Translational Control of Protein Synthesis in Staphylococcus aureus.

The calculated in vivo polypeptide chain growth rate for Staphylococcus auteus MF-31 grown in nutritionally rich medium assuming all the ribosomes were functional was found to be approximately 16 amino acids/s/ribosome, but decreased to 10.2 amino acids/s/ribosome for cells grown in poor medium. An in vitro analysis revealed that 70S ribosomes isolated from rich medium cells were more active than similar 70S ribosomes derived from cells grown in poor medium. The 30S subunit was found responsible for the increased activity of the rich monosomes, whereas the 50S subunit appeared to be capable of either high or low activity.     

The Synthesis In Vitro of Flagellin by a Cell-Free Extract from Bacillus Pumilus

Abstract

Bacterial flagella are constructed mainly, or perhaps exclusively, of protein subunits, the flagellins. Demonstration is given in this report for the in vitro incorporation of radioactive amino acids into flagellin, it also appears that part of such incorporation reflects de novo synthesis of flagellin moleculus. Cell-free extracts were prepared from flagellated cells of Bacillus pumilus, by digestion of the cell wall with lysozyme, lysis in the Standard buffer of NIRENBERG and MATTHAEI (1961), treatment with deoxyribonuclease and centrifugation at 15.000×g. The reaction mixtures contained the cell-free extract, one or more [¹⁴C]-amino acids and the usual components required for cell-free protein synthesis. After incubation at 37° carrier flagellin was added and the pH of the reaction mixture adjusted to 2. Flagellin, which is soluble at this pH, was purified by disc electrophoresis or by reconstitution of flagellar filament at pH 5.4 followed by electrophoresis on a column of ethanolized cellulose. When an amino acid absent from B. pumilus flagellin (such as tyrosine) was used, the amount of radioactivity incorporated into the flagellin fraction was negligible as compared to that incorporated when radioactive leucine, arginine and lysine were used. The identity of the purified radioactive protein was established more conclusively by tryptic digestion and chromatographic separation of the resulting peptides. The ninhydrin positive peaks were shown to be coincident with the radioactive peaks. The radioactive peaks disappeared when a cell-free extract from non-flagellated mutant cells was used. The incorporation of radioactive methionine in the N-terminal position of the molecule indicated that at least some of the molecules had been synthesized de novo.     

GTP and Protein Synthesis in Cell-Free Systems

Abstract

Two supernatant fractions, T1 and T2, have been isolated and partially purified from rat liver and rabbit reticulocytes. In a cell-free system programmed with polyuridylic acid, both fractions are needed for phenylalanine polymerization. T1 factor and GTP are required for the enzymatic binding of phenylalanil-tRNA to template charged ribosomes. In the course of the binding reaction, GTP is hydrolyzed while no dipeptide formation can be detected. T2 factor coincides with a ribosome-linked GTPase, which is not stimulated by the addition of polyuridylic acid and phenylalanil-tRNA.     

Cell-Free Protein Synthesis by Rumen Protozoa

SYNOPSIS. The characteristics of protein synthesis by cell-free extracts of mixed rumen protozoa have been investigated. ATP,¹ GTP, and an energy supply system were necessary for amino acid incorporation which was partially inhibited by cycloheximide but not by chloramphenicol (100 μg/ml). The system was particularly sensitive to the cation concentration of the incubation mixture, maximal incorporation requiring 5 mM Mg⁺⁺ and 50 mM K⁺ Incorporation was further stimulated by the addition of 0.25 mM spermidine or 0.25 mM MnCl₂. Sucrose gradient centrifugation of the cell sap after amino add incorporation showed that most of the incorporated radioactivity was associated with free polysomes. These polysomes contained 82 S ribosomes which dissociated in high Tris concentrations to yield 40 S and 55 S ribosomes.     

Cell Wall Synthesis in Staphylococcus aureus in the Presence of Protein Synthesis Inhibitory Agents

Electron-microscopic and biochemical studies on morphological changes in Staphylococcus aureus following exposure to protein synthesis inhibitory agents such as lincomycin (LCM), clindamycin (CLM), erythromycin (EM), and spiramycin (SP) are presented in this paper. It was demonstrated that bacterial cell walls became extremely thickened usually with the formation of multilayers, when exposed to each of the above-mentioned antibiotics. Furthermore, electron density of the cytoplasm was higher in those cells exposed to drugs than in intact control cells. Incorporations of ¹⁴C-labeled l-lysine into the cell-wall fraction and the protein fraction were measured for biochemical elucidation of these phenomena. Labeled lysine was selectively incorporated into the cell-wall fraction when the test organism was exposed to the respective antibiotics. Uptake at 15 min after exposure was about twice as large as that of intact control cells. SP and CLM inhibited protein synthesis while they stimulated cell-wall synthesis. The evidences for thickening of, and formation of multilayers in the bacterial cell walls following exposure to drugs were closely related to the stimulating action of these antibiotics on the cell-wall synthesizing system. Morphology of resistant clinical isolates following such antibiotic exposure was also investigated using two staphylococcal strains, one resistant to EM alone and the other completely cross-resistant to all the macrolides.     

Exopenicillinase Synthesis in Staphylococcus aureus

In Staphylococcus aureus, penicillinase remaining cell-bound (60 to 75% of original total) after treatment with citrate does not become exopenicillinase. Exopenicillinase in these cells appears only under conditions permitting de novo penicillinase synthesis. By use of (14)C-labeled cells, it was shown that exopenicillinase consists of newly synthesized molecules which have not equilibrated with preformed membrane-bound enzyme.     

Protein Synthesis in Cell-Free Extracts of Streptococcus faecalis

If a cell-free extract of Streptococcus faecalis is fractionated by way of a 10 to 70% sucrose gradient, at least three areas are found capable of protein synthesis having a density greater than can be accounted for by association of individual ribosomes. These areas represent distinct “polysome” peaks rather than random distribution of polymers of varied length. They appear to be membrane subunits. In addition there is a further particle with a density of about 150S, incapable of protein synthesis but which is capable of stimulating protein synthesis in some of the larger fractions found by gradient centrifugation.     

Biphasic Modulation by ACTH-Like Peptides of Protein Synthesis in a Cell-Free System from Rat Brain

Brain protein synthesis in a cell-free system was stimulated by 10(-8) M-ACTH1-24. This stimulatory effect was completely inhibited by aurintricarboxylic acid (ATA), an inhibitor of reinitiation of new peptide chains. The N-terminal peptide sequence 4-10 exerted a biphasic modulation of cell-free protein synthesis, i.e., a stimulation at low concentrations (10(-8) and 10(-10) M) and an inhibition at a high concentration (10(-4) M). The D-isomer, ACTH4-10-7-D-phe, also showed a biphasic modulation that, however, was in a direction opposite to that shown by ACTH4-10-7-L-phe at 10(-8) M and 10(-4) M.     

Protein A-Mediated Multicellular Behavior in Staphylococcus aureus

The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.Staphylococcus aureus is a gram-positive bacterium that lives as part of the normal microflora on the skin and mucous membranes of humans and animals. If S. aureus passes through the epithelial barrier and reaches internal organs, it can cause a variety of diseases, ranging from minor skin infections, such as furuncles or boils, to severe infections, such as bacteremia, pneumonia, osteomyelitis, or endocarditis. Despite the progress with antibiotics in the treatment of bacterial infections over the last 2 decades, the number of infections due to S. aureus has increased (11, 30). The infection rate has been correlated with an increase in the use of prosthetic and indwelling devices in modern medical practices (24, 26). S. aureus, as well as other coagulase-negative staphylococci, displays a strong capacity to irreversibly attach to the surface of implanted medical devices and forms multilayered communities of bacteria, known as biofilms, that grow embedded in a self-produced extracellular matrix (23). The biofilm formation process occurs in two steps: first, bacterial cells irreversibly attach to a surface, and second, they interact with each other and accumulate in multilayered cell clusters embedded in a self-produced extracellular matrix. Primary attachment is mediated by physico-chemical cell surface properties as well as specific factors that mediate the attachment to the host-derived extracellular matrix components that rapidly coat the biomaterial following insertion into the patient. Numerous proteins from the MSCRAMMs family (microbial surface components recognizing adhesive matrix molecules) are involved in the first step of S. aureus biofilm formation, such as clumping factors ClfA (37) and ClfB (41) and fibrinogen and fibronectin binding proteins (FnBPA and FnBPB) (25, 31). Once bacteria accumulate in multilayered cell clusters, most have no direct contact with the surface, and thus cell-to-cell interactions become essential for biofilm development and maintenance. An extracellular polysaccharide intercellular adhesin (PIA, or PNAG), produced by icaADBC operon-encoded enzymes, is currently the best-characterized element mediating intercellular interactions in vitro (8, 23, 34, 35, 38). Alternatively, a number of surface proteins can replace PIA/PNAG exopolysaccharide in promoting intercellular adhesion and biofilm development, including the surface protein Bap (9). All the tested staphylococcal isolates harboring the bap gene were shown to be strong biofilm producers, and inactivation of the icaADBC operon in bap-positive strains had no effect on in vitro biofilm formation (57). Remarkably, proteins homologous to Bap are involved in the biofilm formation process in diverse bacterial species (33). A second surface protein, SasG, as well as its homologous protein in Staphylococcus epidermidis, Aap, also mediates intercellular interactions and biofilm development in the absence of the ica operon (7, 51). More recently, two independent laboratories have shown that fibronectin binding proteins A and B (FnBPA and FnBPB) induce biofilm development of clinical isolates of S. aureus (45, 55). Finally, there is growing evidence that extracellular DNA, despite not being sufficient to replace PIA/PNAG exopolysaccharide, is an important S. aureus biofilm matrix component (50).During the course of a systematic mutagenesis study of the 17 two-component systems of S. aureus that aimed to identify biofilm-negative regulators, we found that S. aureus agr arlRS double mutants developed an alternative, ica-independent biofilm in a chemically defined medium, Hussain-Hastings-White (HHW) medium (56). This study focused on the identification of the proteinaceous compound responsible for the biofilm developed by S. aureus agr arlRS mutants. Here, we show that S. aureus protein A is responsible for the aggregative phenotype and capacity for biofilm formation displayed by this strain. Furthermore, overproduction of protein A in wild-type S. aureus strains or addition of soluble protein A to bacterial growth medium induced aggregation and biofilm development, suggesting that protein A does not need to be covalently linked to the cell wall to promote multicellular behavior. Moreover, deletion of the spa gene significantly decreased the capacity of S. aureus to colonize subcutaneously implanted catheters. Our findings support a novel role for protein A in promoting multicellular behavior and suggest that protein A-mediated biofilm development may have a critical function during the infection process of S. aureus.     

Protein Synthesis by a Cell-Free Preparation from the Bird Malaria,Plasmodium lophurae*

SYNOPSIS. Cytoplasmic polyribosomes were isolated from the avian malaria parasite Plasmodium lophurae by lysis with 0.15% Triton X-100 followed by high speed centrifugation through a discontinuous sucrose gradient. Polyribosomes were protected from nuclease degradation using 100 μg/ml heparin or 50 μg/ml dextran sulfate. Cell-free incorporation of radioisotope-labeled amino acids required a pH 5 fraction (duck reticulocyte), Mg²⁺, and an energy-generating system. The protein synthesizing system was stimulated by the addition of polyuridylic acid. Optimum conditions for protein synthesis by the plasmodial system are described. The effects of drugs on the cell-free protein synthesizing system using duck reticulocyte and plasmodial ribosomes are reported.     

Protein A Mutants of Staphylococcus aureus

Staphylococcus aureus Cowan I was exposed to nitrosoguanidine or ethyl-methanesulfonate, and survivors were screened on nutrient agar plates containing rabbit anti-protein A serum for loss of protein A production. More than half of all protein A-deficient mutants also lacked nuclease, coagulase, alpha hemolysin, fibrinolysin, mannitol utilization, and the phage-type pattern. Mutants with a spectrum of these properties were also isolated. Induced or spontaneous reversions of the mutants were observed. The properties of the protein A-deficient mutants suggest that synthesis or release (or both) of a number of extracellular products of S. aureus is controlled by a common regulatory mechanism.     

Protein Synthesis in Cell-Free Preparations of Crithidia fasciculata*

SYNOPSIS. Cell-free preparations from Crithidia jasciculata carried out protein synthesis as measured by ¹⁴C-leucine uptake (optimum ～ 10 mM Mg⁺⁺) and poly U-directed ¹⁴C-phenylalanine uptake (optimum ～ 16 mM Mg⁺⁺). Characteristics of the system and sucrose density-gradient patterns of ribosomes were investigated. The charging and transfer reactions—the 2 main steps in protein synthesis—were inhibited by stilbamidine, hydroxystilbamidine, pentamidine, quinapyramine (Antrycide), and suramin.     

Inhibition of Cell-Free Protein Synthesis by Hydrostatic Pressure

Pressure inhibition of cell-free polypeptide synthesis is manifested in the same manner as that observed in the intact cell: (i) starting at approximately 200 atm, there is a progressive inhibition with increasing pressures; (ii) there is complete inhibition at 680 atm; (iii) incorporation into polypeptide is instantaneously reversible after pressure release and proceeds at a rate parallel to an atmospheric control; and (iv) the volume change of activation (DeltaV*) is 100 cm(3)/mole. Peptide bond formation per se can occur at a pressure level which is totally inhibitory to polypeptide synthesis. The one investigated step in translation that is inhibited in an identical manner is the binding of aminoacyl-transfer ribonucleic acid (AA-tRNA) to the ribosome-messenger RNA (mRNA) complex. The volume change of activation (DeltaV*) calculated for the binding reaction is also 100 cm(3)/mole. Thus, the inability of AA-tRNA to bind to ribosomes and mRNA under pressure, possibly in conjunction with translocation, appears to be responsible for the observed inhibition of the translational mechanism.     

Characterization of an Initiating Cell-Free Protein Synthesis System Derived from Rabbit Brain

Abstract: Protein synthesis in the brain is known to be affected by a wide range of treatments. The detailed analysis of the mechanisms that are involved would be facilitated by the development of cell-free translation systems derived from brain tissue. To date, brain cell-free systems have not been fully characterized to demonstrate a capacity for initiation of translation. The following criteria were utilized to demonstrate that a cell-free protein synthesis system derived from rabbit brain was capable of initiation in vitro : (a) sensitivity of cell-free translation to the initiation inhibitor aurintricarboxylic acid (ATA); (b) binding of [³⁵S]Met-tRNA_f to 40S and 80S initiation complexes; (c) incorporation of labeled initiation methionine into high-molecular-weight proteins; and (d) the association of labeled exogenous mRNA with polysomes. The optimum conditions for amino acid incorporation in this system were 4 mM-Mg²⁺, 140 mM-K⁺, and pH 7.55. Incorporation was dependent on the addition of ATP, GTP, and an energy-generating system. Cell-free protein synthesis reflected the normal process, since a similar spectrum of proteins was synthesized in vitro and in vivo. This initiating cell-free translation system should have wide application in the analysis of the mechanisms whereby various treatments affect protein synthesis in the brain.     

Protein Synthesis in Cell-Free Systems: an Effect of Interferon

The activity of ribosome and cell-sap fractions from interferon-treated and control chick embryo fibroblasts was compared in mixed chick-mouse and purely chick cell-free systems capable of the synthesis of viral polypeptide(s) in response to viral ribonucleic acid (RNA). Interferon treatment of cells did not affect the intrinsic amino acid incorporation activity of these systems or their response to polyuridylic acid. With encephalomyocarditis (EMC) virus RNA as messenger, however, a fraction of the ribosomes from interferon-treated cells appeared less active than parallel controls. The results obtained with the corresponding cell-sap fractions were variable. Although competition between endogenous and added messengers cannot be excluded in these systems, a reduced level of translation of EMC RNA with interferon-treated cell ribosomes was also suggested by the results of analyses of tryptic digests of the products formed in response to the RNA. In addition, these analyses showed that this reduced activity must reflect a reduction in the rate or frequency of translation rather than a decrease in the length of the EMC RNA translated, for the same polypeptides were synthesized in response to the RNA with material from interferon-treated and control cells. Interferon added directly to the cell-free system was without effect. Although suggestive, these results do not provide definitive evidence for or against the hypothesis that virus protein synthesis is inhibited at the translational level in the interferon-treated cell. Possible alternative interpretations of the data are discussed.