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.     

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.     

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.     

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.     

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.     

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.     

地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。     

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease.

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease has been identified. This pin (proteolysis inhibition) gene was selected for its ability to support plaque formation by a lambda Ots vector at 40 degrees C. Southern blot experiments indicated that this T4 gene is included within the 4.9-kilobase XbaI fragment which contains gene 49. Subcloning experiments showed that T4 gene 49.1 (designated pinA) is responsible for the ability of the Ots vector to form plaques at 40 degrees C. Deficiencies in Lon protease activity are the only changes known in E. coli that permit lambda Ots phage to form plaques efficiently at 40 degrees C. lon+ lysogens of the lambda Ots vector containing pinA permitted a lambda Ots phage to form plaques efficiently at 40 degrees C. Furthermore, these lysogens, upon comparison with similar lysogens lacking any T4 DNA, showed reduced levels of degradation of puromycyl polypeptides and of canavanyl proteins. The lon+ lysogens that contained pinA exhibited other phenotypic characteristics common to lon strains, such as filamentation and production of mucoid colonies. Levels of degradation of canavanyl proteins were essentially the same, however, in null lon lysogens which either contained or lacked pinA. We infer from these data that the T4 pinA gene functions to block Lon protease activity; pinA does not, however, appear to block the activity of proteases other than Lon that are involved in the degradation of abnormal proteins.     

Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain.

The structural gene hap for the extracellular hemagglutinin/protease (HA/protease) of Vibrio cholerae was cloned and sequenced. The cloned DNA fragment contained a 1,827-bp open reading frame potentially encoding a 609-amino-acid polypeptide. The deduced protein contains a putative signal sequence followed by a large propeptide. The extracellular HA/protease consists of 414 amino acids with a computed molecular weight of 46,700. In the absence of protease inhibitors, this is processed to the 32-kDa form which is usually isolated. The deduced amino acid sequence of the mature HA/protease showed 61.5% identity with the Pseudomonas aeruginosa elastase. The cloned hap gene was inactivated and introduced into the chromosome of V. cholerae by recombination to construct the HA/protease-negative strain HAP-1. The cloned fragment containing the hap gene was then shown to complement the mutant strain.     

Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli.

A DNA fragment of Serratia marcescens directing an extracellular serine protease (Mr, 41,000) was cloned in Escherichia coli. The cloned fragment caused specific excretion of the protease into the extracellular medium through the outer membrane of E. coli host cells in parallel with their growth. No excretion of the periplasmic enzymes of host cells occurred. The cloned fragment contained a single open reading frame of 3,135 base pairs coding a protein of 1,045 amino acids (Mr 112,000). Comparison of the 5' nucleotide sequence with the N-terminal amino acid sequence of the protease indicated the presence of a typical signal sequence. The C-terminal amino acid of the enzyme was found at position 408, as deduced from the nucleotide sequence. Artificial frameshift mutations introduced into the coding sequence for the assumed distal polypeptide after the C terminus of the protease caused complete loss of the enzyme production. It was concluded that the Serratia serine protease is produced as a 112-kilodalton proenzyme and that its N-terminal signal peptide and a large C-terminal part are processed to cause excretion of the mature protease through the outer membrane of E. coli cells.     

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.     

Cloning and expression in Escherichia coli of the structural gene coding for the monomeric protein of the S layer of Thermus thermophilus HB8.

The gene coding for the 100 kDa monomeric protein (P100) of the S layer of Thermus thermophilus HB8 has been cloned in the Escherichia coli plasmid vector pUC9. Recombinant plasmids were selected by colony screening with anti-P100 rabbit antiserum. The gene, named slpA (for surface layer protein A), was identified in a bacterial clone harboring a hybrid plasmid, pMF4, with a 5.8-kbp insert. This plasmid consistently expressed a protein specifically recognized by anti-P100 antiserum. Expression was apparently independent of Plac, indicating that the promoter for P100 is functional in E. coli. Most E. coli strains transformed with plasmids containing the 5.8-kbp insert cloned in pMF4 expressed two proteins with apparent masses of 52 and 50 kDa, which were strongly recognized by anti-P100 antiserum in Western immunoblots. The 52-kDa fragment could be overproduced, and the sequence of the N-terminal undecapeptide, determined by microsequencing, indicated that it could correspond to the N-terminal domain of P100. Expression of slpA in lon mutants of E. coli led to accumulation of a protein indistinguishable from native P100, indicating that the complete gene was cloned and that the product of lon, protease La, was involved in proteolytic degradation of P100 synthesized in E. coli.     

Molecular cloning and sequencing of the sppA gene and characterization of the encoded protease IV, a signal peptide peptidase, of Escherichia coli

Clones carrying a gene causing overproduction of protease IV, a signal peptide peptidase of Escherichia coli, were isolated from the Clarke and Carbon's collection. Restriction mapping analysis revealed that pLC7-10 and pLC40-13, thus isolated, shared the same chromosomal DNA region. The 2.3-kilobase RsaI-SalI fragment in this region, which was found to carry the gene, was subjected to nucleotide sequence determination. Only one long open reading frame was found. The hypothetical polypeptide sequence deduced from the DNA sequence has a molecular mass of 67,241 daltons. The putative gene was named sppA. Protease IV was purified to homogeneity from the cytoplasmic membrane of an overproducing strain harboring a sppA gene-carrying plasmid. The purified enzyme gave a single polypeptide band of 67,000-dalton molecular mass on sodium dodecyl sulfate-polyacrylamide gel. This molecular mass and the amino acid composition of the purified enzyme were consistent with the deduced primary structure of the sppA gene product. The molecular mass thus determined was almost twice as large as that previously reported by Pacaud (Pacaud, M. (1982) J. Biol. Chem. 257, 4333-4339). A cross-linking study revealed that protease IV is a tetramer of the polypeptide. From these results, we conclude that protease IV is a tetramer of the sppA gene product.     

Molecular characterization of a cell wall-associated proteinase gene from Streptococcus lactis NCDO763

Streptococcus lactis NCDO763 harbours a plasmid designated pLP763. The cells harbouring pLP763 are able to grow to a higher density in milk because of their proteinase-positive phenotype (Prt+). The 6.2 kb HindIII-PstI fragment from pLP763 was found to be responsible for the Prt+ phenotype. The DNA fragment contains an incomplete large open reading frame (ORF). Further sequence analysis downstream from the PstI site revealed that the ORF consists of 5706 bases. It was found that the deduced amino acid sequence consisting of 1902 amino acid residues was extremely similar to that of the Wg2 proteinase, a serine protease from Streptococcus cremoris, suggesting that both genes were derived from a common ancestral gene.     

马铃薯卷叶病毒中国株（PLRV-Ch）ORF2a基因特征分析

马铃薯卷叶病毒 (ＰＬＲＶ)是正链ＲＮＡ病毒 ,属黄化病毒组[1 ] 严格虫传 ,分布广泛 ,难以控制 ,侵染马铃薯 ,给生产造成巨大损失。ＰＬＲＶ基因全长 6 0ｋｂ ,有 6个读码框架 ,其中ＯＲＦ2ａ是第二读框 ,全长 192 0ｂｐ ,编码一个 70ｋＤ的多肽。另外 ,ＯＲＦ2ａ在与ＯＲＦ2ｂ重叠处可发生移码继续转译 ,直到ＯＲＦ2ｂ的尾 ,转译产物为一条 118ｋＤ的多肽 ,该蛋白的Ｃ端与复制酶的序列具很大的同源性[2～ 4] 。Ｐｒｕｆｅｒ[5] 等和Ｋｕｊａｗａ[6] 等分别研究了ＰＬＲＶ基因组上ＯＲＦ2ａ与ＯＲＦ2ｂ重叠区附近与移码有关的滑动序列及其…