protein engineering is the tool to obtain desirable characters of enzymes. A novel esterase gene was isolated from South China Sea and subjected to a random mutagenesis and site directed mutagenesis for higher activity and thermo-stability compared to wild type. RESULTS: A novel esterase showed the highest hydrolytic activity against p-nitrophenyl acetate (pNPA, C2) and the optimal activity at 40 °C and pH 8.5. It was a cold-adapted enzyme and retained approximately 40% of its maximum activity at 0 °C. A mutant, with higher activity and thermo-stability was obtained by random mutagenesis. Kinetic analysis indicated that the mutant Val29Ala/Tyr193Cys shown 43.5% decrease in K m , 2.6-fold increase in K cat , and 4.7-fold increase in K cat /K m relative to the wild type. Single mutants V29A and Y193C were constructed and their kinetic parameters were measured. The results showed that the values of K m , K cat , and K cat /K m of V29A were similar to those of the wild type while Y193C showed 52.7% decrease in K m , 2.7-fold increase in K cat , and 5.6-fold increase in K cat /K m compared with the wild type. The 3-D structure and docking analysis revealed that the replacement of Tyr by Cys could enlarge the binding pocket. Moreover Y193C also showed a better thermo-stability for the reason its higher hydrophobicity and retained 67% relative activity after incubation for 3 h at 50 °C. CONCLUSIONS: The superior quality of modified esterase suggested it has great potential application in extreme conditions and the mutational work recommended that important information for the study of esterase structure and function.

Kinetics of Sulbactam Hydrolysis by ß-Lactamases, and Kinetics of ß-Lactamase Inhibition by Sulbactam.

Sulbactam is one of four ß-lactamase inhibitors in current clinical use to counteract drug resistance caused by degradation of ß-lactam antibiotics by these bacterial enzymes. As a ß-lactam itself, sulbactam is susceptible to degradation by ß-lactamases. I investigated the Michaelis-Menten kinetics of sulbactam hydrolysis by 14 ß-lactamases, representing clinically widespread groups within all four Ambler classes, i.e., CTX-M-15, KPC-2, SHV-5, and TEM-1 for class A; IMP-1, NDM-1, and VIM-1 for class B; Acinetobacter baumannii ADC-7, Pseudomonas aeruginosa AmpC, and Enterobacter cloacae P99 for class C; and OXA-10, OXA-23, OXA-24, and OXA-48 for class D. All of the ß-lactamases were able to hydrolyze sulbactam, although they varied widely in their kinetic constants for the reaction, even within each class. I also investigated the inactivation kinetics of the inhibition of these enzymes by sulbactam. The class A ß-lactamases varied widely in their susceptibility to inhibition, the class C and D enzymes were very weakly inhibited, and the class B enzymes were essentially or completely unaffected. In addition, we measured the sulbactam turnover number, the sulbactam/enzyme molar ratio required for complete inhibition of each enzyme. Class C enzymes had the lowest turnover numbers, class A enzymes varied widely, and class D enzymes had very high turnover numbers. These results are valuable for understanding which ß-lactamases ought to be well inhibited by sulbactam. Moreover, since sulbactam has intrinsic antibacterial activity against Acinetobacter species pathogens, these results contribute to understanding ß-lactamase-mediated sulbactam resistance in Acinetobacter, especially due to the action of the widespread class D enzymes.

Transglycosylation by a chitinase from Enterobacter cloacae subsp. cloacae generates longer chitin oligosaccharides.

Humans have exploited natural resources for a variety of applications. Chitin and its derivative chitin oligosaccharides (CHOS) have potential biomedical and agricultural applications. Availability of CHOS with the desired length has been a major limitation in the optimum use of such natural resources. Here, we report a single domain hyper-transglycosylating chitinase, which generates longer CHOS, from Enterobacter cloacae subsp. cloacae 13047 (EcChi1). EcChi1 was optimally active at pH 5.0 and 40 °C with a Km of 15.2 mg ml-1, and k cat/Km of 0.011× 102 mg-1 ml min-1 on colloidal chitin. The profile of the hydrolytic products, major product being chitobiose, released from CHOS indicated that EcChi1 was an endo-acting enzyme. Transglycosylation (TG) by EcChi1 on trimeric to hexameric CHOS resulted in the formation of longer CHOS for a prolonged duration. EcChi1 showed both chitobiase and TG activities, in addition to hydrolytic activity. The TG by EcChi1 was dependent, to some extent, on the length of the CHOS substrate and concentration of the enzyme. Homology modeling and docking with CHOS suggested that EcChi1 has a deep substrate-binding groove lined with aromatic amino acids, which is a characteristic feature of a processive enzyme.

Structure and gene cluster of the O-antigen of Enterobacter cloacae C4115.

An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae C4115 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the linear pentasaccharide repeating unit of the O-polysaccharide was established: →2)-α-l-Rhap-(1 â†’ 2)-α-l-Rhap-(1 â†’ 2)-α-l-Rhap-(1 â†’ 2)-α-d-Galp-(1 â†’ 3)-α-d-FucpNAc-(1→ The O-antigen gene cluster of E. cloacae C4115 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in consistence with the O-polysaccharide structure. The O-antigen structure and gene cluster of E. cloacae C4115 are similar to those of E. cloacae G3421 studied by us earlier (Perepelov A.V. et al. Carbohydr. Res. 427 (2016) 55-59).

Structural and genetic characterization of the O-antigen of Enterobacter cloacae C5529 related to the O-antigen of E. cloacae G3054.

On mild acid degradation of the lipopolysaccharide of Enterobacter cloacae C5529, the O-polysaccharide chain was cleaved at the linkages of 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Psep5Ac7Ac). The resultant oligosaccharide and an alkali-treated lipopolysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit of the O-polysaccharide was established: →4)-ß-Psep5Ac7Ac-(2 â†’ 3)-ß-d-Galp-(1 â†’ 6)-ß-d-Galf-(1 â†’ 3)-α-d-Galp-(1→ It differs from a structurally related O-polysaccharide of E. cloacae G3045 studied early (Perepelov, A. V.; Wang, M.; Filatov, A. V.; Guo, X.; Shashkov, A. S.; Wang, L.; Knirel, Y. A. Carbohydr. Res. 2015; 407:59-62) in positions of substitution of ß-Psep5Ac7Ac (O-4 vs. O-8) and ß-Galp (O-3 vs. O-6) and the absence of a side-chain α-Galp residue. The O-antigen gene clusters of E. cloacae C5529 and G3045 are organized identically and include genes with the same putative functions in the O-polysaccharide synthesis. Based on these and serological data, it is suggested to combine E. cloacae C5529 and G3054 in one O-serogroup as two subgroups.

Thermodynamics of an aminoglycoside modifying enzyme with low substrate promiscuity: The aminoglycoside N3 acetyltransferase-VIa.

Kinetic, thermodynamic, and structural properties of the aminoglycoside N3-acetyltransferase-VIa (AAC-VIa) are determined. Among the aminoglycoside N3-acetyltransferases, AAC-VIa has one of the most limited substrate profiles. Kinetic studies showed that only five aminoglycosides are substrates for this enzyme with a range of fourfold difference in kcat values. Larger differences in KM (∼40-fold) resulted in ∼30-fold variation in kcat /KM . Binding of aminoglycosides to AAC-VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (ΔG < 0). A net deprotonation of the enzyme, ligand, or both accompanied the formation of binary and ternary complexes. This is opposite of what was observed with several other aminoglycoside N3-acetyltransferases, where ligand binding causes more protonation. The change in heat capacity (ΔCp) was different in H2 O and D2 O for the binary enzyme-sisomicin complex but remained the same in both solvents for the ternary enzyme-CoASH-sisomicin complex. Unlike, most other aminoglycoside-modifying enzymes, the values of ΔCp were within the expected range of protein-carbohydrate interactions. Solution behavior of AAC-VIa was also different from the more promiscuous aminoglycoside N3-acetyltransferases and showed a monomer-dimer equilibrium as detected by analytical ultracentrifugation (AUC). Binding of ligands shifted the enzyme to monomeric state. Data also showed that polar interactions were the most dominant factor in dimer formation. Overall, thermodynamics of ligand-protein interactions and differences in protein behavior in solution provide few clues on the limited substrate profile of this enzyme despite its >55% sequence similarity to the highly promiscuous aminoglycoside N3-acetyltransferase. Proteins 2017; 85:1258-1265. © 2017 Wiley Periodicals, Inc.

Combining X-ray and neutron crystallography with spectroscopy.

X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H+ ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction.

Structure and gene cluster of the O-antigen of Enterobacter cloacae G3421.

The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G3421 and studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. In addition, partial solvolysis with anhydrous trifluoroacetic acid was applied, which cleaved selectively the α-l-rhamnopyranosidic linkages. The following structure of the branched hexasaccharide repeating unit was established. The O-polysaccharide studied shares the ß-l-Rhap-(1→4)-α-l-Rhap-(1→2)-α-l-Rhap trisaccharide fragment with the O-polysaccharide of Shigella boydii type 18. The O-antigen gene cluster of E. cloacae G3421 was sequenced. Functions of genes in the cluster, including those for glycosyltransferases, were tentatively assigned by a comparison with sequences in the available databases and found to be consistent with the O-polysaccharide structure.