FruR-controlled antisense RNA -downregulation of isocitrate dehydrogenase in Escherichia coli

doi:10.21203/rs.3.rs-4854438/v1

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FruR-controlled antisense RNA -downregulation of isocitrate dehydrogenase in Escherichia coli

https://doi.org/10.21203/rs.3.rs-4854438/v1

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In E. coli, catabolite repressor activator (Cra) protein (formerly called FruR) is known to regulate the expression of many genes positively and negatively; this effect is modulated by intracellular levels of fructose-1-phosphate (F-1-P) and fructose-1,6-bisphopahate (F-1,6-bisP). In this paper, we report conditionally expressed antisense RNA corresponding to 101bp of isocitrate dehydrogenase (icd) gene (as-icd) under Cra (FruR) responsive promoter fruB (P_fruB as-icd construct denoted as pVS2K3) in E. coli K-12 (DH5α) and E. coli B (BL21) strains. Previously studies have shown that ICDH mutants failed to grow on glucose in absence of glutamate and accumulated citrate intracellularly. Hence, a conditional downregulation of icd gene could overcome this lethality and also help in understanding the flux towards citrate accumulation. Effect of P_fruB as-icd (pVS2k3) construct was monitored in E. coli K-12 (DH5α) and E. coli B (BL21) during growth on carbon sources wherein the fruB promoter is active (glucose) or repressed (glycerol). A 3–4 fold decrease in ICDH activity was observed in E. coli DH5α expressing pVS2K3 on glucose but P_fruB as-icd expression differed in E. coli BL21 on glucose. This alteration could be attributed to the anomalous Cra regulation seen in E. coli B strain which could be a crucial factor while choosing fruB promoter for expression studies.

Isocitrate dehydrogenase

conditional down-regulation

antisense RNA

catabolite repressor activator (Cra) protein

E. coli B & K derived strains

Escherichia coli have been genetically engineered so as to reapportion the carbon flux towards secretion of different low molecular weight organic acids. It is an extensively used model system to study the regulation of central carbon metabolism (Zhou et al. 2003; Wendisch et al. 2006). Over-expression of critical genes, an important means of manipulation of metabolic end-products has proved to be highly effective, for instance, in the over-production of succinate (Vemuri et al. 2002; Singh et al. 2011; Wang et al. 2011). Natural antisense RNA regulation is emerging as an important feature of gene regulation in bacteria operating in diverse physiological states (Georg 2011; Saberi et al., 2016). Down-regulation of expression of crucial genes by antisense RNA (asRNA) is a potential tool to modulate microbial metabolism. Several studies have demonstrated the effectiveness of asRNA strategy in prokaryotes (Coleman et al. 1984; Pestka et al. 1984; Ellison et al. 1985; Engdahl et al. 1997; Kernodle et al. 1997) and it was efficiently exploited for over-production of industrially important products (Desai and Papoutsakis 1999). AsRNA technology is considered advantageous than gene inactivation technology since complete loss of protein function is not achieved and as a result possible lethal or detrimental effects associated with gene knockout mutants are avoided. Moreover, expression of asRNAs could decrease target gene expression conditionally (Coleman et al. 1984; Yin and Ji 2002).

The present study tries to understand the possible metabolic changes under conditional downregulation of icd gene. NADP⁺-dependent isocitrate dehydrogenase (ICDH), encoded by icd gene, catalyse the conversion of isocitrate to α-ketoglutarate and CO₂ and regulates the TCA-glyoxylate bypass shift. Mutations within the icd gene result in lethality and manifest an auxotrophic dependency on glutamate, leading to the accumulation of citric acid in both E. coli K and B strains (Lakshmi and Helling, 1976; Helling 1995; Aoshima et al., 2003; Kabir and Shimizu, 2004).Conditional downregulation of icd gene under Catabolite repressor activator (Cra) repressed promoters could help in overcoming the lethal effect and the auxotrophic requirement for glutamate as observed in case of icd mutants and yet allow citric acid accumulation.

Catabolite repressor activator (Cra) protein represses the genes of glycolytic pathway and activates the genes of gluconeogenesis and glyoxalate pathways (Saier and Ramseier, 1996). Fructose-1-phosphate (F-1-P) and fructose-1,6-bisphosphate (F-1,6-P) counteract the function of Cra by binding to Cra protein and prevent it from binding to DNA. One of the Cra repressed promoters is that of the fruBKA operon which is involved in the catabolism of fructose (Ramseier 1996; Chavarria and de Lorenzo 2018). P_fruB is switched on when E. coli is grown on glucose or fructose and is repressed on glycerol in response to the low levels of F-1-P and F-1,6-P (Holms, 2001). The present study hypothesized the use of fruB promoter for conditional down-regulation of icd gene in E. coli K leading to citrate accumulation. Extending the study to E. coli B strain suggested that the differential Cra regulation background could play a crucial role while choosing PfruB promoter for expression studies in E. coli.

2.1 Microbial Strains and Plasmids. Table 1 lists all bacterial strains and plasmids exercised in this study. E. coli DH5α and E. coli BL21(λDE3) and E. coli BL21 were used as representatives of the K and B strains, respectively.

2.2 Media for growth and maintenance. E. coli DH5α, E. coli BL21 and E. coli BL21(λDE3) transformants were maintained on Luria Agar with ampicillin (50 µg/ml).

2.3 PCR primer design for construction of P_fruB as-icd chimera. Four primers were designed, analogous to the upstream and downstream regions of the targeted regions of icd gene and fruB promoter (Table 2). Sequence information of E. coli K-12 strain was used for generating primers. ECfruB L1 primer (5’cggaattcTGCTCATAACTTTACGGCTT 3’) carried an EcoR1 site at the 5’ end (bold highlighted in the sequence) followed by 20 nucleotides corresponding to − 158 to − 139 region of fruB promoter. The EC fruB R1 (5’ GTTCCGGCACAAGGCGGATAACTGGA 3’) primer contained 14 nucleotides corresponding to + 19 to + 33 region of icd gene (underlined in sequence) followed by 11 nucleotides corresponding to + 15 to + 5 of fruB gene. ECicd L1 (5’ ATGTTCCAGTTATCCGCCTTGTGCCGGAA 3’) primer contains 15 bp corresponding to atg + 5 to + 15 (15 nucleotides) of fruB gene at the 5’ region (bold highlighted in sequence) followed by 14 nucleotides corresponding to + 33 to + 19 nucleotides corresponding to icd gene. The EC icdR1 (5’ cgggatccTATCGCAGGACGCAAAC 3’) primer carried the BamH1 site (bold highlighted in the sequence) followed by 17 nucleotides corresponding to − 72 to − 56. Primers ECfruB R1 and ECicd L1 have 14 nucleotides corresponding to icd and 11 nucleotides corresponding to fruB that are complementary to each other (Elias J., (2009, unpublished Ph.D thesis). Taq DNA polymerase, along with its respective buffer, dNTPs, and primers, were sourced from Bangalore Genei Pvt. Ltd., India, and Sigma Chemicals Pvt. Ltd., respectively. These components were employed following the guidelines outlined by the respective manufacturers. The PCR (Techne, USA) was carried out at initial denaturation of 94°C- 5 min, 40cycles of denaturation 94°C- 30 sec, annealing 62°C − 30sec, elongation 72°C − 45sec, final elongation 72°C- 10 min (Elias J., (2009, unpublished Ph.D thesis).

2.4 Construction of pVS2k3 (P_fruB as-icd). Construction of P_fruB as-icd was carried out as depicted schematically in Fig. 1. First, PCR amplification of E. coli DH5α genomic DNA was carried out separately using pairs of ECfruBL1 and ECfruBR1 as well as ECicdL1 and ECicdR1 primers. The fragments corresponding to 192 bp region encompassing the fruB promoter and 122 bp region of the icd gene were obtained. The 192 bp amplicon of fruB contained 170 bp region of fruB promoter, 14 bp of icd region at one end and additional 8 bp containing an EcoR1 site on the other end. Similarly, icd amplicon had 103 bp regions of icd with 11 bp corresponding to fruB and 8 bp extra with a BamH1 recognition site on either end. Recombinant PCR was carried out using the ECfruB L1 and ECicdR1 primers and the previously obtained amplicons. This resulted in a 289 bp fragment containing fruB promoter and as-icd flanked by EcoR1 and BamH1 sites (Fig. 1). The amplified product was inserted into the pTZ57R vector using the InsT/Aclone™ PCR product cloning kit (MBI Fermentas) and subsequently transformed into E. coli DH5α. The presence of the correct plasmid was verified through PCR analysis using ECfruBL1 and ECicd R1 primers. Digestion of the plasmid with EcoR1 released an insert of 290 bp confirming correctness of the construct (data not shown), which was denoted as pVS2K3 (Elias J., (2009, unpublished Ph.D thesis).

2.5 Construction of pJE6 (P_tac as-icd). PCR amplification of E. coli DH5α genomic DNA was carried out using ECicdL1 and ECicdR1 primers (Table 2). The fragments corresponding to 122 bp region of the icd gene were obtained. The, icd amplicon had 103 bp regions of icd with 11 bp corresponding to fruB and 8bp extra with a BamH1 recognition site on either end. The amplified product was cloned into the pTZ57R (pJE4) vector using the InsT/Aclone™ PCR product cloning kit from MBI Fermentas, and then transformed into E. coli DH5α. Verification of the correct plasmid presence was performed through PCR analysis utilizing ECicdL1 and ECicd R1 primers. Digestion of the plasmid with BamH1 released an insert of 125bp corresponding to as-icd which was subcloned into pTTQ18 vector (Elias J., (2009, unpublished Ph.D thesis). Presence of the insert was verified by PCR using ECicdL1 and ECicd R1 primers and by restriction digestion with BamH1. The orientation of the icd gene with respect to P_tac promoter was further confirmed by PCR amplification using 5’GGTGATCAAGCTGTTGACAATTAATCATCGG3' Primer BF1 and ECicd R1.

2.6 Plasmid DNA isolation and transformation. Plasmid DNA was isolated from E. coli and used for transformation by CaCl₂ method using standard protocols (Sambrook et al., 2001).

2.7 Growth experiments. For comparison of growth of transformed and native strains, E. coli cultures (Table 1) grown overnight in Luria-Bertrani (LB) broth for 24 h were washed with 0.9% saline, resuspended in saline solution and used to inoculate (0.1%) media containing M9 minimal media either glucose (50 mM), glycerol (100 mM), acetate (50mM) and following micronutrients (mg/L): ZnSO₄.7H₂O (0.16), H₃BO₃ (0.5), CuSO₄.5H₂O (0.08), FeSO₄.7H₂O (3.5), MnSO₄.4H₂O (0.4) and CaCl₂.2H₂O (0.03) (Elias J., (2009, unpublished Ph.D thesis). Growth was monitored by absorbance at 600 nm. IPTG (1 µg/ml) was used for induction of expression of as-icd in plasmid pJE6. Cultures were nurtured on a rotary shaker at 37⁰C and pH of culture supernatant was recorded at regular intervals.

2.8 Analytical methods. Variations in cell density were used as an indicator of growth, measured spectrophotometrically as absorbance at 600 nm using a Helios λ spectrophotometer (Thermo Spectronics, Cambridge, United Kingdom). As decrease in media pH was associated with acid production, observations were recorded until the media pH dropped below 5 or the optical density (O.D.) at 600 nm exceeded 1.9. Aseptically, 1 ml aliquots were withdrawn at predetermined time intervals and promptly frozen at -20°C for later biochemical analyses. Prior to analysis, the stored samples were centrifuged at 9,200 × g for 1 minute at 4°C using a Heraeus multifuge 15R (rotor-75003348/75002006). The resulting culture supernatants were then utilized to measure residual glucose and organic acids.Organic acids were analyzed using HPLC (Merck-Hitachi L-7100). The culture supernatants were filtered using 0.2 µm nylon membranes (MDI Advanced Microdevices, India) prior to HPLC analysis. The organic acids present in the filtered supernatants were identified and quantified using high-performance liquid chromatography (HPLC) with a Varian Microsorb RP-18 column at ambient temperature. The mobile phase consisted of 0.01 M Na2HPO4 and 5% acetonitrile, delivered at a flow rate of 0.2 ml/min. Detection of the column effluents was carried out using a UV detector set at 210 nm.GOD-POD kit (Enzopak, Reckon Diagnostics Pvt. Ltd, India) was used to determine concentration of residual glucose in the medium (Adhikary et al. 2014)

Table 1

**Bacterial strains and plasmids used in this study.**
Strains or Plasmids	Genotype	References
E. coli Strains
DH5α	(nal[r])F[-]thi[-]recAgyrA	(Sambrook et al. 2001)
BL21(DE3)	F- gal [dcm][lon] ompT hsdSB(rB- mB-); an E. coli B-strain) with DE3 a prophage carries the T7 RNA polymerase gene	(Sambrook et al. 2001)
BL21	F- gal [dcm][lon] ompT hsdSB(rB- mB-)	(Sambrook et al. 2001)
Plasmids
pTZ57R	Cloning vector Amp^r	MBI, fermentas
pVS2K3	Amp^r; P_fruB as-icd in pTZ57R	Present study
pTTQ18	P_tac promoter, Amp^r	(Stark 1987)
pJE6	pTTQ18 carrying as-icd, P_tac promoter, Amp^r	Present study
Amp^r, Ampicilin resistance

Table 2

**Primers used for the construction of** P_fruB **as-i**cd **chimera**
Primers	Sequence	Remarks
ECIcd L1	(5’ atgttccagttatccgccttgtgccggaa 3’)	+ 5 to + 15 atg of the fruB promoter (bold highlighted) followed by + 33 to + 19 region of the icd gene
ECIcd R1	(5’ cgggatcctatcgcaggacgcaaac 3’)	BamH1 site at the 5’end (bold highlighted) followed by − 72 to − 56 region of the icd gene
ECFruB L1	(5’ cggaattctgctcataactttacggctt 3’)	EcoR1 site at 5’end (bold highlighted, -158 to − 139 of fruB promoter.
EcFruB R1	(5’ gttccggcacaaggcggataactgga 3’)	+ 19 to + 33 of icd gene (underlined) followed by + 15 to + 5 of the fruB promoter

The physiological parameters assessed included specific glucose depletion rate, growth rate and biomass yield, as outlined by Chao and Liao (1993). The total glucose and glycerol consumption was calculated by subtracting the final substrate concentrations in the culture medium from the initial concentrations. Organic acid yields were quantified as grams of organic acid produced per gram of glucose consumed per gram of dry cell weight. Statistical analysis of all parameters was performed using GraphPad Prism software (version 3.0).

2.9 Preparation of cells, cell free extracts and enzymatic assays. M9 medium grown cells were collected by centrifugation at 9,200 x g for 2 min at 4^o C at an appropriate growth phase from 30 ml cultures. The cells were resuspended in sonication buffer (10 mM potassium phosphate buffer, pH 7.7 containing 500 mM NaCl and 2 mM MgCl₂) and lysed using a Branson sonifier 450. CS, ICL and ICDH were assayed at late log phase while G-6-PDH was assayed at mid log phase (Elias J., (2009, unpublished Ph.D thesis). The activity of citrate synthase (CS, EC 4.1.3.7) was assessed by measuring the absorbance of 5,5-dithiobis (2-nitrobenzoic acid) at 412 nm, which undergoes a change upon interaction with the sulfhydryl group of coenzyme A (CoA) (Srere 1969; Jain et al. 2013). The 1.0 ml assay mixture consisted of the following components: 93 mM Tris-HCl (pH 8.0), 0.16 mM acetyl CoA, 0.2 mM oxaloacetate, 0.1 mM 5-dithiobis (2-nitrobenzoic acid), and cell lysate. Oxaloacetate was used to initiate the reaction. The molar absorbance coefficient was 13.6 mM^− 1cm^− 1 at 412 nm. Glucose-6-phosphate dehydrogenase (G-6-PDH, EC 1.1.1.49) (Eisenberg and Dobrogosz, 1967) and isocitrate dehydrogenase (ICDH, EC 1.1.1.42) (Garnak and Reeves, 1979) enzyme activities were measured by observing the reduction of NADP at 340 nm. Isocitrate lyase (ICL, EC 4.1.3.1) activity was ascertained by quantifying glyoxylate formation at 324 nm in presence of phenylhydrazine HCl (Dixon 1959). Spectrophotometrically quantification of total protein concentration was determined by the modified method of Folin-Lowry; bovine serum albumin was used as the standard (Peterson, 1979).

2.10 Enzymatic measurement of organic acids. Cell extracts were obtained from stationary phase cultures of E. coli transformants cultivated on M9-glucose and M9-glycerol mediums, following the same preparation steps as for enzyme assays. These extracts were filtered using a 0.2 µm nitrocellulose membrane and were either kept on an ice bath or frozen until further analysis. The filtrates were used to assess intracellular citric acid levels, while the filtered supernatants from the same cultures were used to determine extracellular acetic acid (Buch et al., 2009).

The spectrophotometric measurement of intracellular citric acid was carried out based on the method adapted from Petrarulo et al. (1995), with minor modifications from Elias J's (2009) unpublished Ph.D. thesis. The assay used a 1.0 ml mixture consisting of 50 mM phosphate buffer, 0.02 ml of 246 mM phenylhydrazine, 0.02 ml of citrate lyase (0.27U from a 13.3 units/ml stock), and either citric acid standard or cell extract. The phosphate buffer contained 50 mM buffer (pH 6.5), 0.1 mM ZnSO4·7H2O, and 0.2 g/L sodium azide. A standard curve was created using citric acid standards from 5 µM to 20 µM (Buch et al. 2009). Optical density (O.D.) at 330 nm was recorded after 3 minutes of citrate lyase addition, and the intracellular citrate concentration (in mM) was calculated. The cellular volume was assumed to be 1.63 µl/mg dry cell weight (dcw), according to Emmerling et al. (1999).

3.1 Effect of P _fruB as-icd (pVS2k3) on Isocitrate dehydrogenase (ICDH) activity in E. coli DH5α and E. coli BL21. On glucose P_fruB as-icd expression in E. coli DH5α down-regulated ICD activity by 3–4 fold decrease (Fig. 2a) but no effect was discernible in case of this strain on glycerol (Fig. 2a). The enzyme activity of ICDH activity remained unchanged in E. coli BL21 (λDE3) expressing pVS2k3 (Fig. 2b). To validate that the dysfunction of Cra could be a probable reason for the failure of P_fruB as-icd in BL21(λDE3) as-icd was expressed under IPTG inducible promoter (P_tac). P_tac as-icd expression in E. coli BL21 monitored a 3–4 fold reduction in ICDH activity (Fig. 2b). Higher ICDH activity was observed in E. coli BL21 on both glucose and acetate (Fig. 2c). E. coli DH5α showed no ICDH activity on acetate.

3.2 Effect of P _fruB as-icd on physiological parameters in E. coli DH5α. Reduced ICDH activity by P_fruB as-icd did not alter the growth rate (0.37 ± 0.08 h^− 1) but significantly improved the glucose consumption rate (5.7 ± 1.25 g glc.g^− 1dcw.h^− 1) when compared to the E. coli harboring the control plasmid pTZ57R (Fig. 3, Table 3).

3.3 Effect of P _fruB as-icd on Citrate synthase (CS), Glucose 6 phosphate dehydrogenase (G-6-PDH) and isocitrate lyase (ICL) activities in E. coli DH5α on glucose. Down-regulation of ICDH activity by P_fruB as-icd demonstrated a significant increase in CS activity (0.079 ± 0.008 U) compared to its plasmid control (0.04 ± 0.002 U). G-6-PDH activity significantly decreased in response to the P_fruB as-icd down regulation of icd gene (Fig. 4). No ICL activity was detected on glucose.

3.4 Effect of P _fruB as-icd on organic acid secretion in E. coli DH5α. Antisense RNA mediated down regulation of icd gene was reflected in the increased citrate accumulation in (1.32 ± 0.013 mM) as compared to plasmid control strain (0. 65 ± 0.05 mM) (Table 4). No extracellular citrate was detected in E. coli DH5α

Table 3

**Physiological parameters demonstrating the effect of P**_fruB-asicd **expression in** **E. coli** **DH5α on glucose.**
E. coli strains	Growth rate (µ)h^-1	Specific glucose consumption rate (Q_glc)	Total glucose consumed (mM)	Biomass Y_dcw/glc (g dcw.g^-1.h^-1)
DH5α	0.26 ± 0.02	7.0 ± 1.5	48 ± 2.5	0.023 ± 0.010
DH5α: pTZ57R	0.32 ± 0.08	3.2 ± 1.0	42 ± 5	0.025 ± 0.010
DH5α: pVS2k3	0.37 ± 0.08^ns	5.7 ± 1.25 **	35 ± 1.5	0.033 ± 0.015^a**
The results are expressed as Mean ± SD of 8–10 independent observations. Carbon source used is 50mM glucose for E. coli DH5α. Specific growth rate (µ(h^− 1)), specific glucose consumption rate (Q_Glc) and Biomass (Y_dcw/glc) were determined from mid log phase of each experiment. Total glucose utilized was determined at the end of growth curve. Significance calculated with respect to the respective plasmid control ** p < 0.01, ns- non significant

Table 4

**Organic acid formation in** **E. coli** **DH5α transformants expressing P**_fruB-asicd on **50 mM glucose.**
E. coli strains	Intracellular citrate (mM)	Extracellular Acetate (mM)
DH5α	0.65 ± 0.06	ND
DH5α pTZ57R	0.69 ± 0.1	1.04 ± 0.25
DH5α pVS2k3	1.32 ± 0.13^a**	1.95 ± 0.65^{a ns}
The table depicts the citrate (intracellular) and acetate (extracellular) levels in the late stationary phase cultures of E. coli DH5α transformants (plasmid control and test) grown on M9 minimal media with 50mM as carbon source. All the values are represented as Mean ± SD of n = 8 observations. ** p < 0.01and ^ns non-significant.

Low levels of acetate were found on 50mM glucose in E. coli DH5α. E. coli DH5α pVS2k3 (1.95 ± 0.65 mM) expressing P_fruB as-icd had slight increase in acetate levels compared to its plasmid control bearing E. coli DH5α (1.04 ± 0.25 mM) (Table 4).

Antisense RNA offers a tool to partially down-regulate the expression of bacterial genes thus overcoming the problems encountered with null mutants (Kernodle et al. 1997; Kurreck 2003). Conditional antisense RNA expressing systems are considered to be more effective as they exhibit leaky phenotype. A tetracycline (tet) controlled antisense RNA expressing system was developed; it allowed selective genes of the chromosome to be switched on and off and even control their expression levels. This offered a potential tool to generate a quantitative data of the gene product (Yin and Ji 2002). Parish and Stoker (1997), demonstrated that conditional regulation of antisense RNA expression under an inducible promoter in mycobacteria facilitated the elucidation of the role of essential genes. Present study deals with designing of a conditionally regulated antisense RNA against the icd gene of E. coli where carbon source availability controls the antisense RNA expression. The strategy exploits the natural mechanism of Cra protein-mediated control of fruB promoter which connects carbon source availability through intracellular F-1-P and F-1,6-P concentrations to gene expression (Fig. 5). The conditional antisense RNA strategy employed in this study is novel in that (a) it avoids the use of external inducers for the down- regulation of target genes and (b) depends upon the nature and amount of the available carbon sources. fruB promoter has been used to quantify the amount of nutrients available to the microbial residents in the phyllosphere (Leveau and Lindow 2001). In this study they expressed the promoter from E. coli in Erwinia herbicola as they are closely related bacteria but in the present study, we report that the constitutive Cra expression as seen in E. coli B could restrict its use among E. coli strains.

P_fruB as-icd expression down-regulated ICDH enzyme activity by 3-4fold in E. coli DH5α on glucose but not on glycerol as fruB promoter is known to get de-repressed by increased F-1,6-P levels seen in E. coli when grown on glucose. This de-repression of the fruB promoter produces as-icd RNA (Fig. 5). However, when glycerol is used, the fruB promoter stays inhibited by the Cra protein because of the low levels of fructose-1,6-phosphate, resulting in no alteration in ICDH activity. This finding could be supported with earlier reports that fruB promoter responded to the fructose in the medium but failed to express on galactose (Leveau and Lindow 2001).

E. coli K and B strains are known to differ in the manner in which the regulation of glyoxylate pathway and Cra protein takes place (Phue et al. 2005; Son et al. 2011). E. coli B strains secrete low levels of acetate as compared to E. coli K strains this property supports efficient recombinant protein expression in E. coli B strains (Shiloach et al. 1996). In contrast to E. coli K strain, P_fruB as-icd had no effect on the ICD activity in E. coli B strain when grown on glucose. This suggested that there could be alteration in Cra regulation hence to validate that the dysfunction of Cra could be a probable reason for the failure of P_fruB as-icd in BL21(λDE3) as-icd was expressed under IPTG inducible promoter. P_tac as-icd expression in E. coli BL21 downregulated the ICDH activity by 3-4fold. Moreover the growth of E. coli BL21 on acetate supported the anamalous Cra protein as E. coli Cra mutants could not grow on acetate. These results support that a dysfunctional Cra regulation could restrict the universal use of fruB promoter in E.coli strains. Hence the physiological experiments to monitor the effects of conditional downregulation of icd gene were carried out in E. coli DH5α.

Down-regulation of ICDH by P_fruB as-icd in E. coli DH5α increased CS activity which is in agreement with the earlier results related to E. coli icd mutants (Lakshmi and Helling 1976; Kabir and Shimizu 2004). This increase in icd mutants was proposed to enhance NAD(P)H levels which were generated by ICDH in wild type (Park et al. 1994). Conditional asicd demonstrated increased growth along with high CS activity compared to the E. coli icd mutants (Lakshmi and Helling 1976; Kabir and Shimizu 2004). Lower G-6-PDH activity in E. coli expressing as-icd could be attributed to the partial block that suffices the requirements of NADPH. Absence of ICL activity in E. coli on glucose irrespective of antisense expression is well supported by the reports that on glucose there is no ICL activity in E. coli K-12 derivative (Phue et al. 2005).

P_fruB as-icd expression significantly increased intracellular citrate levels which supports that blocking ICD could be a probable target for citrate accumulation in E. coli strains and these finding were similar to the icd mutant. High acetate levels were observed in E. coli expressing antisense as compared to that of icd mutant (Kabir and Shimizu 2004). E. coli has a reportedly low TCA flux and the partial block in the ICDH activity probably leads to further decrease in the TCA flux resulting in pyruvate accumulation in large amount which could be directed to acetate formation. Absence of citrate in the extracellular medium is due to the absence of a citrate efflux mechanism in E. coli (Lakshmi and Helling 1976).

The present study reports that down regulation of icd gene using antisense technology controlled by fruB (Table 5) was capable of accumulating citrate without altering the growth of E. coli K strain as seen in case of icd mutants (Lakshmi and Helling 1976; Aoshima et al. 2003; Kabir and Shimizu 2004). Cra dysfunction in E. coli BL21was reported earlier (Phue et al. 2005; Son et al. 2011) the present study supports this report as P_fruB was not found to be a suitable promoter for expression studies in E. coli BL21. Thus, E. coli K and B strains differ distinctly in the metabolism, proteome and the genome sequence of B strains (Jeong et al. 2009; Han, 2016) could facilitate in unraveling these differences.

Table 5

**Characteristic differences between an** **E. coli icd** **mutant and** **E. coli** **expressing as-icd** **gene under** **fruB promoter.**
Parameters	E. coli icd mutant (Lakshmi and Helling 1976; Kabir and Shimizu 2004)
Growth rate	Slow	No change
Glucose consumption rate	Low	∼2 fold increase
Glucose consumed	Required	No change
Glucose 6 phosphate dehydrogenase	ND	∼1.2 fold decrease
Citrate Synthase	High	∼2 fold increase
Isocitrate Dehydrogenase	High	∼3–4 fold decrease
Isocitrate lyase	High	ND
Citrate (Intracellular)	11 mM	∼ 1.32 ± 0.13 mM 2 fold increase
Acetate	Low	∼ 2 fold increase
ND- not detected and NA-not available

Three overlapping antisense genes in Escherichia coli O157:H7 EDL933 cause translationally arrest mutant phenotype (Graf et al., 2023). Escherichia coli K12 MG1655 contains 663 overlapping pairs with larger than 30 nucleotides out of them 586 are co-oriented, 75 convergent and 2 divergent (Huvet and Stumpf, 2014). Natural antisense RNAs in bacteria are known to post transcriptional inhibition of mRNA, direct inhibition of translation and also act as riboswitches associated with wide range of bacterial activities viz plasmid replication, metabolite sensing biofilm formation, conjugation, toxin synthesis (Saberi et al., 2016).

Conflict of Interest statement:

none

Funding:

The authors have no relevant financial or non-financial interests to disclose. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Author Contribution

All authors contributed to the study conception and design. Strain developments and plasmid construction were done by Vikas Sharma and Jisha Elias. Growth experiments, analytical, biochemical assays, data collection, and data analysis were performed by Jisha Elias. The first draft of the manuscript was written by Jisha Elias and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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FruR-controlled antisense RNA -downregulation of isocitrate dehydrogenase in Escherichia coli

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