Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features of this gram-positive lactic acid non-colonizing gut bacteria include its generally recognized as safe (GRAS) status, probiotic properties, the absence of inclusion bodies and endotoxins, surface display and extracellular secretion technology, and a diverse selection of cloning and inducible expression vectors. This have made L. lactis a desirable and promising host on par with other well established model bacterial or yeast systems such as Escherichia coli, Salmonella cerevisiae and Bacillus subtilis. In this article, we review recent technological advancements, challenges, future prospects and current diversified examples on the use of L. lactis as a microbial cell factory. Additionally, we will also highlight latest medical-based applications involving whole-cell L. lactis as a live delivery vector for the administration of therapeutics against both communicable and non-communicable diseases. Lactococcus lactis with dairy products, the bacterium was originally isolated from plants where it was believed to be dormant, and only became active and multiplied in the gastrointestinal tract after being consumed by ruminants [Lactococcus genus in 1985, L. lactis is divided into three subspecies namely L. lactis subsp. lactis, L. lactis subsp. cremoris, and L. lactis subsp. hordniae [Despite the common association of uminants . Originahordniae . Phenotyhordniae , 4.Lactococcus lactis has been used for centuries in the fermentation of food especially cheese, yoghurt, sauerkraut and the like, thereby rendering it’s generally recognized as safe (GRAS) status by the Food and Drug Administration (FDA). Apart from imparting flavour, L. lactis being a lactic acid bacteria (LAB) also produces acid which preserves food. Some strains further enhances this preservation property with the production of bacteriocins, thus reinforcing its role in the food industry. Other than its important function in food, L. lactis has become the model LAB when it comes to genetic engineering. Several factors including its small-sized fully sequenced genome (2.3 Mbp), and the development of successfully compatible genetic engineering tools such as cloning and expression systems with customizable options, have rendered it a desirable model. Over the past two decades, L. lactis has vastly extended its application from food to being a successful microbial cell factory  and Zirex system which are both regulated based on zinc availability where the former and latter are repressed and induced by the presence of zinc, respectively [xylT which is induced by xylose [nisR and nisK regulate expression of the nisin genes. NisK is a histidine-protein kinase which resides in the cytoplasmic membrane and acts as a receptor for the nisin molecule. Upon reception of nisin, it activates nisR via phosphorylation, which in turn induces transcription of two promoters in the nisin gene cluster: PnisA and PnisF [In most cases, inducible promoters are preferred over constitutive promoters as they provide better control to the user. There are various lactococcal inducible systems such as Pectively . Other iectively , 12 and y xylose . Howevernd PnisF .L. lactis NZ9000 is a derivative of the nisin-negative MG1363 strain with the nisR and nisK genes inserted into its chromosome [nisA start codon [L. lactis” [® plasmids and compatible host strains developed by NIZO Food Research (Netherlands) are now commercially available, with many derivatives being established using NIZO systems including a system for TA-cloning, designated pNZ-T to facilitate restriction enzyme independent cloning [The host romosome . When a romosome , which ert codon . Other c lactis” . Many of cloning .L. lactis as a host for heterologous protein production [E. coli where secreted proteins are mostly stuck in the periplasm [L. lactis only possesses a single extracellular housekeeping protease, HrtA, thereby reducing the chances of secreted heterologous proteins being degraded [Secretion of heterologous proteins are mostly preferred compared to intracellularly expressed proteins due to advantages such as simpler purification steps, higher yields, and better target interactions . In viewoduction . In addieriplasm . Furtherdegraded , 22.L. lactis, there is only one majorly secreted protein which is Usp45, whose function is still unknown [L. lactis, and was recently engineered through a series of mutations to further increase its secretion efficiency (SE) by 51% [Pediococcus pentasaceus, with the ability to secrete heterologous proteins with efficiencies comparable to Usp45 in L. lactis [Signal peptides (SPs) are N-terminal extensions of a protein which signals the host to target the protein towards the extracellular region by translocation across the cytoplasmic membrane and cell wall. While the sequence of SPs are vastly diversed, they display a common tripartite structure which includes the positively charged N-terminus, the hydrophobic H-region and the negatively charged cleavage region at the C-terminus . In L. l unknown . Neverth) by 51% . More re. lactis . When SP. lactis , thus deL. lactis including the use of synthetic LEISSTCDA propeptide sequence (SPs are followed by a propeptide sequence which is cleaved after translocation to produce the mature protein) [hrtA mutant strains (the only reported cell surface proteolytic housekeeping gene) [L. lactis when co-expressed with B. subtilis PrsA protein, which is a surface anchored protein with chaperon-like functions and have been shown to decrease degradation of exported proteins [Apart from SPs, past literature have reported several other strategies which have proven to improve SE in protein) , and theng gene) . In anotproteins .The thick and rigid cell wall of gram-positive bacteria as well as the lack of an outer membrane envelope has made them suitable for the cell surface display of proteins. Displaying proteins on bacterial cell wall allows the bacteria to act as carriers of proteins, especially antigens, and allow interaction of displayed proteins with targeted environments. There are five different types of protein anchors described in lactic acid bacteria; (1) transmembrane anchors: (2) lipoprotein anchors which binds to the cell membrane; (3) LPXTG-type cell wall anchoring domains; (4) AcmA-repeats anchor domain; (5) S-layer protein attachments which are bound to cell wall components .L. lactis, the most commonly used method for surface display of proteins is through the LPXTG sorting signal of surface-associated proteins which are recognized by the sortase enzyme, and covalently bound to the cell wall. In this method, the anchoring mechanism relies on the sortase activity as this membrane-anchored enzyme cleaves the sorting signal of the target protein at its pentapeptide motif (LPXTG) and promotes covalent anchoring of the target protein to the cell wall [trans surface display, where proteins are displayed from the outside of L. lactis host cells, as we have previously shown [E. coli), purified and bound non-covalently to the lactococcal cell wall simply by mixing the purified heterologous proteins to lactococcal cell cultures. More importantly, this enables the lactococcal cells to carry heterologous proteins without being genetically modified, a method which have also been demonstrated with Newcastle disease virus hemagglutinin-neuraminidase (HN) protein for specific targeting of breast cancer cells [L. lactis for delivery [In ell wall , 30. Howell wall . More inly shown . Using ter cells . In addidelivery . A variadelivery . A simildelivery . The draL. lactis is a strictly homolactic fermentative bacteria which completely converts its carbon source into l-lactate from pyruvate through a very efficient lactate dehydrogenase (LDH) enzyme with a Km value of 1.1 mM [L. lactis and other LAB, under different physiological conditions, three other enzymes apart from LDH also converts pyruvate: (i) α-acetolactate synthase (ALS) which is active at high pyruvate concentrations and low pH (≤6.0) [L. lactis is also a natural factory for the production of many other aromatic acetylated products such as diacetyl, acetaldehyde and acetate, resulting from mixed fermentation. Nevertheless, LDH still dominates with maximal enzymatic activity at high sugar concentrations and high intracellular nicotinamide adenine dinucleotide dehydrogenase (NADH) levels [Naturally, f 1.1 mM . Lactic f 1.1 mM , as an ef 1.1 mM . Additiof 1.1 mM . While lH (≤6.0) ; (ii) pyH (≤6.0) ; and (iiH (≤6.0) . Therefo) levels , 43.L. lactis have focussed primarily on customizing the prioritization of mixed fermentation products by re-routing lactate-pyruvate metabolism towards other industrially important products such as diacetyl, acetaldehyde and acetoin which are important flavour compounds in dairy products. This was achieved through the use of an LDH deficient L. lactis strain which consequently increased the amount of α-acetolactate in place of lactate, where the former is a reduced carboxylated form of diacetyl [To date, metabolic engineering efforts in diacetyl . This todiacetyl .L. lactis involves manipulation of the NADH:NAD+ co-factor ratio which influences fermentation patterns because the in vivo activity of several central redox enzymes, namely glyceraldehyde 3-phosphate dehydrogenase (GADPH), PDH, LDH, alcohol dehydrogenase (ADH) and NADPH oxidase (NOX), are significantly influenced by this ratio. For example, the nox gene which encodes NADH oxidase converts molecular oxygen to water at the expense of NADH. Overexpression of NOX diminishes the NADH pool, and increases NAD+, thereby re-routing pyruvate from the NADH-dependent LDH pathway to either the NADH independent ALS pathway or the NAD+ dependent PDH pathway. This strategy has been shown to be successful in shifting homolactic fermentation to mixed-acid fermentation with acetate and acetoin as main products, while producing α-acetolactate and diacetyl in small amounts [L. lactis has also been engineered to be a factory for the production of sweeteners, including the introduction of heterologous pathways or enzymes such as alanine dehydrogenase from Bacillus sphaericus for the production of l-alanine [Another common metabolic engineering strategy in  amounts . Combini amounts . In fact amounts . On a di-alanine .L. lactis have somewhat shifted towards increasing the production of non-food flavouring metabolites. Examples include the B vitamins, primarily folate (B11) and riboflavin (B2), which were overexpressed in L. lactis using the NICE system [L. lactis as a food-grade platform where the production of multivitamins from guanosine triphosphate (GTP) precursors can be increased by 3 to 10 folds following overexpression of a GTP biosynthetic enzyme (GTP cyclohydrolase I) [More recently, the emphasis of metabolic engineering in E system –51. Thesolase I) , 50.L. lactis in bacteriocin production as a bio-preservative against Listeria monocytogenes [L. lactis include lacticin 3147 [L. lactis and some favourable attributes for applications include acid stability and thermotolerance to high temperatures in addition to improvement in production systems.Other recent studies have shown the potential of ytogenes , 53 and ytogenes  via prevytogenes . Examplecin 3147 , lacticicin 3147  and LsbBcin 3147 . Howevercin 3147  as lanthcin 3147 –62 to chL. lactis has also been engineered to produce ethanol as biofuels when supplemented with cheap renewable feedstock waste products [On another note, products . A summaLactobacillus spp., L. lactis has been used as a vehicle to deliver therapeutics such as cytokines into the human body. The first evidence of such applications was published in Steidler et al. [L. lactis was used to treat inflammatory bowel disease (IBD) in colitis-induced mice. Since then, L. lactis secreting IL-10 has gone into clinical trials and concurrently ushered in the emergence of a genetically modified thymidine auxotrophic L. lactis strain for biological containment which disallows growth of the bacteria unless provided externally with thymidine or thymine [L. lactis , yet not colonize the gut unlike r et al. , where e thymine , 77. Whiis Table  for the L. lactis strains have also been investigated as treatment against food allergy such as cow’s milk allergy [L. lactis delivering IL-10 gastrointestinally prior to sensitization was able to induce immunotolerance towards the allergen, thus reducing food-induced anaphylaxis. Recombinant L. lactis producing IL-12, a T-helper 1 (Th1) bias cytokine has also been investigated for the treatment of asthma, successfully skewing the Th2 dominant immunologic response in murine models of asthma to a Th1 response which simultaneously elevates interferon gamma (IFN-γ) whilst reducing IL-4 levels [L. lactis has been used to co-produce or secrete a wide range of other adjuvants and growth factors. Successful examples include murine IL-12 [When it comes to hypersensitivity, IL-10 secreting  allergy . In this4 levels . To datene IL-12 , transfone IL-12 , insulinne IL-12 , receptone IL-12  and otheL. lactis in delivering cytokines to alleviate allergy symptoms, L. lactis has also been developed as factory for production and purification of the allergen itself. In 2007, Glenting et al. reported the production of immunologically active recombinant peanut allergen Ara 2 in L. lactis with high yields [L. lactis can simultaneously be used to deliver allergens such as the major birch allergen Bet-v1 [L. lactis are naturally great delivery vehicles for allergy immunotherapy as many non-recombinant LAB by itself, including L. lactis, have shown anti-allergic effects through their immunomodulatory effects, owing to their cell wall components and other non-established factors.Apart from the use of h yields . Recombin Bet-v1 , and then Bet-v1  through L. lactis NZ9000 was used to secrete tumour metastasis-inhibiting peptides such as KiSS1 which inhibited HT-29 cell proliferation and migration through the induction of apoptosis pathways and by down regulating matrix metallopeptidase 9 (MMP-9) expression. This suggested a possible role for L. lactis as a cell factory for colorectal cancer therapeutics [L. lactis as a microbial cell factory include subtilisin QK-2 as an anti-thrombotic agent [Recently, in the field of anti-cancer therapeutics, recombinant apeutics . Other eic agent , BT crysic agent , heat shL. lactis. An example is γ-amino butyric acid (GABA), which is a non-protein amino acid with hypotensive, anti-cancer, anti-anxiety and diuretic properties [L. lactis, GABA production can also be used to differentiate between L. lactis ssp. lactis and L. lactis ssp. cremoris as the former produces GABA while the latter does not [L. lactis includes hyaluronic acid, which is a carbohydrate polymer used in wound healing, dermatitis and cosmetic-based applications [In addition to protein- and whole cell-based therapeutics, metabolites with medicinal applications are also produced by operties , 92. Natoperties . In L. ldoes not . Other eications .L. lactis usage is as a factory for antigen production, thus allowing the bacteria to act as live vaccines. Using LAB as vaccine carriers is appealing as they are able to induce both mucosal and systemic immune responses, have adjuvant properties, and is free from risks associated with the use of conventional attenuated live pathogens such as Salmonella spp. and Mycobacterium spp. [L. lactis to surface display antigens also transforms it into the preferred host with increased immunogenicity compared to its intracellularly expressed or secreted counterparts [L. lactis involved expressing tetanus fragment toxin C (TFTC), which was highly successful in eliciting immune responses in mice, especially when administered together with IL-2 and IL-6 adjuvants [L. lactis as detailed in several past reviews with a comprehensive list of LAB-based vaccines [Without doubt, one of the most exciting aspects of modern ium spp. . When itterparts . One of djuvants . Since tvaccines –100, togL. lactis surface displaying the E7 antigen whist secreting IL-12 was shown to provide full prophylactic protection in immunized mice and was also able to induce regression of palpable tumours in tumour-induced mice [L. lactis includes glycosylated tyrosinase related protein-2 (TRP-2) tumour antigen against melanoma  [Over the past decade, the emergence of cancer vaccines developed via a lactococcal platform has also been gaining momentum following the onset of prokaryotic antigen production. These include a vaccine against human papilloma virus type-16 induced tumours where ced mice . Other c trials)  and carc trials) . The latL. lactis was also made possible without the pathogen’s clinical manifestations. A very recent example is the expressive characterization of the Streptococcus mutans surface glycoprotein, Cnm in L. lactis which was found to mediate binding to extracellular matrix (ECM) proteins in a rabbit model of infective endocarditis [L. monocytogenes have been proposed for use in DNA vaccination using L. lactis as hosts for plasmid production [L. lactis strains harbouring viral antigens such as influenza A nucleoprotein (NP) have also been studied, and shown to elicit superior immunogenicity, especially when coupled with oral adjuvants such as cholera toxin B (CTB) subunits [Being capable of heterologous protein expression, characterization of bacterial and viral virulence factors using carditis . In addioduction . Recombisubunits .L. lactis as a vaccine delivery system via oral and intranasal administration routes in chickens and ferrets [L. lactis surface using PgsA anchor motif administered orally together with (CTB) as adjuvant was also found to elicit high antigen-specific cell-mediated responses in mice when challenged with lethal dosages of H5N1 [Campylobacter jejuni [Various lactococcal-based vaccines for animal diseases have also been developed, mostly with favourable results. In poultry diseases, extensive research has been performed against the H5N1 virus, using  ferrets –109. It  of H5N1 . This de of H5N1 , H5N2 [1 of H5N1 , avian i of H5N1  and infer jejuni .L. lactis in the livestock industry was reported a decade ago, where GroEL heat shock protein from Brucella abortus was expressed and secreted as a vaccine candidate. However, its intracellular expression was shown to be unstable with a low secretion efficiency [B. abortus was found to render protective immunity against brucellosis when tested in mice [L. lactis also reported good biological activity, where symptoms and development of dextran sodium sulphate (DSS)-induced colitis in mice were attenuated [L. lactis has also made hallmarks in the aquaculture industry where lactococcal-based vaccines against Aeromonas hydrophila using D1 and D4 aerolysin genes were developed with increased survival in tilapia fish when administered intraperitoneally and orally [Streptococcus iniae membrane protein, has also incurred significant vaccinative and probiotic effects in olive flounders [One of the earliest uses of ficiency . Through in mice . Very retenuated . Use of d orally . Lactocolounders .Staphylococcus aureus which functions to increase immunomodulatory properties of L. lactis strains during mucosal delivery as a live DNA vaccine vector [L. lactis shuttle vectors such as the pNZ:vig [L. lactis as a factory for antigens and adjuvants renders it a very promising live bacterial vaccine host, consequently turning it into one of the most extensively researched areas.At present, enhancements to the lactococcal vaccine delivery system are continuously being carried out, amongst which, includes the recent incorporation of the cell-surface anchored fibronectin binding protein A (FnBPA) from e vector , 120. L. pNZ:vig  and pPER pNZ:vig  for the  pNZ:vig . It is aL. lactis has also been engineered as a cell factory for the production of both plant proteins and bioactive compounds as described in Table Arabidopsis thaliana was the first functional plant protein to be expressed in L. lactis [Pentadiplandra brazzeana, was successfully expressed, albeit in low amounts [L. lactis soon led to the metabolic engineering and consequent production of industrially applicable secondary metabolites. In 2007, alcohol acyltransferase (SAAT) and linalool/nerolidol synthase (FaNES) of strawberry were reportedly expressed in L. lactis, leading to the production of the flavouring and scent compound, linalool [Although unconventional, . lactis . A year  amounts . The estlinalool , which iPersicaria minor) [L. lactis leading to the production of germacrene D and β-sesquiphellandrene, respectively. Interestingly, L. lactis uses the mevalonate pathway (MVA) for terpenoid biosynthesis, a pathway more commonly found in eukaryotes. Most prokaryotes such as E. coli uses another terpenoid biosynthesis pathway called the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, hence almost all metabolic engineering research for heterologous plant terpenoid production have been focussed on the MEP pathway of E. coli or the MVA pathway of yeast. At present, the lactococcal MVA accommodates much room for optimization as initial attempts to metabolically engineer this pathway resulted in more than doubling of sesquiterpenes produced [Our research group has also successfully expressed two plant terpene synthases from orchid  and kesua minor)  in L. laproduced .L. lactis has been proven to be an excellent host for the expression of membrane proteins due to several advantages: (i) they are amino acid auxotrophs allowing incorporation of labels for detection, (ii) they only have a single membrane layer compared to E. coli, (iii) they have a small genome size with little proteolytic activity, and (iv) they come with extensive genetic engineering tools including the highly efficient and well tested NICE system.Membrane proteins are typically difficult proteins to express and purify due to their nature which includes low abundance, relatively high hydrophobicity, instability and various topologies including polytopic proteins with multiple transmembrane regions. Due to these limitations, only about 400 three-dimensional membrane protein structures have been elucidated compared to 40,000 soluble proteins, which accounts for a mere 0.01% . Over thL. lactis using the NICE system alone, including both prokaryotic and eukaryotic membrane proteins [L. lactis as an alternative host for membrane protein overexpression of eukaryotic expression, successfully expressing human Lys–Asp–Glu–Leu KDEL receptor and mitochondrial carriers from yeasts and fungi. Prior to this, only prokaryotic membrane protein expressions were performed in L. lactis, mostly with homologous proteins, some which were able to reach up to 30% of total membrane proteins [L. lactis was successfully used to express rat and human membrane proteins involved in liver detoxification with higher yields than conventional E. coli and Saccharomyces cerevisiae expression systems [To date, there are close to 100 membrane proteins expressed in proteins . Kunji aproteins  were theproteins . Most re systems .Lactococcus lactis has also been developed as an alternative system for the production of plant membrane proteins, using A. thaliana peripheral and intrinsic proteins as a model [A. thaliana cDNA libraries were cloned into Gateway entry vectors and transferred into a destination vector (pBS-RFA) through recombination, thus allowing for proper reading frame preservation. The gene of interest is then excised and cloned into pNZ8148 and expressed as usual using the NICE system. This method allows for the use of Gateway available cDNA libraries, which in essence, cannot be used with the lactococcal NICE system due to host incompatibility. Using this method, six A. thaliana membrane proteins were produced of which three were successfully solubilized and purified with two of them being shown to be functional [ a model . A lactonctional .L. lactis, where Lactococcal-based expression was found to be an efficient and valuable alternative to E. coli, many times complementing proteins which were unsuccessfully produced in the latter [E. coli remains the superior host in terms of production yield in most cases, the fusion of proteins with Mistic, a 13 kDa protein from B. subtilis was reported to facilitate and improve membrane protein production in L. lactis. A more recent research validated the use of Mistic in successfully boosting the expression of both eukaryotic and prokaryotic membrane protein expression in L. lactis [Using modified Gateway-compatible systems, research efforts were extended to the expression of 20 different membrane proteins from plants, human and bacteria in six different hosts including e latter . While E. lactis .L. lactis enables various heterologous genes to be expressed, its yield is very much case-dependant, with lesser problems when it comes to closely related organisms such as Streptococcus spp., Enterococcus spp., Staphylococcus spp. and low-GC Lactobacillus spp. However, the greatest obstacle still revolves around its codon usage and/or distribution of rarely used codons [Lactococcus spp. is still generally less robust when compared to the aerobically growing B. subtilis. Previously, genes encoding toxic gene products typically resulted in unsuccessful cloning attempts, further challenging the already-low transformation rate, but this was overcome by incorporating the nisA promoter in single copy on the chromosome [While manipulations involving d codons , 150. Adromosome , 152.L. lactis owing to the loosely controlled stability and small intestinal absorption of nisin, which ultimately influences the pharmacokinetics and pharmacodynamics interplay [L. lactis which remains unresolved to-date [L. lactis as a cell factory.Following up on the use of NICE system, controlling the consistency of dosages and delivery of therapeutic molecules is also difficult to predict with nterplay . Another to-date , and eluL. lactis designated GRAS status, a study documenting multiple drug efflux proteins attributing resistance to ethidium bromide was discovered in L. lactis subsp. lactis MG1363 [L. lactis as a cell factory involves ingestion or uptake into the host, there lies a risk for horizontal transfer of these efflux pumps to other commensal or potentially pathogenic bacteria. In other words, transit of L. lactis through the GIT which is frequently exposed to antibiotics may cause susceptible gene exchange with the surrounding flora, potentially leading to antibiotic resistant strains. In addition, release of chloramphenicol resistant pNZ-harbouring L. lactis strains into the environment is also a grave concern.In spite of limited systematic studies available on the acquired antibiotic resistance especially from food due to s MG1363 . Since mL. lactis as a cell factory be put in place, especially for new strains, that conform to pre-marketing safety profiling and post-marketing follow-up to determine their acceptability [L. lactis [L. lactis should be used. Examples include basis of delivered molecule, persistence of strain, robustness of the expression system, composition of different molecule subtypes and delivery to specific eukaryotic cells [L. lactis have actually entered human trials.Therefore, it is imperative that guidelines mandating the management of tability . Many la. lactis . Alternaic cells . While iic cells , which aWhen it comes to cancer vaccines, new knowledge and advancements in immunomodulation and microencapsulation technology of live lactococcal delivery vectors coupled with the richness of gut-associated lymphoid tissues (GALTs) have consequently opened up a gateway in exploiting future vaccination efforts via an oral-mucosal route, where we predict future research efforts being streamlined towards the lactococcal-based production of recombinant tumour antigens, especially against gastrointestinal malignancies over the next decade. In essence, this approach slingshots the practical use of peptide-based vaccines by overcoming its existing shortcomings such as its poor plasma stability and systemic half-life when administered in vivo.Lactococcus lactis have come a long way from being a food bacterium to a microbial cell factory for the production of industrially important products with potentially great bio-economic value, especially in the medical field. In spite of its limitations, there is still much room for improvement of the lactococcal system as a microbial cell factory since its molecular toolbox is still relatively limited compared to those available for E. coli. An expansion of said toolbox would be akin to opening a Pandora’s box, thus allowing further potential especially in terms of genetic and metabolic engineering to overcome limitations highlighted above.


Background: The lined seahorse, Hippocampus erectus, is an Atlantic species and mainly inhabits shallow sea beds or coral reefs. It has become very popular in China for its wide use in traditional Chinese medicine. In order to improve the aquaculture yield of this valuable fish species, we are trying to develop genomic resources for assistant selection in genetic breeding. Here, we provide whole genome sequencing, assembly, and gene annotation of the lined seahorse, which can enrich genome resource and further application for its molecular breeding. Findings: A total of 174.6 Gb (Gigabase) raw DNA sequences were generated by the Illumina Hiseq2500 platform. The final assembly of the lined seahorse genome is around 458 Mb, representing 94% of the estimated genome size . The contig N50 and scaffold N50 reached 14.57 kb and 1.97 Mb, respectively. Quality of the assembled genome was assessed by BUSCO with prediction of 85% of the known vertebrate genes and evaluated using the de novo assembled RNA-seq transcripts to prove a high mapping ratio (more than 99% transcripts could be mapped to the assembly). Using homology-based, de novo and transcriptome-based prediction methods, we predicted 20 788 protein-coding genes in the generated assembly, which is less than our previously reported gene number (23 458) of the tiger tail seahorse (H. comes). Conclusion: We report a draft genome of the lined seahorse. These generated genomic data are going to enrich genome resource of this economically important fish, and also provide insights into the genetic mechanisms of its iconic morphology and male pregnancy behavior. Hippocampinae) are a group of popular and iconic species because of their unique body plan and male pregnancy. As an interesting model, seahorses could provide exceptional clues for studying evolution in virtue of their closed brood pouch, male pregnancy, and seasonal migration [Hippocampus comes) [patristacin subfamily of astacin metalloproteases that may be closely related to the unusual male pregnancy in this species since they were expanded and highly expressed in the male brood pouch during mid- and late pregnancy [Syngnathidae, an interesting teleost family, exhibit special morphological innovations and reproductive behavior, and these phenotypes have come into being through long-term molecular evolution , 2. Seahigration , 4. Reces comes)  and provregnancy .H. erectus; Fig. Here, we provide a draft genome of the lined seahorse . All animal experiments conformed to the guidelines of the Animal Ethics Committee and were approved by the Institutional Review Board on Bioethics and Biosafety of BGI . Seven libraries, including three short-insert libraries  and four long-insert libraries , were constructed based on the standard protocol of Illumina  and sequenced using the Illumina HiSeq2500 platform (the read length is 125 bp). Finally, we generated a total of 174.6-Gb raw sequences.These raw sequences contained some sequencing errors, which may reduce the quality of genome assembly. Hence we filtered these raw sequences with the following stringent filtering processes through SOAPfilter (v. 2.2) software : (i) filThe genome size was estimated based on the k-mer spectrum  with theThe generated clean reads were further assembled by SOAPdenovo2 (v. 2.04)  with optBenchmarking Universal Single-Copy Orthologs (BUSCO)  is a sofde novo assembled RNA-seq transcripts from different developmental stages of the lined seahorse  to map nt paper . All thede novo predictions. First, RepeatMask (v. 3.3.0) [de novo repeat library. Then we used RepeatMask [de novo repeat library. All the repeats were finally combined together with a filtering of those redundant repetitive sequences. In total, the lined seahorse genome comprises approximately 30.43% repetitive sequences, in which 28.12% are TEs. Interestingly, the most abundant type of TE is class II DNA transposon, which covered around 15% of the genome. Our data are similar to the report of the tiger tail seahorse [Tandem repeats were searched in the generated genome assembly by utilizing Tandem Repeats Finder (v. 4.04) . Transpo. 3.3.0)  was empl. 3.3.0) . RepeatP. 3.3.0) , an upda. 3.3.0)  and Repe. 3.3.0)  were usepeatMask  to identde novo prediction of repeat-masked genome sequences. Short genes (less than 150 bp) and premature or frame-shifted genes were removed.Repetitive regions in the genome sequence were replaced with “N” to reduce the ratio of pseudogene annotations. Then we chose 1000 full-length but randomly selected genes from zebrafish homology gene set to train the model parameters for AUGUSTUS. We subsequently employed AUGUSTUS 3.0.1  and GenSDanio rerio), medaka (Oryzias latipes), fugu (Takifugu rubripes), stickleback (Gasterosteus aculeatus), and Nile tilapia (Oreochromis niloticus) were downloaded from Ensembl (release 83) [H. comes) were downloaded from our recently published genome data (Bioproject ID: PRJNA314292) [Protein sequences of zebrafish (ease 83) . ProteinA314292) . ProteinA314292)  with E-vA314292)  was applWe downloaded the transcriptome data of the lined seahorse from our previous work . The rawde novo prediction, homology-based annotation, and transcriptome-based prediction were merged to form a comprehensive and non-redundant gene set using GLEAN [The gene models based on ng GLEAN . Finallyng GLEAN .patristacin subfamily of the astacin metalloprotease family may be closely related to the unusual male pregnancy in seahorses since we identified six patristacin genes in the tiger tail seahorse and confirmed their expansion and high expression in the male brood pouch [patristacin in the lined seahorse genome. Related patristacin protein sequences were downloaded from the tiger tail seahorse genome data [patristacin genes in the lined seahorse. The RNA-seq reads were mapped by TopHat [patristacin genes were expressed during pregnancy in the male lined seahorse.The od pouch . We alsoome data  and usedome data . We chosome data  to prediome data  to confiy TopHat , and genThe protein sequences predicted from the lined seahorse genome were aligned to the Swiss-Prot and TrEMBL databases  using BlXiphophorus maculatus), and spotted gar (Lepisosteus oculatus), were downloaded from Ensembl (release 83) [H. comes) were downloaded from our recently published genome data [Sygnathus scovelli) were downloaded from the Cresko Lab web server (http://creskolab.uoregon.edu) [Protein sequences of seven ray-fin fishes, including zebrafish, medaka, fugu, stickleback, Nile tilapia, platyfish (ease 83) . Proteinome data . Proteingon.edu) . The congon.edu)  to clustWe extracted 2812 one-to-one orthologous genes from the above-mentioned gene family set. The protein sequences of each selected family were aligned using MUSCLE (v. 3.8.31)  with theSeahorses are a fascinating teleost group with special morphological innovations and reproductive behavior. In our previous genome paper about the tiger tail seahorse , we paidGigaScience database [Supporting data are available in the database , and theGIGA-D-16-00137_Original_Submission.pdfClick here for additional data file.GIGA-D-16-00137_Revision_1.pdfClick here for additional data file.GIGA-D-16-00137_Revision_2.pdfClick here for additional data file.Response_to_reviewer_comments_Original_Submission.pdfClick here for additional data file.Response_to_reviewer_comments_Revision_1.pdfClick here for additional data file.Reviewer_1_Report_.pdfClick here for additional data file.Reviewer_2_Report_.pdfClick here for additional data file.Reviewer_2_Report_(Revision_1).pdfClick here for additional data file.Reviewer_3_Report_.pdfClick here for additional data file.Reviewer_3_Report_(Revision_1).pdfClick here for additional data file.


Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, and Pneumocystis jirovecii.There is limited information on the association between colonization density of upper respiratory tract colonizers and pathogen-specific pneumonia. We assessed this association for P. jirovecii defined microbiologically confirmed cases for the given pathogens.In 7 low- and middle-income countries, nasopharyngeal/oropharyngeal swabs from children with severe pneumonia and age-frequency matched community controls were tested using quantitative polymerase chain reaction (PCR). Differences in median colonization density were evaluated using the Wilcoxon rank-sum test. Density cutoffs were determined using receiver operating characteristic curves. Cases with a pathogen identified from lung aspirate culture or PCR, pleural fluid culture or PCR, blood culture, and immunofluorescence for H. influenzae were observed in both microbiologically confirmed cases and chest radiograph (CXR)–positive cases compared to controls. Staphylococcus aureus and P. jirovecii had higher densities in CXR-positive cases vs controls. A 5.9 log10 copies/mL density cutoff for H. influenzae yielded 86% sensitivity and 77% specificity for detecting microbiologically confirmed cases; however, densities overlapped between cases and controls and positive predictive values were poor (<3%). Informative density cutoffs were not found for S. aureus and M. catarrhalis, and a lack of confirmed case data limited the cutoff identification for P. jirovecii.Higher densities of H. influenzae colonization density and H. influenzae–confirmed pneumonia in children; the association may be particularly informative in epidemiologic studies. Colonization densities of M. catarrhalis, S. aureus, and P. jirovecii are unlikely to be of diagnostic value in clinical settings.There is evidence for an association between  Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, and Pneumocystis jirovecii [Many pneumonia pathogens can also be upper respiratory tract (URT) colonizers, including irovecii . Obtainiirovecii , 8. In mirovecii .S. pneumoniae is reported elsewhere [Previous studies have suggested that children with pneumonia may have higher pathogen density in the URT compared to children without pneumonia, though there is heterogeneity by study and pathogen , 10–19. lsewhere .Haemophilus influenzae type b vaccine was used routinely at all sites except Thailand, while South Africa, The Gambia, Mali, and Kenya used pneumococcal conjugate vaccine throughout the duration of the study [The PERCH study is a multicountry, standardized evaluation of the etiologic agents causing severe and very severe pneumonia among children in sites in 7 low- and middle-income countries: Dhaka and Matlab, Bangladesh; Basse, The Gambia; Kilifi, Kenya; Bamako, Mali; Soweto, South Africa; Nakhon Phanom and Sa Kaeo, Thailand; and Lusaka, Zambia. he study . PERCH fhe study .P. jirovecii by induced sputum, pleural fluid, or lung aspirate immunofluorescence or toluidine blue staining [Identification and selection of cases and controls have been described previously . In briestaining . AntibioA flocked nasopharyngeal (NP) swab  and a rayon oropharyngeal (OP) swab (Fisher Scientific), transported in universal transport media (Copan) and processed within 24 hours of collection were used for URT pathogen detection by PCR. Blood was collected for culture. Pleural fluid specimens were collected as clinically indicated. Lung aspirates were collected from a subset of cases in The Gambia, South Africa, Mali, and Bangladesh.PERCH employed conventional and molecular diagnostic techniques for the identification of potential pathogens, as described elsewhere , 28–31. P. jirovecii; Mycoplasma pneumoniae; Chlamydophila pneumoniae; S. pneumoniae; H. influenzae type b; H. influenzae species; S. aureus; M. catarrhalis; Bordetella pertussis; Klebsiella pneumoniae; and Salmonella species. Standard curves for quantification were generated using 10-fold serial dilutions of plasmid standards provided by FTD approximately every 3 months and were used to calculate pathogen density (log10 copies/mL) from the sample cycle threshold values; standards were only available for the linear range of detection of the assay from 4.0 to 8.0 log10 copies/mL. Additionally, induced sputum, pleural fluid, and lung aspirate specimens were tested for P. jirovecii by immunofluorescence (South Africa) and toluidine blue staining (Zambia).Respiratory specimens  were evaluated using the Fast Track Diagnostics Respiratory Pathogens 33 test (FTD Resp-33) , a 33-target, 8-multiplex real-time quantitative PCR platform for the detection of selected viruses and the following bacteria and fungi: Blood cultures were incubated using automated systems . Organisms were identified according to standard microbiological methods.P. jirovecii but excluded from all other analyses.We made comparisons between community controls and both microbiologically confirmed pneumonia cases and radiographic pneumonia cases. Microbiologically confirmed cases were not restricted to children with abnormal findings on CXR in order to include children who died before a radiograph was taken or had not developed a positive finding at time of initial radiograph. Human immunodeficiency virus (HIV)–infected cases were included in a supplemental analysis for Among children positive for each organism, mean and median colonization densities were compared using Wilcoxon rank-sum tests. Potential covariates of colonization density were evaluated, including site, age, sex, vaccination status, and prior antibiotic administration . Total bP values are 2 sided.All analyses were performed using SAS software version 9.4  and R Statistical Software 3.2.1 . The PERCH study protocol was approved by the institutional review board or ethical review committee at each of the study site institutions and at the Johns Hopkins Bloomberg School of Public Health. Parents or guardians of all participants provided written informed consent.H. influenzae, M. catarrhalis, S. aureus, or P. jirovecii  were microbiologically confirmed for at least 1 organism of interest, 1733 (44.4%) had an abnormal CXR, and 1700 (43.6%) had an abnormal CXR without microbiologic confirmation of irovecii . Site saH. influenzae  were NP/OP positive for H. influenzae. Among the 21 cases, 14 (66.7%) were CXR positive. Six confirmed M. catarrhalis cases were NP/OP positive, among which 4 (66.7%) were CXR positive. Fifteen confirmed S. aureus cases were NP/OP positive, among which 10 (66.7%) were CXR positive. One confirmed P. jirovecii case was NP/OP positive and was also CXR positive. Only H. influenzae was found at significantly higher median densities in microbiologically confirmed cases compared to controls . Prevalence was similar between CXR-positive cases and controls for S. aureus and P. jirovecii. Moraxella catarrhalis detection was more common among controls than CXR-positive cases, even after adjusting for site and prior antibiotics . Median density (log10 copies/mL) was higher in CXR-positive cases compared to controls for H. influenzae, S. aureus, and P. jirovecii, while M. catarrhalis median density was similar in CXR-positive cases and controls. For all organisms, there was substantial overlap of the distribution of densities between microbiologically confirmed cases, CXR-positive cases, and controls  and M. catarrhalis (5.77 vs 5.21 log10 copies/mL) among CXR-positive cases . There were similar rates of antibiotic use prior to NP/OP specimen collection between microbiologically confirmed cases and nonconfirmed CXR-positive cases. HIV-infected, CXR-positive cases had a higher median density compared to HIV-negative, CXR-positive cases for P. jirovecii (5.03 vs 3.78 log10 copies/mL) .Though densities varied by site, trends between microbiologically confirmed cases, CXR-positive cases, and controls remained and were consistent across age categories, WHO-defined severity categories, and sex . Prior antibiotic use was associated with a lower density for 10 copies/mL), CXR-positive cases (5.81 log10 copies/mL), and controls (5.75 log10 copies/mL), and also similar among CXR-positive cases across age groups, adjusting for prior antibiotic use and site. Evaluating whether the proportion of total bacterial density attributable to specific organisms differed between case and control groups, median relative proportions of H. influenzae tended to be higher in H. influenzae–confirmed cases (41.6%) and CXR-positive cases (7.2%) compared with controls (1.2%). Conversely, median proportional densities of M. catarrhalis were higher in controls compared to CXR-positive cases (14.3% vs 5.0%). Among 100 randomly selected controls, 24% had M. catarrhalis–dominated proportional densities (≥50%) compared with 14%–16% of non–M. catarrhalis confirmed cases . However, there was substantial overlap in the range of proportional densities between microbiologically confirmed cases, CXR-positive cases, and controls.Total bacterial density across all bacterial targets from our PCR panel was similar among consolidated pneumonia cases  was moderately sensitive and specific, the S. aureus cutoff did not significantly improve sensitivity and specificity over qualitative analysis using simply presence or absence of specific organisms.Using density in logy of 77% . Though P. jirovecii (n = 1) and M. catarrhalis (n = 6) cases, we explored the use of NP/OP positive, CXR-positive cases and controls to identify potential cutoffs for P. jirovecii (4.0 log10 copies/mL) and M. catarrhalis (5.0 log10 copies/mL) . Densities among the limited number of microbiologically confirmed cases were consistent with these cutoffs, as 5 of 6 microbiologically confirmed M. catarrhalis cases were above the thresholds. The P. jirovecii cutoff demonstrated 48% sensitivity and 72% specificity for identifying P. jirovecii–positive CXR-positive cases (AUC = 0.58), among cases and controls positive for P. jirovecii (98% specificity including negatives). Moraxella catarrhalis cutoffs did not help differentiate CXR-positive cases from controls (AUC = 0.50).Given the limited numbers of NP/OP-positive microbiologically confirmed H. influenzae (1.01% to 1.75%), S. aureus (1.81% to 2.14%), and P. jirovecii (0.89% to 1.67%), though the PPV is limited by lack of sensitivity in identifying microbiologically confirmed cases. Conversely, negative predictive values for identifying H. influenzae, S. aureus, and P. jirovecii microbiologically confirmed cases remained >99% for any detection in the URT and when utilizing the density cutoff.Positive predictive values (PPVs) for identifying microbiologically confirmed cases from all cases using dichotomous URT positivity were low and improved marginally when density cutoffs were applied to H. influenzae, adjusted for site, age, sex, and prior antibiotic use. Additionally, having a density above the cutoff was associated with case fatality for P. jirovecii .Densities above the cutoffs (compared to low densities and negatives) were associated with hypoxemia , any CXR abnormality , and primary endpoint consolidation  for H. influenzae was significantly higher in microbiologically confirmed cases compared with community controls. Given the high prevalence of H. influenzae as a common colonizer, the improved specificity provided by the optimized cutoff of 5.9 log10 copies/mL helped delineate between common colonization and H. influenzae–mediated pneumonia. However, the overlapping density distributions between all case and control groups, and the low PPV limit the utility of density in individuals for clinical diagnosis. Our findings are similar to results from a study in adult patients in Denmark that yielded 90% sensitivity and 65% specificity using a cutoff at 5.0 log copies/mL [H. influenzae density and radiographic pneumonia; however, findings may have been limited by lack of a confirmed case group [Colonization density of the URT with opies/mL . Anotherse group .P. jirovecii may provide information toward differentiating carriage and pathogen-mediated pneumonia, although our evaluation was limited by a lack of microbiologically confirmed pneumocystis pneumonia (PCP) cases. A potential cutoff at 4.0 log10 copies/mL conferred 50% sensitivity and 72% specificity for radiographic pneumonia; however, the proportion of radiographic pneumonia cases with PCP-mediated pneumonia is unknown, and the PERCH process for identifying CXR abnormality may have been less sensitive for detecting radiographic features uniquely associated with PCP. The potential density cutoff is in line with other studies that have identified cutoffs between approximately 3 and 4.5 log copies/mL using clinically confirmed PCP patients, though we have not standardized the quantitative standards across these different studies [P. jirovecii detection from induced sputum PCR is widely accepted in clinical practice and would have expanded our sample of microbiologically confirmed cases [Colonization density of  studies . Utilizied cases , 36. Howed cases .M. catarrhalis and S. aureus [S. aureus cutoff was specific, the relatively high proportion of NP/OP negatives among the microbiologically confirmed cases precludes the identification of a highly sensitive and specific cutoff. It has been suggested that Moraxella and Corynebacterium/Dolosigranulum-dominated microbiota profiles confer stability and are protective against respiratory disease, while Streptococcus- and Haemophilus-dominated microbiota profiles enhance susceptibility to respiratory infections [M. catarrhalis is supported by our finding of both higher prevalence, proportional density, and absolute density in controls compared to radiographic pneumonia cases.Previous studies have failed to identify an association between density and pathogen-confirmed pneumonia for . aureus . While tfections , 39. TheH. influenzae and M. catarrhalis. However, because prior antibiotic use was more common among cases compared to controls, the bias was toward the null for the analyses. Staphylococcus aureus and P. jirovecii are unlikely to be significantly affected by first-line antibiotics, which was reflected in our findings [Though our analysis benefited from a large sample size enrolled from multiple heterogeneous study sites and comprehensive clinical and laboratory standardization, there were limitations. Despite our large overall sample size, the number of microbiologically confirmed cases was limited. However, leave-one-out cross validation and findings from comparisons of CXR-positive cases vs controls supported findings from microbiologically confirmed cases. While we were able to evaluate co-pathogen interactions and proportional densities between a select number of potential pathogens, a metagenomics or microbiome approach would be better suited to understand the contribution of individual pathogens in the context of the upper respiratory tract microbiome . Furtherfindings . VariabiH. influenzae, although the PPV was limited by poor sensitivity for identifying microbiologically confirmed cases and low prevalence. Regardless, it is likely impossible to identify a URT density cutoff for these colonizers that could be applied as a clinical gold standard because the densities overlap and span the PCR linear range in both cases and controls, precluding the identification of clear cutoffs that would independently guide clinical decisions.While we have provided cutoffs optimized for sensitivity and specificity, the choice of a cutoff can be tailored for specific applications. Identifying a density cutoff that maximizes combined sensitivity and specificity provides additional information from the NP/OP specimen in the PERCH primary etiology analysis; however, the interpretation and application of these cutoffs may not be ideal for use as either a diagnostic or screening tool . Moreover, the PPV using the density cutoff remained low for H. influenzae colonization density and H. influenzae–mediated pneumonia in children, and also a potential association between P. jirovecii colonization density and pathogen-specific pneumonia. The use of molecular diagnostics from URT specimens provides significant advantages in both sensitivity and speed over traditional culture diagnostics, but ascribing lung infection based on detection of colonization in the URT is challenging. Compared to using presence or absence of positivity, utilizing colonization density improves specificity of molecular diagnostics with small reductions in sensitivity and improves information from the URT in the context of population-level epidemiologic studies such as the PERCH integrated analysis, but remains suboptimal for use as a gold standard diagnostic in clinical settings at the individual case level.We found evidence for the relationship between Clinical Infectious Diseases online. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplemental MaterialsClick here for additional data file.


Using a fully factorial precipitation by warming experiment in an old-field ecosystem in the northeastern USA we studied the climatic sensitivity of seedlings of six native tree species. Warm and dry conditions suppressed seedling growth, but affected species differently by increasing mortality, enhancing rates of herbivory or decreasing foliar carbon uptake. Our results indicate that, in the northeastern USA, dry years in a future warmer environment could have damaging effects on the growth capacity of early secondary successional forests, through species-specific effects on leaf production, herbivory and mortality. Acer rubrum, Betula lenta, Prunus serotina, Ulmus americana), primarily as a result of reduced foliar C, not increased foliar N. Interestingly, these conditions also harmed the other two species in different ways, increasing either mortality (Populus grandidentata) or herbivory (Quercus rubra). Specific leaf area and LWC varied across species, but did not show strong treatment responses. Our results indicate that, in the northeastern USA, dry years in a future warmer environment could have damaging effects on the growth capacity of these early secondary successional forests, through species-specific effects on leaf production , herbivory and mortality.Predicting the effects of climate change on tree species and communities is critical for understanding the future state of our forested ecosystems. We used a fully factorial precipitation  by warming  experiment in an old-field ecosystem in the northeastern USA to study the climatic sensitivity of seedlings of six native tree species. We measured whole plant-level responses: survival, total leaf area (TLA), seedling insect herbivory damage, as well as leaf-level responses: specific leaf area (SLA), leaf-level water content (LWC), foliar nitrogen (N) concentration, foliar carbon (C) concentration and C:N ratio of each of these deciduous species in each treatment across a single growing season. We found that canopy warming dramatically increased the sensitivity of plant growth (measured as TLA) to rainfall across all species. Warm, dry conditions consistently reduced TLA and also reduced leaf C:N in four species ( Climate change is warming air temperatures and shifting precipitation patterns , which wStudies have shown that plant tolerances and responses to climatic conditions change over the course of development . AlthougField data from multilevel temperature and precipitation experiments can be used to refine or benchmark the processes within current-generation climate models  area (TLA) and specific leaf area (SLA: leaf area per unit leaf mass) are key indicators of a plant’s carbon and water balance , serve aIn general, drier conditions typically suppress TLA, which reduces water loss, and increase biomass allocation to roots, which increases water uptake capacity , increasChanges in temperature and precipitation regimes are also known to directly influence rates of plant nutrient uptake and allocation of nutrients to leaf tissue . If photAcer rubrum (red maple), Betula lenta (sweet birch), Populus grandidentata (big-toothed aspen), Prunus serotina (black cherry), Quercus rubra (red oak) and Ulmus americana (American elm). Most of these species have large current and projected ranges that span much of the eastern and Midwestern USA , and foliar carbon and nitrogen concentration in seedlings of six tree species: sitivity , as wellsitivity , doubledB. lenta and P. grandidentata; their native ranges are more restricted to cooler and wetter regions  tree seedling survival, (ii) estimated TLA and (iii) leaf herbivory damage. We expected warming to increase these variables under wet and ambient conditions, but decrease them under drier conditions.We also tested the following leaf-level hypotheses: (i) LWC will decline across the gradient of increased warming and with reduced precipitation, and will be lowest with the combination of increased warming and drier conditions. (ii) SLA and (iii) foliar N will increase (lowering C:N ratio) with warming and with added precipitation, but will decline with the combination of increased warming and drier conditions.We expected whole plant- and leaf-level responses to scale linearly with the magnitude of warming.−1 (This research was conducted at the Boston-Area Climate Experiment (BACE), a multifactorial climate manipulation experiment in an old-field ecosystem at the University of Massachusetts’ Suburban Experiment Station in Waltham, MA, USA . The site experiences a mean annual temperature of 9.3 °C, a mean annual precipitation of 1194 mm yearThe BACE utilizes a full-factorial, split-plot, randomized block design with the precipitation treatment as the whole-plot factor and the temperature treatments as subplot factors nested within the precipitation treatment. Four levels of warming crossed with three levels of precipitation produced 12 different climate regimes, each of which was replicated three times for a total of 36 plots (2 m × 2 m in size). The four levels of warming were unwarmed (ambient temperature), low warming (+~1.3 °C multi-year average), medium warming (+~2.7 °C multi-year average) and high warming (target of +4.0 °C), and the three levels of precipitation were ambient rainfall, 50 % reduced rainfall (dry) and 50 % increased rainfall during the growing season (wet). Warming values achieved varied by season, leaf angle, heater response time and vegetation cover. Models project that the northeastern USA will warm by 2–3 °C by the end of the 21st century  mounted 1 m above the ground at each corner of every plot, facing towards the centre of the plot and downward at a 45° angle. The wattage varied by treatment: 200, 600 and 1000 W heaters for the low, medium and high warming treatments, respectively. Plant canopy temperatures of all ambient and high warming plots were monitored every 10 s by infrared radiometers . For each group of three warmed plots within a precipitation zone, power to all heaters was on a single circuit, and was continually adjusted based on the temperature difference between the unwarmed and high warming plots in that zone. The temperature control algorithms were designed to achieve a 4 °C difference between the unwarmed and high warming treatments when possible. Intermediate treatments received proportionally less warming because the heaters surrounding these plots had lower wattages [see Precipitation treatments were achieved using a roof of evenly spaced, clear polycarbonate slats that collected half of the precipitation over the dry treatments (year-round) and immediately delivered it to the wet treatments via an overhead sprinkler system (May–November). Soil moisture was fully recharged to water holding capacity during the winter months in all plots . In ordeWithin each precipitation main plot, the four warming split-plots were arranged linearly from ambient to high, and spaced 1 m apart. In 2007, a 0.6 m deep trench was dug around each split-plot, and lined with polyethylene sheets to prevent lateral movement of water and nutrients between split-plots. All treatments were in effect as of July 2008. The herbaceous vegetation within and around the split-plots was clipped twice per growing season. θR) using pairs of time-domain reflectometry (TDR) waveguides installed vertically to provide integrated measures of volumetric soil moisture in the top 10 cm and top 30 cm; calculations in Within each of the 36 split-plots, soil moisture was monitored weekly as relative extractable water (A. rubrum (red maple), B. lenta (sweet birch), P. grandidentata (big-toothed aspen), P. serotina (black cherry), Q. rubra (red oak), U. americana (American elm), Betula populifolia (grey birch) and Pinus strobus (white pine). Due to time and resource limitations B. populifolia and P. strobus were not measured in this study, leaving six species and 864 tree seedlings that were measured. Whole plant-level measurements  as well as leaf-level responses  were calculated to determine seedling responses to the treatments. All measurements were completed only over the 2011 growing season.Within each of the 36 split-plots, four evenly spaced 0.5 m × 0.5 m subplots were cleared of grasses and forbs and designated for tree seedling planting. One seedling (<20 cm in height) of each of eight tree species was planted into each of these subplots in late April 2011, resulting in a total of 1152 seedlings planted. The eight tree species planted were: Seedling mortality counts were made by comparing the number of seedlings alive in late April as compared to late July. Dead seedlings were not included in the leaf-level analyses. In order to estimate total leaf production without destructively harvesting across the peak growing season, we used length and width measurements to develop species-specific leaf size categories . The mean leaf area for each size category of each species was calculated across all individuals. The estimated TLA for each individual tree seedling was then calculated as the number of leaves observed within each size category multiplied by the species-specific measured mean leaf area for each category, and summed across size categories.Leaf samples were dried to a constant mass at 65 °C to determine dry weight and leaf-level water content (LWC) was calculated by subtracting dry weight from wet weight, and dividing by wet weight. Specific leaf area was measured as leaf area divided by dry weight. One small size category, dried leaf sample from each tree seedling was then ground and analysed for carbon and nitrogen concentration on a Costech ECS 4010 elemental analyzer .2), SLA (cm2 g−1), leaf herbivory (%), LWC (%), leaf tissue percent nitrogen by weight (%N) and leaf C:N ratio (g g−1) were analysed for all species together using linear mixed-effects models. Specific leaf area, LWC and herbivory were averaged for each individual tree. The mixed-effects models included a fully factorial combination of precipitation treatment, warming treatment and species as fixed effects. Block, precipitation treatment within block and warming treatment within precipitation treatment within block were included as random effects in each model to represent the experimental design of BACE. For model fitting, we used the ‘lmer’ function from the lme4 package . To evaluate the level of significance for each fixed effect, we calculated a Wald χ2 statistic and performed a Type-II Wald test using the ‘ANOVA’ function in the ‘car’ package  tree mortality; only 7, 8 and 19, respectively, out of 144 each. Prunus serotina and A. rubrum had moderate levels of mortality (43.1 and 45.8 %) with 82 and 78 of each species surviving. The highest mortality rate (61.9 %) was in P. grandidentata; only 55 out of the 144 seedlings survived. Among the species with >15 % mortality, only P. grandidentata showed a significant response to the treatments. Both warming and reduced precipitation caused greater P. grandidentata mortality , but precipitation did not have an effect on other species (precipitation by species: P < 0.05). Total leaf area was 19 and 58% lower in medium and high warming, respectively, compared to no warming in B. lenta, but warming did not have a direct effect in other species .Total leaf area was reduced by 83 % under the combination of reduced precipitation and warming as compared to controls . Rates of herbivory on Q. rubra were greatest in the combined reduced precipitation, high warming plots, showing an increase of 137 % as compared to the ambient precipitation, no warming plots .Specific leaf area varied by species, with the highest values seen in Q. rubra by 35 % . In addition, the highest level of warming reduced the LWC of B. lenta by 21 % as compared to the unwarmed plots .In general, LWC was relatively unaffected by the treatments. However, dry treatments reduced LWC of U. americana increasing leaf N by 84 % in the driest and warmest plots compared to plots with reduced precipitation . Ulmus americana was also the only species to respond significantly  and reductions in water supply (i.e. soil moisture) . Using fogically . Howeverogically , suggestogically . InteresQ. rubra leaf metabolite responses at BACE, As we did not measure what types of insect herbivores were consuming the leaves, we cannot be sure whether they were attracted to the leaves or present in higher numbers within the high temperature, dry plots. In a study of Under favourable conditions (e.g. warm and wet), plants tend to produce higher SLA . This suUlmus americana had the overall lowest foliar N level, and was the only species to respond to the treatments, with greater leaf N concentrations in the warmer and drier plots. We expected foliar N to increase (and foliar C:N to decrease) with warming and added precipitation. However, the treatments had no consistent effect on foliar percent N . Ulmus aP. grandidentata and Q. rubra  more strongly than the other species, and B. lenta TLA was most strongly suppressed by warming , parallel those of other studies from the BACE site , with further support from NSF (DEB-1146279) and the U.S. Department of Energy’s Office of Science (BER), through the Terrestrial Ecosystem Science programme and the Northeastern Regional Center of the National Institute for Climatic Change Research. Faculty and student summer research funds were provided by the Babson Faculty Research Fund. Partial support for J.S.D.’s participation in this project was provided by the USDA’s National Institute of Food and Agriculture, through Hatch project 1000026 and AFRI grant 2015-67003-23485. This is paper no. 1821 of the Purdue Climate Change Research Center (PCCRC).V.L.R., S.S.H. and J.S.D. conceived of the project. V.L.R., N.G.S. and S.S.H. took the field measurements. V.L.R. prepped lab samples. N.G.S. and S.S.H. analysed the data. All authors contributed to the writing of the article.None declared.The following additional information is available in the online version of this article—Table S1. Results from post hoc Tukey’s tests for TLA, LWC and C:N across precipitation treatments. Key: SE, standard error of the least square means; Ddf, denominator degrees of freedom; lowerCL, lower confidence limit; upperCL, upper confidence limit; Group, indicates statistically different mean values for treatment within species based on Tukey’s HSD (α = 0.05). Ambient denotes treatments receiving ambient precipitation.Table S2. Results from post hoc Tukey’s tests for TLA, LWC and C:N across warming treatments. Key: SE, standard error of the least square means; Ddf, denominator degrees of freedom; lowerCL, lower confidence limit; upperCL, upper confidence limit; Group, indicates statistically different mean values for treatment within species based on Tukey’s HSD (α = 0.05). Control denotes the unwarmed treatments.Table S3. Specific leaf area (SLA) by species (mean ± SE) with different lower case letters indicating significant differences (P < 0.05) from a Tukey’s HSD test.Figure S1. Hourly canopy temperature averaged across plot type for DOY 121-239 in the added precipitation , ambient precipitation  and reduced precipitation  over the course of the experiment. Darker colours within each precipitation treatment indicate higher levels of warming . Means are for the three plot types over 119 days (n = 357).Figure S2. Estimated total leaf (surface) area in late July (±SE) for all species averaged together in the wet (dark grey triangles), ambient (grey circles) and dry (light grey squares) precipitation treatments across the four warming treatments.Figure S3. Leaf level insect herbivory  for A. rubrum, B. lenta, P. grandidentata, P. serotina, Q. rubra and U. americana in the wet (dark triangles), ambient (grey circles) and dry (light squares) precipitation treatments across the four warming treatments measured in late July.Figure S4. Specific leaf area (mean ± SE) for A. rubrum, B. lenta, P. grandidentata, P. serotina, Q. rubra and U. americana in the wet (dark triangles), ambient (grey circles) and dry (light squares) precipitation treatments across the four warming treatments measured in late July.Figure S5. Leaf-level water content  of A. rubrum, B. lenta, P. grandidentata, P. serotina, Q. rubra and U. americana in the wet (dark triangles), ambient (grey circles) and dry (light squares) precipitation treatments across the four warming treatments measured in late July.Figure S6. Foliar carbon by weight  of A. rubrum, B. lenta, P. grandidentata, P. serotina, Q. rubra and U. americana in the wet (dark triangles), ambient (grey circles) and dry (light squares) precipitation treatments across the four warming treatments measured in late July.Supporting InformationClick here for additional data file.


Burkholderia ubonensis is commonly co-isolated from environmental specimens harbouring the melioidosis pathogen, Burkholderia pseudomallei. B. ubonensis has been reported in northern Australia and Thailand but not North America, suggesting similar geographic distribution to B. pseudomallei. Unlike most other Burkholderia cepacia complex (Bcc) species, B. ubonensis is considered non-pathogenic, although its virulence potential has not been tested. Antibiotic resistance in B. ubonensis, particularly towards drugs used to treat the most severe B. pseudomallei infections, has also been poorly characterised. This study examined the population biology of B. ubonensis, and includes the first reported isolates from the Caribbean. Phylogenomic analysis of 264 B. ubonensis genomes identified distinct clades that corresponded with geographic origin, similar to B. pseudomallei. A small proportion (4%) of strains lacked the 920kb chromosome III replicon, with discordance of presence/absence amongst genetically highly related strains, demonstrating that the third chromosome of B. ubonensis, like other Bcc species, probably encodes for a nonessential pC3 megaplasmid. Multilocus sequence typing using the B. pseudomallei scheme revealed that one-third of strains lack the “housekeeping” narK locus. In comparison, all strains could be genotyped using the Bcc scheme. Several strains possessed high-level meropenem resistance (≥32 μg/mL), a concern due to potential transmission of this phenotype to B. pseudomallei. In silico analysis uncovered a high degree of heterogeneity among the lipopolysaccharide O-antigen cluster loci, with at least 35 different variants identified. Finally, we show that Asian B. ubonensis isolate RF23-BP41 is avirulent in the BALB/c mouse model via a subcutaneous route of infection. Our results provide several new insights into the biology of this understudied species.The bacterium  Burkholderia pseudomallei causes the disease melioidosis, which occurs in most tropical regions across the globe. The true burden of melioidosis is unknown but has been predicted to affect 165,000 people every year, resulting in 89,000 deaths. B. pseudomallei is easily confused with its close relative B. ubonensis as both species are frequently found in the same environmental niche and can appear phenotypically identical using serotyping and laboratory culture methods. B. ubonensis is a poorly characterised species but has recently gained interest in the research community as a potential biocontrol agent in B. pseudomallei-endemic regions, and for production of unusual and versatile biocompounds that are now being exploited for industrial applications. B. ubonensis is thought to be non-pathogenic, although other members of the B. cepacia complex to which it belongs are known for their ability to cause clinical disease that can be fatal in immunocompromised patients and people with cystic fibrosis. In this study, we investigated the biology of B. ubonensis to better understand its genetics, genomics, global distribution, virulence potential and antibiotic resistance. We show that this organism is highly genetically diverse, is avirulent in the mouse model, and can naturally encode high levels of meropenem resistance. We also identify B. ubonensis in the Caribbean for the first time, with phylogenomic analysis revealing distinct clades corresponding to geographic origin.The pathogenic bacterium  B. ubonensis is a member of the Burkholderia cepacia complex (Bcc) [B. ubonensis have been proposed as potential agents in biocontrol against Burkholderia pseudomallei [The Gram-negative soil- and water-dwelling bacterium ex (Bcc) , a genetex (Bcc) . The Bccex (Bcc) . Many Bcex (Bcc) . Novel cdomallei  and in bdomallei .B. pseudomallei, the causative agent of the tropical infectious disease melioidosis, is frequently isolated from the same soil samples as B. ubonensis in regions where both species are endemic [B. pseudomallei is not a part of the healthy human flora, the ‘gold standard’ method for melioidosis confirmation is growth of B. pseudomallei from clinical specimens. For maximum isolation of B. pseudomallei from non-sterile sites such as sputum and pus, clinical laboratories require selective culture methods such as Ashdown’s agar containing gentamicin [B. pseudomallei from microbiologically complex environmental samples such as soil and surface water, which would otherwise yield growth and dominance of many other species [B. ubonensis is the most commonly co-isolated species when using B. pseudomallei enrichment methods in the melioidosis-endemic “Top End” of the Northern Territory, Australia, in part due to the indistinguishable nature of certain B. ubonensis and B. pseudomallei morphotypes [B. pseudomallei O-antigen type B is found in 25% of B. ubonensis strains from Australia [ endemic . Melioid endemic . As B. pntamicin  and Ashdntamicin . These m species . We havephotypes . In addiustralia , furtherB. ubonensis, although a clearer picture is emerging. The first B. ubonensis isolate  was reported in 2000 [B. ubonensis genome  was sequenced to closure in 2015 [B. pseudomallei genome. In Bcc species, the third replicon, a megaplasmid called pC3 (formerly chromosome III), has been shown to be important for stress resistance, virulence, and antifungal and proteolytic activity in several strains [B. ubonensis has been achieved in vitro, pC3 loss in wild-type B. ubonensis strains has not yet been identified.Little is currently known about the population biology and genomics of  in 2000 , and the in 2015 . The MSM strains . This reB. pseudomallei isolates have been fully susceptible to meropenem [B. vietnamiensis, B. cepacia and B. cenocepacia [B. pseudomallei [Burkholderia species in the environment and potentially to the globally important pathogen B. pseudomallei.Previous work has shown that Bcc species can encode for innate high-level resistance towards many clinically relevant antibiotics, including the carbapenem antibiotic meropenem . Meropeneropenem , althougeropenem . Certainocepacia , as welldomallei , 24, exhB. ubonensis from Australia and Asia. In addition, we identify the first B. ubonensis isolates from the Caribbean. Using large-scale comparative genome analysis, we interrogated 264 B. ubonensis genomes to better understand the geographic distribution and genetic diversity of this species, including potential loss of the pC3 megaplasmid. We also explored rates of meropenem resistance in Asian and Australian B. ubonensis strains, lipopolysaccharide (LPS) O-antigen cluster prevalence and diversity, and the virulence potential of an Asian B. ubonensis strain in the BALB/c mouse model.The current study describes the first comprehensive analysis of the population biology of B. ubonensis isolates were recovered has been previously described [Procedures and ethics approval for collection of the environmental specimens from which the escribed , 25. TheB. ubonensis isolates examined in this study originated from northern Australia (n = 238), Central Australia (n = 4), Ubon Ratchathani, Thailand (n = 15), Papua New Guinea , and Puerto Rico (n = 6), and were obtained from samples of soil (n = 160), water (n = 15), or plant material (n = 2)  . DNA wastime PCR , which ts MSMB22 ).https://github.com/dsarov/MGAP---Microbial-Genome-Assembler-Pipeline), which incorporates Trimmomatic [https://github.com/tseemann/VelvetOptimiser), GapFiller [B. ubonensis MSMB0022 genome [Genomic data were already publicly available for 230 of the 264 isolates , 27. Formmomatic , Velvet mmomatic , VelvetOapFiller  and SSPAapFiller  into its2 genome  as a ref2 genome . ReferenB. ubonensis genomes for phylogenetic analysis. B. ubonensis MSMB0022 was used as a reference genome for paired-end read alignment. BEDTools [The default settings of SPANDx v3.0  were useBEDTools , which iB. pseudomallei K96243 , B. ubonensis MSMB0057 , B. pseudomallei 576 , B. ubonensis MSMB0122 , B. pseudomallei MSHR0840 , B. thailandensis 82172  and B. humptydooensis MSMB0043 . B. ubonensis type strains for determining the prevalence of novel LPS O-antigen genotypes were: A21, BDU9, BDU12, BDU14, INT1-BP158, MSMB0022, MSMB0054, MSMB0063, MSMB0083, MSMB0103, MSMB0268a, MSMB0609, MSMB0742, MSMB0782, MSMB0827, MSMB1058, MSMB1137, MSMB1172, MSMB1173, MSMB1178, MSMB1189, MSMB1206, MSMB1304, MSMB1471, MSMB1517, MSMB1586, MSMB1591, MSMB2105, MSMB2123, MSMB2166, MSMB2180, MSMB2207, RF23-BP17, and RF32-BP11. Sequence coordinates for these LPS O-antigen clusters were extracted from MGAP-assembled genomes based on Mega BLAST analysis against the MSMB0057 O-antigen biosynthesis cluster [BEDTools was also used to determine LPS O-antigen type based on mapping quality against both known and novel LPS O-antigen clusters. Known clusters included In silico MLST was carried out on all isolates using the Bcc scheme (http://pubmlst.org/bcc/), and on 173 of the 264 B. ubonensis isolates based on the B. pseudomallei scheme . Ninety-one strains could not be genotyped using the B. pseudomallei scheme as they lack the narK housekeeping locus [ng locus . Sequencng locus . ST assihttp://tree.bio.ed.ac.uk/software/figtree/) was used to visualise PAUP outputs.The maximum parsimony function of PAUP v4.0a153  was usedin vitro, phylogenetically unrelated B. ubonensis strains MSMB0782 and MSMB1215 were passaged five times on Ashdown’s agar (37°C for 24-48h), and strains INT1-BP274 and RF23-BP41 were passaged 10 times. MSMB0782 and INT1-BP274 were also subjected to five freeze/thaws ranging from -80°C to room temperature, and INT1-BP274 was passaged seven times at 42°C or room temperature. Eighteen colonies of MSMB2036, which is the same ST as the pC3-negative strain MSMB2035, were then examined for pC3 loss by passaging once on Luria-Bertani agar and growing at 37°C for 48h. DNA from all laboratory-passaged strains was extracted using a chelex heat soak procedure [To promote pC3 loss rocedure  and dilurocedure . MSMB203B. ubonensis strains  were used to determine meropenem MICs in 40  strains . This suB. ubonensis to cause disease via the subcutaneous (sc) route of infection was examined in a murine BALB/c model using a Thai environmental isolate, RF23-BP41  when delivered via the intraperitoneal [ad libitum throughout the study. Mice were lightly anaesthetised with vaporised isoflurane and injected via a single 100μL sc injection in the scruff of the neck. All mice in a single cage received the same infectious dose . Three infection control mice were injected in an identical way, but with 100μL of sterile 1x PBS instead of bacterial culture. Mice were monitored daily for health status and euthanased on day 21 post-injection with CO2 gas followed by exsanguination.The ability of F23-BP41 , collectritoneal , 43 or aritoneal  routes. ritoneal . After sB. ubonensis is not known. To date, strains have only been reported from the environment in Wuhan, China [B. ubonensis in the Caribbean environment for the first time, with six isolates retrieved from soil obtained from the north-central and north-eastern regions of Puerto Rico . A recent study of soil samples in the southern United States to determine the presence of Burkholderia spp., and particularly B. pseudomallei, did not yield a single B. ubonensis or B. pseudomallei isolate, although several other Bcc species were retrieved [B. ubonensis nor B. pseudomallei are naturally found in the environment in North America. It remains to be determined whether B. ubonensis is found in other melioidosis-endemic regions such as Africa, Central America, the Indian Ocean islands, South America or South Asia.The true global distribution of n, China , Ubon Ran, China , northern, China , and PNGn, China . In thisetrieved . It is tB. ubonensis phylogeny was reconstructed from 264 genomes derived from Australian, Thai, PNG and Puerto Rican isolates to determine the existence of a continental phylogeographic signal, a phenomenon that has been described in B. pseudomallei [B. ubonensis isolates (n = 240), whereas Clade I contained all isolates from Thailand (n = 15), the PNG isolate A21, and two Australian strains from the tropical “Top End” region of the Northern Territory, and Clade III was comprised of the six Puerto Rican isolates , with the remaining four comprising all Puerto Rican  and Clade IV (n = 14) isolates, plus 57 isolates within Clade VI that were isolated from various “Top End” locales. These results show that certain B. ubonensis strains cannot be fully genotyped with the B. pseudomallei MLST scheme. However, in three instances where strains could be genotyped, the B. pseudomallei scheme was superior at differentiating strains that were related yet distinct on a genomic level  showed high-level resistance (≥32 μg/mL) towards this antibiotic, including all six Puerto Rican strains. In contrast, two Australian strains (MSMB1215 and MSMB2152) exhibited the lowest MICs at 2–3 μg/mL . Both hinem MICs . Althougnem MICs . The lacB. ubonensis, although the basis for this resistance and its persistence in Bcc populations is not clear. In comparison, the highest meropenem MICs recorded for B. pseudomallei to date are ~4 μg/mL [B. pseudomallei, where human-to-human transmission is exceptionally rare and where infections are almost always acquired from the environment [B. ubonensis infections and the identification of high-level meropenem resistance in environmental samples argue against this route of selection in the context of B. ubonensis. B. pseudomallei does not encode a carbapenamase, which likely explains why high-level resistance has not been reported. However, it is conceivable that B. pseudomallei may acquire a carbapenamase whilst residing in the environment, especially from closely related species that share this niche, such as B. ubonensis or other Bcc species. Determining the molecular basis for high-level meropenem resistance in B. ubonensis and in other Bcc species should be a focus of future studies to not only promote a better understanding of resistance mechanisms in these species, but to also provide a basis for proactive monitoring of B. pseudomallei populations in the event of carbapenamase acquisition.Many other Bcc species strains can exhibit high-level meropenem resistance , 52, ind~4 μg/mL , 54, witironment , Bcc speironment . The selB. ubonensis is a highly diverse species. We found 128 STs among the 173 strains that could be genotyped using the B. pseudomallei MLST scheme, and 182 STs among the 264 strains based on the Bcc scheme, although these numbers underestimate diversity due to multiple related isolates being tested from single environmental specimens in our study  of these STs were found within a single sample; such samples are likely to be identical or clonally related due to their physical proximity. We next examined B. ubonensis diversity within our environmental samples. Of the 51 samples where two or more B. ubonensis isolates were retrieved, 26 (51%) exhibited two or more STs, revealing that multiple B. ubonensis genotypes commonly exist within single environmental samples. This result reflects similar observations made in studies examining B. pseudomallei diversity in environmental samples from Thailand [B. vietnamiensis in the United States [B. cepacia genomovar III (now known as B. cenocepacia) in the United States, Canada and Australia [MLST revealed that ur study . Among tThailand , 58, B. d States , and B. ustralia . Whilst B. ubonensis genomes against the MSMB0022 reference showed that 2.78Mbp (39%) of the B. ubonensis reference genome was variably present, with the remaining 4.41Mbp conserved across these strains. Ten phylogenetically unrelated strains  failed to map reads against the entire sequence for pC3, equating to one-third of the variable regions observed in our dataset  also observed that four of 110 Bcc isolates tested in their study (4%) had lost pC3, with one of these events having been confirmed to have occurred following laboratory passage [B. ubonensis reference genome was variable among the 264 strains.Gene presence/absence analysis of the 264  dataset . Certain dataset . This reB. ubonensis. However, certain growth conditions appear to encourage pC3 segregation, raising the possibility that this replicon may be a megaplasmid [B. ubonensis strains MSMB0782, MSMB1215, INT1-BP274 and RF23-BP41 of pC3 by performing laboratory passage and growth under varying conditions, including multiple freeze/thaws, growth at 42°C and room temperature, or multiple passages. Despite these attempts, none were successful at segregating pC3. To examine whether an insufficient number of colonies were being tested, we next attempted passage of 18 colonies of MSMB2036, which is closely related to the pC3-lacking strain MSMB2035. Four (22%) colonies lost pC3 after a single passage on Luria-Bertani agar at 37°C for 48h, as observed by a lack of amplification using the Bu_pC3 primers. This finding demonstrates that, as with other Bcc species, the third replicon of B. ubonensis is not necessary for the organism’s survival, at least in a laboratory setting. It remains to be determined whether pC3 replicates independently of the two chromosomes in B. ubonensis. It has been proposed that the second (and where applicable) third ‘chromosomes’ found in approximately 10% of bacterial genomes are in fact ‘chromids’, a term used to define replicons that are not strictly chromosomes or plasmids [The conservation of pC3 and its phylogenetic relatedness to chromosomes I and II confirms that pC3 is under strong selection pressure to be maintained in Bcc species, including aplasmid . Based oaplasmid , 16, we plasmids . To mainB. ubonensis pC3 megaplasmid is unusually large, although such size is not unprecedented, with B. cenocepacia H111 encoding a curable 1.04Mbp pC3 megaplasmid [Streptomyces clavuligerus [Azospirillum brasilense [B. ubonensis MSMB0022 failed to be detected as a plasmid using the online PlasmidFinder and VecScreen tools; however, we found that these tools also failed to identify the B. vietnamiensis megaplasmid pBVIE01, possibly because PlasmidFinder has been optimised for plasmid identification in Enterobacteriaceae [parA and parB genes from B. vietnamiensis G4 pBVIE01 showed weak evidence of these partitioning system genes in MSMB0022 pC3, although more solid BLAST hits were obtained with chromosome I genes. This result does not rule out the presence of plasmid maintenance loci encoded on this replicon, but rather demonstrates the difficulties in identifying genetic homology across distantly related species. Similarly, the presence of 5S, 16S and 23S ribosomal RNA-encoding genes on pC3 does not necessarily rule out this replicon as being a megaplasmid [At 920kb, the aplasmid . Larger uligerus , and a 1asilense . The pC3eriaceae . BLAST aaplasmid . Read deaplasmid .B. ubonensis strains possess the unusual B. pseudomallei type B LPS O-antigen [in silico. Due to insufficient contig coverage across the LPS cluster, 19 strains could not be fully genotyped using this approach; however, these strains did not possess clusters matching to other LPS types. Of the remaining 245 strains that could be genotyped, type B LPS was identified in 20 (8%). In total, 35 different LPS types were found, compared with only four LPS types among 477 global B. pseudomallei strains using the same in silico approach. The most abundant LPS type in the B. ubonensis cohort was MSMB0063 Type Novel, with 28 strains having this genotype; in contrast, eleven LPS types were seen in only a single isolate . Given B. ubonensis, we inoculated BALB/c mice via sc injection using 1.7x 104, 105, and 106 CFU of the Thai strain RF23-BP41. To our knowledge, B. ubonensis virulence has not yet been tested in the mouse model. RF23-BP41 was chosen for several reasons. First, its Thai origin maximises the probability of genetic relatedness to the putatively pathogenic LMG 24263 strain. Second, RF23-BP41 was isolated from a region where individuals (particularly rice farmers) regularly come into contact with soil bacteria, increasing the likelihood of successful human infection. Third, this strain demonstrated resistance towards meropenem (MIC 16μg/mL), which would potentially confer a selective advantage during antibiotic treatment. Finally, this strain harbours pC3, which has been shown to impart virulence capacity in other Bcc species [6 CFU, no mice exhibited weight loss or lethargy during the 21-day challenge experiment, with their health status identical to that of the three control mice. The same result was observed in the BALB/c mice subcutaneously injected with B. thailandensis E264 at a similar dosage range [B. thailandensis strains are capable of infecting immunocompromised humans [50 of B. pseudomallei in BALB/c mice was ~1x103 CFU when delivered via the sc route [4 CFU when administered via the intraperitoneal route, with virulence reduced but not abolished in highly laboratory-passaged strains [B. cenocepacia strain J2315 caused universal mortality when inoculated at 103 cfu into gp91phox−/− mice via an intratracheal route, whereas other B. cenocepacia strains were less virulent and B. vietnamiensis strain R2 was avirulent [4 cfu also showed differential virulence within Bcc species, with some mice clearing their infections [To further examine the virulence potential of  species , 16. Evege range . Certaind humans –69, and d humans . In contsc route , and bet strains , 72. Othvirulent . Anotherfections , indicatB. ubonensis strains. Characterising the virulence potential of other B. ubonensis strains may identify unusual pathogenic strains, although we deem this unlikely based on the lack of verified human infections caused by B. ubonensis. In consideration of the IACUC guidelines, we chose not to carry out testing of further strains using the mouse model. We acknowledge that our study only tested B. ubonensis in immunocompetent BALB/c mice via a sc route. The use of immunocompromised or immune-deficient mouse models or infection via different routes may reveal that B. ubonensis can cause disease in such cases. Bcc species carry various virulence factors that are thought to contribute to their pathogenic potential, including extracellular lipases, metalloproteases, serine proteases, flagella, pili, adhesins, toxins, siderophores and lipopolysaccharides [B. ubonensis compared with other Bcc species but doing so may shed further light on its potential virulence capacity. It may be possible to use such in silico methods rather than further animal experiments to determine whether B. ubonensis is unusual compared with other Bcc species due to a lack of key virulence loci or pathways in its genome.Based on the findings of these earlier studies, pathogenicity may also vary among charides . We did B. ubonensis, a largely neglected member of the Bcc due to its ostensibly avirulent nature. Genomic analysis of 264 B. ubonensis strains from Australia, PNG, Puerto Rico and Thailand revealed that B. ubonensis is a genetically highly diverse organism, with at least 26% of its chromosomal DNA variably present among strains. Like B. pseudomallei, B. ubonensis has a distinct phylogeographic signature that can be distinguished at the genomic level. It remains to be determined whether B. ubonensis is found on other continents. ‘Chromosome III’ encodes a ubiquitous yet apparently dispensable pC3 megaplasmid, similarly to other Bcc species, and can segregate in the laboratory setting. Like other Bcc species, we show that B. ubonensis strains exhibit variable levels of meropenem resistance. Determining the molecular mechanism underpinning high-level meropenem resistance in certain B. ubonensis strains will provide a better understanding of the potential transmission of this phenotype to the melioidosis bacterium B. pseudomallei, which frequently co-resides with B. ubonensis in the environment. Finally, using the immunocompetent BALB/c mouse model, we show that an Asian B. ubonensis strain is not likely to cause disease, providing evidence that at least some members of this species are probably avirulent in immunocompetent individuals. Further studies are needed to confirm the avirulent nature of B. ubonensis across a greater strain set using both immunocompetent and immunocompromised or immunodeficient animal models, or in silico analysis of the B. ubonensis genome to identify intact virulence determinants. The apparent non-pathogenic nature of certain B. ubonensis strains may make them amenable to large-scale biotechnological applications, such as biocontrol and biofuel production.The metabolic diversity of Bcc species continues to spur interest in this highly adaptable group of bacteria. Our study provides important new insights into the biology of S1 FigB. ubonensis Clades I-VI are labelled, and B. ubonensis strains from regions other than Australia are noted. Consistency index = 0.36. The tree was rooted with the B. pseudomallei complex species. In total, 277 taxa were used to reconstruct this phylogeny, of which 264 were B. ubonensis.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.