Genes Involved in Yellow Pigmentation of Cronobacter sakazakii ES5 and Influence of Pigmentation on Persistence and Growth under Environmental Stress

Cronobacter spp. are opportunistic food-borne pathogens that are responsible for rare but highly fatal cases of meningitis and necrotizing enterocolitis in neonates. While the operon responsible for yellow pigmentation in Cronobacter sakazakii strain ES5 was described recently, the involvement of additional genes in pigment expression and the influence of pigmentation on the fitness of Cronobacter spp. have not been investigated. Thus, the aim of this study was to identify further genes involved in pigment expression in Cronobacter sakazakii ES5 and to assess the influence of pigmentation on growth and persistence under conditions of environmental stress. A knockout library was created using random transposon mutagenesis. The screening of 9,500 mutants for decreased pigment production identified 30 colorless mutants. The mapping of transposon insertion sites revealed insertions in not only the carotenoid operon but also in various other genes involved in signal transduction, inorganic ions, and energy metabolism. To determine the effect of pigmentation on fitness, colorless mutants (ΔcrtE, ΔcrtX, and ΔcrtY) were compared to the yellow wild type using growth and inactivation experiments, a macrophage assay, and a phenotype array. Among other findings, the colorless mutants grew at significantly increased rates under osmotic stress compared to that of the yellow wild type while showing increased susceptibility to desiccation. Moreover, ΔcrtE and ΔcrtY exhibited increased sensitivity to UVB irradiation.Cronobacter spp. (formerly Enterobacter sakazakii) are opportunistic food-borne pathogens that cause rare but life-threatening cases of meningitis, necrotizing enterocolitis, and septicemia in neonates (7, 30, 39, 40). While the pathogen appears to be ubiquitous, powdered infant formula (PIF) has been implicated as the main source of Cronobacter infection, necessitating effective means of both detecting this organism and preventing contamination in the PIF production environment (14, 26, 40).Although white strains have been observed occasionally, the production of yellow pigment on tryptic soy agar (TSA) is still one of the key discriminative criteria in the identification of presumptive Cronobacter spp. isolates via the ISO/TS 22964 standard protocol (3, 6, 11, 25). Studies of which colorless or cream-white strains of Cronobacter spp. (formerly Enterobacter sakazakii) were identified have reported prevalence rates of 8, 13, and 21.4% (6, 11, 24).The pigment''s carotenogenic nature recently was identified in Cronobacter strain ES5 on a molecular and chemical level (31). Carotenoids are known to stabilize cellular membranes and influence membrane fluidity (13, 22, 48). Functioning as antioxidants, carotenoids scavenge reactive oxygen species (37, 54, 55). Moreover, pigments play a role in the survival of bacteria in harmful environments and have been found to increase the virulence of pathogens such as Staphylococcus aureus and Erwinia chrysanthemi (32, 33, 44, 55). In Cronobacter strain ES5, a gene cluster comprised of seven genes (crtE-idi- crtXYIBZ) was found to be responsible for carotenoid biosynthesis (31). While the study mentioned above identified the operon responsible for carotenoid production, the involvement of other genes in pigment expression has not been investigated.Because no research exists on the influence of pigmentation on the fitness and persistence of Cronobacter spp., the potential implications of failing to detect colorless strains of this organism in the PIF production environment are difficult to assess. Thus, the aim of this study was to further describe the genetic basis of the pigmented phenotype of Cronobacter strain ES5 by isolating and characterizing isogenic white mutants via random transposon mutagenesis and subsequent sequencing, and to identify the impact of pigmentation on persistence and growth under conditions of environmental stress by comparing white mutants to the yellow wild type in a variety of growth and inactivation experiments, a macrophage assay, and a phenotype array.