Inactivation of the Crp/Fnr Family of Regulatory Genes in Listeria monocytogenes Strain F2365 Does Not Alter Its Heat Resistance at 60°Ct

A surprising facet of the Listeria lnonocytogenes genorne is the presence of 15 genes that code for regulators in the Crp/ For family and include the virulence regulator PrfA. The genes under the transcriptional control of these regulators are currently undetermined, with the exception of some genes controlled by the major virulence regulator PrfA. Using 12 strains of L. monocvtogenes, each with an inserted gene cassette that interrupts and renders nonfunctional a different L. monocvtogenes strain F2365 CrplFnr regulator, we heat challenged each strain at 60°C with an immersed-coil heating apparatus, modeled the survivor data to calculate the underlying mean and mode of the heat resistance distribution for each strain, and compared the thermal tolerance of each mutant to the wild-type strain to determine if any of the Crp/Fnr mutants demonstrated altered heat tolerance. All 12 of the Crp/Fnr mutant strains tested had heat resistance characteristics similar to the wild-type strain (P > 0.05), indicating that mutations in these Crp/Fnr genes neither increased nor decreased the sensitivity of L. tn000cvto,qenes strain F2365 to mild heat. The availability of whole genome sequencing data for at least four strains of Listeria inonocyrogenes has created opportunities for exploring unique genomie features for their potential to affect food safety and pathogenesis. One aspect of the whole genome sequence comparisons that we have been particularly interested in is the observation that L. rnonocvro genes has an especially large number of regulatory genes in the Crp/Fnr family (3, 8). In comparison. Escherichia co/i has three Crp/Fnr family regulators, and Bacillus subtilis has only one (4). The presence of so many of these regulatory genes indicates that they most likely play a significant role in L. Inonocvrogenes cellular function during some part of the organism's normal existence. Since the roles of these regulators, the genes that they target, and the conditions under which they are exerting their effects are largely unknown, we used 12 L. /nonocvto genes strain F2365 Crp/Fnr mutants, created through site-specific recombination, to begin studying the behavior of these knockout mutants. These isogenic mutants are being used in comparative studies in an effort to determine if there are phenotypic differences between any of the mutants and the parent strain, which would provide an indication of the functional role for these regulators in Listeria. A previous study indicated a possible oxidative stress response function ° Author for correspondence. Tel: 215-233-6678-. Fax: 215-233-658; E-mail: [email protected]. t Mention 01 trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the VS. Dcpartiiieist of Agriculture. for two of these regulators (11). We chose not to study three of the Crp/Fnr homolog mutants, since one was not able to he integrated (II), one was found to be unstable, and the third, pj'A, has been extensively studied elsewhere. PrfA has been previously shown to he a primary regulator of virulence gene expression in L. monocvlogenes (12) and has also been implicated in the activation and repression of various other genes involved in responding to stress (7). The Crp/Fnr family of regulatory proteins has been recognized as controlling a variety of physiological functions that include aspects of metabolism, catabolism, virulence, and responses to environmental stress (4). Some Crp/Fnr regulators are directly involved in responses to oxidative stress (4), while others (i.e., PrfA) have been implicated in the indirect regulation of genes involved in responding to osmotic stress, cold stress, and other general stresses (7). Since thermal treatments are a commonly used means of bacterial inactivation in foods, we thermally challenged the 12 mutants and the wild-type strain to determine if any of the Crp/Fnr knockout mutants exhibited altered resistance (either increased or decreased) to thermal inactivation and therefore have the potential to serve as targets for food safety interventions. MATERIALS AND METHODS Bacterial strains and culturing. L. /notiocvtogenes strain F2365 (serotype 4h, food isolate, 1985 Mexican-style cheese outbreak) (5) was originally obtained from the Centers for Disease Control and Prevention (Atlanta, Ga.). We previously constructed single transposon mutants for 12 of the 15 known L. tn000cytoJ . 1-v'd I'rol.. \ ol. (,1) ',,. I I lll..\! RIHI \N(I-. ()I ( kl'.I \N \lt IAN I 79 TABLE]. Average modeled heat resistance distribution means and confidence levels (CL) associated with comparisons of the population of cells thermal/v challenged at 60°C to the control L. monocvtogefleS Heat resistance F2365 Crp/Fnr avg distribution Difference of avg Strain family gene locus mean (mm) mean (from control) P° Lower CL" Upper CL5