L. monocytogenes strain EGD-e was used as the reference strain. All Escherichia coli strains were grown at 37°C in LB medium (Oxoid Ltd, Basingstoke, United Kingdom), and all L. monocytogenes strains were grown at 37°C in BHI broth (Oxoid Ltd). E. coli DH5α was used for transformation, and E. coli BL21 was used for protein expression. When needed, the following antibiotics were added to E. coli or L. monocytogenes cultures: 100 µg/mL ampicillin, 10 µg/mL chloramphenicol, or 50 µg/mL kanamycin. Primers were purchased from Tsingke (Tsingke Biotechnology Co., Ltd, Hangzhou, China), and the sequences are shown in Table S1 .

The ΔdegU mutant was constructed by a two-step allelic exchange procedure using the pKSV7 shuttle plasmid as previously described (Cheng et al., 2021). The degU complementation strains were generated using the integrative plasmid pIMK2 as previously described (Zhang et al., 2020). The targeted degU gene was cloned into pIMK2 via a one-step cloning method and then electroporated into competent L. monocytogenes cells. The mutant and complement strains were verified by PCR and DNA sequencing.

A degU-lacZ fusions containing the promoter region of degU and the lacZ gene, was cloned into the Sac I and BamH I sites of the plasmid pIMK2. Then, the recombinant plasmid was introduced into the wild-type EGD-e and the ΔdegU mutant strain. All strains were grown overnight in BHI broth at 37°C, diluted 1:1,000 in fresh BHI broth, and grown at 43°C to an OD600 of 0.6. The collected culture was assayed for β-Galactosidase activity using a β-galactosidase (β-GAL) Activity Assay Kit(Micromethod; Sangon Biotech) according to the manufacturer’s specification.

The wild-type, ΔdegU mutant, and complement strains were grown in BHI broth overnight, diluted 1:1,000 in fresh BHI broth, and incubated at 43°C to an OD₆₀₀ of 0.6. Total bacterial RNA was extracted using the Bacteria Total RNA Isolation Kit (Sangon Biotech, China) and reverse-transcribed into cDNA using reverse transcriptase from TOYOBO. According to the manufacturer’s specifications, real-time qPCR was performed using an Mx3000P qPCR system (Stratagene-Agilent) and SYBR qPCR Mix (TOYOBO). All expression results were normalized relative to the housekeeping gene rpoB. Relative transcription levels were determined using the 2^−ΔΔCt method. RT-PCR was performed as previously described (Liu et al., 2018). The PCR products were electrophoresed on a 1% agarose gel and photographed using a SmartView Pro 2400 system (Major Science, USA).

The degU gene was amplified from genomic DNA of L. monocytogenes EGD-e with the primer pair PdegU-F and PdegU-R and cloned into the Nde I and Xho I sites of the expression vector pET30a. Then, the recombinant plasmid pET30a-degU was transformed into E. coli BL21 to express His6-DegU protein. The expression was induced by isopropyl-β-D-thiogalactopyranoside (0.5 mM) at 16°C for 5 h and purified by Ni-nitrilotriacetic acid (Ni-NTA) resin affinity chromatography. The purified proteins were confirmed by running the SDS-PAGE and stored at -80°C until use.

The recombinant protein DegU was obtained and phosphorylated according to previously described procedures (Goodman et al., 2020). DNA probes were purified with a Gel Extraction Kit (TIANGEN, China) and were labeled using the Biotin Labeling Kit for the EMSA (Beyotime, China). EMSA was performed with the Chemiluminescent EMSA Kit (Beyotime). Samples were analyzed by 4% non-denaturing polyacrylamide gel electrophoresis in 0.5 × TBE buffer. The gel was then transferred to a nylon membrane (Beyotime) followed by UV crosslinking. The bands were detected using the Chemiluminescent EMSA Kit (Beyotime).

DNase I footprinting experiments were carried out as previously described (Li et al., 2018). To prepare the fluorescent FAM-labeled probes, the promoter region of hrcA was PCR amplified using a 2× KOD One PCR Master Mix (TOYOBO) from the plasmid T-hcrA using primers containing 6-FAM at the 5′ end. The labeled probes (300 ng) were then mixed with purified DegU in a 40-µL reaction volume at 25°C for 30 min. Subsequently, 0.015 units of DNase I (Promega) and the reaction buffer were added, followed by incubation for 1 min at 37°C. The reaction was terminated by adding 140 µL of DNase I stop solution. Digested DNA samples were extracted with phenol-chloroform, and pellets containing DNA were resuspended in 30 µL of water. The results were analyzed using Peak Scanner software v1.0 (Applied Biosystems).

Data were analyzed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA) using two-tailed Student’s t-tests and are presented as means ± SD.

To verify whether DegU plays an important role in the heat tolerance of L. monocytogenes, we generated the in-frame deletion mutant strain ΔdegU and the complemented strain CΔdegU. When exposed to heat stress (43°C), the mutant strain ΔdegU showed a significant growth defect on BHI agar plates compared with the wild-type EGD-e and CΔdegU strains ( Figure 1A ). The promoter activity of P_degU-lacZ in the WT and ΔdegU strains had no obvious change under heat stress ( Figure S3 ), which indicated that DegU protein level was not elevated under heat stress. Previous studies have demonstrated that the Pta-AckA pathway plays a role in DegU protein phosphorylation (Gueriri et al., 2008a). The RT-qPCR results showed that the mRNA level of the pta and ackA genes, which are responsible for DegU activation, were significantly elevated under heat stress in the WT strain ( Figure 1B ). Collectively, these results strongly indicated that DegU contributes to the heat resistance of L. monocytogenes.

Many heat-shock-related genes have been previously identified, including hrcA, grpE, dnaK dnaJ, htpX, and groEL (Diamant and Goloubinoff, 1998; Arsene et al., 2000; van der Veen and Abee, 2010). In this study, we examined the link between DegU and the transcript levels of these genes using RT-qPCR. Under heat stress, the transcript levels of the hrcA, grpE, dnaK, and dnaJ genes were significantly lower in the ΔdegU mutant strain than in the wild-type EGD-e and complemented strains ( Figure 2A ), and the htpX and groEL genes were not changed (data not show). These four genes are adjacent to the L. monocytogenes chromosome ( Figure 2B ). To verify if they are co-transcribed as a polycistronic mRNA under heat stress, we performed RT-PCR across the hrcA-grpE, grpE-dnaK, and dnaK-dnaJ junctions. The results confirmed that the hrcA, grpE, dnaK, and dnaJ genes comprise an operon ( Figure 2B ). However, it should be recognized that RT-PCR is not sufficient to conclude that these genes constitute a complete operon, which may be short overlapping transcripts generated from internal promoters and terminators. These findings indicated that DegU is essential for the transcriptional regulation of the hrcA-grpE-dnaK-dnaJ operon.

To further investigate the mechanism involved in the DegU-mediated regulation of the hrcA-grpE-dnaK-dnaJ operon, we determined the binding site for DegU in the promoter regions of hrcA and dnaJ using EMSA, with the degU and groES promoters respectively serving as a positive or negative control for DegU binding. Marked band shifts were observed with the hrcA promoter region but not with that of dnaJ ( Figure 3A ). These EMSA data showed that DegU bound to the promoter region of hrcA but not to that of dnaJ. To map the precise binding sequence of DegU, a DNase I footprinting assay was performed using FAM-labeled probes. As shown in Figure 3B , a 56-bp sequence (3′-AACCGCACTATTTGACCTATTTTGACCAAACAATCCTACTTTAGTCTGAAATCGAG-5′) appeared to be protected from DNase I digestion by DegU binding. To identify the minimum sequence required for DegU binding, the fragment of the hrcA promoter region used for EMSA was divided into segments so that the specific binding site was confined within the remaining 50 bp ( Figure 3C ).

According to previous studies, the TSS (designated as +1) of hrcA was found to be located 45 bp upstream of its start codon and designated as C ( Figure 4 ) (Wurtzel et al., 2012). In addition, analysis of the hrcA promoter region revealed a putative −10 AATTTACCA box and a putative −35 AGTCAA box respectively located at 8 bp and 31 bp downstream of the TSS ( Figure 4 ). Furthermore, the specific DegU binding sequence was mapped from 52 to 101 bp upstream from the hrcA TSS ( Figure 4 ).