Summary: GenAlEx: Genetic Analysis in Excel is a cross-platform package for population genetic analyses that runs within Microsoft Excel. GenAlEx offers analysis of diploid codominant, haploid and binary genetic loci and DNA sequences. Both frequency-based  and distance-based  analyses are provided. New features include calculation of new estimators of population structure: G′ST, G′′ST, Jost’s Dest and F′ST through AMOVA, Shannon Information analysis, linkage disequilibrium analysis for biallelic data and novel heterogeneity tests for spatial autocorrelation analysis. Export to more than 30 other data formats is provided. Teaching tutorials and expanded step-by-step output options are included. The comprehensive guide has been fully revised.Availability and implementation: GenAlEx is written in VBA and provided as a Microsoft Excel Add-in . GenAlEx, and supporting documentation and tutorials are freely available at: http://biology.anu.edu.au/GenAlEx.Contact:rod.peakall@anu.edu.au GenAlEx 6 was originally developed as a teaching tool to facilitate teaching population genetic analysis at the graduate level . GenAlExF-statistics, Nei’s genetic distance, population assignment and relatedness. The latter includes Analysis of Molecular Variance (AMOVA), Principal Coordinates Analysis (PCoA), Mantel tests, TwoGener, multivariate and 2D spatial autocorrelation. Readers are referred to GenAlEx offers population genetic analysis of diploid codominant, haploid, haplotypic and binary genetic data from animals, plants and microorganisms. It accommodates a wide range of genetic markers, including microsatellites (SSRs), single-nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms and DNA sequences. Both allele frequency-based and distance-based analysis options are provided. The former includes estimates of heterozygosity and genetic diversity, GenAlEx 6.5 maintains backward compatibility, but it provides access to the expanded spreadsheet of Excel 2007 onward. Thus, the maximum numbers of loci and samples are vastly expanded and only constrained by memory. More than 30 different Excel graphs summarize the outcomes of genetic analyses. Graphics can be further manipulated with Excel options and easily converted to pdf or other publication-quality formats.FST as a measure of population genetic structure . GenAlEx offers the calculation of a series of Shannon indices, including the mutual information index FST, F′ST, G′ST, G′′ST, Dest and SHUA. This will allow informed comparison of the new estimators of population structure.The Mantel test capability of GenAlEx has been extended to allow multiple comparison among pairwise population statistics such as GenAlEx 6.5 introduces novel heterogeneity tests  around the world.More than 150 pages of documentation are provided. This includes Appendix 1 that outlines the statistical analyses used and their supporting references. The revised guide to GenAlEx 6.5 fully cross-links with the GenAlEx tutorials and Appendix 1.GenAlEx 6.5 offers a wide range of population genetic analysis options for the full spectrum of genetic markers within the Microsoft Excel environment on both PC and Macintosh computers. When combined with its user-friendly interface, rich graphical outputs for data exploration and publication, tools for data manipulation and export options to many other software packages, we believe that GenAlEx offers an ideal launching pad for population genetic analysis by students, teachers and researchers alike.


Trypanosoma (Duttonella) vivax genotypes between West Africa and Southern Africa can provide information on the epidemiology and control of trypanosomosis. Cattle blood samples from Zambia and Ghana were screened for T. vivax infection using specie-specific PCR and sequencing analysis. Substantial polymorphism was obtained from phylogenetic analysis of sequences of cathepsin L-like catalytic domains. T. vivax from Ghana clustered together with West African and South American sequences, while T. vivax from Zambia formed one distinct clade and clustered with East African and Southern African sequences. This study suggests existence of distinct genetic diversity between T. vivax genotypes from West Africa and Zambia as per their geographical origins.Understanding the evolutionary relationships of  T. vivax infection that predominantly affects cattle, buffalo, goats, sheep, and wild bovids [T. vivax is a hemoprotozoan parasite found in Africa, central and South America. In Africa the parasite is transmitted both cyclically by Glossina spp. and mechanically by tabanids and other biting flies, while in South America only mechanical transmission occurs [T. vivax infection.Food and Agriculture Organization (FAO) has estimated that the problem of trypanosomosis costs Africa about US$4.5 billion per year, which includes losses in agricultural production, perennial expenditure on trypanocidal drugs, and other local intervention schemes in attempts to control trypanosomosis , 19. Of d bovids , 18. T. n occurs , 26, 29.T. vivax infection in small ruminants from Africa and South America has been reported to result in a variety of clinical outcomes ranging from acute to chronic or subclinical disease; the course of infection varying depending on the parasite strain, endemicity, and the species and breed of the ruminant host [T. vivax strains are believed to be more pathogenic compared to East African strains, however, severe hemorrhagic outbreaks, with high mortality levels, have been periodically reported in East Africa [T. vivax strains have been shown to have close genetic similarity to West African strains [T. vivax was imported into the New World through infected cattle from West Africa [T. vivax is considered to be an important salivarian trypanosome species because of its wide distribution, pathogenicity to cattle, and its relatively high infection rates in tsetse, it remains highly neglected in the scientific literature. This is attributed to the species being notoriously difficult to work with as isolates are not easily adapted to culture or grown in standard laboratory animals [T. vivax have typically focused on comparison between isolates from across Africa and South America [ant host , 5, 10. t Africa , 21. Sou strains , 14, re-t Africa . Althoug animals . Few stu America .T. vivax circulating in Ghana (West Africa) and Zambia (Southern Africa) and compared their relationship by phylogenetic analysis.The genomes of trypanosomes consist of multiple copies of catL-like genes that vary depending on species. Cathepsin L-like (catL-like) enzymes are cysteine proteases involved in the pathogenicity, immunity, cell differentiation, infectivity, and metabolism of trypanosomes . The potT. vivax infection using the species-specific PCR (designated TviCatL-PCR) employing primers TviCatL1 and DTO155 [T. vivax-positive samples (n = 11) were randomly selected and subjected to PCR amplifying approximately 500 bp of cathepsin L-like gene using primers DTO154 and DTO155 [T. vivax in a previous study [n = 11). PCR was conducted using Amplitaq Gold® 360 reagent  with 35 cycles of amplification to minimize PCR errors. The amplified products were cloned into a pGEM-T vector  and sequenced using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) and an ABI Prism 3130× genetic analyzer according to the manufacturers’ instructions. The DNA sequences obtained were submitted to the DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp) under Accession Nos. AB781071 to AB781078.A total of 100 bovine blood samples from Petauke district in Zambia (Latitude: −14° 14′ 28.5246′′ and Longitude: 31° 19′ 11.4564′′) were screened for T. vivax infection (data not shown). The infection rate (32%) was comparable to that observed in Ghanaian cattle (25.3%) in our previous study [T. vivax from Ghana clustered together with those from West Africa (Burkina Faso) and South America (Brazil and Venezuela)  . T. vivaT. vivax has been reported to be limited [T. vivax with those from West Africa and South America is in agreement with previous findings [T. vivax was introduced into South America with bovines imported from West Africa [T. vivax from Central Africa is reported to share molecular features with both the East and West African isolates [T. vivax populations corroborating the high genetic divergence between West and East African T. vivax strains. This has been revealed according to morphological, immunological, pathological, molecular features, and behavior in the tsetse fly and mammalian hosts.Genetic diversity of  limited . It is,  limited . Clusterfindings , 12, 14,t Africa , 18. T. isolates . Previouisolates , 14, 11 T. vivax using a panel of eight microsatellites [T. vivax-positive samples (data not shown). Such failure has been attributed to a population whose parasitemia is below the threshold for detection by single-locus PCR [T. vivax genotypes from Ghana (West Africa) and Zambia (Southern Africa).An attempt to characterize tellites  failed tocus PCR . Additioocus PCR . In conc


Eucalyptus tetrodonta, a co-dominant tree species of tropical, northern Australian savannas, does not invade adjacent monsoon rain forest unless the forest is burnt intensely. Such facilitation by fire of seedling establishment is known as the "ashbed effect." Because the ashbed effect might involve disruption of common mycorrhizal networks, we hypothesized that in the absence of fire, intact rain forest arbuscular mycorrhizal (AM) networks inhibit E. tetrodonta seedlings. Although arbuscular mycorrhizas predominate in the rain forest, common tree species of the northern Australian savannas (including adult E. tetrodonta) host ectomycorrhizas. To test our hypothesis, we grew E. tetrodonta and Ceiba pentandra (an AM-responsive species used to confirm treatments) separately in microcosms of ambient or methyl-bromide fumigated rain forest soil with or without severing potential mycorrhizal fungus connections to an AM nurse plant, Litsea glutinosa. As expected, C. pentandra formed mycorrhizas in all treatments but had the most root colonization and grew fastest in ambient soil. E. tetrodonta seedlings also formed AM in all treatments, but severing hyphae in fumigated soil produced the least colonization and the best growth. Three of ten E. tetrodonta seedlings in ambient soil with intact network hyphae died. Because foliar chlorosis was symptomatic of iron deficiency, after 130 days we began to fertilize half the E. tetrodonta seedlings in ambient soil with an iron solution. Iron fertilization completely remedied chlorosis and stimulated leaf growth. Our microcosm results suggest that in intact rain forest, common AM networks mediate belowground competition and AM fungi may exacerbate iron deficiency, thereby enhancing resistance to E. tetrodonta invasion. Common AM networks–previously unrecognized as contributors to the ashbed effect–probably help to maintain the rain forest–savanna boundary. Eucalyptus tetrodonta F.Muell. and E. miniata Cunn. ex Schauer are canopy co-dominants of coastal savannas E. tetrodonta seedlings can invade, but otherwise they cannot, even after canopy destruction by tropical cyclones E. tetrodonta are uncertain.Eucalypts predominate across much of monsoon tropical, northern Australia. The apparent necessity of fire to facilitate establishment of some species' seedlings, especially those of eucalypts and pines, is known as the "ashbed effect"  as adults E. tetrodonta seedlings, and that, much like intense burns, soil fumigation or partial sterilization  in the end compartments of rectangular microcosms divided into three equal compartments by nylon mesh root barriers through which AM fungus hyphae could pass (We grew 40 uld pass . In the Ceiba pentandra (L.) Gaertn.  in microcosms to which we applied the same four treatments as for E. tetrodonta. We did not grow C. pentandra together with E. tetrodonta in the same microcosms because we did not wish them to compete with one another .In order to determine if soil fumigation and repeated hypha severing succeeded in diminishing AM formation as intended, we grew 40 seedlings of an AM-responsive We constructed the microcosms from plastic planter boxes  C. B. Rob. (Lauraceae). Before transplant, each sprout's source root was treated by dipping it into powdered rooting hormone and micronutrients ("Clonex"). Then, the compartments were filled with ambient soil topped with 2–3 cm of fumigated soil to reduce the chance of spores splashing into end compartments. We filled both end compartments of 20 microcosms with ambient soil, and filled those of an additional 20 microcosms with fumigated soil. The end compartments were neither actively inoculated with AM fungi, nor were they treated with a microbial filtrate We filled the microcosm central compartments with rain forest soil  collecteL. glutinosa sprouts on 20 June, 1997, we randomized all 40 microcosms with no more than 2 cm separation in two rows on a wire mesh screenhouse bench at the CSIRO Berrimah campus  in Darwin. The screenhouse provided light shade. The nurse plants grew in the microcosms for six months before we transplanted E. tetrodonta and C. pentandra seedlings to begin the experiment. This pre-treatment allowed AM fungus hyphae to extend into stationary end compartments, and allowed hyphae from propagules in stationary, ambient-soil end compartments potentially to reach nurse plant roots. In order to disrupt hypha spread, three days after transplanting the L. glutinosa sprouts, we began lifting the detached end compartments of each microcosm by 8–10 cm every two or three days. We continued this periodic severing of hyphae until all plants were harvested.After transplanting E. tetrodonta obtained from Top End Seeds  and of C. pentandra obtained from the George Brown Botanic Gardens (Darwin) were sown in germination flats of fumigated rain forest soil in the screenhouse. On 20 December, 1997, we transplanted two 1–2 week-old E. tetrodonta or three just-emerged C. pentandra seedlings into both end compartments of separate microcosms. One week later on 28 December, we replaced C. pentandra of which all had died in 22 microcosm ends. A week before the first growth measurement, we clipped the weakest individuals of C. pentandra and E. tetrodonta, leaving only one seedling in each end compartment. Thus, there were 40 individuals (10 per treatment) in 20 microcosms for each species .Seeds of E. tetrodonta seedlings in ambient soil with or without iron fertilization from May through July, 1998, mean monthly rainfall was less than 0.4 mm, and the maximum and minimum mean monthly temperatures were 33.4 and 21.4 °C, respectively .During the rainy season (October–April) all microcosms received natural rainfall and were watered only if necessary, but during the dry season they were watered three times daily by an overhead sprinkler system. Mean monthly rainfall from December, 1997 through April, 1998 ranged from 75 mm to 789 mm, and mean monthly maximum and minimum air temperatures ranged from 31.6 to 33.9 and 24.9 to 25.5 °C, respectively. While we continued to grow E. tetrodonta. We measured L. glutinosa nurse plants only twice, on 22 December, 1997, at the start of the experiment, and on 10 May, 1998. We measured L. glutinosa stem diameter, height from the soil surface to the tallest shoot apex, longest leaf length, and counted the number of leaves.Beginning on 19 January, 1998, one month after transplant, and continuing every two weeks until harvest, we measured height from the soil surface to the shoot apex, length of the longest leaflet or leaf, and counted the number of leaves of every seedling. Additionally, we categorically recorded foliar chlorosis of C. pentandra and only the E. tetrodonta grown in fumigated soil. We harvested at this time because of declining health and mortality of E. tetrodonta, especially in networked, ambient soil. Root systems of both species were extracted from the soil, rinsed gently over a 1 mm sieve, and preserved in 50% ethanol for assessment of mycorrhizal colonization. For E. tetrodonta and their accompanying L. glutinosa nurse plants, we separated leaf blades from petioles and stems, dried all to constant weight in an oven at 60 °C, and then weighed them.On 10 May, 1998, 141 DAT, we harvested all E. tetrodonta seedlings grown in ambient soil when we harvested the seedlings from fumigated soil. Instead, we continued to grow them for an additional 69 days with or without iron fertilization. We randomly assigned half of each group of hyphae-severed and networked seedlings to receive 81 mg of iron  eight times at irregular intervals . This addition was intended transiently to elevate the available iron concentration of rain forest soil approximately to that of savanna soil  with a compound microscope according to the magnified gridline intersection method E. tetrodonta with very small root systems. We excluded colonization data of two small individuals from the fumigated soil, networked treatment because they were outliers (below the lower 99.9% confidence limit for the other plants of that treatment).For both E. tetrodonta leaf tissues (petioles excluded) were analyzed for element concentrations by the Department of Primary Industries & Fisheries, Berrimah Agricultural Research Centre, Chemistry Section (Berrimah). Leaf tissues of as many individuals as possible were analyzed separately, but because some E. tetrodonta had tiny leaves, we composited tissue of 2–4 plants as necessary. There was one composite of 2 fumigated soil, hyphae-severed plants; three composites (two of 2 and one of 3 plants) of fumigated soil, networked plants; one composite of 4 ambient soil, hyphae-severed plants; and two composites of 2 ambient soil, networked plants. The Chemistry Section also analyzed ambient and fumigated composited samples of the rain forest soil, and for comparison, ambient and fumigated samples of savanna soil collected 0.57 km distant from the rain forest. Soil pH and electrical conductivity were determined in a 1:5 soil:water extract; K, Ca, Mg, and Na in ammonium chloride; Colwell P in sodium bicarbonate; S in calcium dihydrogen phosphate; Zn, Cu, Mn, and Fe in DTPA; organic carbon by modified Walkley-Black digestion; and exchangeable Al in calcium chloride.t-tests (approximate t-tests for Cu and exchangeable Al) to compare rain forest and savanna soil (n = 2 for each soil), and we report as significantly different .We used ifferent  those atC. pentandra percentage root length colonized by AM fungi by split-plot, two-factor ANOVA with soil fumigation as the whole-plot factor, but because of different harvest dates, we analyzed E. tetrodonta AM colonization by separate two-sample t-tests for each harvest. Percentage colonized root length was arcsine-square root transformed before analysis. E. tetrodonta dry weight variables were compared by paired t-tests (pairing plants in the same microcosm) for plants in fumigated soil, and by t-tests  for plants in ambient soil after Bonferroni correction for the number of analyzed response variables (P≤0.0125 = 0.05/4 dry weight response variables).We analyzed C. pentandra and E. tetrodonta by split-plot, two-factor, repeated-measures ANOVAs after Levene's test of heteroscedasticity. Applying a Bonferroni correction for three morphometric response variables, we report as significant, effects for which P≤0.017. Mortality of E. tetrodonta in the ambient, networked treatment unbalanced the numbers of seedlings per treatment, so we used JMP Pro v. 10.0.0  with restricted maximum-likelihood calculations to perform the E. tetrodonta repeated-measures analyses.We analyzed morphological responses of L. glutinosa response variables to compare the effects on L. glutinosa of being in a microcosm with C. pentandra versus being with E. tetrodonta. Because L. glutinosa individuals were 15–20 cm tall at transplant, we relativized final morphological measurements by using their differences from initial measurements. We examined hypothesized negative associations of C. pentandra and E. tetrodonta with L. glutinosa for effects of severing common mycorrhizal networks with Pearson's correlations. Because we did not determine the dry weights of C. pentandra, we analyzed relativized final morphological measurements for C. pentandra (longest leaflet length change) versus L. glutinosa number of leaves. For E. tetrodonta versus L. glutinosa, however, we analyzed shoot dry weights. One ambient, networked E. tetrodonta seedling with a dry weight (1.67 g) almost twice the upper 99.9% confidence limit for its treatment was excluded.We used a one-way MANOVA performed with JMP Pro v. 10.0.0 followed by univariate analyses of the four E. tetrodonta to iron fertilization over 69 d following the initial fertilization by repeated-measures ANOVAs performed with JMP Pro v. 10.0.0, followed by Bonferroni correction (P≤0.017) for having examined three response variables. We used Fisher exact tests to examine the effects of iron fertilization on recovery from chlorosis.We analyzed morphological responses of E. tetrodonta foliage across both harvests by one-way ANOVA or non-parametric Kruskal-Wallis tests if variances were heteroscedastic. We used a Bonferroni-corrected P0.0045 (=0.05/11 elements) to assess the significance of these ANOVAs and of Pearson's correlation coefficients between E. tetrodonta leaf dry weights and element concentrations.We analyzed element concentrations in Among the 15 soil attributes shown in C. pentandra AM-colonized root length  but colonization was not affected significantly by severing hyphae  or by the interaction of fumigation and severing hyphae . At 141 DAT, mean seedling height differed by 3.7 cm and mean number of leaves by 0.7 leaves in fumigated versus ambient soil. Increase of longest leaflet length, which appeared to have reached an asymptote at 8.0 cm, was not significantly affected by soil fumigation . We found no significant effects of severing hyphae on C. pentandra growth rates , nor did fumigation, severing, and time interact for any morphological response variable .Fumigation significantly diminished height increase , but in ambient soil 210 DAT, it did not , longest leaf length increase . Hyphae-severed seedlings in fumigated soil exceeded the mean height of seedlings in all other treatments by 3.4 cm, the mean longest leaf length by 2.2 cm, and the mean number of leaves by 3.4. We found no significant effects of fumigation on any morphological variable , nor did fumigation, hyphae severing, and time interact significantly for any variable .Three of ten E. tetrodonta seedlings grown in fumigated soil were significantly increased by severing hyphae, but total root dry weights and fine root-to-leaf dry weight ratio did not differ  when accompanied by C. pentandra than when accompanied by E. tetrodonta. Univariate analyses of L. glutinosa nurse plant height change , largest leaf length change , number of leaves change , and stem diameter change  revealed significant effects on the latter three response variables.C. pentandra longest leaflet length change (a proxy for growth rate that was not affected significantly by soil fumigation) and L. glutinosa number of leaves on 10 May, 1998 (a proxy for plant size), although there was no significant association for hyphae-severed plants  nor number of leaves increase  was affected significantly. At harvest, mean longest leaf length of iron-fertilized plants exceeded that of not-fertilized plants by 2.0 cm, and iron fertilization had remedied chlorosis in all 5 chlorotic plants among the 8 plants randomly allocated to be fertilized. In contrast, all 4 chlorotic among 9 not-fertilized plants remained chlorotic. Although the two random groups did not differ in the proportion of chlorotic plants before fertilization began (Fisher exact two-tail P = 0.637), they differed significantly in the proportion of plants that recovered from chlorosis (Fisher exact two-tail P = 0.008). Iron fertilization, however, did not significantly elevate E. tetrodonta seedling dry weights  had 2.6 times the mean manganese concentration of those in ambient soil. Without Bonferroni correction, boron differed only for fumigated, hyphae-severed versus networked plants . Although not significantly different among these four groups (P = 0.061), iron had a conspicuously high mean concentration in fumigated soil for networked plants which also had the smallest mean leaf dry weight of all groups . There were no significant differences among treatments in the total foliar content of any element.After Bonferroni correction, only leaf tissue manganese concentration was affected significantly among the four treatments shown in l groups . In ambil groups . Among aE. tetrodonta seedlings, and that the detriment involves iron limitation. The detriment likely is a consequence of two complementary phenomena: common mycorrhizal network interconnections mediate plant competition E. tetrodonta, and active AM fungus mycelia may lead to iron sequestration whether or not those mycelia interconnect plants. Although our study did not trace hyphal interconnections between plants, negative associations between C. pentandra or E. tetrodonta and L. glutinosa only when hyphae were not severed are consistent with common mycorrhizal networks being present, as is the enhancement of E. tetrodonta mean root colonization in fumigated soil when networked Our results strongly support our hypotheses that AM fungi are detrimental to E. tetrodonta morphological parameters are somewhat surprising because of the extent to which E. tetrodonta seedlings formed AM . AM improved C. pentandra growth in ambient versus fumigated soil, but in distinct contrast, the growth of E. tetrodonta seedlings with the lowest mean root colonization (29%) in hyphae-severed, fumigated soil exceeded growth in all other treatments. Consequently, little-colonized seedlings might be the most likely to establish successfully in the stressful milieu of rain forest gaps. Indeed, under our experiment's conditions that simulated non-burnt rain forest gaps, three networked E. tetrodonta seedlings in ambient soil died. Although AM previously have been reported to cause growth depressions of plants grown singly in pots, those negative effects usually have reflected AM failing to repay their cost to the host under conditions of low light, extremely low available phosphorus, or high phosphorus fertilization initiated after abundant mycorrhizas already had formed E. tetrodonta growth, because under the same conditions in which AM were detrimental to E. tetrodonta, AM benefitted C. pentandra. Even though mycorrhizas usually are considered to be archetypical mutualisms, our work underscores that mutualism is not constitutive but is context-dependent The negative effects of potentially networked hyphae on the rates of increase of all E. tetrodonta seedlings sustain AM that might contribute to their deaths in rain forest soil. Natural selection may not favor rejection of AM because AM are beneficial in other contexts or if formed by a different suite of fungus species. For instance, many shrub-layer species in Northern Territory savannas form AM E. tetrodonta seedlings could be connected to common AM networks in savanna. Available iron is two-and-a-half times more abundant there than in rain forest soil, however, perhaps mitigating potential negative effects of AM. Alternatively, if E. tetrodonta seedlings quickly form ectomycorrhizas It is peculiar that C. pentandra by fumigation even when hyphae were not severed implies that fumigation diminished viable AM fungus propagules to an extent not entirely compensated by hyphae from the nurse plant. Repeated severing did not significantly affect C. pentandra root colonization, however, probably because roots of these relatively large plants came sufficiently close to mesh root barriers to be colonized in the 2–3 d interval between hypha severing. Once established, colonization could spread within a root system to reach an asymptote E. tetrodonta which was smaller than C. pentandra, thereby probably prolonging the time needed for roots to closely approach mesh barriers. Not severing hyphae in fumigated soil compartments, however, elevated AM colonization of E. tetrodonta at 141 DAT to a level similar to that at 210 DAT, thereby supporting that seedlings and nurse plants indeed may have been connected by common mycorrhizal networks. No effect of hypha severing at 210 DAT, suggests that E. tetrodonta seedlings had attained asymptotic colonization.Our treatments reduced but did not eliminate AM colonization of either host species. Nevertheless, fumigated, hyphae-severed compartments probably had the least AM inoculum of any treatment while, ambient, networked compartments probably had the most inoculum. Reduction of colonization of E. tetrodonta whole plant dry weight versus that of networked plants. Fumigation, however, neither significantly affected E. tetrodonta growth rates , nor did fumigation interact significantly with hypha severing. Therefore, any fertilization effect of fumigation such as N and P release from killed microbes E. tetrodonta of non-networked AM. That differs from the pot experiments reported by Stocker E. tetrodonta growth rates only when combined with initial elimination of AM inoculum by fumigation, so it is possible that in ambient soil there may have been an inhibitory effect of non-networked AM that we could not detect statistically.In fumigated soil, repeated hypha severing doubled C. pentandra and L. glutinosa, and between E. tetrodonta and L. glutinosa which suggest that belowground competition was mediated across common mycorrhizal networks. The belowground competition was not sufficiently strong to produce a significant beneficial effect of hypha severing for C. pentandra, however, because C. pentandra were the largest plants and probably the strongest competitors overall. Aboveground, because of close spacing of the completely randomized microcosms, tall C. pentandra were as likely to shade adjacent E. tetrodonta seedlings as they were to shade their accompanying nurse plants, thereby probably distributing aboveground competition relatively evenly across our entire experiment. Belowground, however, hyphal interconnections likely influenced competition within individual microcosms. Even though root system overlap was prevented by mesh barriers, AM fungus hyphae could cross the mesh and might have redistributed mineral nutrients When hyphae were not severed we found significant negative associations between E. tetrodonta seedlings to iron fertilization of ambient soil unambiguously demonstrated that iron was a growth-limiting mineral nutrient. Fertilization not only stimulated leaf growth but also completely eliminated chlorosis. Iron limitation of E. tetrodonta in rain forest soil is consistent with only two-fifths as much iron being available in rain forest as in savanna soil. Moreover, iron fertilization has been reported to remedy chlorosis of several eucalypt species The response of E. tetrodonta leaf analyses do not suggest that any element other than iron was limiting. Although manganese concentration was highest for all E. tetrodonta grown in fumigated soil, iron-fertilized seedlings at 210 DAT attained the highest mean whole plant dry weight with only one-third the manganese concentration of plants in fumigated soil. Furthermore, no analyzed element's total foliar content differed significantly among treatments. Even though we did not analyze nitrogen, it is unlikely that nitrogen limited growth because iron fertilization stimulated growth without supplemental nitrogen.E. tetrodonta growth was associated with the lowest mean AM colonization. Alternatively, under some conditions AM fungi might exacerbate iron deficiency by producing the glycoprotein glomalin E. tetrodonta seedlings were planted. Such a rapid decline of immunoreactive, easily-extractable glomalin is supported by findings of Preger et al. AM have been reported to improve the iron nutrition of woody plants under some conditions E. tetrodonta seedling growth in fumigated, hyphae-severed rain forest soil, then intense fires must be capable of leading to similar diminution of glomalin in the field. Knorr et al. If the hypothesized decline of glomalin contributed to E. tetrodonta seedlings avoid inclusion in common AM networks that exacerbate belowground competition with rapidly-growing rain forest species. Once ECM fungi invade to associate with establishing seedlings that have survived sufficiently long for the fungi to encounter them If a severe fire burns into rain forest, microbe death and glomalin decline might elevate iron availability, and AM host death might help ECM networks in savanna may enhance the resistance of savanna to invasion by AM rain forest plants. ECM networks probably have far greater potential for hydraulic redistribution E. tetrodonta growth was disruption of common AM networks, not previously recognized as part of the ashbed effect, but a potential consequence of fire-caused mortality of AM hosts.Fire–mycorrhiza–vegetation feedbacks likely provide resilience to rain forest and savanna alternative stable-state systems, although rain forest resilience is overcome by the ashbed effect. The mechanisms of that effect are unresolved, however, and in our experiments, most suggested ashbed mechanisms played no role. No ECM Our results sharply distinguish the possible roles of different mycorrhiza types in influencing plant community composition. ECM and AM should not be viewed simply as alternative plant adaptations that minimize niche overlap and foster coexistence of their hosts. We have shown that common AM networks can be actively antagonistic to an eventual ECM host.Table S1Dry weight responses of Eucalyptus tetrodonta seedlings to soluble iron fertilization and to common mycorrhizal network hypha severing in ambient rain forest soil 210 days after transplant.(DOCX)Click here for additional data file.Table S2Leaf element concentrations of Eucalyptus tetrodonta seedlings in response to soluble iron fertilization and to common mycorrhizal network hypha severing in ambient rain forest soil 210 days after transplant.(DOCX)Click here for additional data file.


HIV-associated KS is the most common reported malignancy in sub-Saharan Africa, and appropriate therapy of KS requires accurate diagnosis. In much of the region, however, KS diagnosis is limited to clinical suspicion without pathologic confirmation. Where pathology is available, specific anti-KSHV stains are rarely available and overall pathologic accuracy for KS has not been evaluated.We introduced skin punch biopsy for KS at HIV/AIDS care clinics in Uganda and Kenya within the East Africa IeDEA Consortium. Clinicians suspecting KS could obtain a biopsy same day without charge. After interpretation by local African pathologists who only had access to routine H&E staining, biopsies were read by dermatopathologists at UCSF who could, at their discretion, recut and restain specimens or stain against latency-associated nuclear antigen (LANA) of KSHV. The interpretation by the U.S. dermatopathologists, who serve a large base of HIV-infected patients, was considered the gold standard.Clinicians at 26 HIV/AIDS clinics in Uganda and Kenya referred 739 patients with clinically suspected KS for skin biopsy. Overall, 77% (95% CI: 74%--80%) of these clinically suspected cases were determined pathologically to be KS after U.S. review; 19% had another diagnosis and 4% were indeterminate. There was no significant difference in the percentage found to be KS between countries (p=0.20) or over time (p=0.11). When KS was not found, a wide variety of other diagnoses, both clinically significant and insignificant, were made by the U.S. dermatopathologists (Table Amongst clinicians at HIV/AIDS clinics in East Africa, clinical suspicion of KS alone is not optimally specific for KS diagnosis. Clinical suspicion alone often either misdiagnoses conditions which are less concerning than KS or misses other serious conditions that require different therapy than KS. Assuming the U.S. interpretation is the gold standard, pathologic determination of KS in East Africa is specific but not optimally sensitive. The findings urge for increased availability of skin punch biopsies for KS diagnosis in Africa and augmentation of pathology services.


Acinetobacter baumannii is an opportunistic pathogen responsible for hospital-acquired infections. A. baumannii epidemics described world-wide were caused by few genotypic clusters of strains. The occurrence of epidemics caused by multi-drug resistant strains assigned to novel genotypes have been reported over the last few years.A. baumannii strains assigned to genotypes ST2, ST25 and ST78, representative of the most frequent genotypes responsible for epidemics in several Mediterranean hospitals, and four complete genome sequences of A. baumannii strains assigned to genotypes ST1, ST2 and ST77. Comparative genome analysis showed extensive synteny and identified 3068 coding regions which are conserved, at the same chromosomal position, in all A. baumannii genomes. Genome alignments also identified 63 DNA regions, ranging in size from 4 o 126 kb, all defined as genomic islands, which were present in some genomes, but were either missing or replaced by non-homologous DNA sequences in others. Some islands are involved in resistance to drugs and metals, others carry genes encoding surface proteins or enzymes involved in specific metabolic pathways, and others correspond to prophage-like elements. Accessory DNA regions encode 12 to 19% of the potential gene products of the analyzed strains. The analysis of a collection of epidemic A. baumannii strains showed that some islands were restricted to specific genotypes.In the present study, we compared whole genome sequences of three A. baumannii provides a scaffold to rapidly evaluate the genomic organization of novel clinical A. baumannii isolates. Changes in island profiling will be useful in genomic epidemiology of A. baumannii population.The definition of the genome components of  Acinetobacter comprises 26 species with valid names and nine genomic species with provisional designations that were defined by DNA-DNA hybridization. Acinetobacter baumannii, A. pittii and A. nosocomialis are the three species more frequently associated with human diseases  [NC_010611] [NC_009085] [NC_010410] [A. baumannii strains ST2 3990 [GenBank:AEOY00000000], ST25 4190 [GenBank:AEPA00000000] and ST78 3909 [GenBank:AEOZ00000000] strains , Acinetobacter sp. DR1 [GenBank:NC_014259], A. calcoaceticus RUH2202 [GenBank:ACPK00000000], A. haemolyticus ATCC19194 [GenBank:ADMT00000000], A. johnsonii SH046 [GenBank:ACPL00000000], A. junii SH205 [GenBank: ACPM00000000], A. lwoffii SH145 [GenBank:ACPN00000000], A. radioresistens SK82 [GenBank:ACVR00000000], Acinetobacter sp. ATCC27244 [GenBank:ABYN00000000], A. nosocomialis RUH2624 [GenBank:ACQF00000000] and A. pittii SH024 [GenBank:ADCH00000000] were also used for comparison. The A. baumannii strains used in PCR analyses of GEIs have been previously described [Comparative genome analysis were performed on whole genome sequences of _011586] , ACICU _010611] , ATCC179_010410]  and drafA. baumannii strain AB0057 used as reference template [A. baumannii AB0057, ACICU, ATCC and ABAYE strains using MAUVE [rpoB gene sequence analysis for genomic species identification was performed as previously described [Gene products putatively encoded by the ST25 4190, ST78 3909 and ST2 3990 strains were identified using xBASE2, comparing the draft genome sequences to the genome of the template . The corng MAUVE . HomologA. baumannii strains was assessed by PCR amplification. PCR reactions were carried out by incubating 20 ng of genomic DNA with 160 ng of each primer in the presence of dXTPs (200 nanomoles), 1.5 mM magnesium chloride and the Taq DNA polymerase Recombinant (Invitrogen). The sequences of the oligomers used as primers, the experimental conditions, the length of the amplimers, the coding regions amplified are all listed in Additional file The conservation of specific GEIs in a set of The co-linearity of contigs and the DNA content of the corresponding chromosomal regions were assessed by sequencing PCR products bridging contig ends.The authors declare that they have no competing interests.Conceived and designed the experiments: PPDN, FR, MG, MT, and RZ. Performed the experiments and analyzed the data: FR, PPDN, and MG. Wrote the paper: PPDN and RZ. All authors read and approved the final manuscript.Structures of plasmids identified in ST2 3990, ST25 4190 and ST78 3909 strains. the figure shows the circular maps of plasmids p1ABST2, p2ABST2, p1ABST25, p2ABST25 and p1ABST78 with relevant features. ORFs and direction of the transcription are represented by arrow-shaped boxes. Plasmid sizes and names of various features are reported.Click here for fileCoding capacity of plasmids carried by strains 3909 3990 and 4190. the table lists ORFs of plasmids p1ABST2, p2ABST2, p1ABST25, p2ABST25 and p1ABST78. Position, number of amino acids and putative function are reported for each ORF.Click here for fileTarget site duplications. sequences duplicated at the ends of GEIs upon genome integration are listed in the table. Base changes in left and right TSDs are marked according to IUB codes. Residues missing in one TSD are in parenthesis. Known target genes are indicated.Click here for fileGEIs organization and ORFs content. the 63 sheets of the EXCEL file correspond to the 63 genomic loci carrying GEIs shown in Figure 2. The ORF number, the amino acid length and the hypothesized function are given in each sheet. For draft genomes, the corresponding contigs are indicated. Identical or closely related ORFs present in different GEIs are positioned in the same row and labelled by the same colour to facilitate view. ORFs denoted as tb were identified by tBLASTn analyses. Grey and orange bars denote closely located ORFs putatively co-expressed. Homologous coding regions are boxed when a single ORF in one strain corresponds to two or more contiguous ORFs in others.Click here for fileMicro-heterogeneity regions. coding regions present/absent in the compared A. baumannnii genomes, denoted in the text as mhrs (micro-heterogeneity regions), and their hypothetical function, are listed in the table. Alternative regions present at the same locus are marked by different colour characters. mhrs containing two or more ORFs are boxed.Click here for fileCryptic prophages. structures of cryptic prophages identified in A. baumannii genomes. Prophage types are boxed to highlight their relatedness as resulting from MAUVE alignment. Different CP1 and CP2 are shown to illustrate the degree of genetic variation of A. baumannii prophage families.Click here for fileGene products putatively encoded by strains 4190, 3909 and 3990. ORFs of strains 4190, 3909 and 3990 and the corresponding contig number are shown.Click here for fileGenomic regions, amplified genes, primers, amplicon sizes and cycling conditions used in PCR surveys. .Click here for file


Natrialba magadii is an aerobic chemoorganotrophic member of the Euryarchaeota and is a dual extremophile requiring alkaline conditions and hypersalinity for optimal growth. The genome sequence of Nab. magadii type strain ATCC 43099 was deciphered to obtain a comprehensive insight into the genetic content of this haloarchaeon and to understand the basis of some of the cellular functions necessary for its survival.Nab. magadii consists of four replicons with a total sequence of 4,443,643 bp and encodes 4,212 putative proteins, some of which contain peptide repeats of various lengths. Comparative genome analyses facilitated the identification of genes encoding putative proteins involved in adaptation to hypersalinity, stress response, glycosylation, and polysaccharide biosynthesis. A proton-driven ATP synthase and a variety of putative cytochromes and other proteins supporting aerobic respiration and electron transfer were encoded by one or more of Nab. magadii replicons. The genome encodes a number of putative proteases/peptidases as well as protein secretion functions. Genes encoding putative transcriptional regulators, basal transcription factors, signal perception/transduction proteins, and chemotaxis/phototaxis proteins were abundant in the genome. Pathways for the biosynthesis of thiamine, riboflavin, heme, cobalamin, coenzyme F420 and other essential co-factors were deduced by in depth sequence analyses. However, approximately 36% of Nab. magadii protein coding genes could not be assigned a function based on Blast analysis and have been annotated as encoding hypothetical or conserved hypothetical proteins. Furthermore, despite extensive comparative genomic analyses, genes necessary for survival in alkaline conditions could not be identified in Nab. magadii.The genome of Nab. magadii is predicted to be metabolically versatile and it could use different carbon and energy sources to sustain growth. Nab. magadii has the genetic potential to adapt to its milieu by intracellular accumulation of inorganic cations and/or neutral organic compounds. The identification of Nab. magadii genes involved in coenzyme biosynthesis is a necessary step toward further reconstruction of the metabolic pathways in halophilic archaea and other extremophiles. The knowledge gained from the genome sequence of this haloalkaliphilic archaeon is highly valuable in advancing the applications of extremophiles and their enzymes.Based on genomic analyses,  Euryarchaeota contains a diverse array of archaea currently classified under eight named classes  and ten orders -sn-glycerol} [Nab. magadii synthesizes and accumulates 2-sulfotrahalose as an osmolyte under hypersaline conditions [Nab. magadi have also been characterized since its discovery [The genus ronments . The typm growth . Natrialm growth . In contmembrane . Furtherlycerol} . Previounditions . The bioiscovery -18.Nab. magadii, a low pass genomic sequence of Nab. asiatica strain ATCC 700177 has been reported [Haloterrigena turkmenica strain DSM 5511, a halophilic archaeon isolated from sulfate saline soil in Turkmenistan, is complete and contains 5,287 protein coding genes [Natronomonas pharaonis DSM 2160, a haloalkaliphilic archaeon isolated from a soda lake in Egypt, contains 2,843 protein coding genes [Nab. magadii at the whole genome level and perform comparative genomic studies with other halophilic as well as haloalkaliphilic archaea. An exploration of the pathways of coenzyme biosynthesis and proteolysis within Nab. magadii was also envisaged.Although knowledge on the biology of halophilic archaea has greatly advanced during the last decade, attempts to understand the physiology and genetics of the haloalkaliphilic archaea are scarce . The stureported . The genng genes . Furtherng genes . In adding genes . The goaNab. magadii was grown at 37°C aerobically in liquid medium containing 20 g/L yeast extract using the method of Tindall et al. [et al. for Halobacterium halobium  [Nab. magadii was sequenced using a combination of both Sanger and 454 technologies. A Sanger whole genome shotgun library, which produced 26,484 reads with an average insert size of 6.5 kb, and a 454 FLX standard library, which generated 96.3 Mbp of data, were constructed for this genome. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov/. The Phred/Phrap/Consed software package (http://www.phrap.com) was used for sequence assembly and quality assessment. After the shotgun stage, reads were assembled with parallel phrap . Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones . Gaps between contigs were closed by editing in Consed, custom primer walk, or PCR amplification . A total of 594 additional custom primer reactions were necessary to close all gaps and raise the quality of the finished sequence. The estimated error rate for the completed genome of Nab. magadii was less than 1 in 100,000. The final assembly was based on 19.1 Mbp of Sanger draft data, which provided 4.3x coverage of the genome, and 96.3 Mbp of 454 draft data, which provided 21.7x coverage of the genome.l et al. , and genlinarum) . Genomichttp://wiki.rzg.mpg.de/HaloferaxWiki. Proteins deemed to be specific to Nab. magadii were compared against the NCBI non-redundant protein database to determine whether they were hypothetical or conserved hypothetical. If there was no adequate alignment with any protein , the translated ORF was named a hypothetical protein.Preliminary automated annotation, prediction of the number of subsystems, and pairwise BLAST comparisons of protein sets within different strains were performed using the Rapid Annotation using Subsystems Technology (RAST), which is a fully automated, prokaryotic genome annotation service . SubsequNab. magadii genome sequence was rearranged to facilitate visual comparison. This was accomplished using the Artemis Comparison Tool to identify a coordinate where the sequence was shifted relative to that of Htg. turkmenica. The coordinate was located at 1961610 bp and the Nab. magadii sequence was cut starting at this coordinate until the end of the sequence and placed at the beginning of the fasta file so that the genome start was near the major origin of replication.Multiple genome comparisons were performed using the ‘progressive alignment’ option available in the program MAUVE version 2.3.0 ,28. DefaNab. magadii sequence was aligned with that of Htg. turkmenica. The Nab. magadii genome project is deposited in the Genomes OnLine Database (GOLD) and the complete genome sequence is available from GenBank/EMBL/DDBJ with accession numbers CP001932, CP001933, CP001934, and CP001935. The genome of Nab. magadii is also accessible through HaloLex  and the UCSC Archaeal genome browser (http://archaea.ucsc.edu/).A synteny plot was generated using the program NUCmer, which uses exact matching, clustering, and alignment extension strategies to create a dot plot based on the number of identical alignments between two genomes . NUCmer Nab. magadii consisted of four replicons . Three of these elements had a GC content of ~61% whereas pNMAG02 had a GC content of 56.82%. A comparison of some of the relevant features of these four elements is shown in Table Nab. magadii. Since halovirus φCh1 has already been described elsewhere [Nab. magadii contained two genes encoding putative replication factor C-like proteins (Nmag_1868 and 1910). The large chromosome, pNMAG01, and pNMAG02 were predicted to replicate using a conserved archaeal mechanism [The complete genome sequence of lsewhere -32, the echanism , since eNab. magadii contained ~36 full-length or truncated genes encoding putative transposases. These insertion sequence elements were scattered throughout the chromosomes and about 20 of these belong to the IS605 OrfB family. The IS605 OrfB transposase  genes were highly diverse, as is typical of halophilic archaea [IS605 OrfA  was identified in the genome. Other transposase genes in Nab. magadii include 7 of the broad category IS4 (3 IS7-type and 4 IS9-type), a single IS240-type, and 4 related to ISSod10. The small number of transposase genes and their heterogeneity may indicate that Nab. magadii is only minimally affected by these elements. The genome also contained several genes related to bacteriophage elements  and a vgr-like gene related to recombination hot spot elements. In addition, there were 13 genes encoding integrase/recombinase-like proteins  with fibrillarin-like RNA methyltransferase as the catalytic component. The small chromosome pNMAG01 contained a copy of 16 S rRNA-tRNAAla-23 S rRNA-5 S rRNA sequence on the minus strand and a copy of 23 S rRNA-5 S rRNA sequence on the plus strand. The three 16 S rRNA-tRNAAla-23 S rRNA-5 S rRNA sequences of Nab. magadii had 99% nucleotide identity to each other. The small chromosome pNMAG01 also contained an orphan 5 S rRNA sequence that had 89% nucleotide identity to the other four 5 S rRNA genes of Nab. magadii. Since pNMAG02 lacked rRNA operons and had a lesser GC content than the large and small chromosomes, this self replicating element could be considered a large plasmid. The heterogeneity of the rRNA operons within Nab. magadii is not a unique feature and the occurrence of such rRNA operons among halobacterial genomes is thought to be due to recombination between rRNA genes of different strains or species [Nab. magadii were closely related to those of Nab. asiatica (97% identity), Htg. turkmenica (96% identity), and Nmn. pharaonis (90% identity). Furthermore, the genome of Nab. magadii was compared to 17 complete haloarchaeal genomes available in the public databases , followed by Halopiger xanaduensis strain SH-6 . There were lesser number of orthologs  in Nmn. pharaonis, which has a relatively smaller genome. However, when the data for the percentage of proteins having a bidirectional best blast hit in Nab. magadii was computed , followed by Hpg. xanaduensis (60%) and Htg. turkmenica (51%). Results from Nmn. pharaonis and Htg. turkmenica are emphasized in this paper since the former was the only other haloalkaliphilic archaeon with a complete genome sequence and the latter contained the highest number of orthologs.Archaeal genomes generally have 1–4 rRNA operons consisting of the 16 S, 23 S, and 5 S rRNA genes with a tRNAd spacer . The lar species . The 16 Nab. magadii was 1.7 Mb larger than the complete genome of Nmn. pharaonis, which consists of three replicons . However, Nab. magadii genome was 1 Mb smaller than the complete genome of Htg. turkmenica, which consists of seven replicons . The GC content (61.42%) of the large chromosome of Nab. magadii was slightly lesser than that of the large chromosomes of Htg. turkmenica (~66% GC) and Nmn. pharaonis (~63% GC). Alignment of the large chromosome of Nmn. pharaonis  with that of Nab. magadii using MAUVE showed the presence of very few short syntenic regions (data not shown), whereas a similar alignment using the large chromosome of Htg. turkmenica  showed the presence of numerous short syntenic regions  with an average nucleic acid identity of 89% . The plus and minus strand matches among the chromosomes of Htg. turkmenica and Nab. magadii generated by NUCmer are shown in Figure The combined size of the complete genome of s Figure . To furtNab. magadii using the TaxPlot tool of NCBI revealed that Htg. turkmenica contained 2387 orthologs, whereas Nmn. pharaonis contained only 426 orthologs . These analyses further confirmed that Nab. magadii was more closely related to Htg. turkmenica than to Nmn. pharaonis. In Nab. magadii, 1518 genes could not be assigned a function based on BLAST analysis and were annotated as encoding hypothetical or conserved hypothetical proteins. The isoelectric point (pI) of most of the predicted proteins of Nab. magadii was in the 3–5 range, indicating that the general proteome is acidic, which is typical of most halophilic archaea. A two-way comparison of the large chromosomes revealed that Nab. magadii contained ~945 putative protein-coding genes that had no homologs in Htg. turkmenica. A vast majority (~75%) of these Nab. magadii-specific genes encoded hypothetical proteins. Other genome-specific genes in Nab. magadii encoded ABC-type transporters, ATPases, kinases, phosphatases, proteases, and oxidoreductases. The genome of Nab. magadii also contained a variety of simple sequence repeats encoding characteristic peptide repeat patterns.A three-way comparison of all predicted protein-coding genes of + concentration exceeds 5 M by pumping Na+ out and K+ into the cell using a variety of cation/proton antiporters [Nab. magadii contained an operon of nine genes encoding a putative pH adaptation K+ efflux system (Nmag_3445-3453). Genes related to this operon were present in several halophilic archaea, indicating that they may not encode a specialized system involved in stress response to alkaline growth conditions. Apart from this operon, the genome contained three other genes encoding putative cation/proton antiporters and a gene encoding a putative OsmC family protein  for osmotic adaptation.Low molecular weight organic compounds such as amino acids, polyols, and sugars facilitate cellular adaptation to high-osmolarity and are referred to as osmoprotectants or compatible solutes . Halophi. magadii. Nab. maNab. magadii contained a cluster of 11 genes encoding putative gas vesicle synthesis family proteins, which were related to the gas vesicle family proteins of Hbt. salinarum. However, Nab. magadii lacked genes related to those encoding the minor gas vesicle protein (GvpC) and the regulators (GvpD and GvpE). The gas vesicle gene clusters of Nab. magadii and Htg. turkmenica were highly similar to each other and appeared to contain a distant homolog of the Hbt. salinarum gvpI gene . Nevertheless, these genes encode putative proteins with an N-terminal extension of more than 200 residues not found in GvpI of Hbt. salinarum. Furthermore, the gas vesicle clusters of Nab. magadii and Htg. turkmenica contained an additional gene  that was absent in Hbt. salinarum. Nab. magadii also contained a gene encoding a hemAT-type aerotactic transducer with a putative globin-coupled sensor protein comprising of a globin fold domain and a methyl-accepting chemotaxis transducer domain  that afford protection against hypoosmotic shock, chaperone proteins DnaJ and DnaK, a thermosome, and heat shock proteins that may participate in protein quality control and cellular response to stress. Nab. magadii also contained 47 genes  encoding putative proteins of various sizes with a universal stress protein (UspA) domain. One of these genes (Nmag_1302) appeared to form an operon with a gene encoding a putative GCN5-related N-acetyltransferase , and a similar gene pair was found in Htg. turkmenica  and Nmn. pharaonis . It is possible that the GNAT is involved in the acetylation of the linked universal stress protein in these species. In addition, the large chromosome of Nab. magadii contained genes encoding a superoxide dismutase (sodA), two catalases , two alkyl-hydroperoxidase-like proteins, a carbonic anhydrase, and methionine sulfoxide reductases , but not in other archaea. These genes may be involved in metal homeostasis in the hypersaline environment that Nab. magadii inhabits. Nab. magadii also encoded DNA methylases, DNA damage repair excinuclease ABC subunits, DNA mismatch repair proteins, and DNA repair/recombination proteins RadA and RadB  encoding putative peptidases/proteases were found in Htg. turkmenica .At least 83 genes encoding various types of peptidases/proteases were identified in the genome of Nab. magadii contained nine genes encoding putative S8 and S53 subtilisin kexin sedolisins , the amino acid motifs containing the catalytic triad (Asp-His-Ser) were conserved in all of them. Six of the predicted subtilisins  of Nab. magadii contained putative targeting signals for translocation through the twin-arginine transport (Tat) pathway, suggesting that these proteases are most likely exported out of the cell. Within this group, Nmag_0715 has been biochemically characterized and designated as the Natrialba extracellular protease (Nep) [Nab. magadii[Sphingomonas, are a specialized group of metalloproteases assigned to M81 family and they participate in the last step of the degradation pathway of microcystin LR [Nmn. pharaonis and Htg. turkmenica  are a large superfamily of functionally diverse endo- and exo-peptidases that occur in prokaryotes and eukaryotes . Nab. mase (Nep) . Nep was. magadii. Interes. magadii. This diHfx. volcanii[Nab. magadii contained an operon encoding putative 20 S proteasome α and β subunits . Apart from this operon, the genome contained separate genes encoding 20 S proteasome α and β subunit homologs . Nab. magadii was also predicted to encode homologs of PAN (Nmag_1362 and 2440) and ubiquitin-like small archaeal modifier proteins . The genome of Nab. magadi contained two genes encoding putative ubiquitin-like activating enzymes of archaea . Furthermore, it also encoded a distant homolog of UbaA (Nmag_0356) containing a C-terminal JAB1/MPN/Mov34 metalloenzyme (JAMM) domain that was predicted to remove SAMPs from target proteins. In contrast, Hfx. volcanii encodes only a single UbaA-type protein that functions in both protein conjugation (sampylation) and sulfur mobilization [Nab. magadii also encoded an archaeal-type LonB protease (Nmag_2822), which was demonstrated in its cell membranes [All archaeal genomes studied to date are predicted to encode self-compartmentalized proteases (20 S proteasomes and Lon-type proteases) likely to function in energy-dependent proteolysis and an ubiquitin-type mechanism for targeting proteins to proteasomes termed sampylation ,55. In a volcanii). The geembranes . While LHar. marismortui[Nab. magadii encodes a homolog of TET , which, in combination with the energy-dependent proteases, may participate in the intracellular protein turnover in this extremophile. Furthermore, similar to the majority of haloarchaea, Nab. magadii appears to encode homologs of the three families of membrane-embedded regulatory proteases denoted as I-CLiPs. These include sppA-type signal peptide peptidases , site-2 protease class of zinc metalloproteases that cleave transmembrane domains , and rhomboids . Furthermore, Nab. magadii contained genes encoding type I signal peptidases  and a type IV prepilin peptidase (Nmag_1752). The type I signal peptidases and the type IV prepilin peptidase are predicted to be involved in the processing of N-terminal signal peptides of exported proteins and flagellin precursors, respectively.The tetrahedral aminopeptidase (TET protease) is an energy-independent protein complex  that was isolated from the neutrophilic haloarchaeon rismortui. It has Nab. magadii. A subtilisin protease inhibitor from this archaeon, denoted NSI, was previously purified and biochemically characterized [Nab. magadii is presented in Figure Nab. magadii are independent of each other, their synergistic action in vivo cannot be ruled out.Cellular protease activity is frequently controlled by endogenous protease inhibitors . Genes ecterized . This prNab. magadii contained genes that encoded putative components of the Sec system  and of the Sec-independent Tat protein translocase complex . While the Tat pathway is commonly used for a small subset of exported proteins in bacteria, it is a dominant export route in halophilic archaea. Many of the exported proteins are subsequently attached to the cell membrane by a lipid anchor and Nab. magadii has 119 genes encoding lipid-modified Tat target proteins, as detected by TatLipo analysis [Nab. magadii contained genes encoding putative components of a type II secretion system (Nmag_3137-3138) and an archaeosortase (Nmag_2750) for which 17 targets with PGF_CTERM motif were identified [Protein translocation across the cell membrane in prokaryotes is facilitated by at least three mechanisms including the general secretion (Sec) system, the specialized Tat system, and the highly ornate, substrate-specific secretion systems for delivering effector proteins to target sites -63. Nab.entified .Nab. magadii contained 23 genes encoding putative GTs. Based on BLASTP analysis on the NCBI database and the presence of conserved domains, these genes were assigned into the GTA  and GTB  superfamilies. One of these genes (Nmag_3015) is in an operon with Nmag_3011 , Nmag_3012 , Nmag_3013 , and Nmag_3014 . Nab. magadii also contained genes encoding a putative oligosaccharyltransferase subunit  and a dolichol kinase-like protein (Nmag_1986). Therefore, Nab. magadii appears to have the genetic potential for N-glycosylation.N-glycosylation in archaea and eukaryotes uses dolichol phosphate as the lipid base for the assembly of oligosaccharides ,67. GlycNab. magadii cells . Other genes in the genome that encoded putative enzymes involved in polysaccharide biosynthesis included six polysaccharide deacetylases , two polyprenyl glycosyl-phosphotransferases , an O-antigen polymerase (Nmag_0143), two UDP-N-acetylglucosamine 2-epimerases (Nmag_0149 and Nmag_0676), an acylneuraminate cytidylyltransferase (Nmag_0148), an O-acetyltransferase (Nmag_0150), a N-acylneuraminate-9-phosphate synthase (Nmag_0151), and two capsule synthesis proteins (Nmag_1511 and 3999). It is possible that some of these genes are involved in the biosynthesis of Nab. magadii exopolysaccharide- or capsule-like material identified in the TEM images.Several species of halophilic archaea are known to produce copious amounts of extracellular polysaccharides . Althougs Figure , purific420 biosynthesis in haloarchaea. Analyses of genes putatively involved in coenzyme biosynthesis in Nab. magadii was performed in light of these new discoveries. This section describes the genes related to the biosynthesis of thiamine, riboflavin, NAD, coenzyme F420, folate, heme, and cobalamin.Archaeal metabolic pathways are unique and diverse, in comparison to those of eubacteria . However1 (thiamine pyrophosphate) is involved in several microbial metabolic functions [de novo or acquire it from their niche [Nab. magadii contained a gene (Nmag_3460) encoding a putative ThiI and several ubiquitin-like β-grasp fold proteins . However, β-grasp fold proteins have multiple functions in halophilic archaea, being involved in sulfur chemistry as well as ubiquitin-like protein modification by SAMPylation [Nab. magadii remains unidentified.Vitamin Bunctions . Prokaryir niche . Thiaminir niche . ThiaminNab. magadii lacked homologs of ThiG, ThiH, and TenI involved in HET-P biosynthesis in bacteria. Interestingly, Nab. magadii encoded a homolog of the yeast HET-P synthase THI4 (Nmag_2419). However, Nmag_2419 is currently annotated as ribose-1,5-bisphosphate isomerase based on the functional characterization of the ortholog MJ0601 from Methanocaldococcus jannaschii. In contrast, the ortholog of Nmag_2419 in Pyrococcus kodokaraensis (TK0434) does not have ribose-1,5-bisphosphate isomerase activity [Nab. magadii contained purM (Nmag_1281) and thiC (Nmag_2593) homologs, which were predicted to be involved in AIR and HMP biosynthesis, respectively (Table 2).Furthermore, activity . BiochemNab. magadii and other halophilic archaea contain both enzymes. Whereas ThiE (Nmag_1811) is a monofunctional protein, ThiN (Nmag_1282) exists as a C-terminal domain in a ThiDN fusion protein. At the last step, thiamine phosphate is predicted to be further phosphorylated to thiamine pyrophosphate by ThiL (Nmag_1515). Therefore, the conversion of AIR to HMP-PP in Nab. magadii appears to be similar to the bacterial pathway and may involve ThiC and ThiD, whereas HET-P biosynthesis in this haloarchaeon appears to be similar to the eukaryotic pathway and may involve Nmag_2419. In addition, Nab. magadii contained genes encoding a HET kinase  and a thiamine transporter .Two distinct proteins, ThiE and ThiN, are known to join HMP-PP and HET-P to generate thiamine phosphate. 2 (riboflavin) is the precursor of coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are cofactors for several biochemical reactions [de novo using one molecule of GTP and two molecules of ribulose 5-phosphate as substrates [M jannaschii[M. jannaschii, GTP cyclohydrolase III, the first enzyme of the riboflavin biosynthesis pathway, produces an archaeal-specific formylated intermediate that requires a subsequent deformylation step [M. jannaschii riboflavin kinase. Conversely, riboflavin synthases of halophilic archaea are related to those of M. jannaschii, but are unrelated to bacterial riboflavin synthases. Overall, six genes encoding putative enzymes of the riboflavin biosynthesis pathway were identified in Nab. magadii and only two of these (Nmag_0941 and 0942) were clustered together (Table 2).Vitamin Beactions . Most babstrates . Riboflaannaschii. In geneion step . However3 (nicotinic acid) is the central component of coenzymes NAD and NADP, which are essential redox cofactors in metabolism. Like most bacteria, halophilic archaea synthesize NAD from aspartate via quinolinate. Nab. magadii contained 7 genes  that were predicted to be involved in NAD biosynthesis.Vitamin B420 is involved in methanogenesis [e.g., aflatoxin reduction in mycobacteria [420 to substrates with electron-deficient ring systems. Furthermore, 5-amino-6-(D-ribitylamino)uracil is an intermediate of coenzyme F420 and riboflavin biosynthesis pathways [420 has been detected in some halophilic archaea [420 biosynthesis genes have been identified among the methanogens [Nab. magadii and other halophilic archaea contained several genes encoding putative enzymes of the coenyzme F420 biosynthesis pathway. These genes were identified based on the presence of their homologs among the methanogens. Furthermore, using SIMBAL analysis [420 dependent enzymes have been predicted in halophilic archaea . Nab. magadii predicted proteins with an assigned F420-related TIGRFam are shown in Table 2. A possible function of coenzyme F420 in the respiratory chain of Nab. magadii is discussed later in this paper.Coenzyme Fogenesis ,77 and obacteria ,79) thatpathways ,77. Alth archaea , and coehanogens , its preanalysis , coenyzmNab. magadii genes putatively involved in folate biosynthesis were generally similar to those described in Nmn. pharaonis, including the archaeal-type GTP cyclohydrolase (Nmag_2853). Nab. magadii contained a pabABC operon (Nmag_2792-2794) and the homologs of these genes were predicted to be involved in para-aminobenzoate biosynthesis in Nmn, pharaonis[Nab. magadii also contained genes encoding a dihydropteroate synthase, a fused dihydropteroate-dihydrofolate synthase, a dihydrofolate reductase, and a methenyltetrahydrofolate cyclohydrolase (Table 2). The latter enzyme is predicted to participate in the conversion of the C1 metabolite attached to tetrahydrofolate. However, none of the other genes encoding C1-converting enzymes identified in Haloquadratum or Haloarcula were found in Nab. magadii and Nmn. pharaonis. Furthermore, Nab. magadii contained three genes encoding putative enzymes of the later stages of the folate biosynthesis pathway . Nevertheless, genes encoding the bacterial homologs of FolQBK, the atypical FolQ described in Desulfovibrio, or the alternative pathway bypassing FolQB  were absent in Nab. magadii and Nmn. pharaonis. Therefore, precise mechanisms of folate biosynthesis in these species remain to be discovered.Tetrahydrofolate participates in a number of biochemical reactions and reduced folate cofactors are required for the biosynthesis of a variety of molecules in both prokaryotes and eukaryotes ,84. The pharaonis. Nab. made novo using a branched pathway and aminolevulinic acid as the precursor [Environmental bacteria utilize a variety of redox molecules such as porphyrins and other modified tetrapyrroles like heme, siroheme, and adenosylcobalamin for catalysis, energy transfer, and signal transduction . These trecursor ,88. In me.g.cobYcbiZ) [e.g.Nmn. pharaonis) use the “anaerobic” branch, which is characterized by an oxygen-independent ring contraction process (cibG) [Halobacterium synthesizes cobalamin de novo under aerobic conditions [Nmn. pharaonis has homologs of the ATP-independent early cobalt chelatase (CbiX) from Bacillus halodurans and Archaeoglobus fulgidus[cbiLcbiHcbiFcbiGcbiEcbiT, and cbiC), but two pathway gaps (corresponding to cbiD/cobF and cbiJ/cobK) still remain. A set of 11 genes is known to be involved in conversion of cobyrinic acid into adenosylcobalamin .The complete pathway for the biosynthesis of adenosylcobalamin from precorrin-2 involves two major branches and several enzymes ,90, someobYcbiZ) ,92. Halos (cibG) . Howevernditions . The “an fulgidus,95. In tNab. magadii was incapable of de novo cobalamin biosynthesis since it lacked the genes encoding enzymes for conversion of precorrin-2 into cobyrinic acid. This is in contrast to Htg. turkmenica, which was predicted to be capable of de novo cobalamin biosynthesis since it contained the corresponding genes. However, Nab. magadii was predicted to be capable of corrinoid salvage since it contained a gene encoding a putative corrinoid transporter. Nab. magadii also contained a set of genes that were predicted to be involved in the conversion of cobyrinic acid into adenosylcobalamin, including a gene (cbiZ) that is specific to the archaeal corrinoid salvage pathway (Table 2).Based on genome analyses, it appeared that Desulfovibrio[Bacillus megaterium SirC) or might also be a ferrochelatase . From comparison of Nab. magadii with other halophilic archaea, another possibility emerges: iron chelation may be performed by one of the proteins annotated as CbiX-type cobalt chelatase. Nmn. pharaonis has three cbiX paralogs , two of which have closely related orthologs in nearly all other halophilic archaea. Htg. turkmenika, probably capable of de novo cobalamin biosynthesis, has orthologs of both cbiX1 and cbiX2. However, Nab. magadii, which is predicted to be incapable of de novo cobalamin biosynthesis, and therefore expected to lack these proposed early cobalt chelatases, surprisingly contained a cbiX2 ortholog (Nmag_3212). It is possible that cbiX2 functions as a ferrochelatase during siroheme/heme biosynthesis rather than as a cobaltochelatase during de novo cobalamin biosynthesis.The heme biosynthesis pathway in archaea involving uroporphyrinogen III, precorrin-2, and siroheme appears to be similar to that of lfovibrio. Conversd1 biosynthesis, is generated by the decarboxylation of siroheme on the C12 and C18 acetyl groups [nirDLGH gene set, which is represented by a pair of two-domain proteins in halophilic archaea . Heme d1 is a coenzyme of dissimilatory nitrite reductase  and is not required by organisms lacking this enzyme. The last steps of heme biosynthesis include the removal of acetyl side chains of Fe-coproporphyrin by AhbC (encoded by Mbar_A1793 in Methanosarcina barkeri) and the oxidative decarboxylation of heme by AhbD (encoded by DVU_0855 in Desulfovibrio) [ahbC and not ahbD. Orthologs encoding AhbC and AhbD were present in Nmn. pharaonis (NP1542A and NP1546A) and Htg. turkmenica (Htur_1726 and 1728), but not in Nab. magadii. The presence of ahbC and ahbD in some halophilic archaea but not in others is believed to be due to metabolic heterogeneity rather than incomplete heme biosynthesis. Conversion of heme  into heme A in Nab. magadii was predicted to be catalyzed by CtaA and CtaB homologs .Didecarboxysiroheme, a common intermediate of heme and heme l groups . Sirohemovibrio)  [gene asde novo using pimelic acid as a precursor, and some others have evolved mechanisms for importing this essential cofactor from their natural environments [Nab. magadii is a biotin auxotroph, Nmn. pharaonis is a biotin prototroph and the genome of this haloalkaliphilic archaeon has been shown to contain at least three genes putatively involved in the biosynthesis of biotin [Nab. magadii was apparent from the analyses of its genome sequence. However, the large chromosome of Nab. magadii contained a locus encoding a putative biotin transporter , which may facilitate the uptake of biotin from the environment.Vitamin H, commonly known as biotin, acts as a coenzyme in several enzyme-catalyzed carboxylation and decarboxylation reactions . Most baronments ,98. Wherf biotin . The absNab. magadii. Additional file Nab. magadii. Genes that encoded putative enzymes for glycerol utilization, aromatic amino acid catabolism, ureagenesis, and urea degradation were also identified in Nab. magadii. Other putative metabolic features of Nab. magadii included xylose isomerases, an alpha amylase, a methylglyoxal synthase, sulfatases, a chlorite dismutase, sarcosine oxidases, and aldehyde dehydrogenases  is essential for the functioning of molybdoenzymes such as dimethylsulfoxide and trimethylamine-N-oxide reductases, formate dehydrogenases, and nitrate reductases ,100. Molubstrate . The larHaloarcula marismortui converts acetyl-CoA to glyoxylate via the key intermediate methylaspartate [Nab. magadii, activities of the enzymes of the methylaspartate cycle, but not those of the key enzymes of the glyoxylate cycle, were detected [Nab. magadii. The square archaeon Haloquadratum walsbyi contains a gene (HQ2709A) encoding a phosphoenolpyruvate-dependent phosphotransferase system (PTS) that is predicted to be involved in the phosphorylation of dihydroxyacetone [Htg. turkmenicaHfx. volcanii, and several other haloarchaeal genomes. However, Nab. magadii and Nmn. pharaonis lacked homologs of these genes encoding PTS components.The haloarchaeon spartate . Glyoxyldetected . An operyacetone . Homolog+ is the hallmark of such an adaptation. Nab. magadii had a cluster of eight genes that form the atpHIKECFAB operon encoding putative ATP synthase subunits (Nmag_1370-1377) and an unlinked atpD homolog (Nmag_1366). Similar gene clusters were found in several halophilic archaea. Ion specificity of the ATP synthase is determined by the c-ring, which is encoded by the atpK gene (Nmag_1375) for A-type ATP synthases. Nab. magadii may have a proton-driven ATP synthase since its predicted AtpK lacks the sequence signature of Na+-dependent ATP synthases [Hfx. volcanii[Hbt. salinarum[Nmn. pharaonis[Running a proton-driven, energy-conserving ATP synthase at high extracellular pH is an obvious challenge. Energy coupling of sodium ions instead of protons was proposed to be an adaptation to alkaliphilic growth conditions and an ATP synthase driven by Naynthases . Instead volcanii, Hbt. sasalinarum, and Nmnpharaonis.Nmn. pharaonis have revealed several novel features, including a gene encoding a type II NADH dehydrogenase (NP3508A) [Acidianus ambivalens was proposed to be involved in NADH reoxidation, feeding into the lipid-soluble quinone pool [Nab. magadii (Nmag_0301) and several other halophilic archaea. Nab. magadii also contained genes encoding a putative nuo complex (Nmag_3245-3255), which was similar to the mitochrondrial NADH dehydrogenase (complex I). Although 13 nuo cluster subunits were conserved among halophilic archaea and E. coli, the nuoEFG subcomplex, which is involved in accepting NADH, was missing in halophilic archaea [Hbt. salinarum[420, which is similar to NADH in its redox potential, may interact with the nuo complex in halophilic archaea. In addition to the NADH dehydrogenases, Nab. magadii and other halophilic archaea are predicted to encode a succinate dehydrogenase that may oxidize succinate and reduce the quinone pool of the electron transport chain.Reduction of oxygen and the associated proton-coupled electron transfer (respiration) is the primary source of energy among aerobic organisms. Respiratory complexes, which include a variety of cytochromes and terminal oxidases, are essential components of this process. Biochemical and comparative genomic analyses of the electron transport chain of NP3508A) ,108. A hone pool . A homol archaea . Furthersalinarum. It is sNab. magadii contained loci encoding putative cytochrome c-type terminal oxidase subunits I and II (Nmag_0263-0264) and cytochrome ubiquinol oxidase subunits I and II (Nmag_1036-1037). Furthermore, pNMAG02 contained an operon encoding putative cytochrome ubiquinol oxidase subunits I and II (Nmag_4038-4039) that were related to the proteins encoded by Nmag_1036-1037 (46% identity at the protein level). The homologs of Nmag_0263-0264 and Nmag_1036-1037 were present in Htg. turkmenica , but not in Nmn. pharaonis. Two cbaDBAC operons (plus a monocistronic cbaE) encoding putative cytochrome ba3 terminal oxidase complexes were identified in pNMAG01 (Nmag_3754-3758 and 3802–3805), and these operons appeared to be related to each other (38-65% identity at the predicted protein level). The homologs of these ORFs were present in Htg. turkmenica (Htur_0462- 0466) and Nmn. pharaonis (NP2960A-NP2968A), the latter of which have been functionally characterized [cbaD is fused to halocyanin in Hbt. salinarum and Har. marismortui[Nab. magadii contained several genes encoding putative halocyanin-like proteins . Halocyanins are coupled to the reduced quinone pool by the cytochrome bc1 complex. Although genes encoding a cytochrome bc1 complex are present in Hbt. salinarumHfx. volcanii, and several other halophilic archaea, they were absent in Htg. turkmenicaNmn. pharaonis, and Nab. magadii. The electron transfer from the reduced lipid-soluble quinone pool to halocyanin remains unresolved in species that lack a cytochrome bc1 complex. Based on genome comparisons, it appeared that the respiratory chain of Nab. magadii was more similar to that of Htg. turkmenica than to Nmn. pharaonis.A number of cytochromes involved in respiratory electron transport have been characterized among the archaea ,111. Tercterized . Halocyacterized ,108. Thirismortui. Nab. maNab. magadii included Nmag_0636 (cytochrome oxidase assembly protein), Nmag_1972 (cytochrome P450), and Nmag_3057 (cytochrome c biogenesis protein). The small chromosome pNMAG01 contained a gene encoding a putative cytochrome c biogenesis protein (Nmag_3708) that had 64% identity to the protein encoded by Nmag_3057. Nab. magadii also contained genes (Nmag_2430-2432) encoding a putative sulfur utilization factor (SUF) system, which was shown to be important for Fe-S cluster biogenesis during stress in E. coli[Nab. magadii include those encoding electron transfer flavoprotein subunits (Nmag_0482-0483 and 1388–1389), SCO1/SenC electron transport proteins , and a redoxin domain protein (Nmag_3709).Other genes encoding putative cytochrome-related proteins in the large chromosome of n E. coli. Other gNab. magadii contained a pair of genes (Nmag_3535-3536) encoding putative HK-RR proteins. Nab. magadii also contained two pairs of genes (Nmag_2042-2043 and Nmag_3740-3741) encoding putative HK-RR proteins with an additional RR domain in the N-terminus of the predicted HK protein. Furthermore, another set of genes (Nmag_1129-1130) encoding a HK-like protein, which was distantly related to CheA of Nmn. pharaonis, and a putative protein containing a RR domain was also predicted in Nab. magadii. Interestingly, Nab. magadii also contained 15 genes encoding putative HK proteins without a cognate RR gene  and 11 genes encoding putative RR proteins without a cognate HK gene .Two-component signal transduction systems consisting of a histidine kinase (HK) and a response regulator (RR) constitute one of the most frequently encountered bacterial and archaeal communication circuits . Nab. maNab. magadii contained a single rhodopsin gene . Nab. magadii also contained 3 genes  encoding distant homologs of rhodopsins that were related to each other (36-49% sequence identity). They were also related to NP1758A from Nmn. pharaonis (33%-42% sequence identity) and predicted to encode distant rhodopsin homologs that lack the Lys residue involved in covalent retinal attachment. Whether these rhodopsin homologs interact with retinal noncovalently, or if they interact with retinal at all, is unknown. Two of the three genes encoding retinal homologs in Nab. magadii (Nmag_2879 and 2881) were located adjacent to genes (Nmag_2880 and 2882) encoding putative methyl-accepting chemotactic transducer proteins. In Nmn. pharaonis, NP1758A and NP3132A, which are homologs of Nmag_2879 and 2881, were also found adjacent to genes  encoding putative methyl-accepting chemotactic transducer proteins [Htg. turkmenica lacked genes encoding bacteriorhodopsin and halorhodopsin, it contained a single gene (Htur_3663) that appeared to be a distant homolog of Nmag_2879 and 2881. Furthermore, similar to Nmn. pharaonisNab. magadii lacked a gene encoding a proton pump bacteriorhodopsin. However, Nab. magadii contained a locus encoding putative phytoene desaturase, UbiA prenyltransferase, carotene biosynthesis protein, and phytoene synthase (Nmag_1001-1004) as well as a gene encoding a putative squalene/phytoene synthase .Halobacterial perception of and response to physical stimulus such as light (phototaxis) is mediated by photoreceptors . The genproteins . TherefoHbt. salinarum and designated Brp and Blh [brp and blh were present in Nmn. pharaonis , they were absent in Nab. magadii. However, Nab. magadii contained a homolog (Nmag_4083) of Hqr. walsbyi HQ2020A, which was predicted to encode a distinct β-carotenase unrelated to those mentioned above [Htg. turkmenica lacked homologs of brp and blh as well as HQ2020A, which is consistent with the absence of all canonical rhodopsins in this organism.Two unrelated enzyme families are used for the cleavage of β-carotene into retinal in halophilic archaea. Distant paralogs, which belong to one of these β-carotenase enzyme families, have been identified in  and Blh . Althouged above . InteresNab. magadii contained two loci encoding putative motility and signal transduction functions. One of them contained exclusively “che genes” in a cheYBACCDR operon (Nmag_3145-3151), which is preceded by two divergently transcribed and distantly related cheW genes (Nmag_3152-3153). The cheYBACCDR operon encodes a very long signal transduction histidine kinase , a response regulator receiver protein (CheY), a CheR-type MCP methyltransferase, and a response regulator receiver-modulated methylesterase (CheB). While CheD has been reported to function as glutamine deamidase in some organisms or as methylesterase in others, CheB functions as both [Htg. turkmenica (Htur_0954-0962) and based on predicted protein homology, it appeared that the two loci were evolutionarily very closely linked. Highly similar gene clusters were also found in the genomes of Halopiger xanaduensisNatrinema pellirubrum, and Natronobacterium gregoryi. The second locus (Nmag_2859-2889) contained “che genes” along with “fla genes” encoding flagellin biosynthesis and assembly functions (see below). The “che genes” in this locus encode putative CheA, CheY, CheR, CheB, CheD, a two-domain CheC, and two CheW proteins. Nab. magadii contained two cheF genes within this locus and homologs of these genes were shown to be involved in chemotaxis in Halobacterium[Nab. magadii include Nmag_0478, 0937, 1253, 1386, 1542, 2639, 3325, 3638, and 3856). Among these, two  were adjacent to genes encoding periplasmic ligand-binding proteins.Microbial response to chemical stimulus  is mediated by chemoreceptors . The lar as both . A similbacterium. This geHbt. salinarum[Nab. magadii is a region  with 13 predicted ORFs encoding putative flagellin biosynthesis and assembly proteins (Nmag_2862-2874). Except Nmag_2871, which encoded a protein of unknown function, all other ORFs were located on the plus strand. This region contains 4 flagellin genes (Nmag_2862-2865), which encode the flagella proteins previously identified [Nab. magadii contained homologs of flaF (Nmag_2866), flaG (Nmag_2867), flaH (Nmag_2873), flaI (Nmag_2874), and flaJ (Nmag_2869). The latter two genes (flaI and flaJ) encode putative proteins homologous to the type II secretion system proteins E and F, respectively. In several archaea, FlaI has been shown to be involved in flagellin assembly [flaHflaI, and flaJ represent a core set of highly conserved genes presumably crucial for archaeal motility.Archaeal flagella are very different in composition and assembly in comparison to bacterial flagella . In contsalinarum. Within entified  and charentified . Furtherassembly ,125, andassembly . The motassembly . NeverthNab. magadii contains distinctive flagella [fla locus (Nmag_2862-2874) of Nab. magadii was involved in flagellin biosynthesis and motility. In addition, the large chromosome of Nab. magadii contained genes encoding a putative full-length PilT protein (Nmag_1543) and a prepilin peptidase (Nmag_1752), whose homologs were found in Nmn. pharaonis (NP0198A and NP1276A) and Htg. turkmenica (Htur_3514 and Htur_0098).Since previous electron microscopic analyses have demonstrated that flagella -129, andNab. magadii contained 90 genes  encoding putative transcriptional regulators (TRs). BLASTP analyses indicated that most of these predicted proteins were related to bacterial TRs. These TRs were categorized into the following families based on their helix-turn-helix (HTH) motifs (numbers in parenthesis indicate the number of proteins in each family): AsnC (16), IclR (13), PadR (10), ArsR/GntR/Fur (11), XRE (6), TrmB (8), AbrB (3), TetR (3), MarR (3), CopG/Arc/MetJ/NikR (10), HxlR (2), TenA (1), ModE (1), and unassigned (3). Apart from these TRs, Nab. magadii also contained 27 genes encoding TRs with an HTH-10 domain, which was also found in bacterio-opsin activators . In addition, Nab. magadii encoded a two-domain archaeal histone (Nmag_2205), a single TATA-binding transcription initiation factor , a single transcription initiation factor TFE (Nmag_3157), and a set of seven transcription initiation factors TFB .Archaeal basal transcription machinery has many similarities to the eukaryotic RNA polymerase II apparatus. However, the mechanisms of transcription regulation and the transcriptional regulators among archaea are distinct from those of eukaryotes ,131. NabNab. magadii, a haloalkaliphilic archaeon that belongs to a physiologically distinct subgroup of halophilic archaea. Although Nab. magadii appears to have developed strategies similar to Nmn. pharaonis to optimally thrive in low water activity and high pH habitats, the genetic architecture of Nab. magadii is more similar to that of Htg. turkmenica than to Nmn. pharaonis. The presence of genes encoding the biosynthesis of the osmoprotectant 2-sulfotrehalose is an uncommon feature among halophilic archaea and this may have contributed to the evolution of Nab. magadii in its natural environment. Nab. magadii has genes encoding a number of cation/proton antiporters as well as pathways for the biosynthesis and/or transport of various cofactors and vitamins. The occurrence of genes encoding enzymes involved in glycolysis, gluconeogenesis, and glycerol utilization suggests that Nab. magadii is metabolically versatile and can use different carbon and energy sources to sustain growth. Furthermore, the large repertoire of genes encoding putative proteases/peptidases and peptide transport systems is indicative of the protein/peptide catabolic potential of Nab. magadii. It also appears that Nab. magadii can perceive and process physical and chemical stimuli, and respond appropriately by moving toward or away from those stimuli using the flagellar apparatus. The information obtained from this comparative genomic analysis contributes to our overall understanding of the biology and diversity of halophilic archaea. In particular, it will guide current and future research on the genetics and physiology of Nab. magadii. Such studies are expected to facilitate the manipulation of this archaeon as a model for haloalkalphilic metabolism and its optimization for biotechnological applications.This report describes the genome sequence of The author(s) declare that they have no competing interests.Nab. magadii proteases/inhibitors (Figure Nab. magadii samples used for TEM analysis. FP participated in the manual curation of Nab. magadii genome, reconstructed several coenzyme biosynthetic pathways, and helped draft the manuscript. RDC and JAM conceived the study, participated in genome analyses, and helped draft the manuscript. All authors read and approved the final manuscript.SS performed the annotation using RAST, planned the comparative analysis, and drafted most of the manuscript. JFC contributed to whole genome comparisons and generated Figure s Figure . RAP preTable S1.Natrialba magadii ATCC 43099 genes discussed in the text. This table lists Nab. magadii ATCC 43099 genes related to bacteriophage and recombination elements, rRNA genes, and genes encoding adaptive features.Click here for fileTable S2. Bidirectional best blast pairs among proteins fromNatrialba magadii and 17 other haloarchaeal genomes. This table lists the number of bidirectional best blast pairs among proteins from Nab. magadii and 17 other halophilic archaea. The first column is the number of total proteins in each genome, the second column is the number of bidirectional best blast pairs, and the third column is the percentage of proteins having a bidirectional best blast hit in Nab. magadii.Click here for fileTable S3.Natrialba magadii ATCC 43099 genes encoding putative peptidases/proteases, protease inhibitors, and regulatory proteins. This table lists Nab. magadii ATCC 43099 genes encoding various types of proteases and peptidases as well as protease inhibitors and regulatory proteins.Click here for fileTable S4. Natrialba magadii ATCC 43099 genes involved in metabolism. This table lists Nab. magadii ATCC 43099 genes encoding molybdenum cofactor biosynthesis and other metabolic functions.Click here for file