Modification of translation factor aIF5A from Sulfolobus solfataricus.

Eukaryotic eIF5A and its bacterial orthologue EF-P are translation elongation factors whose task is to rescue ribosomes from stalling during the synthesis of proteins bearing particular sequences such as polyproline stretches. Both proteins are characterized by unique post-translational modifications, hypusination and lysinylation, respectively, which are essential for their function. An orthologue is present in all Archaea but its function is poorly understood. Here, we show that aIF5A of the crenarchaeum Sulfolobus solfataricus is hypusinated and forms a stable complex with deoxyhypusine synthase, the first enzyme of the hypusination pathway. The recombinant enzyme is able to modify its substrate in vitro resulting in deoxyhypusinated aIF5A. Moreover, with the aim to identify the enzyme involved in the second modification step, i.e. hypusination, a set of proteins interacting with aIF5A was identified.

Holins: form and function in bacteriophage lysis.

During the lytic cycle of most bacteriophages, a phage-encoded peptidoglycan-degrading activity is elaborated. At least four entirely distinct types of enzymes fulfill this role and are given the generic name 'endolysin'. Endolysins characterized to date are synthesized without a signal sequence and thus accumulate fully folded and active in the cytosol during the vegetative phase. Small membrane proteins are required in order for endolysins to gain access to the peptidoglycan. Because the available data suggest that the membrane lesion formed by these proteins is stable and non-specific, these proteins have been given the designation 'holins' ('hole'-formers). Analysis of the primary sequence suggests a simple membrane topology with two or more membrane-spanning helical domains and a highly charged, hydrophilic C-terminus. Comparison of the sequences of holins from phages of Gram-negative hosts suggests there are at least two major holin groups. Putative holin genes have also been found in bacteriophages of Gram-positive bacteria. Altogether, in phages of Eubacteria, 11 or more unrelated gene families which share the functional and structural characteristics of holins have been identified. Genetic and physiological analysis suggest that holins are primarily regulated at the level of function. Holin function is modulated in some cases by a second protein encoded by the holin gene. The primary regulation of holin function, however, appears to be intrinsic to the holin structure itself, since a missense allele of the S holin gene of phage lambda has been found which abolishes the normal delay that allows the vegetative phase to generate a useful number of progeny.

Ternary complex formation on leaderless phage mRNA.

The phage Lambda PRM promoter-derived cI mRNA and phage P2 gene V mRNA are transcribed beginning with the A residue of the AUG start codon. Using lacZ fusion analysis we have assessed the effects of alterations in the immediate downstream coding region on the translational efficiency of these mRNAs. Mutations, including deletions of the putative downstream box of either cI or gene V mRNAs, showed no significant reduction in expression of the different lacZ fusions. Primer extension inhibition analysis suggests a role of ribosomal protein S1 in cI mRNA recognition.

Synthesis of two bacteriophage lambda S proteins in an in vivo system.

Bacteriophage lambda has two genes which are essential for lysis: R, a gene encoding a 158-amino-acid (aa) transglycosylase that attacks the peptidoglycan, and S, a gene encoding two inner-membrane-associating proteins, designated S105 and S107 for their predicted lengths in aa residues. S105 and S107 are thought to have opposing roles in lysis, with the former acting as the lethal lysis effector and the latter as a lysis inhibitor. Here, we used a T7-polymerase-mediated expression system to show that S105 and S107 are synthesized at a constant ratio of about 2.5:1 throughout the period leading up to lysis, indicating that lysis scheduling does not require a translationally controlled switch from inhibitor (S107) to effector (S105) synthesis. However, evidence is presented that the mRNA sequences immediately 5' to the ribosome-binding site (RBS) of the S gene are required for the rather limited translation, but not the stability, of the S mRNA. No difference could be found in the pattern of ternary complex formation over the two S start codons in in vitro toe-printing assays with the wild-type mRNA and with mRNA deleted of the upstream sequences. Nevertheless, these results may suggest a role for translational control in S gene expression, if not in its temporal regulation or in the partition between S105 and S107 production.

Discrimination of 5'-terminal start codons by translation initiation factor 3 is mediated by ribosomal protein S1.

The interrelation between ribosomal protein S1 and IF3 in recognition/discrimination of 5'-terminal start codons by 30S ribosomes has been studied using in vitro toeprinting. The study has been performed with two naturally occurring leaderless mRNAs, lambda cI and phage r1t rro mRNA, as well as with an artificial leaderless mRNA derived from the E. coli ompA gene. We show that in the absence of S1, IF3 does not discriminate against the authentic 5'-terminal start codon of both cI and rro mRNA. Since IF3 was able to exert its proofreading function for initiator tRNA(fMet) on 30S ribosomes lacking S1, this observation cannot be attributed to a lack of binding to or action of IF3 on 30S(-S1) ribosomes. In contrast to leaderless mRNAs, ternary complex formation occurs in the presence of IF3 with 30S ribosomes when the start codon is preceded by a short 20-nucleotide 5'-untranslated region containing a canonical Shine and Dalgarno sequence. This suggests that 5'-terminal start codons are recognised by IF3 as non-standard because of the lack of 16S rRNA-mRNA contacts.

Modulation of ribosomal recruitment to 5'-terminal start codons by translation initiation factors IF2 and IF3.

Sequence determinants and structural features of the RNA govern mRNA-ribosome interaction in bacteria. However, ribosomal recruitment to leaderless mRNAs, which start directly with the AUG start codon and do not bear a Shine-Dalgarno sequence like canonical mRNAs, does not appear to rely on 16S rRNA-mRNA interactions. Here, we have studied the effects of translation initiation factors IF2 and IF3 on 30S initiation at a 5'-terminal AUG and at a competing downstream canonical ribosome binding site. We show that IF2 affects the forward kinetics of 30S initiation complex formation at the 5'-terminal AUG as well as the stability of these complexes. Moreover, the IF2:IF3 molar ratio was found to play a decisive role in translation initiation of a leaderless mRNA both in vitro and in vivo indicating that the translational efficiency of an mRNA is not only intrinsically determined but can be altered depending on the availability of components of the translational machinery.

Phi X174 protein E-mediated lysis of Escherichia coli.

Bacteriophage PhiX174 encodes a single lysis gene, E, the function of which is necessary and sufficient to induce lysis of Escherichia coli. Here we present a novel model for E-lysis: physiological, genetic and biochemical data are presented which suggest that a transmembrane tunnel penetrating the inner and outer membrane is formed during the lytic action of protein E. Moreover, using high magnification scanning and transmission electron microscopy in this study, it was possible to visualize the transmembrane lysis structure directly.

Direct determination of molecular ratios of peptides coupled via N-succinimidyl 3-(2-pyridyldithio)propionate to carrier proteins using high performance liquid chromatography.

A simple, rapid and reproducible method is presented for direct determination of the substitution ratio of a carrier protein with a synthetic nonradioactively labeled peptide. The peptide was covalently linked by a thiol group of a cysteine residue to the immunogenic carrier protein using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. The substitution ratio was determined after reductive cleavage of the intermolecular disulfide bond between peptide and carrier and the amount of carrier and peptide quantitated directly by calibrated HPLC analysis within 15 min.

Non-specific hole formation in the Escherichia coli inner membrane by lambda S proteins in independent of cellular secY and secA functions and of the proportion of membrane acidic phospholipids.

Formation of the lesion in the Escherichia coli inner membrane caused by lambda lysis protein S was examined by electron microscopy. We also show that macromolecules exceeding the size of the lambda R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery.

The hydrophilic C-terminal part of the lambda S holin is non-essential for intermolecular interactions.

Available evidence indicates that oligomerization of the bacteriophage lambda S holin leads to a non-specific lesion in the cytoplasmic membrane which permits transit of the phage encoded transglycosylase to the periplasm. In an attempt to locate an intermolecular interaction domain in S a chimeric protein comprising the N-terminal 32 aa of phage PhiX174 lysis protein E and the last 75 aa of lambda S has been constructed. We report that the E phi S fusion protein is stable, membrane bound, and inhibits S-mediated lysis in trans. C-terminal truncations of the E phi S fusion protein indicated that the hydrophilic C-terminal end of S (i.e. the last 15 aa) is non-essential for oligomerization.

Expression of the Alcaligenes eutrophus phbA gene in Escherichia coli using a positive selection vector based on phage Lambda lysis genes.

A new positive selection vector, pGS23, based on the Lambda lysis cassette has been designed for efficient expression of homologous and heterologous genes in Escherichia coli. The plasmid permits controlled expression of a gene of interest under transcriptional control of the lac promoter with translation initiation of coding sequences directed by the phage T7 gene 10 ribosome binding site. The application of the vector system was tested for high level expression of the heterologous phbA gene of Alcaligenes eutrophus in E. coli.

Evaluation of the E. coli ribosomal rrnB P1 promoter and phage-derived lysis genes for the use in a biological containment system: a concept study.

A concept study devised for the development of a biological containment system has been conducted. We show that the lysis genes of different phage origin function in a variety of bacteria. They may therefore be suited for conditional suicide cassettes. Moreover, we tested whether the Escherichia coli rrnB P1 promoter could function as an environmentally responsive element sensing poor growth conditions expected after an accidental release of E. coli production strains from a bioreactor. Mimicking poor nutrient conditions by production of the alarmone guanosine tetraphosphate (ppGpp) with a plasmid encoded ppGpp synthetase I, the rrnB P1 promoter activity was completely turned off. These experiments suggested that the rrnB P1 promoter may be used as an efficient biosensor for altered growth conditions. A concept for a conditional suicide system employing the rrnB P1 promoter and phage-derived lysis genes as key components is discussed.

Dizziness and vertigo in a department of emergency medicine.

Dizziness is a common and vexing diagnostic problem in emergency departments. The term is rather undefinite and often misused, but can in practice be classified into four categories: fainting, disequilibrium, vertigo and miscellaneous syndromes. Vertigo is the most common category of dizziness. Classification of vertigo can be based either on chronological criteria (acute, recurrent or chronic vertigo) or on topographical criteria (peripheral or central vertigo). Physicians working in emergency departments must be able to rapidly identify patients with potentially serious forms of vertigo, which could cause death or disability, and patients with mild conditions, that can be effectively treated. Previous studies and the experience of the authors have shown that reliable diagnostic hypotheses can be generated by taking a proper clinical history (focused on the onset and duration of the disease, the circumstances causing the vertigo and associated otological or neurological symptoms) and performing an accurate physical examination (evaluation of neurological defects and spontaneous or provoked nystagmus), supplemented by few laboratory tests and diagnostic procedures. Therapy of vertigo in emergency settings is mainly symptomatic and based on sedation and use of vestibulosuppressant drugs (antihistamines, phenothiazines).

[Influence of high environmental temperature on various parameters of blood coagulation in healthy subjects and in thrombosis risk patients]. / Influenza dell'elevata temperatura ambientale su alcuni parametri della coagulazione in soggetti sani ed in pazienti a rischio trombotico.

The effects of environmental hyperthermia (exposure to a hot, dry microclimate) on the human body were investigated with particular reference to certain clotting parameters in healthy subjects and patients at risk of thrombosis. The study covered 70 volunteers, 10 of them clinically healthy (6 males and 4 females) aged 37.7 +/- 9.7 and 60 patients at risk of thrombosis aged 18-60 and divided according to pathology as follows: 26 with ischaemic cardiopathy, 22 with metabolic disorders (12 diabetics, 8 with dyslipidaemia, 2 with hyperuricaemia) and 12 with obliterating arteriopathies of the lower extremities (Fontaine stage 2 and 3). The following standardised protocol was adopted: 2 hours exposure in a controlled climate chamber (40 degrees C, 40-50% humidity, standard air speed 4 m/min, barometric pressure 760 mmHg) for a total of 8 exposures (2 per week for 1 month). This approach was adopted in order to assess not only the effect of each single exposure but also the role of any adaptation to heat. Three blood samples were taken from each subject for each session: the first in basal conditions in a comfortable environment, the second at the end of the 2 hour exposure; the third 30 minutes after the end of the session. Simultaneously samples of arterial blood were taken for pH assays and a spleen echography was performed in basal conditions and at the end of the session for each subject. Each blood sample was tested for several parameters essentially attributable to blood concentration for a broader view of the biological effects of exposure to heart (Ht, blood protein, Nat, K+). The clotting factors under specific study were also assessed (platelet count and volume, beta-thromboglobulin, PF4, von Willebrand Factor VIII, thromboxane B2, fibronectin). Body weight, blood pressure and oral temperature were also measured in all subjects before and after each session. In all subjects both healthy and at risk of thrombosis oral temperature increased (1 +/- 0.4 degrees); on average blood pressure was already higher in basal conditions in the patient group; body weight fell by 900 +/- 120 G in both groups. Ht and blood protein increased significantly in both groups while electrolyte changes were insignificant and blood pH showed a tendency towards acidosis. Clotting parameters revealed a tendency towards thrombophilia in all subjects: platelet count and volume were already higher in the patient group in basal conditions and increased after exposure to hyperthermia. Beta-thromboglobulin, FP4, Factor VIII, thromboxane B2 and fibronectin all increased.(ABSTRACT TRUNCATED AT 400 WORDS)

The RNA chain elongation rate of the lambda late mRNA is unaffected by high levels of ppGpp in the absence of amino acid starvation.

In this study, the effects of high levels of guanosine tetraphosphate (ppGpp) on the decay and RNA chain elongation kinetics of the bacteriophage lambda late transcript in Escherichia coli were examined in the absence of amino acid starvation. The accumulation, mRNA decay kinetics, and RNA chain elongation rate of the lambda late mRNA were determined after heat induction of lambdacI857 lysogens in the presence of high levels of ppGpp induced from a RelAalpha fragment-overproducing plasmid. The accumulation kinetics and elongation rate determinations of the late mRNA were made at long times after induction to allow a new steady state of transcriptional activities under conditions of elevated intracellular levels of ppGpp. The results indicate no prolonged or significant effect on either mRNA decay or the RNA chain elongation rate of the late mRNA as a result of elevated ppGpp levels. Surprisingly, the RNA chain elongation rate determinations indicate an RNA polymerase processivity of approximately 90-100 nucleotides/s for the lambda late transcript despite the presence of high levels of ppGpp. The results are discussed in terms of various models for regulation of stable and messenger RNA synthesis in E. coli.

Influence of C-terminal modifications of phi X174 lysis gene E on its lysis-inducing properties.

The phi X174 gene E product (gpE) causes lysis of Escherichia coli by inducing the host autolytic system. Experiments were carried out to ascertain which part of the 91 amino acid polypeptide carries the functional site for this process. For this purpose fusion genes were created comprising the first 51 codons of gene E and unrelated sequences coding for 102 or 33 amino acids respectively. The chimeric protein of 153 amino acids consisting of the N-terminal part of gpE and a fragment of beta-galactosidase, was neither able to lyse E. coli nor to restore beta-galactosidase activity by alpha-complementation. Expression of the 84 amino acid polypeptide, however, was able to induce lysis of E. coli. It is therefore concluded that the functional lysis-inducing site of gpE is located within the cloned N-terminal part of gene E. In the shorter chimeric protein the sequence following the functional site was tolerated or necessary for stabilization, but in the longer chimeric protein, the C-terminal sequence disturbed the lysis-inducing conformation.

Functional assembly of the lambda S holin requires periplasmic localization of its N-terminus.

Bacteriophage-lambda-induced host-cell lysis requires two phage-encoded proteins, the S holin and the R transglycosylase. At a specific time during infection, the holin forms a lesion in the cytoplasmic membrane that permits access of the R protein to its substrate, the peptidoglycan. The lambda S gene represents the prototype of holin genes with a dual-start motif; they encode two proteins, a lysis effector and a lysis inhibitor. Although the two S proteins differ only by two amino acids (Met-1 and Lys-2) at the N-terminus, the longer product (S107) acts as an inhibitor of the lysis effector (S105). The functional difference between the proteins has been previously ascribed to the Lys-2 residue in S107. It was therefore of interest to determine the subcellular localization of the N-terminus of either S protein. To study the membrane topology of the S proteins, we used the topology probe TEM beta-lactamase and an N-terminal tag derived from the Pseudomonas aeruginosa phage Pf3 coat protein. We show that both S proteins have a type III (Nout/Cin) topology. The results provide insight into the regulatory mechanism imposed by the dual-start motif and will be discussed in terms of a model for temporal regulation of the S-dependent "hole" in the membrane.http://link.springer-ny. com/link/service/journals/00203/bibs/172n1p31.html

Molecular function of the dual-start motif in the lambda S holin.

The lambda S gene represents the prototype of holin genes with a dual-start motif, which leads to the synthesis of two polypeptides, S105 and S107. They differ at their N-terminus by only two amino acids, Met-1 and Lys-2, at the beginning of the longer product. Despite the minor difference, the two proteins have opposing functions in lysis, with protein S107 being an inhibitor and protein S105 being an effector of 'hole formation' in the inner membrane. Here, we have studied the molecular mechanism underlying the 'lysis clock' contributed by the dual-start motif. We have used protein fusions in which the secretory signal sequence of the M13 procoat protein VIII has been abutted to the N-terminal Met residues of S105 and S107 respectively. S-dependent 'hole formation' required removal of the signal sequence in both fusion proteins, as both the VIII-S105 and the VIII-S107 fusion proteins were non-functional when leader peptidase cleavage was inhibited. These results strongly supported the hypothesis that functional assembly of S proteins requires translocation of their N-terminus to the periplasm. Using signal sequence cleavage as a measure of translocation, we observed that the translocation kinetics of the N-terminus of the S107 moiety was reduced about threefold when compared with the N-terminus of the S105 moiety. Moreover, depolarization of the membrane resulted in an immediate cleavage of the signal sequence and 'hole formation' exerted by the S107 moiety of the VIII-S107 fusion protein. A model is presented in which S107 with a reversed topology of its N-terminus interacts with S105 and poisons 'hole formation'. Upon depolarization of the membrane, translocation of the N-terminus of S107 to the periplasm results in the functional assembly of S proteins, i.e. 'hole formation'.

Two beginnings for a single purpose: the dual-start holins in the regulation of phage lysis.

For most large phages of both Gram-positive and Gram-negative bacteria, there appears to be a single pathway for achieving disruption of the host envelope, requiring at least two phage-encoded lysis functions (a holin and an endolysin). The holin is a small membrane protein which causes a non-specific lesion in the cytoplasmic membrane, which allows the endolysin to gain access to its substrate, the peptidoglycan. The scheduling of host lysis is effected by regulatory mechanisms which govern the synthesis and activity of the holin protein accumulating in the membrane. Accordingly, aspects of expression and function of holin genes are considered here, focusing mainly on the lambdoid S genes. This group of genes, of which lambda S is the prototype, are characterized by a dual-start motif consisting of two Met start codons separated by one or two codons, at least one of which specifies Arg or Lys. Two protein products are elaborated, differing only by two or three N-terminal residues but apparently possessing opposing functions: the shorter polypeptide is the active holin, or lysiseffector, whereas the longer polypeptide apparently acts as an inhibitor of holin function. Models will be considered which may account for the ability of the holin to form a 'hole' in the cytoplasmic membrane at a programmed time, as well as for the inhibitory properties of the longer product. Finally, we discuss recent results suggesting that the dual-start motif can be viewed as a level of regulation superimposed on a timing function intrinsic to the canonical holin structure.

Genetically modified filamentous phage as bactericidal agents: a pilot study.

AIMS: To evaluate the ability of a filamentous phage encoding lethal proteins to kill bacteria without host-cell lysis. METHODS AND RESULTS: Bacterial survival was determined after infection of a growing Escherichia coli culture with phage M13 encoding either the restriction endonuclease BglII gene or modified phage lambda S holin genes. The genetically engineered phage exerted a high killing efficiency while leaving the cells structurally intact. When compared with a lytic phage, the release of endotoxin was minimized after infection with the genetically modified phages. CONCLUSIONS: Genetically engineered phage can be used for efficient killing, concomitantly minimizing endotoxin release. SIGNIFICANCE AND IMPACT OF THE STUDY: This feasibility study provides a possible strategy for the use of genetically engineered phage as bactericidal agents by optimizing the advantages and minimizing potential risks such as release of pyrogenic cell wall components.