Isolation of thermophilic mutants of Bacillus subtilis and Bacillus pumilus and transformation of the thermophilic trait to mesophilic strains

Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10 degrees C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 degrees C and 70 degrees C at a frequency of less than 10(-10). The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10(-3) to 10(-2) for single markers and about 10(-7) for two unlinked markers. With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10(-7), suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.     

Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria

One of the well-known observations of proteins from thermophilic bacteria is the bias of the amino acid composition in which charged residues are present in large numbers, and polar residues are scarce. On the other hand, it has been reported that the molecular surfaces of proteins are adapted to their subcellular locations, in terms of the amino acid composition. Thus, it would be reasonable to expect that the differences in the amino acid compositions between proteins of thermophilic and mesophilic bacteria would be much greater on the protein surface than in the interior. We performed systematic comparisons between proteins from thermophilic bacteria and mesophilic bacteria, in terms of the amino acid composition of the protein surface and the interior, as well as the entire amino acid chains, by using sequence information from the genome projects. The biased amino acid composition of thermophilic proteins was confirmed, and the differences from those of mesophilic proteins were most obvious in the compositions of the protein surface. In contrast to the surface composition, the interior composition was not distinctive between the thermophilic and mesophilic proteins. The frequency of the amino acid pairs that are closely located in the space was also analyzed to show the same trend of the single amino acid compositions. Interestingly, extracellular proteins from mesophilic bacteria showed an inverse trend against thermophilic proteins (i.e. a reduced number of charged residues and rich in polar residues). Nuclear proteins from eukaryotes, which are known to be abundant in positive charges, showed different compositions as a whole from the thermophiles. These results suggest that the bias of the amino acid composition of thermophilic proteins is due to the residues on the protein surfaces, which may be constrained by the extreme environment.     

Protein thermostability: structure-based difference of amino acid between thermophilic and mesophilic proteins

Structural distributions of each amino acid were compared between 20 pairs of thermophilic and mesophilic proteins to obtain thermostable factors. Five kinds of residual structure states such as fully-exposed, exposed, partially exposed (or partially buried), buried, well-buried states were considered for analyzing the structural patterns of amino acids. The statistical tests revealed that lower frequency in partially exposed state of SER, lower frequency in exposed state and higher frequency in well-buried state of ALA, higher frequency in buried state of GLU, higher frequency in exposed state of ARG, etc. could be critical factors related with protein thermostability.     

Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase.

The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms possesses very different thermal properties. The B. caldolyticus enzyme has optimal activity at 60-65 degrees C and a half-life of 26 min at 65 degrees C, compared to values of 46 degrees C and 60 s at 65 degrees C, respectively, for the B. subtilis enzyme. Chemical cross-linking shows that both enzymes are hexamers. Vmax is determined as 440 micromol.min(-1).mg protein(-1) and Km values for ATP and ribose 5-phosphate are determined as 310 and 530 microM, respectively, for the B. caldolyticus enzyme. The enzyme requires 50 mM Pi as well as free Mg2+ for maximal activity. Manganese ion substitutes for Mg2+, but only at 30% of the activity obtained with Mg2+. ADP and GDP inhibit the B. caldolyticus enzyme in a cooperative fashion with Hill coefficients of 2.9 for ADP and 2.6 for GDP. Ki values are determined as 113 and 490 microm for ADP and GDP, respectively. At low concentrations ADP inhibition is linearly competitive with respect to ATP. A predicted structure of the B. caldolyticus enzyme based on homology modelling with the structure of B. subtilis 5-phospho-alpha-D-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure.     

On the origin of synonymous codon usage divergence between thermophilic and mesophilic prokaryotes

Synonymous codon usage analysis between thermophilic and mesophilic prokaryotes has gained wide attention in recent years. Although it is known that thermophilic and mesophilic prokaryotes use different subset of synonymous codons, no reason for this difference is known so far. In the present communication, by analyzing a large number of thermophilic and mesophilic prokaryotes, we provide evidence that bias in the selection of synonymous codons between thermophilic and mesophilic prokaryotes is related to differential folding pattern of mRNA secondary structures. Moreover, we observe that error-minimizing property has significant influence in differentiating the synonymous codon usage between thermophilic and mesophilic prokaryotes. Biological implications of these results are discussed.     

Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis

The ability of a Bacillus subtilis strain to grow and produce biosurfactant on different carbon and nitrogen sources under thermophilic conditions (45°C) was studied. The strain was able to reduce surface tension to 34 dynes cm⁻¹ on 2% sucrose, and 32 dynes cm⁻¹ on starch after 96 h of growth. The biosurfactant was stable at 100°C and within a wide pH range (3.0–11.0). Biosurfactant formation at mesophilic conditions (30°C) was also studied. The organism was able to produce the maximum amount of biosurfactant when nitrate ions were supplied as the nitrogen source. The potential application of the biosurfactant in oil recovery from desert oil fields, acidic and alkaline environments is demonstrated. The biosurfactant was identical to surfactin as confirmed by TLC and IR analysis. Received 29 May 1997/ Accepted in revised form 03 October 1997     

Support vector machine for discrimination of thermophilic and mesophilic proteins based on amino acid composition

The identification of the thermostability from the amino acid sequence information would be helpful in computational screening for thermostable proteins. We have developed a method to discriminate thermophilic and mesophilic proteins based on support vector machines. Using self-consistency validation, 5-fold cross-validation and independent testing procedure with other datasets, this module achieved overall accuracy of 94.2%, 90.5% and 92.4%, respectively. The performance of this SVM-based module was better than the classifiers built using alternative machine learning and statistical algorithms including artificial neural networks, Bayesian statistics, and decision trees, when evaluated using these three validation methods. The influence of protein size on prediction accuracy was also addressed.     

Genes and derived amino acid sequences of S-layer proteins from mesophilic,thermophilic, and extremely thermophilic methanococci

Cells of methanococci are covered by a single layer of protein subunits (S-layer) in hexagonal arrangement, which are directly exposed to the environment and which cannot be stabilized by cellular components. We have isolated S-layer proteins from cells of Methanococcus vannielii ( T(opt.)=37 degrees C), Methanococcus thermolithotrophicus ( T(opt.)=65 degrees C), and Methanococcus jannaschii ( T(opt.)=85 degrees C). The primary structure of the S-layer proteins was determined by sequencing the corresponding genes. According to the predicted amino acid sequence, the molecular masses of the S-layer proteins of the different methanococci are in a small range between 59,064 and 60,547 Da. Compared with its mesophilic counterparts, it is worth noting that in the S-layer protein of the extreme thermophile Mc. jannaschii the acidic amino acid Asp is predominant, the basic amino acid Lys occurs in higher amounts, and Cys and His are only present in this organism. Despite the differences in the growth optima and the predominance of some amino acids, the comparative total primary structure revealed a relatively high degree of identity (38%-45%) between the methanococci investigated. This observation indicates that the amino acid sequence of the S-layer proteins is significantly conserved from the mesophilic to the extremely thermophilic methanococci.     

Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins

Summary. Thermophilic proteins show substantially higher intrinsic thermal stability than their mesophilic counterparts. Amino acid composition is believed to alter the intrinsic stability of proteins. Several investigations and mutagenesis experiment have been carried out to understand the amino acid composition for the thermostability of proteins. This review presents some generalized features of amino acid composition found in thermophilic proteins, including an increase in residue hydrophobicity, a decrease in uncharged polar residues, an increase in charged residues, an increase in aromatic residues, certain amino acid coupling patterns and amino acid preferences for thermophilic proteins. The differences of amino acids composition between thermophilic and mesophilic proteins are related to some properties of amino acids. These features provide guidelines for engineering mesophilic protein to thermophilic protein. Authors’ addresses: Yuan-Jiang Pan, Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Zhejiang University Road 38, Hangzhou 310027, China; Wei-Fen Li, Microbiology Division, College of Animal Science, Zhejiang University, Hangzhou 310029, China     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, IV. The primary structure of the mesophilic lactate dehydrogenase from Bacillus subtilis

The complete amino-acid sequence of lactate dehydrogenase from the mesophilic Bacillus subtilis (B. X1) was determined. Approximately 70% of the sequence was obtained by sequence analysis of intact protein (N-terminal sequence) and of four CNBr fragments (CNBr3, CNBr4, CNBr5 and CNBr6). Sequences overlapping the CNBr fragments were determined from polypeptide fragments obtained by cleavage using o-iodosobenzoic acid (cleavage at Trp) or clostripain (cleavage at Arg). The C-terminal amino-acid residue (Asn) was detected by carboxypeptidase Y-degradation. Lactate dehydrogenase from B. subtilis shows a 69% sequence homology to that from the thermophilic strain B. stearothermophilus, and a 34% sequence homology to those from higher organism. The homology of these enzymes is particularly high at the active site regions (the coenzyme and substrate binding sites). The relatively high sequence conservation of the lactate dehydrogenases from B. subtilis and B. stearothermophilus (and from other bacilli) allows a structural comparison of this temperature variants.     

Cloning,expression, and characterization of the gsdA gene encoding thermophilic glucose-6-phosphate dehydrogenase from Aquifex aeolicus

The gsdA gene of the extreme thermophilic bacterium Aquifex aeolicus, encoding glucose-6-phosphate dehydrogenase (G6PDH), was cloned into a high-expression vector and overexpressed as a fusion protein in Escherichia coli. Here we report the characterization of this recombinant thermostable G6PDH. G6PDH was purified to homogeneity by heat precipitation followed by immobilized metal affinity chromatography on a nickel-chelate column. The data obtained indicate that the enzyme is a homodimer with a subunit molecular weight of 55 kDa. G6PDH followed Michaelis-Menten kinetics with a K(M) of 63 micro M for glucose-6-phosphate at 70 degrees C with NADP as the cofactor. The enzyme exhibited dual coenzyme specificity, although it showed a preference in terms of k(cat)/ K(M) of 20.4-fold for NADP over NAD at 40 degrees C and 5.7-fold at 70 degrees C. The enzyme showed optimum catalytic activity at 90 degrees C. Modeling of the dimer interface suggested the presence of cysteine residues that may form disulfide bonds between the two subunits, thereby preserving the oligomeric integrity of the enzyme. Interestingly, addition of dithiothreitol or mercaptoethanol did not affect the activity of the enzyme. With a half-life of 24 h at 90 degrees C and 12 h at 100 degrees C, this is the most thermostable G6PDH described.     

Interconversion of functional motions between mesophilic and thermophilic adenylate kinases

Dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (AK), for which the open/closed transition limits the rate of catalytic turnover. Here, we compare our previously published coarse-grained (double-well Gō) simulation of mesophilic AK from E. coli (AKmeso) to simulations of thermophilic AK from Aquifex aeolicus (AKthermo). In AKthermo, as with AKmeso, the LID domain prefers to close before the NMP domain in the presence of ligand, but LID rigid-body flexibility in the open (O) ensemble decreases significantly. Backbone foldedness in O and/or transition state (TS) ensembles increases significantly relative to AKmeso in some interdomain backbone hinges and within LID. In contact space, the TS of AKthermo has fewer contacts at the CORE-LID interface but a stronger contact network surrounding the CORE-NMP interface than the TS of AKmeso. A "heated" simulation of AKthermo at 375K slightly increases LID rigid-body flexibility in accordance with the "corresponding states" hypothesis. Furthermore, while computational mutation of 7 prolines in AKthermo to their AKmeso counterparts produces similar small perturbations, mutation of these sites, especially positions 8 and 155, to glycine is required to achieve LID rigid-body flexibility and hinge flexibilities comparable to AKmeso. Mutating the 7 sites to proline in AKmeso reduces some hinges' flexibilities, especially hinge 2, but does not reduce LID rigid-body flexibility, suggesting that these two types of motion are decoupled in AKmeso. In conclusion, our results suggest that hinge flexibility and global functional motions alike are correlated with but not exclusively determined by the hinge residues. This mutational framework can inform the rational design of functionally important flexibility and allostery in other proteins toward engineering novel biochemical pathways.