Dual Positive Feedback Regulation of Protein Degradation of an Extra-cytoplasmic Function σ Factor for Cell Differentiation in Streptomyces coelicolor

Here we report that in Streptomyces coelicolor, the protein stability of an ECF σ factor SigT, which is involved in the negative regulation of cell differentiation, was completely dependent on its cognate anti-σ factor RstA. The degradation of RstA caused a ClpP/SsrA-dependent degradation of SigT during cell differentiation. This was consistent with the delayed morphological development or secondary metabolism in the ΔclpP background after rstA deletion or sigT overexpression. Meanwhile, SigT negatively regulated clpP/ssrA expression by directly binding to the clpP promoter (clpPp). The SigT-clpPp interaction could be disrupted by secondary metabolites, giving rise to the stabilized SigT protein and retarded morphological development in a non-antibiotic-producing mutant. Thus a novel regulatory mechanism was revealed that the protein degradation of the ECF σ factor was initiated by the degradation of its anti-σ factor, and was accelerated in a dual positive feedback manner, through regulation by secondary metabolites, to promote rapid and irreversible development of the secondary metabolism. This ingenious cooperation of intracellular components can ensure economical and exquisite control of the ECF σ factor protein level for the proper cell differentiation in Streptomyces.     

Inflammation in insects: The release of a plasmatocyte depletion factor following interaction between bacteria and haemocytes

Injection of a suspension of the Gram-positive bacterium Bacillus cereus into the haemocoel of Galleria mellonella larvae caused a rapid decrease in the number of circulating plasmatocytes. A similar phenomenon was observed following the injection of cell-free haemolymph collected from larvae previously challenged with bacteria, indicating that plasmatocyte depletion was mediated by a humoral factor released into the haemolymph. The release of plasmatocyte depletion factor could be effected in vitro by incubating adherent haemocytes with a suspension of B. cereus. The in vitro production of the factor did not require the presence of exogenous humoral components, was dose-dependent, was inhibited at low temperatures, and showed the same specificity with respect to bacterial species as observed in vivo. Activity of the plasmatocyte depletion factor destroyed by a 15-min incubation at 56°C. The release of the factor appears to be an early biochemical signal in the complex cellular response of this insect to bacterial infection.     

Functional characterization of single-chain factor X from rat liver

¹⁴C-Labeled single-chain factor X prepared by vitamin K-dependent carboxylation in vitro was partially purified by adsorption to BaSO₄ and chromatography on DEAE-Sephacel. Known activators of factor X were analyzed for their effect on the single-chain molecule. ¹⁴C-Labeled factor X antigens were recovered immunochemically from incubation mixtures and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation with trypsin resulted in the generation of factor Xa clotting activity, and the ¹⁴C-labeled product migrated after reduction with an apparent molecular weight of 22,500 ± 1500 (mean ± 1 SD). The light chain produced by factor Xa was similar to that produced by trypsin (M_r 24,500 ± 1500; mean ± 1 SD). Incubation of single-chain factor X with factor VII and thromboplastin, factor IXa, or the factor X activating enzyme from Russell's viper venom gave a reducible product with a light chain of higher apparent molecular weight (M_r 37,000–38,000). Incubation with factor VII and thromboplastin also resulted in the generation of factor Xa clotting activity. Incubation of single-chain factor X with platelets resulted in the binding of about 20% of the ¹⁴C. The bound ¹⁴C-labeled factor X antigen released by freezing and thawing in the presence of EDTA was reduced to give a ¹⁴C-labeled polypeptide with M_r 31,000. Walker 256 tumor cells bound about 30% of the ¹⁴C. The bound material, after reduction, gave a ¹⁴C-labeled polypeptide with M_r 23,000.     

Inhibition of Clostridium perfringens by a Novel Strain of Bacillus subtilis Isolated from the Gastrointestinal Tracts of Healthy Chickens

The objectives of this study were to isolate beneficial strains of microorganisms from the gastrointestinal tracts of healthy chickens and to screen them against Clostridium perfringens, a causative agent of necrotic enteritis in poultry. One of the bacteria isolated, a strain of Bacillus subtilis, was found to possess an anticlostridial factor that could inhibit the C. perfringens ATCC 13124 used in this study. The anticlostridial factor produced by B. subtilis PB6 was found to be fully or partially inactivated in the presence of pronase, trypsin, and pepsin. In contrast, the antimicrobial activity of the anticlostridial factor was not affected by treatment at 100 or 121°C or by treatment with any of the organic solvents used in the study. The optimum growth temperature and optimum pH for production of the anticlostridial factor were 37°C and 6.20, respectively. Using the mass spectroscopy-mass spectroscopy technique, the apparent molecular mass of the anticlostridial factor was estimated to be in the range from 960 to 983 Da. In terms of the antimicrobial spectrum, the anticlostridial factor was inhibitory toward various strains of C. perfringens implicated in necrotic enteritis in poultry, Clostridium difficile, Streptococcus pneumoniae, Campylobacter jejuni, and Campylobacter coli.     

F factor mobilization of non-conjugative chimeric plasmids in Escherichia coli: General mechanisms and a role for site-specific recA-independent recombination at oriV1

Plasmid pSC101 is neither self-transmissible nor efficiently mobilized (made to transfer) by the Escherichia coli F factor. When fragments of F factor DNA were inserted into pSC101 the resulting chimeric plasmids were mobilized by the F factor at enhanced frequencies. These chimeric plasmids, which were not self-transmissible, fell into three classes according to their relative ability to be mobilized by an autonomous or integrated F factor: (1) class I pSC101-F chimeric plasmids contain the origin of transfer of the F factor (oriT) and were mobilized in trans at an efficiency nearly equal to that of F factor transfer; (2) class II pSC101-F chimeric plasmids lacked both oriT and the origin of vegetative F replication (oriV1), and were mobilized in cis via fusion with the F factor in a recA-dependent recombination to yield a transferable co-integrated plasmid; (3) class III pSC101-F chimeric plasmids lacked oriT but contained oriV1 and were mobilized in cis via co-integration with the F factor probably at oriV1 in a recA-independent recombination. A fourth class of mobilization event, not exhibited by pSC101-F chimeric plasmids, was also observed. Mobilization of pBR322 and pSC101 occurred in cis via transposon-mediated recA-independent fusion with F. On the basis of these results we present a general classification scheme of non-conjugative plasmids and also suggest mechanisms for their mobilization.     

Isolation and sequence of a Mycobacterium tuberculosis sigma factor

A DNA segment from Mycobacterium tuberculosis containing a gene for a putative sigma factor was isolated and sequenced. The protein encoded by this gene is 92% similar to the Mycobacterium smegmatis sigma factor MysB, and has been designated Mtu SigB. A Mycobacterium leprae homologue of mysB and mtu sigB was identified in the database.     

USLE/RUSLE中植被覆盖与管理因子研究进展

通用土壤流失方程(USLE)及修正通用土壤流失方程(RUSLE)是世界范围内应用最广泛的土壤侵蚀预报模型,模型中C因子表示植被覆盖和管理措施对土壤侵蚀的作用,是人为控制土壤侵蚀的重要因子。回溯了C因子发展演变历程,依据国内外最新研究成果,系统阐述了不同尺度C因子估算方法。在小区、坡面、小流域尺度上,C因子确定主要依赖于野外实验观测,研究条件的一致性尤其是标准小区的统一是C因子值可比性的前提。流域、区域尺度C因子确定通常需要利用遥感影像,遥感技术的发展促进了流域、区域尺度C因子估算方法的进步,使提取的C因子图更加精细、准确,但是使用遥感数据全面刻画C因子含义仍然是一大挑战,因此仍需加强C因子相关研究。共归纳了10种确定C因子的方法,介绍了不同方法的优缺点及适用条件,提出了我国C因子研究应加强的工作,希望为相关领域研究者提供参考。     

Drug Resistance of Enteric Bacteria VIII. Chromosomal Location of Nontransferable R Factor in Escherichia coli

The R₂₁(TC) factor, obtained by transduction of the R₁₀(TC.CM.SM.SA) factor with phage ε to group E Salmonella, is not transferable by the normal conjugal process. However, when R₂₁(TC)⁺ transductants are infected with the F₁₃ factor, the nontransferable R₂₁(TC) factor acquires transmissibility by conjugation. R₂₁(TC)⁺ conjugants of Escherichia coli K-12, to which only the R₂₁(TC) factor was transmitted by cell-to-cell contact from an F′ R⁺ donor, were still unable to transfer their R₂₁(TC) factor by conjugation. In crosses between Hfr and F⁻E. coli K-12 strains containing R₂₁(TC), the gene responsible for tetracycline resistance was located on the E. coli K-12 chromosome between lac and pro, near lac.     

Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents

1. o-Iodosobenzoate and 2,2′-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol.2. The inhibition of the Ca²⁺-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2′-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide.3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher K_m for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

Molecular changes during the activation of coagulation factor V by snake venom proteases

Thrombin activation of factor V constitutes an important feedback reaction in the regulation of coagulation. We therefore examined the details of activation of bovine factor V by two purified snake venom proteolytic enzymes, factor V-activating protease from Russell's viper venom and a platelet-aggregating enzyme, thrombocytin, fromBothrops atrox venom. The reactions were followed by changes in factor V coagulant activity, immunoelectrophoresis, and electrophoresis of radiolabeled factor V in sodium dodecylsulfate under reducing conditions. When factor V (M _r 330,000) was exposed to factor V-activating protease at an enzyme-to-substrate ratio of 1:35 at 37°, cleavage occurred in 1 min, with formation of an intermediate (M _r 250,000) coincident with a nine-fold activity increase. By 2 min, additional cleavage occurred, with disappearance of the intermediate and formation of two final fragments (M _r 150,000 and 100,000) but no further change in coagulant activity. The concentration of these components remained unchanged from 5 to 15 min. Immunoelectrophoresis against antiserum directed against factor V confirmed cleavage of the molecule. Incubation of factor V with thrombocytin at 37° for 1 min resulted in a four-fold increase of factor V activity, with the formation of an intermediate (M _r 220,000). By 2 min, a 7.5-fold activation was found, with a decline in the concentration of the intermediate; the predominant species hasM _r =130,000. At 5 min the intermediate disappeared and a second, final fragment ofM _r of ～150,000 appeared without further change in coagulant activity. Immunoelectrophoresis again confirmed selective proteolysis. Thus, incubation of factor V-activating protease or thrombocytin with factor V results in different molecular alterations associated with an increase in the coagulant activity of this clotting factor.     

Specificity of the transhydrogenase factor for chromatophores of Rhodopseudomonas spheroides and Rhodospirillum rubrum

Extensive washing of chromatophores of Rhodospirillum rubrum and Rhodopseudomonas spheroides with dilute buffer results in a complete loss of the energylinked transhydrogenase activities of Rsp. rubrum but only a partial loss of the light-driven reaction in chromatophores of Rps. spheroides. It was not possible to reactivate the Rps. spheroides transhydrogenation with the Rsp. rubrum transhydrogenase factor nor with a protein fraction of Rps. spheroides isolated by procedures identical to that used for the isolation of the Rsp. rubrum transhydrogenase factor. The Rsp. rubrum factor is highly specific and cannot be replaced by a number of sulfhydryl compounds tested for reconstitution of Rsp. rubrum transhydrogenation. A published procedure for the isolation of a “transhydrogenase factor” from Rps. spheroides chromatophores yields a preparation having energy-dependent transhydrogenation when supplemented with dithiothreitol in the absence of added chromatophores.     

A comparative study of the bactericidal activity of horseshoe crab (Limulus polyphemus) hemolymph and vertebrate serum

This paper describes a partially heat-labile, naturally occurring bactericidal factor in cell-free hemolymph preparations obtained from Limulus polyphemus. This bactericidal activity has been shown to be directed against two Gram-negative bacteria, Escherichia coli and Klebsiella pneumoniae, whereas it had no effect on the Gram-positive bacteria tested, Micrococcus lysodeikticus and Staphylococcus aureus. Maximal bactericidal activity of this factor was observed at 30°C and pH 6.0. Since complement and antibody are required for antimicrobial activity in vertebrate sera, the activity of this factor in the presence of various complement inhibitors was assayed. The bactericidal activity of Limulus hemolymph is abolished by treatment with endotoxin; however, other anticomplementary substances were without effect. Limulus amebocyte lysate is known to contain protein which may be precipitated by endotoxin; it is possible that the reduction of bactericidal activity produced by endotoxin treatment may be caused by the denaturation of a bactericidal protein moiety produced by the hemocytes.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase: localization of substrate binding sites.

Butanedione in the presence of borate buffer reversibly inhibits Rhodospirillum rubrum chromatophore transhydrogenase complex and the separated membrane-bound and soluble factor components of the complex. NADP⁺ completely protected against inactivation of the membrane-bound component, whereas NAD⁺ was without effect. Soluble factor was maximally protected only partially by either NAD⁺ or NADP⁺, but a mixture of the substrates afforded complete protection. NADP⁺-dependent association of soluble factor with factor-depleted membranes was markedly decreased after incubation of membranes with butanedione in the absence, but not in the presence, of NADP⁺. Soluble factor was bound to agarose-NAD and was eluted by NAD⁺, but not by NADP⁺. These results demonstrate the presence of at least three nicotinamide adenine dinucleotide binding sites on R. rubrum transhydrogenase complex, including separate NADP and NAD binding sites on soluble factor and a NADP binding site on the membrane-bound component.     

Interaction between fibronectin and purified staphylococcal clumping factor

Clumping of Staphylococcal aureus was observed in the presence of fibrinogen as well as fibronectin. In order to elucidate the mechanism of this clumping, binding of radiolabelled fibrinogen and fibronectin to S. aureus cultures was studied. Cultures of S. aureus reacted with ¹²⁵I-labelled fibrinogen as well as fibronectin. The binding of labelled fibrinogen to S. aureus could be completely inhibited by unlabelled fibronectin, whereas the binding of labelled fibronectin was only partially inhibited by unlabelled fibrinogen. This suggested an interaction of fibronectin with clumping factor which is the binding protein for fibrinogen in staphylococci. The clumping factor was purified from S. aureus strain K 807 by affinity chromatography on fibrinogen-Sepharose followed by HPLC. The purified clumping factor inhibited the binding of fibrinogen and fibronectin to staphylococci. In western blots the purified clumping factor reacted with fibrinogen as well as fibronectin. Thus, the direct interaction of clumping factor with fibronectin might be responsible for the clumping of staphylococci in fibrinogen depleted plasma or serum.     

Purification and characterization of alkane solubilizing factor produced by Pseudomonas PG-1

The normal hexadecane emulsifying and solubilizing factor (PG-1 ESF C₁₆) produced by Pseudomonas PG-1 during growth on n-hexadecane was isolated and purified. The factor was composed of protein, carbohydrate and lipid, which were largely undialyzable. Ca²⁺ was necessary for activation and heat stability of the factor. Particle size of the factor was less than 10 nm. All the protein along with 68–74% of the carbohydrate in the factor was obtained in a single protein peak by gel filtration chromatography using Biogel P-30. The isolated protein fraction showed a 1–5 fold increase in n-hexadecane solubilizing activity. The isolated protein was shown to be a homogeneous, monomeric protein with a molecular weight of approximately 11,000 daltons by SDS-PAGE. The protein and carbohydrate moieties in the isolate were separated by DEAE-cellulose chromatography. Neither purified protein nor carbohydrate showed n-hexadecane solubilizing activity separately, but when these were mixed full activity was restored. Hydrocarbon emulsifying activity was confined to the lipid fraction, which was isolated to the extent of 85% from the Biogel P-30 column by ethyl ether extraction.     

Enzymatic and immunological characterization of the conditioning factor for epidermal outgrowth in the cockroach Leucophaea maderae

A partial characterization of a haemocyte derived conditioning factor which permits rapid in vitro outgrowth of insect epidermis (Leucophaea maderae) was attempted. The factor resisted denaturating agents such as heat (100°C), ethanol, acetone, formol, and acetic acid. It was not extractable by hot pyridine or detergent, nor destroyed by lipase, RNase, DNase, or glycolytic enzymes. The factor was, however, completely inactivated by all four proteolytic enzymes tested. Rabbit antisera were produced against a haemocyte homogenate and tested in vitro. One of the antisera contained antibodies against the conditioning factor. The nature and origin of the conditioning factor are discussed in some detail.     

Schistosoma mansoni: reduction in clinical manifestations and in worm burdens conferred by serum and transfer factor from immune or normal rhesus monkeys.

Although delayed hypersensitivity to Schistosoma mansoni was conferred on rhesus monkeys (Macaca mulatta) by means of dialyzable transfer factor prepared from peripheral leukocytes or lymph node cells of infected immune donors, when such animals were challenged with 1000 cercariae of S. mansoni they developed worm burdens similar to those of nontreated controls. However, recipients of transfer factor that, in addition, received hyperimmune serum showed minimal clinical symptoms and significantly reduced worm burdens after subsequent infection with S. mansoni irrespective of whether the donors used for the transfer factor were immune or uninfected. A significant but lower degree of protection was conferred by combinations of either S. mansoni transfer factor or normal transfer factor and normal serum. Neither transfer factor nor hyperimmune serum alone conferred protection to recipients. Susceptibility to infection was assessed by observing the signs of the disease, determining the worm burdens by perfusion 10 weeks after exposure, and by observing the appearance of the intestine at autopsy. The animals which received transfer factor and immune serum were protected against clinical disease. Good correlation between worm burdens and severity of disease was observed.