scholarly journals In VivoTitration of Folate Pathway Enzymes

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Deepika Nambiar ◽  
Timkhite-Kulu Berhane ◽  
Robert Shew ◽  
Bryan Schwarz ◽  
Michael R. Duff ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Dihydrofolate Reductase ◽  
Environmental Changes ◽  
Stress Conditions ◽  
Antibacterial Drug ◽  
Content Type ◽  
E Coli ◽  
Knockout Strain ◽  
R67 Dihydrofolate Reductase

ABSTRACTHow enzymes behave in cells is likely different from how they behave in the test tube. Previousin vitrostudies find that osmolytes interact weakly with folate. Removal of the osmolyte from the solvation shell of folate is more difficult than removal of water, which weakens binding of folate to its enzyme partners. To examine if this phenomenon occursin vivo, osmotic stress titrations were performed withEscherichia coli. Two strategies were employed: resistance to an antibacterial drug and complementation of a knockout strain by the appropriate gene cloned into a plasmid that allows tight control of expression levels as well as labeling by a degradation tag. The abilities of the knockout and complemented strains to grow under osmotic stress were compared. Typically, the knockout strain could grow to high osmolalities on supplemented medium, while the complemented strain stopped growing at lower osmolalities on minimal medium. This pattern was observed for an R67 dihydrofolate reductase clone rescuing a ΔfolAstrain, for a methylenetetrahydrofolate reductase clone rescuing a ΔmetFstrain, and for a serine hydroxymethyltransferase clone rescuing a ΔglyAstrain. Additionally, an R67 dihydrofolate reductase clone allowedE. coliDH5α to grow in the presence of trimethoprim until an osmolality of ∼0.81 is reached, while cells in a control titration lacking antibiotic could grow to 1.90 osmol.IMPORTANCEE. colican survive in drought and flooding conditions and can tolerate large changes in osmolality. However, the cell processes that limit bacterial growth under high osmotic stress conditions are not known. In this study, the dose of four different enzymes inE. coliwas decreased by using deletion strains complemented by the gene carried in a tunable plasmid. Under conditions of limiting enzyme concentration (lower than that achieved by chromosomal gene expression), cell growth can be blocked by osmotic stress conditions that are normally tolerated. These observations indicate thatE. colihas evolved to deal with variations in its osmotic environment and that normal protein levels are sufficient to buffer the cell from environmental changes. Additional factors involved in the osmotic pressure response may include altered protein concentration/activity levels, weak solute interactions with ligands which can make it more difficult for proteins to bind their substrates/inhibitors/cofactorsin vivo, and/or viscosity effects.

scholarly journals Genotoxic, Metabolic, and Oxidative Stresses Regulate the RNA Repair Operon ofSalmonella entericaSerovar Typhimurium

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Jennifer E. Kurasz ◽  
Christine E. Hartman ◽  
David J. Samuels ◽  
Bijoy K. Mohanty ◽  
Anquilla Deleveaux ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Stress Responses ◽  
Nitrogen Limitation ◽  
Selective Advantage ◽  
Stress Conditions ◽  
Rna Repair ◽  
Content Type ◽  
E Coli ◽  
Oxidative Stresses

ABSTRACTThe σ54regulon inSalmonella entericaserovar Typhimurium includes a predicted RNA repair operon encoding homologs of the metazoan Ro60 protein (Rsr), Y RNAs (YrlBA), RNA ligase (RtcB), and RNA 3′-phosphate cyclase (RtcA). Transcription from σ54-dependent promoters requires that a cognate bacterial enhancer binding protein (bEBP) be activated by a specific environmental or cellular signal; the cognate bEBP for the σ54-dependent promoter of thersr-yrlBA-rtcBAoperon is RtcR. To identify conditions that generate the signal for RtcR activation inS. Typhimurium, transcription of the RNA repair operon was assayed under multiple stress conditions that result in nucleic acid damage. RtcR-dependent transcription was highly induced by the nucleic acid cross-linking agents mitomycin C (MMC) and cisplatin, and this activation was dependent on RecA. Deletion ofrtcRorrtcBresulted in decreased cell viability relative to that of the wild type following treatment with MMC. Oxidative stress from peroxide exposure also induced RtcR-dependent transcription of the operon. Nitrogen limitation resulted in RtcR-independent increased expression of the operon; the effect of nitrogen limitation required NtrC. The adjacent toxin-antitoxin module,dinJ-yafQ, was cotranscribed with the RNA repair operon but was not required for RtcR activation, although YafQ endoribonuclease activated RtcR-dependent transcription. Stress conditions shown to induce expression the RNA repair operon ofEscherichia coli(rtcBA) did not stimulate expression of theS. Typhimurium RNA repair operon. Similarly, MMC did not induce expression of theE. colirtcBAoperon, although when expressed inS. Typhimurium,E. coliRtcR responds effectively to the unknown signal(s) generated there by MMC exposure.IMPORTANCEHomologs of the metazoan RNA repair enzymes RtcB and RtcA occur widely in eubacteria, suggesting a selective advantage. Although the enzymatic activities of the eubacterial RtcB and RtcA have been well characterized, the physiological roles remain largely unresolved. Here we report stress responses that activate expression of the σ54-dependent RNA repair operon (rsr-yrlBA-rtcBA) ofS. Typhimurium and demonstrate that expression of the operon impacts cell survival under MMC-induced stress. Characterization of the requirements for activation of this tightly regulated operon provides clues to the possible functions of operon componentsin vivo, enhancing our understanding of how this human pathogen copes with environmental stressors.

scholarly journals Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis

2020 ◽  
Vol 89 (1) ◽  
pp. e00401-20
Author(s):  
Raghuveer Singh ◽  
Jessica A. Slade ◽  
Mary Brockett ◽  
Daniel Mendez ◽  
George W. Liechti ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Diaminopimelic Acid ◽  
Competition Strategy ◽  
Content Type ◽  
E Coli ◽  
Glutamate Racemase ◽  
Heterologous System ◽  
Substrate Competition

ABSTRACTThe Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapFCt function in vivo in C. trachomatis. We reasoned that, because DapFCt utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapFCt DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapFCt is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis. Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.

Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Nirbhay Singh ◽  
Anu Chauhan ◽  
Ram Kumar ◽  
Sudheer Kumar Singh
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Low Ph ◽  
Stress Conditions ◽  
Starvation Stress ◽  
Content Type ◽  
E Coli ◽  
Link Type

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

scholarly journals A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Erin M. Nawrocki ◽  
Hillary M. Mosso ◽  
Edward G. Dudley
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Microbial Interactions ◽  
Severe Illness ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

scholarly journals In Vivo Bioluminescent Monitoring of Therapeutic Efficacy and Pharmacodynamic Target Assessment of Antofloxacin against Escherichia coli in a Neutropenic Murine Thigh Infection Model

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Yu-Feng Zhou ◽  
Meng-Ting Tao ◽  
Yu-Zhang He ◽  
Jian Sun ◽  
Ya-Hong Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Subcutaneous Administration ◽  
Free Drug ◽  
Infection Model ◽  
Bioluminescent Imaging ◽  
Time Curve ◽  
Content Type ◽  
E Coli ◽  
Tract Infections

ABSTRACT Antimicrobial resistance among uropathogens has increased the rates of infection-related morbidity and mortality. Antofloxacin is a novel fluoroquinolone with broad-spectrum antibacterial activity against urinary Gram-negative bacilli, such as Escherichia coli. This study monitored the in vivo efficacy of antofloxacin using bioluminescent imaging and determined pharmacokinetic (PK)/pharmacodynamic (PD) targets against E. coli isolates in a neutropenic murine thigh infection model. The PK properties were determined after subcutaneous administration of antofloxacin at 2.5, 10, 40, and 160 mg/kg of body weight. Following thigh infection, the mice were treated with 2-fold-increasing doses of antofloxacin from 2.5 to 80 mg/kg administered every 12 h. Efficacy was assessed by quantitative determination of the bacterial burdens in thigh homogenates and was compared with the bioluminescent density. Antofloxacin demonstrated both static and killing endpoints in relation to the initial burden against all study strains. The PK/PD index area under the concentration-time curve (AUC)/MIC correlated well with efficacy (R 2 = 0.92), and the dose-response relationship was relatively steep, as observed with escalating doses of antofloxacin. The mean free drug AUC/MIC targets necessary to produce net bacterial stasis and 1-log10 and 2-log10 kill for each isolate were 38.7, 66.1, and 147.0 h, respectively. In vivo bioluminescent imaging showed a rapid decrease in the bioluminescent density at free drug AUC/MIC exposures that exceeded the stasis targets. The integration of these PD targets combined with the results of PK studies with humans will be useful in setting optimal dosing regimens for the treatment of urinary tract infections due to E. coli.

scholarly journals In Vivo mRNA Profiling of Uropathogenic Escherichia coli from Diverse Phylogroups Reveals Common and Group-Specific Gene Expression Profiles

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Piotr Bielecki ◽  
Uthayakumar Muthukumarasamy ◽  
Denitsa Eckweiler ◽  
Agata Bielecka ◽  
Sarah Pohl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Specific Gene ◽  
Content Type ◽  
E Coli ◽  
Human Host ◽  
Tract Infections

ABSTRACTmRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression ofEscherichia colipathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associatedE. coliisolates to different phylogenetic groups. Whereas thein vivogene expression profiles of the majority of genes were conserved among 21E. colistrains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribedin vivorelative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease.IMPORTANCEUrinary tract infections (UTI) are very common; at least half of all women experience UTI, most of which are caused by pathogenicEscherichia colistrains. In this study, we applied massive parallel cDNA sequencing (RNA-seq) to provide unbiased, deep, and accurate insight into the nature and the dimension of the uropathogenicE. coligene expression profile during an acute UTI within the human host. This work was undertaken to identify key players in physiological adaptation processes and, hence, potential targets for new infection prevention and therapy interventions specifically aimed at sabotaging bacterial adaptation to the human host.

scholarly journals Role of Urinary Cathelicidin LL-37 and Human β-Defensin 1 in Uncomplicated Escherichia coli Urinary Tract Infections

2014 ◽  
Vol 82 (4) ◽  
pp. 1572-1578 ◽  
Author(s):  
Karen L. Nielsen ◽  
Pia Dynesen ◽  
Preben Larsen ◽  
Lotte Jakobsen ◽  
Paal S. Andersen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Adult Women ◽  
Content Type ◽  
E Coli ◽  
Innate Defense ◽  
High Level ◽  
Tract Infections

ABSTRACTCathelicidin (LL-37) and human β-defensin 1 (hBD-1) are important components of the innate defense in the urinary tract. The aim of this study was to characterize whether these peptides are important for developing uncomplicatedEscherichia coliurinary tract infections (UTIs). This was investigated by comparing urinary peptide levels of UTI patients during and after infection to those of controls, as well as characterizing the fecal flora of participants with respect to susceptibility to LL-37 andin vivovirulence. Forty-seven UTI patients and 50 controls who had never had a UTI were included. Participants were otherwise healthy, premenopausal, adult women. LL-37 MIC levels were compared for fecalE. coliclones from patients and controls and were also compared based on phylotypes (A, B1, B2, and D).In vivovirulence was investigated in the murine UTI model by use of selected fecal isolates from patients and controls. On average, UTI patients had significantly more LL-37 in urine during infection than postinfection, and patient LL-37 levels postinfection were significantly lower than those of controls. hBD-1 showed similar urine levels for UTI patients and controls. FecalE. coliisolates from controls had higher LL-37 susceptibility than fecal and UTIE. coliisolates from UTI patients.In vivostudies showed a high level of virulence of fecalE. coliisolates from both patients and controls and showed no difference in virulence correlated with the LL-37 MIC level. The results indicate that the concentration of LL-37 in the urinary tract and low susceptibility to LL-37 may increase the likelihood of UTI in a complex interplay between host and pathogen attributes.

scholarly journals Can Pathogenic and Nonpathogenic Bacteria Be Distinguished by Sensory Protein Abundance?

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Subhrajit Bhar ◽  
Tungadri Bose ◽  
Sharmila S. Mande
Keyword(s):  
Escherichia Coli ◽  
Genome Size ◽  
Ecological Niche ◽  
Environmental Changes ◽  
Ecological Niches ◽  
Content Type ◽  
E Coli ◽  
Depth Analysis ◽  
Component Systems ◽  
The Relationship

ABSTRACT Signal transduction systems are essential for microorganisms to respond to their ever-changing environment. They can be distinguished into one-component systems, two-component systems, and extracytoplasmic-function σ factors. Abundances of a few signal-transducing proteins, termed herein as sensory proteins (SPs), have previously been reported to be correlated with the genome size and ecological niche of certain Gram-positive bacteria. No such reports are available for Gram-negative bacteria. The current study attempts to investigate the relationship of the abundances of SPs to genome size in Escherichia coli, and the bacterial pathotypes or phylotypes. While the relationship between SP abundance and genome size could not be established, the sensory protein index (SPI), a new metric defined herein, was found to be correlated with E. coli virulence. In addition, significant association was observed among the distribution of SPs and E. coli pathotypes. Results indicate that such associations might be due to genomic rearrangements to best utilize the resources available in a given ecological niche. Overall, the study provides an in-depth analysis of the occurrence of different SPs among pathogenic and nonpathogenic E. coli strains. Possibilities of using the SPI as a marker for identifying pathogenic strains from among an organism complex are also discussed. IMPORTANCE Sensory proteins (SPs) act as sensors and actuators for a cell and participate in important mechanisms pertaining to bacterial survival, adaptation, and virulence. Therefore, bacterial species residing in similar ecological niches or those sharing common pathotypes are expected to exhibit similar SP signatures. We have investigated profiles of SPs in different species of Escherichia coli and present in this article the sensory protein index (SPI), a metric for quantifying the abundance and/or distribution of SPs across bacterial genomes, which could indicate the virulence potency of a bacterium. The SPI could find use in characterizing uncultured strains and bacterial complexes, as a biomarker for disease diagnostics, evaluating the effect of therapeutic interventions, assessing effects of ecological alterations, etc. Grouping the studied strains of E. coli on the basis of the frequency of occurrence of SPs in their genomes could potentially replicate the stratification of these strains on the basis of their phylotypes. In addition, E. coli strains belonging to the same pathotypes were also seen to share similar SP signatures. Furthermore, the SPI was seen to be an indicator of pathogenic potency of E. coli strains. The SPI metric is expected to be useful in the (pathogenic) characterization of hereto uncultured strains which are routinely sequenced in host microbiome analysis projects, or from among an ensemble of microbial organisms constituting a biospecimen. Thus, the possibilities of using the SPI as a biomarker for diagnosis of a disease or the outcome of a therapeutic intervention cannot be ruled out. Further, SPIs obtained from longitudinal ecological samples have the potential to serve as key indicators of environmental changes. Such changes in the environment are often detrimental to the resident biome and methods for timely detection of environmental changes hold huge socioeconomic benefits.

scholarly journals Magnesium Sensing Regulates Intestinal Colonization of Enterohemorrhagic Escherichia coli O157:H7

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Yutao Liu ◽  
Runhua Han ◽  
Junyue Wang ◽  
Pan Yang ◽  
Fang Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Intestine ◽  
Intestinal Tract ◽  
Regulatory System ◽  
Regulatory Pathway ◽  
Content Type ◽  
E Coli ◽  
Low Magnesium ◽  
Enterocyte Effacement

ABSTRACT The large intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 detects host cues to regulate virulence gene expression during colonization and infection. However, virulence regulatory mechanisms of EHEC O157:H7 in the human large intestine are not fully understood. Herein, we identified a virulence-regulating pathway where the PhoQ/PhoP two-component regulatory system senses low magnesium levels and signals to the O island 119-encoded Z4267 (LmiA; low magnesium-induced regulator A), directly activating loci of enterocyte effacement genes to promote EHEC O157:H7 adherence in the large intestine. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, feeding mice a magnesium-rich diet significantly reduced EHEC O157:H7 adherence in vivo. This LmiA-mediated virulence regulatory pathway is also conserved among several EHEC and enteropathogenic E. coli serotypes; therefore, our findings support the use of magnesium as a dietary supplement and provide greater insights into the dietary cues that can prevent enteric infections. IMPORTANCE Sensing specific gut metabolites is an important strategy for inducing crucial virulence programs by enterohemorrhagic Escherichia coli (EHEC) O157:H7 during colonization and infection. Here, we identified a virulence-regulating pathway wherein the PhoQ/PhoP two-component regulatory system signals to the O island 119-encoded low magnesium-induced regulator A (LmiA), which, in turn, activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence in the low-magnesium conditions of the large intestine. This regulatory pathway is widely present in a range of EHEC and enteropathogenic E. coli (EPEC) serotypes. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, mice fed a magnesium-rich diet showed significantly reduced EHEC O157:H7 adherence in vivo, indicating that magnesium may help in preventing EHEC and EPEC infection in humans.

scholarly journals The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Minho Lee ◽  
Minju Joo ◽  
Minji Sim ◽  
Se-Hoon Sim ◽  
Hyun-Lee Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Environmental Changes ◽  
Oxygen Availability ◽  
Rnase Iii ◽  
Biochemical Adaptation ◽  
E Coli ◽  
Mrna Transcripts ◽  
Nuclease Mapping ◽  
Rnase G

AbstractRapid modulation of RNA function by endoribonucleases during physiological responses to environmental changes is known to be an effective bacterial biochemical adaptation. We report a molecular mechanism underlying the regulation of enolase (eno) expression by two endoribonucleases, RNase G and RNase III, the expression levels of which are modulated by oxygen availability in Escherichia coli. Analyses of transcriptional eno-cat fusion constructs strongly suggested the existence of cis-acting elements in the eno 5′ untranslated region that respond to RNase III and RNase G cellular concentrations. Primer extension and S1 nuclease mapping analyses of eno mRNA in vivo identified three eno mRNA transcripts that are generated in a manner dependent on RNase III expression, one of which was found to accumulate in rng-deleted cells. Moreover, our data suggested that RNase III-mediated cleavage of primary eno mRNA transcripts enhanced Eno protein production, a process that involved putative cis-antisense RNA. We found that decreased RNase G protein abundance coincided with enhanced RNase III expression in E. coli grown anaerobically, leading to enhanced eno expression. Thereby, this posttranscriptional up-regulation of eno expression helps E. coli cells adjust their physiological reactions to oxygen-deficient metabolic modes. Our results revealed a molecular network of coordinated endoribonuclease activity that post-transcriptionally modulates the expression of Eno, a key enzyme in glycolysis.

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