Lon Gene Research Articles

Combined effect of polyphosphate kinase and lon protease in Streptomyces coelicolor A3(2) antibiotic production.

The bacterial stringent response is a global regulatory process in which polyphosphate kinase (Ppk) and lon protease are important players. Previous studies have shown that overexpression of the lon gene and deletion of the ppk gene significantly increased actinorhodin production in Streptomyces coelicolor (SCO). In this study, a recombinant SCOΔppk-lon cell, expressing the extra lon gene in Δppk cells, was simulated using a modified in silico (computational) model, ecSco-GEM, and the negative effect of Ppk on actinorhodin production was confirmed. In addition, we identified key enzymes that play a positive role in actinorhodin production. Of these, NADH dehydrogenase/complex-I, beta-ketoacyl-[acyl-carrier-protein] synthase III, glycine cleavage system, and superoxide dismutase were identified as the most significant. By confirming these results with experiments, we have shown that GEMs can be a reliable starting point for in vitro (lab-based) studies of Streptomyces..

Impact of a random TN5 mutation on endoglucanase secretion in ruminal cellulolytic Escherichia coli

ObjectiveMost protein secretion systems are found in gram-negative bacteria, but the mechanism of endoglucanase (BcsZ) secretion in Escherichia coli (E. coli) remains unclear. MethodsIn this study, we used JBZ-DH5α (which overexpresses BcsZ on the E. coli DH5α genome) as the initial strain. A mutant library was created by randomly inserting the TN5 transposon into the genome, and mutants with reduced transparent circles were identified on Congo red plates. The insertion sites of transposons in the genome were determined through whole-genome sequencing. ResultsThe results revealed that the genes rnc, lon, and suhB, which encode RNC-ribonuclease III (RNC), LON-protease (LON), and SuhB-inositol phosphatase (SuhB), respectively, were disrupted. BcsZ secretion decreased in E. coli DH5α when the lon, rnc, or suhB genes were deleted, but the overexpression of these genes restored their secretion levels. ConclusionThese findings suggest that the lon, rnc, and suhB genes play a role in BcsZ secretion in E. coli, potentially enhancing our knowledge of BcsZ secretion and offering a strategy to increase protein secretion in E. coli as a cell factory.

Intracellular Survival and Pathogenicity Modulation of Salmonella Lon, CpxR, and RfaL Mutants Used as Live Bacterial Vectors under Abiotic Stress, Unveiling the Link between Stress Response and Virulence in Epithelial Cells.

In the current study, two Salmonella Typhimurium strains, JOL 912 and JOL 1800, were engineered from the wild-type JOL 401 strain through in-frame deletions of the lon and cpxR genes, with JOL 1800 also lacking rfaL. These deletions significantly attenuated the strains, impairing their intracellular survival and creating unique immunological profiles. This study investigates the response of these strains to various abiotic stress conditions commonly experienced in vivo, including temperature, acidity, osmotic, and oxidative stress. Notably, cold stress induced a non-significant trend towards increased invasion by Salmonella compared to other stressors. Despite the observed attenuation, no significant alterations in entry mechanisms (trigger vs. zipper) were noted between these strains, although variations were evident depending on the host cell type. Both strains effectively localized within the cytoplasm, demonstrating their ability to invade and interact with the intracellular environment. Immunologically, JOL 912 elicited a robust response, marked by substantial activation of nuclear factor kappa B (NF-kB), and chemokines, interleukin 8 (CXCL 8) and interleukin 10 (CXCL 10), comparable to the wild-type JOL 401 (over a fourfold increase compared to JOL 1800). In contrast, JOL 1800 exhibited a minimal immune response. Additionally, these attenuations influenced the expression of cyclins D1 and B1 and caspases 3 and 7, indicating cell cycle arrest at the G2/M phase and promotion of the G0/G1 to S phase transition, alongside apoptosis in infected cells. These findings provide valuable insights into the mechanisms governing the association, internalization, and survival of Salmonella mutants, enhancing our understanding of their regulatory effects on host cell physiology.

Kosakonia radicincitans with hypervirulent lON genes causes human bloodstream infections.

Kosakonia radicincitans is a species within the new genus Kosakonia, which is typically a plant pathogen, with rare reports of human infection. The number of human infections may be underestimated because this new genus is under-represented among diagnostic tools. This report describes a case of bloodstream infection caused by K. radicincitans. The pathogen was identified by matrix-assisted laser desorption/ionization-TOF mass spectrometry and 16S rRNA gene sequencing. The hypervirulent human pathogenicity gene LON, which has not been described before, was detected in the bacterial genome by gene annotation. Thus, this discovery provides a new reference for studying the pathogenic mechanism of this rare pathogen.

Validation of Lon Gene Disruption using Linear DNA Cassette by Crelox Mechanism in E. coliStrains: To Achieve Better Solubility of Putrescine Monooxygenase.

The online version contains supplementary material available at 10.1007/s12088-023-01056-x.

Prospective lipid-A altered live attenuated Salmonella Gallinarum confers protectivity, DIVA capability, safety and low endotoxicity against fowl typhoid

The present study describes creating an attenuated Salmonella Gallinarum (SG) strain with reduced endotoxicity to prevent fowl typhoid. The strain was attenuated by deleting the lon, cpxR, and rfaL virulence-related genes. Endotoxicity was reduced by deleting the pagL open reading frame and replacing it with the lpxE gene derived from Francisella tularencis. Both events, (1) deletion of the pagL and (2) introduction of the lpxE genes, conferred reduced endotoxicity by detoxifying the lipid A structure. The detoxified SG strain (SGVSdt) was well tolerated in 7-day-old chicks when administered orally at 1 × 108 CFU/bird and in 14-day-old birds administered 1 × 107 CFU/bird subcutaneously. Parenteral immunization of detoxified vaccine strain was completely safe in birds and free of environmental contamination. Subcutaneous immunization conferred disease protection and induced humoral and cell-mediated immune responses marked by Th1-skewed patterns similar to those produced by the commercial SG9R vaccine strain. Compared with the SG9R-based vaccine, the SGVSdt construct generated significantly fewer inflammatory TNF-α responses while significantly inducing IFN-γ cytokine levels as an indication of an adaptive antibacterial response. The differentiating infected from vaccinated animals (DIVA) capability was on par with the predecessor SGVS. This study presents an appealing biological strategy to minimize lipid A-mediated endotoxicity without compromising protective efficacy against the SG challenge. Reduced endotoxicity permits the utilization of higher inoculation doses to maximize protection against fowl typhoid.

Emergence of a Hypervirulent Tigecycline-Resistant Klebsiella pneumoniae Strain Co-producing bla NDM-1 and bla KPC-2 With an Uncommon Sequence Type ST464 in Southwestern China.

Emergence of blaNDM–1 and blaKPC–2 co-producing Klebsiella pneumoniae strains is currently attracting widespread attention, but little information is available about their tigecycline resistance, virulence, and prevalence in Southwest China. In July 2021, an extensively drug-resistant K. pneumoniae strain AHSWKP25 whose genome contained both blaNDM–1 and blaKPC–2 genes was isolated from the blood of a patient with the malignant hematological disease in Luzhou, China. We investigated the resistance profiles of AHSWKP25 using microbroth dilution, agar dilution, modified carbapenemase inactivation (mCIM), and EDTA-modified carbapenemase inactivation methods (eCIM). The virulence of AHSWKP25 was assessed through string tests, serum killing assays, and a Galleria mellonella larval infection model. Conjugation and plasmid stability experiments were conducted to determine the horizontal transfer capacity of plasmids. And efflux pump phenotype test and real-time quantitative reverse transcription-PCR (RT-PCR) were used to determine its efflux pump activity. Sequencing of AHSWKP25 determined that AHSWKP25 belonged to ST464, which is resistant to antibiotics such as carbapenems, tetracycline, fluoroquinolones, tigecycline, and fosfomycin. The efflux pump phenotype tests and RT-PCR results demonstrated that efflux pumps were overexpressed in the AHSWKP25, which promoted the tigecycline resistance of the bacteria. AHSWKP25 also showed hypervirulence and serum resistance in vitro model. AHSWKP25 carried several different plasmids that contained blaNDM–1, blaKPC–2, and mutated tet(A) genes. Sequence alignment revealed that the plasmids carrying blaNDM–1 and blaKPC–2 underwent recombination and insertion events, respectively. We demonstrated that an X3 plasmid carrying blaNDM–1 was transferred from pSW25NDM1 to E. coli J53. We also identified missense mutations in the ramR, rcsA, lon, and csrD genes of AHSWKP25. Our results highlighted the potential of blaNDM–1 and blaKPC–2 co-producing K. pneumoniae strains to further develop antimicrobial resistance and hypervirulent phenotypes, but measures should be taken to closely monitor and control the spread of superbugs with multidrug-resistant phenotypes and hypervirulence.

Combinatorial metabolic engineering of Escherichia coli for de novo production of 2′-fucosyllactose

2′-Fucosyllactose (2′-FL) is a kind of fucosylated lactose of human milk oligosaccharides (HMOs). In this work, Escherichia coli MG1655 was metabolically engineered to increase 2′-FL production. The 2′-FL titer was raised from 0.0118 to 0.8062 g/L by increasing three gene copies of α1,2-fucosyltransferase (FutC) and by deleting the lon, wcaJ, lacZ, and lacI genes in the competitive pathways. Additionally, the 2′-FL titer was raised to 2.9 g/L by fusing a TrxA tag at the N-terminus of FutC, and then to 3.4 g/L by deleting glutathione reductase (Gor). Finally, the 2′-FL reached 3.3 g/L in the final strain E. coli MG27 through the de novo pathway in shake flask, and reached 10.3 g/L in a 3-L fermentor.

Genetic interference exerted by Salmonella-delivered CRISPR/Cas9 significantly reduces the pathological burden caused by Marek\u2019s disease virus in chickens

Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek’s disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek’s disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.

Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae.

The highly conserved ATP-dependent Lon protease plays important roles in diverse biological processes. The lon gene is usually nonessential for viability; however, lon mutants of several bacterial species, although viable, exhibit cellular defects. Here, we show that a lack of Lon protease causes pleiotropic effects in the rice pathogen Burkholderia glumae. The null mutation of lon produced three colony types, big (BLONB), normal (BLONN), and small (BLONS), in Luria-Bertani (LB) medium. Colonies of the BLONB and BLONN types were re-segregated upon subculture, while those of the BLONS type were too small to manipulate. The BLONN type was chosen for further studies, as only this type was fully genetically complemented. BLONN-type cells did not reach the maximum growth capacity, and their population decreased drastically after the stationary phase in LB medium. BLONN-type cells were defective in the biosynthesis of quorum sensing (QS) signals and exhibited reduced oxalate biosynthetic activity, causing environmental alkaline toxicity and population collapse. Addition of excessive N-octanoyl-homoserine lactone (C8-HSL) to BLONN-type cell cultures did not fully restore oxalate biosynthesis, suggesting that the decrease in oxalate biosynthesis in BLONN-type cells was not due to insufficient C8-HSL. Co-expression of lon and tofR in Escherichia coli suggested that Lon negatively affects the TofR level in a C8-HSL-dependent manner. Lon protease interacted with the oxalate biosynthetic enzymes, ObcA and ObcB, indicating potential roles for the oxalate biosynthetic activity. These results suggest that Lon protease influences colony morphology, growth, QS system, and oxalate biosynthesis in B. glumae.

Effect of lon Protease Overexpression on Endotoxin Production and Stress Resistance in Bacillus thuringiensis.

Lon protease, an intracellular protease, plays a key role in cell homeostasis in bacteria and is involved in numerous physiological processes. In this work, we aimed to study the impact of Lon on the production of endotoxins and stress response in Bacillus thuringiensis, which is an important bioinsecticide alternative for toxic chemicals. For this purpose, lon gene was cloned into a multi-copy vector with its original promoter and transcriptional terminator and expressed in B. thuringiensis serovar israelensis ATCC 35,646. Our results showed that the recombinant lon gene transcribed and translated efficiently and the resulting protein was active. Although the sporulation efficiency of the recombinant strain was found to be reduced and its mobility impaired, overexpression of the lon gene triggered the production of endotoxin. Together with increased biofilm formation, recombinant strain exhibited significantly better adaptation to osmotic and heat shock stresses and UV exposure compared to wild type and the control strain with empty plasmid. This study suggested a possible link between Lon protease and the production of insecticide and stress response in B. thuringiensis and provides a platform for future studies focusing on enhancing bio-insecticidal production using this bacterium.

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.

Evaluation of Putative Type II Toxin-Antitoxin Systems and Lon Protease Expression in Shigella flexneri Following Infection of Caco-2 Cells

: Shigella flexneri causes bacillary dysentery in developing countries. Due to recent reports regarding antimicrobial resistance in human S. flexneri, finding alternative therapeutics is of vital importance. Toxin-antitoxin (TA) systems have recently been introduced as antimicrobial targets owing to their involvement in bacterial survival in stress conditions and “persister” cell formation. In this study, the presence of four TA loci were studied in S. flexneri ATCC 12022. The presence of genes coding for the identified TA loci and Lon protease were confirmed by the PCR method using specific primers. Caco-2 cell lines were then infected with this standard strain, and 8 and 24 h post-infection, expression levels of genes coding for the studied TA loci, and Lon protease were evaluated using a real-time PCR method. Expression of mazF, GNAT (Gcn5-related N-acetyltransferase), yeeU, pfam13975, and Lon genes showed 5.4, 9.8, 2.3, 2.7, and 13.8-fold increase, respectively, 8 h after bacterial invasion of the Caco-2 cell line. In addition, the expression of the aforementioned genes showed 4.8, 10.8, 2.3, 3.7, and 16.8-fold increase after 24 h. The GNAT and lon genes showed significantly higher expression levels compared to the control (P value < 0.05). However, the increase in the expression level of yeeU was the same at 8 h and 24 h post-infection. In addition, mazF expression level showed a slight decrease at 24 h compared to 8h post-infection. Genes coding for GNAT and Lon protease showed a significantly higher expression after invading the Caco-2 cell line. Therefore, targeting GNAT or Lon protease can be taken into consideration for finding novel antimicrobial drug strategies. The exact functions and mechanisms of TA systems in S. flexneri isolates are suggested to be experimentally determined.

Deletion of the lon gene augments expression of Salmonella Pathogenicity Island (SPI)-1 and metal ion uptake genes leading to the accumulation of bactericidal hydroxyl radicals and host pro-inflammatory cytokine-mediated rapid intracellular clearance

ABSTRACT In the present study, we characterized the involvement of Lon protease in bacterial virulence and intracellular survival in Salmonella under abiotic stress conditions resembling the conditions of a natural infection. Wild type (JOL401) and the lon mutant (JOL909) Salmonella Typhimurium were exposed to low temperature, pH, osmotic, and oxidative stress conditions and changes in gene expression profiles related to virulence and metal ion uptake were investigated. Expression of candidate genes invF and hilC of Salmonella Pathogenicity Island (SPI)-1 and sifA and sseJ of SPI-2 revealed that Lon protease controls SPI-1 genes and not SPI-2 genes under all stress conditions tested. The lon mutant exhibited increased accumulation of hydroxyl (OH·) ions that lead to cell damage due to oxidative stress. This oxidative damage can also be linked to an unregulated influx of iron due to the upregulation of ion channel genes such as fepA in the lon mutant. The deletion of lon from the Salmonella genome causes oxidative damage and increased expression of virulence genes. It also prompts the secretion of host pro-inflammatory cytokines leading to early clearance of the bacteria from host cells. We conclude that poor bacterial recovery from mice infected with the lon mutant is a result of disrupted bacterial intracellular equilibrium and rapid activation of cytokine expression leading to bacterial lysis.

Lon Protease Is Important for Growth Under Stressful Conditions and Pathogenicity of the Phytopathogen, Bacterium Dickeya solani.

The Lon protein is a protease implicated in the virulence of many pathogenic bacteria, including some plant pathogens. However, little is known about the role of Lon in bacteria from genus Dickeya. This group of bacteria includes important potato pathogens, with the most aggressive species, D. solani. To determine the importance of Lon for pathogenicity and response to stress conditions of bacteria, we constructed a D. solani Δlon strain. The mutant bacteria showed increased sensitivity to certain stress conditions, in particular osmotic and high-temperature stresses. Furthermore, qPCR analysis showed an increased expression of the lon gene in D. solani under these conditions. The deletion of the lon gene resulted in decreased motility, lower activity of secreted pectinolytic enzymes and finally delayed onset of blackleg symptoms in the potato plants. In the Δlon cells, the altered levels of several proteins, including virulence factors and proteins associated with virulence, were detected by means of Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) analysis. These included components of the type III secretion system and proteins involved in bacterial motility. Our results indicate that Lon protease is important for D. solani to withstand stressful conditions and effectively invade the potato plant.

Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli.

To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli. Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined. Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)=16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX=32-64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline. Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.

One Extra Copy of lon Gene Causes a Dramatic Increase in Actinorhodin Production by Streptomyces coelicolor A3(2).

ATP-dependent Lon protease plays important roles in different physiological processes, including cellular differentiation of the bacteria and is a part of an important stress response regulon (HspR/HAIR). In Streptomyces, biosynthesis of secondary metabolites starts with cellular differentiation and stress is one of the factor that affect metabolite production. To clarify the effect of Lon protease on secondary metabolite production, we constructed a recombinant strain of Streptomyces coelicolor A3(2) that has one extra copy of lon gene with its own promoter and transcriptional terminator in its genome. Expression of lon gene in the recombinant strain was determined by quantitative real time (RT-qPCR). Actinorhodin and undecylprodigiosin production of the recombinant cell was measured in liquid R2YE and it was found to produce about 34 times more actinorhodin and 9 times more undecylprodigiosin than the wild-type at 168h of growth. Development of stable Streptomyces strains capable of producing high amounts of secondary metabolites is valuable for biotechnology industry. One extra copy of lon gene is enough to boost antibiotic production by S. coelicolor A3(2) and this change do not cause any metabolic burden in the cell.

Improvement of pyoluteorin production in Pseudomonas protegens H78 through engineering its biosynthetic and regulatory pathways.

Pyoluteorin (Plt) is a PKS-NRPS hybrid antibiotic that is produced by Pseudomonas spp. and shows strong antifungal and antibacterial activities. Pseudomonas protegens H78, which was isolated from the rape rhizosphere in Shanghai, can produce a large array of secondary metabolites, including antibiotics and siderophores. Plt is produced at low levels in the H78 wild-type strain. This study aimed to improve Plt production through combinatory genetic engineered strategies. Plt production was significantly enhanced (by14.3-fold) in the strain engineered by the following steps: (1) deletion of the translational repressor gene rsmE in the Gac/Rsm-RsmE pathway; (2) deletion of the ATP-dependent protease gene lon that encodes a potential enzyme that degrades positive regulators; (3) deletion of the negative regulatory gene pltZ of the Plt ABC-type transporter operon pltIJKNOP; (4) deletion of an inhibitory sequence within the operator of the transcriptional activator gene pltR; and (5) overexpression of the pltIJKNOP transport operon. The Plt production of the final engineered strain was increased to 214 from 15μgml-1 in the H78 wild-type strain. In addition, the pltA gene in the pltLABCDEFG biosynthetic operon was characterized as the gene encoding the rate-limiting enzyme in the Plt biosynthetic pathway of H78. However, overexpression of the rate-limiting enzyme gene pltA or the transcriptional activator gene pltR did not further improve Plt biosynthesis in the above multiple-gene knockout strains.

LoiA directly represses lon gene expression to activate the expression of Salmonella pathogenicity island-1 genes

Salmonella enterica serovar Typhimurium uses virulence factors encoded by Salmonella pathogenicity island 1 (SPI-1) to invade the intestinal epithelium. The low oxygen-induced transcriptional regulator LoiA is established to be a positive regulator of SPI-1 genes and can also repress the expression of the ATP-dependent Lon protease, a negative regulator of SPI-1 genes. Whether the repression of lon by LoiA is associated with its regulation of SPI-1 genes, as well as the regulatory mechanism involved, is unknown. In this study, we showed that LoiA directly represses the expression of the lon gene during invasion by binding to the promoter region of lon. The activation of S. Typhimurium SPI-1 gene expression by LoiA is associated with its repression of lon. Moreover, through invasion and mouse infection assays, we found that the repression of lon by LoiA contributes to S. Typhimurium invasion of intestinal epithelial cells and virulence in mice. However, LoiA does not influence lon expression during S. Typhimurium growth in macrophages, which may be associated with the low expression level of loiA in macrophages. Collectively, these findings establish the direct regulation of lon by LoiA and describe a novel mechanism by which LoiA regulates SPI-1 gene expression.

Comprehensive analysis of Lon proteases in plants highlights independent gene duplication events.

The degradation of damaged proteins is essential for cell viability. Lon is a highly conserved ATP-dependent serine-lysine protease that maintains proteostasis. We performed a comparative genome-wide analysis to determine the evolutionary history of Lon proteases. Prokaryotes and unicellular eukaryotes retained a single Lon copy, whereas multicellular eukaryotes acquired a peroxisomal copy, in addition to the mitochondrial gene, to sustain the evolution of higher order organ structures. Land plants developed small Lon gene families. Despite the Lon2 peroxisomal paralog, Lon genes triplicated in the Arabidopsis lineage through sequential evolutionary events including whole-genome and tandem duplications. The retention of Lon1, Lon4, and Lon3 triplicates relied on their differential and even contrasting expression patterns, distinct subcellular targeting mechanisms, and functional divergence. Lon1 seems similar to the pre-duplication ancestral gene unit, whereas the duplication of Lon3 and Lon4 is evolutionarily recent. In the wider context of plant evolution, papaya is the only genome with a single ancestral Lon1-type gene. The evolutionary trend among plants is to acquire Lon copies with ambiguous pre-sequences for dual-targeting to mitochondria and chloroplasts, and a substrate recognition domain that deviates from the ancestral Lon1 type. Lon genes constitute a paradigm of dynamic evolution contributing to understanding the functional fate of gene duplicates.