Codon usage bias of goose circovirus and its adaptation to host.

Goose circovirus (GoCV), a potential immunosuppressive virus possessing a circular single-stranded DNA genome, is widely distributed in both domesticated and wild geese. This virus infection causes significant economic losses in the waterfowl industry. The codon usage patterns of viruses reflect the evolutionary history and genetic architecture, allowing them to adapt quickly to changes in the external environment, particularly to their hosts. In this study, we retrieved the coding sequences (Rep and Cap) and the genome of GoCV from GenBank, conducting comprehensive research to explore the codon usage patterns in 144 GoCV strains. The overall codon usage of the GoCV strains was relatively similar and exhibited a slight bias. The effective number of codons (ENC) indicated a low overall extent of codon usage bias (CUB) in GoCV. Combined with the base composition and relative synonymous codon usage (RSCU) analysis, the results revealed a bias toward A- and G-ending codons in the overall codon usage. Analysis of the ENC-GC3s plot and neutrality plot suggested that natural selection plays an important role in shaping the codon usage pattern of GoCV, with mutation pressure having a minor influence. Furthermore, the correlations between ENC and relative indices, as well as correspondence analysis (COA), showed that hydrophobicity and geographical distribution also contribute to codon usage variation in GoCV, suggesting the possible involvement of natural selection. In conclusion, GoCV exhibits comparatively slight CUB, with natural selection being the major factor shaping the codon usage pattern of GoCV. Our research contributes to a deeper understanding of GoCV evolution and its host adaptation, providing valuable insights for future basic studies and vaccine design related to GoCV.

Epidemiology and Genetic Diversity of PCV2 Reveals That PCV2e Is an Emerging Genotype in Southern China: A Preliminary Study.

Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus type 2 (PCV2), has ravaged the pig industry, causing huge economic loss. At present, PCV2b and PCV2d are highly prevalent genotypes worldwide, while in China, in addition to PCV2b and PCV2d, a newly emerged PCV2e genotype detected in the Fujian province has attracted attention, indicating that PCV2 genotypes in China are more abundant. A preliminary study was conducted to better understand the genetic diversity and prevalence of PCV2 genotypes in southern China. We collected 79 random lung samples from pigs with respiratory signs, from 2018 to 2021. We found a PCV2-positivity rate of 29.1%, and frequent co-infections of PCV2 with PCV3, Streptococcus suis (S. suis), and other porcine pathogens. All PCV2-positive samples were sequenced and subjected to whole-genome analysis. Phylogenetic analysis, based on the PCV2 ORF2 gene and complete genomes, found that PCV2 strains identified in this study belonged to genotypes PCV2a (1), PCV2b (6), PCV2d (10), and PCV2e (6). Importantly, PCV2e was identified for the first time in some provinces, including Guangdong and Jiangxi. Additionally, we found two positively selected sites in the ORF2 region, located on the previously reported antigenic epitopes. Moreover, codon 63, one of the positively selected sites, has different types of amino acids in different genotypes. In conclusion, this study shows that PCV2e is an emerging genotype circulating in southern China, which warrants urgent, specific surveillance to aid the development of prevention and control strategies in China.

sRNA23, a novel small RNA, regulates to the pathogenesis of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (S. suis 2) is an important ubiquitous zoonotic pathogen. To date, regulatory factors and their implication in S. suis pathogenesis are not fully understood. Small non-coding RNAs (sRNAs) have been proven to function as important regulatory factors in bacterial pathogenesis and stress adaptation. Here, we identified a differentially downregulated S. suis 05ZYH33 sRNA after iron starvation by RNA-seq, which we named sRNA23. The presence of sRNA23 was further confirmed by RACE and Northern blot. Expression of sRNA23 was significantly altered under different environmental stresses such as nutritional starvation, osmotic pressure, oxidative stress, and lysozymal exposure. A sRNA23-deleted mutant exhibited relatively shorter streptococcal chains and weakened biofilm-forming ability. The mutation also resulted in decreased adherence of the S. suis 05ZYH33 to human laryngeal epidermoid carcinoma (HEp-2) cells, increased sensitivity to phagocytosis by RAW264.7 macrophages, and significantly reduced hemolytic activity. Furthermore, we observed that a sRNA23-deleted mutant had a low survival rate in pig whole blood and attenuated virulence in a mouse model. Moreover, based on RNA pull-down and electrophoretic mobility shift assay, we found that sRNA23 can directly bind to two proteins involved in adhesion and biofilm formation, namely, moonlighting protein FBA (fructose diphosphate aldolase) and rplB (50S ribosomal protein L2), respectively. Collectively, sRNA23 enhances S. suis 2 pathogenicity and the binding between sRNA23 and FBA/rplB might play an essential role in the adherence and biofilm-forming ability of S. suis 2.

Effect of interfacial dipole on heterogeneous ice nucleation.

In this work, we performed molecular dynamics simulations of ice nucleation on a rigid surface model of cubic zinc blende structure with different surface dipole strength and orientation. Our results show that, although substrates are excellently lattice-matched to cubic ice, ice nucleation merely occurred as the interfacial water molecules (IWs) show identical or similar orientations to that of water molecules in cubic ice. Free energy landscapes revealed that, as substrates have non-suitable dipole strength/orientation, there exist large free energy barriers for rotating dipole IWs to the right orientation to trigger ice formation. This study stresses that, beyond the traditional view of lattice match and the similarity of lattice length between the substrate and new-formed crystal, the similarity between molecular orientations of interfacial component and component in the specific new-formed crystalline face is also critical for promoting ice nucleation.

Genome-wide analysis of the synonymous codon usage pattern of Streptococcus suis.

Streptococcus suis (S. suis) is a gram-positive coccus that causes disease in humans and animals. The codon usage pattern of bacteria reveals a range of evolutionary changes that assist them to enhance tolerance to environments. To better understand the genetic features during the evolution of S. suis, we performed codon usage analysis. Nine pathogenic strains of different serotypes and different geographical distribution were analyzed to better understand the differences in their evolutionary process. Nucleotide compositions and relative synonymous codon usage (RSCU) analysis revealed that A/T-ending codons are dominant in S. suis. Neutrality analysis, correspondence analysis and ENC-plot results revealed that natural selection is the predominant element prompting codon usage. Cluster analysis based on RSCU was roughly consistent with the dendrogram rooted genomic BLAST analysis. Comparison of synonymous codon usage pattern between S. suis and susceptible hosts (H. sapiens and S. scrofa) revealed that the codon usage of S. suis is separated from the synonymous codon usage of susceptible hosts. The CAI values implied that S. suis includes a series of predicted highly expressed coding sequences contained in metabolism and transcriptional regulation, revealing the necessity of this pathogen to deal with various environmental conditions. The study of codon usage in S. suis may provide evidence involving the molecular evolution of bacteria and a better understanding of evolutionary relationships between S. suis and its corresponding hosts.

Establishing a System for Functional Characterization of Full-Length cDNAs of Camellia sinensis.

: Tea (Camellia sinensis) is enriched with bioactive secondary metabolites, and is one of the most popular nonalcoholic beverages globally. Two tea reference genomes have been reported; however, the functional analysis of tea genes has lagged, mainly due to tea's recalcitrance to genetic transformation and the absence of alternative high throughput heterologous expression systems. A full-length cDNA collection with a streamlined cloning system is needed in this economically important woody crop species. RNAs were isolated from nine different vegetative tea tissues, pooled, then used to construct a normalized full-length cDNA library. The titer of unamplified and amplified cDNA library was 6.89 × 106 and 1.8 × 1010 cfu/mL, respectively; the library recombinant rate was 87.2%. Preliminary characterization demonstrated that this collection can complement existing tea reference genomes and facilitate rare gene discovery. In addition, to streamline tea cDNA cloning and functional analysis, a binary vector (pBIG2113SF) was reengineered, seven tea cDNAs isolated from this library were successfully cloned into this vector, then transformed into Arabidopsis. One FL-cDNA, which encodes a putative P1B-type ATPase 5 (CsHMA5), was characterized further as a proof of concept. We demonstrated that overexpression of CsHMA5 in Arabidopsis resulted in copper hyposensitivity. Thus, our data demonstrated that this represents an efficient system for rare gene discovery and functional characterization of tea genes. The integration of a tea FL-cDNA collection with efficient cloning and a heterologous expression system would facilitate functional annotation and characterization of tea genes.

Evolution and Genetic Diversity of Porcine Circovirus 3 in China.

The identification of a new circovirus (Porcine Circovirus 3, PCV3) has raised concern because its impact on swine health is not fully known. In Fujian Province in eastern China, even its circulating status and genetic characteristics are unclear. Here, we tested 127 tissue samples from swine from Fujian Province that presented respiratory symptoms. All of the PCV3 positive samples were negative for many other pathogens involved in respiratory diseases like PCV2, PRRSV, and CSFV, suggesting that PCV3 is potentially pathogenic. From phylogenetic analysis, PCV3 strains are divided into two main clades and five sub-clades; PCV3a-1, PCV3a-2, PCV3a-3, PCV3b-1, and PCV3b-2. Our identified strains belong to genotypes PCV3a-1, PCV3a-2, PCV3a-3, and PCV3b-2, indicating a high degree of genetic diversity of PCV3 in Fujian province until 2019. Interestingly, we found the time of the most recent common ancestor (tMRCA) of PCV3 was dated to the 1950s, and PCV3 has a similar evolutionary rate as PCV2 (the main epidemic genotypes PCV2b and PCV2d). In addition, positive selection sites N56D/S and S77T/N on the capsid gene are located on the PCV3 antigen epitope, indicating that PCV3 is gradually adaptive in swine. In summary, our results provide important insights into the epidemiology of PCV3.

N-Acyl-Homoserine Lactone Quorum Sensing Switch from Acidogenesis to Solventogenesis during the Fermentation Process in Serratia marcescens MG1.

N-acyl-homoserine lactone quorum sensing (AHL-QS) has been shown to regulate many physiological behaviors in Serratia marcescens MG1. In the current study, the effects of AHL-QS on the biosynthesis of acid and neutral products by S. marcescens MG1 and its isogenic ∆swrI with or without supplementing exogenous N-hexanoyl-L-homoserine lactone (C6-HSL) were systematically investigated. The results showed that swrI disruption resulted in rapid pH drops from 7.0 to 4.8, which could be restored to wild type by supplementing C6-HSL. Furthermore, fermentation product analysis indicated that ∆swrI could lead to obvious accumulation for acidogenesis products such as lactic acid and succinic acid, especially excess acetic acid (2.27 g/l) produced at the early stage of fermentation, whereas solventogenesis products by ∆swrI appeared to noticeably decrease by an approximate 30% for acetoin during 32-48 h and by an approximate 20% for 2,3-butanediol during 24-40 h, when compared to those by wild type. Interestingly, the excess acetic acid produced could be removed in an AHL-QS-independent manner. Subsequently, quantitative real-time PCR was used to determine the mRNA expression levels of genes responsible for acidogenesis and solventogenesis and showed consistent results with those of product synthesis. Finally, by close examination of promoter regions of the analyzed genes, four putative luxI box-like motifs were found upstream of genes encoding acetyl-CoA synthase, lactate dehydrogenase, α-acetolactate decarboxylase, and Lys-like regulator. The information from this study provides a novel insight into the roles played by AHL-QS in switching from acidogenesis to solventogenesis in S. marcescens MG1.

Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Senecavirus A evolution.

Senecavirus A (SVA) infection was recently confirmed in pigs in Brazil, United States of America and Canada. To better understand the molecular characteristics of isolated SVA genomes, we first reported genome-wide comprehensive analyses of codon usage and various factors that have contribute to the molecular evolution in SVA. The effective number of codons (ENC) ranged from 54.51 to 55.54 with an average of 54.87 ± 0.285, which reveals a relatively stable nucleotide composition. We found that codon usage bias of the SVA was low. Mutational pressure acted as an increasingly dominant factor for the evolution of the virus compared with the natural selection. Notably, codon usage bias was also affected by the geographic distribution and isolated time. The first systemic analysis on the codon usage bias of the SVA provides important information for the understanding of the evolution of the SVA and has fundamental and theoretical benefits.

Analysis of the codon usage pattern in Middle East Respiratory Syndrome Coronavirus.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which first broken out in Jeddah in 2012, causes a severe acute respiratory illness with a high mortality rate. To better understand the molecular characteristics of isolated MERS-CoV genomes, we first analysed the codon usage pattern of the zoonotic MERS-CoV strains comprehensively to gain an insight into the mechanism of cross-species transmission. We found that MERS human/camel isolates showed a low codon usage bias. Both mutation and nature selection pressure have contributed to this low codon usage bias, with the former being the main determining factor. We also observed that gene function, evolution time and the different host species of the virus all contributed to the bias of MERS-CoV, to some extent. Additionally, the codon usage pattern of MERS-CoV isolates is different from other related Nidovirales viruses isolated from bats and hedgehogs. In the future, more epidemiological surveys are required to examine the factors that resulted in the emergence and outbreak of this virus.

Mechanism of 2,3-butanediol stereoisomers formation in a newly isolated Serratia sp. T241.

Serratia sp. T241, a newly isolated xylose-utilizing strain, produced three 2,3-butanediol (2,3-BD) stereoisomers. In this study, three 2,3-butanediol dehydrogenases (BDH1-3) and one glycerol dehydrogenase (GDH) involved in 2,3-BD isomers formation by Serratia sp. T241 were identified. In vitro conversion showed BDH1 and BDH2 could catalyzed (3S)-acetoin and (3R)-acetoin into (2S,3S)-2,3-BD and meso-2,3-BD, while BDH3 and GDH exhibited the activities from (3S)-acetoin and (3R)-acetoin to meso-2,3-BD and (2R,3R)-2,3-BD. Four encoding genes were assembled into E. coli with budA (acetolactate decarboxylase) and budB (acetolactate synthase), responsible for converting pyruvate into acetoin. E. coli expressing budAB-bdh1/2 produced meso-2,3-BD and (2S,3S)-2,3-BD. Correspondingly, (2R,3R)-2,3-BD and meso-2,3-BD were obtained by E. coli expressing budAB-bdh3/gdh. These results suggested four enzymes might contribute to 2,3-BD isomers formation. Mutants of four genes were developed in Serratia sp. T241. Δbdh1 led to reduced concentration of meso-2,3-BD and (2S,3S)-2,3-BD by 97.7% and 87.9%. (2R,3R)-2,3-BD with a loss of 73.3% was produced by Δbdh3. Enzyme activity assays showed the decrease of 98.4% and 22.4% by Δbdh1 and Δbdh3 compared with the wild strain. It suggested BDH1 and BDH3 played important roles in 2,3-BD formation, BDH2 and GDH have small effects on 2,3-BD production by Serratia sp. T241.

Cloning, expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30.

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation-reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe(2+) and for (3S/3R)-acetoin reduction in the presence of Mn(2+), while several cations inhibited its activity, particularly Fe(2+) and Fe(3+) for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .

A new NAD(H)-dependent meso-2,3-butanediol dehydrogenase from an industrially potential strain Serratia marcescens H30.

The budC gene coding for a new meso-2,3-butanediol dehydrogenase (BDH) from Serratia marcescens H30 was cloned and expressed in Escherichia coli BL21(DE3), purified, and characterized for its properties. The recombinant BDH with a molecular weight of 27.4 kDa exhibited a reversible transformation between acetoin and 2,3-butanediol. In the presence of NADH, BDH could catalyze the reduction of diacetyl and (3R)-acetoin to (3S)-acetoin and meso-2,3-butanediol, respectively, while (3S)-acetoin as a substrate could be further transformed into (2S, 3S)-2,3-butanediol at pH 9.0. For diol oxidation reactions, (3R)-acetoin and (3S)-acetoin were obtained when meso-2,3-butanediol and (2S,3S)-2,3-butanediol were used as the substrates with BDH and NAD(+). (2R,3R)-2,3-butanediol was not a substrate for the BDH at all. The low K m value (4.1 mM) in meso-2,3-butanediol oxidation reaction and no activity for diacetyl, acetoin, and 2,3-butanediol as the substrates with NADP(+)/NADPH suggested that the budC gene product belongs to a NAD(H)-dependent meso-2,3-BDH. Maximum activities for diacetyl and (3S/3R)-acetoin reduction were observed at pH 8.0 and pH 5.0 while for meso-2,3-butanediol oxidation it was pH 8.0. However, the optimum temperature for oxidation and reduction reactions was about 40 °C. In addition, the BDH activity for meso-2,3-butanediol oxidation was enhanced in the presence of Fe(2+) and for diacetyl and (3S/3R)-acetoin reduction in the presence of Mg(2+) and Mn(2+), while several metal ions inhibited its activity, particularly Fe(3+) for reduction of diacetyl and acetoin. Sequence analysis showed that the BDH from S. marcescens H30 possessed two conserved sequences including the coenzyme binding motif (GxxxGxG) and the active-site motif (YxxxK), which are present in the short-chain dehydrogenase/reductase superfamily.