Abstract
Cronobacter species can cause necrotizing enterocolitis and meningitis in neonates and infants, their infection is closely relevant to their responses to extreme growth conditions. In this study, the response of Cronobacter species to amino acid deficiency has been investigated. Four Cronobacter species formed smooth colonies when grown on the solid LB medium, but formed mucoid colonies when grown on the amino acid deficient M9 medium. When the mucoid colonies were stained with tannin mordant, exopolysaccharide around the cells could be discerned. The exopolysaccharide was isolated, analyzed, and identified as colanic acid. When genes wcaD and wcaE relevant to colanic acid biosynthesis were deleted in Cronobacter sakazakii BAA-894, no exopolysaccharide could be produced, confirming the exopolysaccharide formed in C. sakazakii grown in M9 is colanic acid. On the other hand, when genes rcsA, rcsB, rcsC, rcsD, or rcsF relevant to Rcs phosphorelay system was deleted in C. sakazakii BAA-894, colanic acid could not be produced, suggesting that the production of colanic acid in C. sakazakii is regulated by Rcs phosphorelay system. Furthermore, C. sakazakii BAA-894 grown in M9 supplemented with amino acids could not produce exopolysaccharide. Transcriptomes of C. sakazakii BAA-894 grown in M9 or LB were analyzed. A total of 3956 genes were differentially expressed in M9, of which 2339 were up-regulated and 1617 were down-regulated. When C. sakazakii BAA-894 was grown in M9, the genes relevant to the biosynthesis of exopolysaccharide were significantly up-regulated; on the other hand, the genes relevant to the flagellum formation and chemotaxis were significantly down-regulated; in addition, most genes relevant to various amino acid biosynthesis were also significantly regulated. The results demonstrate that amino acid deficiency has a global impact on C. sakazakii cells.
Highlights
Cronobacter species are food-borne opportunistic pathogens that can cause necrotizing enterocolitis and meningitis in neonates and infants (Feeney et al, 2014; Forsythe, 2017)
The ABC transporter complex LivFGHMJ can transport Leu, Ile, or Val, FIGURE 9 | (A) Transcriptomic analysis showed that most genes involved in flagellum formation and chemotaxis were down-regulated in C. sakazakii BAA-894 grown in M9 medium. (B)
The lower expression of genes emrA, emrD, emrE, and emrR encoding the multidrug resistance pumps C. sakazakii BAA-894 grown in M9 might be attributable to glucose, considering the increased antibiotic sensitivity of C. sakazakii BAA-894 grown in M9 does not change after supplemented with 5 mM amino acids (Figure 7)
Summary
Cronobacter species are food-borne opportunistic pathogens that can cause necrotizing enterocolitis and meningitis in neonates and infants (Feeney et al, 2014; Forsythe, 2017). Response of Cronobacter sakazakii to Amino Acid Deficiency than other Enterobacteriaceae to environmental stresses (Dancer et al, 2009), which might be associated with its pathogenesis (Alvarez-Ordóñez et al, 2014). The shortage of amino acids, the most important nitrogen source for bacteria, can lead to a coordinated regulation of metabolism, which is very important for the bacteria adapting to the new environment (Kuroda et al, 1999). In Escherichia coli, amino acid starvation results in the phosphorylation of the response regulator NtrC, which activates RelA (Shyp et al, 2012). Amino acid starvation can result in the coordinate inhibition of a variety of metabolic activities in E. coli, such as fatty acid and phospholipid biosynthesis (Podkovyrov and Larson, 1996). Understanding the responses of C. sakazakii to amino acid starvation might provide important information for C. sakazakii infection
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