Orientia tsutsugamushi, during feeding. Co-existence of multiple O. tsutsugamushi strains within infected mites has previously been reported in naturally infected, laboratory-reared mite lines using molecular methods to characterize the 56-kDa type-specific antigen (TSA) gene. In the current study, more advanced next-generation sequencing technology was used to reveal the heterogeneity of O. tsutsugamushi genotypes in field-collected trombiculid mites from rodents and small mammals in scrub typhus-endemic areas of Thailand. Twenty-eight trombiculid mites collected from 10 small mammals were positive for O. tsutsugamushi, corresponding to a prevalence rate of 0.7% within the mite population. Twenty-four of the infected mites were Leptotrombidium spp., indicating that this genus is the main vector for O. tsutsugamushi transmission in Thailand. In addition, O. tsutsugamushi was detected in the mite genera Ascoschoengastia, Blankaartia, Gahrliepia, and Lorillatum. Of the 10 infested small animal hosts, six had 2–10 infected mites feeding at the time of collection. Deep sequencing was used to characterize mixed infections , and 5 of the 28 infected mites (17.9%) contained mixed infections. Additionally, 56-kDa TSA gene sequence analysis revealed identical bacterial genotypes among co-feeding mites with single or mixed infections. These results suggest that co-feeding transmission may occur during the feeding process, and could explain the occurrence of mixed infections in individual mites, as well as the recovery of multiple infected mites from the same host. This study also revealed highly diverse within-host O. tsutsugamushi genotypes. The occurrence of multiple O. tsutsugamushi genotypes within individual mites has important implications, and could provide a mechanism for pathogen evolution/diversification in the mite vector.Trombiculid mites are the vectors of scrub typhus, with infected larval mites (chiggers) transmitting the causative agent,  Orientia tsutsugamushi (OT), which is transferred through the bite of an infected mite. Previously, we reported the co-existence of multiple OT strains within laboratory-reared mites. Here, we extended our investigation into wild trombiculid mite populations collected from areas of Thailand where scrub typhus is endemic. More advanced next-generation sequencing (NGS) technology was used to examine the presence and abundance of mixed infection in individual mites based on sequence analysis of the 56-kDa type-specific antigen gene. NGS data revealed heterogeneity of OT genotypes in field-collected mites, and mixed infection with two to three OT genotypes was consistently observed . Moreover, the identical OT genotype detected among co-feeding mites with single or mixed infections suggesting that co-feeding transmission may occur during the feeding process. This result could explain the occurrence of mixed infections in individual mites, as well as the recovery of multiple infected mites from the same host.Scrub typhus is a leading cause of undifferentiated febrile illness, putting 1 billion people at risk of infection. Trombiculid mites are the major vectors of the causative agent of scrub typhus,  Orientia, although O. tsutsugamushi was thought to be the only causative agent until recently. Orientia species have been separated from the genus Rickettsia on the basis of differences in genome arrangement and cell wall structure, as well as the substantial genetic distance between the two genera [Orientia and the presence of the pathogen in locations outside of the previously described endemic region of the Tsutsugamushi triangle have been documented [Orientia species in both the Middle East and South America, with infected patients presenting with symptoms similar to those of scrub typhus [O. tsutsugamushi antigens in these patients. Comparison of the 16S rRNA, 47-kDa HtrA, and 56-kDa type-specific antigen (TSA) gene sequences against the GenBank database showed that the causative agents were most closely related to, but clearly separated from, O. tsutsugamushi. Based on these findings, one of the isolates was proposed as a new species of Orientia, and named Candidatis Orientia chuto [Scrub typhus is caused by intracellular Gram-negative bacteria belonging to the genus o genera , 2. Receo genera , 4. Envio genera –7. In adcumented . Recent b typhus , 10. SerO. tsutsugamushi, especially species belonging to the genus Leptotrombidium, including L. arenicola, L. deliense, L. pallidum, L. fletcheri, L. scutellare, L. chiangraiensis, L. imphalum, and L. akamushi [L. pallidum and L. scutellare are the predominant vector species, and are distributed throughout the islands [L. deliense is the main vector [Blankaartia and Ascoschoengastia; however, O. tsutsugamushi carriage has not been as deeply investigated in these genera [O. tsutsugamushi are widely distributed throughout Asia, northern Australia, and the western Pacific Islands, placing an estimated one billion people at risk of scrub typhus [Currently, there is no vaccine available for scrub typhus infection. The prophylactic treatment with antibiotic as a prevention method is recommended by World Health Organization (WHO) under special circumstances in endemic areas. Other general protective measures include avoiding exposure conditions, wearing appropriate clothing, and using insect and spatial repellents to prevent the chigger bites , 12. Patakamushi –20. In S islands –23, whiln vector , 25. Othe genera , 27. In b typhus .O. tsutsugamushi isolates serotyping was developed utilizing serum raised in laboratory animals to individual orientia isolates. The produced antiserum or antisera reactive to new orientia isolates by IFA indicated how closely related the new isolate was to known isolates used to produce the antisera and consequently the lack of reactivity indicated the new isolate was antigenically distinct from the isolate(s) that produced the sera. Early on three prototypes  strains were identified, Karp, Kato and Gilliam. Subsequently other antigenically distinct isolates were identified. Utilizing the serotyping technique it was found that Karp was the most prevalent O. tsutsugamushi serotype of isolates found in rodents and vectors in Thailand, with other serotypes appearing to be less common [To assess the diversity of s common .O. tsutsugamushi isolates using hyperimmune serum raised against prototype strains of the genotypes  Karp, Gilliam, Kato, and TA716, resulted in cross-reactivity against genetically diverse isolates suggesting that many serotypes existed [O. tsutsugamushi isolates from patients and rodent hosts in many parts of Thailand contrary to the serotyping study just mentioned above and that many of these new isolates were found to have much less similarity to the prototype strains than previously thought using serotyping techniques [With advances in genetic characterization of bacteria the relationship between serotyping and genotyping of orientia isolates was assessed. Early studies showed that serotyped  existed , 31. Intchniques , 32, 33.O. tsutsugamushi is maintained through both transovarial and transstadial transmission processes in the mite population, with these mechanisms thought to be the main ways in which the bacterium is maintained in the wild [O. tsutsugamushi from an infected host to naïve mites, although the bacteria failed to transmit transovarially [O. tsutsugamushi from infected mite(s) to co-feeding naïve mites of the same or different species has been demonstrated in a laboratory setting; however, the study did not confirm whether this type of infection resulted in subsequent transstadial and transovarial transmission [Borrelia burgdorferi and Borrelia afzelii, can be transmitted from infected nymphal Ixodes ricinus L. ticks to co-fed uninfected larval ticks [Rickettsia has been observed among ticks, with uninfected Rhipicephalus sanguineus ticks being much more likely to acquire a R. conorii infection when feeding in close proximity to infected ticks, compared to feeding on an infected animal host [Rickettsia felis from infected cat fleas  to naïve cat fleas or rat fleas (Xenopsylla cheopis) on a vertebrate host, and this mode of transmission could be crucial for the maintenance of R. felis within the vector population [O. tsutsugamishi has not been well documented, this mode of transmission might play an important role in maintaining O. tsutsugamushi in nature.the wild , 34–36. varially –39. Moresmission . In ticksmission –44. Thisal ticks , 46. Altmal host . A recenpulation . AlthougO. tsutsugamushi genotypes in single laboratory-reared mite lines [Leptotrombidium mites (L. chiangraiensis (Lc), L. imphalum (Li), and L. deliense (Ld)) were studied. An O. tsutsugamushi Karp-like genotype was found as a single genotype in the L. chiangraiensis line, but as part of a co-infection with an UT302-like genotype in L. imphalum lines or with a Gilliam-like genotype in the L. deliense line. Moreover, the co-existing genotypes were well maintained through transovarial and transstadial transmission in L. imphalum mites [O. tsutsugamushi sequence types was observed in 25% of patients in a study conducted by Sonthayanon et al. [O. tsutsugamushi isolates recovered from patients [O. tsutsugamushi shows a high level of plasticity, with 50% of the genome containing repetitive sequences derived from integrative and conjugative elements. Amongst these, several hundred transposases, phage integrases, and transposable elements have been identified, as well as a massive duplication of components of the conjugative type IV secretion system (359 tra genes), which is distributed throughout the genome [O. tsutsugamushi genomic diversity and complexity. As O. tsutsugamushi is an obligate intracellular parasite, recombination would occur during coinfection of different bacterial strains within the same mammalian host or vector, resulting in the exchange of genetic material between the bacterial strains. The co-existence of O. tsutsugamushi strains of different genotypes in a single trombiculid mite is confirmation that these mechanisms are likely to contribute to the genomic diversity/complexity of the bacterial pathogen.Our previous study showed the co-existence of two te lines . Twelve um mites . Co-infen et al. . The stupatients –51. Indee genome , 53. TheO. tsutsugamushi genotypes in individual field-collected mites in high and low scrub typhus-endemic areas of Thailand. Next generation sequencing (deep sequencing) of the 56-kDa TSA gene was used to accurately determine the presence and abundance of each O. tsutsugamushi genotype in individual infected mites. Additionally, the evidence for horizontal transmission of O. tsutsugamushi between co-feeding mites was discussed based on the study data. The results presented in this study represent an epidemiological assessment of O. tsutsugamushi carriage in a wild vector population in an area with active scrub typhus transmission.In this study, we aimed to examine the prevalence of mixed infection of Rattus rattus sensu stricto, is not found in Thailand. However, three morphologically similar species are present; Rattus tanezumi, Rattus sakeratensis, and an additional mitochondrial lineage of unclear taxonomic status referred to as ‘Rattus R3’ [Rattus rattus complex. Meanwhile, all other members of the genus Rattus are identified to species.Rodents and mites were collected during the wet season (Jun–Aug) in the northeastern (Sisaket and Loei provinces), western (Tak Province), and southern (Pang Nga and Chumphon provinces) regions of Thailand in 2015 . All stuttus R3’ . These tGenomic DNA was extracted from rodent spleen samples using a Wizard Genomic DNA Purification Kit  according to the manufacturer’s instructions, with some modifications as per a previously published protocol . BrieflyO. tsutsugamushi quantitative polymerase chain reaction (qPCR) screening assays. Chigger cytochrome oxidase subunit 1 (host COI gene) was examined by conventional PCR on all mite samples before being subjected to Otsu47 qPCR assay as describe previously [Mites in their larval stage (chigger) were collected from rodent ears by paintbrush under the stereomicroscope and stored separately. Each mite was individually identified to the genus level under a high resolution microscope  using a taxonomic key . Mites weviously .O. tsutsugamushi by qPCR analysis. The primers and probe were designed to detect a portion of the 47-kDa HtrA gene [Field-collected mites and animal tissue (spleen) samples were screened for the presence of trA gene . The reaO. tsutsugamushi was then used as the basis for amplification and next-generation sequencing (NGS) of the 56-kDa TSA gene from O. tsutsugamushi. A fragment of the 56-kDa TSA gene  was amplified in a first-round PCR using primers RTS-8 and RTS-9 [2, 1× PCR buffer, and 0.4 U of iProof High-Fidelity DNA Polymerase . Amplification was performed using a DNA thermal cycler under the following conditions: initial denaturation at 98°C for 2 min; 30 cycles of 98°C for 10 s, 45°C for 20 s, and 72°C for 45s; and a final extension at 72°C for 10 min. The second amplification was performed using fusion primers for NGS. The fusion primers comprised three parts: (i) 21-bp A and B sequencing adapters plus 4 bp of key sequence (bold letters) , (ii) 10-bp multiplex identifier (MID) sequences (with a unique MID for each sample), and (iii) 56-kDa TSA gene-specific sequences RTS-6 and RTS-7 [2, 200 μM dNTPs, 0.3 μM each primer, and 0.5 U of iProof High-Fidelity DNA Polymerase. Five microliter of diluted product from the first amplification (1:50) was used as template. The thermal cycler parameters consisted of 98°C for 2 min, followed by 30 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 30 s. The resulting amplicons were purified using AMPure beads , and amplicon concentrations were measured using the Quant-iT PicoGreen dsDNA Assay (Invitrogen) according to the manufacturer’s protocol. Each PCR amplicon was then diluted to 109 copies/μl and pooled (10–15 amplicons per pool). Sample pools were amplified by emulsion PCR using a GS Junior+ emPCR Kit (Lib-A) with a ratio of 0.5 copies per bead. Amplicons were then sequenced in both forward and reverse directions at 5000× coverage per sample on a 454 GS Junior Genome Sequencer Instrument using the GS Junior+ Sequencing Kit XL+ chemistry . The average read length obtained from the NGS runs was 633 nucleotides, with a median number of reads per sample of 10,198, .Trombiculid mites or animal tissue found to be positive for the presence of O. tsutsugamushi genotype, the number of reads aligned to each reference had to be at least 5% of the total reads. The extracted reads per reference were assembled using de novo assembly, and the consensus sequence was extracted. The GenBank accession numbers for consensus sequences reported in this study were provided at the end of this article. The consensus sequences were aligned with reference sequences retrieved from the GenBank database using the MUSCLE codon alignment algorithm [Raw reads were quality filtered and de-multiplexed with standard quality filtering parameters using CLC Genomics Workbench v 9.0 software (Qiagen). After quality trimming, reads < 500 bp and > 800 bp in length were discarded. All trimmed reads were then de-multiplexed using the MID barcodes. All reads from each sample were mapped to a full reference set  using thlgorithm . A maximlgorithm  S1 Fig)O. tsutsuEscherichia coli DH5a-T1R as per the manufacturer’s instructions (Invitrogen). Transformants were randomly selected and screened by nested PCR. Between 6 and 28 clones from individual chiggers appeared to contain the correct 56-kDa TSA gene insert fragment. Plasmids containing the correct 56-kDa TSA gene insert were then purified from the E. coli host using a QIAprep Spin Miniprep Kit (Qiagen), and sent for DNA sequencing (Sanger method) by AITBiotech (Singapore).The 56-kDa TSA gene was amplified by nested polymerase chain reaction using our previously designed primer  for the Gplots and applying complete linkage clustering and Euclidean distances computed among 36 genotypes from 28 infected mites [All statistical analyses (Chi-square tests) and graphical illustrations presented in this study were performed in the R environment for statistical computing –66. A nued mites .Erinaceomorpha, Soricomorpha, Scandentia, Macroscelidea, and Rodentia) Populations to Support Zoonotic Diseases Surveillance and Ectoparasite Collection” (PN# 12–06), reviewed and approved by the USAMC-AFRIMS Institutional Animal Care and Use Committee (IACUC). All sampling procedures and experimental manipulations were reviewed and approved as part of obtaining the animal collection protocol (PN# 12–06). Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals, and adhered to principles outlined in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition.Rodents were trapped according to the institutional animal collection protocol titled “Field Sampling of Small Mammal (Orders: http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the 56-kDa TSA gene described in this paper are: MH290189 (Lep.DS092.9c), MH290190 (Lep.DS092.8c), MH290191 (Lep.DS021.b), MH290192 (Lep.DS016.b), MH290193 (Lep.DS092.6b), MH290194 (Lep.DS020.2a), MH290195 (Lep.DS092.8a), MH290196 (Lep.DS021.1h), MH290197 (Lep.DS016.e), MH290198 (Lep.DS092.8e), MH290199 (Lep.DS092.9e), MH290200 (Lep.DS092.10e), MH290201 (Lep.DS123.1e), MH290202 (Lep.DS078.d), MH290203 (Lep.DS021.3f), MH290204 (Lep.DS092.7g), MH290205 (Lep.DS092.5g), MH290206 (Lep.DS092.4g), MH290207 (Lep.DS092.2g), MH290208 (Lep.DS092.1g), MH290209 (Lep.DS020.1g), MH290210 (Lep.DS021.4g), MH290211 (Lep.DS123.2g), MH290212 (Lep.DS027.g), MH290213 (Lep.DS030.g), MH290214 (Asc.MS651.g), MH290215 (Lep.DS024.b), MH290216 (Bla.DS123.a), MH290217 (Lep.DS092.3e), MH290218 (Bla.DS123.e), MH290219 (Lep.DS024.d), MH290220 (Lep.DS024.g), MH290221 (Bla.DS123.g), MH290222 (Gah.DS024.g), MH290223 (Lep.DS021.2g), and MH290224 (Lor.MS651.g).The GenBank (O. tsutsugamushi in Chumphon and Pang Nga provinces (southern Thailand), while low prevalence rates were observed in Sisaket and Loei (northeastern Thailand) and Tak (western Thailand) provinces. Therefore, we decided to survey both high and low prevalence areas for comparison. Small mammals and rodents were collected during the rainy season (Jun–Aug) of 2015. In total, 275 small mammals and rodents were collected from five provinces in three regions of Thailand (R. rattus complex (n = 113), Bandicota indica , and Mus caroli  were the most abundant species sampled in this study, accounting for 41.1%, 19.6%, and 15.3% of total rodents collected, respectively. R. rattus complex were the most abundant species in all provinces (34.5–65.3%) except Tak Province, in which M. caroli (46.2%) was the most abundant species collected.Collection sites were selected based on our annual surveillance data (2012–2016) on scrub typhus prevalence in small mammals and trombiculid mites in Thailand . The datThailand . The colThailand . The greR. rattus complex , B. indica , and Tupaia glis . Overall, mite infestation rates among animals collected from each of the provinces ranged from 33–86.7%. Of the infested animals, those from Sisaket, Chumphon, and Pang Nga provinces were heavily infested, with a chigger index  ranging from 19.1–21.4 (Leptotrombidium (49.1%), followed by Gahrliepia (24.4%) and Ascoschoengastia (20.4%). Overall, a significantly greater proportion of Leptotrombidium mites were collected relative to other mite genera , with the abundance of Leptotrombidium spp. clearly observed in Pang Nga and Chumphon provinces (southern Thailand) , B. indica , and T. glis , with mean chigger index scores of 25.1 , 18.3 (987/54), and 12.2 (223/13), respectively . The majority of animals were infested with mites belonging to one  or two  different genera, while only 27 (15.3%) and two (1.1%) animals were infested with three or four different mite genera, respectively, with R. rattus complex accounting for the majority of these animals   small mammals and rodents examined in this study . The inf9.1–21.4 . Howeverhailand) . The greectively . However, 65.5%) .O. tsutsugamushi in the animal populations was very low . The three infected rodents were identified as R. rattus complex (n = 2) and B. indica (n = 1), and were collected from Pang Nga Province (southern Thailand)  . HoweverO. tsutsugamushi using the Otsu47 qPCR assay revealed positive results for 28 mites . Twenty-four of the 28 infected mites were Leptotrombidium spp. (85.7%), while the remaining four mites were Ascoschoengastia spp., Lorillatum spp., Gahrliepia spp., and Blankaartia spp. . However, tissue samples from all 10 animals were negative for O. tsutsugamushi infection using the same assay. The overall infection rate among mites collected from these 10 animals was 8.4% (28/333), whereas the O. tsutsugamushi prevalence among mites per host varied from 2.2–21.3%. Prevalence rates for infection among the same mite species per host ranged from 0–21.3% , with only five mites  showing mixed infection with 2–3 different O. tsutsugamushi genotypes. The most prevalent O. tsutsugamushi genotype found in the mites was TA763 B (44.4%), followed by Kato B (19.4%)  . The majO. tsutsugamushi genotype infections (n = 5) were identified in animals from Pang Nga Province. Four infected mites were Leptotrombidium spp., and one was Blankaartia spp. Two Leptotrombidium mites had a mixed infection with two O. tsutsugamushi genotypes, whereas the rest of the mites (two Leptotrombidium spp. and one Blankaartia spp.) showed mixed infection with three genotypes. The genotype composition and the relative abundance of each of the genotypes were determined by the number of NGS reads, or by the number of clones for those sequences amplified using the cloning technique . However, when a host was infested with several mite genera, the number of infected mites proportionately reflected the relative abundance of each of the mite genera. For example, 90 mites were collected from R. rattus complex individual MS0651, and belonged to three different genera: 71 Ascoschoengastia spp., 16 Lorillatum spp., and 3 Gahrliepia spp. Of these 90 mites, only three were infected with O. tsutsugamushi, two of which were Ascoschoengastia spp. and one was Lorillatum spp. No infection was detected in Gahrliepia spp. mites, which were present on the host in the lowest numbers. The same situation occurred with mites collected from R. rattus complex individual DS024 (n = 19), which showed an almost equal prevalence of Leptotrombidium spp. (n = 10) and Gahrliepia spp. (n = 9). In this case, one infected mite was identified from each genus. Also, among mites collected from B. indica individual DS123 (n = 36), two of the infected mites were Leptotrombidium spp. (n = 26), while the remaining infected mite was Blankaartia spp. (n = 10).Of the 10 small mammal and rodent hosts infested with llection . Three oO. tsutsugamushi genotypes detected in mites collected from the same animal host , each of which carried the TA763 B genotype. An interesting case was observed in a B. indica rat  had the Kato B and TA763 B genotypes, respectively, while the third infected mite  contained both genotypes. A similar situation was observed in another B. indica rat  infested with 10 infected Leptotrombidium mites. Eight mites had a single infection, while two had mixed infection. Among the mites with a single infection, five shared the same TA763 B genotype and two had the Kato B genotype. The latter genotype was also detected in two infected mites with mixed infection, and both of these mites also shared the Karp C genotype. In contrast, in a common tree shrew  shared the same genotype (TA763 B). Likewise, two infected Leptotrombidum spp. infested on another common tree shrew . The heatmap shown in O. tsutsugamushi 56-kDa TSA gene genotypes detected in 28 infected mites. Alignment and identity analysis of the 56-kDa TSA gene sequences belonging to the same genotypes revealed a high level of similarity for each of the genotypes, with percent identities ranging from 98–100%, except for the Kato A genotype, where only 89% identity was observed.A network graph was generated to determine the relationships between mal host . From hoame host . For exaO. tsutsugamushi genotypes in field-collected trombiculid mites using NGS based on the 56-kDa TSA gene (variable domains I–III). These findings build on our previous research showing that different O. tsutsugamushi strains can co-exist in laboratory-reared Leptotrombidium mites maintained in an ABSL-3 facility [O. tsutsugamushi genotypes were detected among co-feeding trombiculid mites, which may indicate co-feeding transmission. Co-feeding transmission of O. tsutsugamushi occurs among laboratory-reared infected and uninfected mites (Leptotrombidium spp. and Blankaartia spp.) that had co-fed on ICR mice [O. tsutsugamushi genotypes. This conclusion is supported by two main observations. First, a single O. tsutsugamushi genotype was detected in multiple mites belonging to different genera. Second, one O. tsutsugamushi genotype was detected in a mite with mixed infection, with the same genotype also found in a co-feeding mite with single infection  were found on hosts infested with more than two infected mites. Thus, a higher number of infected mites per host increases the likelihood of an uninfected mite acquiring O. tsutsugamushi during co-feeding [This study is the first to provide evidence of the co-existence of multiple facility , 36. Co-ICR mice . The curnfection ; Fig 2A.-feeding .T. glis) were predominantly found on the ventral and genital areas, with few mites in the inner earlobe. This is probably because the skin in the inner ear of shrews is very thick compared to rodents, and is not conducive to chigger feeing. The observation that mites were more widely dispersed on shrews and did not feed in tight clusters within the inner earlobe could be one possible hypothesis to explain why infected mites on shrews did not have identical O. tsutsugamushi genotypes, and showed no evidence of co-feeding transmission.Among the rodents collected in the current study, mites were mostly collected from the inner earlobe, and some hosts were heavily infested (as many as 181 mites/host). Only a small number of mites were found on the soft tissues of the ventral and genital areas. From previous field experience, clusters of mites are rarely found on the ventral surfaces of rodents, possibly because rodents are able to groom these areas; whereas, they cannot groom inside their ears and clusters of mites are able to form. In contrast, mites on the common tree shrew  feeding in the close proximity on the same host. This could explain the finding of an identical O. tsutsugamushi genotype in Gahrliepia spp. and Leptotrombidium spp. mites on R. rattus complex individual DS024, and in a Blankaartia spp. mite on B. indica individual DS123. However, the Blankaartia mite collected from DS123 also carried the Gilliam genotype, which was not detected in its co-feeding Leptotrombidium mites. So, the question remains of how the Blankaartia mite acquired the Gilliam genotype. A similar observation was made among Ascoschoengastia and Lorillatum mites co-feeding on R. rattus complex MS651, where both mites carried TA763 B genotype bacteria. However, because all of these conclusions are based solely on data from field-collected mites, where transmission could not be monitored, other unknown factors could have influenced the results. For example, Gilliam genotype detected in Blankaartia mite infested on host DS123 may have been present in the mite prior to attachment to this host . Further study is therefore needed to investigate whether other genera are capable of transmitting O. tsutsugamushi in nature, and to confirm whether co-feeding transmission results in mixed infection of O. tsutsugamushi genotypes. Such co-feeding transmission studies can be conducted using laboratory-reared infected mites and animal models, allowing for more controlled experimentation.In the high-prevalence area (Pang Nga Province), smission , 70–73. Thailand . Howevertellaris . The autl et al. , might bl et al. . HoweverLeptotrombidium mites or a higher chigger index does not necessarily directly correspond to an increased prevalence of O. tsutsugamushi in mite populations. This was the case in Chumphon Province, which had the second highest abundance of mites, and where the majority of mites were Leptotrombidium spp. However, no infected mites were detected in this area. In addition, two infected mites (Ascoschoengastia spp. and Lorillatum spp.) were collected from a R. rattus complex in Sisaket Province; however, no Leptotrombidium spp. mites were collected from animals captured in this area. Based on our study data, Pang Nga Province could be considered a hotspot for scrub typhus transmission within mite populations, as multiple infected mites were usually found on each animal host. Interestingly, during the study period, 211 human cases of scrub typhus were reported in Pang Nga Province by the Thai Ministry of Public Health. The current study also revealed highly diverse within-host O. tsutsugamushi genotypes, with as many as three different O. tsutsugamushi genotypes found in individual mites. The ecological dynamics of multiple O. tsutsugamushi genotypes in one host could drive pathogen evolution/diversification in the vector. This may be consistent with the hypothesis that genetic recombination among different O. tsutsugamushi genotypes can occur in the mite vector [It is worth noting that an abundance of e vector .S1 FigA maximum likelihood tree was constructed using the GTR+G model of nucleotide substitution in the MEGA 6 program with bootstrapping (1000 replicates). Percent abundance of each genotype detected in individual mites is indicated in parenthesis after each sequence.(TIF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 TableAnalysis was performed using CLC Genomics Workbench.(PDF)Click here for additional data file.S2 TableA nucleotide distance matrix was generated using “DNADist DNA Distance Matrix” in BioEdit.(PDF)Click here for additional data file.


Helicobacter pylori (H. pylori) are closely associated with the pathogenicity of bacteria. However, the significance of H. pylori babA, homB, aspA, and sabA genes is not clear in phenotypic characteristics of virulence. This study aimed to investigate the frequency and importance of these genes in patients with H. pylori positive peptic ulcer (PU). The cag A and vac A genes of H pylori were characterized by urease, catalase, oxidase test, and gram staining. Genomic DNA was extracted and stored. The babA, homB, aspA, and sabA genes were determined by using polymerase chain reaction analysis. Patients with a PU or nonulcer dyspepsia (NUD) based on the upper gastrointestinal (UGI) endoscopy findings were included in the study. Biopsy samples from antrum and corpus were cultured into Columbia agar. babA , homB , aspA , and sabA  genotypes was not different between PU and NUD patients. There were some correlations between the presences of these genes. A total 214 patients were included (99 PU and 115 NUD) and H. pylori could be isolated in 82 patients (36 PU and 46 NUD). The frequency of the babA, homB, aspA, and sabA genes of H. pylori by PCR. However, the frequency of these factors was not different in patients with PU and NUD. There is no role of babA, homB, aspA, and sabA genes for the development of peptic ulcer in Turkish population. This study managed to determine H. pylori) is a gram-negative, spiral-shaped, 4-6-flagellated mobile bacterium that grows in the digestive tract and microaerophilic environment at 37°C in culture. The coccoid form of H. pylori is also called sleeping form. H. pylori produces urease that catalyzes the hydrolysis of urea to yield ammonia and carbonic acid. Flagella, urease, and adhesins are all essential factors for H. pylori to colonize the gastric mucosa [Helicobacter pylori  and the sialic acid-binding adhesion (sabA) are considered to have a significant function on initial colonization of H. pylori [. pylori , 8.H. pylori outer-membrane proteins (hom) family is a small protein family including the C-terminal hydrophobic motif and signal sequences of outer membrane proteins. The hom family is one of the outer-membranes coding gene family that is divided into two families: homA and homB which are 90% identical; the difference is related to central domain [ H. pylori have reported that babA promotes attachment of H. pylori to the gastric epithelial cells. The babA facilitates entry of cagA and vacA virulence factors into host cells [Recent studies on adherence features ofst cells , 11. sabA first identified in the babA-mutant H. pylori strain [ sabA binds to sialylated carbohydrates on the surface of neutrophils. From this perspective, sabA is thought to promote immune response [The second adhesion isi strain . The sabresponse . H. pylori babA, homB, aspA, and sabA genes and to identify the rate of these virulence genes in the biopsy samples by PCR analysis.The aim of this study was to determineA total of 214 patients were included in this study: 115 nonulcer dyspepsia and 99 peptic ulcer. The patients were from south east part of Turkey undergoing upper gastrointestinal endoscopy at the endoscopy unit of the Department of Gastroenterology, University of Gaziantep. During endoscopy, biopsy samples were taken and the obtained tissues were placed into 0.8% serum physiologic solution and then cultured immediately. Informed consent was taken from all patients and The Ethics Committee of Medical School of University of Gaziantep approved the study. Results were confirmed both clinically and microbiologically. H. pylori selective supplement  to eliminate another bacterial contamination, and then incubated under anaerobic conditions, 5% CO2 at 37°C for 4-6 days.In order to prevent contamination, aseptic conditions were provided. The obtained tissues were immediately placed into a liquid 0.8% serum physiologic solution and inoculated into Columbia agar with 5% sheep blood , containing H. pylori, catalase , urease , and oxidase  tests were performed and also H. pylori morphology was identified.To prove existence of H. pylori under the light microscope, gram staining method was performed. Crystal violet  was applied to heat-fixed smear of bacterial culture. Lugol  that binds crystal violet was added. To decolorize it, ethanol  was added and then stained with safranin .To observeGenomic DNA was extracted from histopathologically confirmed cases of nonulcer dyspepsia and peptic ulcer using Qiagen DNA isolation kit Qiagen, QIAmp DNA Mini Kit  according to manufacturer's instructions. The DNA was stored at -20°C until used for molecular studies.μl master mixture that contained 100 ng of genomic DNA, 10 pmole each of primers, 10X buffer, 2 mM each of nucleotides , and 0.5 units of Taq Polymerase. PCR annealing temperatures for primers of aspA, babA, cagA, homB, sabA, and vacA were 59°C, 55°C, 58°C, 58°C, 56°C, and 55°C, respectively. PCR products were then electrophoresed for 45 min at 130 Volt on 1% agarose gel in the presence of 0.5g/mL of ethidium bromide  and illuminated under UV light .Touchdown PCR protocols were performed using Dream Taq DNA Polymerase  kit. PCR amplifications were performed on 50  p values <0.05 were considered significant.Comparisons of variables were performed with the chi-square test, One-way ANOVA test, and Tukey's Multiple Comparison Test (GraphPad Prism 5) to compare the differences among nonulcer dyspepsia and peptic ulcer patients. H. pylori by urease, catalase, and oxidase tests. As a result of these tests, H. pylori were detected in 82 patients (38.32%), whereas bacteria could not be detected in 132 patients (61.68%). All H. pylori-positive patients (82) were further analyzed for the presence of H. pylori virulence factors by PCR using babA, homB, aspA, and sabA-specific primers encoding  and 36 peptic ulcer ) were enrolled in this study (p<0.001). However, there were no significant differences in mean age (p>0.05).A total of 82is study . The mea(i) Blood Group Antigen-Binding Adhesin, babA. The babA gene of H. pylori was determined in 16 patients (19.51%), whereas 66 patients (80.49%) were classified as babA-negative  were from nonulcer dyspepsia patients and 9 of them (56.25%) were from peptic ulcer patients  and peptic ulcer (p < 0.001) (negative . Out of patients . The pre< 0.001)   .(ii) Helicobacter Outer Membrane Family Member, homB. The 1005-bp PCR product indicating the presence of homB gene was detected in 3 patients (3.66%), whereas 79 patients (96.34%) were negative for homB gene  were from nonulcer dyspepsia patient and 1 isolate (33.3%) was from patient diagnosed with peptic ulcer disease. The presence of homB was associated with the presence of aspA (p <0.001) and babA (p <0.05)  and peptic ulcer patients (p < 0.001) (omB gene . Out of p <0.05) . A stati< 0.001)   .(iii) Aspartate Ammonia-Lyase, aspA. Fifty-nine biopsies that were obtained from different patients (71.94%) were positive for the aspA gene, with the remaining 23 (28.04%) being aspA-negative as a result of the 1401-bp PCR product  were from nonulcer dyspepsia patients, and 25 isolates (42.38%) were from patients diagnosed with peptic ulcer disease. The frequency of aspA (71.95%) (p<0.0001) was significantly higher compared to the frequency of babA (19.51%), homB (3.65%), and sabA (7.31%) in nonulcer dyspepsia and peptic ulcer patients. The presence of aspA was associated with the presence of babA, homB, and sabA (p < 0.0001)  and peptic ulcer (p < 0.001) ( product . Out of  0.0001) . There w< 0.001)   .(iv) Sialic Acid-Binding Adhesin, SabA. The 187-bp PCR product indicating the presence of sabA gene of H. pylori was determined in 6 patients (7.31%), whereas 76 patients (92.69%) were classified as sabA-negative  were from nonulcer dyspepsia patients and 1 of them (16.66%) was from peptic ulcer patient. The presence of sabA was just associated with the presence of aspA (p < 0.001). Furthermore, the presence of sabA gene had significant effect on nonulcer dyspepsia (p < 0.001) and peptic ulcer (p < 0.001) (negative . Out of < 0.001)   .Possible combinations of all of these virulence factors were determined in Turkish population Figures .H. pylori is a gram-negative bacillus which causes gastritis, peptic ulcer, and gastric cancer [ H. pylori depends on geographic regions, age, social and economic status, occupation, and living environment [ H. pylori have genetically diverse strains, and the strains differ in virulence [c cancer . The preironment , 13. H. irulence . aspA, babA, homB, and sabA genes in H. pylori isolated from patients suffering from gastroduodenal diseases in Turkey determined using PCR analysis and the relationship between these virulence factors was assessed.In this study, the distribution of babA-positive H. pylori and gastric inflammation in humans. Furthermore, the babA-positive H. pylori increased risk of peptic ulcer and gastric cancer in humans [Studies have reported that there is a relationship betweenn humans , 16. babA gene has been detected on the outer membrane of the H. pylori strain. It has been shown that the babA is able to induce DNA double-strand breaks (DSBs) in the cells, but DSBs are the strictest type of DNA destruction and can cause chromosomal aberrations, such as deletions, insertions, and translocations resulting in loss of heterozygosity which are hallmarks of gastric cancer [Thec cancer .H. pylori strains that were isolated from East Asia expressed babA gene but H. pylori strains from 24 western countries did not express babA gene. These bacterial strains caused mild gastric problem. A meta-analysis review revealed that the existence of babA is correlated with high risk of peptic ulcer (OR = 2.069), especially the duodenal ulcer (OR = 1.588). This type of association was observed only in Western countries and not in Asian countries [ountries . H. pylori infection, the babA expression disappeared in the experimental animals [During the first 2-12 weeks during the experimental animals , 20.H. pylori that was isolated from patient samples showed incredible variety at the babA locus, which can translate distinct adhesin that binds only blood group  [ B/BLeb) . babA expression is a dynamic process. The vigorous and variety nature of host glycosylation adds extra complexity. It has been shown that loss of babA expression associated with gender. For this conclusion, the mice model has been used [ oorA, scoD, aroQ, fld A, and aspA of H. pylori proteins are thought to hypothetically interact. It is deduced that these proteins also play a role in oxidation reduction [We have also observed the same results as documented in literatures. Theeen used . The ooreduction .aroQ, aspA, fldA, icd, OorA, and scoB) and high acid environment adaptation proteins (katA and napa) in H. pylori has been shown to be high [ amiE and amiF and aspA. These enzymes can help neutralize the protons entering under acidic environmental conditions by producing intrabacterial ammonia. This study revealed that aspA is taking part in intrabacterial ammonia production [Significant increase in protein with antioxidant activity ( be high . It has oduction .cagA) and vacuolating cytotoxin (vacA) are H. pylori virulence factors and associated with gastric ulcer, gastric cancer [ alpA, alpB, babA, homB, hopZ, oipA, and sabA are all correlated with variable disease outcomes [ cagA and vacA are polymorphic genes. The outer membrane proteins families are strictly correlated paralogs. For instance, the bab- genes family consists of three paralogs babA, babB, and babC. These paralog H. pylori genes can be located at three different chromosomal loci [The cytotoxin-associated gene  and babA (p< 0.05) gene was detected in this study. On the other hand, there was no significant relationship between homB and sabA gene according to this study [During infection and an increase ofis study . hom gene family is a small paralogous protein. The homB and homA genes are almost the same which are 90% [ homB was observed more often than homA in East Asia. The homB was correlated with an enlarged risk of peptic ulcer disease in East Asia. The homB has a role in proinflammation. The homB was important for bacterial attachment to host cell surface [The are 90% . The hom H. pylori to bind carbohydrates. The sabA has vital and important role in the primary colonization of H. pylori. H. pylori sabA proteins are also taking part in stable infections and development of chronic inflammation which directs to tissue damage [There are many different adhesion components existing one damage .H. pylori sabA is a diversity gene. The sabA gene has been associated with different stomach diseases such as 100% in gastric cancer, 86.7% in gastric ulcer, and 83.3% in gastritis and duodenal ulcer [al ulcer . H. pylori gene sequences might differ markedly from other regions or other countries. These differences are detectable by PCR analysis and sequencing. These data suggest that virulence factor variants may present new markers for other factors involved in gastric carcinogenesis or probably influencing the result of H. pylori infection [In conclusion, virulence factors ofnfection .


Adelphocoris suturalis, A. fasciaticollis, A. lineolatus) and one zoophytophagous mirid bug (Nesidiocoris tenuis) to detect whether the duplication, molecular evolution, and expression levels of PGs were related to host range expansion in mirid bugs.Horizontal gene transfer and gene duplication are two major mechanisms contributing to the evolutionary adaptation of organisms. Previously, polygalacturonase genes (PGs) were independently horizontally transferred and underwent multiple duplications in insects . Here, we chose three phytozoophagous mirid bugs  in mirid bugs. Most PGs (> 17) were highly expressed in the three phytozoophagous mirid bugs, while only one PG was relatively highly expressed in the zoophytophagous mirid bug. Natural selection analysis clearly showed that a significant relaxation of selection pressure acted on the PGs in zoophytophagous mirid bugs  rather than in phytozoophagous mirid bugs , suggesting a function constraint of PGs in phytozoophagous mirid bugs.By RNA-seq, we reported 30, 20, 19 and 8 PGs in Taken together with gene duplication, molecular evolution, and expression levels, our results suggest that PGs are more strictly required by phytozoophagous than by zoophytophagous mirid bugs and that the duplication of PGs is associated with the expansion of host plant ranges in mirid bugs.The online version of this article (10.1186/s12862-019-1351-1) contains supplementary material, which is available to authorized users. Horizontal gene transfer (HGT) and gene duplication are two major mechanisms contributing to the evolutionary adaptation of organisms –6. HGT rLygus spp.) [As one of a group of plant cell wall-degrading enzymes (PCWDEs), polygalacturonase (PG) is ubiquitous in fungi, bacteria, and plants, catalyzing hydrolysis of α-1, 4-glycosidic linkages in polygalacturonic (pectic) acid . Interesus spp.) –20. Subsus spp.) , 16, 21.us spp.) . Thus, tus spp.) , 15.Apolygus lucorum [A. lucorum, Adelphocoris suturalis, A. fasciaticollis and A. lineolatus [Nesidiocoris tenuis) [A. suturalis, A. fasciaticollis, A. lineolatus and N. tenuis by RNA-seq. We also investigated the expression levels of PG genes in A. suturalis and N. tenuis. Our results indicate that the phytozoophagous mirid bugs possessed more PG genes and higher expressed levels of these genes than did the one of zoophytophagous species examined, suggesting that the gene duplication of horizontally transferred PG genes may have been part of what allowed the host range expansion toward mixed phytozoophagy in mirid bugs.Previously, we demonstrated the duplication of PG genes in  lucorum . Accordi lucorum , 24. Thuneolatus , 26) tha tenuis) . Here, wWith permission, we captured the insects in experiment stations of Chinese Academy of Agricultural Sciences. No permits were required for the described insect collection and experimentation.A. suturalis, A. fasciaticollis, and A. lineolatus were collected from a cotton field at the Langfang Experimental Station of the Chinese Academy of Agricultural Sciences  in 2015. Adults of N. tenuis were collected from a tobacco field at the Jimo Experimental Station of the Chinese Academy of Agricultural Sciences  in 2014. Field-collected insects were used for genetic analyses.Adults of A. suturalis = 3 groups, A. fasciaticollis = 1 group, A. lineolatus = 1 group, and N. tenuis = 3 groups) were used to isolate total RNA with Trizol reagent  following the manufacturer’s instructions. The cDNA library was constructed, sequenced and analyzed as described by Xu et al. [blastx BLAST based on the databases of Nr (NCBI non-redundant protein sequences) (https://www.ncbi.nlm.nih.gov/genbank/ and https://www.ncbi.nlm.nih.gov/protein/), String  (https://string-db.org/), Swissprot  (http://www.ebi.ac.uk/uniprot/) and KEGG (Kyoto Encycloedia of Genes and Genomes) (https://www.genome.jp/kegg/) for functional annotation. The e-value cut-off was set at 1e-5 for further analysis.Compared with nymphs, adults of mirids can feed on a wider diversity of plant species because their ability to fly allows them to move from species to species as plants flower. Therefore, we chose adults to investigate the PG genes in this study. Fifty adults for each group , Joint Genome Institute (JGI) and GenBank at National Center for Biotechnology Information (NCBI)  and 14 cous work . Then, BA. lucorum and Lygus lineolaris from Genbank and then constructed a dataset containing 147 PG genes for subsequent evolutionary analysis. The sequence alignment was performed using the codon model as implemented in PRANK [Beside our self-sequenced PG genes, we also downloaded PG genes of in PRANK . Phylogein PRANK  with 100in PRANK .dn/ds). To investigate the drive force behind the shift toward plant feeding, we searched for potential selections acting on PG genes in mirids. Here we mainly focus on the transformation between zoophytophagous (N. tenuis) and phytozoophagous (the other mirids). We use an ML approach [dn/ds ratio for all branches ; (2) dn/ds ratio = 1 for all branches ; (3) zoophytophagous and phytozoophagous lineages have a different dn/ds ratio ; and finally (4) neutral evolution for zoophytophagous lineage (ω2 = 1). A likelihood ratio test (LRT) was employed to determine if the alternative model, indicating positive selection, was superior to the null model. The newly developed RELAX method [After duplication, genes tended to have a different selective pressure measured as a ratio between synonymous and non-synonymous substitution  , and only one PG in A. suturalis and the PG in N. tenuis contained one intron in the open reading frame region . Also our analysis suggests that the zoophytophagous lineages, which experienced serious loss of PGs, also evolved according to the non-neutral pattern . Given this result, we further specifically tested whether the selection pressure differed between predatory and phytozoophagous mirid bugs. When we set zoophytophagous lineages as foreground and phytozoophagous branches as reference, RELAX estimated the selection intensity parameter value of K = 0.546 , and the alternative relaxation model significantly outperformed the null model with a P-value = 1.58 × 10− 4, which is consistent with the results assessed with the likelihood ratio test. However, this was not the case when we set phytozoophagous lineages as the foreground , which suggest that a relaxation of selection pressures occurred on the zoophytophagous branches.The codeml branch model significantly rejected the neutral evolutionary hypothesis for the whole phylogeny of mirid PGs  and used the CDS sequence of PGs for counting FPKM value. In A. suturalis, 29 of 30 PGs were highly expressed  and the expression level of these 30 PGs showed statistically significant differences     by causing plant stunting and fruit abscission . To feedA. suturalis and N. tenuis. One intron was found in the CDS region of PG in N. tenuis. There were no introns in the CDS regions of six PGs. Although we found introns in PGs of mirid bugs, we could not exclude the possibility that the duplication of PGs in mirid bugs derived from retrotransposition.In this study, we determined the expressed PG genes in three phytozoophagous and one zoophytophagous mirid bugs by RNA-seq technology. Phytozoophagous mirid bugs usually have a broad host-plant range, while zoophytophagous mirid bugs feed on only a few plants –43. InteN. tenuis from the Bryocorinae) were more ancient than phytozoophagous mirid bugs , suggesting a host-expansion from zoophytophagous to phytozoophagous mirid bugs [Theoretically, neutral evolution and natural selection drive ecological population divergence and the speciation of organisms –50. As mrid bugs . Indeed,rid bugs . Taken tA. suturalis (three replicates), N. tenuis (three replicates), A. fasciaticollis (no replicates) and A. lineolatus (no replicates) using FPKM values from of RNA-seq. Our results clearly showed that almost all PGs were highly expressed in the three phytozoophagous mirid bugs (> 17), but only one PG was relatively highly expressed in zoophytophagous mirid bugs with huge fluctuation, which most likely resulted from that PGs were needed more by phytozoophagous than zoophytophagous mirid bugs. The other reason that feeding on cotton caused the upregulation of PGs could not be completely excluded because the samples used in this study of phytozoophagous mirid bugs were collected in cotton field and the zoophytophagous mirid bugs were collected in tobacco field. Controversially, N. tenuis has been used as a natural enemy for pest management because of its zoophytophagy [N. tenuis as a natural enemy due to the relaxed selection and only one PG expressed in N. tenuis, but with controlled density because of occasionally high expression level.Adaptive evolution at a molecular level includes two mechanisms to regulate gene function: (1) nucleotide/amino acid sequence variation which could be detected with selection pressure as described above, and (2) expression levels which are essential for gene function  –55. To dytophagy –58. TakiWe identified the number of expressed PGs in three phytozoophagous and one zoophytophagous mirid bug and found a significant, positive correlation between the numbers of PGs and host plants. Natural selection analysis suggested the PGs of zoophytophagous mirid bug were under a significantly relaxed selection. More than 17 PGs were highly expressed in each of the three species of phytozoophagous mirid bugs, but only one PG was relatively highly expressed in predatory mirid bugs. Taken together with evidence of gene copy number, molecular evolution and gene expression levels, our results suggested that PGs were more needed by phytozoophagous than zoophytophagous mirid bugs and the duplication of PGs promoted the host-expansion of mirid bugs. This research suggests that PGs are target genes for the management of phytozoophagous mirid bugs (e.g. RNAi).Additional file 1:Table S1. Primers used in this study. Table S2. Summary of the sequence assembly after Illumina sequencing. As = Adelphocoris suturalis, Af = A. fasciaticollis, Al = A. lineolatus, Nt = Nesidiocoris tenuis. Table S3. Identification of polygalacturonase genes in Adelphocoris suturalis. Table S4. Identification of polygalacturonase genes in Adelphocoris fasciaticollis. Table S5. Identification of polygalacturonase genes in Adelphocoris lineolatus. Table S6. Identification of polygalacturonase genes in Nesidiocoris tenuis. (XLSX 24 kb)Additional file 2:Figure S1. The discription of RNA-seq in Adelphocoris suturalis. (a) The distribution of sequences length. (b) The E-value distribution of the top matches in the nr database. (c) The species distribution of the matches in the nr database. (d) The sequence similarity distribution. Figure S2. The discription of RNA-seq in Adelphocoris fasciaticollis. (a) The distribution of sequences length. (b) The E-value distribution of the top matches in the nr database. (c) The species distribution of the matches in the nr database. (d) The sequence similarity distribution. Figure S3. The discription of RNA-seq in Adelphocoris lineolatus. (a) The distribution of sequences length. (b) The E-value distribution of the top matches in the nr database. (c) The species distribution of the matches in the nr database. (d) The sequence similarity distribution. Figure S4. The discription of RNA-seq in Nesidiocoris tenuis. (a) The distribution of sequences length. (b) The E-value distribution of the top matches in the nr database. (c) The species distribution of the matches in the nr database. (d) The sequence similarity distribution. Figure S5. The genome structure of polygalacturonase gene (m_50643) in Nesidiocoris tenuis. The intron was showed using shade. Figure S6. The expression levels of PGs in Adelphocoris fasciaticollis. Figure S7. The expression levels of PGs in Nesidiocoris tenuis. (DOCX 825 kb)Additional file 3:The coding sequence of PGs identified in this study. (ZIP 71 kb)


Rhodococcus aetherivorans JCM 14343T, which possesses the versatile ability to degrade recalcitrant noncyclic and cyclic ether compounds. The 4.2-Mbp genome of this bacterium contains alkane hydroxylase and propane monooxygenase genes involved in the degradation of noncyclic and cyclic ethers, respectively.Here, we report the draft genome sequence of  Rhodococcus aetherivorans JCM 14343T, which possesses the versatile ability to degrade recalcitrant noncyclic and cyclic ether compounds. The 4.2-Mbp genome of this bacterium contains alkane hydroxylase and propane monooxygenase genes involved in the degradation of noncyclic and cyclic ethers, respectively.Here, we report the draft genome sequence of  Rhodococcus possess the versatile ability to catabolize a variety of xenobiotic organic pollutants and are useful for the bioremediation of polluted environments , was originally isolated from petrochemical biotreater sludge as a methyl tert-butyl ether (MTBE)-degrading strain , Japan. After cultivation in malt extract-glucose-yeast extract (MGY) medium  and purified with ethanol precipitation and using the NucleoSpin gDNA cleanup kit (Macherey-Nagel). The purified DNA was fragmented and prepared into a sequence library using the TruSeq Nano DNA LT library preparation kit . This library was sequenced with 101-bp paired-end sequencing using the Illumina HiSeq 2500 system, which produced 22,532,896 reads with a yield of 2,276 Mbp. Unless otherwise noted, default parameters were used for all software tools. From the obtained sequence reads, the adapter sequences were trimmed using Cutadapt v. 1.1, and low-quality-value regions were further trimmed using Trimmomatic v. 0.32 (http://www.usadellab.org/cms/?page=trimmomatic). The trimmed reads were assembled onto the draft genome sequence using Velvet v. 1.2.08 (https://www.ebi.ac.uk/~zerbino/velvet/). The draft genome sequence had a total length of 6,442,200 bp with a G+C content of 70.2% and a genome coverage of 353× and is based on 265 contigs with an N50 value of 159,427 bp and a maximum contig length of 504,978 bp.The sequenced strain, ) medium , total gR. aetherivorans JCM 14343T contains alkane hydroxylase genes  identical to those of Rhodococcus spp. The alkane hydroxylase is involved in the initial MTBE oxidation , leading to the cleavage of their high-energy C-O bond (The genome was annotated with Rapid Annotations using Subsystems Technology . Totals xidation . AdditioC-O bond .R. aetherivorans JCM 14343T has been deposited at DDBJ/EMBL/GenBank under the accession number BLAH00000000. The version described in this paper is the first version, BLAH01000000, and consists of sequences BLAH01000001 to BLAH01000265. The raw sequencing reads were deposited under accession number DRR201558. The BioProject and BioSample numbers are PRJDB8788 and SAMD00186790, respectively, and the assembled and annotated set of contigs is publicly available under assembly accession number GCA_009176285.The whole-genome shotgun project of


We sampled 91 individuals from both atractaspidine genera (Atractaspis and Homoroselaps). Additionally, we used ancestral-state reconstructions to investigate fang and diet evolution within Atractaspidinae and its sister lineage . Our results indicated that current classification of atractaspidines underestimates diversity within the group. Diversification occurred predominantly between the Miocene and Pliocene. Ancestral-state reconstructions suggest that snake dentition in these taxa might be highly plastic within relatively short periods of time to facilitate adaptations to dynamic foraging and life-history strategies.Atractaspidines are poorly studied, fossorial snakes that are found throughout Africa and western Asia, including the Middle East. We employed concatenated gene-tree analyses and divergence dating approaches to investigate evolutionary relationships and biogeographic patterns of atractaspidines with a multi-locus data set consisting of three mitochondrial ( Atractaspis, renowned for their unique and exceptionally long and mobile fangs , ND4 [679 bp], cyt b [1094 bp], c-mos [605 bp], and RAG1 [1009 bp]). Individuals with missing data were included in the concatenated sequence analyses, because placement of individuals that are missing a significant amount of sequence data can be inferred in a phylogeny, given an appropriate amount of informative characters [Our data set consisted of 3933 base pairs , ND4 1st codon position (GTR+G), ND4 2nd codon position (TVM+G), and ND4 3rd codon position (HKY+I+G); cyt b 1st codon position (TVM+G), cyt b 2nd codon position (HKY+I+G) and cyt b 3rd codon position (GTR+G); c-mos and RAG1 1st, 2nd and 3rd codon positions (HKY+I). Preferred topologies for the ML and BI analyses were identical, with similar, strong support values for most clades  Atractaspis andersonii, (2) Atractaspis aterrima, (3) A. bibronii, (4) A. bibronii rostrata, (5) A. cf. bibronii rostrata, (6) A. boulengeri, (7) A. congica, (8) A. corpulenta corpulenta, (9) A. corpulenta kivuensis, (10) A. dahomeyensis, (11) A. duerdeni, (12) A. engaddensis, (13) A. irregularis, (14) A. cf. irregularis, (15) A. reticulata heterochilus, and (16) A. microlepidota. There was strong support for a western Asia/Middle East and Africa clade containing A. andersonii, A. engaddensis, A. microlepidota, A. micropholis, A. watsoni, and A. sp. Atractaspis andersonii did not form a monophyletic group, because one of the samples from Oman (AF471127) was recovered as sister to a clade of A. engaddensis with strong support . A well-supported clade of Atractaspis irregularis samples was partitioned by strongly supported central (A. cf. irregularis) and western African (A. irregularis) subclades. Atractaspis duerdeni was recovered within a well-supported A. bibronii complex. Atractaspis bibronii rostrata samples were partitioned into two highly divergent clades from southeastern DRC and Tanzania/Mozambique.The following models of nucleotide substitution were selected by PartitionFinder for BI analyses: t clades , and sinter taxa . The gen support . The wesFor the analyses including all atractaspidine and aparallactine samples available to us , preferrA. corpulenta corpulenta/A. corpulenta kivuensis as sister to A. congica/A. dahomeyensis with strong support . Results recovered a fixed front fang condition for the ancestor of all Homoroselaps (99.8%). The ancestor to all aparallactines was inferred to have a rear fang condition (99.6%), and this remained consistent throughout most aparallactine nodes with the exception of Polemon  and Aparallactus modestus .X-ray computer tomography of collared snakes and burrowing asps can be seen in Figs Lycodonomorphus) subsequently adopted a more specialized diet of amphibians, reptiles, and fish. The common ancestor for aparallactines and atractaspidines was inferred to have a generalist diet of rodents, reptiles, and amphibians (92.4%). Results recovered a more specialized ancestral diet of snakes and lizards (64.5%) for aparallactines, which was favored over a generalist diet (27.7%). The condition of a snake and lizard diet (79.9%) was favored over a generalist diet (16.2%) for the ancestor of Polemon/Chilorhinophis and Amblyodipsas/Macrelaps/Xenocalamus. The latter dietary condition was retained for the ancestor of Polemon/Chilorhinophis (79.4%) and the ancestor of Amblyodipsas/Macrelaps/Xenocalamus (87.6%). Specialized dietary conditions were recovered for the genera Aparallactus , Polemon , and Xenocalamus . Results suggested a generalist diet for Atractaspidinae (92.3%). The ancestor of Homoroselaps was inferred to have a diet consisting of mostly lizards and snakes (99.9%), whereas the ancestor of Atractaspis was inferred to have a broader diet of rodents, reptiles, and amphibians (99.2%).For the analyses with diet data, likelihood reconstructions inferred a generalist diet of rodents, reptiles, and amphibians for the ancestral condition of all lamprophiids (99.7%) . SeveralAtractaspis that are found in western Asia/Middle East  [Atractaspis is an African origin with a vicariance or dispersal event into the western Asia/Middle East region in the late Miocene (Atractaspis from western Asia/Middle East and Africa last shared a common ancestor during the late Miocene around 12.1 mya (7.8–17.6). Other studies of African-western Asian/Middle Eastern complexes  recovered similar dates during the late Miocene, with the Red Sea proving to be a strong biogeographic barrier [Varanus from Africa and the Middle East split from each other 6.9 mya [Bitis arietans last shared a common ancestor around 4 mya [Atractaspis species from the Horn of Africa, and future studies should include samples of A. fallax, A. magrettii, A. leucomelas, and A. scorteccii to improve understanding of likely Africa–Asia biogeographic patterns in atractaspidines.Atractaspidines are distributed throughout sub-Saharan Africa except for three species of epidota) , 29–31.  Miocene . Atracta barrier –69. Howe 6.9 mya , and Afrnd 4 mya . These dnd 4 mya –71. Our Atractaspis began to diversify around the mid-Oligocene simultaneously with many aparallactine genera [Adolfus, Atheris, Boaedon, Naja, Kinyongia, and Panaspis) and frogs , have shown similar trends of species diversification during the late Miocene [e genera . Many ofe genera –73. Othe Miocene , 74–78.Atractaspis was most likely a consequence of increasingly xeric conditions during the Miocene, when forest and other moist habitats were fragmented [Atractaspis were likely isolated in fragmented patches of forest during the mid- to late Miocene. Atractaspis irregularis is partitioned clearly by western African and central African lineages that diverged in the mid-Miocene, similar to Aparallactus modestus [Atractaspis also diversified. Atractaspis from the Near and Middle East  and southern Africa (A. bibronii and A. duerdeni) are not tropical forest species, and they inhabit deserts or semi-desert savannas and dry woodland [Atractaspis, to disperse into these habitats during the dry conditions of the mid- to late Miocene. Studies on mammals and birds show most diversification events during the Pliocene [Atractaspis aterrima, A. congica, A. dahomeyensis, and populations of South African A. bibronii , and the streaky canary (Serinus striolatus) [Atractaspis bibronii rostrata, the hyperoliid reed frog Hyperolius spinigularis and the aparallactine Aparallactus guentheri showed no clear biogeographic patterns between populations in different areas of the Eastern Arc Mountains. These results support the hypothesis that the evolutionary history of species from the Eastern Arc Mountains is lineage specific [Atractaspis bibronii rostrata inhabit low-elevation woodlands and grasslands, and transitional habitats, rather than montane forest  [Atractaspis bibronii rostrata to continuously disperse between the different mountains of the Eastern Arcs, despite increased aridity. Additionally, ecological niche requirements may also explain the different biogeographic patterns seen in Aparallactus jacksonii and Atractaspis bibronii rostrata. Atractaspis bibronii has a generalist diet  and could have exploited more habitats than Aparallactus jacksonii, which is a centipede specialist [In contrast to ountains . Aparall aridity . Other violatus) , 85. Butcksonii) . This woecialist .Atractaspis and Homoroselaps are strongly supported as monophyletic sister taxa. Results from Figueroa et al. [Atractaspis . This sample was excluded from our analyses, because the only sequence available for this taxon was from BDNF, a gene not used herein. The results from Figueroa et al. [BDNF nuclear gene. Results from our study indicate that A. irregularis is a monophyletic lineage within a strongly supported, monophyletic Atractaspis.Our results indicate that both a et al.  recoverea et al.  may be aAtractaspis: (1) the ‘bibronii’ group , characterized by a single posterior supralabial, three anterior infralabials, normal-sized venom glands, and a sub-Saharan distribution; and (2) the ‘microlepidota’ group , characterized by two anterior temporals, highly elongated venom glands, and a North African/Near and Middle Eastern distribution. Whereas our study did not include genetic samples of all known species of Atractaspis, results herein  of Laurent [A. micropholis/A. microlepidota/A. watsoni clade recovered by Moyer and Jackson [Underwood and Kochva  recognizs herein  support d Kochva . Our resd Kochva ) and a ‘ion; and  the ‘mic Laurent  and the  Jackson . However Jackson , most re Jackson , and the Jackson –9 and Vi Jackson .Atractaspis seem to be cryptic complexes of species. Because of the extensive geographic distribution of A. bibronii in central, eastern and southern Africa, it is unsurprising to find several highly divergent lineages that likely represent cryptic species. Given the proximity (ca. 167–333 km) of our Tanzanian localities of A. bibronii rostrata  to the insular type locality for this taxon , the morphological similarity between our voucher specimens and the types [A. bibronii  and P. fulvicollis laurenti (DRC) [Atractaspis corpulenta and eastern DRC populations of A. corpulenta kivuensis also showed marked genetic divergences between each other  [Atractaspis bipostocularis from Mt. Kenya, was named for its two postocular scales, which distinguishes it from the single postocular of topotypic A. irregularis [A. cf. irregularis from the Albertine Rift have a single postocular (EG pers. obs.), A. bipostocularis is likely a distinct species that is endemic to the central Kenyan highlands. Other synonyms of A. irregularis that have one postocular and type localities in or near the Albertine Rift are likely attributable to our well-supported clade of A. cf. irregularis  , Gabonesch other . The wel, Ghana) , whereasegularis . Becauseegularis , and morFig 2 in ), and inTanzania ), A. schana, DRC ), and A.nia, DRC ). Additinia, DRC –93 and tAtractaspis in particular have very distinctive fangs  that have made their taxonomic history complicated  [Homoroselaps resemble fangs of elapids more than vipers. In contrast, aparallactines tend to have rear fangs  that foraged above ground or in burrows of other organisms, and these taxa subsequently evolved into more specialized forms with specialized diets. Several aparallactines are dietary specialists [Aparallactus specialize on centipedes and possibly other invertebrates like earthworms; Chilorhinophis and Amblyodipsas consume snakes and other small, fossorial reptiles; Polemon are ophiophagous [Macrelaps consume reptiles, amphibians, and rarely mammals [Xenocalamus consume amphisbaenians [Burrowing asps and collared snakes have unique ecologies, particularly in terms of dietary preferences. iperids) , 31, 94.ngs Figs  [18, 25,ophagous , but mayAtractaspis are dietary generalists that consume a diverse variety of squamates, rodents (particularly nestling rodents), and occasionally amphibians [Atractaspis are anatomically distinct from those of other front-fanged snakes such as viperids and elapids, because atractaspidines lack a distinct accessory gland and the presence of mucous-secreting cells at the end of each serous tubule [Atractaspis from western and northern African, and western Asia/Middle East species. These glands may be up to 12 cm long in A. engaddensis and 30 cm long in A. microlepidota [Atractaspis is clearly partitioned according to venom gland length and geographic distribution  [Atractaspis may have evolved to facilitate the predation of rodent nestlings or squamates in tight burrows. Preying on animals in tight burrows limits mobility of the predator, because the body of the prey item can serve as a physical barrier, stopping the predator from further pursuit. Many lizards can detach their tails if a predator grabs the tails from behind. Shine et al. [Atractaspis. Deufel and Cundall [Atractaspis was likely the result of the following advantages: (1) greater envenomation efficiency resulting from the longer fangs; (2) closed mouth venom delivery system, allowing envenomation during head contact with any part of the prey; (3) capacity to quickly envenomate and release prey; and (4) potential for effective defense against adult rodents. Most prey consumed by Atractaspis  [Amblyodipsas, Chilorhinophis, Homoroselaps, Macrelaps, Polemon, and Xenocalamus [Atractaspis. However, rodents and other mammals are not commonly preyed on by other burrowing asps and collared snakes [Atractaspis, but the success and wide distribution of this genus may be partially attributed to mammalian prey. Unlike aparallactines, Atractaspis can quickly envenomate and dispatch all rodents in a nest [Atractaspis. Interestingly, in a feeding experiment, Atractaspis never attempted to ingest snake prey until the prey stopped reacting to fang pricks [Atractaspis will not risk injury until prey are completely immobilized. The unique fang and predatory behavior of Atractaspis has its functional trade-offs; Atractaspis lack large mandibular and maxillary teeth that allow snakes to quickly consume prey [Atractaspis forage, kill, and consume prey in the soil and below the surface, there were likely no negative selective pressures acting against slow ingestion of prey. Because they are fossorial, Atractaspis may be relatively safe from predators while feeding, which is when non-fossorial snakes may be vulnerable to predation or attacks from other animals [Unlike several aparallactines, phibians , 96–100.s tubule , 101–103lepidota . Phylogelapidae) . The unie et al.  postulat Cundall  hypothes snakes)  are alsoocalamus , 31, 97.d snakes . Deufel d snakes  stated tn a nest . A rear g pricks . This obume prey , and the animals , 33.Aparallactus modestus clearly lacks enlarged fangs , explaitus Figs  and 6.Polemon fangs are not easily classified. The fangs of Polemon are located on the anterior half of the maxilla, rather than the more typical posterior end  more effectively than they would with a rear-fang condition like Aparallactus. Snakes with rear fangs must typically chew in a forward orientation until the rear fang can penetrate the flesh of the prey item [Micrurus and Ophiophagus). The front-fang condition may be a favorable trait to feed on snakes, in order to immobilize and kill more quickly.end Figs  and 6. T in size , 95, 107rey item . SeveralXenocalamus, similar selective pressures  that led to the evolution of fang and predatory behaviors in Atractaspis, may have led to the evolution of its unique quill-shaped snout [Amblyodipsas polylepis, Xenocalamus possess relatively large maxillary teeth that gradually increase in size from the anterior to posterior side of the maxilla Click here for additional data file.


Brettanomyces naardenensis is a spoilage yeast with potential for biotechnological applications for production of innovative beverages with low alcohol content and high attenuation degree. Here, we present the first annotated genome of B. naardenensis CBS 7540. The genome of B. naardenensis CBS 7540 was assembled into 76 contigs, totaling 11,283,072 nucleotides. In total, 5168 protein-coding sequences were annotated. The study provides functional genome annotation, phylogenetic analysis, and discusses genetic determinants behind notable stress tolerance and biotechnological potential of B. naardenensis. Brettanomyces  are of  be used ,3,4,5. Bed beers ,6. B. naardenensis was previously shown to spoil carbonated beverages and soft drinks with pH ranging 2.6–3.2 [B. naardenensis strain CBS 7540 was isolated in 1990 from a soft drink in South Africa . Occurrence of B. naardenensis in Cabernet Sauvignon wine was also reported [Brettanomyces, to assimilate soluble starch. The broad substrate range of Brettanomyces can contribute to reduction of residual sugar content in beverages production. This property can be used for the production of super-attenuated and lower-calorie beers [Brettanomyces in highly dosed hopped wort and also during beer maturation. Hops are a natural source of nitrate, which can be used as a nitrogen source by Brettanomyces species. in beer brewing, and thus result in their proliferation, at the same time reducing the nitrate content. Nitrate removal from drinks may decrease cancer and other health risks [The acetate-producing, Crabtree-negative yeast  2.6–3.2 ,8. The Breported  and, unlie beers . The abiie beers , may in th risks .Brettanomyces yeasts showed that this group of yeasts is not only a source for deterioration of the sensory qualities of drinks, but in some cases can also provide beneficial aromas for craft and specialty beverages [Brettanomyces aroma wheel was recently developed [Brettanomyces yeasts contributes to tropical fruit and pineapple-like flavors of lambic and gueuze beers. Esterification of cheesy-flavored long-chain fatty acids  by Brettanomyces yeasts leads to generation of the grape-like beer flavor [Brettanomyces contributes to generation of flavor-active aglycones, such as benzaldehyde, linalool, or eugenol from glucosides present in hops or fruits [Brettanomyces was also explored for production of the antiaging compound, resveratrol [Negative health and economic consequences of alcohol consumption have awoken an interest in the production of non-alcoholic beverages by the brewing industry using non-conventional yeasts. Studies on everages . The Breeveloped . Ethyl ar flavor . Beta-glr fruits . Beta-glveratrol . Brettanomyces yeasts can be considerably facilitated by knowledge of the whole genome sequence. Up to now, mainly the genomes of strains of Brettanomyces bruxellensis—isolated from wine, lambic beer, and distilleries [Brettanomyces custersianus and Brettanomyces anomalus are also available [B. naardenensis. Exploring the full biotechnological potential of illeries ,25,26,27vailable . This stB. naardenensis culture grown in 100 mL YPD at 25 °C to OD 10 were harvested. Aerobic batch cultivation of the strain for RNA isolation was performed using defined minimal media supplemented with 2% glucose in 1L Multifors fermenters . Two milliliters of B. naardenensis CBS 7540 was prepared using a standard zymolyase and phenol-chloroform extraction. A sample quality test was made for DNA sample, and the qualified sample was used to construct libraries. Covaris or Bioruptor was used to break the genomic DNA into smaller fragments . T4 DNA Polymerase, Klenow DNA Polymerase, and T4 PNK were used for end repair of the small DNA fragments. Three prime end A addition and adapter ligation were performed using taq-polymerase. The target DNA fragments were size-selected by electrophoresis and enrichment was performed using PCR amplification and purification. The qualified library was cluster prepared and sequenced. Genome sequencing was performed using the Illumina GA II  by generating multiplexed paired-end libraries (an average insert size of 500 bp). Raw fluorescent images and call sequences was processed using a base-calling pipeline (Solexa Pipeline-version 1.0). The genomic DNA of Isolation and processing of RNA from triplicates of batch cultures were performed as previously described . The RNAThe raw data (fastq format) were cleaned by FastQC and fastx_toolkit. The read size was 250 × 2 bp. We used SOAPdenovo1.05 to assemble the cleaned reads . All rawB. naardenensis CBS 7540 genome assembly was built in three steps: Prediction of candidate cDNAs using transcriptome data (assembled de-novo with Trinity and genome-guided with tophat and cufflinks) within the PASA package, followed by training of the SNAP ab-initio gene finder, and finally the actual gene build combining evidence alignments and ab-initio predictions using the Maker2 package. The annotation was deposited into European Nucleotide Archive (ENA) using EMBLmyGFF3 [B. naardenensis CBS 7540 was deposited in the European Nucleotide Archive (ENA) with accession numbers CAACVR010000001 to CAACVR010000076 under project number PRJEB30032The annotation of the BLmyGFF3 . The annhttp://www.blast2go.com/b2ghome), based on best BLAST matches of predicted cDNAs against the RefseqP fungal reference dataset. Annotation of B. naardenensis CBS 7540 was performed by searching the KOG (available online: http://www.ncbi.nlm.nih.gov/COG/) database using BLAST software.Functional genome annotation. Functional annotation of transcripts was performed using the Blast2Go pipeline  and the sample purity was determined using NanoDrop. Libraries with Illumina-compatible adapters were constructed using the KAPA HyperPlus Library Prep Kit  by following the instructions of the manufacturer. The finished libraries were quantified with Qubit dsDNA HS Assay Kit (Invitrogen) and the average library size was determined using DNF-473 Standard Sensitivity NGS Fragment Analysis Kit (1 bp–6000 bp); (Agilent). The generated multiplexed paired-end libraries had an average library size of 584 bp. Whole-genome sequencing was performed using the MiSeq Reagent Kit v2, 500 Cycles (Illumina) on the MiSeq sequencing platform  with 10 pM flow cell loading. The sequencing chemistry employed 4-channel sequencing-by-synthesis (SBS) technology. Reads generated by CBS 7540 genome resequencing were mapped to the genome assembly of B. naardenensis CBS 7540 by using BWA version 0.7.4 [To generate reads for analysis of variants, we resequenced the genome of on 0.7.4 . Identifon 0.7.4 . Debaryomyces hansenii, Kluyveromyces lactis, Ogataea polymorpha, Pichia kudriavzevii, Yarrowia lipolytica, Komagataella pastoris, Candida albicans, and Saccharomyces cerevisiae were downloaded from Ensembl and NCBI databases. We first built the orthologous groups of within 12 yeast genomes. Then, 2328 gene groups, which had a strict and phylogeny-based one-to-one orthology relationship in all species included in the phylome, were respectively aligned by ClustalOmega, and trimmed using trimAl , and then concatenated into a single alignment. Finally, a maximum likelihood tree was constructed by MEGA. All nodes received the highest support in terms of approximate likelihood ratio tests and of a bootstrap analysis of 100 replications.The proteomes of B. naardenensis and B. bruxelensis was done using program BLAST 2.2.29+ as described earlier [Comparison of the gene content between genomes of  earlier . B. naardenensis CBS 7540 was assembled into 76 contigs. The details of genome assembly are summarized in B. naardenensis CBS 7540 genome assembly was found to be 44.5%, which is comparable to that of B. bruxellensis [The genome of ellensis .B. bruxellensis CBS 2499 [B. naardenensis CBS 7540 genome are presented in Brettanomyces yeasts genomes. An advanced annotation approach was used to increase gene prediction accuracy . In totaCBS 2499 . The annB. naardenensis CBS 7540 genome based on Blast2Go and KOG database is presented is Functional genome annotation of the B. bruxellensis CBS 11270 [Analysis of polymorphisms of the CBS 7540 genome was performed by mapping the CBS 7540 reads to the de novo assembly of the CBS 7540 genome. Reads generated by CBS 7540 genome resequencing mapped to assembly sequence of the CBS 7540 genome with 100 x times coverage. We detected 851 variants , which iWe detected 323 single-nucleotide polymorphisms (SNPs), constituting 0.002% of the genome size. Transitions  were detected more frequently than transversions . DEKNAAT104779 (24 variants), DEKNAAT104433 (14 variants), DEKNAAT102160 (12 variants), and DEKNAAT103651 (10 variants). The corresponding best BLAST hits in B. bruxellensis AWRI1499 were a predicted subunit of the CCR4-NOT complex (AWRI1499_4082), a predicted iron homeostasis modulator (AWRI1499_1657), hypothetical protein (AWRI1499_4852), and a predicted CCR4-NOT transcription subunit 3 (AWRI1499_3655). An additional 45 genes had between one and nine variants ; a putative xylulokinase (DEKNAAC100346); a putative xylose/arabinose reductase (DEKNAAC102931); and a putative NADPH-dependent D-xylose reductase (DEKNAAC104786). It has previously been shown that the CBS 6042 strain of B. naardenensis could ferment xylose with 1.8 g/L of ethanol produced from 20 g/L of xylose [The CBS 7540 genome included a number of hypothetical genes predicted to be involved in xylose metabolism, including a putative D-xylulose reductase (f xylose . Broad sf xylose . NIT genes) encoding for the nitrate assimilation pathway: Nitrate transporter (YNT1); nitrate reductase (YNR1); nitrite reductase (YNI1) in the genome of B. naardenensis CBS 7540. This is different to some B. bruxellensis strains [B. naardenensis in a nitrate-rich environment such as hopped beer.Investigation of alternative nitrogen metabolism genes did not result in identification of the gene cluster ) but not URA1 (oxygen-independent cytoplasmic DHOD) [B. bruxellensis could use for uracil biosynthesis under anaerobic conditions [S. cerevisiae, which has lost URA9 and employs URA1 for uracil synthesis under anaerobic conditions.ic DHOD) . It remanditions . This isB. naardenensis CBS 7540 genome: DEKNAAC101096, DEKNAAC103026, and DEKNAAC103616; ADH3 DEKNAAC105038 and a NADPH-dependent medium-chain ADH with broad substrate specificity DEKNAAC104540. Similar to B. bruxellensis, B. naardenensis has several independently duplicated ADH and ADH-like genes, which are presumably involved in metabolism of alcohols, including ethanol, and also other aromatic compounds [B. bruxellensis, one ADH gene is involved in both ethanol production and consumption [Scheffersomyces stipitis [Wickerhamomyces anomalus [We have annotated genes of five alcohol dehydrogenases (ADH) in the ompounds . Interesstipitis  and Wickanomalus .B. bruxellensis AWRI1499, we identified two beta-glucosidase candidate genes in the genome of B. naardenensis CBS 7540: unnamed proteins DEKNAAC101161  and DEKNAAC101664 . This indicates that B. naardenensis has genetic potential for production of flavor-active aglycones. Further metabolic conversions of monoterpenes by complex brewing cultures can contribute to the generation of fruity or floral odors [The enzyme beta-glucosidase hydrolyzes glucose from glycosides derived from hops or fruit ingredients of beverage forming volatile aglycones. Using blast search versus beta-glucosidase AWRI1499_4190 of al odors .ATF1 and ATF2 genes involved in production of acetate esters disables Brettanomyces from producing the banana-flavored isoamyl acetate and honey-flavored 2-phenylacetate. However, the presence of esterase could potentially enable the formation of ethyl esters with tropic fruit-like odors: Ethyl acetate, hexanoate, and octanoate. Esterase DEKNAAC103380 was annotated in the genome of B. naardenensis CBS 7540. Esterification of cheesy-flavored long-chain fatty acids  by Brettanomyces yeasts was suggested to contribute in formation of grape-like beer flavor [DEKNAAC104471 and DEKNAAC100147, have high sequence similarity to DEKNAAC103380 and could potentially encode additional esterases.The absence of r flavor . Two othS. cerevisiae and B. bruxellensis can convert cinnamic acids derived from grape skin into hydroxycinnamic acids using esterase following conversion into hydroxysterene using cinnamate decarboxylase. However, unlike S. cerevisiae, B. bruxellensis displays vinyl phenol reductase (VPR) activity, which catalyzes further conversion of hydroxystyrene into a volatile ethyl derivate [B. naardenensis CBS 7540 genome with the B. bruxellensis ST05.12/26 VPR sequence identified a likely orthologous gene , which suggested that this enzyme may play a role in off-odors formation in B. naardenensis as well.Both derivate . Ethyl pderivate . A BLASTB. bruxellensis, which was reported as a potent acetate producer, B. naardenensis was described to generate moderate amounts of acetate under aerobic conditions [DEKNAAC103751, DEKNAAC103492, and DEKNAAC101110; aldehyde dehydrogenase (NADP+) Ald4 DEKNAAC100618; and mitochondrial aldehyde dehydrogenase DEKNAAC103573 were annotated in the genome of B. naardenensis CBS 7540. We identified several B. naardenensis CBS 7540 gene candidates for acetyl-CoA synthetase, DEKNAAC103441, DEKNAAC102074, DEKNAAC102090.Sensation of acetate is a matter for discussion—its presence is desirable for certain brewing styles and in other beverages is perceived as a sign of spoilage. In contrast to nditions . The reanditions ,46. SeveBrettanomyces/Dekkera genus [B. bruxellensis [The inhibition of alcoholic fermentation under anaerobic conditions—commonly known as the Custer effect—characteristic of yeasts belonging to the ra genus  is assocellensis  is not sDEKNAAC101381 likely encodes AOX in B. naardenensis CBS 7540 . Alternative respiration involving salicylhydroxamic acid (SHAM)-sensitive alternative oxidase (AOX) was suggested to present an additional mechanism behind the Custer effect . The hypB. naardenensis was shown to survive in pH ranging from 2.6–3.2 [ 2.6–3.2 ,8. The m 2.6–3.2 , and inh 2.6–3.2 . Plasma  2.6–3.2 .S. cerevisiae determined several genes conferring high acetic acid tolerance: A transcriptional activator involved in the response to weak acid stress, HAA1; CUP2, paralog of HAA1; glyoxylase gene GLO1 ; oxidative stress-resistance gene DOT5 encoding nuclear thiol peroxidase, and vacuolar membrane Atpase VMA7 [B. naardenensis CBS 7540 were identified as DEKNAAC104025, DEKNAAC102923, DEKNAAC104208, and DEKNAAC101910, respectively.Studies on ase VMA7 . The corB. naardenensis has remarkably low thermotolerance, which could limit its dissemination in human-related habitats. B. naardenensis was able to grow at 25 °C but not 30 °C. This is different to B. bruxellensis, which can grow at 37 °C [B. naardenensis are scarce, it is unclear if ethanol levels can act as additional limiting factor.On other hand, at 37 °C . Since dBrettanomyces species research, the literature on B. naardenensis is rather scarce. This study represents the first genomic investigation of the B. naardenensis. Genomes of most Brettanomyces species became recently available [Brettanomyces nanus, which was repeatedly isolated from breweries at different sites in Sweden [Despite the recent interest in vailable ,26,27,28n Sweden ,54,55, rB. naardenensis CBS 7540 is in the range of that determined for other Brettanomyces species [B. naardenensis, CBS 6040, CBS 6041, CBS 6042, CBS 6107, and CBS 6117, have been isolated from carbonated soft drinks in USA, Belgium, Netherlands, Norway, and Denmark, respectively [B. bruxellensis population, suggesting an anthropic influence on the spatial biodiversity of this microorganism [B. bruxellensis, almost no isolates from “natural” non-human related habitats were reported for B. naardenensis. All above-mentioned B. naardenensis strains were described as soft drink spoilage microorganisms, suggesting notable species tolerance to low pH and osmotic stress. The low number of reports on the occurrence of B. naardenensis in wine [B. bruxellensis, B. anomala, and B. custersianus [B. bruxellensis wine strains could confer a selective advantage of this species in the presence of the high levels of sulfur dioxide, a preservative used in winemaking [B. naardenensis in soft drinks. The genome size 11.3 Mbp of  species ,26,27,28ectively . CBS 754organism . Similar in wine  and beerersianus . A genomnemaking ,23,58. IB. naardenensis genes putatively involved in metabolism of alternative carbon sources, which could contribute to production of innovative beverages with high attenuation degree and low alcohol content. A number of identified candidate genes responsible for generation of volatile compounds suggests that further substrate optimizations might allow for suppressing undesirable and enhancing pleasant aromas generated by B. naardenensis.The recent rise in craft and specialty beer production has awoken an interest in non-conventional yeast species and resulted in more careful consideration of organoleptic properties of fermented beverages flavor . OrganolB. bruxellensis, while the other species such as B. naardenensis are still respiratory, i.e., they are only forming ethanol under oxygen limitation. In contrast to the Saccharomyces line, development of fermentative lifestyle in B. bruxellensis was not preceded by a whole genome duplication. There are indications that promoter re-wiring and amplification of fermentative genes such as alcohol dehydrogenase genes played a role in developing the fermentative live style [B. bruxellensis [B. bruxellensis, a low number of identified variants points towards high genome stability of B. naardenensis CBS 7540. Low genetic polymorphism could limit the physiological plasticity of the species abolishing evolution towards fermentative lifestyle—this is consistent with preservation of respiratory phenotype of B. naardenensis. Interestingly, the fermentative lifestyle has evolved in one species of this genus, ve style . Those rellensis . In contB. naardenensis [Brettanomyces species. A high level of homozygosity between Saccharomyces strains was shown to be associated with sporulation and selfing phenomena [B. naardenensis diverged from other Brettanomyces species earliest, and therefore molecular physiology of this species might be distinct from other species of this genus. The teleomorphic state of denensis  was showdenensis . The highenomena ,62. PhylB. naardenensis provides a basis for further research efforts to understand notable stress tolerance and uncover the full biotechnological potential of this yeast in the production of new-generation beverages.The genome sequence of B. naardenensis from food-related environments, this species remains poorly investigated. Genome survey of B. naardenensis provides the first insights into the genetic landscape of this yeast. We present highly refined RNA-guided genome annotation. Additionally, functional annotations provide tools for further system biology studies on this species. Genome size and gene content of B. naardenensis is comparable to other annotated Brettanomyces species. However, high genome homozygosity is different from dynamic heterozygous genome of B. bruxellensis. Apart from highly diverged DNA and protein sequences between these two Brettanomyces species, promotor rewiring boosted differentiation of B. naardenensis and B. bruxellensis lifestyles into respiratory and fermentative, respectively. Our analysis supports distant phylogenetic relations between these species. Identification of genes involved in tolerance to low pH explains ability of B. naardenensis to spoil soft drinks. We have determined B. naardenensis genes that could contribute to production of innovative beverages with high attenuation degree and low alcohol content. Candidate genes responsible for generation of volatile compounds were identified, which provides the basis for future exploration the full potential of B. naardenensis.Despite frequent isolation of