LuxR family transcriptional repressor YjjQ modulates the biofilm formation and motility of avian pathogenic Escherichia coli
Introduction
Avian pathogenic Escherichia coli (APEC), a type of extraintestinal pathogenic E. coli (ExPEC), can invade the respiratory tract of birds, causing acute septicemia and sudden death in poultry, which can lead to serious economic losses in the poultry industry (Breland et al., 2017; Moulin-Schouleur et al., 2007). APEC strains contain many transcriptional regulatory proteins belonging to different families, such as Gluconate operon transcriptional regulator (GntR) family, Luminescence regulator (LuxR) family, Multiple antibiotic resistance regulator (MarR) family, Mercuric-responsive transcriptional regulator (MerR) family, and Nitrogen regulatory protein C (NtrC) family. Among these, the family of LuxR-type transcriptional regulators, such as RcsB, BglG, YjjQ, LeuO, and DctR, are widely found in most Gram-negative bacteria, where they stimulate or inhibit the expression of virulence factors, and regulate various bacterial functions, including cell motility, biofilm formation, quorum sensing, and antibiotic production (Venkatesh et al., 2010). RcsB is the response regulator of the Rcs genes and is involved in motility, biofilm formation, and acid stress responses (Howery et al., 2016). The global regulator LeuO is involved in the biofilm formation and virulence of Acinetobacter baumannii, V. cholerae, and E. coli, regulating the expression of Salmonella enterica pathogenicity island 1(SPI-1) (Espinosa and Casadesús, 2014; Islam et al., 2021).
YjjQ, a member of the LuxR family of transcriptional regulators, consists of 242 amino acids and contains a helix-turn-helix (HTH) DNA binding domain (Doktorgrades and Dreck, 2013). In APEC IMT5155, YjjQ is involved in the iron uptake system through the regulation of iron uptake genes, such as ireA, fyuA, sitA, and chuA, and plays an important role in colonization in vivo (Li et al., 2008). In E. coli K-12 and uropathogenic E. coli (UPEC) strains, YjjQ binds to the flagellar master regulator flhDC operon and inhibits motility (Wiebe et al., 2015). A few putative YjjQ target loci encoding membrane proteins, such as bcs, ompC, gfc, kpsMT, and yfiRNB, further interfere with the binding of other transcriptional regulators (Doktorgrades and Dreck, 2013).
Biofilm formation contributes to the persistence of bacterial infection, motility, drug resistance, and resistance to external environmental stress (Barnhart and Chapman, 2006; Ciornei et al., 2010). Some E. coli strains require motility in the early stages of biofilm formation (Pratt and Kolter, 1998). In Y. enterocolitica and B. cereus, flagella and motility are necessary for biofilm formation under variable culture conditions (Kim et al., 2008; Liaqat et al., 2018). Bacterial flagella, as an important virulence factor, also participate in the virulence of pathogenic bacteria, including adhesion, colonization, biofilm formation, virulence factor secretion, and modulation of the immune system of eukaryotic cells (Duan et al., 2013; Erhardt and Dersch, 2015; Josenhans and Suerbaum, 2002; Merino et al., 2006). In APEC, the flagellar rotor protein FliG contributes to adherence and invasion of DF-1 cells, and virulence in a chick infection model (Yin et al., 2021). It has been shown that many transcription factors (TFs) in APEC directly or indirectly affect biofilm formation. McbR, a GntR/FadR family regulator, plays an important role in the biofilm formation of APEC and regulates the expression of the biofilm-associated genes bcsA, fliC, wcaF, and fimA (Yu et al., 2019). The transcriptional regulator PhoP can directly bind to the outer membrane protein tolC gene operon and affect APEC biofilm formation and pathogenicity (Li et al., 2020; Yin et al., 2019). Thus, it is essential to understand the TFs that influence the formation of biofilm to prevent it effectively.
RNA-Seq technology (also known as transcriptome sequencing technology), studies gene expression at the RNA level. Based on information about gene expression, the function of unknown genes can be inferred and the mechanism of action of specific regulatory genes can be revealed, allowing for the identification of cellular phenotypic attribution and disease diagnosis (Costa-Silva et al., 2017; Hrdlickova et al., 2017; Mutz et al., 2013). In this study, we demonstrated the role of YjjQ in the biofilm formation and motility of APEC. RNA-seq data investigated the mechanism that regulates YjjQ in APEC. YjjQ can alter the expression of a variety of biofilm formation and flagellar genes. Therefore, this study further investigated YjjQ as a repressor in APEC biofilm formation and motility, providing new data for the prevention and control of APEC.
Section snippets
Bacterial strains, plasmids, and growth conditions
The clinical strain APEC27 was isolated from the lung of a dead chicken with septicemic symptoms of colibacillosis in Anhui, China. The bacterial strains and plasmids used in this study are listed in Table 1. All bacteria were cultured at 37 °C in Luria-Bertani (LB) medium (Sangon, Shanghai, China) with antibiotics added as necessary at the following concentrations: ampicillin (100 μg/mL), chloramphenicol (30 μg/mL) (Sangon). Each liter of SOB plus glycerol (SOBG) medium contains 20 g of
Absence of YjjQ did not affect the growth of AE27
The schematic diagram of the strategy for deleting the yjjQ gene in AE27 is shown in Fig. 1A. The mutant strain AE27ΔyjjQ and the complement strain AE27ΔyjjQ-comp were successfully constructed by the lambda red recombinase system (Fig. 1B), and the growth curves of the mutant strain AE27ΔyjjQ and the complement strain AE27ΔyjjQ-comp in LB medium were similar to that of the wild-type AE27 (Fig. 1C).
Mutation of YjjQ increased pellicle and biofilm formation of APEC
We studied the role of transcription factor YjjQ on pellicle and biofilm formation in a SOBG
Discussion
Studies have shown that the transcriptional regulators YjjQ contribute to motility and biofilm formation in E. coli K12 and that YjjQ influences the colonization of APEC (Doktorgrades and Dreck, 2013). It is well known that biofilm formation is an important virulence factor for persistent bacterial infection. Bacteria attach to the medium via biofilm formation, and flagellar-mediated motility plays an important role in this process (Prigent-Combaret et al., 2000; Guttenplan and Kearns, 2013).
Declaration of Competing Interest
The authors have no competing interests.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (Grant No. 31772707, 31972644), the University Synergy Innovation Program of Anhui Province (Grant No. GXXT-2019-035).
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These authors contributed equally to this work.