Lon Protease Research Articles

Lon protease downregulates phenazine-1-carboxamide biosynthesis by degrading the quorum sensing signal synthase PhzI and exhibits negative feedback regulation of Lon itself in Pseudomonas chlororaphis HT66.

Pseudomonas chlororaphis HT66 exhibits strong antagonistic activity against various phytopathogenic fungi due to its main antibiotic phenazine-1-carboxamide (PCN). PCN gene cluster consists of phzABCDEFG, phzH, phzI, and phzR operons. phzABCDEFG transcription is activated by the PhzI/R quorum sensing system. Deletion of the lon gene encoding an ATP-dependent protease resulted in significant enhancement of PCN production in strain HT66. However, the regulatory pathway and mechanism of Lon on PCN biosynthesis remain unknown. Here, lon mutation was shown to significantly improve antimicrobial activity of strain HT66. The N-acyl-homoserine lactone synthase PhzI mediates the negative regulation of PCN biosynthesis and phzABCDEFG transcription by Lon. Western blot showed that PhzI protein abundance and stability were significantly enhanced by lon deletion. The in vitro degradation assay suggested that Lon could directly degrade PhzI protein. However, Lon with an amino acid replacement (S674 -A) could not degrade PhzI protein. Lon-recognized region was located within the first 50 amino acids of PhzI. In addition, Lon formed a new autoregulatory feedback circuit to modulate its own degradation by other potential proteases. In summary, we elucidated the Lon-regulated pathway mediated by PhzI during PCN biosynthesis and the molecular mechanism underlying the degradation of PhzI by Lon in P.chlororaphis HT66.

Fifteen days of moderate normobaric hypoxia does not affect mitochondrial function, and related genes and proteins, in healthy men.

To investigate within the one study potential molecular and cellular changes associated with mitochondrial biogenesis following 15 days of exposure to moderate hypoxia. Eight males underwent a muscle biopsy before and after 15 days of hypoxia exposure (FiO2 = 0.140-0.154; ~ 2500-3200 m) in a hypoxic hotel. Mitochondrial respiration, citrate synthase (CS) activity, and the content of genes and proteins associated with mitochondrial biogenesis were investigated. Our main findings were the absence of significant changes in the mean values of CS activity, mitochondrial respiration in permeabilised fibers, or the content of genes and proteins associated with mitochondrial biogenesis, after 15 days of moderate normobaric hypoxia. Our data provide evidence that 15 days of moderate normobaric hypoxia have negligible influence on skeletal muscle mitochondrial content and function, or genes and proteins content associated with mitochondrial biogenesis, in young recreationally active males. However, the increase in mitochondrial protease LON content after hypoxia exposure suggests the possibility of adaptations to optimise respiratory chain function under conditions of reduced O2 availability.

Low Cytoplasmic Magnesium Increases the Specificity of the Lon and ClpAP Proteases.

Proteolysis is a fundamental property of all living cells. In the bacterium Salmonella enterica serovar Typhimurium, the HspQ protein controls the specificities of the Lon and ClpAP proteases. Upon acetylation, HspQ stops being a Lon substrate and no longer enhances proteolysis of the Lon substrate Hha. The accumulated HspQ protein binds to the protease adaptor ClpS, hindering proteolysis of ClpS-dependent substrates of ClpAP, such as Oat, a promoter of antibiotic persistence. HspQ is acetylated by the protein acetyltransferase Pat from acetyl coenzyme A (acetyl-CoA) bound to the acetyl-CoA binding protein Qad. We now report that low cytoplasmic Mg2+ promotes qad expression, which protects substrates of Lon and ClpSAP by increasing HspQ amounts. The qad promoter is activated by PhoP, a regulatory protein highly activated in low cytoplasmic Mg2+ that also represses clpS transcription. Both the qad gene and PhoP repression of the clpS promoter are necessary for antibiotic persistence. PhoP also promotes qad transcription in Escherichia coli, which shares a similar PhoP box in the qad promoter region with S. Typhimurium, Salmonella bongori, and Enterobacter cloacae. Our findings identify cytoplasmic Mg2+ and the PhoP protein as critical regulators of protease specificity in multiple enteric bacteria. IMPORTANCE The bacterium Salmonella enterica serovar Typhimurium narrows down the spectrum of substrates degraded by the proteases Lon and ClpAP in response to low cytoplasmic Mg2+, a condition that decreases protein synthesis. This control is exerted by PhoP, a transcriptional regulator activated in low cytoplasmic Mg2+ that governs proteostasis and is conserved in enteric bacteria. The uncovered mechanism enables bacteria to control the abundance of preexisting proteins.

One Bout of Aerobic Exercise Elicits Alterations in The Expression of Mitochondrial Unfolded Protein Response (UPRmt) Markers in Skeletal Muscle

Muscle contractions during aerobic exercise stimulate molecular pathways to promote the production of new mitochondria, and improve their function. These adaptations translate into improved oxygen consumption and metabolic capacity of skeletal muscle. However, the increased drive for mitochondrial biogenesis during contractile activity causes an accumulation of misfolded proteins, disrupting mitochondrial homeostasis. The action of the UPRmt, a cytoprotective stress response pathway, is required to mitigate the associated proteotoxicity. This protein quality control mechanism favours the transcription of mitochondrial chaperones and proteases to refold and degrade the defective proteins, while inhibiting global protein translation. However, the regulation and activity of the UPRmt in helping skeletal muscle adapt to exercise stress during and after an acute bout of exercise has yet to be determined. Thus, to investigate this, mice were separated into sedentary (SED), acute exercise (AE) and acute exercise with recovery (AER) groups (n=4/group). The exercise consisted of 90-minutes of treadmill running at 15m/min. Tibialis anterior muscles were extracted from the AE groups immediately following exercise and from the AER group 3 hours post-exercise. Total RNA was isolated and mRNA levels were measured using qPCR. In line with previous data, PGC-1α mRNA increased by 50% in the AE group and increased further to 3.4-fold in the AER group. Activating transcription factor 5 (ATF5), an important regulator of UPRmt gene expression, did not change with acute exercise or recovery. ATF4, another transcription factor for stress-responsive genes, was also unchanged at the end of exercise, but was upregulated by 28% in the recovery period. mRNA levels of downstream targets of ATF5, Lon protease (LonP) and chaperones (HSP60 and GRP75) were upregulated by 18%, 24% and 77%, respectively. HSP60 mRNA returned to control levels during recovery, while GRP75 and LonP mRNAs remained elevated in the AER group. Our data indicate that acute exercise influences the expression of genes involved in both mitochondrial biogenesis and those of the UPRmt, contributing to the maintenance of organelle proteostasis in exercised muscle. Investigating the regulatory mechanisms governing this acute mitochondrial stress response is critical, as preservation of cellular function in the face of exercise-induced stress is essential for metabolic adaptations in muscle and thus, for whole-body homeostasis.

LONP1 Regulates Mitochondrial Accumulations of HMGB1 and Caspase-3 in CA1 and PV Neurons Following Status Epilepticus.

Lon protease 1 (LONP1) is a highly conserved serine peptidase that plays an important role in the protein quality control system in mammalian mitochondria. LONP1 catalyzes the degradation of oxidized, dysfunctional, and misfolded matrix proteins inside mitochondria and regulates mitochondrial gene expression and genome integrity. Therefore, LONP1 is up-regulated and suppresses cell death in response to oxidative stress, heat shock, and nutrient starvation. On the other hand, translocation of high mobility group box 1 (HMGB1) and active caspase-3 into mitochondria is involved in apoptosis of parvalbumin (PV) cells (one of the GABAergic interneurons) and necrosis of CA1 neurons in the rat hippocampus, respectively, following status epilepticus (SE). In the present study, we investigated whether LONP1 may improve neuronal viability to prevent or ameliorate translocation of active caspase-3 and HMGB1 in mitochondria within PV and CA1 neurons. Following SE, LONP1 expression was up-regulated in mitochondria of PV and CA1 neurons. LONP1 knockdown deteriorated SE-induced neuronal death with mitochondrial accumulation of active caspase-3 and HMGB1 in PV cells and CA1 neurons, respectively. LONP1 knockdown did not affect the aberrant mitochondrial machinery induced by SE. Therefore, our findings suggest, for the first time, that LONP1 may contribute to the alleviation of mitochondrial overloads of active caspase-3 and HMGB1, and the maintenance of neuronal viability against SE.

Evidence for Involvement of the Salmonella enterica Z-Ring Assembly Factors ZapA and ZapB in Resistance to Bile.

Genes annotated as ygfE and yiiU in the genome of Salmonella enterica serovar Typhimurium encode proteins homologous to Escherichia coli cell division factors ZapA and ZapB, respectively. ZapA− and ZapB− mutants of S. enterica are bile-sensitive. The amount of zapB mRNA increases in the presence of a sublethal concentration of sodium deoxycholate (DOC) while zapA mRNA remains unaffected. Increased zapB mRNA level in the presence of DOC is not caused by upregulation of zapB transcription but by increased stability of zapB mRNA. This increase is suppressed by an hfq mutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. The ZapB protein is degraded in the presence of DOC, and degradation appears to involve the Lon protease. We propose that increased stability of zapB mRNA in the presence of DOC may counter degradation of bile-damaged ZapB, thereby providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of bile.

A Novel, Universally Active C-terminal Protein Degradation Signal Generated by Alternative Splicing

Proteome integrity is crucial for cellular homeostasis and adaptation to stress conditions such as hypoxia. One mechanism for rapid adaptation of the proteome in response to changing environmental signals is alternative splicing. In addition to generating different protein isoforms, alternative splicing is also capable of controlling total protein levels by the regulated synthesis of non-productive mRNA isoforms. The hypoxia-induced isoform E of the tumor suppressor MAX is produced by retention and translation of the last intron. This leads to an alternative C-terminus that harbors a potent C-degron, the isoE degron. Strikingly, the isoE degron represents a universal protein degradation signal that is not only functional in mammalian cells, but also in yeast and even in bacteria. Essential for efficient protein decay is a conserved (F/W)xxW motif. Degradation of isoE tagged proteins is mediated by the proteasome in eukaryotes and Lon protease in bacteria. Thus, the isoE degron is a broadly applicable and highly efficient tool in protein analyses.

A bacterial protease depletes c-MYC and increases survival in mouse models of bladder and colon cancer.

Is the oncogene MYC upregulated or hyperactive? In the majority of human cancers, finding agents that target c-MYC has proved difficult. Here we report specific bacterial effector molecules that inhibit cellular MYC (c-MYC) in human cells. We show that uropathogenic Escherichia coli (UPEC) degrade the c-MYC protein and attenuate MYC expression in both human cells and animal tissues. c-MYC protein was rapidly degraded by both cell-free bacterial lysates and the purified bacterial protease Lon. In mice, intravesical or peroral delivery of Lon protease delayed tumor progression and increased survival in MYC-dependent bladder and colon cancer models, respectively. These results suggest that bacteria have evolved strategies to control c-MYC tissue levels in the host and that the Lon protease shows promise for therapeutic targeting of c-MYC in cancer.

Lon Protease- and Temperature-Dependent Activity of a Lysis Cassette Located in the Insecticidal Island of Yersinia enterocolitica.

The Yersinia genus comprises pathogens that can adapt to an environmental life cycle stage as well as to mammals. Yersinia enterocolitica strain W22703 exhibits both insecticidal and nematocidal activity conferred by the tripartite toxin complex (Tc) that is encoded on the 19-kb pathogenicity island Tc-PAI Ye All tc genes follow a strict temperature regulation in that they are silenced at 37°C but activated at lower temperatures. Four highly conserved phage-related genes, located within the Tc-PAI Ye , were recently demonstrated to encode a biologically functional holin-endolysin gene cassette that lyses its own host W22703 at 37°C. Conditions transcriptionally activating the cassette are not yet known. In contrast to Escherichia coli, the overproduction of holin and endolysin did not result in cell lysis of strain W22703 at 15°C. When the holin-endolysin genes were overexpressed at 15°C in four Y. enterocolitica biovars and in four other Yersinia spp., a heterogenous pattern of phenotypes was observed, ranging from lysis resistance of a biovar 1A strain to the complete growth arrest of a Y. kristensenii strain. To decipher the molecular mechanism underlying this temperature-dependent lysis, we constructed a Lon protease-negative mutant of W22703 in which the overexpression of the lysis cassette leads to cell death at 15°C. Overexpressed endolysin exhibited a high proteolytic susceptibility in strain W22703 but remained stable in the W22703 Δlon strain or in Y. pseudotuberculosis Although artificial overexpression was applied here, the data indicate that Lon protease plays a role in the control of the temperature-dependent lysis in Y. enterocolitica W22703.IMPORTANCE The investigation of the mechanisms that help pathogens survive in the environment is a prerequisite to understanding their evolution and their virulence capacities. In members of the genus Yersinia, many factors involved in virulence, metabolism, motility, or biofilm formation follow a strict temperature-dependent regulation. While the molecular mechanisms underlying the activation of determinants at body temperature have been analyzed in detail, the molecular basis of low-temperature-dependent phenotypes is largely unknown. Here, we demonstrate that a novel phage-related lysis cassette, which is part of the insecticidal and nematocidal pathogenicity island of Y. enterocolitica, does not lyse its own host following overexpression at 15°C and that the Lon protease is involved in this phenotype.

The Glyoxysomal Protease LON2 Is Involved in Fruiting-Body Development, Ascosporogenesis and Stress Resistance in Sordaria macrospora.

Microbodies, including peroxisomes, glyoxysomes and Woronin bodies, are ubiquitous dynamic organelles that play important roles in fungal development. The ATP-dependent chaperone and protease family Lon that maintain protein quality control within the organelle significantly regulate the functionality of microbodies. The filamentous ascomycete Sordaria macrospora is a model organism for studying fruiting-body development. The genome of S. macrospora encodes one Lon protease with the C-terminal peroxisomal targeting signal (PTS1) serine-arginine-leucine (SRL) for import into microbodies. Here, we investigated the function of the protease SmLON2 in sexual development and during growth under stress conditions. Localization studies revealed a predominant localization of SmLON2 in glyoxysomes. This localization depends on PTS1, since a variant without the C-terminal SRL motif was localized in the cytoplasm. A ΔSmlon2 mutant displayed a massive production of aerial hyphae, and produced a reduced number of fruiting bodies and ascospores. In addition, the growth of the ΔSmlon2 mutant was completely blocked under mild oxidative stress conditions. Most of the defects could be complemented with both variants of SmLON2, with and without PTS1, suggesting a dual function of SmLON2, not only in microbody, but also in cytosolic protein quality control.

Cleavage of PrePL by Lon promotes growth and pathogenesis in Magnaporthe oryzae.

ATP-dependent Lon proteases function in bacterial pathogenesis by regulating the expression of the Type III secretion system; however, little is known about how Lon proteases regulate fungal pathogenesis. We previously investigated Lon-binding proteins involved in fungal pathogenesis that interact with PrePL, the smallest Magnaporthe oryzae Lon-binding protein. Here, we show that Lon cleaves PrePL and produces Pc, an extracellular 11-kDa isoform with catalase and peroxidase activity. The ΔPrePL loss-of-function strain showed stronger sporulation and accelerated disease development, suggesting a temporally specific negative regulatory mechanism controlled by PrePL in disease progression. Neither the truncated Pc, nor the full-length PrePL missing the Lon cleavage site complemented the ΔPrePL phenotype, suggesting that full-length PrePL and Pc both function in fungal development. PrePL targeted to the mitochondria undergoes hydrolysis by Lon to produce Pc, which accumulates in the fungal apoplast. Importantly, recombinant Pc induced plant defence responses and cell death after being infiltrated into selected plant leaves, indicating that it functions as an avirulence factor. This work thus reveals a novel pathogenic factor in the fungal Lon-mediated pathway. Additionally, our results provide new insight into the functions of a full-length protein and its cleaved isoform in fungal pathogenesis.

Real-time in situ monitoring of Lon and Caspase-3 for assessing the state of cardiomyocytes under hypoxic conditions via a novel Au–Se fluorescent nanoprobe

Myocardial dysfunction caused by cardiomyocyte apoptosis under ischemic and hypoxic conditions is the pathological basis of most cardiovascular diseases. Current diagnosis of myocardial dysfunction still focuses on the symptomatic stage, usually after the occurrence of the irreversible remodelling and functional impairment. Thus, early stage identification of the apoptotic cardiomyocytes induced by hypoxia is highly significant for preventing the onset and delaying the progression of myocardial dysfunction. Herein, a novel Au–Se nanoprobe with strong anti-interference capability was developed for simultaneous real-time in situ monitoring the expression of Lon protease (Lon) and Caspase-3 with high-fidelity in living cardiomyocytes. As Lon upregulation plays a major role in the initiation of hypoxia-induced apoptosis and Caspase-3 is a marker protein for apoptosis, the nanoprobe has been successfully applied for imaging the activation of Lon-Caspase-3 apoptotic signalling pathway and assessing the state of cardiomyocytes under hypoxic conditions. Furthermore, combining with mitochondrial H2O2 probe-MitoPY1, the nanoprobe was also used to confirm the synergistic effect of Lon and ROS on hypoxia-induced apoptosis of cardiomyocytes and evaluate the function of ROS scavenger on attenuating such apoptosis. This work proposed a promising strategy for early diagnosis, prevention and treatment of hypoxic-ischemic myocardial dysfunction.

Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance.

Aims: Ischemia-reperfusion injury (IRI)-induced acute kidney injury (IRI-AKI) is characterized by elevated levels of reactive oxygen species (ROS), mitochondrial dysfunction, and inflammation, but the potential link among these features remains unclear. In this study, we aimed to investigate the specific role of mitochondrial ROS (mtROS) in initiating mitochondrial DNA (mtDNA) damage and inflammation during IRI-AKI.Methods: The changes in renal function, mitochondrial function, and inflammation in IRI-AKI mice with or without mtROS inhibition were analyzed in vivo. The impact of mtROS on TFAM (mitochondrial transcription factor A), Lon protease, mtDNA, mitochondrial respiration, and cytokine release was analyzed in renal tubular cells in vitro. The effects of TFAM knockdown on mtDNA, mitochondrial function, and cytokine release were also analyzed in vitro. Finally, changes in TFAM and mtDNA nucleoids were measured in kidney samples from IRI-AKI mice and patients.Results: Decreasing mtROS levels attenuated renal dysfunction, mitochondrial damage, and inflammation in IRI-AKI mice. Decreasing mtROS levels also reversed the decrease in TFAM levels and mtDNA copy number that occurs in HK2 cells under oxidative stress. mtROS reduced the abundance of mitochondrial TFAM in HK2 cells by suppressing its transcription and promoting Lon-mediated TFAM degradation. Silencing of TFAM abolished the Mito-Tempo (MT)-induced rescue of mitochondrial function and cytokine release in HK2 cells under oxidative stress. Loss of TFAM and mtDNA damage were found in kidneys from IRI-AKI mice and AKI patients.Conclusion: mtROS can promote renal injury by suppressing TFAM-mediated mtDNA maintenance, resulting in decreased mitochondrial energy metabolism and increased cytokine release. TFAM defects may be a promising target for renal repair after IRI-AKI.

Reduced peroxisomal import triggers peroxisomal retrograde signaling

Maintaining organelle function in the face of stress is known to involve organelle-specific retrograde signaling. Using Caenorhabditis elegans, we present evidence of the existence of such retrograde signaling for peroxisomes, which we define as the peroxisomal retrograde signaling (PRS). Specifically, we show that peroxisomal import stress caused by knockdown of the peroxisomal matrix import receptor prx-5/PEX5 triggers NHR-49/peroxisome proliferator activated receptor alpha (PPARα)- and MDT-15/MED15-dependent upregulation of the peroxisomal Lon protease lonp-2/LONP2 and the peroxisomal catalase ctl-2/CAT. Using proteomic and transcriptomic analyses, we show that proteins involved in peroxisomal lipid metabolism and immunity are alsoupregulated upon prx-5(RNAi). While the PRS can be triggered by perturbation of peroxisomal β-oxidation, we also observed hallmarks of PRS activation upon infection with Pseudomonas aeruginosa. We propose that the PRS, in addition to a role in lipid metabolism homeostasis, may act as a surveillance mechanism to protect against pathogens.

DNA damage-signaling, homologous recombination and genetic mutation induced by 5-azacytidine and DNA-protein crosslinks in Escherichia coli

Covalent linkage between DNA and proteins produces highly toxic lesions and can be caused by commonly used chemotherapeutic agents, by internal and external chemicals and by radiation. In this study, using Escherichia coli, we investigate the consequences of 5-azacytidine (5-azaC), which traps covalent complexes between itself and the Dcm cytosine methyltransferase protein. DNA protein crosslink-dependent effects can be ascertained by effects that arise in wild-type but not in dcmΔ strains. We find that 5-azaC induces the bacterial DNA damage response and stimulates homologous recombination, a component of which is Dcm-dependent. Template-switching at an imperfect inverted repeat (“quasipalindrome”, QP) is strongly enhanced by 5-azaC and this enhancement was entirely Dcm-dependent and independent of double-strand break repair. The SOS response helps ameliorate the mutagenic effect of 5-azaC but this is not a result of SOS-induced DNA polymerases since their induction, especially PolIV, seems to stimulate QP-associated mutagenesis. Cell division regulator SulA was also required for recovery of QP mutants induced by 5-azaC. In the absence of Lon protease, Dcm-dependent QP-mutagenesis is strongly elevated, suggesting it may play a role in DPC tolerance. Deletions at short tandem repeats, which occur likewise by a replication template-switch, are elevated, but only modestly, by 5-azaC. We see evidence for Dcm-dependent and-independent killing by 5-azaC in sensitive mutants, such as recA, recB, and lon; homologous recombination and deletion mutations are also stimulated in part by a Dcm-independent effect of 5-azaC. Whether this occurs by a different protein/DNA crosslink or by an alternative form of DNA damage is unknown

Article of Significant Interest in This Issue

In bacteria, the Lon protease destroys poor-quality polypeptides to prevent the toxic accumulation of misfolded proteins. However, Lon also degrades essential regulatory proteins, such as DnaA in Caulobacter crescentus; hence, activity must be carefully monitored. Barros et al. (e00344-20) show that the Lon protease is itself degraded in vivo. This process requires active Lon proteases and requires carboxy-terminal residues also found in various Lon orthologs. In Caulobacter, expression of stabilized Lon drastically reduces DnaA levels, leading to cell death. They suggest that this posttranslational control balances the requirement for Lon with the toxicity associated with unconstrained proteolysis.

Reduced Lon protease 1 expression in podocytes contributes to the pathogenesis of podocytopathy

Emerging evidence has shown that mitochondrial dysfunction is closely related to the pathogenesis of podocytopathy, but the molecular mechanisms mediating mitochondrial dysfunction in podocytes remain unclear. Lon protease 1 is an important soluble protease localized in the mitochondrial matrix, although its exact role in podocyte injury has yet to be determined. Here we investigated the specific role of this protease in podocyte in glomerular injury and the progression of podocytopathy using podocyte-specific Lon protease 1 knockout mice, murine podocytes in culture and kidney biopsy samples from patients with focal segmental glomerular sclerosis and minimal change disease. Downregulated expression of Lon protease 1 was observed in glomeruli of kidney biopsy samples demonstrating a negative correlation with urinary protein levels and glomerular pathology of patients with focal segmental glomerular sclerosis and minimal change disease. Podocyte-specific deletion of Lon protease 1 caused severe proteinuria, impaired kidney function, severe kidney injury and even mortality in mice. Mechanistically, we found that continuous podocyte Lon protease 1 ablation induced mitochondrial homeostasis imbalance and dysfunction, which then led to podocyte injury and glomerular sclerosis. Invitro experiments implicated the kidney protective effect of Lon protease 1, which inhibited mitochondrial dysfunction and podocyte apoptosis. Thus, our findings suggest that the regulation of Lon protease 1 in podocytes may provide a novel therapeutic approach for the podocytopathy.

LysR-type transcriptional regulator of high temperature inducible class A β-lactamase gene controls pathogenic R-body production genes in Azorhizobium caulinodans

ABSTRACT Azorhizobium caulinodans ORS571 is a rhizobium of the tropical legume Sesbania rostrata. This bacterium possesses the reb operon, which is associated with pathogenic R-bodies. To establish symbiosis with host plants, the expression of reb operon is suppressed at a low level by multiple regulators, such as PraR transcription factor and Lon protease. The reb operon is highly expressed in the presence of 2-oxoglutarate at temperatures lower than the optimum growth temperatures (37–38°C). 2-oxoglutarate has been found to inhibit the binding of PraR to the promoter region of the reb operon, but it is still unclear how temperature effects reb operon expression. Therefore, the purpose of our research is to clarify the temperature-depending expression mechanism. In this study, we found that PenR, which is a LysR-type transcriptional regulator of a class A β-lactamase gene (penA), is involved in reb operon expression. The reb operon of the praR mutant was highly expressed at 37°C, but not at 38°C, while the reb operon of the praR penR mutant was highly expressed even at 38°C. We also found that the expression of penA is temperature-responsive and under the control of Lon protease. penA of the wild type (WT) strain was more highly expressed at higher growth temperatures in the range of 25°C to 41°C, and the expression levels of penA in the lon mutant were lower than those in the WT strain at any growth temperatures. The penA of the penR mutant was hardly expressed irrespective of growth temperatures. Electrophoretic mobility shift assay, using His6-tagged PenR, revealed PenR bound to the promoter region of penA, but not to that of the reb operon. From these results, it was concluded that, in response to temperature, PenR represses the expression of the reb operon in an indirect manner, and induces penA in a direct manner, respectively.

Transcriptomic Analysis of Shiga Toxin-Producing Escherichia coli during Initial Contact with Cattle Colonic Explants.

Foodborne pathogens are a public health threat globally. Shiga toxin-producing Escherichia coli (STEC), particularly O26, O111, and O157 STEC, are often associated with foodborne illness in humans. To create effective preharvest interventions, it is critical to understand which factors STEC strains use to colonize the gastrointestinal tract of cattle, which serves as the reservoir for these pathogens. Several colonization factors are known, but little is understood about initial STEC colonization factors. Our objective was to identify these factors via contrasting gene expression between nonpathogenic E. coli and STEC. Colonic explants were inoculated with nonpathogenic E. coli strain MG1655 or STEC strains (O26, O111, or O157), bacterial colonization levels were determined, and RNA was isolated and sequenced. STEC strains adhered to colonic explants at numerically but not significantly higher levels compared to MG1655. After incubation with colonic explants, flagellin (fliC) was upregulated (log2 fold-change = 4.0, p < 0.0001) in O157 STEC, and collectively, Lon protease (lon) was upregulated (log2 fold-change = 3.6, p = 0.0009) in STEC strains compared to MG1655. These results demonstrate that H7 flagellum and Lon protease may play roles in early colonization and could be potential targets to reduce colonization in cattle.

Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering

Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY+. This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY+ was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression.