A Novel LysR Family Factor STM0859 is Associated with The Responses of Salmonella Typhimurium to Environmental Stress and Biofilm Formation

Salmonella is a gram-negative intracellular pathogenic bacterium (Richardson et al. 2018), widely present in contaminated animal-derived foods or drinking water. This bacterium frequently infects humans due to ingestion of contaminated foods (Jajere 2019). The infection of this zoonotic pathogen will result in various clinical symptoms, which are characterized by diarrhea, abdominal pain, and vomiting. Especially in immunocompromised individuals (e.g., infants, children, or the elderly), Salmonella can cause serious syndromes and even death, leading to serious global food safety and public health issues (Zishiri et al. 2016).

As a representative strain of Salmonella enterica subsp. enterica, Salmonella Typhimurium (ST) can survive under unfavorable environmental conditions, such as dryness, extreme temperatures, antibiotics, and disinfectants. What is more, it can form a biofilm (BF) (Borges et al. 2018), which enables ST to acquire the capability of long-term survival in the contaminated eggs, meat, milk, and other animal-derived food sources, causing frequent occurrences of human food-borne salmonellosis (Lamas et al. 2018; Rohren et al. 2019). Currently available studies have shown that ST needs several transcriptional factors to regulate the expression of specific genes to adapt to the complex and changeable environments to withstand various adverse environmental stresses (Suar and Ryan 2015).

Among the transcriptional factors found in prokaryotes, LysR is an important transcription factor family, which was proved to be related to bacterial quorum sensing, response to environmental stress, biofilm (BF) formation, and regulation of virulence (Gebhardt et al. 2020). Through bioinformatics analyses, we have revealed that STM0859 protein is a novel member of LysR transcription factor family of ST. However, so far, the regulatory roles of STM0859 in ST response to environmental stress and BF formation remain unclear.

The main purpose of this study was to clarify the regulatory role of a novel transcription factor STM0859 of the LysR family in response to environmental stress and BF formation. Using λ-Red homologous recombination technology, we knocked out the STM0859 gene and carried out the detection of responses to environmental stress, BF formation capability, and the expression levels of related genes of STM0859 gene-deficient strains, aiming to provide new insights into the regulatory mechanism of STM0859 for the response to environmental stress in ST.

As shown in Fig. 1, STM0859 protein had a characteristic helix-turn-helix (HTH) DNA-binding and LysR substrate-binding domains (Fig. 1A). Besides, STM0859 protein was composed of α-helices, β-turns, and random coils, which formed a cage-like shape in 3D structure (Fig. 1B).

Fig. 1.

PCR showed that the homogeneous recombination strain SL1344-pKD46 was successfully constructed (Fig. S1A). The target fragment was amplified with R3/R4 primer and transferred into SL1344-pKD46 to generate ST-ΔSTM0859 strain (Fig. S1B). PCR verification of ST-ΔSTM0859 strain carrying pCP20 plasmid was performed with the primer pair R7/R8 (Fig. S2A). The fragments of 1,146 bp and 220 bp were amplified from ST-SL1344 and ST-ΔSTM0859 strains, respectively (Fig. S2B). The constructed ST-ΔSTM0859 gene-deficient strain was verified by DNA sequencing, and the complementation strain ST-ΔSTM0859/STM0859 was confirmed by PCR (Fig. S3).

The growth of ST-SL1344, ST-ΔSTM0859 and ST-ΔSTM0859/STM0859 were consistent under 37°C culture condition. There was no significant difference in growth rate between the three strains in the stress environment of 4% NaCl, 1 mM H₂O₂, 0.3% cholate, and pH 10 (Fig. 2), indicating that the deficiency of STM0859 gene did not affect ST growth under the stressful environmental conditions, such as alkaline, high salt, oxidation and ethanol stresses. However, as compared with that of ST-SL1344, the growth rate of ST-ΔSTM0859 in the acidic stress environment of pH 4 (Fig. 2B), and the movement ability (Fig. S4) of the semi-solid culture base were significantly reduced (p < 0.01, Fig. 3), indicating that the STM0859 gene deficiency can lead to a decrease in ST’s acid resistance.

Fig. 2.

Fig. 3.

BF detection indicated that ST-SL1344, ST-ΔSTM0859, and ST-ΔSTM0859/STM 0859 strains could form BF at 30 h and 48 h. However, the BF growth ability of ST-ΔSTM0859 was significantly declined than that of ST-SL1344 and ST-ΔSTM0859/STM0859 when observed under the inverted microscope (LEICA, Germany) (Fig. 4A). There was a significant difference in BF formation between ST-ΔSTM0859 and ST-SL1344 (p < 0.01, Fig. 4B), indicating that STM0859 can promote BF formation.

Fig. 4.

The transcription levels of the genes rcsB and rpoS in ST-ΔSTM0859 were significantly reduced when compared with those of ST-SL1344, while the transcription levels of these genes mentioned above were restored in ST-ΔSTM0859/STM0859 (Fig. 5), indicating that the STM0859 gene deficiency reduced the expression levels of genes (rcsB and rpoS) related acid stress and BF formation in ST.

Fig. 5.

The pET-STM0859 recombinant vector was successfully constructed by double enzymatic digestion with EcoR I. and Xho I (Fig. S5). After being induced by IPTG, SDS-PAGE and Western blot confirmed that the recombination protein STM0859 with a molecular weight of 48.6 kDa was obtained (Fig. S6). The results of the gel retardation assay (EMSA) showed that the DNA fragment of the rcsB gene promoter (Fig. 6A and 6B) displayed apparent band retardation after being bound by STM0859 protein (Fig. 6C). In contrast, the migration of the DNA fragment of the rcsB gene promoter with the unrelated protein BSA (control group) was not retarded (Fig. 6D), indicating that the STM0859 protein could specifically bind with the promoter of rcsB gene.

Fig. 6.

As an important foodborne pathogen, ST can infect humans through animal food and cause poisoning, posing a serious threat to food safety and public health. Moreover, ST can survive in stressful environments such as low temperature, hyperosmolarity, and acidity, and form biofilms under certain conditions to resist unfavorable internal and external environments (Kim et al. 2013; Ramachandran et al. 2016). It has been proven that many factors are associated with the responses of Salmonella Typhimurium to environmental stress and biofilm formation (Eran et al. 2020).

Available studies have confirmed that the transcription factor is an essential regulatory molecule in ST, regulating virulence and environmental stress-related gene expression (Song et al. 2020). Among them, the members of LysR family are the transcription factors widely found in prokaryotes (Yang et al. 2020), playing important regulatory roles in the life activities of bacteria. As a LysR transcription factor family member, ST-STM0859 contains a DNA binding domain and an HTH-18 domain consisting of a helix-turn-helix in molecular structure, similar to that of E. coli and Acinetobacter. Moreover, sequence analysis of the STM0859 gene showed that this gene is highly conservative among ST strains (data not shown).

It has been shown that there are significant differences in the expression levels of LysR family proteins in different growth environments, suggesting that they are involved in the regulation of the bacterial responses to environmental stresses (Zhang et al. 2019). (Srinivasan et al. 2013) revealed that oxyRKP was involved in regulating the response to oxidative stresses, drug resistance, and virulence of Klebsiella pneumoniae. (Fragel et al. 2019) confirmed that ST LysR transcription factors OxyR, SpvR, LeuO, CysB were involved in the regulation of oxidative stress and toxicity. (Fu et al. 2019) revealed that four LysR-type transcriptional regulator family proteins (LTTRs) were related to antibiotic resistance in Aeromonas hydrophila. Herein, we confirmed that the acid stress tolerance and BF formation ability were significantly reduced after deleting the ST STM0859 gene, suggesting that STM0859 was involved in regulating acid stress tolerance and BF formation of ST.

The current researches have found that Salmonella has evolved a complex acid-resistant mechanism involving the regulation of transcription factors and the synthesis of acid shock proteins in the acid stress environments (Lang et al. 2020). When ST infects host cells, the first pressure is the host’s stomach acid (Lee and Kim 2017). Therefore, sensing and responding to pH changes is critical to the survival of ST. It has been proven that fur, rcsB and rpoS play important roles in modulating the adaptability of Salmonella Typhimurium to environmental stresses such as low temperature, hyperosmolarity or acidity, and biofilm formation (Knudsen et al. 2014; Leclerc et al. 2017). Here, we confirmed that the transcription levels of genes rcsB were significantly reduced in the STM0859 gene deficiency strain.

Based on the analysis of the transcription levels of those genes related to acid response and biofilm formation using qRT-PCR, we further explored the interaction between the STM0859 protein and the promoter DNA fragment of rcsB gene whose expression was significantly reduced. It was confirmed that the STM0859 protein could modulate the mRNA level of the rcsB gene by binding with its promoter. Given that rcsB plays an important role in the adaptive response to acid stress (Lehti et al. 2012); thus, it is reasonable to speculate that the STM0859 protein can indirectly regulate the response to acid stress and BF formation through the interaction with the promoter of the rcsB gene.

However, the acid stress response is a highly complex process in Salmonella, which is involved acid resistance systems and a complicated regulation network (Kenney 2019). The mechanism insights into the regulation of acid stress response at the molecular level will contribute to developing novel antibiotics to deal with Salmonella Typhimurium infection (Zhao and Houry 2010).

Taken together, this study confirmed that the novel LysR family factor STM0859 was involved in the acid stress tolerance and BF formation of ST. Moreover, the STM0859 could bind the promoter of the rcsB gene and alter its expression, thus indirectly modulating the response to acid stress and BF formation of ST.