Preferred amino acids and thermostability

Most organisms grow at temperatures from 20 to 50 degrees C, but some prokaryotes, including Archaea and Bacteria, are capable of withstanding higher temperatures, from 60 to >100 degrees C. Their biomolecules, especially proteins, must be sufficiently stable to function under these extreme conditions; however, the basis for thermostability remains elusive. We investigated the preferential usage of certain groupings of amino acids and codons in thermally adapted organisms, by comparative proteome analysis, using 28 complete genomes from 18 mesophiles (M), 4 thermophiles (T), and 6 hyperthermophiles (HT). Whenever the percent of glutamate (E) and lysine (K) increased in the HT proteomes, the percent of glutamine (Q) and histidine (H) decreased, so that the E + K/Q + H ratio was >4.5; it was <2.5 in the M proteomes, and 3.2 to 4.6 in T. The E + K/Q + H ratios for chaperonins, potentially thermostable proteins, were higher than their proteome ratios, whereas for DNA ligases, which are not necessarily thermostable, they followed the proteome ratios. Analysis of codon usage revealed that HT had more AGR codons for Arg than they did CGN codons, which were more common in mesophiles. The E + K/Q + H ratio may provide a useful marker for distinguishing HT, T and M prokaryotes, and the high percentage of the amino acid couple E + K, consistently associated with a low percentage of the pair Q + H, could contribute to protein thermostability. The preponderance of AGR codons for Arg is a signature of all HT so far analyzed. The E + K/Q + H ratio and the codon bias for Arg are apparently not related to phylogeny. HT members of the Bacteria show the same values as the HT members of the Archaea; the values for T organisms are related to their lifestyle (intermediate temperature) and not to their domain (Archaea) and the values for M are similar in Eukarya, Bacteria and Archaea

Sequence analysis of the translational elongation factor 3 from Saccharomyces cerevisiae.

The gene YEF-3 encoding the elongation factor for protein synthesis in Saccharomyces cerevisiae is an essential gene as shown by one-step gene disruption and is located on chromosome XII as determined by orthogonal field alternation gel electrophoresis. The nucleotide sequence of the gene was determined from a sequential series of subclones generated from the YEF-3 gene cloned into bacteriophage M13. The HOMOL1 sequence and the RPG box, which are considered to be enhancer elements involved in coordinate regulation of transcription of the genes coding for yeast ribosomal proteins and protein synthesis factors, are found in the 5'-flanking region of the gene. A dyad symmetry that enables hairpin loop formation in the DNA molecule is found in the 3'-terminal at the termination site of transcription. An open reading frame of 3132 nucleotides codes for a deduced protein of 115,860 Da. A striking feature of the elongation factor 3 deduced polypeptide is the internal repeat of a region with approximately 200 amino acids which includes an ATP-binding site and shares similarity with some transport and drug-resistant proteins. Another characteristic is the presence of a highly charged C-terminal region composed of three basic polylysine blocks, suggesting interaction with RNA. The sequence supports the hypothesis that YEF-3 encodes a protein synthesis factor and suggests that its main role may be to transduce nucleoside triphosphate energy into mechanical energy for translocation during translation.

Effect of heat shock on S6 phosphorylation during the development of Blastocladiella emersonii.

Changes in phosphorylation of ribosomal protein S6 during heat shock, induction of thermotolerance and recovery from heat shock at different stages of Blastocladiella emersonii development were investigated. Independently of the initial state of S6 phosphorylation (maximal or intermediate), a rapid and complete dephosphorylation of S6 is induced by heat shock and S6 remains unphosphorylated during the acquired thermotolerance. During recovery from heat shock rephosphorylation of S6 occurs always to the levels characteristic of that particular stage, coincidently with the turn off of heat shock protein synthesis.

Acquisition of thermotolerance during development of Blastocladiella emersonii.

In the fungus Blastocladiella emersonii the synthesis of heat-shock proteins is developmentally regulated; particular subsets of heat-shock proteins are induced by heat shock during sporulation, germination and growth and some heat shock-related proteins are spontaneously expressed during sporulation (Bonato et al., 1987, Eur. J. Biochem., in press). Nevertheless, acquisition of thermotolerance can be induced at any stage of the life cycle. The development of thermotolerance is correlated with the enhanced synthesis of some heat-shock proteins: hsp 82a, hsp 82b, hsp 76, hsp 70, hsp 60, hsp 25, hsp 17b. Other hsps are not specifically involved in thermotolerance.

Differential expression of heat-shock proteins and spontaneous synthesis of HSP70 during the life cycle of Blastocladiella emersonii.

The heat-shock response in Blastocladiella emersonii is dependent on the developmental stage. Cells exposed to elevated temperatures at different stages of the life cycle (sporulation, germination or growth) show a differential synthesis of heat-shock proteins (hsps). Of a total of 22 polypeptides induced, particular subsets of hsps appear in each phase, demonstrating a non-coordinate heat-shock gene expression. In contrast, heat-shock-related proteins (hsp76, hsp70, hsp39a) are spontaneously expressed at a high level during sporulation. By the criteria of two-dimensional gel electrophoresis and partial proteolysis mapping, the 70,000-Da protein, whose synthesis is induced spontaneously during sporulation, is indistinguishable from the heat-inducible hsp70. The techniques of in vitro translation, and Northern analysis using a Drosophila hsp70 probe, demonstrated that enhanced synthesis of hsp70, which occurs during heat-shock treatment and spontaneously during sporulation, is associated with an accumulation of hsp70 mRNA. These observations suggest that hsp70 gene expression is induced during sporulation.

Analysis of Blastocladiella emersonii ribosomal proteins in four two-dimensional gel electrophoresis systems.

Ribosomal proteins of the aquatic fungus Blastocladiella emersonii were isolated and characterized on four different two-dimensional polyacrylamide gel electrophoresis systems. 40S and 60S ribosomal subunit proteins from zoospores were identified. The position of every protein was determined in each electrophoretic system using the "four-corners" method (Madjar et al., Molecular and General Genetics, 171: 121-134, 1979). Thirty-two and 39 proteins were identified in the 40S and 60S ribosomal subunits, respectively. The molecular weights of individual proteins in the 40S subunit ranged from 10 000 to 37 000, with a number-average molecular weight of 20 000. The molecular weight range for the 60S subunit was 13 000-51 000 with a number-average molecular weight of 21 000. Proteins from ribosomes of different cell types were compared and found to be qualitatively indistinguishable. The only consistent difference in the patterns of proteins was in the S6 protein of the 40S subunit, which is the major phosphoprotein of Blastocladiella ribosomes.

Influence of RP4 plasmid on extracellular protease secretion by Proteus mirabilis.

The transference by conjugation of protease genetic information between Proteus mirabilis strains only occurs upon mobilization by a conjugative plasmid such as RP4 (Inc P group). Upon receiving the RP4 plasmid, the level of proteolytic activity of the protease-excreting P. mirabilis is reduced to about 50%. A similar phenomenon occurs when the protease character is mobilized by the RP4 plasmid from the above transconjugant to a non-protease-excreting recipient strain. The molecular mechanism underlying the interference of R plasmids with proteolytic activity remains to be elucidated but there is evidence suggesting that some alteration in the bacterial envelope might be involved.

Phosphorylation of ribosomal protein S6 in the aquatic fungus Blastocladiella emersonii.

The changes in the degree of phosphorylation of ribosomal protein S6 during the life cycle of the aquatic fungus Blastocladiella emersonii were analyzed by two-dimensional gel electrophoresis. Three phosphorylated derivatives of S6 are present throughout the entire life cycle. However, under certain germination conditions, more highly phosphorylated derivatives of S6 appear. Nonetheless, the resumption of protein synthesis that occurs during germination is not dependent on those highly phosphorylated derivatives of S6. The pattern and sites of phosphorylation of S6 labelled in vivo with [32P]orthophosphate have been compared with those of 40S ribosomal subunit labelled in vitro by partially purified protein kinases. Three major phosphopeptides were found in S6 isolated from the zoospore, while six phosphopeptides were found after zoospore germination (in germling cells). The phosphopeptide patterns of S6 phosphorylated by the cAMP-dependent protein kinase and by casein kinases I and II were completely distinct. Only the cAMP-dependent protein kinase gives rise to a phosphopeptide found in 32P-labelled cells, indicating that one of sites phosphorylated in vivo is also phosphorylated in vitro by the cAMP-dependent protein kinase.

Sensibilidade de linhagens de Proteus mirabilis portadores do plasmidio R ao desoxicolato de sodio. / Sensitivity of Proteus mirabilis strains carrying plasmid R to sodium deoxycholate

A presenca de plasmidio R, em clones excretores e nao excretores da protease, de uma linhagem de Proteus mirabilis, confere maior sensibilidade ao desoxicolato de sodio

Acao de alguns agentes curagenicos na perda da excrecao de protase em Proteus mirabilis. / Effects of some cu excretion of Proteus mirabilis

Drogas curagenicas, como brometo de etidio acriflavina e mitomicina C, aumentam grandemente, a conversao de celulas excretoras de protease instaveis de Proteus mirabilis em celulas nao excretoras. Esse efeito nao ocorre sobre celulas excretoras estaveis de protease. A rifampicina apenas seleciona celulas protease-negativas, por eliminacao preferencial de celulas excretoras.Temperaturas superiores a fisiologica nao sao efetivas na perda de excrecao de protease em linhagens de P. mirabilis que excretam protease de maneira instavel

Cyclic nucleotide-independent protein kinases from ribosomes and phosphorylation of a single 40S ribosomal subunit protein in zoospores of Blastocladiella emersonii.

Cyclic nucleotide-independent protein kinase (EC 2.7.1.37) activity was found in the nuclear cap organelle, within which ribosomes of zoospores of Blastocladiella emersonii are sequestered. Two protein kinase activities were resolved from the high-salt wash fraction of zoospore ribosomes by selective adsorption to DEAE-cellulose. Both enzymes phosphorylated in vitro a 32,000 Mr protein of the 40S ribosomal subunit. Phosphorylation of this ribosomal protein, which exhibits electrophoretic properties similar to those of mammalian ribosomal protein S6, was also observed in vivo in 32P-labeled zoospores.