An analysis of determinants of amino acids substitution rates in bacterial proteins

The variation of amino acid substitution rates in proteins depends on several variables. Among these, the protein's expression level, functional category, essentiality, or metabolic costs of its amino acid residues may play an important role. However, the relative importance of each variable has not yet been evaluated in comparative analyses. To this aim, we made regression analyses combining data available on these variables and on evolutionary rates, in two well-documented model bacteria, Escherichia coli and Bacillus subtilis. In both bacteria, the level of expression of the protein in the cell was by far the most important driving force constraining the amino acids substitution rate. Subsequent inclusion in the analysis of the other variables added little further information. Furthermore, when the rates of synonymous substitutions were included in the analysis of the E. coli data, only the variable expression levels remained statistically significant. The rate of nonsynonymous substitution was shown to correlate with expression levels independently of the rate of synonymous substitution. These results suggest an important direct influence of expression levels, or at least codon usage bias for translation optimization, on the rates of nonsynonymous substitutions in bacteria. They also indicate that when a control for this variable is included, essentiality plays no significant role in the rate of protein evolution in bacteria, as is the case in eukaryotes.     

A similarity ring for amino acids based on their evolutionary substitution rates

A similarity ring for amino acids is obtained by reordering the rows and columns of their substitution probability matrix so as to minimize the variance of the matric elements about the main diagonal.     

Zooplankton induced changes in dissolved free amino acids and in production rates of freshwater bacteria

This study examined the importance of zooplankton in the flux of dissolved free amino acids (DFAA) in the water and into bacteria. DFAA release rates were followed in laboratory grazing experiments usingDaphnia galeata andEudiaptomus graciloides as grazers, andScenedesmus acutus andSynechococcus elongatus as food sources. Except for minor initial peaks, DFAAs were released continuously during the first 2 hours and made up 6–12% (in one experiment 50%) of the calculated ingestion rates. During three diel studies in lakes, effects of removal and increase of the density of zooplankton (>200m) on the pools of DFAA as well as on the bacterial production were followed. During two of the diel studies, higher DFAA pools were measured when 3–4 times the natural zooplankton density was present, and in one study a minor increase also occurred in the bacterial production, compared with results from experiments without zooplankton and with a natural zooplankton density. The increase in bacterial growth coincided with a decline in DFAA. During the third study, neither DFAA nor the bacterial production changed significantly when the zooplankton density was increased 3 times. Removal of zooplankton, however, caused a decline in both DFAA and bacterial production. Our data suggest a close relationship between occurrence of zooplankton and release of DFAA, but the factors regulating the amount of DFAA released and its effect on bacterial growth are not yet understood.     

Specific uptake rates of amino acids by attached and free-living bacteria in a mesotrophic lake

Seasonal and spatial patterns of specific uptake rates of amino acids by bacteria in Lake Constance were studied. The total bacterial population was divided into small (0.2- to 1.0-micron) and large (1.0- to 3.0-micron) free-living bacteria and attached bacteria by fractionated filtration. Data for attached bacteria, received by retention on 3.0-micron-pore Nuclepore filters, were corrected for free-living bacteria in this fraction. Specific uptake rates based on autoradiography were also recorded. Specific uptake rates for attached bacteria ranged from 9.41 X 10(-11) to 6.11 X 10(-8) ng of C h-1 cell-1 and were therefore significantly greater than those for free-living bacteria during most time periods. However, they were not significantly different from those for cells proven to be active by autoradiography. Specific uptake rates for small free-living bacteria ranged between 7.68 X 10(-11) and 4.60 X 10(-9) ng of C h-1 cell-1. They were nearly in the same range of those for large free-living bacteria (5.10 X 10(-11) to 1.07 X 10(-8) ng of C h-1 cell-1), although both fractions exhibited pronounced differences in their seasonal and vertical distributions.     

Variability of pressure adaptation in deep sea bacteria

Isolations of pressure-adapted deep sea bacteria from depths of 1,400 to 5,100 m resulted in a variety of psychrophilic barotolerant and barophilic strains. Growth rates determined at different pressures indicated a gradual transition between the two types of pressure-adapted isolates. The presence of barotolerant bacteria in deep water, sustained by sinking particulate matter, causes the nonbarophilic response of natural populations, i.e., increased growth after decompression. With increasing pressure-adaptation in barophilic isolates the maximum growth rates at optimum pressures decrease. Thus, the observed general slow-down of microbial activity in the deep sea takes effect regardless of the common occurrence of psychrophilic and barophilic bacteria. The highest degree of barophilism was observed in isolates from nutrient-rich habitats such as intestinal tracts of deep sea animals or decaying carcasses. Detailed studies with an isolate, growing barophilically on a complex as well as a single-carbon-source medium, showed that (1) culturing at pressures lower than optimal for growth resulted in the formation of cell filaments, (2) growth was unaffected by repeated compression/decompression cycles and (3) no perceptible differences in the distribution of radiolabeled carbon from an amino acid mixture occurred in cells grown at, below and above the pressure optimal for growth.Dedicated to Professor Dr. Hans G. Schlegel on the occasion of his 60th birthday in recognition of his broad microbiological interests and in special appreciation of his lasting support for the Marine Microbiology Course at the Stazione Zoologica (Naples, Italy) now for almost 25 years Non-standard abbreviations. The traditional use of atm as a unit of pressure (=10 m of water column, =1.013 bar, =101.3 kN/m²) is retained here in view of the important relation between water depth and hydrostatic pressure in the present study. Due to the compression of seawater and the geographic variability of gravity, there is a progressive deviation of the actual pressure with depth amounting to +4.9 atm at 5,000 m and a latitude of 30°. EPC, cell counts obtained by epifluorescence microscopy. PY, peptone yeast extract medium     

Specific uptake rates of amino acids by attached and free-living bacteria in a mesotrophic lake.

Seasonal and spatial patterns of specific uptake rates of amino acids by bacteria in Lake Constance were studied. The total bacterial population was divided into small (0.2- to 1.0-micron) and large (1.0- to 3.0-micron) free-living bacteria and attached bacteria by fractionated filtration. Data for attached bacteria, received by retention on 3.0-micron-pore Nuclepore filters, were corrected for free-living bacteria in this fraction. Specific uptake rates based on autoradiography were also recorded. Specific uptake rates for attached bacteria ranged from 9.41 X 10(-11) to 6.11 X 10(-8) ng of C h-1 cell-1 and were therefore significantly greater than those for free-living bacteria during most time periods. However, they were not significantly different from those for cells proven to be active by autoradiography. Specific uptake rates for small free-living bacteria ranged between 7.68 X 10(-11) and 4.60 X 10(-9) ng of C h-1 cell-1. They were nearly in the same range of those for large free-living bacteria (5.10 X 10(-11) to 1.07 X 10(-8) ng of C h-1 cell-1), although both fractions exhibited pronounced differences in their seasonal and vertical distributions.     

Genome bias influences amino acid choices: analysis of amino acid substitution and re-compilation of substitution matrices exclusive to an AT-biased genome

The genomic era has seen a remarkable increase in the number of genomes being sequenced and annotated. Nonetheless, annotation remains a serious challenge for compositionally biased genomes. For the preliminary annotation, popular nucleotide and protein comparison methods such as BLAST are widely employed. These methods make use of matrices to score alignments such as the amino acid substitution matrices. Since a nucleotide bias leads to an overall bias in the amino acid composition of proteins, it is possible that a genome with nucleotide bias may have introduced atypical amino acid substitutions in its proteome. Consequently, standard matrices fail to perform well in sequence analysis of these genomes. To address this issue, we examined the amino acid substitution in the AT-rich genome of Plasmodium falciparum, chosen as a reference and reconstituted a substitution matrix in the genome's context. The matrix was used to generate protein sequence alignments for the parasite proteins that improved across the functional regions. We attribute this to the consistency that may have been achieved amid the target and background frequencies calculated exclusively in our study. This study has important implications on annotation of proteins that are of experimental interest but give poor sequence alignments with standard conventional matrices.     

Switch from an aquaporin to a glycerol channel by two amino acids substitution

The MIP (major intrinsic protein) proteins constitute a channel family of currently 150 members that have been identified in cell membranes of organisms ranging from bacteria to man. Among these proteins, two functionally distinct subgroups are characterized: aquaporins that allow specific water transfer and glycerol channels that are involved in glycerol and small neutral solutes transport. Since the flow of small molecules across cell membranes is vital for every living organism, the study of such proteins is of particular interest. For instance, aquaporins located in kidney cell membranes are responsible for reabsorption of 150 liters of water/day in adult human. To understand the molecular mechanisms of solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located at the same positions in glycerol channels. Here, we show that substitution of a tyrosine and a tryptophan by a proline and a leucine, respectively, in the sixth transmembrane helix of an aquaporin leads to a switch in the selectivity of the channel, from water to glycerol.     

Modification in amino acids of dead sea scroll parchments

Fragments of Dead Sea Scroll Parchments were extracted for collagen and subjected to amino acid analysis. In modern parchment samples, 90% or more of the protein could be extracted in hot aqueous solution as collagen. In the ancient specimens, 70% or less was extractable. The hot-solution insoluble material was analyzed for collagen. In the soluble extract, the quantity of tyrosine, histidine, and methionine was reduces. Dityrosine was dected. The need to extend such studies is discussed.     

Secretion of amino acids by bacteria: Physiology and mechanism

Abstract: Although representing a common property of microorganisms and being widely used for biotechnological purposes, solute secretion has been relatively poorly studied in terms of biochemistry. In this review, various examples of metabolite secretion processes by bacteria are discussed with the emphasis on the mechanisms of amino acid secretion by coryneform bacteria. Among the metabolic concepts which may be applied to explain the physiological meaning of metabolite secretion, mainly two concepts are dealt with, i.e. the so-called 'overflow metabolism' on the one hand and the situation where non-metabolizable intermediates are accumulated and finally secreted on the other. In the central part of this review, the different concepts are discussed which have been put forward to mechanistically explain amino acid secretion under particular metabolic conditions and in particular strains of bacteria, i.e. secretion mediated (i) by diffusion, (ii) by the participation of amino acid uptake systems, and (iii) by the use of specific secretion systems. These concepts are then applied to amino acid secretion in Corynebacterium glutamicum , and emplified by detailed studies on the mechanism and regulation of the secretion of lysine, isoleucine and glutamate by C. glutamicum .     

Higher rates of amino acid substitution in rodents than in humans.

An analysis of 54 protein sequences from humans and rodents (mice or rats), with the chicken as an outgroup, indicates that, from the common ancestor of primates and rodents, 35 of the proteins have evolved faster in the lineage to mouse or rat (rodent lineage) whereas only 12 proteins have evolved faster in the lineage to humans (human lineage). The average rate of amino acid substitution is significantly faster in the rodent lineage than in the human lineage. In addition, the average rate of insertion/deletion is also faster in rodents than in humans and there is a positive correlation between the rate of amino acid substitution and the rate of insertion/deletion in a protein sequence.     

Protein synthesis: twenty three amino acids and counting

The genetic code can be interpreted during translation as 21 amino acids and three termination signals. Recent advances at the interface of chemistry and molecular biology are extending the genetic code to allow assignment of new amino acids to existing codons, providing new functional groups for protein synthesis.     

Nonuniformity of nucleotide substitution rates in molecular evolution: Computer simulation and analysis of 5S ribosomal RNA sequences

Summary The effects of temporal (among different branches of a phylogeny) and spatial (among different nucleotide sites within a gene) nonuniformities of nucleotide substitution rates on the construction of phylogenetic trees from nucleotide sequences are addressed. Spatial nonuniformity may be estimated by using Shannon's (1948) entropy formula to measure the Relative Nucleotide Variability (RNV) at each nucleotide site in an aligned set of sequences; this is demonstrated by a comparative analysis of 5S rRNAs. New methods of constructing phylogenetic trees are proposed that augment the Unweighted Pair-Group Using Arithmetic Averages (UPGMA) algorithm by estimating and compensating for both spatial and temporal nonuniformity in substitution rates. These methods are evaluated by computer simulations of 5S rRNA evolution that include both kinds of nonuniformities. It was found that the proposed Reference Ratio Method improved both the ability to reconstruct the correct topology of a tree and also the estimation of branch lengths as compared to UPGMA. A previous method (Farris et al. 1970; Klotz et al. 1979; Li 1981) was found to be less successful in reconstructing topologies when there is high probability of multiple mutations at some sites. Phylogenetic analyses of 5S rRNA sequences support the endosymbiotic origins of both chloroplasts and mitochondria, even though the latter exhibit an accelerated rate of nucleotide substitution. Phylogenetic trees also reveal an adaptive radiation within the eubacteria and another within the eukaryotes for the origins of most major phyla within each group during the Precambrian era.     

Extracellular amino acids of aerobic spore-forming bacteria]

An ability to synthesize and accumulate in the growth medium for extracellular amino acids of 108 strains of 13 aerobic sporeforming bacteria species at deep cultivation on the simple synthetic glucose-mineral liquid nutrient medium optimized for the representatives of this genus. Has been studied the bacillus strains studied in the present conditions synthesize 19 amino acids and 2 amino carbons in different quantities and combinations. Amino acids being synthesized the most and least by these microorganisms have been determined in quantitative expression. Extracellular amino acid accumulation was proved to be a strain property not of genus. In general, the genus studied was evaluated as active one in amino acid production. The most active strains may present an interest as initials for further selective obtaining of producers of separate amino acids. The outlook for aerobic bacilli strains having marked amino-synthesizing properties and complex of other biological activities is being discussed for construction of medicinal and prophylactic of live microbial culture preparations.     

Industrial production of amino acids by coryneform bacteria

In the 1950s Corynebacterium glutamicum was found to be a very efficient producer of L-glutamic acid. Since this time biotechnological processes with bacteria of the species Corynebacterium developed to be among the most important in terms of tonnage and economical value. L-Glutamic acid and L-lysine are bulk products nowadays. L-Valine, L-isoleucine, L-threonine, L-aspartic acid and L-alanine are among other amino acids produced by Corynebacteria. Applications range from feed to food and pharmaceutical products. The growing market for amino acids produced with Corynebacteria led to significant improvements in bioprocess and downstream technology as well as in molecular biology. During the last decade big efforts were made to increase the productivity and to decrease the production costs. This review gives an overview of the world market for amino acids produced by Corynebacteria. Significant improvements in bioprocess technology, i.e. repeated fed batch or continuous production are summarised. Bioprocess technology itself was improved furthermore by application of more sophisticated feeding and automatisation strategies. Even though several amino acids developed towards commodities in the last decade, side aspects of the production process like sterility or detection of contaminants still have increasing relevance. Finally one focus of this review is on recent developments in downstream technology.     

Rapid microbial metabolism of non-protein amino acids in the sea

The non-protein amino acids, -alanine and -aminobutyric acid, frequently dominate the amino acid composition of deep-sea sediments. This accumulation is most likely due to the slower decomposition of non-protein amino acids by microorganisms or to the preferential adsorption of non-protein amino acids by clay minerals. We investigated relative rates of heterotrophic uptake of alanine, -ala, and -aba in sea water to see if there were different rates of microbial assimilation and respiration between these protein and non-protein amino acids. Heterotrophic uptake was rapid for all three amino acids with turnover times of hours in productive coastal waters and days in more oligotrophic open-ocean waters. Uptake of the non-protein amino acids was significantly slower than uptake of alanine, particularly in anoxic waters. However, the difference in uptake rates is probably not great enough to cause significant preferential accumulation of non-protein amino acids.     

Effects of genotype on rates of substitution during experimental evolution

Rates of molecular evolution may vary widely between populations, yet the causes of this variation are still incompletely understood. Genetic differences between populations may make an important contribution to variation in rates of evolution, owing to differences in fitness, population size, mutation rates, or in the distribution of fitness effects (DFEs) of available beneficial mutations. By whole genome sequencing of Escherichia coli populations experimentally evolved in the presence of a quinolone antibiotic, we found that rates of substitution varied by genotype, with evidence for a contribution from a genotype's starting fitness. Subsequent targeted sequencing showed that genotypes with high average substitution rates were more likely to undergo the simultaneous fixation of several mutations, consistent with theoretical models of multiple mutation dynamics. Moreover, patterns of substitution were indicative of epistatic relationships between known resistance mutations.