The setup allows a rapid screening for effects in the mutual species interaction. Furthermore, it is easy to handle, inexpensive, and reproducible.Caries and periodontitis are important human diseases associated with formation of multi-species biofilms. The involved bacteria are intensively studied to understand the molecular basis of the interactions in such biofilms. This study established a basic Streptococcus mitis, S. salivarius and S. sanguinis, typical inhabitants of the healthy oral cavity, S. mutans as main carriogenic species, and Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra, S. intermedius and Aggregatibacter actinomycetemcomitans as periodontitis-associated bacteria, were investigated for their biofilm forming ability. Different liquid growth media were evaluated. Safranin-staining allowed monitoring of biofilm formation under the chosen conditions. Viable counts and microscopy permitted investigation of biofilm behavior in mixed-species and transwell setups.S. mitis, F. nucleatum, P. gingivalis and P. micra failed to form biofilm structures. S. mutans, S. sanguinis, S. intermedius and S. salivarius established abundant biofilm masses in CDM/sucrose. A. actinomycetemcomitans formed patchy monolayers. For in depth analysis S. mitis, S. mutans and A. actinomycetemcomitans were chosen, because i) they are representatives of the physiological-, cariogenic and periodontitis-associated bacterial flora, respectively and ii) their difference in their biofilm forming ability. Microscopic analysis confirmed the results of safranin staining. Investigation of two species combinations of S. mitis with either S. mutans or A. actinomycetemcomitans revealed bacterial interactions influencing biofilm mass, biofilm structure and cell viability.S. mitis influence, and allows a fast decision about the most important bacterial interactions which should be investigated in depth.This setup shows safranin staining, microscopic analysis and viable counts together are crucial for basic examination and evaluation of biofilms. Our experiment generated meaningful results, exemplified by the noted  Caries and Periodontitis are extremely frequent human diseases with high socioeconomic impact. They are associated with several potentially severe complications due to bacterial invasion of neighbouring anatomical structures or haematogenous spreading and purulent infections at distant sites. If not managed by appropriate therapies, both diseases are chronically progressive. Their pathogenesis is explained by a locally disturbed microecology within the bacterial biofilms covering the surfaces of the teeth and the subgingival sulci.Bacterial biofilms are able to form and spread on the surfaces of the teeth in healthy oral cavities. Such biofilms display typical structural features such as i) a chemically conditioned support, ii) pioneer bacteria firmly adhering to the support's surface, iii) microcolony formation and production of macromolecular extracellular substances, iv) attachment of secondary colonizer binding to the growing biofilm, v) a predefined maximum thickness due to a balance between biofilm production and detachment (i.e. maturation) processes. These biofilms may contain up to several hundred bacterial species. These bacterial consortia are inconsistent between individual sites in one oral cavity and even more between diverse oral cavities Because of the extensive species variation between human individuals the concept of specific indicator bacteria for physiological and pathological biofilms in oral cavities is currently modified in vitro biofilms that truly reflect the natural situation. However, establishing of in vitro biofilms is still necessary to investigate substances suitable for the suppression of caries or periodontitis.The involvement of many species and their constant qualitative and quantitative variations makes it extremely complicated to setup in vitro experiments have to rely on classical culture methods. It is currently impossible to grow representative in vitro multi-species biofilms resembling those encountered during caries or periodontitis. However, mixing a few important species to mimic biofilms encountered during complete health, transition to disease, developing disease, and finally, in deep lesions appears to be feasible. Such studies were performed in many laboratories in vivo biofilm niche, 3) the documentation of biofilm formation, biofilm mass and biofilm maturation, and 4) the quantification of the individual species contained in the biofilms. Thus, a setup which could be used in many labs and which proves useful for many different scientific questions would be beneficial to basically investigate and understand biofilm formation and bacterial interaction in biofilm structures.While the epidemiology of the microflora in healthy and diseased oral cavities has greatly been promoted by the introduction of advanced microscopic and molecular techniques, Streptococcus mitis, S. salivarius, S. mutans, S. sanguinis, S. intermedius, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and Parvimonas micra as bacteria associated with a healthy oral cavity, caries, and periodontitis, respectively. Based on the experimental protocols used in this study, we could demonstrate in mixed species assays as well as in assays employing separating filter membranes between the partners, increased or decreased contribution of single species to biofilm formation and effects on viability exclusively in one co-incubation partner.In the present study, we intended to set up conditions for the investigation of bacterial biofilm formation and combined three complimentary methods  as a basis for multi-species culture investigation. We used in vitro simulation of biofilm generation. It is assumed that not much liquid exchange occurs during periodontitis in vivo, thus static conditions best mimic this situation and also allow the action of potential signalling molecules in mixed species cultures. Six different media were examined for their effect on mono-species biofilm formation for a time period up to five days. Safranin staining was employed as an easy read-out approach. This method is used for the determination of biofilm mass, comprising bacterial cells and extrapolymeric substances. Typically, an OD492 nm with a value of more than 0.05 is required to indicate biofilm formation. Lower values are mostly caused by scattered bacteria in monolayers (data not shown). For comparison, also the growth curves of planktonic cells were recorded for each culture medium. The S. mitis, S. mutans and A. actinomycetemcomitans as representatives of the physiological, cariogenic and periodontitis-associated oral microflora, respectively.Investigation of biofilm structures and bacterial interaction required establishment of reliable biofilm setup protocols. For this purpose, different culture media were tested in a static biofilm setup to evaluate the best conditions for S. mitis, S. mutans and A. actinomycetemcomitans mono-species cultures for biofilm mass over a period of five days showed that biofilm formation of S. mutans occurred within the first 24 hours of incubation time in CDM without glucose , CDM supplemented with 50 mM glucose (CDM/glc) or sucrose (CDM/suc). The highest amounts of biofilm cell mass was formed in CDM/sucrose. The biofilm mass reached at this time point remained stable during the residual observation period. For A. actinomycetemcomitans, scattered patches of monolayers appeared after one day of incubation in CDM/glucose or CDM/sucrose. No multi-layered structures were observed in any tested medium. S. mitis bacteria failed to establish biofilms in all tested media . Planktonic A. actinomycetemcomitans did not grow in this medium . Of note, within these mixed communities no colony forming units of S. mitis were detectable, whereas S. mutans numbers and viability were unchanged compared to single species settings .Combining comitans . Concominditions . FurtherA. actinomycetemcomitans cells with few S. mitis cells on top of their partner cells. Finally, as determined by live/dead stain and viable counts, cells of A. actinomycetemcomitans died faster in the presence of S. mitis compared to mono-species cultures . As shown in S. mutans to S. mitis led to biofilm formation, dominated by S. mutans and its extracellular matrix structures . In the reverse order, the inoculation of S. mitis to S. mutans showed again the integration of S. mitis into the S. mutans biofilm, which also consisted of abundant extracellular matrix , but values were still higher as for the A. actinomycetemcomitans mono-species culture .Of note, viable cell counts for both bacteria decreased in this experiment, similar to the results obtained for the simultaneously seeded two-species culture . FluoresWe next employed transwell assays to investigate if a change in biofilm mass and/or viable counts as seen in simultaneous and successive seeding experiments was caused by direct cell-cell contact or by soluble substances secreted by the tested species. Therefore, experiments were repeated in the above-mentioned combinations, but the bacteria were separated by a membrane with 0.2 µm sized pores.S. mutans biofilm formation was stimulated in the presence of S. mitis  or carbohydrates, respectively, in biofilm mass and structure.S. mutans mono-species biofilms, an increase of biofilm mass could be observed when DNase was added to the 2 and 3 days old biofilm, whereas pronase treatment had such effects only on 2-day-old biofilms. The addition of sodiummetaperiodate led to a decreased biofilm mass of 1-day-old S. mutans biofilms, whereas treatment at days 2 or 3 caused no change. The analysis of A. actinomycetemcomitans monolayers with these substances revealed a significant decrease of safranin staining intensity when pronase was added to 1, 2 or 3 day old monolayers while the other substances had no obvious effects .For A. actinomycetemcomitans, S. mitis, and S. mutans in a three-species culture revealed safranin values only marginally increased compared to the S. mutans mono-species culture  the introduction of a combination of complementary methods to substantiate results, and iii) the demonstration of its usefulness for investigating bacteria from the oral cavity of healthy subjects and/or patients with periodontitis .The aims of this study were i) the establishment of an Initially, we sought the best suited medium for biofilm formation of the chosen bacterial species. Brain heart infusion medium (BHI) supplemented with human serum or saliva-like medium (SLM) was studied because of its similarity to sulcus fluid. Both media preparations hardly supported biofilm formation. The included human serum constituents S. mutans, S. salivarius, S. sanguinis, S. intermedius and the monolayer formation of A. actinomycetemcomitans. One major advantage of this medium is that its composition is known in detail, allowing an easy modulation of the presence and/or concentration of amino acids, phosphate or sugar. Unfortunately, the composition of CDM is more remote from sulcus fluid than that of the complex BHI medium. This potential disadvantage could not be resolved by the addition of human serum, due to the negative influence of the supplement on biofilm formation. Yet, a sucrose (or glucose) supplement has been established as an important substrate for the synthesis of extracellular polysaccharides, which in turn are crucial components of streptococcal biofilms Next, a chemically defined medium without glucose (CDM) supplemented with sucrose was tested and found to be optimal for the biofilm formation of Although more closely reflecting the natural situation, fibronectin coating of surfaces did not significantly influence the biofilm forming ability of the tested mono-species as compared to uncoated supports.et al.Introduced washing steps in biofilm formation experiments are critically discussed When examining bacterial biofilms, several qualitative or quantitative measurements are established. Safranin-staining predominantly detects extracellular substances and is commonly used to quantify biofilm mass A. actinomycetemcomitans and S. mitis behaved contrary in their planktonic growth and biofim behavior. Similar observations were previously reported by Fine and colleagues A. actinomycetemcomitans did not form multi-layered biofilms but covered the plastic surface by monolayers of viable but not multiplying cells. Several A. actinomycetemcomitans strains have been tested for biofilm formation with varying results A. actinomycetemcomitans biofilm formation A. actinomycetemcomitans strains vary in their biofilm growth capabilities, with smooth colony formers growing to less biofilm mass and different biofilm structures A. actinomycetemcomitans strain formed smooth colonies. The tendency of such strains to develop monolayers of viable cells for extended incubation periods has not been described so far. In general A. actinomycetemcomitans is known for its dependence on K+ ion concentration Only by employing these three complementary methods, it became evident that S. mitis no biofilm formation could be observed under all tested conditions. Based on electron microscopy observations, Cowan et al. S. mitis produced few, extremely long fibrils. These fibrils obviously enable the bacteria to adhere to the underlying substrate S. mitis differs from other oral streptococci in its content of nitrogen and oxygen rich polysaccharides S. mitis to form biofilm structures under the chosen conditions. The plastic surfaces of the used 96- and 24-well plates obviously did not support S. mitis adhesion. Previous studies demonstrated a dependence of S. mitis biofilm formation on the presence of acquired pellicle and lectins S. oralis strain C104 formed only small biofilm mass, leading to the conclusion that this species lacks effective colonization factors for binding to abiotic surfaces but can participate in complex biofilms by binding to more successful initial colonizers S. mitis/S. mutans biofilms.For S. mitis has been described as a bacterium with an ecological control function in the oral cavity. Precisely, S. mitis could inhibit A. actinomycetemcomitans colonization S. mitis to both the initial step as well as the ensuing multiplication ability of A. actinomycetemcomitans. In literature nutrient depletion and/or pH shift are discussed mechanisms for bacterial inhibitory effects S. mitis on A. actinomycetemcomitans is currently unknown.A notable result of this study is the obvious change in biofilm mass and viable counts in the two-species combinations compared to single-species settings. S. mitis with S. mutans always resulted in biofilm formation, although the final mass was determined by the timing of the bacterial adherence. Previous studies of van Hoogmoed et al. S. mutans NS adhesion by biosurfactant-releasing S. mitis strains (S. mitis BA and S. mitis BM). These authors found a release of maximal amounts of biosurfactants, identified to be glycolipids, when the S. mitis strains were grown in the presence of sucrose. However, preliminary results from our laboratory indicate that this biosurfactant production could be a strain specific feature (data not shown). In order to introduce spacing as parameter in the line of investigation, transwell experiments were performed. These studies uncovered discrepancies between cfu values and counts of live/dead-stained cells, suggesting the adoption of a VBNC-status of A. actinomycetemcomitans in indirect contact to S. mitis. Furthermore, the experiments with S. mutans as well as A. actinomycetemcomitans in the remote presence of S. mitis suggested a control function for S. mitis under both conditions. At least for the remote effects, production of secreted substances is the most obvious explanation. The chemical nature of these substances needs to be determined. However, it is known from literature that production of detergents, toxic substances as hydrogen peroxide and bacteriocins or bacteriocin-like inhibitory substances are likely candidates for this effect Timing and spacing are two critical parameters in the development of mixed-species biofilms. Combinations of S. mutans mono-species biofilms in the presence of protein-, DNA- or carbohydrate-degrading substances showed an unexpected effect, i.e. an incubation time-dependent increase of biofilm mass induced by DNase and pronase. Others have shown that presence of DNase during biofilm development leads to a significant disturbance of biofilm formation S. mitis in transwell experiments, i.e. a secretion of enzymes with proteolytic or DNase activity.Analysis of In summary, the combined analysis of biofilm formation via safranin stain, determination of cfu and fluorescence microscopy after live/dead-stain allowed fast, unambiguous and reproducible results. Scanning electron- and confocal laser scanning microscopy complemented these results. The whole setup for mono-species cultures could be applied to two- and three-species combinations. This allowed first insights in interactions of chosen bacteria, precisely a mutual influence on biofilm formation and structure, as well as on different levels of viability.Streptococcus mitis ATCC 11843, Streptococcus mutans DSM 20523, Streptococcus sanguinis DSM 20567, Fusobacterium nucleatum DSMZ 25586, Porphyromonas gingivalis W83 ATCC BAA-308, Parvimonas micra ATCC 33270 and Aggregatibacter actinomycetemcomitans (A. actinom.) DSMZ 11123 were purchased from commercial providers . Streptococcus intermedius AC 3105 was obtained from the strain collection of the university Aachen, Germany. S. salivarius K12 was kindly provided by Dr. J. Tagg, New Zealand. Unless otherwise specified, all streptococci and A. actinomycetemcomitans were cultured in brain heart infusion medium  at 37°C under a 5% CO2 – 20% O2 atmosphere. P. gingivalis was cultivated in BHI supplemented with 5 µg/ml hemin and 50 mM galactose, and F. nucleatum and P. micra were cultured in BHI supplemented 0.25% glutamate. The latter three species were grown at 37°C under an anaerobic atmosphere (10% CO2 - 10% H2 – 80% N2).The bacterial strains 2PO4, 1.5 mM K2HPO4, 0.05% urea, pH 6.7) (1∶3). Alternatively, a chemically defined medium  was supplemented with human serum  (4∶1) or a saliva-like medium , 6 mM NaCl, 2.7 mM KCl, 3.5 mM KHium CDM,  was used600 to obtain 1×10∧8 cells ml∧-1. Subsequently, each bacterial suspension was diluted 10-fold in culture medium and inoculated in polystyrene 24-well-plates . The bacteria were cultivated alone to establish mono-species biofilms. Alternatively, S. mutans or A. actinomycetemcomitans were cultivated in combination with S. mitis resulting in two-species biofilms.Bacteria were grown in BHI to stationary phase, washed with phosphate buffered saline , and adjusted to a strain specific OD2, 10% CO2, 10% H2) at 37°C for periods up to 5 days under static conditions (unless otherwise indicated). The atmosphere of the incubator was saturated with water vapor to prevent exsiccation of the cultures and was constantly exposed to a platinum catalyst to decrease the content of short-chained fatty acids in the atmosphere.Biofilm-cultures were grown in an anaerobic incubator under an appropriate atmosphere  were employed. The plastic surfaces of the 96-well plates were either used uncoated or were coated with human fibronectin (Roche) at a concentration of 50 µg/ml∧-1 overnight at 4°C. Prior to the inoculation of the bacteria, fibronectin was removed and wells were washed and air-dried. After incubation of the bacterial cultures, liquid medium was removed and wells were washed gently with PBS in order to remove non-adherent sedimented cells.For determination of biofilm mass, wells were stained with 0.1% safranin for 15 min, washed with PBS and air-dried. Biofilm mass was quantified in the air-dried wells by measuring the absorbance at 492 nm with a microplate reader (Tecan reader).Viable cell numbers from biofilm bacteria were obtained by thorough scraping and washing of the wells with PBS. The resulting suspensions were serially diluted in PBS and plated in 100 µl aliquots on BHI-agar. Colony forming units (cfu) were counted after two days of incubation. The distinct colony morphology allowed for differentiation between the species.Mono- or two-species biofilms were cultured in uncoated 24-well polystyrene cell culture plates , each well containing a sterile, uncoated 13-mm-diameter plastic microscope coverslip . After one to five days of incubation under anaerobic conditions, biofilms were gently washed with PBS, stained with BacLight Live/Dead  and inspected by fluorescence microscopy . Visible biofilms were documented with an attached digital camera .2, sputter-coated with gold (thickness approx. 10 nm), and examined with a Zeiss DSM 960A electron microscope.In parallel experiments, samples were prepared for scanning electron microscopy (SEM) studies as follows: biofilms on the coverslips were fixed for 24 h in a solution containing 2.5% glutardialdehyde. The coverslips were washed with 0.1 M Na-acetate buffer (pH 7.3) and dehydrated in a graded series of ethanol. Subsequently, coverslips were subjected to critical point drying with COFor confocal laser scanning microscopy (CLSM) studies, biofilms were grown in glass-bottom chamber slides (Nunc) and cultured for up to three days under anaerobic conditions. Following incubation, biofilms were gently washed with PBS and stained with BacLight Live/Dead . Preparations were inspected with a Zeiss inverted microscope attached to a Leica TCS SP2 AOBS laser scanning confocal imaging system with an Argon laser at 488-nm excitation wavelength and an Helium/Neon laser at 546-nm excitation wavelength. 3D images were obtained using the IMARIS x64 software.For transwell studies, uncoated 24-transwell polystyrene cell culture plates (Corning) with one coverslip per well were inoculated with 600 µl (10∧7 cfu/ml) of the first bacterial species in the lower compartment and 200 µl (10∧7 cfu/ml) of the second bacterial species in the upper compartment (transwell inserts). Furthermore, uncoated 96 transwell polystyrene microtiter plates were used, containing 200 µl of the first/50 µl of the second bacterial species in the lower/upper compartment, respectively. After one to three days of incubation, transwell inserts and liquid medium were removed. The wells were gently washed with PBS, and biofilms were analyzed for biofilm mass, cell number, as well as biofilm structure using microscopic techniques (see above).S. mitis were inoculated as first bacterial species in an uncoated 96- or 24-well polystyrene plate, the latter with one coverslip per well, and incubated for 24 h under anaerobic conditions at 37°C. Following this incubation time, the liquid medium (containing remaining planktonic bacteria) was removed and 10∧7 S. mutans or A. actinomycetemcomitans suspended in growth medium were inoculated as the second bacterial species into the wells. Subsequently, well plates were again incubated anaerobically at 37°C for up to two days. Biofilm formation was analyzed on a daily basis for two consecutive days by determination of biofilm mass via safranin stain, cell number by counting of cfu/ml and fluorescence microscopy after staining with BacLight Live/Dead . This assay was also performed in reverse order with S. mitis as the second bacterial species inoculated.Medium suspensions with 10∧7 The disorganization of biofilm was performed as described by Inoue et al. Each assay was performed in at least 3 wells at a given time  and was repeated on at least 3 independent occasions . Where appropriate, statistical parameters  were determined employing the Windows Excel program and the Mann-Whitney U Test. P-values less than 0.05 were considered as significant.Figure S1A. actinomycetemcomitans, S. mitis, and S. mutans in CDM/sucrose. Growth was monitored by OD600 nm measurements in hourly intervals. One representative experiment of at least three replicates is shown.Regular growth curves of (0.20 MB TIF)Click here for additional data file.Figure S2Results of safranin-staining assay for the mono-species biofilms on a fibronectin-coated surface. The graph shows the result of safranin-staining assay for the tested mono-species on uncoated and fibronectin-coated surfaces. Fn - fibronectin.(0.15 MB TIF)Click here for additional data file.Figure S3Results of safranin-staining assay for the mono-species biofilm disorganization with pronase, DNase and sodiummetaperiodate. A), B) and C) Results for 500 µg/ml pronase, 90 units DNase, 10 mM sodiummetaperiodate, respectively. SMP - sodiummetaperiodate, * means significance of p<0.05 and ** means significance with p<0.01. PBS was used as control.(0.38 MB TIF)Click here for additional data file.Figure S4S. mitis/S. mutans/A. actinomycetemcomitans three-species combination. A) Safranin-staining assay of the mono- and three species-biofilms of S. mitis, S. mutans and A. actinomycetemcomitans. B) Number of colony forming units for the mono- and three-species cultures. Bacteria in brackets were the corresponding combination partner in the three-species culture. C–D) SEM and fluorescence microcopy of the S. mitis/S. mutans/A. actinomycetemcomitans combination. White arrow: S. mitis; Black arrow: S. mutans.Results of Safranin-staining assay, number of colony froming units and microscopic analysis of the (4.10 MB TIF)Click here for additional data file.Table S1Summarized results for planktonic and biofilm growth of the different species in the tested media.(0.09 MB DOC)Click here for additional data file.Table S2Number of colony forming units obtained for the mono- and two-species cultures in CDM/sucrose.(0.03 MB DOC)Click here for additional data file.Table S3Number of colony forming units for the successive seeding experiment.(0.02 MB DOC)Click here for additional data file.Table S4Number of colony forming units obtained in transwell experiments.(0.02 MB DOC)Click here for additional data file.


Pseudomonas putida is the best studied pollutant degradative bacteria and is harnessed by industrial biotechnology to synthesize fine chemicals. Since the publication of P. putida KT2440's genome, some in silico analyses of its metabolic and biotechnology capacities have been published. However, global understanding of the capabilities of P. putida KT2440 requires the construction of a metabolic model that enables the integration of classical experimental data along with genomic and high-throughput data. The constraint-based reconstruction and analysis (COBRA) approach has been successfully used to build and analyze in silico genome-scale metabolic reconstructions.P. putida KT2440's metabolism, iJN746, which was constructed based on genomic, biochemical, and physiological information. This manually-curated reconstruction accounts for 746 genes, 950 reactions, and 911 metabolites. iJN746 captures biotechnologically relevant pathways, including polyhydroxyalkanoate synthesis and catabolic pathways of aromatic compounds , not described in other metabolic reconstructions or biochemical databases. The predictive potential of iJN746 was validated using experimental data including growth performance and gene deletion studies. Furthermore, in silico growth on toluene was found to be oxygen-limited, suggesting the existence of oxygen-efficient pathways not yet annotated in P. putida's genome. Moreover, we evaluated the production efficiency of polyhydroxyalkanoates from various carbon sources and found fatty acids as the most prominent candidates, as expected.We present a genome-scale reconstruction of P. putida, a biotechnologically interesting all-surrounder. Taken together, this work illustrates the utility of iJN746 as i) a knowledge-base, ii) a discovery tool, and iii) an engineering platform to explore P. putida's potential in bioremediation and bioplastic production.Here we presented the first genome-scale reconstruction of  Pseudomonas putida is a non-pathogenic member of rRNA group I of the genus Pseudomonas that colonizes many different environments and is well known for its broad metabolic versatility and genetic plasticity [P. putida KT2440 is a TOL plasmid cured, spontaneous restriction deficient derivative of P. putida mt-2 [asticity ,2. P. puida mt-2 ,4. This ida mt-2 -8. Conseida mt-2 ,10; ii) ida mt-2 ; iii) biida mt-2 ,12-14; iida mt-2 -17; and ida mt-2 ,19.P. putida KT2440's genome [in silico analyses of its metabolic and biotechnological capacities have been published [P. putida KT2440 requires the construction of a more comprehensive model enabling the integration of the canonical experimental data along with genomic and high-throughput data in a hierarchical and coherent fashion [Since the publication of s genome , our knos genome  and varis genome ,23, prots genome ,26. Subsublished ,28. Howe fashion .in silico. Many of the published mathematical tools have been reviewed [in silico reconstructions for representatives of archaea  approach is one possible modeling approach that uses stoichiometric information about biochemical transformation taking place in a target organism to construct the model. While a metabolic reconstruction is unique to the target organism one can derive many different condition-specific models from a single reconstruction. This conversion of a metabolic reconstruction of an organism into models requires the imposition of physicochemical and environmental constraints to define systems boundaries -32. The reviewed  and encoreviewed . A largereviewed ,32 has b barkeri ), of bac[E. coli ; B. subtsubtilis ; H. pylo. pylori ; M. tuberculosis ,40; S. a. aureus ,42; L. l. lactis ), and of. lactis ). The nu. lactis ); ii) pr. lactis -48; iii). lactis ; and iv). lactis -53. A re methods .Pseudomonas putida KT2440. Based on the naming convention for metabolic networks [iJN746, where i stands for in silico, JN are the initials of the constructor, and 746 corresponds to the number of included metabolic genes. The reconstruction was built using the COBRA approach [in silico metabolic network was further evaluated by comparing i) predicted growth rate capacities in different carbon sources and ii) predicted essential genes with experimental data from P. putida KT2440 and P. aeruginosa. Finally, we show the utility of the P. putida reconstruction to analyze its biodegradative (i.e. toluene degradation) and biotechnological (i.e. bioplastic production) capacities.Here, we describe a highly detailed, genome-scale, metabolic reconstruction of networks , this geapproach ,32 and vapproach ). The inP. putida KT2440, iJN746, was constructed based on its annotated genome sequence [Pseudomonas-specific [The metabolic reconstruction of sequence , primarysequence , PSEUDOCsequence , and SYSsequence ), and bispecific  and biociJN746 accounts for 746 open reading frames (ORF), whose corresponding gene products are involved in 810 metabolic and transport reactions , which are lipid related polymers, from a wide range of carbon sources [The reactions included in e Figure . In gene. putida  and succproducts . Reactios genome . Moreoven source ,60. A thpacities . Finally sources ,61. This sources ,63) and P. putida metabolic network  some reactions had to be added to the network for which no genetic or experimental evidence could be found. Those reactions represent modeling hypotheses, which need further experimental validation and thus received a confidence score of 1. Upon completion, the reconstruction had an overall average confidence score of 2.83. In fact, two thirds of P. putida's metabolic pathways have been very well or well studied, while only a third of the subsystems were primarily based on the genome annotation  [P. putida compared to the other reconstructions. In summary, this comparison indicates that the overall context coverage in iJN746 is comparable with other high-quality network reconstructions when the amount of available literature is considered.The properties of iAF1260, ), B. sub (iYO844 ), M. tub (iNJ661 ), and P.. Despite being close relatives, these two representatives have significant differences in lifestyle and metabolic capabilities. Subsequently, the two metabolic reconstructions have significant differences, emphasizing the importance of organism-specific reconstructions. For instance, the P. aeruginosa reconstruction contains pathways necessary for growth and production of common virulence factors, including alginate, rhamnolipids, phenezines, and quorum-sensing molecules [P. putida's metabolic network. In contrast, P. aeruginosa's metabolic network does not account for pathways necessary to degrade aromatic compounds.A metabolic reconstruction for another representative of the recently . A compaed Table . In contda Table . As a coolecules , which ain silico growth performance with experimental data allows for the assessment of the predictive potential of the metabolic reconstruction and thus represents a valuable tool for network evaluation. Furthermore, in silico growth analysis may expand the known array of carbon-, nitrogen-, and energy sources of the reconstructed organism. In this study, the aerobic growth capabilities of iJN746 in iM9 medium substituted with different carbon sources were determined qualitatively .Flux balance analysis (FBA ) can proly Table  and quanly Table . The groum Table . Furtherzosphere , which izosphere ,67. This46 Table . Of partiJN746, as expected, as only exchange reactions were included in the reconstruction for metabolites, which have been reported to be taken up or secreted by P. putida KT2440. In contrast, some disagreements, such as false negatives, were observed despite a good overall agreement with the in vivo data [P. putida can use L-alanine as a carbon- and nitrogen-source [iJN746 cannot use this compound as a carbon or nitrogen source. This disagreement could not be resolved. In contrast, iJN746 was initially unable to use choline-O-sulphate, choline, or glycine betaine as carbon- and nitrogen-sources despite experimental evidence [iJN746 to use these metabolites as carbon- and nitrogen-sources through the glycine metabolism. In addition, choline-O-sulphate could also be used as sulfur source [see Additional file in silico predictions and physiological properties can be used to drive new discoveries, as was shown for E. coli [No false positive carbon, nitrogen, or sulfur sources were found in ivo data  [Additioivo data . For exan-source  but iJN7evidence . However E. coli .P. putida KT2440, like other Pseudomonas species and rhizosymbionts, has an incomplete glycolytic pathway because of a missing 6-phosphofructokinase [P. putida KT2440 has a complete Entner-Doudoroff pathway, which allows for the utilization of glucose and other sugars as carbon sources . Interestingly, iJN746 grew faster in glucose than experimental in vivo data suggested for P. putida KT2442   Table . In the E. coli corresponding to the normal oxygen diffusion rate under atmospheric oxygen conditions [in silico growth rate.Linear Programming (LP) problems have two parameters, shadow price and reduced cost, which can be used to characterize the optimal solution. While shadow prices are associated with each network metabolite, reduced costs are associated with each network reaction. The reduced cost signifies the amount by which the objective function (e.g. growth rate) would increase when the flux rate through a chosen reaction was increased by a single unit . Analysenditions . Mathema-1) [We performed a phase plane analysis to further elucidate the correlation between toluene uptake, OUR, and biomass production rate Figure . We anal-1)  was achiP. putida KT2440 under toluene-dependent growth conditions, we carried out in vivo experiments to determine the OUR of P. putida KT2440 harboring the TOL plasmid (see Methods). As expected, the OUR in toluene growing cells was higher than glucose or octanoate growing cells; 20.93 compared to 15.34 and 14.88 mmol oxygen/gDW/h, respectively . In fact, oxygen dependent growth of toluene grown cells has been described for other P. putida strains. For example, Alagappan and Cowan reported a 10× higher oxygen-half saturation of P. putida F1 grown on toluene than other aerobic organisms [P. putida when grown on octanoate [In order to better understand this situation and since no detailed information about OUR was found for rganisms . Furtherrganisms ,80; howerganisms , indicatctanoate .P. putida metabolic network is incomplete. In fact, the current information and results suggest that the network is missing one or more reactions enabling a more oxygen-efficient catabolism of toluene and other highly reduced carbon sources (e.g. other aromatic compounds or fatty acids). This analysis represents a nice example of the broad range of applications for which iJN746 can be used to evaluate the consistency of experimental data and in silico prediction. iJN746 can serve as a platform to derive hypotheses about metabolic capabilities or missing functions in the network which can be ultimately tested in the laboratory. Hence, the metabolic reconstruction can help to increase our understanding and knowledge about this biotechnologically important organism.Taken together, our analysis suggests that the current iJN746 was used as a framework to analyze candidate essential genes in P. putida KT2440 in LB rich medium. Therefore, the network reaction(s) associated with each gene was individually "deleted" by setting the flux to 0 and optimizing for the biomass function [in silico essentiality predictions with experimental data to assess the predictive potential of the model. However, no large-scale, experimental gene essentiality data are available for P. putida; the information can only be found for its phylogenetic relative P. aeruginosa PAO1 and P. aeruginosa PA14 [P. putida and P. aeruginosa PAO1 genomes identified 3,143 potential orthologous pairs corresponding to 60% of P. putida's total ORFs, as well as large sections of conserved gene order (synteny) [in silico single gene deletion results with the 335 essential metabolic and non-metabolic genes of P. aeruginosa [iJN746 were predicted to be essential in iLB medium [see Additional file iJN746 agreed with essential genes of P. aeruginosa [see Additional file iJN746 [see Additional file E. coli [function . We wishosa PA14 ,83. A resynteny) . Thereforuginosa ,83. Abou E. coli .fabB gene was predicted to be only essential in iJN746; however, after carrying out a detailed search on Pseudomona's genomes using "The Pseudomonas Genome Database V2"  we found putative ORFs in the KT2440 and PA01 genome. These ORFs were annotated as alternative loci that could substitute a fabB deletion. Both, P. putida and P. aeruginosa have one copy of the fabB gene encoding for the 3-oxoacyl-(acyl-carrier-protein)synthase I . In addition, both strains have a copy of the fabF gene encoding for the 3-oxoacyl-(acyl-carrier-protein) synthase II (PP_1916 (40.92% identity with fabB-KT gene) and PA2965 (42.34% identity with fabB-PAO1 gene). Moreover, in the P. putida and P. aeruginosa genome, some ORFs were annotated putatively to encode for a 3-oxoacyl-(acyl-carrier-protein) synthase II (PP_3303 (35.94% identity) and PP_2780 (27.32% identity) in KT2440, and PA_1373 (36.17% identity) in PAO1 strain. These putative ORFs were not included in iJN746 due to the lack of supporting evidence for their metabolic function, but this analysis showed that i) PAO1 has an isozyme present in its genome, and ii) KT2440 is very likely to have at least one other ORF encoding this or a similar function. In a similar way, the discrepancy between in silico essentiality prediction and in vitro observation for msbA gene could be explained. The gene product of msbA encodes for a transporter of phosphatidylethanolamine, which is known to have a genetic redundancy in Pseudomonas sp. taking into account the Pseudomonas annotation present in "The Pseudomonas Genome Database V2". However, the supporting evidence for alternative ORFs was not strong enough to be included into iJN746.The disagreement between the experimental and computational results can reveal possible errors in the experimental data as well as in the reconstructed network. The disagreements might be caused by low experimental or sequence evidences, each of which would have hindered the inclusion of the information into the reconstruction. For example, the iJN746 but they were reported as essential genes in P. aeruginosa [glyA (PP_0322 or PP_0671), fold (PP_1945 or PP_2265), fabZ (PP_4174 or PP_1602), and pyrH (PP_1771 or 1593), have at least one isozyme in KT2440 which were also accounted for in iJN746. For many remaining incorrectly predicted non-essential genes, the in silico deletion had a significant effect on the growth rate, reflecting their important roles in iJN746 metabolism [see Additional file Finally, 37 genes were not predicted to be essential in ruginosa  [see Addruginosa  but are in vivo or in vitro procedure to enable the usage of the generated data for in silico predictions and comparison.In general, many of these discrepancies suggest that metabolites enabling growth in the knock-outs might be imported from the external rich media since the exact composition of LB medium is not known ,38. Thiset al. reported a detailed amino acid auxotroph study in P. aeruginosa PA01 using a minimal medium [iM9 medium and compared the results with this PA01 study. Here, we found an absolute agreement between in vivo and in silico gene essentiality for six amino acids, namely arginine, histidine, isoleucine, valine, leucine, and tryptophan  the basic msc-PHA production processes in KT2440 are well known [In the previous section, we used the metabolic reconstruction to assess the current knowledge of gn tools  in additgn tools . Here, wgn tools . The biogn tools . These pgn tools . The medll known ,61, ii) iJN746 accounts for msc-PHAs ranging from C6 to C14, including two unsaturated msc-PHAs and a mixed msc-PHA polymer consisting of C8 to C12 chains. We tested the msc-PHA production capability of iJN746 from the different carbon- and energy sources listed in Table -1). Many of these metabolites have been reported to yield in PHA production in Pseudomonas [see Additional file iJN746 as well  precursors includes i) L-branched-chain amino acids , ii) some aromatic compounds metabolized via β-ketoadipate pathway , and iii) other (phenylacetic acid or glycerol)  Figure . Intereslacetate .et al. used inhibitors of fatty acid metabolism and demonstrated that, depending on the nature of the substrate, precursors for PHA synthesis could be derived from either beta-oxidation or fatty acid biosynthesis, and interestingly, when hexanoate was used as carbon source for msc-PHA accumulation, both routes can operate simultaneously [8 and C10 monomers. The model, in contrast, is able to produce the full range of msc-PHAs from carbohydrates  for production of the different msc-PHA monomers or msc-PHA mixtures. Furthermore, computational tools such as OptKnock[E. coli [M. succiniciproducens [Taken together, this example illustrates how  OptKnock or OptSt OptKnock could he[E. coli  or succiroducens .P. putida, a biotechnologically interesting all-surrounder. iJN746 is a highly detailed reconstruction of the P. putida KT2440 metabolic network that captures the important biotechnological capabilities, such as biodegradation of aromatic compounds, of this paradigmatic bacterium. Moreover, iJN746 represents a comprehensive knowledge base summarizing and categorizing the information currently available for P. putida KT2440. This study evaluated the metabolic network content and showed some examples of how iJN746 could be used for biotechnological purposes. Taken together, our results underlined the value of iJN746 as a suitable tool to study of P. putida's metabolism and its biotechnical applications by the P. putida community.Here, we presented the first genome-scale reconstruction of P. putida KT2440 harboring the TOL plasmid was used for in vivo determination of oxygen consumption experiments. The bacterium was grown at 30°C in M9 minimal medium [P. putida KT2440 strain grown in each carbon source was diluted until the turbidity at 600 nm (OD600) was 0.05 in fresh M9 minimal medium with the appropriate carbon source, samples were then incubated until the culture reached a turbidity at 600 nm of 0.6 for glucose or octanoate growing cells and 0.45 in toluene growing cells. Aliquots of 2 ml were taken for OUR determination; the cells were harvested by centrifugation, washed twice and re-suspended in 1 ml of fresh medium containing the appropriate carbon source using the above concentrations. The OUR was measured by monitoring the substrate-dependent oxygen consumption rate at 30°C using an oxygen electrode  in 1-ml assay mixture. Cellular dry weight (CDW) was determined using previously published methods [g. The pellets were washed with 0.9% NaCl and then dried at 105°C for 24 h to a constant weight using pre-dried and weighed 2-ml Eppendorf cups.l medium  with oct methods , using aP. putida KT2440 was obtained from TIGR  and was used as the framework of the network reconstruction. P. putida-specific primary and review literature and books were used to retrieve information about every network reaction: i) substrate specificity, ii) coenzyme specificity, iii) reaction directionality, iv) enzyme and reaction localization, and v) gene-protein-reaction (GPR) association. Relevant references were associated with every network reaction [see Additional files +), water (H2O), and various co-factors ). No gene-associated reactions were included when no corresponding gene was annotated in P. putida's genome but physiological or experimental data supported the presence of the biochemical transformation being part of P. putida's metabolism. Finally the reversibility was determined from primary literature data for each particular enzyme/reaction, if available. This literature search resulted in a first manually-curated reconstruction specific to P. putida's metabolism based on genome annotation and available biochemical evidence. However, this list is normally incomplete and will contain network gaps that may need to be filled depending on supporting evidence. This step requires manual effort again by searching the scientific literature for supporting information. If no P. putida-specific experimental evidence could be found for a transport reaction or biochemical transformation of a metabolite, no reaction or transporter was added to the network. Finally, the network capabilities were evaluated and compared with experimental data as described in Reed et al. [The reconstruction process was done as described previously . BrieflyGR , 06/2/2007 andd et al. . DetaileSimPheny  software was used for the reconstruction and gap evaluation process.v =  to a vector of time derivatives of the concentration vector x =  as vi,min≤ vi ≤ vi,max for reaction i. In fact, for every irreversible network reaction i, the lower bound was defined as vi,min ≥ 0 and the upper bound was defined as vi,max ≥ 0.The reconstructed metabolic network is often represented in a tabular format, listing all network reactions and metabolites in a human-readable manner along with confidence scores and comments  reactions define the presence of media components as if one would add metabolites into an in silico flask.Exchange reactions, which supply the network with nutrients or remove secretion products from the medium, were defined for all known medium components  or different genetic background (e.g. enzyme-deficient mutant) allow the transition from metabolic network reconstruction to condition-specific model. Note that the metabolic network reconstruction is unique to the target organism (and defined by its genome) while it can give rise to many different models by applying condition-specific constraints. All flux rates, P. putida's biomass composition are available, the biomass composition from E. coli [iJN746's biomass function. However, data from P. putida were added,  -39. Sinc E. coli ,93 was uposition ), when ain silico M9 minimal medium (iM9) and in silico Luria-Bertani medium (iLB) [iM9 simulation, and according to the well described M9 minimal medium [2, Co2 +, Fe2 +, H+, H2O, Na2 +, Ni2 +, NH4, Pi and SO4 were allowed to enter and leave the network by setting the constraints on the corresponding exchange reactions (i) to vi,min≥ -106 mmol/gDW/h and to vi,max≤ 106 mmol/gDW/h. The uptake rate for each carbon source was constrained to vi,min≥ -10 mmol/gDW/h and vi,max≤ 0 mmol/gDW/h. The oxygen uptake rate (OUR) was limited to vi,min≥ -18.5 mmol/gDW/h  . For iM9l medium , the fololi data ), if not methods .iM9 minimal medium (as described above) and setting the bounds of toluene uptake between vi,min≥ -11.9 mmol/gDW/h  was carried out using SimPheny . The underlying algorithm was described elsewhere ,97. The ement by  and vi,mvi) and represents the amount by which the objective function (e.g. growth rate) could be increased when the flux rate through this reaction was increased by a single unit [iM9 medium with toluene as carbon source. The constraints were set as described above and linear programming was employed to solve the optimization problem (maximizing growth).Reduced cost is a parameter of linear programming (LP) problems which is associated with each network reaction  iLB rich medium for general gene essentially experiment and ii) glucose-iM9 minimal medium for auxotrophy experiments (See above). The glucose uptake rate was fixed to vi,min = vi,max = -6.3 mmol/gDW/h in the latter study. OUR was set to be vi,min≥ -18.5 mmol/gDW/h in both cases.In order to determine the effect of a single gene deletion, all the reactions associated with each gene in function . A lethaiM9 medium was determined by setting the growth rate to vgrowth,min = vgrowth,max 0.2 gDW/gDW/h. The lower bound of each carbon uptake reaction was set to vi,min≥ -10 mmol/gDW/h and the upper bound was set to be vi,max≤ 0 mmol/gDW/h. The lower bound of the oxygen uptake rate was set to vi,min≥ -20 mmol/gDW/h for all simulations. In iJN746, six types of msc-PHAs are defined as well as msc-PHA compounds consisting of four different carbon chains [see Figure The msc-PHA production from each possible carbon source Table  in iM9 mAll computational simulations were performed using Matlab  if not stated otherwise. TomLab  was used as linear programming solver. Optimization formulations and the gene deletion studies employed the Matlab-based COBRA toolbox .Pseudomonas putida KT2440. JN and IT performed the analyses. JN, IT, BOP designed the study and wrote manuscript. All authors read and approved the final manuscript.All authors conceived the study. JN carried out the reconstruction of Table S1. Carbon, nitrogen, and sulfur sources, which enabled growth of iJN746.Click here for fileTable S2. Essentials genes predicted correctly in iJN746 compared with experimental data of P. aeruginosa.Click here for fileFigure S1. Schematic representation of in silico gene essentiality in iJN746 (iLB medium) compared experimental data of gene essentiality in P. aeruginosa [ruginosa .Click here for fileTable S3. False-positive essential genes in iJN746 when compared with P. aeruginosa's experimental data [tal data .Click here for fileTable S4. False-negative essential genes in iJN746. Genes that were not predicted to be essential in iJN746 but were reported as essential genes in P. aeruginosa [ruginosa .Click here for fileTable S5. Predicted essential genes in Glucose-iM9 minimal medium. Not shown are genes that were also predicted to be essential in iLB rich medium.Click here for fileTable S6. PHA polymer composition found in different Pseudomonas strains sorted by carbon sources.Click here for fileTable S7. List of metabolites in iJN746. The file contains a detail list of metabolites present in the metabolic reconstruction. The molecular formulae, the charge as well as the KeggID are shown.Click here for fileTable S8. List of the reactions contain in iJN746. The file details the reactions account in the metabolic reconstruction. The official name, the equation of the reaction, the subsystem, the EC number and de GPR association is shown.Click here for fileTable S9. List of biomass components in iJN746. This file contains the complete list of compounds which are part of Pseudomonas putida biomass.Click here for file


Tragopogon mirus and T. miscellus are allotetraploids (2n = 24) that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA) following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12) from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA) of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis). We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids.in situ hybridization (FISH), we analyzed copy numbers and distribution of these highly reiterated genes in seven lines of synthetic T. mirus  and four lines of synthetic T. miscellus . Variation among diploid parents accounted for most of the observed gene imbalances detected in F1 hybrids but cannot explain frequent deviations from repeat additivity seen in the allotetraploid lines. Polyploid lineages involving the same diploid parents differed in rDNA genotype, indicating that conditions immediately following genome doubling are crucial for rDNA changes. About 19% of the resynthesized allotetraploid individuals had equal rDNA contributions from the diploid parents, 74% were skewed towards either T. porrifolius or T. pratensis-type units, and only 7% had more rDNA copies of T. dubius-origin compared to the other two parents. Similar genotype frequencies were observed among natural populations. Despite directional reduction of units, the additivity of 35S rDNA locus number is maintained in 82% of the synthetic lines and in all natural allotetraploids.Using Southern blot hybridization and fluorescent Tragopogon allopolyploids. The extent of these rDNA changes was generally higher in natural populations than in the synthetic lines. We hypothesize that locus-specific and chromosomal changes in early generations of allopolyploids may influence patterns of rDNA evolution in later generations.Uniparental reductions of homeologous rRNA gene copies occurred in both synthetic and natural populations of  Chromosome counts suggest that between 30 and 100% of angiosperm species are polyploids  propose In plants, nuclear ribosomal DNA (rDNA) units occur in tandem arrays at one or several loci . EachThe behavior of rDNA in allopolyploids has attracted considerable attention because it is used as a molecular and cytogenetic marker of allopolyploidy . Indeed,Cardamine schulzii dCTP . The ITS-1 probe was a BstNI fragment from the cloned 18S-ITS-5.8S subregion of T. mirus rDNA (GenBank: AY458586). The 18S rDNA probe was a cloned 1.7-kb fragment of the tomato 18S rRNA gene [Southern blotting followed the protocol described in  using rDRNA gene , and theRNA gene . HybridiFor rDNA quantification, 17.5-200 ng of DNA were denatured in 0.2 M NaOH and loaded on a nylon membrane  using a slot blot apparatus . The membranes were hybridized with respective DNA probes in a Church Gilbert buffer . RadioacStatistical calculations were carried out using a chi-square function implemented in MicroSoft Excel.T. dubius, T. porrifolius, and T. pratensis  and maintained through selfing. "Lineages" were obtained by selfing of plants from the same cross but from different F1 seeds.The generation of allotetraploid lines and phenotypic and karyological analysis were described elsewhere . Brieflyis Table . In thisT. dubius 2613 and T. porrifolius 2607; these were scored as "high-copy" accessions. On the other hand, T. dubius 2615 and T. porrifolius 2611 showed relatively weak hybridization signals and were scored as "low-copy" accessions. Variation within populations and among progeny was low (< 15%) or negligible. The copy number estimates for each population are given in Table Because estimates of rDNA amounts may vary among populations of diploid species, we determined gene copy number of diploid parents using slot blot hybridization. Two to six individuals from each population were analyzed. The DNA was hybridized on blots with the 18S rDNA probe and the amount of radioactivity estimated. An example of our hybridization analysis is shown in Additional file BstNI restriction site polymorphism that distinguishes the diploid parents. In T. dubius, ITS-1 contains a BstNI site close to the 5.8S gene while there is no BstNI site in the ITS-1 of T. porrifolius and T. pratensis [T. pratensis and T. porrifolius and to the ~ 500-bp fragment in T. dubius  and one pair of 5S rDNA loci at metaphase . T. prae Figure . Both 35 Figures . Chromos1 Figure  than in 1 Figure . The FIST. mirus) and 5B (T. miscellus); the averaged values for each line are summarized in Figure We carried out a population-level study of 35S rRNA gene copy number using Southern blot hybridization using the ITS-1 probe Figures , 3. The 1 hybrid plants. In 11 F1 individuals (cross 47) from a single cross involving a "high-copy" T. dubius 2613 paternal parent and a "low-copy" T. porrifolius 2611 maternal parent  forming a lower molecular weight band, while the upper band of T. porrifolius-origin rDNA units (PO) was significantly weaker. Radioactivity scanning revealed that on average 60% of the total hybridization signal occurred in the lower molecular weight band, representing rDNA units of T. dubius origin. This value is close to the expected ratio considering the unit copy numbers in the diploid parents  was stronger than the band inherited from T. dubius. Again, F1 hybrids generally showed the expected number of genes inherited from their parents  and "high-copy" T. dubius 2613 . Thirty-seven individuals obtained from four lineages  were analyzed by Southern blot hybridization using the ITS-1 probe   in the S1 generation while one S2 individual gained a strong site likely on chromosome F  and "high-copy" T. dubius 2613 , but different individuals were used as parents than in crosses 73 and 98. On the Southern blots, the ratios of parental bands were mostly balanced  and "high-copy" T. dubius 2613 . Different individuals were used as parents than in the previous two lines. On blots, the probe showed a much stronger hybridization to the band corresponding to the T. porrifolius-origin homeolog than to the homeolog of T. dubius origin  are smaller and highly decondensed  showed only partial decondensation.In this cross we used "high-copy"  Figures , 5. FISHd Figure . On the T. dubius 2615 as the maternal parent and a "high-copy" T. porrifolius 2607 as a donor of the paternal genome. The gene ratio of all eight allotetraploid individuals was slightly shifted towards the T. porrifolius homeolog  and a "high-copy" T. dubius 2613  and yielded two lineages (134-15 and 134-16). On the blots, individuals from both lines showed a relatively weak T. dubius-origin band and a strong T. porrifolius-origin band . While both lineages 135-1 and 135-2 had gene ratios skewed towards  Figures , 5. Ther Figures . However Figures .T. pratensis 2609 was used as the maternal genome donor and "low-copy" T. dubius 2616 as the paternal genome donor. There was little or no variation in the gene ratios among the individuals analyzed, and all individuals had reduced Southern hybridization signal against the T. dubius-origin units  while most other members of this line had a dominant band of T. pratensis origin  and "high-copy" T. dubius 2613  were used as genome donors. This combination of parents yielded the largest number of allotetraploid individuals (35) out of the T. miscellus crosses attempted. Lineages 111-4, 111-5, and 111-7 had relatively uniform profiles of Southern hybridization bands with ratios skewed away from the units of T. dubius origin  exhibited balanced rDNA genotypes, 69 individuals (63%) showed more 35S rDNA of T. porrifolius origin than expected, and only 9 plants (8%) had more T. dubius-origin rDNA  had balanced gene ratios while 65 individuals (92%) inherited more T. pratensis-origin units than expected. The genetic variation in copy ratios among the progeny of a single cross ranged from low or negligible  to high   Figure . Some crTragopogon species. The differences in copy number were larger between populations of the same species than between species. These data indicate that shifts in rDNA array sizes occur at the lineage level. Similar levels of interpopulation variability were reported among Arabidopsis accessions [1 diploid hybrids. For example, a "low-copy" T. dubius accession 2615 combined with a "high-copy" T. porrifolius 2607 should generate skewed 1:2 DU/PO ratios in a hybrid  resulting from the cross T. dubius 2613 × T. porrifolius 2611 showed altered homeolog gene ratios from Mendelian expectation . In general, deviation from repeat additivity occurred more frequently in lines resulting from crosses involving T. dubius 2613 as a parent than any other accession  were sampled to allow firm conclusion on this topic. Variation within lines was generally lower than that between the lines. Nevertheless, members of lineage 111-1 (synthetic T. miscellus) did show much variation . High transcriptional activity from an rDNA locus with reduced rRNA gene copy numbers has also been reported previously for natural T. mirus and T. miscellus individuals [T. miscellus had two small and two large rDNA loci  revealed a number of abnormalities, including multivalent formation, lagging chromosomes, and aneuploidy [T. dubius 2613 (70 and 98) showed higher frequencies of both meiotic irregularities and rDNA rearrangements than lines derived from other accessions. There seems to be a good correlation between meiotic pairing abnormalities and frequency of rDNA changes in Tragopogon. Ribosomal RNA gene copy number losses may occur through unequal recombination .Meiotic aberrations have been implicated in rRNA gene imbalances in natural populations of n . Indeedeuploidy . Chromos0 plants , arguing that meiotic irregularities may not be the only mechanism responsible for rDNA rearrangements, but mechanisms acting during or immediately after genome doubling could also be involved. Such mechanisms include recombination between rDNA loci (at homologous loci or otherwise), aneuploidy, and chromosome loss in somatic cells, the latter two having been reported in interspecific Arabidopsis hybrids and allopolyploids [T. mirus synthetic lines 70 and 73  have nearly two-fold higher rDNA copy number compared to T. porrifolius (2611) at the same location. Yet, in both natural populations of T. mirus allotetraploids sampled, the T. dubius rDNA represents only 20-25% of the total rDNA [du repeats have been lost in the approximately 30-40 generations since the polyploids  formed at these locations. We were surprised to discover that one (98) out of three lines of T. mirus synthesized from Pullman parents showed a genotype that closely resembled that of both natural populations. The gene imbalances were, however, less pronounced in the other two lines . In line 73 some individuals even had more T. dubius units, indicating that array size could be both maintained and altered during allopolyploidy. Interestingly, one natural population of T. mirus  also contained individuals with ratios skewed away from T. porrifolius  homogenized ITS nearly to completion another old allotetraploid, Tragopogon castellanus, retained equivalent amounts of both parental ITS types [Glycine [It is assumed that, as time passes, homogenization processes such as unequal crossing over continue to gradually replace parental rDNA arrays with novel allopolyploid species arrays. However, the time factor does not seem to be the only player. For example, while most Old World TS types . SimilarTS types  and Glyc[Glycine  most, buTragopogon allopolyploids, indicating that some aspects of genome evolution might be repeatable. The biological significance of gene losses as well as their potential adaptive significance is unclear. Uniparental deletions  would not affect fitness because there is a large excess of genes in the partner genome. One possibility is that intralocus rearrangements  preclude interlocus homogenization in older allopolyploids. The latter process is frequently associated with reduction of loci and repeats [Tragopogon populations remains to be analyzed. Finally, rDNA arrays were recently shown to influence gene expression at ectopic positions in Drosophila [We observed similar reductions of homeologous rRNA gene copies in both synthetic and natural, 80-year-old populations of  repeats . It is t repeats  proposedosophila , and locin situ hybridization; NOR: nucleolar organizer region; Organisms: T. dubius: Tragopogon dubius; T. mirus: Tragopogon mirus; T. miscellus: Tragopogon miscellus; T. porrifolius: Tragopogon porrifolius; T. pratensis: Tragopogon pratensisITS: internal transcribed spacer; IGS: intergenic spacer; FISH: fluorescent HM carried out most of the molecular biology and cytogenetic experiments. AK, DS, PS, and AL designed the study. AK wrote and drafted the paper. RM participated in the DNA analysis. AL carried out some FISH experiments. JT made the crosses and prepared synthetic lines. All authors read and approved the final manuscript.Slot blot quantification of gene copies in parental diploids. The DNA amounts are indicated above each lane. The blot was hybridized with the 32P - labeled 18S rDNA probe. Experiments I and II were carried out in this study; experiment III is from [ is from .Click here for fileT. mirus (S1 generation)Analysis of intergenic rDNA spacer polymorphisms in DNA of synthetic . Genomic DNA was digested with BstYI and SspI restriction enzymes. Southern blot hybridization was carried out using the 26S rDNA probe.Click here for file2 generation of synthetic T. mirusSouthern blot analysis of the S. Individuals were the progenies of three lineages from line 73.Click here for file2 generation of synthetic T. mirusFISH analysis of the S. The same plants as in Additional file po locus left after the deletion of the majority of genes. Note fusion of subtelomeric NORs at the chromatids  and considerable variability in condensation of rDNA chromatin among sister plants (A-C). The following individuals are shown: (A) - 73-14-6A, (B) - 73-14-6B, (C) - 73-14-6C,  - 73-1-3 D, (F) - 73-2-8B.Click here for fileT. mirus (line 116)Expression analysis of rDNA in synthetic . RNA isolation and RT-CAPS assay were carried out as described in [ribed in . Note tyClick here for fileT. dubius 2613 and T. porrifolius 2611Analysis of IGS subrepeats in . We used a dot plot alignment tool at http://www.vivo.colostate.edu/molkit/dnadot/, self (x-axis) to self (y-axis) alignment (Window size: 9. Mismatch limit: 0). The IGSs were amplified using primers designed to conserved regions in 26S rDNA and 18S rDNA [18S rDNA . BrieflyClick here for file


Parthenium argentatum (guayule) is an industrial crop that produces latex, which was recently commercialized as a source of latex rubber safe for people with Type I latex allergy. The complete plastid genome of P. argentatum was sequenced. The sequence provides important information useful for genetic engineering strategies. Comparison to the sequences of plastid genomes from three other members of the Asteraceae, Lactuca sativa, Guitozia abyssinica and Helianthus annuus revealed details of the evolution of the four genomes. Chloroplast-specific DNA barcodes were developed for identification of Parthenium species and lines.P. argentatum is 152,803 bp. Based on the overall comparison of individual protein coding genes with those in L. sativa, G. abyssinica and H. annuus, we demonstrate that the P. argentatum chloroplast genome sequence is most closely related to that of H. annuus. Similar to chloroplast genomes in G. abyssinica, L. sativa and H. annuus, the plastid genome of P. argentatum has a large 23 kb inversion with a smaller 3.4 kb inversion, within the large inversion. Using the matK and psbA-trnH spacer chloroplast DNA barcodes, three of the four Parthenium species tested, P. tomentosum, P. hysterophorus and P. schottii, can be differentiated from P. argentatum. In addition, we identified lines within P. argentatum.The complete plastid genome of P. argentatum chloroplast will enrich the sequence resources of plastid genomes in commercial crops. The availability of the complete plastid genome sequence may facilitate transformation efficiency by using the precise sequence of endogenous flanking sequences and regulatory elements in chloroplast transformation vectors. The DNA barcoding study forms the foundation for genetic identification of commercially significant lines of P. argentatum that are important for producing latex.The genome sequence of the  Parthenium argentatum Gray, commonly known as guayule, is a shrub in the Asteraceae that is native to the southwestern United States and northern Mexico. Parthenium argentatum produces high quality rubber in bark tissue, which is under development for biomedical uses. The U.S. Food and Drug Administration recently approved the first medical device made from P. argentatum natural rubber. Products made from P. argentatum latex are designed for people who have Type I latex allergies, induced by natural rubber proteins from Hevea brasiliensis. In addition to biomedical products, natural rubber is essential and irreplaceable in many industrial and consumer applications, and the price is rising under heavy demand, making natural rubber increasingly more precious. As an industrial crop that grows in temperate climates, P. argentatum represents a viable alternative source of high quality natural rubber.P. argentatum, is through chloroplast engineering [One strategy for improving crops, such as the rubber-producing ineering -3. Transineering , enhanciineering .P. argentatum, transgene containment is important because it is currently cultivated as an industrial crop in its native region in the southwestern United States.Importantly from the point of view of minimizing environmental impact, expressing foreign proteins in the chloroplast results in transgene containment ,7. It isConstruction of vectors for chloroplast transformation requires some knowledge of the chloroplast genome sequence to identify insertion sites. To date, just short of one hundred plastid genomes from angiosperms have been completely sequenced. The sequences are highly conserved . InteresChloroplast sequences are useful for identification of species, using a particular sequence as a DNA tag or barcode . An ideaP. argentatum as a commercial source of natural rubber. Breeding efforts would be enhanced by informative chloroplast DNA barcodes. Because a very small amount of tissue is required for barcode analysis, purity of breeding lines can be determined at an early stage of seedling growth. In addition, barcodes would allow breeders and seed producers to discover seed lot contamination before advancing breeding lines for latex production. Having the ability to removing contaminating lines, especially when they represent lower rubber lines, would improve the efficacy of breeding efforts.At present, classical breeding is being used to improve P. argentatum to enhance its commercial viability. We have chosen two approaches, biotechnology through chloroplast metabolic engineering and marker-assisted breeding. The P. argentatum chloroplast genome sequence that we report herein, supports our efforts in both approaches. In this article, we report the complete sequence of the chloroplast genome of P. argentatum and describe the development of DNA barcodes. The complete sequence of the P. argentatum chloroplast genome has enabled us to construct chloroplast transformation vectors based on the exact sequence of the large inverted regions, and to identify novel insertion sites in non-essential, non-coding regions. Barcode analysis with the matK gene and psbA-trnH spacer sequence allowed us to discriminate three of four Parthenium species from each other and from P. argentatum, and a subset of the P. argentatum lines from each other. These barcodes will be used in our breeding program.The focus of our research program is improvement of Parthenium argentatum is represented in a circular map . It is 152,803 bp in size and includes a duplicated region of inverted repeats (IR) of 24,424 bp. The IR are separated by small single copy (SSC) and large single copy (LSC) regions of 19,390 bp and 84,565 bp, respectively. The total G+C content of the whole chloroplast genome is 37.6%. The gene content and arrangement were observed to be similar to those in Lactuca sativa and Helianthus annuus [Guitozia abyssinica (NC_010601), including one large (Inv1) and one small inversion (Inv2) in the LSC region. There are 85 genes coding for proteins  and H. annuus (98.4%), than in L. sativa (97.2%). The greater average identity in G. abyssinica than in H. annuus is in large part due to deletions in the two copies of the ycf2 loci in H. annuus, otherwise, H. annuus is more similar overall than G. abyssinica. Fourteen genes in H. annuus and G. abyssinica were 100% identical to those in P. argentatum, compared to only four genes in L. sativa  and three cultivated lines of P. argentatum  (data not shown). The best differentiation of Parthenium species and lines within P. argentatum was obtained with the psbA-trnH spacer region barcode. There were 5 indel sites in 400 bp of DNA in the six lines tested. When 1000 bp of the matK DNA barcode were analyzed, a total of 12 indel sites were found. In 600 bp from the trnL-UAA intron region, only one indel site was observed. Obtaining good sequence from the rpoC spacer region was difficult, but in 500 bp, four indel sites were identified. Therefore, due to the higher number of informative sites, the matK and psbA-trnH DNA barcodes were used for further studies of Parthenium taxa.To differentiate  studies ,18,23,24matK, an efficient barcode for Parthenium species was defined. Using the Parth-matK-F and Parth-matK-R primers, matK DNA sequences were examined in Parthenium species, lines of P. argentatum and AZ101, a hybrid of P. argentatum cv. 11591 × P. tomentosum. We sampled 601 nucleotides in the matK gene, which yielded fourteen potentially informative, variable positions (2.3%), with eight nucleotide substitutions (1.3%) and six length mutations (indels) (1.0%). Although the psbA-trnH spacer region in P. integrifolium DNA did amplify with the psbA-trnH barcode primers, the matK locus did not amplify with the matK-barcode primers. This matK barcode was effective at differentiating P. schottii, P. hysterophorus, and P. tomentosum from each other and from a group that included P. incanum, P. argentatum lines and one hybrid , with eleven nucleotide substitutions (2.4%) and three length mutations (0.7%). First of all, we found that there was 100% consensus in the barcode sequence among samples tested of line AZ1 (n = 21), AZ4 (n = 15), Cal6 (n = 17), AZ101 (n = 3), P. incanum (n = 6) and P. tomentosum (n = 5). On the other hand, there was a second barcode sequence within line AZ2 , AZ3 , AZ5 , AZ6  and 11591 . The minority or alternative barcodes differed from the corresponding common barcode by one to three bases.The non-coding spacer region between es Table . A 469 bpsbA-trnH spacer barcode differentiated P. hysterophorus, P. integrifolium and P. schottii from each other and from all the other species and lines. The psbA-trnH spacer barcode of P. argentatum cultivar 11591 and the two breeding lines C156 and C86 was different from those of the remaining P. argentatum lines, P. tomentosum and P. incanum. The barcode of AZ101, which is a hybrid between P. argentatum cultivar (cv.) 11591 and P. tomentosum, is similar to or identical to that of P. tomentosum. Parthenium incanum's barcode clustered with two AZ2 variants and a plant of unknown parentage, indicating their close relationship. Analysis with both the psbA-trnH spacer and matK barcodes provided further differentiation , to maximize the possibility of successful recombination. Improving crop plants via chloroplast transformation is a viable strategy [Metabolic engineering of plants by inserting transgenes in the chloroplast would potentially be made more efficient with knowledge of chloroplast sequences, based on the conclusions of one group that chloroplast transformation efficiency was significantly enhanced when vectors were constructed with 100% homologous sequences . Other gstrategy  that wilmatK barcode contained sufficient information to differentiate three Parthenium species  from each other and from P. argentatum and P. incanum. However, the matK-barcode did not differentiate P. incanum from P. argentatum or P. agentatum lines from each other  have a different psbA-trnH barcode than the common DNA barcode sequence of AZ2 , suggest that chloroplasts are not present in the mature pollen and thereby are likely to be maternally inherited in  species , as wellthaliana ,9, it isP. argentatum chloroplast will enrich the sequence resources of plastid genomes in commercial crops. The availability of the complete plastid genome sequence may facilitate improved transformation efficiency by using the precise endogenous flanking sequences and regulatory elements in chloroplast transformation vectors. The DNA barcoding study forms the foundation for genetic identification of commercially important lines of P. argentatum that are producing natural rubber latex for biomedical applications.The genome sequence of the Parthenium argentatum line AZ2 plant was placed in the dark for 2-days before harvesting young leaves. Chloroplasts were isolated from leaves using a 30-52% sucrose-gradient according to both Palmer [EcoRI and BstBI and examined by agarose gel electrophoresis to confirm the clear banding pattern, which indicated that the amplification product was chloroplast and not nuclear DNA.A mature, greenhouse-grown h Palmer  and Jansh Palmer . GenomicParthenium argentatum chloroplast genome sequencing was carried out using 454 Sequence Technology . Random sequences were assembled into a draft genome sequence using Newbler as described by Chaisson et al. [P. argentatum and the other members of the Asteraceae, H. annuus (NC_007977), L. sativa (NC_007578), and G. abyssnica (NC_010601), the percent identity of nucleotide sequences within the open reading frame was calculated based on alignments made with ClustalW [P. argentatum were identified by comparing the sequence in the inversion region [L. sativa, H. annuus and Nicotiana tabacum (NC_001879). The end points of the inversion were determined as described by Timme et al. [P. argentatum  to idenva NC_0078, and G.ClustalW . Inversin region  with thae et al. . The mVIe et al.  was usedm Figure .Parthenium species and lines, a chloroplast DNA barcode system was developed. Four regions of the Parthenium chloroplast genome were explored, including the intron in trnL-UAA, the rpoC and matK genes, and the non-coding spacer between psbA-trnH. Plant genomic DNA was isolated from young plants (3-4 weeks old) of available Parthenium species, cultivars, and lines using DNeasy Plant Mini Kit . PCR was carried out with Phusion DNA Polymerase according to manufacturer's instructions . The primers, TrnL-F, 5'-CGAGTTGGGGATAGAGGGACTTGAAC-3' and TrnL-R, 5'-GATATGGCGAAATAGGTAGACGCTACGGAC-3' were used to amplify trnL-UAA; for rpoC, rpoC1-F, 5'-CATAGGAGTTGCTAAGAGTCAAATTCGG-3' and rpoC2-R, 5'-CCTTTTCTAGATCTTGATTCACGTAGAAATTCCGC-3'; for matK, matK-F, 5'-GAATTTCAAATGGAGAATTCCAAAGC-3' and matK-end-R, 5'-CGAGCTAAAGTTCTAGCACAAGAAAGTCG-3'; and for psbA-trnH, psbA-F, 5'-GGAAGTTATGCATGAACGTAATGCTC-3' and trnH-R, 5'-CGCGCATGGTGGATTCACAATC-3'. PCR products were sequenced in both directions. Sequences were compared and any sequences with differences from the majority sequence were re-sequenced in both directions. Barcode differentiations were visualized using the UPMGA best tree method in Jukes-Cantor mode and then bootstrapped with 1000 replicates according to manufacturer's instructions in MacVector . Helianthus annuus was included as an outgroup.To differentiate various Parthenium, the central region of the matK gene was the best for finding divergence in Parthenium species. DNA from P. schottii, P. tomentosum, P. incanum, a cultivar of P. argentatum cv. 11591, nine lines of P. argentatum  and AZ101 (a hybrid of P. argentatum cv 11591 × P. tomentosum) was amplified via PCR with a 60°C annealing temp, using primers Parth-matK-F, 5'-CAAGCTCATCTGGAAATCTTGGTTCAGGCTC-3' and Parth-matK-R, 5'-GCCAACGATCCAACCAGAGGCATAATTGG-3'. The PCR products were sequenced in both directions using the same primers. In addition, the non-coding spacer region between the two genes psbA-trnH (500 bp) was used to further differentiate the Parthenium taxa. DNA was amplified with the PCR using primers psbA-F and trnH-R at an annealing temperature of 58°C. PCR products were sequenced in both directions with the following primers, psbAF1-seq, 5'-GCTGCTATTGAAGCTCCATC-3' and Rev1-seq-trnh Gua, 5'-CCTTGATCCACTTGGCTACATCCG-3'.Based on preliminary analysis of selected taxa of IR: inverted repeat; SSC: small single copy; LSC: large single copy; bp: base pair; kb: kilobase pair; INV: inverted region.SK designed and performed all aspects of the laboratory research, isolated chloroplasts, assembled the genome sequence, compared the coding sequences in the four genomes, designed and performed all barcode amplifications and sequencing, aligned the sequences, and wrote the first draft. FMH conceived of and participated in the sequencing of the chloroplast genome. CMM facilitated all aspects of the laboratory work and revised the manuscript. KC conceived this study, provided the plant lines, and revised the manuscript. MCW supervised the work, assisted in the design of this study, with SK interpreted all data, performed analysis of barcode sequence alignments, and revised all versions of the manuscript. All authors read and approved the final manuscript.Parthenium argentatum (Genbank Accession Location of 1230297) chloroplast genes in the genome sequence. The coordinates of genes in the chloroplast genome of Parthenium argentatum and comparison of the sequence of these genes (% identity) with those in Helianthus annuus, Guitozia abyssinica and Lactuca sativa.Click here for file


To the Editor:Mycobacterium colombiense is a new species belonging to the M. avium complex (MAC). It is characterized by a unique internal transcribed spacer sequence and causing respiratory tract and disseminated infection in HIV-infected patients in Colombia  and an erythrocyte sedimentation rate of 18 mm/h.A 25-month-old girl with an unremarkable medical history was hospitalized in the pediatric department of Timone Hospital, Marseille, France, due to development of swelling in a right subclavicular lymph node over a 1-month period. A 5-day course of oxacillin, which was administered orally, had been unsuccessful in alleviating the symptoms. The patient’s general condition was excellent, and results of a physical examination were normal, with the exception of a 2-cm hard, immobile, yet painless, noninflammatory, enlarged lymph node. Due to the presence of the enlarged lymph node, a chest radiograph was performed, and results were normal. A hemogram indicated a hemoglobin concentration of 113 g/L, a leukocyte count 8.3 × 102 at 37°C yielded acid-fast bacilli after a 7-day incubation period.Fine-needle aspiration of the lymph node showed necrosis and mature, activated lymphocytes. These results suggested a possible diagnosis of lymphoma, and a surgical excision biopsy was subsequently performed. Direct microscopic examinations were carried out after results obtained by Gram and Ziehl-Neelsen staining showed that the lymph node was negative for acid-fast bacilli. Histopathologic analysis indicated epithelioid cell granulomas containing giant cells and caseous necrosis without altered leukocytes, all of which are compatible with tuberculosis. Culturing of the biopsy specimen in BACTEC broth  at 5% COrpoB gene (M. colombiense CIP108962T (M. colombiense from other MAC species (After inactivating the cells and extracting the DNA by using a previously described method, we identified the isolate by PCR sequencing of the M. colombiense was shown to be responsible for an enlarged lymph node in 1 child from Spain who did not show any evidence of HIV infection (M. colombiense–infected lymph nodes also yield clinical and histopathologic features evocative of tuberculosis. Indeed, MAC organisms remain the most prevalent agents demonstrated in diseased lymph nodes in children (Recently, M. colombiense may have been underestimated. In children, M. hemophilum (M. avium subsp. avium (M. avium subsp. hominissuis (M. lentiflavum (M. bohemicum (M. simiae (Mycobacterium species is absolutely necessary. This identification should use PCR sequencing of selected universal molecular targets, including the 16S rRNA and rpoB genes (Because modern molecular tools used for the description of emerging MAC species have not been available in most previous reports, the real prevalence of . simiae . BecauseMycobacterium spp. implicated in lymphadenopathy in children, France*