Minimal transcriptional changes occur in C41(DE3) and C43(DE3) compared to their parental strain BL21(DE3).
We first wanted to investigate what changes occur in C41(DE3) and C43(DE3) compared to the parental strain BL21(DE3) in the absence of protein expression. Each strain was grown in rich media (Luria Broth, LB) to mid-log phase (OD600nm = 0.6) at 37 °C. RNA was then isolated and subjected to transcriptional analysis. Each experiment was performed in biological triplicate. RNA libraries were prepared and sequenced. The analysis was performed using RNAsik (26), mapping all reads to the reference strain BL21(DE3) CP001509.3, which was recently updated (27), and the analysis performed in Degust (28). The data quality was confirmed using Degust (Table S1). Using a log2 fold change (log2FC) ≥|1| and a false discovery rate (FDR) of less than 0.01 as the cut-off for significant differential expression, a total of 115 genes were differentially expressed in C41(DE3) from which 68 genes were upregulated, and 47 genes were downregulated (Supp Fig. S1a). In C43(DE3) a total of 239 genes were differentially expressed under the same conditions, where 140 genes were upregulated and 99 were downregulated (Supp Fig. S1b). Remarkably, both strains had the same proportion of genes upregulated (59%) and downregulated (41%) overall. C43(DE3) has twice as many genes differentially expressed compared to C41(DE3), however, there was a 33% overlap in upregulated genes (i.e. 52 of 156 unique genes) (Fig. 1a) and a 21% overlap in downregulated genes (i.e. 25 of 121 unique genes) demonstrating some similiarities between the two derivative strains (Fig. 1b).
Transformed strains, induced with IPTG.
Given the use of the strains for recombinant protein production, we assessed the gene expression profiles when the strains were transformed with a plasmid, pACYCDuet-1, and grown in the presence of the inducer IPTG. The pACYCDuet-1 plasmid is a derivative of the P15A miniplasmid (29). It has a copy number of ~10, carries the lacI gene to provide control over gene expression and a gene that confers chloramphenicol resistance for plasmid selection. We reasoned that the production of a membrane protein target would significantly influence the cell’s transcriptomic response. Therefore, theassessment was made that the host E. coli strains would not contain any specific “gene of interest” cloned into the plasmid, so as not to mask the features of C41(DE3) and C43(DE3) that optimize them for membrane protein expression.
Cells containing the pACYCDuet-1 vector were grown in LB growth medium (with chloramphenicol) to mid-log phase at 37 °C, before the addition of IPTG. Cells were then grown for a further two hours at 37 °C. At this point, the cells were collected and RNA was isolated and subjected to transcriptional analysis. Each experiment was performed in biological triplicate. All strains grew similarly before and after the addition of IPTG (Fig. 2a). To delineate between the two different experimental parameters, the strains in this experiment were named BL21(DE3)EV+IPTG, C41(DE3)EV+IPTG and C43(DE3)EV+IPTG.
RNA libraries were prepared and sequenced, the transcripts analysed with Degust, and the data quality assessed statistically (Table S3). Significant changes were observed with a total of 2018 genes identified as differentially expressed in C41(DE3)EV+IPTG where 1024 genes were upregulated and 994 genes were downregulated as defined by a change in expression of log2FC ≥1 and FDR ≥0.01. In C43(DE3)EV+IPTG a total of 1646 genes were differentially expressed under the same conditions where 827 genes were upregulated and 819 were downregulated (Fig. 2). Comparison of the differential gene expression in C41(DE3) EV+IPTG and C43(DE3) EV+IPTG shows largely similar expression profiles in volcano plots (Fig. 2b and 2c). Venn diagrams demonstrate the majority of genes that are differentially expressed are common to both of the strains: making up 60% of differentially expressed genes in C41(DE3) (1206 of 2018 unique genes) and 73% of genes in C43(DE3) (1206 of 1646 unique genes) (Fig. 2d and 2e). Despite this, there are no similarities or overall patterns with respect to the largest fold change of differentially expressed genes between C41(DE3) and C43(DE3), (Supp. Table S4).
Metabolism pathways are significantly changed in C41(DE3)EV+IPTGand C43(DE)EV+IPTG.
Many of the functional pathways appear to be very similar between C41(DE3) and C43(DE3). The differentially expressed genes in C41(DE3)EV+IPTG and C43(DE)EV+IPTG were classified according to their COG pathways (Fig. 3) and also using their KEGG annotations (Supp Fig. 2). Genes that were significantly changed in both C41(DE3)EV+IPTG and C43(DE)EV+IPTG were identified. The large majority of differentially expressed genes encode proteins involved in metabolism, particularly energy production and conversion (C) including TCA cycle genes (sucABCD), ATP biosynthesis (atpAGH) and respiration (nuoABCEFGIJKLM). A range of genes involved in amino acids metabolism were upregulated. These include genes in the biosynthetic pathways and metabolism of cysteine, methionine, tryptophan, tyrosine, phenylalanine, alanine, proline; amino acids utilized particularly for membrane protein biogenesis. We note that intermediates from TCA cycle also feed directly into the pathways for leucine, isoleucine and valine biosynthesis (30).
Other areas significantly upregulated include carbohydrate transport and metabolism (G), including melibiose transporters (melA, melB), trehalose/glucose metabolism (otsA, otsB, treC) and pyruvate metabolism (pykA) and inorganic ion transport and metabolism (P) inclusing taurine transport (tauA, tauD) and oligopeptide ABC transporters (oppB, oppC, oppD). Many of these genes encode membrane proteins, which we hypothesized might place demands to increased capacity in the membrane protein biogenesis pathway. In addition, activation of genes mediating transcription/translation processes (K) and cell wall/membrane/envelope biogenesis (M) are upregulated log2FC>2 with FDR<0.01 (Fig. 3). We find that in many of these pathways the same proportion of genes are being upregulated and downregulated concomitantly. This suggests there are global changes in play within specific pathways that are unique to the derivative strains possibly affecting their response to membrane protein biogenesis (Fig. 3).
Adaptations for inner membrane biogenesis in C41(DE3) and C43(DE3)
We discovered that genes encoding several molecular chaperones and components of the membrane biogenesis pathway are transcribed at higher levels in C41(DE3)EV+IPTG and C43(DE3)EV+IPTG compared to BL21(DE3)EV+IPTG (Table 1). In gram-negative bacteria, membrane protein biogenesis relies on protein translocases in the inner membrane (the SecYEG translocase for unfolded polypeptides or the TAT for folded proteins) and the outer membrane protein BAM, the core b-barrel assembly machinery and TAM,the translocation and assembly module of the b-barrel assembly machinery. Periplasmic intermediates are maintained by a series of chaperones and proteases. Proteins destined for the inner membrane rely on cytoplasmic chaperones and protease inhibitors to ensure the translocation pathway remains efficient. The schematic in Figure 4 illustrates genes involved in this pathway of protein targeting to the Sec translocon for membrane protein folding and assembly. Many of these genes are upregulated in C41(DE3) and C43(DE3), demonstrating how these derivative strains can manage an onslaught of gene expression that would otherwise be toxic to the cell (Table 1, Fig. 4) (13, 31).
Table 2 shows no significant changes were seen in genes involved in the membrane-embedded components of the SecYEG machinery, although there was some significant upregulation of some of the TAT translocon components (tatC, tatD, tatE). The TAM components of the outer membrane protein assembly machinery were downregulated, while the BAM components remained unchanged.
Genes encoding for the two chaperones of the Sec translocation pathway, SecA and SecB, are both upregulated as are molecular chaperones located in the cytoplasm (groEL, groES, dnaK and ybbN; Table 1) and periplasm (degP, degQ and fkpA; Table 2). . The increased abundance of these chaperones could provide capacity to collect nascent membrane proteins prior to engagement with the membrane translocases, and to assist in the folding of the domains of the membrane proteins that protrude into the cytoplasm and periplasm.
Several genes involved in polysaccharide biosynthesis were upregulated, which is essential for building the outer leaflet of the outer membrane surface (32). Genes in the retrograde phospholipid trafficking pathway mlaABCD were significantly downregulated. It remains untested but possible that these changes might reorganise membrane structure to be permissive for enhanced inner membrane protein accumulation.
Functionally unknown, unassigned and uncharacterized
Many of the genes that were differentially expressed are categorised as “functionally unknown” (S) using the COG annotation (Fig. 3), or as “unassigned” using the KEGG annotators (Supp Fig. 2). Since the COG annotations have not been updated for several years, we were interested in confirming the number of genes with still unknown function. The genes were compared to a recently published Y-ome: an updated list of every uncharacterized gene in E. coli K-12 MG1655 (33). From the total of 1024 genes upregulated in C41(DE3)EV+IPTG , 226 of these were assigned to group [S], the “uncharacterized” COG identifier (Supp Table S6). From these 226 genes, 138 remain uncharacterized evidenced by their presence in the E. coli MG1655 Y-ome list of uncharacterized genes. This accounts for 14-18% of all differentially expressed genes in C41(DE3)EV+IPTG and C43(DE3) EV+IPTG (Supp Table S6). . Further characterisation of these various genes will aid in the overall understanding of cellular responses to enhance membrane protein expression.
How do the genetic mutations in C41(DE3) and C43(DE3) affect their transcriptome profile?
The genomes of C41(DE3) and C43(DE3) are published confirming the known mutations present in the T7RNAP and also identifying several other changes (14). We were interested in determining if these mutations affected the expression of their corresponding genes. Both strains contain mutations in the lacUV5 promoter region of the T7RNAP that revert it back to a weaker form. A downregulation of T7RNAP is observed in all strains compared to their respective BL21(DE3) controls (Supp. Table S5). As discussed earlier the rbsD IS3 excision causes upregulation of the rbs operon.
Genomic sequencing of C41(DE3) identified four unique changes not passed onto C43(DE3) (14); Supp. Table S5). Of the three genes containing a single amino acid change, there is a significant upregulation of melB and yhhA in our analysis in both C41(DE3) EV+IPTG and C43(FDE3)EV+IPTG in comparison to BL21(DE3)EV+IPTG but no significant change in ycgO expression (Supp. Table S5). MelB is a sugar transporter of melibiose coupled with cation exchange (34) that has been shown to be affected by membrane composition of the inner, thus changes may merely reflect a cellular response to an altered membrane environment (35). YhhA is an uncharacterised protein that contains a signal sequence suggesting it localizes to the cell envelope; however, nothing more has been reported about this gene.
C43(DE3) contains mutations in the genes dcuS, fur, cydA, yibJ, yjcO, lon and lacI (14). The majority of mutations do not invoke any significant changes in their gene expression compared to the relevant BL21(DE3) controls. The gene encoding the ATP-dependent protease Lon, is significantly upregulated due to the excision of an IS4 element that restores expression of lon (Supp. Table S5) (14, 16). The Lon protease is associated with regulated protein degradation for the purpose of protein quality control (17, 18). A point mutation in the lac repressor, lacI present in the DE3 region of C43(DE3) results in the downregulation of lacI expression in C43(DE3) compared to BL21(DE3) albeit not significant according to our set parameters (log2FC -1.3, FDR 0.03; Supp Fig. S5). This mutation in C43(DE3) has previously been suggested to be less responsive to its inducer allolactose (13) and subsequently results in superior repression of the lac operon. In the presence of the vector pACYCDuet-1 and induction with IPTG, the lacI expression observed is masked by the contribution of the plasmid encoded lacI expression.