The purified colicin S8 is a multimeric protein.

Bacteriocins have been isolated both as simple proteins and as proteins in association with carbohydrates, lipids, etc. Colicins are commonly inducible and extracellular. Their molecular masses range from 30 to 90 kDa. Pure colicin S8 was obtained in three steps from supernatant of induced cells: (i) Ammonium sulfate precipitation; (ii) anion exchange chromatography; and (iii) phenyl-Sepharose hydrophobic chromatography, either by preparative or fast performance liquid chromatography (FPLC) analytical purification procedure. In our hands, purified colicin S8 was an aggregation of extremely related polypeptides. Composition of those active fractions was the same: five polypeptides of molecular weight around 55 kDa. Behavior on molecular filtration indicated a molecular weight higher than 200 kDa. Similar results were obtained when purification was carried out through FPLC. Producing strains contain a single plasmid that encodes colicin S8; in minicells, this plasmid was shown to specify a 60 kDa polypeptide. We conclude that more than one form of colicin S8 exists. The forms are structurally related and can be recognized by antibodies raised against one of the polypeptides. Consistent with this conclusion, comparison of peptides produced after hydrolysis with chlorosuccinamide indicated that the active proteins contained both shared and unique components.     

Nucleotide sequence and physical map of kanamycin-resistant plasmid pUB110 from Staphylococcus aureus

The complete nucleotide sequence of Staphylococcus aureus plasmid pUB10 was determined. The sequence consists of 4545 b.p. and contains 64% A-T and 36% G-C pairs. pUB110 was found to contain four open reading frames, capable of coding for polypeptides having more than 80 amino acids. All the putative polypeptides are coded for by one DNA strand. The molecular weights of four putative polypeptides are (in kilodaltons): A-49.5; B-38.8; C-28.8 and D-9.5. Polypeptide C is involved in kanamycin resistance. Polypeptide B is, possibly, involved in pUB110 replication. No role has yet been established for polypeptides A and D, since deletions in their coding sequences have no detectable effect on any properties of pUB110 plasmid.     

Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus

pT181 is a naturally occurring Staphylococcus aureus plasmid, encoding inducible resistance to tetracycline. The plasmid has a copy number of about 20 per cell, and belongs to the incompatibility group inc3. The complete nucleotide sequence of pT181 has been determined and consists of 4437 bp. The nucleotide sequence contains 69.8% A-T and 30.2% G-C pairs. pT181 was found to contain four open reading frames capable of coding for polypeptides containing more than 50 amino acids. All the putative polypeptides are coded by one strand. The molecular weights of the four putative polypeptides are (in daltons): A, 37,500; B, 35,000; C, 23,000, and D, 18,000. Polypeptide A corresponds to the repC protein, earlier shown to be specifically required for pT181 replication. Polypeptide B (and possibly polypeptide D) are involved in tetracycline resistance. No role has yet been established for polypeptide C; deletion of the coding sequence for the C polypeptide has no detectable effect on any property of the pT181 plasmid. A region consisting of about 1200 bp contains information for the replication and copy number control of this plasmid. The sequencing results are discussed in relation to the replication properties and tetracycline resistance associated with the pT181 plasmid.     

Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34.

From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 [ORF]; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression. ORF0ab and ORF1 exhibit a codon usage that is not typical for A. eutrophus. The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis. Among 72 insertion mutants, the majority were nickel sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30% similarity, respectively, with the corresponding proteins of czc.     

pS86, A New Theta-Replicating Plasmid from Enterococcus faecalis

The complete nucleotide sequence of the small (5149 bp) and cryptic plasmid pS86 from Enterococcus faecalis ssp. faecalis S-86 has been determined. Sequence analysis revealed six putative open reading frames (ORFs) encoding polypeptides of 28.3, 11.5, 8.4, 65.1, 7.3, and 11.96 kDa each. Based on sequence similarity, two cassettes have been identified in pS86: ORF1 codes for the replication initiation protein (Rep); ORF4 codes for a putative mobilization protein that shows similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. No function could be assigned to the other putative ORFs found. According to our results, pS86 plasmid could use a theta-mode of replication, similar to the recently described theta-type replicons from pUCL287 (Tetragenococcus halophila) and pLA1 or pLA105 (Lactobacillus acidophilus) plasmids. Received: 24 November 1999 / Accepted: 26 April 2000     

Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg(2+)-inducible, NADH dependent and also required 2-mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and merC (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hgr Z. mobilis but not with the Hgs strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hgr Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21.     

Salt-induced changes in protein composition in light-grown callus of Mesembryanthemum crystallinum

The halophyte Mesembryanthemum crystallinum (ice plant) has been suggested as a model for salt-tolerance in higher plants. To investigate salt-induced changes in polypeptide patterns at the cellular level, a light-grown callus of M. crystallinum with substantial chlorophyll content, was established and the effect of NaCl on the composition of phenol-extracted protein was examined by SDS- and 2D-polyacrylamide gel electrophoresis (PAGE). SDS-PAGE showed the accumulation of five polypeptides with estimated molecular masses of 40, 34, 32, 29 and 14 kDa was enhanced by the addition of 200 m M NaCl to the culture media. The addition of ABA (10 μ M ) or mannitol (400 m M ) did not elicit the same degree of accumulation of these salt-specific proteins. These polypeptides were classified into two groups according to their course of induction: early responsive (40, 34, 29 kDa) and late-responsive (32, 14 kDa) proteins. In addition, two polypeptides (20, 18 kDa) were transiently accumulated during salt treatment. Further separation of soluble proteins by 2-D gel electrophoresis, either isoelectric focusing (IEF) or non-equilibrium pH-gradient electrophoresis (NEPHGE) followed by SDS-PAGE, showed more alterations in accumulation of polypeptides by NaCl than 1-D gel electrophoresis. Overall, levels of more than 30% of basic polypeptides, detected by NEPHGE/SDS-PAGE, were altered by 200 m M NaCl treatment, while only 10% of neutral and acidic polypeptides, detected by IEF/SDS-PAGE, were changed. The enhanced expression of these proteins by salt in cultured cells is most likely related to the cellular responses to salinity, and not to the mechanism of CAM induction in this facultative halophyte.     

Isolation of genes required for hydrogenase synthesis in Escherichia coli

A mutant strain of Escherichia coli, strain AK23, is devoid of hydrogenase activity when grown anaerobically on glucose and cannot grow on H2 plus fumarate. From E. coli chromosomal DNA library, a plasmid, pAK23, was isolated which restored hydrogenase activity in this strain. Two smaller plasmids, pAK23C and pAK23S, containing different parts of the insert DNA fragment of plasmid pAK23, were isolated. The former plasmid restored activity in strain AK23 while the latter did not. The smallest active DNA fragment in plasmid pAK23C was 0.9 kb. This gene is designated hydE. Plasmids pAK23 and pAK23S restored activity in another hydrogenase-negative strain, SE-3-1 (hydB), while plasmid pAK23C did not, suggesting that plasmid pAK23 contains two genes required for hydrogenase expression. Strain AK23 was also devoid of formate hydrogenlyase and formate dehydrogenase activities and these activities were restored by some of the plasmids. Hydrogenase and formate-related activities in strain AK23 were restored by growth of cells in a high concentration of nickel. Plasmid pAK23C led to synthesis of a polypeptide of subunit molecular mass 36 kDa and plasmid pAK23S led to synthesis of polypeptides of subunit molecular masses 30 and 41 kDa.     

Characterization of a putative colonization factor (PCFO166) of enterotoxigenic Escherichia coli of serogroup O166

Enterotoxigenic Escherichia coli (ETEC) of serogroup O166 gave mannose-resistant haemagglutination (MRHA) with bovine and human erythrocytes. The strains did not react with antisera prepared against the known colonization factors CFA/I, CFA/II, CFA/III, CFA/IV and PCFO159:H4. Strain E7476 of serotype O166:H27, which produced heat-stable enterotoxin (ST), was examined initially. It produced fimbriae about 7 nm in diameter. On SDS-PAGE two possible fimbrial polypeptides of molecular mass 15.5 and 17.0 kDa were seen. When variants of strain E7476 were isolated, loss of ST and MRHA together was associated with loss of a 98 MDa plasmid, while loss of ST alone correlated with plasmid deletion. An absorbed anti-strain E7476 antiserum reacted specifically with the 15.5 and 17.0 kDa polypeptides in Western immunoblotting and bound to the intact fimbriae by immuno-electron microscopy. When this antiserum was used in an ELISA to examine other strains of serogroup O166, a positive reaction was obtained with all the ST- and MRHA-positive strains. One strain of serotype O71:H27 and two strains of serotype O98:H- also reacted with the absorbed anti-strain E7476 antiserum. The antiserum did not react with ETEC carrying known colonization factors. E. coli K12 and a number of E. coli of different serotypes carrying a plasmid coding for ST transferred from strain E7476, all gave MRHA and reacted with the absorbed anti-strain E7476 antiserum. The term putative colonization factor O166 (PCFO166) is proposed to describe the adhesive factor(s) on ETEC of serogroup O166 because of the similarity of properties with those of known colonization factors.     

Immune haemolymph proteins in response to bacterial infection and identification of a putative bacteria binding protein in malaria vector Anopheles stephensi

Induction of haemolymph proteins in mosquito A. stephensi due to wounding or bacterial infection (E. coli) was analyzed using SDS-PAGE. Wounding response of pupa revealed subsequent induction of two polypeptides (21 and 74 kDa). Two other polypeptides (44 and 57 kDa) were induced commonly in both pupa and adult female haemolymph upon bacterial infection. In vitro binding assay revealed identification of 44 kDa, a putative bacterial binding protein, a more relevant protein for further elucidation of molecular mechanism involved in host parasite interactions.     

HtpG,the prokaryotic homologue of Hsp90, stabilizes a phycobilisome protein in the cyanobacterium Synechococcus elongatus PCC 7942

HtpG, a homologue of HSP90, is essential for thermotolerance in cyanobacteria. It is not known how it plays this important role. We obtained evidence that HtpG interacts with linker polypeptides of phycobilisome in the cyanobacterium Synechococcus elongatus PCC 7942. In an htpG mutant, the 30 kDa rod linker polypeptide was reduced. In vitro studies with purified HtpG and phycobilisome showed that HtpG interacts with the linker polypeptide as well as other linker polypeptides to suppress their thermal aggregation with a stoichiometry of one linker polypeptide/HtpG dimer. We constructed various domain‐truncated derivatives of HtpG to identify putative chaperone sites at which HtpG binds linker polypeptides. The middle domain and the N‐terminal domain, although less efficiently, prevented the aggregation of denatured polypeptides, while the C‐terminal domain did not. Truncation of the C‐terminal domain that is involved in the dimerization of HtpG led to decrease in the anti‐aggregation activity, while fusion of the N‐terminal domain to the middle domain lowered the activity. In vitro studies with HtpG and the isolated 30 kDa rod linker polypeptide provided basically similar results to those with HtpG and phycobilisome. ADP inhibited the anti‐aggregation activity, indicating that a compact ADP conformational state provides weaker aggregation protection compared with the others.     

Characterization of seminal plasma proteins and sperm proteins in ejaculates from normospermic bulls and bulls with thermally-induced testicular degeneration

Semen was collected from 5 mature beef bulls by electroejaculation before, during, and after 20 days of scrotal insulation. Thermally-induced testicular degeneration was irreversible in 3 of the bulls. Analysis of sperm and seminal plasma polypeptides revealed that 15 to 30 sperm polypeptides and 25 to 30 seminal plasma polypeptides were indistinguishable between bulls prior to the insulation treatment. Changes in the sperm polypeptides pattern appeared as early as 2 days after the insulation treatment and persisted for at least 11 months in 2 of the bulls. In the spermatozoa, there was a detectable loss of 31, 34, 49 and 58 kDa polypeptides and an appearance of 6 to 8 new major polypeptides, ranging from 32 to 83 kDa. The 83 kDa polypeptide was most prominent in the 2 bulls that regained normal sperm motility and morphology following the insulation period. The post-insulation appearance of a seminal plasma polypeptide (circa 60 kDa) was also identified in these 2 bulls. Seminal plasma polypeptides remained qualitatively unaltered by the insulation treatment in the 3 bulls with irreversible testicular degeneration.     

Photoaffinity labeling of soluble auxin-binding proteins.

The photoaffinity labeling agent azido-IAA (5-N3-[7-3H]indole-3-acetic acid), a biologically active analogue of the endogenous auxin indole-3-acetic acid, was used to search for auxin-binding proteins in the soluble fraction of Hyoscyamus muticus cells. Azido-IAA became covalently attached to three polypeptides with a high specific activity. The labeling was specific for IAA and not due to random tagging. Two polypeptides with molecular masses of 31 and 24 kDa in the 0-30% ammonium sulfate fraction were labeled after UV photolysis at 0 degree C but not at -196 degrees C, and appeared to have a high affinity indole-binding site(s) for which active, non-indole auxins were not good ligands. A third polypeptide with a molecular mass of 25 kDa present in the 50-60% ammonium sulfate fraction labeled exclusively at -196 degrees C and had a significant affinity for active auxins but not for inactive indoles. The azido-IAA labeling pattern, pI, competition results, and immunoprecipitation all indicate that the 31- and 24-kDa polypeptides are related to the basic form of endo-1,3-beta-glucanase (EC 3.2.1.39). Azido-IAA labeling polypeptides equivalent to the 31- and 24-kDa species were apparently also present in the cell wall. The low pH optimum for binding of azido-IAA to the 25-kDa polypeptide suggests the location of the active protein in a compartment such as the vacuole or a transport vesicle rather than in the cytosol.     

In vivo newly translated polypeptides are sequestered in a protected folding environment.

Molecular chaperones play a fundamental role in cellular protein folding. Using intact mammalian cells we examined the contribution of cytosolic chaperones to de novo folding. A large fraction of newly translated polypeptides associate transiently with Hsc70 and the chaperonin TRiC/CCT during their biogenesis. The substrate repertoire observed for Hsc70 and TRiC is not identical: Hsc70 interacts with a wide spectrum of polypeptides larger than 20 kDa, while TRiC associates with a diverse set of proteins between 30 and 60 kDa. Overexpression of a bacterial chaperonin 'trap' that irreversibly captures unfolded polypeptides did not interrupt the productive folding pathway. The trap was unable to bind newly translated polypeptides, indicating that folding in mammalian cells occurs without the release of non-native folding intermediates into the bulk cytosol. We conclude that de novo protein folding occurs in a protected environment created by a highly processive chaperone machinery and is directly coupled to translation.