, the causative agent of plague, can be transmitted by fleas by two different mechanisms: by early-phase transmission (EPT), which occurs shortly after flea infection, or by blocked fleas following long-term infection. Efficient flea-borne transmission is predicated upon the ability of to be maintained within the flea. Signature-tagged mutagenesis (STM) was used to identify genes required for maintenance in a genuine plague vector, . The STM screen identified seven mutants that displayed markedly reduced fitness in fleas after 4 days, the time during which EPT occurs. Two of the mutants contained insertions in genes encoding glucose 1-phosphate uridylyltransferase () and UDP-4-amino-4-deoxy--arabinose-oxoglutarate aminotransferase (), which are involved in the modification of lipid A with 4-amino-4-deoxy--arabinose (Ara4N) and resistance to cationic antimicrobial peptides (CAMPs). These mutants were more susceptible to the CAMPs cecropin A and polymyxin B, and produced lipid A lacking Ara4N modifications. Surprisingly, an in-frame deletion of retained modest levels of CAMP resistance and Ara4N modification, indicating the presence of compensatory factors. It was determined that WecE, an aminotransferase involved in biosynthesis of enterobacterial common antigen, plays a novel role in Ara4N modification by partially offsetting the loss of These results indicated that mechanisms of Ara4N modification of lipid A are more complex than previously thought, and these modifications, as well as several factors yet to be elucidated, play an important role in early survival and transmission of in the flea vector.


Article metrics loading...

Loading full text...

Full text loading...



  1. Anisimov A. P., Dentovskaya S. V., Titareva G. M., Bakhteeva I. V., Shaikhutdinova R. Z., Balakhonov S. V., Lindner B., Kocharova N. A., Senchenkova S. N. & other authors (2005). Intraspecies and temperature-dependent variations in susceptibility of Yersinia pestis to the bactericidal action of serum and to polymyxin B. Infect Immun 73, 73247331. [View Article][PubMed] [Google Scholar]
  2. Bacot A. W., Martin C. J. (1914). LXVII. Observations on the mechanism of the transmission of plague by fleas. J Hyg (Lond) 13 (Suppl), 423439.[PubMed] [Google Scholar]
  3. Barańska-Rybak W., Sonesson A., Nowicki R., Schmidtchen A. (2006). Glycosaminoglycans inhibit the antibacterial activity of LL-37 in biological fluids. J Antimicrob Chemother 57, 260265. [View Article][PubMed] [Google Scholar]
  4. Barua S., Yamashino T., Hasegawa T., Yokoyama K., Torii K., Ohta M. (2002). Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157 : H7. Mol Microbiol 43, 629640. [View Article][PubMed] [Google Scholar]
  5. Breazeale S. D., Ribeiro A. A., Raetz C. R. (2003). Origin of lipid A species modified with 4-amino-4-deoxy-l-arabinose in polymyxin-resistant mutants of Escherichia coli. An aminotransferase (ArnB) that generates UDP-4-deoxyl-l-arabinose. J Biol Chem 278, 2473124739. [View Article][PubMed] [Google Scholar]
  6. Bulet P., Stöcklin R. (2005). Insect antimicrobial peptides: structures, properties and gene regulation. Protein Pept Lett 12, 311. [View Article][PubMed] [Google Scholar]
  7. Burroughs A. L. (1947). Sylvatic plague studies: the vector efficiency of nine species of fleas compared with Xenopsylla cheopis . J Hyg (Lond) 45, 371396. [View Article][PubMed] [Google Scholar]
  8. Choi K. H., Gaynor J. B., White K. G., Lopez C., Bosio C. M., Karkhoff-Schweizer R. R., Schweizer H. P. (2005). A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2, 443448. [View Article][PubMed] [Google Scholar]
  9. Chouikha I., Hinnebusch B. J. (2012). Yersinia–flea interactions and the evolution of the arthropod-borne transmission route of plague. Curr Opin Microbiol 15, 239246. [View Article][PubMed] [Google Scholar]
  10. Cullen T. W., O’Brien J. P., Hendrixson D. R., Giles D. K., Hobb R. I., Thompson S. A., Brodbelt J. S., Trent M. S. (2013). EptC of Campylobacter jejuni mediates phenotypes involved in host interactions and virulence. Infect Immun 81, 430440. [View Article][PubMed] [Google Scholar]
  11. Danese P. N., Oliver G. R., Barr K., Bowman G. D., Rick P. D., Silhavy T. J. (1998). Accumulation of the enterobacterial common antigen lipid II biosynthetic intermediate stimulates degP transcription in Escherichia coli . J Bacteriol 180, 58755884.[PubMed] [Google Scholar]
  12. Darby C., Ananth S. L., Tan L., Hinnebusch B. J. (2005). Identification of gmhA, a Yersinia pestis gene required for flea blockage, by using a Caenorhabditis elegans biofilm system. Infect Immun 73, 72367242. [View Article][PubMed] [Google Scholar]
  13. Datsenko K. A., Wanner B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 66406645. [View Article][PubMed] [Google Scholar]
  14. Derbise A., Lesic B., Dacheux D., Ghigo J. M., Carniel E. (2003). A rapid and simple method for inactivating chromosomal genes in Yersinia . FEMS Immunol Med Microbiol 38, 113116. [View Article][PubMed] [Google Scholar]
  15. Domka J., Lee J., Wood T. K. (2006). YliH (BssR) and YceP (BssS) regulate Escherichia coli K-12 biofilm formation by influencing cell signaling. Appl Environ Microbiol 72, 24492459. [View Article][PubMed] [Google Scholar]
  16. Eisen R. J., Gage K. L. (2012). Transmission of flea-borne zoonotic agents. Annu Rev Entomol 57, 6182. [View Article][PubMed] [Google Scholar]
  17. Eisen R. J., Bearden S. W., Wilder A. P., Montenieri J. A., Antolin M. F., Gage K. L. (2006). Early-phase transmission of Yersinia pestis by unblocked fleas as a mechanism explaining rapidly spreading plague epizootics. Proc Natl Acad Sci U S A 103, 1538015385. [View Article][PubMed] [Google Scholar]
  18. Eisen R. J., Lowell J. L., Montenieri J. A., Bearden S. W., Gage K. L. (2007). Temporal dynamics of early-phase transmission of Yersinia pestis by unblocked fleas: secondary infectious feeds prolong efficient transmission by Oropsylla montana (Siphonaptera: Ceratophyllidae). J Med Entomol 44, 672677. [View Article][PubMed] [Google Scholar]
  19. Eisen R. J., Vetter S. M., Holmes J. L., Bearden S. W., Montenieri J. A., Gage K. L. (2008). Source of host blood affects prevalence of infection and bacterial loads of Yersinia pestis in fleas. J Med Entomol 45, 933938. [View Article][PubMed] [Google Scholar]
  20. Felek S., Muszyński A., Carlson R. W., Tsang T. M., Hinnebusch B. J., Krukonis E. S. (2010). Phosphoglucomutase of Yersinia pestis is required for autoaggregation and polymyxin B resistance. Infect Immun 78, 11631175. [View Article][PubMed] [Google Scholar]
  21. Fernández L., Alvarez-Ortega C., Wiegand I., Olivares J., Kocíncová D., Lam J. S., Martínez J. L., Hancock R. E. (2013). Characterization of the polymyxin B resistome of Pseudomonas aeruginosa . Antimicrob Agents Chemother 57, 110119. [View Article][PubMed] [Google Scholar]
  22. Flashner Y., Mamroud E., Tidhar A., Ber R., Aftalion M., Gur D., Lazar S., Zvi A., Bino T. & other authors (2004). Generation of Yersinia pestis attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates. Infect Immun 72, 908915. [View Article][PubMed] [Google Scholar]
  23. Gilbreath J. J., Colvocoresses Dodds J., Rick P. D., Soloski M. J., Merrell D. S., Metcalf E. S. (2012). Enterobacterial common antigen mutants of Salmonella enterica serovar Typhimurium establish a persistent infection and provide protection against subsequent lethal challenge. Infect Immun 80, 441450. [View Article][PubMed] [Google Scholar]
  24. Gozdziewicz T. K., Lugowski C., Lukasiewicz J. (2014). First evidence for a covalent linkage between enterobacterial common antigen and lipopolysaccharide in Shigella sonnei phase II ECALPS. J Biol Chem 289, 27452754. [View Article][PubMed] [Google Scholar]
  25. Gunn J. S., Lim K. B., Krueger J., Kim K., Guo L., Hackett M., Miller S. I. (1998). PmrA–PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Mol Microbiol 27, 11711182. [View Article][PubMed] [Google Scholar]
  26. Gunn J. S., Ryan S. S., Van Velkinburgh J. C., Ernst R. K., Miller S. I. (2000). Genetic and functional analysis of a PmrA–PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium. Infect Immun 68, 61396146. [View Article][PubMed] [Google Scholar]
  27. Hensel M., Shea J. E., Gleeson C., Jones M. D., Dalton E., Holden D. W. (1995). Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400403. [View Article][PubMed] [Google Scholar]
  28. Hinnebusch B. J., Perry R. D., Schwan T. G. (1996). Role of the Yersinia pestis hemin storage (hms) locus in the transmission of plague by fleas. Science 273, 367370. [View Article][PubMed] [Google Scholar]
  29. Hinnebusch J., Cherepanov P., Du Y., Rudolph A., Dixon J. D., Schwan T., Forsberg A. (2000). Murine toxin of Yersinia pestis shows phospholipase D activity but is not required for virulence in mice. Int J Med Microbiol 290, 483487. [View Article][PubMed] [Google Scholar]
  30. Hinnebusch B. J., Rudolph A. E., Cherepanov P., Dixon J. E., Schwan T. G., Forsberg A. (2002). Role of Yersinia murine toxin in survival of Yersinia pestis in the midgut of the flea vector. Science 296, 733735. [View Article][PubMed] [Google Scholar]
  31. Hoang T. T., Karkhoff-Schweizer R. R., Kutchma A. J., Schweizer H. P. (1998). A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212, 7786. [View Article][PubMed] [Google Scholar]
  32. Houhamdi L., Raoult D. (2008). Different genes govern Yersinia pestis pathogenicity in Caenorhabditis elegans and human lice. Microb Pathog 44, 435437. [View Article][PubMed] [Google Scholar]
  33. Hwang B. Y., Lee H. J., Yang Y. H., Joo H. S., Kim B. G. (2004). Characterization and investigation of substrate specificity of the sugar aminotransferase WecE from E. coli K12. Chem Biol 11, 915925. [View Article][PubMed] [Google Scholar]
  34. Jarrett C. O., Deak E., Isherwood K. E., Oyston P. C., Fischer E. R., Whitney A. R., Kobayashi S. D., DeLeo F. R., Hinnebusch B. J. (2004). Transmission of Yersinia pestis from an infectious biofilm in the flea vector. J Infect Dis 190, 783792. [View Article][PubMed] [Google Scholar]
  35. Jiang S. S., Lin T. Y., Wang W. B., Liu M. C., Hsueh P. R., Liaw S. J. (2010). Characterization of UDP-glucose dehydrogenase and UDP-glucose pyrophosphorylase mutants of Proteus mirabilis: defectiveness in polymyxin B resistance, swarming, and virulence. Antimicrob Agents Chemother 54, 20002009. [View Article][PubMed] [Google Scholar]
  36. Karlyshev A. V., Pallen M. J., Wren B. W. (2000). Single-primer PCR procedure for rapid identification of transposon insertion sites. Biotechniques 28, 10781082.[PubMed] [Google Scholar]
  37. Kartman L., Prince F. M., Quan S. F., Stark H. E. (1958). New knowledge on the ecology of sylvatic plague. Ann N Y Acad Sci 70, 668711. [View Article][PubMed] [Google Scholar]
  38. Kawasaki K. (2009). Alternative procedures for analysis of lipid A modification with phosphoethanolamine or aminoarabinose. J Microbiol Methods 76, 313315. [View Article][PubMed] [Google Scholar]
  39. Klein K. A., Fukuto H. S., Pelletier M., Romanov G., Grabenstein J. P., Palmer L. E., Ernst R., Bliska J. B. (2012). A transposon site hybridization screen identifies galU and wecBC as important for survival of Yersinia pestis in murine macrophages. J Bacteriol 194, 653662. [View Article][PubMed] [Google Scholar]
  40. Lane M. C., Alteri C. J., Smith S. N., Mobley H. L. (2007). Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract. Proc Natl Acad Sci U S A 104, 1666916674. [View Article][PubMed] [Google Scholar]
  41. Lehane M. J., Wu D., Lehane S. M. (1997). Midgut-specific immune molecules are produced by the blood-sucking insect Stomoxys calcitrans . Proc Natl Acad Sci U S A 94, 1150211507. [View Article][PubMed] [Google Scholar]
  42. Mäkelä P. H., Mayer H. (1976). Enterobacterial common antigen. Bacteriol Rev 40, 591632.[PubMed] [Google Scholar]
  43. Mazurkiewicz P., Tang C. M., Boone C., Holden D. W. (2006). Signature-tagged mutagenesis: barcoding mutants for genome-wide screens. Nat Rev Genet 7, 929939. [View Article][PubMed] [Google Scholar]
  44. McCoy A. J., Liu H., Falla T. J., Gunn J. S. (2001). Identification of Proteus mirabilis mutants with increased sensitivity to antimicrobial peptides. Antimicrob Agents Chemother 45, 20302037. [View Article][PubMed] [Google Scholar]
  45. Mei J. M., Nourbakhsh F., Ford C. W., Holden D. W. (1997). Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis. Mol Microbiol 26, 399407. [View Article][PubMed] [Google Scholar]
  46. Merrell D. S., Hava D. L., Camilli A. (2002). Identification of novel factors involved in colonization and acid tolerance of Vibrio cholerae . Mol Microbiol 43, 14711491. [View Article][PubMed] [Google Scholar]
  47. Minato Y., Ghosh A., Faulkner W. J., Lind E. J., Schesser Bartra S., Plano G. V., Jarrett C. O., Hinnebusch B. J., Winogrodzki J. & other authors (2013). Na+/H+ antiport is essential for Yersinia pestis virulence. Infect Immun 81, 31633172. [View Article][PubMed] [Google Scholar]
  48. Muszyński A., Rabsztyn K., Knapska K., Duda K. A., Duda-Grychtoł K., Kasperkiewicz K., Radziejewska-Lebrecht J., Holst O., Skurnik M. (2013). Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O : 3. Microbiology 159, 17821793. [View Article][PubMed] [Google Scholar]
  49. Nichols R. J., Sen S., Choo Y. J., Beltrao P., Zietek M., Chaba R., Lee S., Kazmierczak K. M., Lee K. J. & other authors (2011). Phenotypic landscape of a bacterial cell. Cell 144, 143156. [View Article][PubMed] [Google Scholar]
  50. Noland B. W., Newman J. M., Hendle J., Badger J., Christopher J. A., Tresser J., Buchanan M. D., Wright T. A., Rutter M. E. & other authors (2002). Structural studies of Salmonella typhimurium ArnB (PmrH) aminotransferase: a 4-amino-4-deoxy-l-arabinose lipopolysaccharide-modifying enzyme. Structure 10, 15691580. [View Article][PubMed] [Google Scholar]
  51. Ohshima N., Yamashita S., Takahashi N., Kuroishi C., Shiro Y., Takio K. (2008). Escherichia coli cytosolic glycerophosphodiester phosphodiesterase (UgpQ) requires Mg2+, Co2+, or Mn2+ for its enzyme activity. J Bacteriol 190, 12191223. [View Article][PubMed] [Google Scholar]
  52. Otto M. (2006). Bacterial evasion of antimicrobial peptides by biofilm formation. In Antimicrobial Peptides and Human Disease, pp. 251258. Edited by Shafer W. M. . Berlin: Springer. [View Article] [Google Scholar]
  53. Park P. W., Pier G. B., Hinkes M. T., Bernfield M. (2001). Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411, 98102. [View Article][PubMed] [Google Scholar]
  54. Perry R. D., Pendrak M. L., Schuetze P. (1990). Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis . J Bacteriol 172, 59295937.[PubMed] [Google Scholar]
  55. Raetz C. R., Reynolds C. M., Trent M. S., Bishop R. E. (2007). Lipid A modification systems in gram-negative bacteria. Annu Rev Biochem 76, 295329. [View Article][PubMed] [Google Scholar]
  56. Ravi C., Jeyashree A., Renuka Devi K. (2011). Antimicrobial peptides from insects: an overview. Res Biotech 2, 17. [Google Scholar]
  57. Rebeil R., Ernst R. K., Gowen B. B., Miller S. I., Hinnebusch B. J. (2004). Variation in lipid A structure in the pathogenic yersiniae. Mol Microbiol 52, 13631373. [View Article][PubMed] [Google Scholar]
  58. Rebeil R., Jarrett C. O., Driver J. D., Ernst R. K., Oyston P. C., Hinnebusch B. J. (2013). Induction of the Yersinia pestis PhoP–PhoQ regulatory system in the flea and its role in producing a transmissible infection. J Bacteriol 195, 19201930. [View Article][PubMed] [Google Scholar]
  59. Reynolds C. M., Kalb S. R., Cotter R. J., Raetz C. R. (2005). A phosphoethanolamine transferase specific for the outer 3-deoxy-d-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant. J Biol Chem 280, 2120221211. [View Article][PubMed] [Google Scholar]
  60. Rick P. D., Silver R. P. (1996). Enterobacterial common antigen and capsular polysaccharides. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 104122. Edited by Neidhardt F. C. . Washington, DC: American Society for Microbiology. [Google Scholar]
  61. Rick P. D., Hubbard G. L., Kitaoka M., Nagaki H., Kinoshita T., Dowd S., Simplaceanu V., Ho C. (1998). Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis. Glycobiology 8, 557567. [View Article][PubMed] [Google Scholar]
  62. Rudolph A. E., Stuckey J. A., Zhao Y., Matthews H. R., Patton W. A., Moss J., Dixon J. E. (1999). Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member. J Biol Chem 274, 1182411831. [View Article][PubMed] [Google Scholar]
  63. Sambrook J., Fritsch E. F., Maniatis T. (1989).Molecular Cloning: A Laboratory Manual, 2nd edn.Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. [Google Scholar]
  64. Schmidtchen A., Frick I. M., Björck L. (2001). Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial alpha-defensin. Mol Microbiol 39, 708713. [View Article][PubMed] [Google Scholar]
  65. Simon R., Priefer U., Puhler A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat Biotechol 1, 784791. [View Article] [Google Scholar]
  66. Skurnik M., Peippo A., Ervelä E. (2000). Characterization of the O-antigen gene clusters of Yersinia pseudotuberculosis and the cryptic O-antigen gene cluster of Yersinia pestis shows that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype O : 1b. Mol Microbiol 37, 316330. [View Article][PubMed] [Google Scholar]
  67. Vadyvaloo V., Jarrett C., Sturdevant D. E., Sebbane F., Hinnebusch B. J. (2010). Transit through the flea vector induces a pretransmission innate immunity resistance phenotype in Yersinia pestis . PLoS Pathog 6, e1000783.[CrossRef] [Google Scholar]
  68. Vetter S. M., Eisen R. J., Schotthoefer A. M., Montenieri J. A., Holmes J. L., Bobrov A. G., Bearden S. W., Perry R. D., Gage K. L. (2010). Biofilm formation is not required for early-phase transmission of Yersinia pestis . Microbiology 156, 22162225. [View Article][PubMed] [Google Scholar]
  69. Viau C., Le Sage V., Ting D. K., Gross J., Le Moual H. (2011). Absence of PmrAB-mediated phosphoethanolamine modifications of Citrobacter rodentium lipopolysaccharide affects outer membrane integrity. J Bacteriol 193, 21682176. [View Article][PubMed] [Google Scholar]
  70. Vinogradov E. V., Knirel Y. A., Thomas-Oates J. E., Shashkov A. S., L’vov V. L. (1994). The structure of the cyclic enterobacterial common antigen (ECA) from Yersinia pestis . Carbohydr Res 258, 223232. [View Article][PubMed] [Google Scholar]
  71. Zhou P., Altman E., Perry M. B., Li J. (2010). Study of matrix additives for sensitive analysis of lipid A by matrix-assisted laser desorption ionization mass spectrometry. Appl Environ Microbiol 76, 34373443. [View Article][PubMed] [Google Scholar]

Data & Media loading...


Supplementary Data


Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error