Lon Gene Research Articles

Mutants of Burkholderia cenocepacia with a change in synthesis of N-acyl-homoserine lactones—Signal molecules of quorum sensing regulation

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.

A genetically engineered derivative of Salmonella Enteritidis as a novel live vaccine candidate for salmonellosis in chickens

To construct a novel live Salmonella Enteritidis (SE) vaccine candidate, SE was genetically engineered using the allelic exchange method to delete two virulence genes, lon and cpxR. The lon gene deletion is essential to impair Salmonella replication and avoid overwhelming systemic disease in the host. The cpxR gene deletion is needed to enhance the ability of bacteria to adhere and invade the host cell. Scanning electron microscopy revealed that the derivatives JOL917 (Δlon), JOL918 (ΔcpxR), and JOL919 (Δlon/ΔcpxR) had increased surface fimbrial filamentous structures. Significant elevations of extracellular polysaccharide and FimA expression were observed for the derivatives compared to the parental wild type JOL860, while biochemical properties of the derivatives were not altered. In the safety examination by inoculation of the derivatives in chickens, gross lesion scores of the liver, spleen, kidney, small intestine and caecal tonsils were moderate in the JOL917 and JOL918 groups, and significantly lower in the JOL919 group than those of the JOL860. Bacterial counts from the spleen and caeca of the JOL917 and JOL918 groups were moderate, and significantly reduced in the JOL919 group compared to the JOL860 group. In addition, only the JOL919 group showed significantly lower bacterial counts in the faecal samples than those of the JOL860 group. Significant elevations of IgG and secretory IgA levels observed in the derivative groups, while the JOL919 and JOL860 groups showed a potent lymphocyte proliferation response as compared to those of the control group. In the protection efficacy examination, JOL919 immunized group showed significantly lower depression, lower gross lesion in the liver and spleen, and lower number of the SE positive internal organs than those of the control group against a virulent wild type SE challenge.

The multifaceted role of Lon proteolysis in seedling establishment and maintenance of plant organelle function: living from protein destruction

Intracellular selective proteolysis is an important post-translational regulatory mechanism maintaining protein quality control by removing defective, damaged or even deleterious protein aggregates. The ATP-dependent Lon protease is a key component of protein quality control that is highly conserved across the kingdoms of living organisms. Major advancements have been made in bacteria and in non-plant organisms to understand the role of Lon in protection against protein oxidation, ageing and neurodegenerative diseases. This review presents the progress currently made in plants. The Lon gene family in Arabidopsis consists of four members that produce distinct protein isoforms localized in several organelles. Lon1 and Lon4 that potentially originate from a recent gene duplication event are dual-targeted to mitochondria and chloroplasts through distinct mechanisms revealing divergent evolution. Arabidopsis mutant analysis showed that mitochondria and peroxisomes biogenesis or maintenance of function is modulated by Lon1 and Lon2, respectively. Consequently, the lack of Lon selective proteolysis leading to growth retardation and impaired seedling establishment can be attributed to defects in the oil reserve mobilization pathway. The current progress in Arabidopsis research uncovers the role of Lon in the proteome homeostasis of plant organelles and stimulates biotechnology scenarios of plant tolerance against harsh abiotic conditions because of climate instability.

Negative regulation of pathogenesis in Pseudomonas syringae pv. tabaci 11528 by ATP-dependent Lon protease.

Pseudomonas syringae pv. tabaci causes wildfire disease in tobacco plants. The hrp pathogenicity island (hrp PAI) of P. syringae pv. tabaci encodes a type III secretion system (TTSS) and its regulatory system, which are required for pathogenesis in plants. Three important regulatory proteins-HrpR, HrpS, and HrpL-have been identified to activate hrp PAI gene expression. The bacterial Lon protease regulates the expression of various genes. To investigate the regulatory mechanism of the Lon protease in P. syringae pv. tabaci 11528, we cloned the lon gene, and then a Δlon mutant was generated by allelic exchange. lon mutants showed increased UV sensitivity, which is a typical feature of such mutants. The Δlon mutant produced higher levels of tabtoxin than the wild-type. The lacZ gene was fused with hrpA promoter and activity of β-galactosidase was measured in hrp-repressing and hrp-inducing media. The Lon protease functioned as a negative regulator of hrp PAI under hrp-repressing conditions. We found that strains with lon disruption elicited the host defense system more rapidly and strongly than the wild-type strain, suggesting that the Lon protease is essential for systemic pathogenesis.

Immunogenicity of a Salmonella Enteritidis mutant as vaccine candidate and its protective efficacy against salmonellosis in chickens

A novel Salmonella Enteritidis (SE) vaccine candidate strain, JOL919 was constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. The study was carried out to evaluate the strain as a vaccine candidate against salmonellosis. The strain showed the enhanced macrophage invasion, early bacterial clearance and higher immune responses as compared to the other mutants, JOL917 ( Δlon) and JOL918 ( ΔcpxR), and the wild type. In further analysis, the chickens immunized with JOL919 showed a significant increase in plasma IgG and intestinal secretory IgA levels, which was an indication of robust humoral and mucosal immune responses induced by the candidate. The lymphocyte proliferation response and CD45 +CD3 + T cells, associated with an activation of T helper and cytotoxic cells, were also significantly increased in the immunized group, which indicated that the candidate also induced cellular immune responses. The immune cell influx into caecal tissues analyzed by immunohistochemistry showed that CD8 + T cells were predominated in the immunized group, suggesting that the candidate can clear the invaded pathogen in the intestines by a more direct way involving cytotoxic activity. By the examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate can provide an efficient protection upon virulent challenge.

Lon Protease Quality Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40) Chaperones

Various environmental insults result in irreversible damage to proteins and protein complexes. To cope, cells have evolved dedicated protein quality control mechanisms involving molecular chaperones and proteases. Here, we provide both genetic and biochemical evidence that the Lon protease and the SecB and DnaJ/Hsp40 chaperones are involved in the quality control of presecretory proteins in Escherichia coli. We showed that mutations in the lon gene alleviate the cold-sensitive phenotype of a secB mutant. Such suppression was not observed with either clpP or clpQ protease mutants. In comparison to the respective single mutants, the double secB lon mutant strongly accumulates aggregates of SecB substrates at physiological temperatures, suggesting that the chaperone and the protease share substrates. These observations were extended in vitro by showing that the main substrates identified in secB lon aggregates, namely proOmpF and proOmpC, are highly sensitive to specific degradation by Lon. In contrast, both substrates are significantly protected from Lon degradation by SecB. Interestingly, the chaperone DnaJ by itself protects substrates better from Lon degradation than SecB or the complete DnaK/DnaJ/GrpE chaperone machinery. In agreement with this finding, a DnaJ mutant protein that does not functionally interact in vivo with DnaK efficiently suppresses the SecB cold-sensitive phenotype, highlighting the role of DnaJ in assisting presecretory proteins. Taken together, our data suggest that when the Sec secretion pathway is compromised, a pool of presecretory proteins is transiently maintained in a translocation-competent state and, thus, protected from Lon degradation by either the SecB or DnaJ chaperones.

Changes of physiological and biochemical properties of Salmonella enterica serovar Typhimurium by deletion of cpxR and lon genes using allelic exchange method

To construct a novel Salmonella attenuated live vaccine, the cpxR and lon genes were deleted from a wild-type Salmonella enterica serovar Typhimurium ( S. Typhimurium) using allelic exchange method, resulting in S. Typhimurium CK31 (Δ cpxR), CK38 (Δ lon), and CK111 (Δ cpxR/ lon). These mutated strains were grown normally, as was the wild-type strain. The biochemical properties of the mutants remained highly similar to those of the wild-type. In comparison with the wild-type, 1.5 to 3.3-fold increases of fimbrial products such as Agf, Fim, and Pef fimbria in the mutants CK31, CK38, and CK111 were observed by using a transmission electron microscope and dot blotting. Furthermore, CK38 and CK111 morphologically appeared elongated in shape and produced 2.0- and 3.2-fold increases, respectively, of capsular polysaccharide, which is a major antigenic component. Approximately 10 4-fold attenuation assessed by analysis of LD 50 of BALB/c mouse was observed by deleting the lon/cpxR (CK111) genes. This result indicated that deletion of lon and cpxR genes induced significant attenuation.

Comparative analysis of Salmonella enterica serovar Enteritidis mutants with a vaccine potential

If any new live Salmonella vaccine is introduced in the future, it is quite probable that detailed characterisation of its attenuation will be required. In this study we therefore compared 34 isogenic mutants of S. Enteritidis in aroA, aroD, galE, ssrA, sseA, phoP, rpoS, ompR, htrA, clpP, lon, rfaL, rfaG, rfaC, hfq, sodCI, hilA, sipA, avrA, sopB, sopA, sopE, sifA, shdA, fliC, fur, relA, spoT, rel-spoT, misL, rmbA, STM4258, STM4259 and spvBC genes for their resistance to stresses likely to be expected in the host and for their virulence and immunogenicity in Balb/C mice. We found that the cold and bile resistances essentially did not correlate with the resistances to other stress factors. Resistance to acid pH, heat, polymyxin and serum correlated with each other and also with the attenuation. When the residual virulence and immunogenicity were both considered, mutants in htrA, ompR, aroA, aroD and lon performed the best in mice. Furthermore, when a detailed comparison of polymyxin and serum sensitive mutants was performed, the serum sensitive mutants were more immunogenic.

Regulation of biofilm formation in Escherichia coli K12: Effect of mutations in the genes HNS, STRA, LON, and RPON

More than 99% of bacteria exist in natural ecosystems as specifically organized biofilms adhering to solid surfaces. Biofilms have a typical architecture and are enclosed in exopolymeric matrix. Bacteria living in biofilms are extremely resistant to antibacterial factors. In this work, we studied the role of some global regulators of gene expression during biofilm formation by cells of Escherichia coli K12. The histone-like proteins H-NS and StpA were shown to play an essential role in the regulation of biofilm formation. Mutant strains deficient in H-NS or StpA generated biofilms poorly comparing with the wild-type isogenic strain. A double mutant deficient in both proteins was almost unable to the biofilm formation. Mutations in the rpoN gene, which encodes for sigma N subunit of RNA-polymerase, and the lon gene, which encodes for Lon-proteinase, increase the biofilm formation by 40–60%.

Influence of mutations in genes of global transcriptional regulators on production of autoinducer AI-2 in the Escherichia coli Quorum Sensing system

The control of gene expression in response to an increase in the bacterial population density (Quorum Sensing) involves low-molecular-weight signal molecules (autoinducers, AI). AI-2 and synthase LuxS mediating its synthesis are widely distributed in Gram-negative and Gram-positive bacteria. In this work, the data were obtained on the role of global regulators of gene expression in AI-2 synthesis in Escherichia coli cells. The mutation inactivating gene rpoS (encodes sigma S subunit of RNA polymerase) was shown to drastically decrease an amount of active AI-2 in the culture medium. Mutations in gene rpoN that encodes sigma N subunit of RNA polymerase and also in gene lon, which encodes Lon proteinase, on the contrary, increase an amount of active AI-2 in supernatants of cultures. Mutant strains lacking histone-like proteins H-NS and StpA accumulate a slightly higher amount of AI-2 than the isogenic wild-type strain: however, an amount of AI-2 decreased in the culture medium of the double mutant devoid of both these proteins.

Isolation and Characterization ofEscherichia coliMutants Exhibiting Altered Response to Thymol

The antimicrobial mode of action of the plant essential oil thymol was studied with Escherichia coli. Random transposon-insertion mutants were screened for altered response to thymol. Of four mutants showing more sensitivity, three were found in rfaQ, whose product is involved in lipopolysaccharide biosynthesis, and the fourth in the quorum-sensing gene qseC. Mutants showing more resistance had mutations in genes whose products are involved in the degradation of short-lived regulatory and abnormal proteins (the lon gene), menaquinone biosynthesis (menA), an unknown function of a putative membrane protein (yagF), synthesis of a small hypothetical protein (an intergene region between the two small genes yiiE and yiiF), and the efflux pump of cadaverine and lysine (cadB). The antibacterial activities of carvacrol, menthol, and cymene, essential oils structurally similar to thymol, were also determined. Although the level of resistance toward thymol was conserved in the respective mutants qseC, menA, and cadB, knockout mutants displayed different levels of tolerance to carvacrol; inconsistencies in resistance levels were also noted in mutants challenged with menthol. Wild-type and mutant E. coli responded to thymol exposure with a massive potassium efflux that generally corresponded to the resistant rate. The verity of the loci accounting for E. coli response suggests a multitarget mode of the antimicrobial activity of thymol and multitolerance mechanisms.

Control of Bacteriophage Mu Lysogenic Repression

The transposable and temperate phage Mu infects Escherichia coli where it can enter the lytic life-cycle or reside as a repressed and integrated prophage. The repressor protein Rep is the key element in the lysis-lysogeny decision. We have analyzed the fate of Rep in different mutants by Western blotting under two conditions that can induce a lysogen: high temperature and stationary phase. We show that, unexpectedly, Rep accumulates under all conditions where the prophage is completely derepressed, and that this accumulation is ClpX-dependent. An analysis of the degradation kinetics shows that Rep is a target of two protease systems: inactivation of either the clpP or lon gene results in a stabilization of Rep. Such a reaction scheme explains the counterintuitive observation that derepression is correlated with high repressor concentration. We conclude that under all conditions of phage induction the repressor is sequestered in a non-active form. A quantitative simulation accounts for our experimental data. It provides a model that captures the essential features of Mu induction and explains some of the mechanisms by which the physiological signals affecting the lysis-lysogeny decision converge onto Rep.

DNA methylation affects the cell cycle transcription of the CtrA global regulator in Caulobacter.

The Caulobacter chromosome changes progressively from the fully methylated to the hemimethylated state during DNA replication. These changes in DNA methylation could signal differential binding of regulatory proteins to activate or repress transcription. The gene encoding CtrA, a key cell cycle regulatory protein, is transcribed from two promoters. The P1 promoter fires early in S phase and contains a GAnTC sequence that is recognized by the CcrM DNA methyltransferase. Using analysis of CcrM mutant strains, transcriptional reporters integrated at different sites on the chromosome, and a ctrA P1 mutant, we demonstrate that transcription of the P1 promoter is repressed by DNA methylation. Moreover moving the native ctrA gene to a position near the chromosomal terminus, which delays the conversion of the ctrA promoter from the fully to the hemimethylated state until late in the cell cycle, inhibited ctrA P1 transcription, and altered the time of accumulation of the CtrA protein and the size distribution of swarmer cells. Together, these results show that CcrM-catalyzed methylation adds another layer of control to the regulation of ctrA expression.

Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism.

Mitochondrial aconitase is sensitive to oxidative inactivation and can aggregate and accumulate in many age-related disorders. Here we report that Lon protease, an ATP-stimulated mitochondrial matrix protein, selectively recognizes and degrades the oxidized, hydrophobic form of aconitase after mild oxidative modification, but that severe oxidation results in aconitase aggregation, which makes it a poor substrate for Lon. Similarly, a morpholino oligodeoxynucleotide directed against the lon gene markedly decreases the amount of Lon protein, Lon activity and aconitase degradation in WI-38 VA-13 human lung fibroblasts and causes accumulation of oxidatively modified aconitase. The ATP-stimulated Lon protease may be an essential defence against the stress of life in an oxygen environment. By recognizing minor oxidative changes to protein structure and rapidly degrading the mildly modified protein, Lon protease may prevent extensive oxidation, aggregation and accumulation of aconitase, which could otherwise compromise mitochondrial function and cellular viability. Aconitase is probably only one of many mitochondrial matrix proteins that are preferentially degraded by Lon protease after oxidative modification.

The lon gene, encoding an ATP-dependent protease, is a novel member of the HAIR/HspR stress-response regulon in actinomycetes.

Members of a family of ATP-dependent proteases related to Lon from Escherichia coli are present in most prokaryotes and eukaryotes. These proteases are generally reported to be heat induced, and various regulatory systems have been described. The authors cloned and disrupted the lon gene and studied the regulation of its expression in Streptomyces lividans. lon is negatively regulated by the HspR/HAIR repressor/operator system, suggesting that Lon is produced concomitantly with the other members of this regulon, DnaK and ClpB. The lon mutant grew more slowly than the wild-type and spore germination was impaired at high temperature. Nevertheless its cell cycle was not greatly affected and it sporulated normally.

Cloning and characterization of a LON gene homologue from the human pathogen Paracoccidioides brasiliensis.

A LON gene homologue from the human pathogen Paracoccidioides brasiliensis (PbLON) has been cloned, sequenced and characterized. It encodes a putative ATP-dependent proteinase Lon, which in Saccharomyces cerevisisae (PIM1) is a heat-inducible protein involved in the degradation of abnormal or short-lived proteins in the mitochondria. The PbLON ORF is within a 3369 bp fragment interrupted by two introns located in the 3'segment. The 5' and 3' regions flanking the ORF contain sequences which resemble known transcription elements. Several transcription binding factor motifs have also been found, including sites for heat shock/stress response and nitrogen control. The deduced protein consists of 1063 residues containing a mitochondrial import signal at the N-terminus and conserved ATP-binding (GPPGVGKT) and serine catalytic (KDGPSAG) sites. It shares high identity with Lon homologues from S. cerevisiae (73%), Homo sapiens (62%) and Escherichia coli (56%). In P. brasiliensis, an MDJ1 putative gene has also been partially sequenced adjacent to PbLON, possibly sharing divergently orientated promoter elements. This chromosomal organization is interesting, since Mdj1p is a heat shock chaperone essential for substrate degradation by PIM1 in yeast.

Metabolic instability of Escherichia coli cyclopropane fatty acid synthase is due to RpoH-dependent proteolysis.

Cyclopropane fatty acids (CFAs) are generally synthesized as bacterial cultures enter stationary phase. In Escherichia coli, the onset of CFA synthesis results from increased transcription of cfa, the gene encoding CFA synthase. However, the increased level of CFA synthase activity is transient; the activity quickly declines to the basal level. We report that the loss of CFA activity is due to proteolytic degradation dependent on expression of the heat shock regulon. CFA synthase degradation is unaffected by mutations in the lon, clpP, and groEL genes or by depletion of the intracellular ATP pools. It seems likely that CFA synthase is the target of an unidentified energy-independent heat shock regulon protease. This seems to be the first example of heat shock-dependent degradation of a normal biosynthetic enzyme.

The ++Sinorhizobium meliloti lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa.

While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lon mutant total EPS was 5.0 +/- 0.2 (mean +/- standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 +/- 0.5. These data suggested that in addition to EPSI, the lon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced by S. meliloti, but typically is expressed only under conditions of phosphate limitation. (13)C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present in trans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.

Cloning and expression of the Campylobacter jejuni lon gene detected by RNA arbitrarily primed PCR

Fingerprinting of RNA by arbitrarily primed PCR was used to identify a heat-inducible gene in Campylobacter jejuni. Comparing RNA fingerprints from C. jejuni cells before and after 20 min of heat shock at 48°C, a differentially amplified PCR product was identified which displayed a high degree of homology to bacterial lon genes. By screening C. jejuni genomic libraries, the entire lon gene was cloned and sequenced. It encodes a protein of 791 amino acids with a calculated molecular mass of 90.2 kDa. Alignment of the Lon amino acid sequence with that of other bacterial species revealed an overall identity of up to 56.6% ( Helicobacter pylori). Northern and RNA dot blot experiments confirmed heat induction of the C. jejuni lon gene, revealing a maximum 6–8-fold increase in the level of specific mRNA.

The isolated proteolytic domain of Escherichia coli ATP-dependent protease Lon exhibits the peptidase activity

Selective protein degradation is an energy-dependent process performed by high-molecular-weight proteases. The activity of proteolytic components of these enzymes is coupled to the ATPase activity of their regulatory subunits or domains. Here, we obtained the proteolytic domain of Escherichia coli protease Lon by cloning the corresponding fragment of the lon gene in pGEX-KG, expression of the hybrid protein, and isolation of the proteolytic domain after hydrolysis of the hybrid protein with thrombin. The isolated proteolytic domain exhibited almost no activity toward protein substrates (casein) but hydrolyzed peptide substrates (melittin), thereby confirming the importance of the ATPase component for protein hydrolysis. Protease Lon and its proteolytic domain differed in the efficiency and specificity of melittin hydrolysis.