Lon Gene Research Articles

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 degrees 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.

Sequence and transcriptional analysis of clpX, a class-III heat-shock gene of Bacillus subtilis

The nucleotide sequence of clpX, which is localized between the tig (trigger factor) and the lon (ATP-dependent protease) genes at 245° on the standard Bacillus subtilis (Bs) genetic map, was determined. The putative clpX gene codes for a 46-kDa protein of 421 amino acid (aa) residues. A comparison of the deduced aa sequence with those of the recently described bacterial clpX gene products from Synechocystis sp., Escherichia coli (Ec), Haemophilus influenzae and Azotobacter vinelandii revealed strong similarities. However, in contrast to Ec, clpX and clpP of Bs are located at different loci on the chromosome and are transcribed as monocistronic genes. A heat-inducible σ A-like promoter was mapped upstream of the clpX structural gene, but no CIRCE element, characteristic of class-I heat-shock genes (e.g., groESL and dnaK), was found between the transcriptional and translational start sites. Although the majority of the heat-inducible general stress genes in Bs are under the control of the alternative sigma factor, σ B, the heat induction of clpX appears to be σ B-independent. The latter indicates that clpX belongs to class-III heat-inducible genes.

Cloning, nucleotide sequencing, and expression of the Azospirillum brasilense lon gene: involvement in iron uptake.

The lon gene of Escherichia coli encodes the lon (La) protease, which is associated with cellular protein degradation. A lon gene homolog from Azospirillum brasilense, a nitrogen-fixing soil bacterium which lives in association with the roots of cereal grasses, was cloned and characterized. The nucleotide sequence of the A. brasilense lon gene was determined. It contains an open reading frame that encodes a protein of 810 amino acids with a predicted molecular mass of about 90 kDa. The deduced amino acid sequence showed a high level of homology with the sequences of all the known lon gene products. An open reading frame homologous to the E. coli clpX gene was found in front of the lon gene. Transcriptional analysis showed that the lon gene of A. brasilense is induced by heat shock and that the mRNA is monocistronic. An A. brasilense mutant, with Tn5 inserted in the lon gene, was shown to be defective in iron uptake and failed to express two membrane proteins that are induced by iron starvation in the parental strain.

Physical mapping of several heat-shock genes in Pseudomonas aeruginosa and the cloning of the mopA (GroEL) gene.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide or amino acid motifs in heat-shock genes/proteins, we have physically mapped the dnaK, lon, and hptG genes of Pseudomonas aeruginosa. Hybridization data suggest that there is a single copy of the mopBA (GroES/GroEL) operon but several additional copies of mopA. In addition, the map coordinates for the rpoD, rpoS, and rpoH genes were determined. The mopA gene from the mopBA operon was cloned and sequenced. The protein product of this gene showed 79% amino acid identity to the Escherichia coli GroEL and 98% identity to the GroEL sequence from P. aeruginosa ATCC 27853. A number of discrepancies were found with the latter sequence.

Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene

The lon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of sigma 32-dependent heat shock genes. In this paper, we report the cloning and characterization of the lon gene from the gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the lon locus, which is localized upstream of the hemAXCDBL operon, was determined. The lon gene codes for an 87-kDa protein consisting of 774 amino acid residues. A comparison of the deduced amino acid sequence with previously described lon gene products from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins. Like the E. coli lon gene, the B. subtilis lon gene is induced by heat shock. Furthermore, the amount of lon-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin. The potential promoter region does not show similarities to promoters recognized by sigma 32 of E. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase E sigma A of B. subtilis. A second gene designated orfX is suggested to be transcribed together with lon and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.

Erratum: Requirement for the Yeast Gene LON in Intramitochondrial Proteolysis and Maintenance of Respiration

Figures 1 and 2B (p. 274) of the report "Requirement for the yeast gene LON in intramitochondrial proteolysis and maintenance of respiration" by C. K. Suzuki et al. (8 Apr., p. 273) were printed incorrectly. The areas indicated as shaded in the sequence shown in Fig. 1 were incorrectly blackened, and the background of the gel shown in Fig. 2B was incorrectly photographed. The correct figures are shown below. It should also have been noted that the sequence shown in Fig. 1 has been deposited in the Genome Sequence Databank, the EMBL Data Library, and the DNA Databank of Japan under accession number L28110. [See figure in the PDF file]

Requirement for the Yeast Gene LON in Intramitochondrial Proteolysis and Maintenance of Respiration

The role of protein degradation in mitochondrial homeostasis was explored by cloning of a gene from Saccharomyces cerevisiae that encodes a protein resembling the adenosine triphosphate (ATP)-dependent bacterial protease Lon. The predicted yeast protein has a typical mitochondrial matrix-targeting sequence at its amino terminus. Yeast cells lacking a functional LON gene contained a nonfunctional mitochondrial genome, were respiratory-deficient, and lacked an ATP-dependent proteolytic activity present in the mitochondria of Lon+ cells. Lon- cells were also impaired in their ability to catalyze the energy-dependent degradation of several mitochondrial matrix proteins and they accumulated electron-dense inclusions in their mitochondrial matrix.

Controlled high-level expression of the lon gene of Escherichia coli allows overproduction of Lon protease

Lon protease from Escherichia coli is an ATP-dependent protease which plays important roles in regulating the levels of specific proteins and in eliminating abnormal proteins. A major problem of working with Lon protease, the inability to substantially overproduce the enzyme, has been overcome by placing the lon gene under the control of an inducible trp promoter within a copy-number-controllable plasmid. Induction resulted in higher levels of production of the protease ( ~ 100 μg/ml of cell culture) than were previously possible. The enzyme has been purified to apparent homogeneity and shown to possess the characteristic ATP-dependent proteolytic activity. Sequence verification during DNA manipulations revealed differences from two previously published sequences for the lon gene.

An Imbalance of HU Synthesis Induces Mucoidy in Escherichia coli

Mutations in a number of loci, including the lon gene, dramatically increase the production of colanic acid capsular polysaccharide and render Escherichia coli K-12 mucoid. The lon gene, which encodes an ATP-dependenty protease, is localized at ten minutes on the E. coli map and is very closely linked to the hupB gene coding for one of the two subunits of the histone-like protein HU. Surprisingly the introduction of a multi-copy plasmid carrying either the hupB or hupA gene into a wild-type E. coli strain, results in the overproduction of one of the HU subunits and repression of the synthesis of the other without changing the overall concentration of HU, also renders the cells mucoid. As in a lon strain, the transcription of the cps genes, the structural genes for the synthesis of colanic acid, is induced dramatically. Protease Lon negatively regulates cps genes by destabilizing RcsA, a positive regulator of capsule synthesis. Regulation of HU synthesis does not affect the steady state level of Lon, as judged by Western blotting. The UV sensitivity of the hup transformed lon + bacteria is identical to the lon + parental strain, suggesting that Lon activity for the degradation of SulA in these cells is normal. Using lac operon fusions to cps gene promoters and to the rcsA promoter we show that the deregulation of HU synthesis does not by pass the positive regulatory action of RcsA and RcsB for the expression of cps genes but functions by stimulating RcsA synthesis.

The lonD gene is homologous to the lon gene encoding an ATP-dependent protease and is essential for the development of Myxococcus xanthus

Myxococcus xanthus contains two genes (lonV and lonD) homologous to the Escherichia coli lon gene for an ATP-dependent protease. We found that the lonD gene encodes a 90-kDa protein consisting of 827 amino acid residues. The lonD gene product shows 49, 48, and 52% sequence identity to the products of the M. xanthus lonV, E. coli lon, and Bacillus brevis lon genes, respectively. When a lonD-lacZ fusion was used, lonD was expressed during both vegetative growth and development. However, while lonD-disrupted strains were able to grow normally vegetatively, the development of M. xanthus was found to be arrested at an early stage in these strains. The mutant strains were able to form neither fruiting bodies nor myxospores.

Myxococcus xanthus encodes an ATP-dependent protease which is required for developmental gene transcription and intercellular signaling

The bsgA gene of Myxococcus xanthus plays an essential role in the regulation of early gene expression during fruiting body formation and sporulation. bsgA mutants behave as though unable to initiate a required cell-cell interaction and consequently fail to transcribe normal levels of many developmentally induced genes. We determined the nucleotide sequence of bsgA, which predicts a single gene encoding a 90.4-kDa protein. The deduced BsgA protein shares 45 and 48% amino acid identity with the lon genes of Escherichia coli and Bacillus brevis, respectively. The cloned bsgA gene was expressed in E. coli, and the BsgA protein was partially purified and found, like its E. coli homolog, to be an ATP-dependent protease. Thus, the basis for the phenotype of bsgA mutants is likely to be a defect in intracellular proteolysis.

Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth.

The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.

Protease II from Escherichia coli: Sequencing and Expression of the Enzyme Gene and Characterization of the Expressed Enzyme1

Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.

Aerobic inactivation of Rhizobium meliloti NifA in Escherichia coli is mediated by lon and two newly identified genes, snoB and snoC

The Rhizobium meliloti NifA protein is an oxygen-sensitive transcriptional regulator of nitrogen fixation genes. Regulation of NifA activity by oxygen occurs at the transcriptional level through fixLJ and at the posttranslational level through the sensitivity of NifA to oxygen. We have previously reported that the NifA protein is sensitive to oxygen in Escherichia coli as well as in R. meliloti. To investigate whether the posttranslational regulation of NifA is dependent on host factors conserved between R. meliloti and E. coli, we carried out a Tn5 mutagenesis of E. coli and isolated mutants with increased NifA activity under aerobic conditions. Fifteen insertion mutations occurred at three unlinked loci. One locus is the previously characterized lon gene; the other two loci, which we have named snoB and snoC, define previously uncharacterized E. coli genes. The products of snoC and lon affect the rate of NifA degradation, whereas the product of snoB may affect both NifA degradation and inactivation. A snoB lon double mutant showed a higher level of NifA accumulation than did a lon mutant, suggesting that the snoB product affects the ability of NifA to be degraded by a lon-independent pathway. The effects of a snoC mutation and lon mutation were not additive, suggesting that the snoC and lon products function in the same degradative pathway.

A Novel Expression System for Production of a Labile Protein in Escherichia coli by infection with Cytosin-substituting T4 Phage.

A novel expression system was developed for the high level production of a labile protein in Escherichia coli. The regulatory signal of bacteriophage T4 uvsY gene was fused in frame with the coding region of human ventricular myosin alkali light chain (VLC1) gene. Expression from the regulatory signal was enhanced and continued in a lysis-inhibition state by infection with a cytosine-substituting T4 phage mutant. VLC1 protein was produced at a low level without infection because of its instability in the cells. Although the productivity was partly improved in a lon-deficient mutant without infection, it was improved about 100-fold with T4 phage infection. T4 phage produces protease inhibitor(s) (pin gene product) against proteases of host cell including the lon gene product (protease La).

Role of instability in the cis action of the insertion sequence IS903 transposase.

An unusual subset of DNA-binding proteins, termed cis-acting proteins, has been shown to act preferentially at their site of synthesis; the transposases of several bacterial insertion sequences (ISs) fall into this class. The transposase of IS903 exhibits a strong preference for action in cis: complementation of defective transposons in trans occurs at less than 1%. Furthermore, transposition mediated by transposase acting in cis is extremely sensitive to the distance between the 3' end of the transposase gene and the nearest transposon inverted repeat; we find that an insertion of 1 kilobase of DNA reduces transposition to 1-2% of control levels. Here we show that there is a strong correlation between the stability of transposase and its ability to act in trans. We found that the wild-type transposase is a very unstable protein with a physical half-life of about 3 min. However, a transposase-beta-galactosidase fusion protein has a much greater half-life and can act equally well in cis or in trans. In addition, the native transposase is stabilized in lon- strains of Escherichia coli, and, in these protease-deficient strains, trans action of transposase is increased 10- to 100-fold. These results suggest that instability of the IS903 transposase is a major determinant of its cis action and that the La protease, product of the lon gene, is an important determinant of transposase instability.

Regulation of breakdown of canavanyl proteins inEscherichia coliby growth conditions inIon+andIon−cells

In vivo rates of proteolysis of canavanyl proteins were compared in lon+ and lon- Escherichia coli strains following growth in a variety of media. Both lon+ and lon- cells grown rapidly in complex media possessed higher levels of constitutive degradative activity than when cultured in minimal media. Pre-growth of lon+ cells in the presence of canavanine induced proteolytic activity following growth in minimal media as did stress agents such as heat, alcohol and puromycin: the lon mutant did not show the increased activity following canavanine treatment. The results suggest the presence of a proteolytic activity which selectively degrades aberrant proteins which does not involve protease La, the product of the lon gene, and which furthermore is regulated in part by growth conditions independently of the stress response.

GroE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis

Overexpression of the SOS-inducible umuDC operon of Escherichia coli results in the inability of these cells to grow at 30 degrees C. Mutations in several heat shock genes suppress this cold sensitivity. Suppression of umuD+C+-dependent cold sensitivity appears to occur by two different mechanisms. We show that mutations in lon and dnaK heat shock genes suppress cold sensitivity in a lexA-dependent manner. In contrast, mutations in groES, groEL, and rpoH heat shock genes suppress cold sensitivity regardless of the transcriptional regulation of the umuDC genes. We have also found that mutations in groES and groEL genes are defective in umuDC-dependent UV mutagenesis. This defect can be suppressed by increased expression of the umuDC operon. The mechanism by which groE mutations affect umuDC gene product function may be related to the stability of the UmuC protein, since the half-life of this protein is shortened because of mutations at the groE locus.

Unusual observations during construction of a new cloning vector providing lon gene expression in Escherichia coli

The plasmid pJMC40 containing the lon gene was transformed into E. coli HB101 leading to clones with either pJMC40 or a large plasmid designated pJMC40ins. pJMC40ins has a 1.3 kb DNA insert outside of the lon operon. Attempts to reduce the size of pJMC40 through circularization of a 6.7 kb EcoRI fragment containing the lon operon and larger parts of pBR322 failed. Circularization of an 8.0 kb EcoRI fragment of pJMC40ins, consisting of the 6.7 kb fragment and the 1.3 kb insert, produced a new plasmid which is useful as a small vector with a functional lon operon. The lon gene was cloned in vitro into the vector pUC18 setting it under the control of the lac promotor. Different attempts to transform this construct into E. coli failed. We suggest that the expression level of lon is very crucial for the viability of E. coli cells. Changes in regulation of lon expression expected to elevate La activity are lethal for E. coli.

Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La.

To learn more about the mechanism and regulation of the ATP-dependent protease La in Escherichia coli, the lon gene was completely sequenced using the dideoxy method on fragments generated by Bal31 digestion. The predicted amino acid composition based on the DNA sequence agreed well with the composition of the acid-hydrolyzed protease. The predicted NH2-terminal amino acid sequence, tryptophan content, and the carboxyl terminus also agreed with experimental data. However, the molecular weight of 87,000 (783 amino acids) calculated from the DNA sequence was lower than prior estimates. The tetrameric enzyme contains four binding sites for ATP, a DNA-binding domain, a proteolytic site, and a regulatory site that binds unfolded polypeptides. An ATP-binding pocket exists on each subunit as shown by consensus sequences and elements of secondary structure resembling those on other nucleotide-binding proteins (e.g. adenylate kinase, RecA). For this purpose, improved consensus patterns for identifying ATP-binding domains were developed. Computer-assisted comparisons, however, failed to demonstrate any regions homologous to sequences in other polypeptides including proteases or DNA-binding proteins. This enzyme also contains an unusual highly acidic domain surrounded by very basic sequences. Protease La is the first ATP-dependent protease sequenced and seems to represent a new type of enzyme. The promoter sequence was similar to consensus sequences for other heat-shock promoters. Using site-directed mutagenesis, alterations were introduced into the putative promoter sequence. Mutations upstream of -35 had little effect, but alterations immediately upstream of -10 lowered basal transcription of a lon-lacZ operon fusion and reduced its response to inducers of the heat-shock response.