Function and mechanism of action of the small regulatory RNA ArcZ in Enterobacterales

Abstract

ArcZ is a small regulatory RNA conserved in Enterobacterales It is an Hfq-dependent RNA that is cleaved by RNase E in a processed form of 55-60 nucleotides. This processed form is highly conserved for controlling the expression of target mRNAs. ArcZ expression is induced by abundant oxygen levels and reaches its peak during the stationary growth phase. This control is mediated by the oxygen-responsive two-component system ArcAB, leading to the repression of arcZ transcription under low-oxygen conditions in most bacteria in which it has been studied. ArcZ displays multiple targets, and it can control up to 10% of a genome and interact directly with more than 300 mRNAs in Escherichia coli and Salmonella enterica ArcZ displays a multifaceted ability to regulate its targets through diverse mechanisms such as RNase recruitment, modulation of ribosome accessibility on the mRNA, and interaction with translational enhancing regions. By influencing stress response, motility, and virulence through the regulation of master regulators such as FlhDC or RpoS, ArcZ emerges as a major orchestrator of cell physiology within Enterobacterales.

Keywords: ArcZ; RNase E; posttranscriptional regulation; sRNA; stress response; virulence.

FIGURE 1.

ArcZ, a cleaved sRNA that is highly conserved in its 3′ part. (A) Alignment of ArcZ sequence from Pantoea ananatis (NZ_CM012203), Escherichia coli (NC_000913.3), Salmonella typhimurium (NC_003197.2), Klebsiella pneumoniae (NC_009648), Photorhabdus laumondii (CP024901.1), Xenorhabdus nematophila (CP060401.1), Dickeya dadantii (CP002038.1), Pectobacterium carotovorum (CP051652.1), Yersinia enterocolitica (CP107102.1), Serratia marcescens (CP139958.1), Proteus mirabilis (CP045257.1), Erwinia amylovora (FN666575.1), Providencia alcalifaciens (CP084296.1), Edwardsiella tarda (CP084506.1), Shigella sonnei (CP026802.1), Citrobacter freundii (CP049015.1), Enterobacter cloacae (CP001918.1), and Cronobacter sakazakii (CP011047.1). Conservation score is plotted below, and the conserved region is colored in red. This alignment was carried out using ClustalW and Jalview (Thompson et al. 2003; Clamp et al. 2004; Troshin et al. 2011). The red squares correspond to known transcription starts. (B) Synteny analysis of chromosomal regions surrounding ArcZ was performed using AnnoView (Wei et al. 2024). The same genome accession numbers as A were used. (C) Model of ArcZ maturation in E. coli. The stem–loop represents the Rho-independent transcriptional termination site of arcZ. The 3′ region of ArcZ is recognized by Hfq through binding. RNase E cleaves ArcZ at a consensus sequence, producing a mature processed form of ArcZ that binds to target mRNA.

FIGURE 2.

ArcZ targets in Escherichia coli. Green arrows indicate activation, red arrows indicate repression, and black arrows indicate either translation or activity. CyaR is an sRNA capable of repressing the translation of nadE mRNA, which encodes an enzyme involved in NAD⁺ biosynthesis but is also capable of repressing the translation of rpoS mRNA. ArcZ degrades CyaR via RNase E, thereby increasing the translation of nadE and the availability of NAD⁺ and rpoS. ArcZ enhances the translation of rpoS mRNA, which induces the Gad pathway, leading to better acid stress resistance. Additionally, ArcZ reduces the translation of mutS mRNA, both directly and indirectly, by activating the translation of rpoS, which in turn transcribes SdsR. SdsR directly represses mutS translation. ArcZ also directly represses flhDC translation and competes with the McaS sRNA, which has a common flhDC mRNA pairing site with ArcZ. McaS activates flhDC translation, while ArcZ inhibits LPS modification by repressing eptB mRNA translation. Figure was created with BioRender.com.

FIGURE 3.

ArcZ targets in Erwinia amylovora. Green arrows indicate activation, red arrows indicate repression, and black arrows indicate either translation or activity. ArcZ plays a role in regulating the response to oxidative stress by modulating the levels of the antioxidant enzymes KatA and Tpx. The abbreviation NC stands for “Noncharacterized” protein. ArcZ modulates motility and biofilm formation through the control of flhDC and lrp translation. Figure was created with BioRender.com.

FIGURE 4.

ArcZ targets in Salmonella typhimurium. Green arrows indicate activation, red arrows indicate repression, and black arrows indicate either translation or activity. ArcZ represses the expression of STM3216, a potential chemoreceptor acquired through horizontal gene transfer. As previously described, ArcZ modulates the response to oxidative stress by repressing tpx mRNA. Additionally, ArcZ represses the translation of sdaC mRNA, which is involved in serine catabolism. ArcZ and FnrS inhibit the translation of hilD, which is responsible for activating the T3SS synthesis. However, fnrS is transcribed under anoxic conditions due to Fnr, whereas arcZ is transcribed under strong aerobic conditions. Therefore, the activation of T3SS is limited by the presence of oxygen. Figure was created with BioRender.com.

FIGURE 5.

Regulation of hexA/pecT by ArcZ in Dickeya and Photorhabdus. Direct and indirect targets of ArcZ are shown in Dickeya (green panel) and Photorhabdus (brown panel). Green arrows indicate activation, red arrows indicate repression, and black arrows indicate either translation or activity. In Dickeya, ArcZ inhibits the translation of pecT, which prevents the inhibition of rsmB transcription by PecT. RsmB is a small Hfq-independent regulatory RNA that binds to RsmA and prevents it from repressing the expression of T3SS and pectinases. In Photorhabdus, ArcZ inhibits the translation of hexA and thus enhances the production of secondary metabolites, which are essential for nematode symbiosis. Figure was created with BioRender.com.

FIGURE 6.

ArcZ's modes of action. ArcZ can activate the translation of mRNA (green panel). (A) ArcZ interacts with the 5′-UTR region of the rpoS mRNA and releases its ribosome-binding site (RBS), which is initially involved in a hairpin structure, allowing for the translation of rpoS. (B) Additionally, during the transcription of rpoS, ArcZ prevents the Rho termination factor from binding to the mRNA, thus blocking premature termination. ArcZ can also repress the translation of mRNA (red panel), such as with mutS or flhDC. (C) ArcZ can repress translation by binding to the RBS of the mRNA, preventing access of the ribosome. Additionally, it can bind upstream of the RBS, such as with pecT or flhD, forming a secondary structure that is incompatible with translation. (D) ArcZ can also bind to the coding region of the mRNA or sRNA, making it more susceptible to RNase such as with tpx and cyaR. Figure was created with BioRender.com.

FIGURE 7.

rpoS and flhD, two ArcZ targets conserved in Enterobacterales. Alignment of (A) rpoS mRNA sequence and (B) flhD mRNA sequence from Pantoeae ananatis, Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Photorhabdus laumondii, Xenorhabdus nematophila, Dickeya dadantii, Pectobacterium carotovorum, Serratia marcescens, Proteus mirabilis, Erwinia amylovora, Providencia alcalifaciens, Edwardsiella tarda, Shigella sonnei, Citrobacter freundii, Enterobacter cloacae, and Cronobacter sakazakii (genomes are the same as those used in Fig. 1). Conservation score is plotted below, and on this plot the interaction zone between ArcZ and rpoS/ArcZ and flhD shown in E. coli and E. amylovora is colored red. The interaction of E. coli ArcZ (in red) with rpoS and flhD mRNA and of E. amylovora ArcZ (in green) with flhD mRNA is shown (Mandin and Gottesman 2010; De Lay and Gottesman 2012; Schachterle et al. 2019b). The red squares correspond to ArcZ binding site regions conserved between E. coli and other bacteria. Alignments were performed using MUSCLE (Edgar 2004) and processed with Jalview (Clamp et al. 2004).

FIGURE 8.

Alignments of mutated arcZ alleles in Enterobacterales. A BLASTN analysis was conducted on the enterobacterales NCBI RefSeq genomes using the conserved nucleotide sequence that contains the mutation (A) G to A or (B) G to U at position 90, in comparison with the Escherichia coli MG1655 reference genome. The mutation is indicated by a red arrow and surrounded by a red rectangle. Alignments were performed using MUSCLE (Edgar 2004) and processed with Jalview (Clamp et al. 2004).