Amino Acid Substitution Patterns Research Articles

Escherichia coli non-coding regulatory regions are highly conserved.

Microbial genome sequences are rapidly accumulating, enabling large-scale studies of sequence variation. Existing studies primarily focus on coding regions to study amino acid substitution patterns in proteins. However, non-coding regulatory regions also play a distinct role in determining physiologic responses. To investigate intergenic sequence variation on a large-scale, we identified non-coding regulatory region alleles across 2350 Escherichia coli strains. This 'alleleome' consists of 117781 unique alleles for 1169 reference regulatory regions (transcribing 1975 genes) at single base-pair resolution. We find that 64% of nucleotide positions are invariant, and variant positions vary in a median of just 0.6% of strains. Additionally, non-coding alleles are sufficient to recover E. coli phylogroups. We find that core promoter elements and transcription factor binding sites are significantly conserved, especially those located upstream of essential or highly-expressed genes. However, variability in conservation of transcription factor binding sites is significant both within and across regulons. Finally, we contrast mutations acquired during adaptive laboratory evolution with wild-type variation, finding that the former preferentially alter positions that the latter conserves. Overall, this analysis elucidates the wealth of information found in E. coli non-coding sequence variation and expands pangenomic studies to non-coding regulatory regions at single-nucleotide resolution.

Amino‐Acid Side‐Chain Nanoarchitectonics for Tuning the Chiroptical Properties and Supramolecular Structure of Pentameric Oligothiophenes

AbstractOligothiophenes with specific photophysical properties and molecular organization are of great interest, since this class of materials are used in organic electronics and bioelectronics, as well as biosensing. Herein, 8 different pentameric oligothiophenes, denoted proteophenes, with different amino acid substitution patterns at distinct positions along the thiophene backbone were investigated. Spectroscopic and microscopic studies of the ligands revealed the formation of optically active self‐assembled materials under acidic or basic conditions. The distinct photophysical characteristics, including induced circular dichroism, as well as the supramolecular structures of the assemblies deduced from light scattering and transmission electron microscopy, were highly influenced by the positioning of distinct amino acid moieties along the thiophene backbone. Proteophenes functionalized with only glutamate residues or these functionalities in combination with hydrophobic valine moieties formed fibrillar structures with excellent chiroptical properties under acidic conditions. In addition, the amino acid functionality at the β‐position of distinct thiophene moieties influenced the induced circular dichroism pattern observed from the proteophenes. Overall, the obtained results demonstrate how changes in the position of various amino acid functionalities, as well as the chemical nature of the amino acid side chain functionality greatly affect the optical properties as well as the architecture of the self‐assembled materials.

Genomic analysis of SARS-CoV-2 variants: diagnosis and vaccination challenges

SARS-CoV-2 put a heavy financial burden on the healthcare system, with millions of laboratory-confirmed cases and deaths worldwide in the last 2 years. During the seventh wave of this pandemic, the continuously evolving nature of SARS-CoV-2 resulted in the emergence of new variants that harbor different mutations. Mutations are associated with changes in the virus behavior, including increased transmissibility, increased virulence, and evasion of neutralizing antibodies. Currently, we need detailed and comprehensive genomic information on all SARS-CoV-2 variants. One of the key points in this study was the genome survey of mutation profiles across variants as a genomic data source, to determine the efficiency of RT-qPCR assays. We also used the source to calculate the binding affinity changes of neutralizing antibodies–mutant receptor binding domain (RBD) complexes and determine vaccine efficacy. Our result revealed that the number of nucleotide mismatches is variable in the WHO-recommended primer-probe sets. Mismatches located at the 3′ ends of the oligonucleotide, may lead to false-negative results. Only the primer-probe sets designed by the Ministry of Public Health of Thailand were exclusive and cannot detect the omicron variant reliably. Binding affinity changes showed that E484K was more deleterious than other mutations and decreased stability between the mutant RBD protein and neutralizing antibodies. The Omicrons show the highest change in binding affinity which may lead to immune escape and increase transmissibility. Additionally, the 7D6 monoclonal antibody in the 7eam complex could neutralize all variants of SARS-CoV-2. We strongly recommend creating and improving a matrix accuracy by processing a large number of SARS-CoV-2 sequences to update RT-qPCR assays and identified immunogenic residues among conserved RBD. Also, a detail computational analysis is needed to investigate distinctive amino acid substitution patterns which may be foundational in the vaccines. Finally, designing in-vitro studies can help confirm the present study and manage COVID-19 patients. Communicated by Ramaswamy H. Sarma

Pan-cancer surveys indicate cell cycle-related roles of primate-specific genes in tumors and embryonic cerebrum

BackgroundDespite having been extensively studied, it remains largely unclear why humans bear a particularly high risk of cancer. The antagonistic pleiotropy hypothesis predicts that primate-specific genes (PSGs) tend to promote tumorigenesis, while the molecular atavism hypothesis predicts that PSGs involved in tumors may represent recently derived duplicates of unicellular genes. However, these predictions have not been tested.ResultsBy taking advantage of pan-cancer genomic data, we find the upregulation of PSGs across 13 cancer types, which is facilitated by copy-number gain and promoter hypomethylation. Meta-analyses indicate that upregulated PSGs (uPSGs) tend to promote tumorigenesis and to play cell cycle-related roles. The cell cycle-related uPSGs predominantly represent derived duplicates of unicellular genes. We prioritize 15 uPSGs and perform an in-depth analysis of one unicellular gene-derived duplicate involved in the cell cycle, DDX11. Genome-wide screening data and knockdown experiments demonstrate that DDX11 is broadly essential across cancer cell lines. Importantly, non-neutral amino acid substitution patterns and increased expression indicate that DDX11 has been under positive selection. Finally, we find that cell cycle-related uPSGs are also preferentially upregulated in the highly proliferative embryonic cerebrum.ConclusionsConsistent with the predictions of the atavism and antagonistic pleiotropy hypotheses, primate-specific genes, especially those PSGs derived from cell cycle-related genes that emerged in unicellular ancestors, contribute to the early proliferation of the human cerebrum at the cost of hitchhiking by similarly highly proliferative cancer cells.

Structural topological analysis of spike proteins of SARS-CoV-2 variants of concern highlight distinctive amino acid substitution patterns

Since the onset of pandemic in 2019, SARS-CoV-2 has diverged into numerous variants driven by antigenic and infectivity-oriented selection. Some variants have accumulated fitness-enhancing mutations, evaded immunity and spread despite global vaccination campaigns. The spike (S) glycoprotein of SARS-CoV-2 demonstrated the greatest immunogenicity and amino acid substitution diversity owing to its importance in the interaction with human angiotensin receptor 2 (hACE2). The S protein consistently emerges as an amino acid substitution (AAS) hotspot in all six lineages, however, in Omicron this enrichment is significantly higher. This study attempts to design and validate a method of mapping S-protein substitution profile across variants to identify the conserved and AAS regions. A substitution matrix was created based on publicly available databases, and the substitution localization was illustrated on a cryo-electron microscopy generated S-protein model. Our analyses indicated that the diversity of N-terminal (NTD) and receptor-binding (RBD) domains exceeded that of any other regions but still contained extended low substitution density regions particularly considering significantly broader substitution profiles of Omicron BA.2 and BA.4/5. Finally, the substitution matrix was compared to a random sample alignment of variant sequences, revealing discrepancies. Therefore, it was suggested to improve matrix accuracy by processing a large number of S-protein sequences using an automated algorithm. Several critical immunogenic and receptor-interacting residues were identified in the conserved regions within NTD and RBD. In conclusion, the structural and topological analysis of S proteins of SARS-CoV-2 variants highlight distinctive amino acid substitution patterns which may be foundational in predicting future variants.

Delayed respiratory syncytial virus outbreak in 2020 in Taiwan was correlated with two novel RSV-A genotype ON1 variants.

BackgroundHuman respiratory syncytial virus (RSV) is a leading pathogen of acute respiratory tract disease among infants and young children. Compared with previous seasons, RSV outbreaks in Taiwan during the 2020–2021 season were delayed because of COVID‐19 mitigation measures. We conducted this study to determine the association of viral factors with clinical characteristics of preschool children with RSV infection.MethodsWe performed a molecular epidemiology analysis of RSV among inpatient preschool children in Taiwan. In 80 nasopharyngeal samples positive for RSV, we sequenced and analyzed viral genotypes according to patient data. Patients' clinical data were obtained from medical files, and their clinical profiles were compared with those of RSV cases recorded during the 2014–2017 seasons.ResultsPhylogenetic analysis revealed that among the RSV‐positive samples, all RSV strains identified during the 2020–2021 season belonged to the ON1 genotype. Most of the Taiwan ON1 strains were categorized into two well‐supported clusters with distinct G protein amino acid substitution patterns that had never been demonstrated previously. Furthermore, the proportion of cases among children aged >24 months increased (P < 0.001). Compared with patients infected during the 2014–2017 seasons, patients infected during the 2020–2021 season were hospitalized for shorter days from hospital admission to dereference (P = 0.004) and had a greater need for oxygen supplements (P = 0.021) and systemic steroid therapy (P = 0.026).ConclusionThe delayed 2020–2021 RSV outbreak in Taiwan was caused by two novel RSV ON1.1 variants. How the change in RSV epidemiology affects future RSV outbreaks warrants exploration.

A thermodynamic model of protein structure evolution explains empirical amino acid substitution matrices.

Proteins evolve under a myriad of biophysical selection pressures that collectively control the patterns of amino acid substitutions. These evolutionary pressures are sufficiently consistent over time and across protein families to produce substitution patterns, summarized in global amino acid substitution matrices such as BLOSUM, JTT, WAG, and LG, which can be used to successfully detect homologs, infer phylogenies, and reconstruct ancestral sequences. Although the factors that govern the variation of amino acid substitution rates have received much attention, the influence of thermodynamic stability constraints remains unresolved. Here we develop a simple model to calculate amino acid substitution matrices from evolutionary dynamics controlled by a fitness function that reports on the thermodynamic effects of amino acid mutations in protein structures. This hybrid biophysical and evolutionary model accounts for nucleotide transition/transversion rate bias, multi‐nucleotide codon changes, the number of codons per amino acid, and thermodynamic protein stability. We find that our theoretical model accurately recapitulates the complex yet universal pattern observed in common global amino acid substitution matrices used in phylogenetics. These results suggest that selection for thermodynamically stable proteins, coupled with nucleotide mutation bias filtered by the structure of the genetic code, is the primary driver behind the global amino acid substitution patterns observed in proteins throughout the tree of life.

Haemophilus influenzae one day in Denmark: prevalence, circulating clones, and dismal resistance to aminopenicillins.

Haemophilus influenzae is a common cause of mucosal infections that warrants accurate surveillance. We aimed to assess the prevalence of the species in clinical specimens, and characterise population structure and resistance to aminopenicillins by whole genome sequencing.We assessed the point prevalence by entering the database records of 1 day in Denmark and examined the genome sequences of nationwide, collected isolates from the same day. The prevalence of H. influenzae in clinical samples on the 10th of January 2018 was 1.78 per 100,000 person-days (all samples), and 2.47 per 1000 hospital bed-days (hospital samples). Of 2009 bacteria deemed clinically relevant and collected in a concerted action by the Danish departments of clinical microbiology, 62 (3.1%) were H. influenzae. All 62 isolates belonged to phylogenetic group I and were unencapsulated. Three strains from separate Danish regions had identical core genome sequences, but a small number of intergenic mutations testified to circulating clones, rather than individual cases of patient-to-patient transmission. The TEM-1 β-lactamase gene was present in 24 strains, while 13 strains were genetically categorised as ampicillin-resistant due to substitutions in penicillin-binding protein 3; shared patterns of amino acid substitutions in unrelated strains indicated putative lateral transfer of chromosomal resistance. Circulating clones of H. influenzae are frequent, and host factors, rather than direct transmission of epidemic strains, may be the primary cause of infection. The bleak presence of ampicillin resistance revealed by sequencing of point prevalence strains underscores the necessity for close examination of testing methods.

Some like it hot, others not

Enzyme Evolution Enzymes strike a delicate balance between features that enhance chemical reactivity and those that contribute to stable structure. Both features are important and can be unrelated or antagonistic. Pinney et al. combined rich experimental work on thermophilic and mesophilic variants of the enzyme ketosteroid isomerase (KSI) with bioinformatic data from a diverse set of bacterial enzymes to reveal the molecular determinants of thermal adaptation in enzymes. For KSI, they observed a trade-off between activity and thermal stability that comes down to a single active-site residue. With their larger dataset, they identified patterns of individual amino acid substitutions that are favored at higher temperatures, and also consider how networks of stabilizing interactions develop. Science , this issue p. [eaay2784][1] [1]: /lookup/doi/10.1126/science.aay2784

Parallel molecular mechanisms for enzyme temperature adaptation.

The mechanisms that underly the adaptation of enzyme activities and stabilities to temperature are fundamental to our understanding of molecular evolution and how enzymes work. Here, we investigate the molecular and evolutionary mechanisms of enzyme temperature adaption, combining deep mechanistic studies with comprehensive sequence analyses of thousands of enzymes. We show that temperature adaptation in ketosteroid isomerase (KSI) arises primarily from one residue change with limited, local epistasis, and we establish the underlying physical mechanisms. This residue change occurs in diverse KSI backgrounds, suggesting parallel adaptation to temperature. We identify residues associated with organismal growth temperature across 1005 diverse bacterial enzyme families, suggesting widespread parallel adaptation to temperature. We assess the residue properties, molecular interactions, and interaction networks that appear to underly temperature adaptation.

Identification of novel protein-coding sequences in Eucalyptus grandis plants by high-resolution mass spectrometry

Eucalyptus species are widely used in the forestry industry, and a significant increase in the number of sequences available in database repositories has been observed for these species. In proteomics, a protein is identified by correlating the theoretical fragmentation spectrum derived from genomic/transcriptomic data against the experimental fragmentation mass spectrum acquired from large-scale analysis of protein mixtures. Proteogenomics is an alternative approach that can identify novel proteins encoded by regions previously considered as non-coding. This study aimed to confidently identify and confirm the existence of previously unknown protein-coding sequences in the Eucalyptus grandis genome. To this end, we used a modified spectral correlation strategy and a dedicated de novo peptide sequencing pipeline. Upon the strategy used here, we confidently identified 41 novel peptide forms and six peptides containing at least one single amino acid substitution. The most representative genomic class of novel peptides was identified as originating from alternative reading frames. In contrast, no clear single amino acid substitution pattern was identified. Validation of the identifications was carried out using a parallel reaction monitoring approach that provided further mass spectrometry support for the existence of the novel peptide sequences. Data are available via ProteomeXchange with identifier PXD022110.

Altered Drug Transport by Plasmodium falciparum Chloroquine Resistance Transporter Isoforms Harboring Mutations Associated with Piperaquine Resistance.

Patterns of multiple amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT, UniProtKB Q8IBZ9) have previously been shown to mediate chloroquine resistance in P. falciparum malarial parasites. Recent reports suggest that novel mutations in PfCRT may mediate resistance to piperaquine (PPQ), which is used extensively as a partner drug in one prominent artemisinin combination therapy. How these novel PfCRT isoforms might mediate PPQ resistance (PPQR) is not known. Using codon optimization and other previously perfected methods for PfCRT analysis in yeast, we have expressed all known PPQR-associated PfCRT isoforms in Saccharomyces cerevisiae yeast and tested whether these isoforms catalyze PPQ transport. Relationships between relative PPQ and CQ transport are analyzed for these isoforms versus other previously recognized drug resistance-associated PfCRT isoforms.

Molecular characterization, catalytic, kinetic and thermodynamic properties of protease produced by a mutant of Bacillus cereus-S6-3

The proteolytic strain Bacillus cereus-S6-3 was subjected to mutagenic treatments viz. UV irradiations and methyl methane sulfonate (MMS). The obtained mutant strain, B. cereus-S6-3/UM90 showed 1.34 fold over the parent strain. Molecular characterization of proteases from the parent (PP/S6-3) and mutant (PM/UM90) strains indicated that they were consisted of two domains and binds a zinc ion and 4 calcium ions in the active site. Amino acid sequence alignment of PM/UM90 protease showed 19 amino acid residues were substituted compared to that of the wild-type enzyme. However, both proteases contained equal number of aromatic and hydrophobic amino acids. Protease from PM/UM90 showed an effective improvement in thermal properties in terms of reaction temperature, t1/2, the values of kd, activation energy (Ea), and decimal reduction time (D) within the temperature range from 60 to 80 °C. In addition, the kinetic and thermodynamic parameters for substrate hydrolysis (i.e., Km, Vmax, ΔH*, ΔG*, ΔS*, kcat, Vmax/Km, kcat/Km, ΔG*E–T and ΔG*E–S) showed a significant improvement of the catalytic efficiency for PM/UM90 protease. Furthermore, the correlation between thermodynamic properties and the patterns of amino acid substitution of wild-type enzyme to has been discussed.

FLAVI: An Amino Acid Substitution Model for Flaviviruses.

Amino acid substitution models represent substitution rates among amino acids during the evolution. The models play an important role in analyzing protein sequences, especially inferring phylogenies. The rapid evolution of flaviviruses is expanding the threat in public health. A number of models have been estimated for some viruses, however, they are unable to properly represent amino acid substitution patterns of flaviviruses. In this study, we collected protein sequences from the flavivirus genus to specifically estimate an amino acid substitution model, called FLAVI, for flaviviruses. Experiments showed that the collected dataset was sufficient to estimate a stable model. More importantly, the FLAVI model was remarkably better than other existing models in analyzing flavivirus protein sequences. We recommend researchers to use the FLAVI model when studying protein sequences of flaviviruses or closely related viruses.

Lower respiratory tract isolates of non-typeable Haemophilus influenzae in Western Sichuan, China: Antimicrobial susceptibility, mechanisms of β-lactam resistance and decade changes

ObjectivesThe aims of this study were to analyse the serotypes of epidemic Haemophilus influenzae and changes in mechanisms of β-lactam resistance over the past decade. ResultsHaemophilus influenzae isolates in Western Sichuan from 2013–2014 were non-typeable H. influenzae (NTHi). β-Lactam MICs for NTHi isolated during 2013–2014 were significantly higher than those from 2003–2004 (P < 0.05). Of 274 NTHi, 141 (51.5%) were β-lactamase-positive (TEM-1 type). There were 35 amino acid (AA) substitutions in ftsI among NTHi isolated from 2013–2014. However, NTHi isolates from 2003–2004 had only nine AA substitutions. Ordered multiple classification logistic regression analysis showed that different AA substitution patterns in ftsI had different effects on β-lactam MICs. The main factors affecting the ampicillin MIC were the mutations R517H (OR = 6.999), L389F (OR = 7.128), N526K (OR = 4.660) and D350N (OR = 0.450). The main factor influencing the amoxicillin/clavulanic acid MIC was an N526K mutation (OR = 9.349). The main factors affecting the cefuroxime MIC were the mutations S357N (OR = 37.453) and N526K (OR = 14.816). Compared with 2003–2004, gBLNAR and gBLPAR isolated from 2013–2014 increased significantly from 13.0% (7/54) and 9.3% (5/54) to 38.2% (84/220) and 45.5% (100/220), respectively (P < 0.001). In the ‘others’ group of ftsI gene mutations, 13 NTHi had the same ftsI gene mutation pattern and 24 AA substitutions. ConclusionThese results confirm that β-lactam-resistant NTHi isolates increased rapidly. AA substitutions in ftsI were more complex and diversified in 2013–2014.

715. Bulgecin A lowers Imipenem MICs in Clinical MDR Acinetobacter baumannii (ACB) Strains

BackgroundGenetic screening of ACB has revealed genes that confer increased susceptibility to β-lactams when these genes are disrupted suggesting novel drug targets. One such target is lytic transglycosylase, LT. Bulgecin A (BlgA) is a natural product of Paraburkholderia acidophila and an LT inhibitor that potentiates the activity of β-lactams.MethodsBroth microdilution MICs were performed using carbapenem-resistant (CR)-colisitin susceptible (ColS) and CR-ColR clinical ACB strains, with a variety of resistance mechanisms, previously studied via whole-genome sequencing. A fixed concentration of pure BlgA at 100 μg/mL was combined with varying concentrations of imipenem. Sequences of the putative LTs in ACB strains were analyzed to look for amino-acid substitutions and correlated with the MIC lowering effect of BlgA. Homology models of the LTs of AB0057 ACB strain were generated.ResultsACB MltE most resembles soluble LTs of other species with 22.39% sequence identity and 92% query coverage to Slt70 of E. coli. MIC results and amino-acid sequence variations in MltE LT of ACB are shown. There were no clear amino-acid substitution patterns to account for differences in BlgA effect. The mutations are distal to the BlgA binding site at residue E506.ConclusionThese are the first MICs reported for BlgA in combination with a carbapenem demonstrating reduction in the MIC by four-fold for several MDR ACB clinical strains. Mutations in MltE Lt do not account for variations in the MICs observed. Changes in the MICs may be related to other factors such as drug penetration and alterations in PBP expression. Additional studies are underway to examine these factors. Disclosures All authors: No reported disclosures.

Weakly haemolytic variants of Brachyspira hyodysenteriae newly emerged in Europe belong to a distinct subclade with unique genetic properties

Brachyspira (B.) hyodysenteriae is widespread globally, and can cause mucohaemorrhagic colitis (swine dysentery, SD) with severe economic impact in infected herds. Typical strains of B. hyodysenteriae are strongly haemolytic on blood agar, and the haemolytic activity is believed to contribute to virulence in vivo. However, recently there have been reports of atypical weakly haemolytic isolates of B. hyodysenteriae (whBh). In this study, 34 European whBh and 82 strongly haemolytic isolates were subjected to comparative genomic analysis. A phylogenetic tree constructed using core single nucleotide polymorphisms showed that the whBh formed a distinct sub-clade. All eight genes previously associated with haemolysis in B. hyodysenteriae were present in the whBh. No consistent patterns of amino acid substitutions for all whBh were found in these genes. In contrast, a genome region containing six coding sequences (CDSs) had consistent nucleotide sequence differences between strongly and whBh isolates. Two CDSs were predicted to encode ABC transporter proteins, and a TolC family protein, which may have a role in the export of haemolysins from B. hyodysenteriae. Another difference in this region was the presence of three CDSs in whBh that are pseudogenes in strongly haemolytic isolates. One of the intact CDSs from whBh encoded a predicted PadR-like transcriptional repressor that may play a role in repression of haemolysis functions. In summary, a sub-clade of whBh isolates has emerged in Europe, and several genomic differences, that potentially explain the weakly haemolytic phenotype, were identified. These markers may provide targets for discriminatory molecular tests needed in SD surveillance.

PoSE: visualization of patterns of sequence evolution using PAML and MATLAB

BackgroundDetermining patterns of nucleotide and amino acid substitution is the first step during sequence evolution analysis. However, it is not easy to visualize the different phylogenetic signatures imprinted in aligned nucleotide and amino acid sequences.ResultsHere we present PoSE (Pattern of Sequence Evolution), a reliable resource for unveiling the evolutionary history of sequence alignments and for graphically displaying their contents. Substitutions are displayed by category (transitions and transversions), codon position, and phenotypic effect (synonymous and nonsynonymous). Visualization is accomplished using MATLAB scripts wrapped around PAML (Phylogenetic Analysis by Maximum Likelihood), implemented in an easy-to-use graphical user interface. The application displays inferred substitutions estimated by baseml or codeml, two programs included in the PAML software package. PoSE organizes patterns of substitution in eleven plots, including estimated non-synonymous/synonymous ratios (dN/dS) along the sequence alignment. In addition, PoSE provides visualization and annotation of patterns of amino acid substitutions along groups of related sequences that can be graphically inspected in a phylogenetic tree window.ConclusionsPoSE is a useful tool to help determine major patterns during sequence evolution of protein-coding sequences, hypervariable regions, or changes in dN/dS ratios. PoSE is publicly available at https://github.com/CDCgov/PoSE

Thermosynechococcaceae as a source of thermostable C-phycocyanins: properties and molecular insights

This study describes characterisation of three thermophilic strains of cyanobacteria belonging to Thermosynechococcaceae and their utilisation as a source of thermostable C-phycocyanin. Three strains NIES 2133, NIES 2134 and PCC 6715 have been cultivated in two types of growth media and two temperatures: 45 and 50 °C and assessed for C-phycocyanin productivity. Among these strains Synechococcus lividus sp. PCC 6715, phylogenetically closer to Thermosynechococcus sp. clade than other Synechococcaceae, has been selected for subsequent study to characterise its C-phycocyanin. The strain showed faster growth rates than the other two and appeared to utilise different bicarbonate uptake mechanism based on SbtA, whilst BCT1 mechanism was found in NIES2133 and 2134. Higher growth rates translated to larger overall C-phycocyanin productivity. C-phycocyanin has been purified using chromatographic methods and its thermostability and pH stability has been assessed against that of Spirulina. Results have shown that PCC 6715 C-phycocyanin is one of the most stable proteins of this type reported to date. It exhibits 90% stability after 5-hour incubation at 50 °C and about 70% stability after 2-week incubation at the same temperature. PCC 6715 phycocyanins show high stability at acidic pH at both 4 °C and 50 °C, whilst at 50 °C maintain 100% stability during 4-hour incubation at pH 4.0 to 8.0. PCC 6715 phycocyanin shows good long-term stability characteristics and could be considered as a thermostable replacement for Spirulina phycocyanin. Analysis of molecular model of PCC 6715 C-phycocyanins and crystal structures of other phycocyanins reveals interesting pattern of amino acid substitutions that are present in thermophilic proteins that could have significant impact on their significantly higher thermostability. Phycocyanins from thermostable cyanobacteria exhibit evolutionarily conserved amino acid substitutions localised in the single structural hotspot of the protein that result in additional stabilising interactions within and between protein subunits when compared to proteins from mesophilic counterparts.

Genetic signatures of lipid metabolism evolution in Cetacea since the divergence from terrestrial ancestor.

In mammalian evolutionary history, Cetacea (whales, dolphins and porpoises) achieved astonishing success by adapting to an aquatic environment. One unique characteristic of cetaceans, contributing to this adaptive success, is efficient lipid utilization. Here, we report a comparative genetic analysis of five aquatic and five terrestrial Cetartiodactyla species using 144 genes associated with lipid metabolism. Mutation ratio (dN /dS ), amino acid substitution in functional domains and metabolic pathways were evaluated using branch-site model in PAML, Pfam and KEGG, respectively. Our tests detected 20 positively selected genes in Cetacea compared to 11 in Bovidae with little overlap between the lineages. We identified lineage-specific patterns of amino acid substitutions and functional domains that were mutually exclusive between cetaceans and bovids, supporting divergent evolution of lipid metabolism since the divergence of these taxa from a common ancestor. Moreover, a pathway analysis showed that the identified genes in cetaceans were associated with lipid digestion, lipid storage and energy-producing pathways. This study emphasizes the evolutionary context of lipid metabolism modification of cetaceans and provides a foundation for future studies of elucidating the adapted biological mechanisms of cetacean lipid metabolism and a framework for incorporating ecological context into studies aimed at investigating adaptive evolution.