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.     

Cloning, expression, and characterization of the lon gene of Erwinia amylovora: evidence for a heat shock response.

The gene encoding the Lon protease of Erwinia amylovora has been cloned by complementation of an Escherichia coli lon mutant. Analysis of the determined nucleotide sequence of the lon gene revealed extensive homology to the nucleotide sequences of cloned lon genes from E. coli, Myxococcus xanthus, and Bacillus brevis. The predicted amino acid sequence of the E. amylovora Lon protease was 94, 59, and 54% identical to the predicted amino acid sequences of the Lon proteases of E. coli, M. xanthus, and B. brevis, respectively. The -10 and -35 promoter regions of the cloned lon gene had extensive homology to the respective consensus sequences of E. coli heat shock promoters. Promoter mapping of the lon gene located the start site 7 bases downstream of the -10 region. Cloning of the lon promoter upstream of a cat reporter gene demonstrated that expression of the E. amylovora lon gene was inducible by a heat shock. This is the first demonstration of a heat shock-regulated gene in E. amylovora. Site-directed mutagenesis of the -10 region of the lon promoter confirmed that the heat shock expression of the E. amylovora lon gene may be mediated by a sigma 32-like factor. Insertional inactivation of the E. amylovora chromosomal lon gene confirmed that the lon gene was not essential for either vegetative growth or infection of apple seedlings. E. amylovora lon mutants had increased sensitivity to UV irradiation and elevated levels of extracellular polysaccharide, suggesting comparable roles for the Lon proteases in both E. amylovora and E. coli.     

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.     

Cloning, characterization, and inactivation of the Bacillus brevis lon gene.

A gene of Bacillus brevis HPD31 analogous to the Escherichia coli lon gene has been cloned and characterized. The cloned gene (B. brevis lon gene) encodes a polypeptide of 779 amino acids with a molecular weight of 87,400 which resembles E. coli protease La, the lon gene product. Fifty-two percent of the amino acid residues of the two polypeptides were identical. The ATP-binding sequences found in E. coli protease La were highly conserved. The promoter of the B. brevis lon gene resembled that recognized by the major RNA polymerase of Bacillus subtilis and did not contain sequences homologous to the E. coli heat shock promoters. The B. brevis lon gene was inactivated by insertion of the neomycin resistance gene. A mutant B. brevis carrying the inactivated lon gene showed diminished ability for the degradation of abnormal polypeptides synthesized in the presence of puromycin.     

lon gene product of Escherichia coli is a heat-shock protein

The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins. Purified Lon protease migrated with the prominent cellular protein H94.0 in E. coli K-12 strains. Peptide map patterns of Lon protease and H94.0 were identical. A mutant form of the protease had altered mobility during gel electrophoresis. An E. coli B/r strain that is known to be defective in Lon function contained no detectable H94.0 protein under normal growth conditions. Upon a shift to 42 degrees C, however, the Lon protease was induced to high levels in K-12 strains and a small amount of protein became detectable at the H94.0 location in strain B/r. Heat induction of Lon protease was dependent on the normal allele of the regulatory gene, htpR, establishing lon as a member of the high-temperature-production regulon of E. coli.     

Cloning, structure and expression of the full-size lon gene in Escherichia coli coding for ATP-dependent La-proteinase

Assembly of the full Escherichia coli K-12 lon gene from the EcoRI--SphI fragment of the bacterial DNA ("modified" gene) cloned and sequenced earlier and the PstI fragment of the same DNA containing 3'-terminal region of the lon gene has been performed. Both "modified" and full genes showed all phenotype properties of lon gene. The complete nucleotide sequence of the gene (2770 bp) coding for the 784 amino acid sequence of protease La was determined. Location of catalytically active serine, histidine and aspartic acid residues was suggested, and ATP-binding site found. The lon gene and protease La structures we found are compared with those described independently and differences observed are discussed.     

Regulatory region of the heat shock-inducible capR (lon) gene: DNA and protein sequences.

The CapR protein is an ATP hydrolysis-dependent protease as well as a DNA-stimulated ATPase and a nucleic acid-binding protein. The sequences of the 5' end of the capR (lon) gene DNA and N-terminal end of the CapR protein were determined. The sequence of DNA that specifies the N-terminal portion of the CapR protein was identified by comparing the amino acid sequence of the CapR protein with the sequence predicted from the DNA. The DNA and protein sequences established that the mature protein is not processed from a precursor form. No sequence corresponding to an SOS box was found in the 5' sequence of DNA. There were sequences that corresponded to a putative -35 and -10 region for RNA polymerase binding. The capR (lon) gene was recently identified as one of 17 heat shock genes in Escherichia coli that are positively regulated by the product of the htpR gene. A comparison of the 5' DNA region of the capR gene with that of several other heat shock genes revealed possible consensus sequences.     

Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable.

The lon gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in the intracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, such as UV sensitivity and overproduction of capsular polysaccharide. We have isolated lambda transducing phage carrying the lon gene and used the lon phage as a target for insertional mutagenesis by a defective transposon Tn10 to produce lon::delta 16 delta 17Tn10 derivatives. The delta 16 delta 17Tn10 (hereafter called delta Tn10) elements were inserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distance from the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different lambda lon::delta Tn10 phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. The lon::delta Tn10 mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis of intact Lon protein, as assayed by antibody on Western blots. An analysis of the protein-degradative ability of lon::delta Tn10 cells suggests that although the insertions in lon caused a reduction in ATP-dependent protein degradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::delta Tn10 mutations and a lon deletion retaining only the amino-terminal 25% of the gene did not affect the energy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in the ATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provide clear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli.     

Cloning and DNA sequence of the gene coding for the major sigma factor from Myxococcus xanthus.

The gene for a sigma factor (rpoD) was cloned from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon starvation for nutrients. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide of 708 amino acid residues (Mr = 80,391) was identified. Except for the amino-terminal sequence consisting of 100 residues, the M. xanthus sigma factor (sigma-80) showed extensive similarity with Escherichia coli sigma-70 as well as Bacillus subtilis sigma-43. In particular, the carboxy-terminal sequence of 242 residues that is known to be required for promoter recognition and core recognition showed 78 and 72% amino acid sequence identity with the E. coli and B. subtilis sigma factors, respectively. The putative RpoD protein was detected at the position of an apparent molecular weight of 86,000 by Western blot (immunoblot) analysis by using antiserum against B. subtilis sigma-43, which agreed well with the position of a vegetative sigma factor of M. xanthus previously identified by Rudd and Zusman (K. Rudd and D. R. Zusman, J. Bacteriol. 151:89-105, 1982).     

Initiation factor 2 of Myxococcus xanthus, a large version of prokaryotic translation initiation factor 2

We have isolated the structural gene for translation initiation factor IF2 (infB) from the myxobacterium Myxococcus xanthus. The gene (3.22 kb) encodes a 1,070-residue protein showing extensive homology within its G domain and C terminus to the equivalent regions of IF2 from Escherichia coli. The protein cross-reacts with antibodies raised against E. coli IF2 and was able to complement an E. coli infB mutant. The M. xanthus protein is the largest IF2 known to date. This is essentially due to a longer N-terminal region made up of two characteristic domains. The first comprises a 188-amino-acid sequence consisting essentially of alanine, proline, valine, and glutamic acid residues, similar to the APE domain observed in Stigmatella aurantiaca IF2. The second is unique to M. xanthus IF2, is located between the APE sequence and the GTP binding domain, and consists exclusively of glycine, proline, and arginine residues.     

Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin inhibitor-deficient mutant of Streptomyces albogriseolus S-3253.

An extracellular serine protease produced by a mutant, M1, derived from Streptomyces albogriseolus S-3253 that no longer produces a protease inhibitor (Streptomyces subtilisin inhibitor [SSI]) was isolated. A 20-kDa protein was purified by its affinity for SSI and designated SAM-P20. The amino acid sequence of the amino-terminal region of SAM-P20 revealed high homology with the sequences of Streptomyces griseus proteases A and B, and the gene sequence confirmed the relationships. The sequence also revealed a putative amino acid signal sequence for SAM-P20 that apparently functioned to allow secretion of SAM-P20 from Escherichia coli carrying the recombinant gene. SAM-P20 produced by E. coli cells was shown to be sensitive to SSI inhibition.     

Identification of a vegetative promoter in Myxococcus xanthus. A protein that has homology to histones

A physical map of 330 x 10(3) base-pairs near the replication origin of Myxococcus xanthus chromosome has been established already. Using DNA fragments from this region, Northern blot hybridization analysis was carried out in order to identify the genes expressed during vegetative growth. One of the genes, tentatively designated as vegA, was cloned and its entire DNA sequence was determined. The amino acid sequence of the gene product deduced from the DNA sequence reveals that the VegA protein is a very basic protein with a molecular weight of 18,700. The gene was expressed in Escherichia coli using an expression vector, and its gene product was identified using SDS/polyacrylamide gel electrophoresis. From the results of S1 nuclease mapping, the vegA promoter was found to contain the sequence TAGACA at the -35 region and the sequence AAGGGT at the -10 region. These two regions are separated by 18 nucleotides. Genetic analysis suggests that the vegA gene may be essential for the growth of M. xanthus. From a computer-aided search for homologies to know protein structures, it was found that the VegA protein has homologies to histone H4 of Tetrahymena thermophila and histone H2B of sea urchin.     

Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.

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.     

GidA is an FAD-binding protein involved in development of Myxococcus xanthus

A gene encoding a homologue of the Escherichia coli GidA protein (glucose-inhibited division protein A) lies immediately upstream of aglU, a gene encoding a WD-repeat protein required for motility and development in Myxococcus xanthus. The GidA protein of M. xanthus shares about 48% identity overall with the small (approximately equal to 450 amino acid) form of GidA from eubacteria and about 24% identity overall with the large (approximately equal to 620 amino acid) form of GidA from eubacteria and eukaryotes. Each of these proteins has a conserved dinucleotide-binding motif at the N-terminus. To determine if GidA binds dinucleotide, the M. xanthus gene was expressed with a His6 tag in E. coli cells. Purified rGidA is a yellow protein that absorbs maximally at 374 and 450 nm, consistent with FAD or FMN. Thin-layer chromatography (TLC) showed that rGidA contains an FAD cofactor. Fractionation and immunocytochemical localization show that full length GidA protein is present in the cytoplasm and transported to the periplasm of vegetative-grown M. xanthus cells. In cells that have been starved for nutrients, GidA is found in the cytoplasm. Although GidA lacks an obvious signal sequence, it contains a twin arginine transport (Tat) motif, which is conserved among proteins that bind cofactors in the cytoplasm and are transported to the periplasm as folded proteins. To determine if GidA, like AglU, is involved in motility and development, the gidA gene was disrupted. The gidA- mutant has wild-type gliding motility and initially is able to form fruiting bodies like the wild type when starved for nutrients. However, after several generations, a stable derivative arises, gidA*, which is indistinguishable from the gidA- parent on vegetative medium, but is no longer able to form fruiting bodies. The gidA* mutant releases a heat-stable, protease-resistant, small molecular weight molecule that acts in trans to inhibit aggregation and gene expression of wild-type cells during development.     

Molecular cloning of the Lon protease gene from Thermus thermophilus HB8 and characterization of its gene product.

The gene encoding Lon protease was isolated from an extreme thermophile, Thermus thermophilus HB8. Sequence analysis demonstrated that the T. thermophilus Lon protease gene (TT-lon) contains a protein-coding sequence consisting of 2385 bp which is approximately 56% homologous to the Escherichia coli counterpart. As expected, the G/C content of TT-lon was 68%, which is significantly higher than that of the E. coli lon gene (52% G/C). The amino acid sequence of T. thermophilus Lon protease (TT-Lon) predicted from the nucleotide sequence contained several unique sequences conserved in other Lon proteases: (a) a cysteine residue at the position just before the putative ATP-binding domain; (b) motif A and B sequences required for composition of the ATP-binding domain; and (c) a serine residue at the proteolytic active site. Expression of TT-lon under the control of the T7 promoter in E. coli produced an 89-kDa protein with a yield of approximately 5 mg.L-1. Recombinant TT-Lon (rTT-Lon) was purified to homogeneity by sequential column chromatography. The peptidase activity of rTT-Lon was activated by ATP and alpha-casein. rTT-Lon cleaved succinyl-phenylalanyl-leucyl-phenylalanyl-methoxynaphthylamide much more efficiently than succinyl-alanyl-alanyl-phenylalanyl-methoxynaphthylamide, whereas both peptides were cleaved with comparable efficiencies by E. coli Lon. These results suggest that there is a difference between TT-Lon and E. coli Lon in substrate specificity. rTT-Lon most effectively cleaved substrate peptides at 70 degrees C, which was significantly higher than the optimal temperature (37 degrees C) for E. coli Lon. Together, these results indicate that the TT-lon gene isolated from T. thermophilus HB8 actually encodes an ATP-dependent thermostable protease Lon.     

Role for tandem duplication and lon protease in AcrAB-TolC- dependent multiple antibiotic resistance (Mar) in an Escherichia coli mutant without mutations in marRAB or acrRAB

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.     

Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.