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Genetics and Molecular Biology

Molecular Analysis of the Enterobacter sakazakii O-Antigen Gene Locus

N. Mullane, P. O'Gaora, J. E. Nally, C. Iversen, P. Whyte, P. G. Wall, S. Fanning
N. Mullane
Centres for Food Safety and Food-borne Zoonomics, UCD Veterinary Sciences Centre
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P. O'Gaora
Conway Institute of Biomolecular & Biomedical Research
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J. E. Nally
School of Agriculture, Food Science & Veterinary Medicine
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C. Iversen
Institute for Food Safety & Hygiene, Vetsuisse Faculty, University of Zurich, CH-8057 Zurich, Switzerland
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P. Whyte
Centres for Food Safety and Food-borne Zoonomics, UCD Veterinary Sciences Centre
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P. G. Wall
School of Public Health & Population Sciences, University College Dublin, Belfield, Dublin 4, Ireland
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S. Fanning
Centres for Food Safety and Food-borne Zoonomics, UCD Veterinary Sciences Centre
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  • For correspondence: sfanning@ucd.ie
DOI: 10.1128/AEM.02302-07
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  • FIG. 1.
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    FIG. 1.

    Restriction length profiles of rfb-encoding locus of E. sakazakii following MboII digestion. Lane 1, ATCC BAA-894; lane 2, E824; lane 3, E825; lane 4, NCTC 11467; lane 5, NCTC 8155; lane 6, 336; lane 7, 344; lane 8, 109; lane 9, CFS06; lane 10, E787; lane 11, 82; lane 12, 102; lane M1, 100-bp DNA ladder (New England Biolabs, Hertfordshire, England); lane M2, DNA molecular weight marker XVI (Roche, Mannheim, Germany). Black and gray boxes indicate profiles of strains corresponding to O:1 and O:2 serotypes, respectively.

  • FIG. 2.
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    FIG. 2.

    Dendrogram generated using BioNumerics software program at a similarity of 80% of MboII restriction digests. Dotted lines indicate uncertain bands. Proposed new species names are included and are marked by asterisks (20). The black and gray boxes in this figure indicate profiles of strains corresponding to serotypes O:1 and O:2, respectively, in the dendrogram (see Fig. 1).

  • FIG. 3.
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    FIG. 3.

    Genetic organization of the NCTC 11467 (O:1) (a) or NCTC 8155 (O:2) (b) O-antigen gene clusters. Arrows labeled with the corresponding gene names represent individual ORFs and the direction of transcription.

  • FIG. 4.
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    FIG. 4.

    A 1.5% agarose gel image of specific-serotype O:1 (top panel) or serotype O:2 (bottom panel) amplicons, generated by PCR. Lane 1, CFS131; lane 2, E824; lane 3, CFS06; lane 4, 44; lane 5, 93; lane 6, E891; lane 7, E784; lane 8, CFS153; lane 9, 88; lane 10, 305N; lane 11, ATCC BAA 893; lane 12, ATCC BAA 894; lane 13, E899; lane 14, 343; lane 15, 80; lane 16, E775; lane 17, 71; lane 18, E846; lane 19, E830; lane 20, 228N; lane 21, CFS136; lane 22, CFS149; lane 23, no-template control; lane M, 100-bp DNA ladder (New England Biolabs, Hertfordshire, England). Dashed-line box indicates serotype O:1 strains, while continuous-line box indicates serotype O:2 strains.

  • FIG. 5.
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    FIG. 5.

    Silver-stained SDS-PAGE of purified LPS samples. The relative positions of the O-antigen side chain and lipid A core are indicated by the brackets. (a) Lane M, rainbow molecular weight marker (high range; RPN 800) (Amersham Life Science, Buckinghamshire, England); lane 1, S. enterica serotype Minnesota LPS (5 μg); lane 2, ATCC BAA-894 (O:1); lane 3, E901 (O:2); lane 4, CFS1001 (O:1); lane 5, CFS10 (O:2); lane 6, 90; lane 7, CFS149 (O:1); lane 8, CFS173 (neither O:1 nor O:2); lane 9, undigested E901. Asterisks indicate O-antigen bands of similar mobilities. (b) Lane M, molecular marker, lane 1, S. enterica serotype Minnesota LPS (5 μg); lane 2, ATCC 12868 (O:2); lane 3, NCTC 8155 (O:2); lane 4, E844 (O:2); lane 5, E892 (O:2); lane 6, 228N (O:2); lane 7, undigested ATCC 12868. Asterisks indicate O-antigen bands of different mobilities in digested NCTC 12868.

Tables

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  • TABLE 1.

    Members of the Enterobacteriaceae used in this study

    SpeciesStrainOriginSerotype
    Enterobacter sakazakii (“Cronobacter” spp.)
        C. sakazakiiCFS06Acid casein powder1
        C. sakazakiiCFS10Acid casein powder2
        C. sakazakiiCFS104Milk powder1
        C. sakazakiiCFS122Environmental1
        C. malonaticus*aCFS129EnvironmentalNDb
        C. sakazakiiCFS131Milk powder1
        C. sakazakiiCFS136Environmental2
        C. sakazakiiCFS149Environmental2
        C. sakazakiiCFS153Environmental1
        C. sakazakiiCFS164Environmental1
        C. sakazakiiCFS173Milk powderND
        C. sakazakiiCFS175Environmental2
        C. sakazakiiCFS176Environmental2
        C. dublinensis*CFS237Milk powderND
        C. sakazakiiCFS1001Environmental1
        C. sakazakii44Environmental1
        C. sakazakii71Environmental2
        C. sakazakii79Environmental2
        C. sakazakii80Environmental2
        C. sakazakii82EnvironmentalND
        C. sakazakii88Environmental1
        C. sakazakii90Environmental1
        C. sakazakii93Environmental1
        C. sakazakii102EnvironmentalND
        C. sakazakii109Environmental2
        C. sakazakii228NMilk powder2
        C. sakazakii305NMilk powder1
        C. sakazakii343Environmental2
        C. sakazakii344EnvironmentalND
        C. sakazakii366EnvironmentalND
        C. sakazakiiE604Clinical2
        C. sakazakiiE632Infant food1
        C. sakazakiiE770Milk powder1
        C. sakazakiiE775Milk powder2
        C. sakazakiiE784Neonate1
        C. sakazakiiE787Clinical2
        C. sakazakiiE824 (CDC 0996-77)Human spinal (fluid)1
        C. malonaticus*E825 (CDC 1058-77Human (breast abscess)ND
        C. sakazakiiE826 (CDC 1059-77)UnknownND
        C. sakazakiiE827 (CDC 0407-77)Human (septum)1
        C. sakazakiiE828 (CDC 3128-77)Human (septum)1
        C. malonaticus*E829 (CDC 1716-77)Human (Blood)ND
        C. sakazakiiE830 (CDC 9369-75)Unknown2
        C. sakazakiiE837Clinical2
        C. sakazakiiE839NeonateND
        C. sakazakiiE844Neonate2
        C. sakazakiiE846Neonate2
        C. turicensis*E866NeonateND
        C. sakazakiiE891Clinical1
        C. sakazakiiE892Clinical2
        C. sakazakiiE899 (CDC 0289-81)Clinical2
        C. sakazakiiE900 (CDC 0322-78)Clinical2
        C. sakazakiiE901 (CDC 1123-79)Clinical2
        C. sakazakiiATCC 12868Unknown2
        C. sakazakiiNCTC 8155 (CDC 9081-75)Milk powder2
        C. sakazakiiNCTC 9238Human (abdominal puss)2
        C. sakazakiiNCTC 29004Unknown2
        C. genomospecies 1*NCTC 9529WaterND
        C. sakazakiiNCTC 11467 (ATCC 29544)Human (throat)1
        C. sakazakiiATCC BAA 893Milk powder1
        C. sakazakiiATCC BAA 894Neonate1
        C. muytjensii*ATCC 51329UnknownND
    Non-Enterobacter sakazakii (“Cronobacter” spp.)
    Escherichia coli (EPEC)NDND
    Escherichia coli (ETEC)NDND
    Escherichia coliK12 NC10538:06NDND
    Salmonella enterica serovar AnatumNDND
    Salmonella enterica serovar AgonaNCTC11377NDND
    Salmonella enterica serovar TyphimuriumLT2 NCTC12416NDND
    Salmonella enterica serovar Enterica PT4NCTC13349NDND
    Citrobacter youngaeNDND
    Citrobacter sedlakiiNDND
    Citrobacter braakiiNDND
    Citrobacter koseriNDND
    Citrobacter freundiiATCC 8090NDND
    Citrobacter koseriNCTC10768NDND
    Klebsiella oxytocaATCC 43086NDND
    Klebsiella pneumoniaeATCC 13883NDND
    Yersinia enterocoliticaNDND
    Enterobacter helveticusNDND
    Escherichia hermanniiNDND
    Enterobacter hormaecheiNDND
    Enterobacter cancerogenesNDND
    Escherichia vulnerisNDND
    Enterobacter cloacaeNCTC11933NDND
    Enterobacter aerogenesNCTC10006NDND
    Enterobacter gergoviaeNCTC11434NDND
    Enterobacter agglomeransNCTC9381NDND
    • ↵a *, proposed species names for taxonomic change according to Iversen et al. (20).

    • ↵b ND, not determined.

  • TABLE 2.

    Putative genes in E. sakazakii O:1 or O:2 O-antigen gene cluster

    ORFLocation in sequence (bp)G+C content (%)Size (aaa)Similar proteinSource (accession no.)% Identical/% similar (no. of aa)Putative function
    O:1 cluster
        orf11-89153.4296GalFSalmonella enterica (AE017220)87/95 (295)UDP-glucose pyrophosphorylase
        orf21282-236150.3282RfbBShigella flexnerii (NP_707936)87/93 (273)dTDP-d-glucose 4,6 dehydratase
        orf32358-323040.9290RmlAEscherichia coli (AAT77170)81/90 (287)dTDP-glucose pyrophosphorylase
        orf43232-363335.8133WbtAEscherichia coli (ABK27315)55/73 (131)Putative isomerase
        orf53626-407831.8150FdtCEscherichia coli (AAT77171)49/58 (147)Acetyltransferase
        orf64079-501434.4311Xanthomonas axonopodis pv. Citri (NP_642021)45/62 (306)Hypothetical protein
        orf75011-611436.7367FdtBEscherichia coli (AAT77174)58/73 (365)Aminotransferase
        orf86111-737337.3420WzxEscherichia coli (AAT77175)46/67 (413)O-antigen flippase
        orf97373-831732.5314WbtEEscherichia coli (AAS73170)28/50 (254)Putative glycosyltransferase
        orf108356-963631.9426WzyEscherichia coli (AAZ85713)19/43 (272)O-antigen polymerase
        orf119623-1050136.7292WbgQShigella boydii (AAL27350)32/51 (281)Putative glycosyltransferase
        orf1210491-1125534.0254Shewanella baltica (ZP_01435692)55/68 (243)Glycosyltransferase
        orf1311469-1287554.3468GndEscherichia coli (ZP_00926655)93/97 (469)6-Phosphogluconate dehydrogenase
    O:2 cluster
        orf11-59455.1197GalFEscherichia coli (AAS99158)83/90 (197)UDP-glucose pyrophosphorylase
        orf2986-206850.3360RmlBEscherichia coli (ABI98981)84/91 (360)dTDP-glucose 4,6-dehydratase
        orf32065-296749.5300RmlDShigella boydii (AAY23334)80/88 (298)dTDP-4-dehydrorhamnose reductase
        orf43017-389546.6292RmlASalmonella enterica (P55254)91/96 (292)dTDP-glucose synthase
        orf53899-444135.9180RfbCShigella boydii (YP_407363)81/90 (180)dTDP-4-dehydrorhamnose 3,5-epimerase
        orf64488-574430.2418RfbXEscherichia coli (NP_416541)27/52 (406)Putative O-antigen transporter
        orf75741-671833.2325Escherichia coli (ZP_01699967)22/42 (280)Glycosyltransferase
        orf86730-748529.4251WbsVShigella boydii (AAS98030)30/48 (241)Glycosyltransferase
        orf97475-847331.0332WzyShigella boydii (AAS98031)27/43 (317)O-antigen polymerase
        orf108493-933228.9279WbsWShigella boydii (AAS98032)30/47 (268)Glycosyltransferase
        orf119334-1034131.9335WbgMEscherichia coli (YP_541302)30/46 (110)Putative galactosyltransferase
        orf1210331-1109529.9254WbbLEscherichia coli (P36667)40/58 (257)Putative lipopolysaccharide biosynthesis glycosyltransferase
        orf1311462-1286854.4468GndEscherichia coli (ZP_00926655)93/97 (469)6-Phosphogluconate dehydrogenase
    • ↵a aa, amino acids.

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Molecular Analysis of the Enterobacter sakazakii O-Antigen Gene Locus
N. Mullane, P. O'Gaora, J. E. Nally, C. Iversen, P. Whyte, P. G. Wall, S. Fanning
Applied and Environmental Microbiology Jun 2008, 74 (12) 3783-3794; DOI: 10.1128/AEM.02302-07

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Molecular Analysis of the Enterobacter sakazakii O-Antigen Gene Locus
N. Mullane, P. O'Gaora, J. E. Nally, C. Iversen, P. Whyte, P. G. Wall, S. Fanning
Applied and Environmental Microbiology Jun 2008, 74 (12) 3783-3794; DOI: 10.1128/AEM.02302-07
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