Evolutionary divergence of TLR9 through ancestral sequence reconstruction.

The transmembrane pattern recognition receptor, Toll-like receptor (TLR), are best known for their roles in innate immunity via recognition of pathogen and initiation of signaling response. Mammalian TLRs recognize molecular patterns associated with pathogens and initiate innate immune response. We have studied the evolutionary diversity of mammalian TLR genes for differences in immunological response. Reconstruction of ancestral sequences is a key aspect of the molecular evolution of TLR to track changes across the TLR genes. The comprehensive analysis of mammalian TLRs revealed a distinct pattern of evolution of TLR9. Various sequence-based features such as amino acid usage, hydrophobicity, GC content, and evolutionary constraints are found to influence the divergence of TLR9 from other TLRs. Ancestral sequence reconstruction analysis also revealed that the gradual evolution of TLR genes in several ancestral lineages leads to the distinct pattern of TLR9. It demonstrates evolutionary divergence with the progressive accumulation of mutations results in the distinct pattern of TLR9.

Emerging resistome diversity in clinical Vibrio cholerae strains revealing their role as potential reservoirs of antimicrobial resistance.

BACKGROUND: This is a unique and novel study delineating the genotyping and subsequent prediction of AMR determinants of Vibrio cholerae revealing the potential of contemporary strains to serve as precursors of severe AMR crisis in cholera. METHODS AND RESULTS: Genotyping of representative strains, VC1 and VC2 was undertaken to characterize antimicrobial resistance genes (ARGs) against chloramphenicol, SXT, nalidixic acid and streptomycin against which they were found to be resistant by antibiogram analysis in our previous investigation. strAB, sxt, sul2, qace∆1-sul1 were detected by PCR. Genome annotation and identification of ARGs with WGS helped to detect the presence of almG, varG, strA (APH(3'')-Ib), strB (APH(6)-Id), sul2, catB9, floR, CRP, dfrA1 genes. Signatures of resistance determinants and protein domains involved in antimicrobial resistance, primarily, efflux of antibiotics were identified on the basis of 30-100% homology to reference proteins. These domains were predicted to be involved in other metabolic functions on the basis of 100% identity with 100% coverage with reference protein and nucleotide sequences and were predicted to be of a diverse taxonomic origin accentuating the influence of the microbiota on AMR acquisition. Sequence analysis of QRDR (quinolone resistance-determining region) revealed SNPs. Cytoscape v3.8.2 was employed to analyse protein-protein interaction of MDR proteins, MdtA and EmrD-2, with nodes of vital AMR pathways. Vital nodes involved in efflux of different classes of antibiotics were found to be absent in VC1 and VC2 justifying the sensitivity of these strains to most antibiotics. CONCLUSIONS: The study helped to examine the resistome of VC isolated from recent outbreaks to understand the underlying reason of sensitivity to most antibiotics and also to characterize the ARGs in their genome. It revealed that VC is a reservoir of signatures of resistance determinants and serving as precursors for severe AMR crisis in cholera. This is the first study, to our knowledge, which has scrutinized and presented systematically, information on prospective domains which bear the potential of serving as AMR determinants in VC with the help of bioinformatic tools. This pioneering approach may help in the prediction of AMR landfalls and benefit epidemiological surveillance and early warning systems.

Antimicrobial potential of Streptomyces sp. NP73 isolated from the forest soil of Northeast India against multi-drug resistant Escherichia coli.

This study reports the isolation and characterization of a Streptomyces sp. from soil, capable of producing bioactive secondary metabolites active against a variety of bacterial human pathogens. We targeted the antimicrobial activity against Escherichia coli ATCC-BAA 2469, a clinically relevant strain of bacteria harbouring resistance genes for carbapenems, extended spectrum beta-lactams, tetracyclines, fluoroquinones, etc. Preliminary screening using the spot inoculation technique identified Streptomyces sp. NP73 as the potent strain among the 74 isolated Actinomycetia strain. 16S rRNA gene and whole genome sequencing (WGS) confirmed its taxonomical identity and helped in the construction of the phylogenetic tree. WGS revealed the predicted pathways and biosynthetic gene clusters responsible for producing various types of antibiotics including the isolated compound. Bioactivity guided fractionation and chemical characterization of the active fraction, carried out using liquid chromatography, gas chromatography-mass spectrometry, infra-red spectroscopy, and nuclear magnetic resonance spectroscopy, led to the tentative identification of the active compound as Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-, a diketopiperazine molecule. This compound exhibited excellent antimicrobial and anti-biofilm properties against E. coli ATCC-BAA 2469 with an MIC value of 15.64 µg ml-1, and the low cytotoxicity of the compound identified in this study provides hope for future drug development.

Phylogeny and structural insights of lipase from Halopseudomonas maritima sp. nov., isolated from sea sand.

A Gram-negative, aerobic bacterial strain RR6T was isolated from the sea sand to produce lipase and proposed as a novel species of Halopseudomonas. The optimum growth occurred at 28-37 °C, and the pH was 6.0-8.0. The optimum growth occurred at 3.0 -6.5% (w/v) NaCl. The major cellular fatty acids were C10:0 3OH, C12:0, C16:1 ω7c/16:1 ω6c, 18:1 ω7c and/or 18:1 ω6c, and C16:0. The predominant polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, unidentified phospholipid, and unidentified lipids. The genome is 3.93 Mb, and the G + C content is 61.3%. The 16S rRNA gene sequences shared 99.73-99.87% sequence similarity with the closely related type strains of Halopseudomonas. The average nucleotide identity and average amino acid identity of strain RR6T with reference type strains were below 95-96%, and the corresponding in-silico DNA-DNA hybridization values were below 70%. Strain RR6T clustered with Halopseudomonas gallaeciensis V113T and Halopseudomonas pachastrellae CCUG 46540 T in the phylogenetic tree. Further, lipase produced by this bacterium belongs to α/ß hydrolase lipase family and exhibits structural similarity to the lactonizing lipase. Based on the polyphasic analysis, the new isolates RR6T represent a novel species of Halopseudomonas for which Halopseudomonas maritima sp. nov. is proposed. The type strain is RR6T (= NBRC 115418 T = TBRC 15628 T).

Underlying selection for the diversity of spike protein sequences of SARS-CoV-2.

The global spread of SARS-CoV-2 is fast moving and has caused a worldwide public health crisis. In the present article, we analyzed spike protein sequences of SARS-CoV-2 genomes to assess the impact of mutational diversity. We observed from amino acid usage patterns that spike proteins are associated with a diversity of mutational changes and most important underlying cause of variation of amino acid usage is the changes in hydrophobicity of spike proteins. The changing patterns of hydrophobicity of spike proteins over time and its influence on the receptor binding affinity provides crucial information on the SARS-CoV-2 interaction with human receptor. Our results also show that spike proteins have evolved to prefer more hydrophobic residues over time. The present study provides a comprehensive analysis of molecular sequence data to consider that mutational variants might play a crucial role in modulating the virulence and spread of the virus and has immediate implications for therapeutic strategies.

Barley beta-Glucan and Zymosan induce Dectin-1 and Toll-like receptor 2 co-localization and anti-leishmanial immune response in Leishmania donovani-infected BALB/c mice.

Toll-like receptors (TLRs), TLR2 in particular, are shown to recognize various glycans and glycolipid ligands resulting in various immune effector functions. As barley ß-glucan and zymosan are the glycans implicated in immunomodulation, we examined whether these ligands interact with Dectin-1, a lectin-type receptor for glycans, and TLR2 and induce immune responses that can be used against Leishmania infection in a susceptible host. The binding affinity of barley ß-glucan and zymosan with Dectin-1 and TLR2 was studied in silico. Barley ß-glucan- and zymosan-induced dectin-1 and TLR2 co-localization was studied by confocal microscopy and co-immunoprecipitation. These ligands-induced signalling and effector functions were assessed by Western blot analyses and various immunological assays. Finally, the anti-leishmanial potential of barley ß-glucan and zymosan was tested in Leishmania donovani -infected macrophages and in L. donovani-infected BALB/c mice. Both barley ß-glucan and zymosan interacted with TLR2 and dectin-1, but with a much stronger binding affinity for the latter, and therefore induced co-localization of these two receptors on BALB/c-derived macrophages. Both ligandsactivated MyD88- and Syk-mediated downstream pathways for heightened inflammatory responses in L. donovani-infected macrophages. These two ligands induced T cell-dependent host protection in L. donovani-infected BALB/c mice. These results establish a novel modus operandi of ß-glucans through dectin-1 and TLR2 and suggest an immuno-modulatory potential against infectious diseases.

An adhesion protein of Salmonella enterica serovar Typhi is required for pathogenesis and potential target for vaccine development.

More than half of all Salmonella enterica serovar Typhi genes still remain unannotated. Although pathogenesis of S. Typhi is incompletely understood, treatment of typhoid fever is complicated by the emergence of drug resistance. Effectiveness of the currently available vaccines is also limited. In search of novel virulence proteins, we have identified several putative adhesins of S. Typhi through computational approaches. Our experiment shows that a 27-kDa outer membrane protein (T2544) plays a major role in bacterial adhesion to the host through high-affinity binding to laminin. Its role in bacterial pathogenesis is underscored by reduced systemic invasion and a 10-fold higher LD(50) of the mutant bacteria in mice. T2544 is strongly immunogenic as revealed by the detection of sustained high titers of serum IgG and intestinal secretory IgA in the immunized mice. In vitro, T2544 antiserum enhanced uptake and clearance of Salmonella by macrophages and augmented complement-mediated lysis, indicating a contribution of T2544-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with recombinant T2544 or passively immunized with T2544 antiserum against subsequent bacterial challenge, suggesting that T2544-specific antibodies are involved in protection. The present study describes an adhesion protein of S. Typhi that contributes to bacterial pathogenesis. Protective antibodies in mice, rapid seroconversion of naturally infected individuals with increasing titers of anti-T2544 IgG from acute to convalescent sera suggesting antibody response in humans, and wide distribution and conservation of the cell-surface adhesin in the clinical isolates of different Salmonella serovars make T2544 a potential vaccine candidate.

Emergence of Genomic Diversity in the Spike Protein of the "Omicron" Variant.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus) has constantly been evolving into different forms throughout its spread in the population. Emerging SARS-CoV-2 variants, predominantly the variants of concern (VOCs), could have an impact on the virus spread, pathogenicity, and diagnosis. The recently emerged "Omicron" variant has exhibited rapid transmission and divergence. The spike protein of SARS-CoV-2 has consistently been appearing as the mutational hotspot of all these VOCs. In order to determine a deeper understanding of the recently emerged and extremely divergent "Omicron", a study of amino acid usage patterns and their substitution patterns was performed and compared with those of the other four successful variants of concern ("Alpha", "Beta", "Gamma", and "Delta"). We observed that the amino acid usage of "Omicron" has a distinct pattern that distinguishes it from other VOCs and is significantly correlated with the increased hydrophobicity in spike proteins. We observed an increase in the non-synonymous substitution rate compared with the other four VOCs. Considering the phylogenetic relationship, we hypothesized about the functional interdependence between recombination and the mutation rate that might have resulted in a shift in the optimum of the mutation rate for the evolution of the "Omicron" variant. The results suggest that for improved disease prevention and control, more attention should be given to the significant genetic differentiation and diversity of newly emerging variants.

Streptomyces sp. MNP32, a forest-dwelling Actinomycetia endowed with potent antibacterial metabolites.

The Actinomycetia isolate, MNP32 was isolated from the Manas National Park of Assam, India, located in the Indo-Burma biodiversity hotspot region of Northeast India. Morphological observations and molecular characterization revealed its identity to be Streptomyces sp. with a 99.86% similar to Streptomyces camponoticapitis strain I4-30 through 16S rRNA gene sequencing. The strain exhibited broad-spectrum antimicrobial activity against a wide range of bacterial human pathogens including WHO-listed critical priority pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii. The ethyl acetate extract was found to disrupt the membrane of the test pathogens which was evidenced through scanning electron microscopy, membrane disruption assay and confocal microscopy. Cytotoxicity studies against CC1 hepatocytes demonstrated that EA-MNP32 had a negligible effect on cell viability. Chemical analysis of the bioactive fraction using gas chromatography-mass spectrometry (GC-MS) showed the presence of 2 major chemical compounds that include Phenol, 3,5-bis(1,1-dimethylethyl)- and [1,1'-Biphenyl]-2,3'-diol, 3,4',5,6'-tetrakis(1,1-dimethylethyl)- which have been reported to possess antimicrobial activity. The phenolic hydroxyl groups of these compounds were proposed to interact with the carbonyl group of the cytoplasmic proteins and lipids leading to destabilization and rupture of the cell membrane. These findings highlight the potential of exploring culturable actinobacteria from the microbiologically under-explored forest ecosystem of Northeast India and bioactive compounds from MNP32 which can be beneficial for future antibacterial drug development.

Development and evaluation of a simple PCR assay and nested PCR for rapid detection of clarithromycin-resistant Helicobacter pylori from culture and directly from the biopsy samples in India.

BACKGROUND: Eradication of Helicobacter pylori provides the most effective treatment for gastroduodenal diseases caused by H. pylori infection. Clarithromycin, a member of the macrolide family, still remains the most important antibiotic used in H. pylori eradication treatment. But the increasing prevalence of clarithromycin resistant H. pylori strains due to point mutations in the V region of the 23S rRNA, poses a great threat in treating the ailing patients. So, we aimed for PCR-mediated rapid detection of the point mutation at 2143 position of 23S rRNA gene in H. pylori that is relevant to clarithromycin resistance from culture and simultaneously from biopsy specimens to avoid the empirical treatment. RESULTS: Newly developed PCR assay using DNA of pure culture detected point mutation in 23S rRNA gene in 21 (8.04%) of 261 clinical strains tested. The agar dilution method showed that all these 21 strains were resistant to clarithromycin indicating the perfect match of the PCR based results. Additionally, the sequencing study also identified the A to G mutation at 2143 position in 23S rRNA gene of the resistant strains only. Consequently, the newly developed Nested-ASP-PCR dealing directly with 50 biopsy specimens demonstrated 100% sensitivity and specificity with the findings of agar dilution method taken as Gold standard. Bioinformatics based analysis such as accessibility analysis and dot plot clearly stated that the base pairing probability has increased due to mutation. Computational studies revealed that the point mutation confers more stability in secondary structure due to conversion of loop to stem. Furthermore, interaction studies showed binding affinity of the CLR to the mutant type is weaker than that to the wild type. CONCLUSION: This assay outlines a rapid, sensitive and simple approach to identify point mutation that confers clarithromycin resistance as well as clarithromycin sensitive strains, providing rapid initiation of effective antibiotic treatment. Additionally, it is simple to adopt for hospital based diagnostic laboratories to evaluate the degree of regional clarithromycin resistance from biopsy specimens itself. Furthermore, in silico studies provide evidence or a signal that the prevalence of clarithromycin resistance may rise in the near future as a result of this point mutation.

Theoretical Study of Dynamical and Electronic Properties of Noncentrosymmetric Superconductor NbReSi.

The noncentrosymmetric NbReSi superconductor with Tc≃6.5 K is characterized by the relatively large upper critical magnetic field. Its multigap features were observed experimentally. Recent studies suggested the realization of P6¯2m or Ima2 symmetry. We discuss the dynamical properties of both symmetries (e.g., phonon spectra). In this paper, using the ab initio techniques, we clarify this ambiguity, and conclude that the Ima2 symmetry is unstable, and P6¯2m should be realized. The P6¯2m symmetry is also stable in the presence of external hydrostatic pressure. We show that NbReSi with the P6¯2m symmetry should host phonon surface states for (100) and (110) surfaces. Additionally, we discuss the main electronic properties of the system with the stable symmetry.

Natural selection shaped the evolution of amino acid usage in mammalian toll like receptor genes.

Toll-like receptors (TLRs) are important as they are able to sense diverse set of pathogens associated molecular patterns (PAMPs) as ligands. These receptors are involved in functions such as immune response, development of signaling process and cell adhesion. In the present study we are interested to analyze the influence of evolutionary selection pressure on the mutational diversity of mammalian TLR genes. We observed differential patterns of amino acid usage between primate and non-primate mammalian TLR genes. GC-content of TLR genes and hydrophobicity of the encoded proteins are the most influential factors correlated with the differential pattern of amino acid usage.The influence of the subcellular location on the amino acid usage pattern of TLRs is evident in present study. Purifying selection is uniformly present on TLR genes, positively selected sites are mostly located over the ligand binding domain. Our study clearly demonstrates that natural selection has shaped the evolution of primate and non-primate mammalian TLR genes.

Enablers and barriers towards ensuring routine immunization services during the COVID-19 pandemic: findings from a qualitative study across five different states in India.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has led to disruption in delivering routine healthcare services including routine immunization (RI) worldwide. Understanding the enablers and barriers for RI services during a pandemic is critically important to develop context-appropriate strategies to ensure uninterrupted routine services. METHODS: A community-based, cross-sectional descriptive study was conducted in five different states of India, nested within an ongoing multicentric study on RI. Telephone in-depth interviews among 56 health workers were carried out and the data were analyzed using a content analysis method. RESULTS: During the COVID-19 pandemic, healthcare providers encountered many challenges at the health system, community and individual level when rendering RI services. Challenges like the limited availability of personal protective equipment and vaccines, deployment for COVID-19 duty at system level, the difficulty in mobilizing people in the community, fear among people at community level, mobility restrictions and limited family support, as well as the stress and stigma at individual level, were barriers to providing RI services. By contrast, the issuing of identification cards to health staff, engaging community volunteers, the support given to health workers by their families and training on COVID-19, were factors that enabled health workers to maintain RI services during the pandemic. CONCLUSIONS: When addressing the COVID-19-related public health emergency, we should not lose sight of the importance of services like RI.

HIV long-term non-progressors share similar features with simian immunodeficiency virus infection of chimpanzees.

HIV-1 infection in human beings has been an outcome of cross-species transmission event of simian immunodeficiency virus from chimpanzees (SIVcpz). Present study reveals differential features of envelope genes representing different categories of HIV-1 disease progression in human beings, namely, rapid progressors (RP), slow progressors (SP) and long-term non-progressors (LTNP) with respect to SIVcpz, based on their amino acid usage patterns. It was evident that SP, LTNP and SIVcpz envelope genes displayed similar patterns of amino acid usage which strongly contrasted with the features exhibited by the envelope genes representing RP category. Robust analysis revealed that selection constraint of human host on SP and LTNP associated envelope genes and chimpanzee host on SIVcpz envelope genes were more severe compared to selection pressure operational on RP associated envelope genes. Evolutionary forces of selection appeared to be comparatively more relaxed on the RP envelope genes in contrast to SP, LTNP and SIVcpz types. Better binding of RP envelope glycoprotein 120 (gp120) compared to envelope gp120 representing SP, LTNP and SIVcpz with host cellular receptor CD4, as inferred employing molecular docking approaches, promises to confer meaningful insights into the event of speedy progression of HIV in rapid progressors. It was interesting to note that envelope glycoprotein exhibited a tendency of hindering proper interaction of host (human/chimpanzee) CD4 and major histocompatibility complex II (MHC II), with a better efficacy in rapid progressors, thus, facilitating highest degrees of immune suppression. Proper identification of the contrasting features might confer a scope to modulate rapid progression of HIV to a long-term non-progressive controlled case, as observed in LTNP and SIVcpz infection, simultaneously aiding therapeutic research against AIDS targeted at drug and vaccine development.Communicated by Ramaswamy H. Sarma.

Characterization of non-typhoidal Salmonella isolates from children with acute gastroenteritis, Kolkata, India, during 2000-2016.

Non-typhoidal Salmonella (NTS) is an important cause of acute gastroenteritis in children. The study was undertaken to determine the isolation rate, serovar prevalence, antimicrobial resistance (AMR) profiles, and molecular subtypes of NTS from a hospital-based diarrheal disease surveillance in Kolkata, India. Rectal swabs were collected from children (< 5 years of age) with acute gastroenteritis from 2000 to 2016. Samples were processed following standard procedures for identification of NTS. The isolates were tested for antimicrobial susceptibility, AMR genes, plasmid profiles, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE) subtypes. A total of 99 (1.0%) Salmonella isolates were recovered from 9957 samples processed. Of the 17 Salmonella serovars identified, S. Worthington (33%) was predominant followed by S. Enteritidis (13%), S. Typhimurium (12%), and others. The isolates showed high resistance towards nalidixic acid (43%), ampicillin (34%), third-generation cephalosporins (32%), and azithromycin (25%), while low resistance was observed for fluoroquinolones (2%). Extended-spectrum beta-lactamase production (blaCTX-M-15 and blaSHV-12 genes) and azithromycin resistance (mphA gene) were common in S. Worthington, while fluoroquinolone resistance (gyrA and parC mutations) was found in S. Kentucky. Diverse plasmid profiles were observed among the isolates. PFGE analysis identified genetically related strains of each serovar in circulation. MLST also revealed phylogenetically clonal isolates of which S. Worthington ST592 and ciprofloxacin-resistant S. Kentucky ST198 were not reported earlier from India. NTS resistant to current drugs of choice poses a potential public health problem. Continuous monitoring of AMR profiles and molecular subtypes of NTS serovars is recommended for controlling the spread of resistant organisms.

Influence of domain architecture and codon usage pattern on the evolution of virulence factors of Vibrio cholerae.

Cholera remains a heavy burden to human health in some developing countries including India where sanitation is poor and health care is limited. After the publication of the complete genome sequence of Vibrio cholerae, the etiological agent of cholera, extensive possibilities, earlier unavailable, have opened up to understand the genetic organization of V. cholerae. In the present study, we analyzed all the pathogenic non-horizontally transferred genes of V. cholerae to know the ancestral relationship and how the pathogenic genes have been evolved in V. cholerae genome. We observed that protein domain has important role in developing pathogenicity, and codon usage pattern of the pathogenic protein domain is also subject to selection. The present study unambiguously depict that the patterns of synonymous codon usage within a protein domain can change dramatically during the course of evolution to give rise to pathogenicity.

Evolutionary complexities of swine flu H1N1 gene sequences of 2009.

A recently emerged novel influenza A (H1N1) virus continues to spread globally. The pandemic caused by this new H1N1 swine influenza virus presents an opportunity to analyze the evolutionary significance of the origin of the new strain of swine flu. Our study clearly suggests that strong purifying selection is responsible for the evolution of the novel influenza A (H1N1) virus among human. We observed that the 2009 viral sequences are evolutionarily widely different from the past few years' sequences. Rather, the 2009 sequences are evolutionarily more similar to the most ancient sequence reported in the NCBI Influenza Virus Resource Database collected in 1918. Analysis of evolutionary rates also supports the view that all the genes in the pandemic strain of 2009 except NA and M genes are derived from triple reassorted swine viruses. Our study demonstrates the importance of using complete-genome approach as more sequences will become available to investigate the evolutionary origin of the 1918 influenza A (H1N1) swine flu strain and the possibility of future reassortment events.

Delving deeper into the unexpected correlation between gene expressivity and codon usage bias of Escherichia coli genome.

Biased usage of synonymous codons has been elucidated under the perspective of cellular tRNA abundance and more recently by the mRNA secondary structure folding stability of the corresponding genes. Taking advantage of publicly available gene expression data for Escherichia coli, a comprehensive investigation of the three classes of genes having different codon usage biases was performed from the standpoint of tRNA abundance, mRNA secondary structure folding stability, and translational error minimization procedure. We detected the different evolutionary forces for translational and/or transcriptional regulation of highly expressed genes depending upon their codon bias. Additionally, the possible role of mRNA folding stability in maintaining the overall high expressivity of the set of lowly biased genes has been articulated. These novel findings certainly strengthen the understanding of the codon usage bias in the Escherichia coli genome.

Nature of selective constraints on synonymous codon usage of rice differs in GC-poor and GC-rich genes.

Synonymous codon usage and cellular tRNA abundance are thought to be co-evolved in optimizing translational efficiencies in highly expressed genes. Here in this communication by taking the advantage of publicly available gene expression data of rice and Arabidopsis we demonstrated that tRNA gene copy number is not the only driving force favoring translational selection in all highly expressed genes of rice. We found that forces favoring translational selection differ between GC-rich and GC-poor classes of genes. Supporting our results we also showed that, in highly expressed genes of GC-poor class there is a perfect correspondence between majority of preferred codons and tRNA gene copy number that confers translational efficiencies to this group of genes. However, tRNA gene copy number is not fully consistent with models of translational selection in GC-rich group of genes, where constraints on mRNA secondary structure play a role to optimize codon usage in highly expressed genes.