Article Figures & Data
Figures
- Figure 1. Taxonomic analysis of mscL and arfA prokaryotic organisms.
(A) Cladogram of prokaryotic organisms obtained from NCBI representative and reference complete genome/proteome dataset, branches are coloured according to phyla/class, inner-ring indicates presence-absence of mscL and arfA genes, middle-ring indicates intergenic localisation of mscL and arfA genes and outer ring indicates taxonomic annotation. (B) Genomic co-localization and taxonomic annotation within mscL–arfA-containing organisms. The species are clustered according the mscL and arfA intergenic distance of x nucleotides: overlap (x ≤ 0), proximal (0 < x ≤ 110), and distal (x > 110). See Supplemental Data 1 for genomic and taxonomic data.
- Figure S1. Taxonomic analysis of bacteria in which only arfA, mscL, or neither of them are present.
See Supplemental Data 1 for genomic and taxonomic data.
- Figure 2. mscL and arfA proximal gene arrangement and expression.
(A) Convergent tail-to-tail gene organisation of mscL and arfA in the genome of E. coli, including the mscL 3′ UTR (+1 to +64 nucleotides) and the complementary arfA 3′ CDS. (B) Steady-state level of mscL RNA using qRT-PCR in wt, arfA-deleted (ΔarfA), and full-length arfA restored (pFL) E. coli cells grown in minimal media with an osmolality of 215 mOsm. (C, D) sfGFP expression driven by PmscL (C) and ParfA (D) in wt and ΔrpoS strains during cell growth in minimal medium (215 mOsm) at exponential and stationary phases, respectively. The data are shown as relative RNA abundance calculated from Ct values of detected mscL normalised to the transcript level of most stable housekeeping genes (see the Materials and Methods section), and as relative fluorescence units (RFU) normalised to OD600 (RFU/OD). The error bars represent the SD of at least three biological replicates; multi-comparison ANOVA analysis was performed (P < 0.001***). ns, no significance; a.u., arbitrary units.
- Figure S2. Design of arfA- and mscL-deleted (ΔarfA) strains.
The 3′ region of arfA CDS (70 nucleotides) was maintained in ΔarfA strains, as the genomic region is a part of mscL 3′ UTR. The kanamycin selection cassette (kanR) was inserted in the opposite orientation to the target gene to avoid read-through transcription. The direction of transcription of genes is indicated by the arrows.
- Figure 3. Transcriptional and post-transcriptional control of mscL and arfA expression.
(A, B) sfGFP expression driven by mscL (A upper panel) and arfA promoter (B upper panel) and steady-state transcript level of mscL (A lower panel) and arfA (B lower panel) measured in wt, arfA, and mscL-deleted (ΔarfA and ∆mscL) E. coli strains grown under different osmotic conditions in minimal media (215 and 764 mOsm). Samples were collected during exponential growth. The data are shown as relative fluorescence units (RFU) normalised to OD600 (RFU/OD) and as relative RNA abundance calculated from Ct values of detected mscL or arfA normalised to the transcript level of most stable housekeeping genes. The error bars represent the SD of at least three biological replicates; multi-comparison ANOVA analysis was performed (P < 0.01**, < 0.001***, < 0.0001****). a.u., arbitrary units.
- Figure S3. Effect of media osmolality and trans-translation upon mscL and arfA expression.
(A) Representative E. coli growth curve at 30°C in rich media (TB) and the decline in media osmolality (ΔmOsm) caused by the cell growth (OD600). (B, C, D) Relative mscL and arfA RNA abundance (RA) measured in strains grown in TB media pre (∆mOsm = 0, OD600 ≈ 4) and post (∆mOsm∼70, OD600 ≈ 12) drop in osmolality for (B) wt, (C) wt and arfA deleted (∆arfA), and (D) wt and smpB deleted (∆smpB) E. coli K12 strains. Ct values were normalised to the RNA abundance of the most stable housekeeping genes of E. coli. (B, C, D) The error bars represent the SD of at least three biological replicates; unpaired t test analysis was performed between pre versus post osmolality drop (B) and wt versus gene-specific deleted strain (C, D) (P < 0.05*, < 0.01**, < 0.001***, < 0.0001****). ns, no significance; a.u., arbitrary units.
- Figure S4. Analysis of mscL RNA and recombinant protein localisation in E. coli BL21(DE3)::mscLHis.
(A, B) Relative mscL abundance (RA) and localisation of recombinant sfGFP (B) detected in E. coli BL21(DE3) wt and::mscLHis cells grown in TB media pre (∆mOsm = 0, OD600 ≈ 4) and post (∆mOsm∼70, OD600 ≈ 12) drop in media osmolality. PP, periplasmic fraction; Ext, extracellular fraction. (C) Relative arfA abundance (RA) measured in E. coli BL21(DE3)::mscLHis cells grown in TB media post drop in osmolality (∆mOsm∼70, OD600 ≈ 12) in absence or presence of IPTG. (A, C) Unpaired t test analysis was performed in (A, C). (P < 0.05*). a.u, arbitrary units.
- Figure 4. mscL regulation mediated by arfA sRNA controls MscL protein level and excretion activity.
(A) Extracellular localisation of sfGFP expressed as %ECP (RFU media/[RFU intracellular+RFU media]) (upper panel), the corresponding mscL transcript abundance (middle panel), and the MscL protein levels (lower panel) from E. coli BL21(DE3)::mscLHis wt and arfA-deleted (ΔarfA) strains expressing sfGFP, after 15 h of growth in rich media (ΔmOsm ∼ −100). MscL Western blot signal was normalised to the signal of RNAP β-polymerase (internal loading control), detected with the anti-β-pol antibody. (B) The predicted stem-loop at 3′ arfA CDS showing the two RNaseIII cut sites at nucleotide position 165 and 189. The arfA sRNAs are shown in red and green. (C) Schematic representation of the arfA variant pCAN constructs used for the rescue experiments. (C, D) mscL transcript abundance in wt and arfA-deleted (ΔarfA) strains bearing an empty pCAN plasmid or one of the constructs in panel (C), grown in minimal media (215 mOsm). (D) Analysis of recombinant protein extracellular localisation (upper panel) and mscL abundance (lower panel) in wt and arfA-deleted (ΔarfA) E. coli BL21(DE3) bearing an empty pCAN plasmid, and arfA-deleted E. coli BL21(DE3) encoding full-length arfA (pFL) or arfA sRNA (psRNA) during growth in rich media. Samples were collected after osmolality drop (∆mOsm ∼70). A representative SDS–PAGE of the media fractions is shown as inset. (A, D, E) The error bars represent the SD of three biological replicates, statistical analysis was performed by unpaired t test (A, D), and by one-way ANOVA multi-comparison test (E). (P < 0.05*, P < 0.01**, < 0.001***, < 0.0001****). ns, no significance statistic; a.u., arbitrary units.
- Figure 5. Effect of arfA and RNaseIII upon mscL transcript stability and expression.
(A) Effect of arfA on the stability of mscL RNA determined by plotting the relative abundance of mscL versus time post rifampicin treatment. Total RNA was isolated from parental (wt) and arfA-deleted (ΔarfA) E. coli K12 cells. The abundance of M1 gene (RNA component of RNaseP) was used as internal standard. Half-life in minutes was determined by one phase decay non-linear fit in GraphPad Prism (version 9). (B, C) Steady-state level of mscL RNA in a nonfunctional RNaseIII genetic background (Δrnc and ΔrncΔarfA) versus wt (B), and in ΔrncΔarfA bearing either empty plasmid (pCan), expressing arfA sRNA (pCan-sRNA), or arfA FL (pCan-FL) (C). (D upper panel) Cleavage assay of in vitro transcribed mscL_FL and arfA RNAs incubated with E. coli RNaseIII. The red asterisks indicate the known sRNAs released by the cleavage of arfA and the major cleavage product released from mscL_FL. (D lower panel) Northern blot analysis of the mscL_FL cleavage reaction using 3′ UTR mscL-specific biotin labelled probe (spanning from nucleotide at position +13 to +38). IRDye streptavidin antibody was used to detect the signal, and imaging was performing using an Odyssey CLx instrument. Statistical analysis was performed by unpaired t test. The error bars represent the SD of minimal three biological replicates. (P < 0.001***). a.u., arbitrary units.
- Figure S5. Analysis of arfA RNA.
Native and episomal arfA relative abundance (RA) measured in wt and ∆RNC∆arfA E. coli cells, bearing an empty and pCan-FL palsmid, respectively, grown in LB media.
- Figure S6. The predicted secondary strucure of mscL 3′ UTR and arfA 3′ CDS.
(A) The mscL 3′ UTR stem-loop secondary structure was predicted using the RNA Vienna RNAfold web server submitting the mscL 3′ UTR sequence between the stop codon (UAA) and +70 nucleotide. (B) The arfA 3′ CDS stem-loop secondary structure was predicted using the RNA Vienna RNAfold web server submitting the arfA CDS sequence between nucleodite 144 and stop codon (UAA). The complementary region between arfA sRNA released by the cleavage of RNAseIII and mscL 3′ UTR is shown in red and green, respectively.
- Figure S7. RNaseIII in vitro assay.
(A, B) RNaseIII cleavage assay with the in vitro synthesized transcripts representing the (A) full-length transcript (mscL_FL) including the 3′ UTR and (B) a truncated CDS only transcript (mscL_TAA). Cleavage reactions were incubated for 7.5, 15, 30, and 60 s at 37°C. The red dashed box highlights the ∼25 nt sRNA released from mscL_FL, but not from mscL_TAA. arfA transcript (arfA), which releases 24 and 30 nt sRNAs after cleavage by RNAseIII, was used as positive control.
- Figure 6. Proposed regulation of mscL and arfA expression, antisense arfA mechanism, and the impact upon MscL-dependent protein excretion of CPs.
(A) Regulation of mscL and arfA expression at the transcriptional and post-transcriptional level. mscL promoter activity is stimulated under the condition of high external osmolality (Osm), stationary phase growth, and the stress sigma factor (σS/RpoS). arfA is constitutively transcribed under the regulation of σ70/RpoD sigma factor. No evidence of cross-regulation between mscL and arfA expression at the promoter level was observed in our experiments. arfA asRNA is produced upon cleavage of arfA transcript by RNaseIII, which is more active under low osmolality (40). mscL transcript levels are post-transcriptional down-regulated by the presence of the arfA asRNA, resulting in a synergistic effect in combination with the low osmolality. However, RNaseIII processing of mscL transcript within the 3′ UTR (demonstrated here in vitro) would avoid this antisense-mediated down-regulation. arfA transcript levels are down-regulated by the trans-translation ribosome–rescue system (tmRNA), and up-regulated when tmRNA is overloaded. (B) Regulation of MscL gating and excretion activity. MscL channel gating is regulated by lateral force induced by turgor pressure within the membrane (41) and by the quaternary protein aggregation (32). During hyperosmotic stress, MscL protein levels are increased, and MscL channels are present in aggregate/ungated cluster–like forms. Under hyposmotic stress condition, MscL protein levels are decreased, and therefore MscL channels are less clustered/sub-states, and low ECP occurs. During the combination of hyposmotic and translational stress, MscL protein level is further decreased which leads to the increase of MscL channel monomers which are capable of fully gating to enable high levels of ECP. During the combination of hyposmotic and translational stress, but in the absence of ArfA (ΔarfA), intermediate numbers of MscL channels are present and moderate ECP occurs.
Supplementary Materials
Supplemental Data 1.
[LSA-2023-01954_Supplemental_Data_1.xlsx]Full_genome_leve: The 3822 prokaryotic proteomes recovered from NCBI on 15/3/22 with full taxonomic annotation. Intergenic_analysis: Metadata and the calculated intragenic distance between mscL and arfA genes found in the same genome. arfA_synteny: Metadata and the calculated integenic distance between arfA and other co-located genes.
In this Issue
Related Articles
Cited By...
- No citing articles found.