Exploration of factors in response to low acid tolerance using random mutagenesis in Cronobacter malonaticus

Cronobacter species are associated with rare but severe infections in newborns, and their tolerance to environmental stress such as acid stress has been described. However, the factors involved in low acid tolerance in Cronobacter are poorly understood. Here, a transposon mutagenesis approach was used to explore the factors involved in acid tolerance in C. malonaticus. Eight mutants from mutant library (n = 215) were successfully screened through a comparison of growth with wild type (WT) strain under acid stress (pH 4.0). Eight mutating sites including glucosyltransferase MdoH, extracellular serine protease, sulfate transporter, phosphate transporter permease subunit PstC, lysine transporter, nitrogen regulation protein NR (II), D-alanine-D-alanine ligase, glucan biosynthesis protein G were successfully identified by arbitrary polymerase chain reaction and sequencing. The biomass of biofilm of eight mutants were significantly reduced using crystal violet staining (CVS) compared with that of WT. furthermore, the more compact biofilms of WT was observed than those of eight mutants through scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Disassembly of biofilms appeared among mutants and WT strain from 48 h to 72 h through the increasing of dead cells and reduction of viable cells and exopolysaccharide. The study reveals the molecular basis involved in acid tolerance of C. malonaticus and a possible relationship between biofilm formation and acid tolerance, which provides valuable information for survival of C. malonaticus under acid stress.