Transposase Gene Research Articles

Molecular epidemiology of Coxiella Brunetii in small ruminants in Punjab, Pakistan: a novel reporting analytical cross sectional study.

Coxiella burnetii, an intracellular zoonotic bacterium, causes query (Q) fever in ruminants. Its role has never been elucidated in small ruminants from Pakistan. The current study is designed to (a) determine the prevalence of coxiellosis in small ruminants, (b) evaluate the association of various potential risk factors and biomarkers in the occurrence of Coxiella burnetii, (c) and determine phylogeny and genetic variability of its various isolates identified during the study. For this purpose, 320 blood samples from sheep (n = 160) and goats (n = 160) were collected from 9 Union Councils of district Kasur, Punjab, and processed for DNA extraction. C. burnetii was confirmed by amplification of IS1111 transposase gene with an amplicon size of 294bp. The results showed that the overall positive percentage of C. burnetii is 36.87% (sheep: 46.9% and goats: 30%). The phylogenetic tree was also constructed which described the possible origin of this pathogen from environment. Besides, after translation into amino acid, the resultant alignment showed several unique changes at position numbers 18 and 27 in the isolates from goats and at 27 and 66 from those of sheep. These mutations can have major impact on the infectious characteristics of this pathogen. Furthermore, different potential risk factors and clinical biomarkers like age, tick infestation, abortion, mastitis, and infertility were also studied and found that these are significantly (p< 0.05) associated with the occurrence of coxiellosis. It is concluded from the study that C. burnetii is endemic in small ruminants in Punjab, Pakistan. The outcomes of this study are alarming for scientific community as well as for policy makers because coxiellosis is an emerging threat to both humans and animals in this region due to its interspecies transmission ability.

Molecular diagnosis of Coxiella burnetii in culture negative endocarditis and vascular infection in South Korea

Background Q fever endocarditis is a major cause of culture-negative endocarditis. The role of Coxellia burnetii is underestimated because it is difficult to diagnose. We investigated the significance of C. burnetii as the cause of culture-negative endocarditis and vascular infection by examining blood and tissue specimens using serological testing and polymerase chain reaction (PCR). Methods All patients with infective endocarditis or large vessel vasculitis were prospectively enrolled at a tertiary-care hospital from May 2016 through September 2020. Q fever endocarditis and vascular infection were diagnosed based on: (1) positive PCR for a cardiac valve or vascular tissue, (2) positive PCR for blood or phase I immunoglobulin G (IgG) ≥ 6400, or (3) phase I IgG ≥ 800 and < 6400 with morphologic abnormality. PCR targeted C. burnetii transposase gene insertion element IS1111a. Results Of the 163 patients, 40 (25%) had culture-negative endocarditis (n = 35) or vascular infection (n = 5). Of the 40 patients, 24 (60%) were enrolled. Eight (33%) were diagnosed with Q fever endocarditis or vascular infection. Of these 8 patients, 6 had suspected acute Q fever endocarditis or vascular infection with negative phase I IgG. Six patients were not treated for C. burnetii, 4 were stable after surgery. One patient died due to surgical site infection after 5 months post-operatively and one died due to worsening underlying disease. Conclusions Approximately one-third of patients with culture-negative endocarditis and vascular infection was diagnosed as Q fever. Q fever endocarditis and vascular infection may be underestimated in routine clinical practice in South Korea. KEY MESSAGE Q fever endocarditis and vascular infection may be underestimated in routine clinical practice, thus, try to find evidence of C. burnetti infection in suspected patients by all available diagnostic tests including PCR.

Serological and Molecular Phylogenetic Detection of Coxiella burnetii in Lactating Cows, Iraq

This study is carried out to investigate the prevalence of Coxiella burnetii (C. burnetii) infections in cattle using an enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assay targeting IS1111A transposase gene. A total of 130 lactating cows were randomly selected from different areas in Wasit province, Iraq and subjected to blood and milk sampling during the period extended between November 2018 and May 2019. ELISA and PCR tests revealed that 16.15% and 10% of the animals studied were respectively positive. Significant correlations (P&lt;0.05) were detected between the positive results and clinical data. Two positive PCR products were analyzed phylogenetically, named as C. burnetii IQ-No.5 and C. burnetii IQ-No.6; and then recorded in the National Center for Biotechnology Information (NCBI) under an accession numbers of MN473204.1 and MN473205.1. Comparative identity of the local strains with NCBI-BLAST strains/isolates revealed 97% similarity and 0.1-0.6% of total genetic mutations/changes. NCBI-BLAST Homology Sequence reported high significant identity (P&lt;0.05) between the local, C. burnetii IQ-No.5 and C. burnetii IQ-No.6; strains and C. burnetii 3345937 (CP014354.1) Netherlands isolate at 99.10% and 99.06%, respectively. The current study concluded that the percentage of infected cows with coxiellosis is relatively high, and Coxiella should be listed as abortive pathogen. Therefore, additional studies should be performed including different animals, samples, and regions.

Genetic Diversity and Distribution of Korean Isolates of Burkholderia glumae.

Burkholderia glumae causes panicle blight of rice (grain rot in Japan and Korea), and the severity of damage is increasing worldwide. During 2017 and 2018, 137 isolates of B. glumae were isolated from symptomatic grain rot of rice cultivated in paddy fields throughout South Korea. Genetic diversity of the isolates was determined using transposase-based PCR (Tnp-PCR) genomic fingerprinting. All 138 isolates, including the B. glumae BGR1 strain, produced toxoflavin in various amounts, and 17 isolates produced an unidentified purple or orange pigment on Luria-Bertani medium and casamino acid-peptone-glucose medium, respectively, at 28°C. Transposase-based PCR genomic fingerprinting was performed using a novel primer designed based on transposase (tnp) gene sequences located at the ends of the toxoflavin efflux transporter operon; this method provided reliable and reproducible results. Through Tnp-PCR genomic fingerprinting, the genetic groups of Korean B. glumae isolates were divided into 11 clusters and three divisions. The Korean B. glumae isolates were mainly grouped in division I (73%). Interestingly, most of the pigment-producing isolates were grouped in divisions II and III; of these, 10 were grouped in cluster VIII, which comprised 67% of this cluster. Results of a phylogenetic analysis based on tofI and hrpB gene sequences were consistent with classification by Tnp-PCR genomic fingerprinting. The BGR1 strain did not belong to any of the clusters, indicating that this strain does not exhibit the typical genetic representation of B. glumae. B. glumae isolates showed diversity in the use of carbon and nitrogen sources, but no correlation with genetic classification by PCR fingerprinting was found. This is the first study to analyze the geographical distribution and genetic diversity of Korean B. glumae isolates.

Evolution along the parasitism-mutualism continuum determines the genetic repertoire of prophages.

Integrated into their bacterial hosts’ genomes, prophage sequences exhibit a wide diversity of length and gene content, from highly degraded cryptic sequences to intact, functional prophages that retain a full complement of lytic-function genes. We apply three approaches—bioinformatics, analytical modelling and computational simulation—to understand the diverse gene content of prophages. In the bioinformatics work, we examine the distributions of over 50,000 annotated prophage genes identified in 1384 prophage sequences, comparing the gene repertoires of intact and incomplete prophages. These data indicate that genes involved in the replication, packaging, and release of phage particles have been preferentially lost in incomplete prophages, while tail fiber, transposase and integrase genes are significantly enriched. Consistent with these results, our mathematical and computational approaches predict that genes involved in phage lytic function are preferentially lost, resulting in shorter prophages that often retain genes that benefit the host. Informed by these models, we offer novel hypotheses for the enrichment of integrase and transposase genes in cryptic prophages. Overall, we demonstrate that functional and cryptic prophages represent a diversity of genetic sequences that evolve along a parasitism-mutualism continuum.

Comparative genomics in \u201cCandidatus Kuenenia stuttgartiensis\u201d reveal high genomic plasticity in the overall genome structure, CRISPR loci and surface proteins

BackgroundAnaerobic ammonium oxidizing bacteria (anammox bacteria) are contributing significantly to the nitrogen cycle and are successfully used in wastewater treatment. Due to the lack of complete genomes in the databases, little is known about the stability and variability of their genomes and how the genomes evolve in response to changing environments.ResultsHere we report the complete genome of the anammox bacterium “Candidatus Kuenenia stuttgartiensis” strain CSTR1 which was enriched planktonically in a semi-continuous stirred-tank reactor. A comparison of the genome of strain CSTR1 with the genome of “Ca. Kuenenia stuttgartiensis” MBR1 and the draft genome of KUST showed > 99% average nucleotide identity among all. Rearrangements of large genomic regions were observed, most of which were associated with transposase genes. Phylogenetic analysis suggests that strain MBR1 is more distantly related to the other two strains. Proteomic analysis of actively growing cells of strain CSTR1 (growth rate ~ 0.33 d− 1) failed to detect the annotated cytochrome cd1-type nitrite reductase (NirS) although in total 1189 proteins were found in the proteome. Yet, this NirS was expressed when strain CSTR1 was under stress or starvation (growth rate < 0.06 d− 1). We also observed large sequence shifts in the strongly expressed S-layer protein compared to other “Ca. Kuenenia” strains, indicating the formation of hybrids of genes encoding the surface proteins.Conclusions“Ca. Kuenenia” strains appear to be relatively stable in their basic physiological traits, but show high variability in overall genome structure and surface proteins.

Intruder (DD38E), a recently evolved sibling family of DD34E/Tc1 transposons in animals

BackgroundA family of Tc1/mariner transposons with a characteristic DD38E triad of catalytic amino acid residues, named Intruder (IT), was previously discovered in sturgeon genomes, but their evolutionary landscapes remain largely unknown.ResultsHere, we comprehensively investigated the evolutionary profiles of ITs, and evaluated their cut-and-paste activities in cells. ITs exhibited a narrow taxonomic distribution pattern in the animal kingdom, with invasions into two invertebrate phyla (Arthropoda and Cnidaria) and three vertebrate lineages (Actinopterygii, Agnatha, and Anura): very similar to that of the DD36E/IC family. Some animal orders and species seem to be more hospitable to Tc1/mariner transposons, one order of Amphibia and seven Actinopterygian orders are the most common orders with horizontal transfer events and have been invaded by all four families (DD38E/IT, DD35E/TR, DD36E/IC and DD37E/TRT) of Tc1/mariner transposons, and eight Actinopterygii species were identified as the major hosts of these families. Intact ITs have a total length of 1.5–1.7 kb containing a transposase gene flanked by terminal inverted repeats (TIRs). The phylogenetic tree and sequence identity showed that IT transposases were most closely related to DD34E/Tc1. ITs have been involved in multiple events of horizontal transfer in vertebrates and have invaded most lineages recently (< 5 million years ago) based on insertion age analysis. Accordingly, ITs presented high average sequence identity (86–95%) across most vertebrate species, suggesting that some are putatively active. ITs can transpose in human HeLa cells, and the transposition efficiency of consensus TIRs was higher than that of the TIRs of natural isolates.ConclusionsWe conclude that DD38E/IT originated from DD34E/Tc1 and can be detected in two invertebrate phyla (Arthropoda and Cnidaria), and in three vertebrate lineages (Actinopterygii, Agnatha and Anura). IT has experienced multiple HT events in animals, dominated by recent amplifications in most species and has high identity among vertebrate taxa. Our reconstructed IT transposon vector designed according to the sequence from the “cat” genome showed high cut-and-paste activity. The data suggest that IT has been acquired recently and is active in many species. This study is meaningful for understanding the evolution of the Tc1/mariner superfamily members and their hosts.

Analysis of CACTA transposase genes unveils the mechanism of intron loss and distinct small RNA silencing pathways underlying divergent evolution of Brassica genomes.

In comparison with retrotransposons, DNA transposons make up a smaller proportion of most plant genomes. However, these elements are often proximal to genes to affect gene expression depending on the activity of the transposons, which is largely reflected by the activity of the transposase genes. Here, we show that three AT-rich introns were retained in the TNP2-like transposase genes of the Bot1 (Brassica oleracea transposon 1) CACTA transposable elements in Brassica oleracea, but were lost in the majority of the Bot1 elements in Brassica rapa. A recent burst of transposition of Bot1 was observed in B. oleracea, but not in B. rapa. This burst of transposition is likely related to the activity of the TNP2-like transposase genes as the expression values of the transposase genes were higher in B. oleracea than in B. rapa. In addition, distinct populations of small RNAs (21, 22 and 24 nt) were detected from the Bot1 elements in B. oleracea, but the vast majority of the small RNAs from the Bot1 elements in B. rapa are 24 nt in length. We hypothesize that the different activity of the TNP2-like transposase genes is likely associated with the three introns, and intron loss is likely reverse transcriptase mediated. Furthermore, we propose that the Bot1 family is currently undergoing silencing in B. oleracea, but has already been silenced in B. rapa. Taken together, our data provide new insights into the differentiation of transposons and their role in the asymmetric evolution of these two closely related Brassica species.

Antibiotic resistance genes and bacterial community on the surfaces of five cultivars of fresh tomatoes.

Antibiotic resistance genes and bacteria (ARGs and ARB) in vegetable or fruit pose risks to ecological environment health. However, the assessment of ARGs and ARB from one popular vegetable, fresh tomato, has not been carried out before. In this study, high-throughput quantitative PCR and 16S rRNA gene Illumina sequencing technology were used to explore the antibiotic resistance characteristics of bacteria on five common cultivars of fresh tomatoes from supermarket. A total of 191 ARGs and 10 mobile genetic elements (MGEs) were detected on the tomato surfaces. The distribution profile of ARGs and MGEs was different among samples, with the organic tomatoes showing more ARGs and MGEs number and relative abundance. Aminoglycoside resistance genes strA and strB, sulfonamide resistance gene sul1, and multidrug resistance gene qacΔ1-01 were the predominant ARGs. Dominant MGEs were transposase genes, which might promote horizontal gene transfer (HGT) of ARGs. Network analysis indicated that fifteen bacterial families might be the potential hosts of ARGs, and the detected MGEs might have positive correlation with ARGs. These results revealed the bacterial ARGs and MGEs from fresh tomato, which might help guide human to pay more attention to ecological environment impacts of ARGs and ARB on the surfaces of vegetable or fruit.

Long-term biogas slurry application increased antibiotics accumulation and antibiotic resistance genes (ARGs) spread in agricultural soils with different properties

Animal manures are commonly applied to soil which possibly promote the spread of antibiotic resistance from soil to human beings via food chains. Biogas slurry is an end product of anaerobic digestion of animal manures, which has been widely applied as fertilizers in the agricultural soil. However, effect of long-term biogas slurry application on the soil antibiotic resistance and the associated mechanism still remains unclear. The present study characterized antibiotics, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and bacterial community, in different agricultural soils unamended (BS-) and amended (BS+) with biogas slurry (8–18 years) in five field experiments. Our results indicated that long-term application of biogas slurry largely increased the concentrations of tetracyclines in soils, and greatly increased the abundances of ARGs, transposase gene (Tn916/1545) and ARGs-associated bacteria. Long-term application of biogas slurry led to tetracyclines accumulation and ARGs enrichment in agricultural soil, and the selection pressure from tetracyclines and the increase of Tn916/1545 abundace become potential contributors for the increase of soil antibiotic resistance via promoting the enrichment of ARG-associated bacteria. The results of the present study should be taken into consideration to develop policy and practice for mitigating the enrichment and spread of antibiotic resistance during the recycling of biogas slurry into agricultural soil.

Molecular characteristics of optrA-carrying Enterococcus faecalis from chicken meat in South Korea

The purpose of this study was to identify the genetic environment of optrA gene in linezolid (LZD)-resistant Enterococcus faecalis from chicken meat and to describe the probable mechanism of dissemination of the optrA gene through plasmid or chromosomal integration. Whole genome sequencing and analysis revealed that all 3 E. faecalis isolates confirmed as LZD- and chloramphenicol-resistant carried fexA adjacent to the optrA gene as well as a variety of resistance genes for macrolides, tetracyclines, and aminoglycosides, simultaneously. But, the other genes conferring LZD resistance, cfr and poxtA, were not detected in those strains. Two isolates harboring the optrA gene in their chromosomal DNA showed >99% similarity in arrangement to the transposon Tn6674 and the transposase genes, tnpA, tnpB, and tnpC and were located in the first open reading frame for transposase. One isolate harboring an optrA-carrying plasmid also showed >99% similarity with the previously reported pE439 plasmid but had 2 amino acid changes (Thr96Lys and Tyr160Asp) and a higher minimum inhibitory concentration against LZD of 16 mg/L than that of pE439 (8 mg/L). Mobile genetic elements such as transposons or plasmids flanking the optrA gene conduct a crucial role in the dissemination of antimicrobial resistance genes. Further investigations are required to identify the way by which optrA is integrated into chromosomal DNA and plasmids.

Evolution of pogo, a separate superfamily of IS630-Tc1-mariner transposons, revealing recurrent domestication events in vertebrates

sBackgroundTc1/mariner and Zator, as two superfamilies of IS630-Tc1-mariner (ITm) group, have been well-defined. However, the molecular evolution and domestication of pogo transposons, once designated as an important family of the Tc1/mariner superfamily, are still poorly understood.ResultsHere, phylogenetic analysis show that pogo transposases, together with Tc1/mariner, DD34E/Gambol, and Zator transposases form four distinct monophyletic clades with high bootstrap supports (> = 74%), suggesting that they are separate superfamilies of ITm group. The pogo superfamily represents high diversity with six distinct families (Passer, Tigger, pogoR, Lemi, Mover, and Fot/Fot-like) and wide distribution with an expansion spanning across all the kingdoms of eukaryotes. It shows widespread occurrences in animals and fungi, but restricted taxonomic distribution in land plants. It has invaded almost all lineages of animals—even mammals—and has been domesticated repeatedly in vertebrates, with 12 genes, including centromere-associated protein B (CENPB), CENPB DNA-binding domain containing 1 (CENPBD1), Jrk helix–turn–helix protein (JRK), JRK like (JRKL), pogo transposable element derived with KRAB domain (POGK), and with ZNF domain (POGZ), and Tigger transposable element-derived 2 to 7 (TIGD2–7), deduced as originating from this superfamily. Two of them (JRKL and TIGD2) seem to have been co-domesticated, and the others represent independent domestication events. Four genes (TIGD3, TIGD4, TIGD5, and POGZ) tend to represent ancient domestications in vertebrates, while the others only emerge in mammals and seem to be domesticated recently. Significant structural variations including target site duplication (TSD) types and the DDE triad signatures (DD29–56D) were observed for pogo transposons. Most domesticated genes are derived from the complete transposase genes; but CENPB, POGK, and POGZ are chimeric genes fused with additional functional domains.ConclusionsThis is the first report to systematically reveal the evolutionary profiles of the pogo transposons, suggesting that pogo and Tc1/Mariner are two separate superfamilies of ITm group, and demonstrating the repeated domestications of pogo in vertebrates. These data indicate that pogo transposons have played important roles in shaping the genome and gene evolution of fungi and animals. This study expands our understanding of the diversity of pogo transposons and updates the classification of ITm group.

Comparative genomics reveals different population structures associated with host and geographic origin in antimicrobial-resistant Salmonella enterica.

Genetic variation in a pathogen, including the causative agent of salmonellosis, Salmonella enterica, can occur as a result of eco-evolutionary forces triggered by dissimilarities of ecological niches. Here, we applied comparative genomics to study 90 antimicrobial resistant (AMR) S. enterica isolates from bovine and human hosts in New York and Washington states to understand host- and geographic-associated population structure. Results revealed distinct presence/absence profiles of functional genes and pseudogenes (e.g., virulence genes) associated with bovine and human isolates. Notably, bovine isolates contained significantly more transposase genes but fewer transposase pseudogenes than human isolates, suggesting the occurrence of large-scale transposition in genomes of bovine and human isolates at different times. The high correlation between transposase genes and AMR genes, as well as plasmid replicons, highlights the potential role of horizontally transferred transposons in promoting adaptation to antibiotics. By contrast, a number of potentially geographic-associated single-nucleotide polymorphisms (SNPs), rather than geographic-associated genes, were identified. Interestingly, 38% of these SNPs were in genes annotated as cell surface protein-encoding genes, including some essential for antibiotic resistance and host colonization. Overall, different evolutionary forces and limited recent inter-population transmission appear to shape AMR S. enterica population structure in different hosts and geographic origins.

Evolution and domestication of Tc1/mariner transposons in the genome of African coelacanth (Latimeria chalumnae).

Here, we comprehensively analysed the abundance, diversity, and activity of Tc1/mariner transposons in African coelacanth (Latimeria chalumnae). Fifteen Tc1/mariner autonomous transposons were identified and grouped into six clades: DD34E/Tc1, DD34D/mariner, DD35D/Fot, DD31D/pogo, DD30-31D/pogo-like, and DD32-36D/Tigger, belonging to three known families: DD34E/Tc1, DD34D/mariner, and DD×D/pogo (DD35D/Fot, DD31D/pogo, DD30-31D/pogo-like, and DD32-36D/Tigger). Thirty-one miniature inverted-repeat transposable element (MITE) transposons of Tc1/mariner were also identified, and 20 of them display similarity to the identified autonomous transposons. The structural organization of these full Tc1/mariner elements includes a transposase gene flanked by terminal inverted repeats (TIRs) with TA dinucleotides. The transposases contain N-terminal DNA binding domain and a C-terminal catalytic domain characterized by the presence of a conservative D(Asp)DE(Glu)/D triad that is essential for transposase activity. The Tc1/mariner superfamily in coelacanth exhibited very low genome coverage (0.3%), but it experienced an extraordinary difference of proliferation dynamics among the six clades identified; moreover, most of them exhibited a very recent and current proliferation, suggesting that some copies of these transposons are putatively active. Additionally, at least four functional genes derived from Tc1/mariner transposons were found. We provide an up-to-date overview of Tc1/mariner in coelacanth, which may be helpful in determining genome and gene evolution in this living fossil.

Inactivation of antibiotic resistant Escherichia coli and degradation of its resistance genes by glow discharge plasma in an aqueous solution

Emerging contaminants such as antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) are becoming a global environmental problem. In this study, the glow discharge plasma (GDP) was applied for degrading antibiotic resistant Escherichia coli (E. coli) with resistance genes (tetA, tetR, aphA) and transposase gene (tnpA) in 0.9% sterile saline. The results showed that GDP was able to inactivate the antibiotic resistant E. coli and remove the ARGs and reduce the risk of gene transfer. The levels of E. coli determined by 16S rRNA decreased by approximately 4.7 logs with 15 min of discharge treatment. Propidium monoazide - quantitative polymerase chain reaction (PMA-qPCR) tests demonstrated that the cellular structure of 4.8 more logs E. coli was destroyed in 15 min. The reduction of tetA, tetR, aphA, tnpA genes was increased to 5.8, 5.4, 5.3 and 5.5 logs with 30 min discharge treatment, respectively. The removal of ARGs from high salinity wastewater was also investigated. The total abundance of ARGs was reduced by 3.9 logs in 30 min. Scavenging tests indicated that hydroxyl radicals (·OH) was the most probable agents for bacteria inactivation and ARGs degradation. In addition, the active chlorine (Cl· and Cl2) which formed during the discharge may also contribute to the inactivation and degradation.

Novel heavy metal resistance gene clusters are present in the genome of Cupriavidus neocaledonicus STM 6070, a new species of Mimosa pudica microsymbiont isolated from heavy-metal-rich mining site soil

BackgroundCupriavidus strain STM 6070 was isolated from nickel-rich soil collected near Koniambo massif, New Caledonia, using the invasive legume trap host Mimosa pudica. STM 6070 is a heavy metal-tolerant strain that is highly effective at fixing nitrogen with M. pudica. Here we have provided an updated taxonomy for STM 6070 and described salient features of the annotated genome, focusing on heavy metal resistance (HMR) loci and heavy metal efflux (HME) systems.ResultsThe 6,771,773 bp high-quality-draft genome consists of 107 scaffolds containing 6118 protein-coding genes. ANI values show that STM 6070 is a new species of Cupriavidus. The STM 6070 symbiotic region was syntenic with that of the M. pudica-nodulating Cupriavidus taiwanensis LMG 19424T. In contrast to the nickel and zinc sensitivity of C. taiwanensis strains, STM 6070 grew at high Ni2+ and Zn2+ concentrations. The STM 6070 genome contains 55 genes, located in 12 clusters, that encode HMR structural proteins belonging to the RND, MFS, CHR, ARC3, CDF and P-ATPase protein superfamilies. These HMR molecular determinants are putatively involved in arsenic (ars), chromium (chr), cobalt-zinc-cadmium (czc), copper (cop, cup), nickel (nie and nre), and silver and/or copper (sil) resistance. Seven of these HMR clusters were common to symbiotic and non-symbiotic Cupriavidus species, while four clusters were specific to STM 6070, with three of these being associated with insertion sequences. Within the specific STM 6070 HMR clusters, three novel HME-RND systems (nieIC cep nieBA, czcC2B2A2, and hmxB zneAC zneR hmxS) were identified, which constitute new candidate genes for nickel and zinc resistance.ConclusionsSTM 6070 belongs to a new Cupriavidus species, for which we have proposed the name Cupriavidus neocaledonicus sp. nov.. STM6070 harbours a pSym with a high degree of gene conservation to the pSyms of M. pudica-nodulating C. taiwanensis strains, probably as a result of recent horizontal transfer. The presence of specific HMR clusters, associated with transposase genes, suggests that the selection pressure of the New Caledonian ultramafic soils has driven the specific adaptation of STM 6070 to heavy-metal-rich soils via horizontal gene transfer.

Molecular Investigation of the Status of Ticks on Infected Cattle for Coxiella burnetii in India.

In Indian subcontinent, the epidemiological studies on the status of ticks in the transmission of Coxiella burnetii have not been explored comprehensively. The objective of the present study was to investigate the status of ticks for C. burnetii among coxiellosis positive cattle. The present study was carried out in three locations of the northern states of India. A total of 1648 tick samples were collected from the tick infested cattle (n = 146) that were tested positive for coxiellosis by indirect serum-ELISA assay and/or the trans-PCR assay. The tick samples were screened using the trans-PCR assay targeting species-specific IS1111 transposase gene of C. burnetii. The sequencing of PCR products was planned to differentiate C. burnetii and Coxiella-like bacteria (CLB). The collected ticks were identified as Rhipicephalus microplus (n = 1049), Hyalomma anatolicum (n = 416), and Hyalomma spp. (n = 183). On molecular investigation, none of the collected tick samples were found to be positive for the IS1111gene. The findings of the present study ruled out the involvement of ticks in circulation of the pathogen within the cattle population that were screened. However, extensive epidemiological studies are needed to conclusively rule out or establish the role of ticks as a competent vector for C. burnetii transmission in cattle and other hosts.

Phylogenetic relationships and genetic divergence of paralytic shellfish toxin- and cylindrospermopsin- producing Cylindrospermopsis and raphidiopsis

Cylindrospermopsis and Raphidiopsis (C/R group) are closely related species responsible for cyanobacterial blooms worldwide. Paralytic shellfish toxins (PSTs) and cylindrospermopsins (CYNs) have been identified in different C/R group strains. However, the evolutionary relationship between PST- and CYN-producing strains has not been systematically evaluated. In this study, C/R group strains and their toxin biosynthesis genes were evaluated by phylogenetic analysis and sequence comparison. None of the tested strains are able to produce PSTs and CYNs simultaneously. The C/R group strains were clustered into five clades, including two non-toxic, two CYN-producing and one PST-producing clades. A high degree of similarity was observed for rpoC1 (> 96%) and ITS-L (> 97%) sequences within each clade with the exception of the ITS-L (87% to 100%) region in CYN-producing R. curvata, which has been shown to contain variable sequence insertions. Genomic analysis revealed that sxtY and sxtZ could be found in both toxic and non-toxic strains. The transposase gene IS4 was only observed in strains from the PST-producing clade. The sxt and cyr gene clusters share five gene families with similar functions. The amino acid sequences of the adenylyl-sulfate kinase genes, sxtO and cyrN, are more similar (45% to 81%) than other pairs of genes (8.0% to 40%). SxtO and CyrN proteins from C/R group strains forms an independent clade on the phylogenetic tree with a high degree of sequence similarity (78% to 100%). In conclusion, PST- and CYN- producing C/R group species can be classified into different clades based on their phylogenetic profile. The sxtO and cyrN genes have probably diverged from a single ancestral adenylyl-sulfate kinase gene, and may be specifically used for toxin biosynthesis in C/R group species.

MOLECULAR DETECTION AND IDENTIFICATION OF COXIELLA BURNETII IN ABORTED SHEEP AND GOATS IN SULAIMANI PROVINCE, KURDISTAN-IRAQ

Q fever is an almost ubiquitous zoonotic disease with a worldwide distribution, caused by Coxiella burnetii which can infect different types of animal species such as cattle, sheep and goats, as well as humans. Infection with C. burnetii in small ruminants, are mostly sub-clinical, although, abortions and stillbirths can occur. The aim of this study was to provide molecular evidence of C. burnetii in aborted small ruminants in different districts of Sulaimani province, Kurdistan-Iraq. Blood and faecal samples were collected from 180 aborted sheep and goats (90 samples each) and analyzed by conventional Polymerase Chain Reaction (PCR) for DNA detection of transposase gene (IS1111) of C. burnetii from February to June 2019. (Comparison sequence analysis exhibit that field isolates highest identities of (Iraq, Iran and China) strains with the rate of 99.84%). Shedding of C. burnetii by aborted sheep and goats was found only in (5.55%) faecal samples investigated. Only 10 out of 60 faecal samples (16.66%) were positive. Six out of 48 faecal samples (12.5%) and 5 out of 12 faecal samples (41.66%) were positive in aborted sheep and goats respectively. None of the blood samples revealed positive amplification for C. burnetii DNA. This paper documents the first molecular detection of C. burnetii in aborted small ruminants in Sulaimani province Kurdistan-Iraq.

An Avirulent Ralstonia Solanacearum Strain Undergoes Phenotype Conversion from a Pathogenic Strain Under Natural Environment

An avirulent R. solanacearum strain named FJAT-1458 was isolated from living tomato vessel and it showed no toxicity to tomato, pepper and eggplant. Multilocus sequence analysis (MLSA) based on eight genes (egl, hrpB, mutS, pehA, recA, rpoA, rpoB and rpoC) and whole genome average nucleotide identity (ANI) analysis suggested that strain FJAT-1458 belong to phylotype I. Genome sequence of the strain FJAT-1458 revealed a circular chromosome and a circular megaplasmid with whole genome size of 6,059,899 bp and GC content of 66.78%. Functional annotation of FJAT-1458 showed a total of 5,442 genes, with 5,166 protein-encoding genes, 202 pseudogenes and 74 noncoding RNA genes. Among which, 3,938 protein-coding genes can be assigned to 23 COG families, and 1,521 of them had KEGG orthologs. Prophage prediction using PHASTER revealed 12 prophages, including 7 intact, 1 questionable and 4 incomplete prophages. Comparative genome analyses between GMI1000 and FJAT-1458 showed that most of the virulence factors were well conserved and only small portion of them were distinct between them. Two genes, including a methyltransferase and an ISL3 family transposase genes, were identified to be inserted immediately upstream (141 bp) of phcA gene, which assumed to be responsible for avirulence of strain FJAT-1458. It is suggested that strain FJAT-1458 was originated from a wild-type pathogenic strain through an accident phenotype conversion, which is like those when cultured under experimental conditions. Our study provides new insight into the evolution of virulence in R. solanacearum strain under natural environment.