Sexual Isolation in Acinetobacter baylyi Is Locus-Specific and Varies 10,000-Fold Over the Genome

Naturally transformable bacteria acquire chromosomal DNA from related species at lower frequencies than from cognate DNA sources. To determine how genome location affects heterogamic transformation in bacteria, we inserted an nptI marker into random chromosome locations in 19 different strains of the Acinetobacter genus (>24% divergent at the mutS/trpE loci). DNA from a total of 95 nptI-tagged isolates was used to transform the recipient Acinetobacter baylyi strain ADP1. A total of >1300 transformation assays revealed that at least one nptI-tagged isolate for each of the strains/species tested resulted in detectable integration of the nptI marker into the ADP1 genome. Transformation frequencies varied up to ∼10,000-fold among independent nptI insertions within a strain. The location and local sequence divergence of the nptI flanking regions were determined in the transformants. Heterogamic transformation depended on RecA and was hampered by DNA mismatch repair. Our studies suggest that single-locus-based studies, and inference of transfer frequencies from general estimates of genomic sequence divergence, is insufficient to predict the recombination potential of chromosomal DNA fragments between more divergent genomes. Interspecies differences in overall gene content, and conflicts in local gene organization and synteny are likely important determinants of the genomewide variation in recombination rates between bacterial species.HORIZONTAL gene transfer (HGT) contributes to bacterial evolution by providing access to DNA evolved and retained in separate species or strains (Cohan 1994a,b; Bergstrom et al. 2000; Ochman et al. 2000; Feil et al. 2001; Koonin 2003; Lawrence and Hendrickson 2003; Fraser et al. 2007). Multilocus sequence typing (MLST) has provided strong evidence for frequent transfer and recombination of chromosomal DNA between related bacterial strains within the same species (Maiden et al. 1998; Enright et al. 2002). HGT occurring by natural transformation allows bacteria to exploit the presence of nucleic acids in their environment for the purposes of nutrition, DNA repair, reacquisition of lost genes, and/or acquisition of novel genetic diversity (Redfield 1993; Mehr and Seifert 1998; Dubnau 1999; Claverys et al. 2000; Szöllösi et al. 2006; Johnsen et al. 2009). It can be inferred from observations of the presence of extracellular DNA in most environments that bacteria are constantly exposed to DNA from a variety of sources, without such exposure necessarily producing observable changes in the genetic compositions of bacterial populations over evolutionary time (Thomas and Nielsen 2005; Nielsen et al. 2007a,b).The absence of sequence similarity between the donor DNA and the DNA of the recipient bacterium is the strongest barrier to the horizontal acquisition of chromosomal genes in bacteria (Matic et al. 1996; Vulic et al. 1997; Majewski 2001; Townsend et al. 2003) as illegitimate recombination occurs only at extremely low frequencies in bacteria (Hülter and Wackernagel 2008a). Single-locus transfer models have been extensively applied and have demonstrated a log-linear decrease in recombination frequencies with increasing sequence divergence for Bacillus subtilis (Roberts and Cohan, 1993; Zawadzki et al. 1995), Acinetobacter baylyi (Young and Ornston 2001), Escherichia coli (Shen and Huang 1986; Vulic et al. 1997), and Streptococcus pneumoniae (Majewski et al. 2000). For instance, heterogamic transformation between nonmutator isolates at the rpoB locus of B. mojavensis is undetectable at sequence divergences >16.7% (Zawadzki et al. 1995) and between S. pneumoniae isolates with sequence divergences >18% (Majewski et al. 2000). In A. baylyi, the nonmutator sequence divergence limit for detectable transformation at the pcaH locus of strain ADP1 was found to be 20% (Young and Ornston 2001), and up to 24% overall divergence yielded transformants at 16S rRNA loci in strain DSM587 (Strätz et al. 1996).Several recent studies also show that short stretches (<200 bp) of DNA sequence identity can facilitate additive or substitutive integration of longer stretches (>1000 bp) of heterologous DNA in bacteria (Prudhomme et al. 1991, 2002; de Vries and Wackernagel 2002; Hülter and Wackernagel 2008a). Thus, the uptake of DNA in bacteria can facilitate larger substitutions within gene sequences and the integration of additional DNA material on the basis of recombination initiated in flanking DNA stretches (either at one or both ends) with high sequence similarity (Nielsen et al. 2000). On the other hand, segments of heterologous DNA interrupting the synteny of homologous DNA have also been shown to be a barrier in intraspecies transformation in S. pneumoniae (Pasta and Sicard 1996, 1999).The various studies of the interspecies transfer potential of single genes demonstrate that the immediate local sequence divergence of the transferred locus is of high importance in determining recombination frequencies in hosts up to 20% divergent (at the housekeeping gene level). However, it can be hypothesized that the broader structural, organizational, and biochemical properties of the genome region surrounding a particular locus will determine its transfer potential to more divergent host species (Cohan 2001; Lawrence 2002). The interspecies transfer potential of various genome regions/loci between more diverged species (>20% at the housekeeping gene level) may therefore differ substantially from a log-linear model (determined experimentally for more closely related species) as local gene organization becomes less conserved with evolutionary time. The barriers to gene exchange between divergent bacterial species is likely a combination of inefficient recombination due to both mismatched base pairs (the main determinator in the log-linear model) and conflicting gene order and organization across the local recombining DNA regions. In addition, selective barriers due to negative effects on host fitness of the transferred DNA regions may become increasingly important for the removal of recombination events from the bacterial population. Recent bioinformatics-based genome analysis of E. coli and Salmonella genomes suggests various parts of the bacterial genome may have different suceptibilities to undergo evolutionarily successful recombination leading to temporal fragmentation of speciation (Lawrence 2002; Retchless and Lawrence 2007). Nevertheless, few studies have experimentally tested the effect of variable species and chromosome locations of genes on their transfer potential between bacteria (Ravin and Chen 1967; Ravin and Chakrabarti 1975; Siddiqui and Goldberg 1975; Cohan et al. 1991; Huang et al. 1991; Fall et al. 2007).Here, we determine to what extent genome location contributes to sexual isolation between the recipient A. baylyi strain ADP1 and 19 sequence divergent (24–27% divergent at the mutS/trpE loci) donor Acinetobacter strains and species (carrying a selectable nptI gene in a total of 95 random genome locations).     

Influence of sea temperature and initial marine feeding on survival of Atlantic salmon Salmo salar post-smolts from the Rivers Orkla and Hals, Norway

The abundance of returning adult Atlantic salmon Salmo salar, in the River Orkla in mid‐norway (1 sea‐winter, SW, fish) and River Hals in north Norway (1–3 SW fish), was tested against the early marine feeding and the seawater temperature experienced by their corresponding year classes of post‐smolts immediately after entry into the Trondheimsfjord (Orkla smolts, 22 years of data) and Altafjord (Hals smolts, 17 years of data). In both river–fjord systems, there was a significant positive correlation between the abundance of returning S. salar and the mean seawater temperature at the time of smolts descending to the sea. The number of 1SW fish reported caught in River Orkla was positively correlated to the proportion of fish larvae in the post‐smolt stomachs in Trondheimsfjord. The abundance of returning S.salar was, however, neither correlated to forage ratio (R_F) nor other prey groups in post‐smolt stomachs in the two fjord systems. In the Altafjord, the post‐smolts fed mainly on pelagic fish larva (70–98%) and had a stable R_F (0·009–0·023) over the 6 years analysed. In the Trondheimsfjord, however, there was a higher variation in R_F (0·003–0·036), and pelagic fish larvae were dominant prey in only two (50 and 91%) of the 8 years analysed. These 2 years also showed the highest return rates of S. salar in River Orkla. These results demonstrate that the thermal conditions experienced by post‐smolts during their early sea migration may be crucial for the subsequent return rate of adults after 1–3 years at sea. Pelagic marine fish larvae seem to be the preferred initial prey for S. salar post‐smolts. As the annual variation in abundance of fish larvae is related to seawater temperature, it is proposed that seawater temperature at sea entry and the subsequent abundance of returning adult S. salar may be indirectly linked through variation in annual availability of pelagic fish larvae or other suitable food items in the early post‐smolt phase.     

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii

The pathway of d-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198–6207). Here we report a comprehensive study of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following ¹³C-labeling patterns of proteinogenic amino acids after growth on [¹³C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that d-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate α-ketoglutarate, involving d-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and α-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal d-xylose degradation pathway that differs from the classical d-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to α-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus.d-Xylose, a constituent of the polymer xylan, is the major component of the hemicellulose plant cell wall material and thus one of the most abundant carbohydrates in nature. The utilization of d-xylose by microorganisms has been described in detail in bacteria and fungi, for which two different catabolic pathways have been reported. In many bacteria, such as Escherichia coli, Bacillus, and Lactobacillus species, xylose is converted by the activities of xylose isomerase and xylulose kinase to xylulose 5-phosphate as an intermediate, which is further degraded mainly by the pentose phosphate cycle or phosphoketolase pathway. Most fungi convert xylose to xylulose 5-phosphate via xylose reductase, xylitol dehydrogenase, and xylulose kinase. Xylulose 5-phosphate is also an intermediate of the most common l-arabinose degradation pathway in bacteria, e.g. of E. coli, via activities of isomerase, kinase, and epimerase (1).Recently, by genetic evidence, a third pathway of xylose degradation was proposed for the bacterium Caulobacter crescentus, in analogy to an alternative catabolic pathway of l-arabinose, reported for some bacteria, including species of Azospirillum, Pseudomonas, Rhizobium, Burkholderia, and Herbasprillum (2, 3). In these organisms l-arabinose is oxidatively degraded to α-ketoglutarate, an intermediate of the tricarboxylic acid cycle, via the activities of l-arabinose dehydrogenase, l-arabinolactonase, and two successive dehydration reactions forming 2-keto-3-deoxy-l-arabinoate and α-ketoglutarate semialdehyde; the latter compound is further oxidized to α-ketoglutarate via NADP⁺-specific α-ketoglutarate semialdehyde dehydrogenase (KGSADH).² In a few Pseudomonas and Rhizobium species, a variant of this l-arabinose pathway was described involving aldolase cleavage of the intermediate 2-keto-3-deoxy-l-arabinoate to pyruvate and glycolaldehyde, rather than its dehydration and oxidation to α-ketoglutarate (4). Because of the presence of some analogous enzyme activities in xylose-grown cells of Azosprillum and Rhizobium, the oxidative pathway and its variant was also proposed as a catabolic pathway for d-xylose. Recent genetic analysis of Caulobacter crecentus indicates the presence of an oxidative pathway for d-xylose degradation to α-ketoglutarate. All genes encoding xylose dehydrogenase and putative lactonase, xylonate dehydratase, 2-keto-3-deoxylonate dehydratase, and KGSADH were found to be located on a xylose-inducible operon (5). With exception of xylose dehydrogenase, which has been partially characterized, the other postulated enzymes of the pathway have not been biochemically analyzed.The pathway of d-xylose degradation in the domain of archaea has not been studied so far. First analyses with the halophilic archaeon Haloarcula marismortui indicate that the initial step of d-xylose degradation involves a xylose-inducible xylose dehydrogenase (6) suggesting an oxidative pathway of xylose degradation to α-ketoglutarate, or to pyruvate and glycolaldehyde, in analogy to the proposed oxidative bacterial pentose degradation pathways. Recently, a detailed study of d-arabinose catabolism in the thermoacidophilic crenarchaeon Sulfolobus solfataricus was reported. d-Arabinose was found to be oxidized to α-ketoglutarate involving d-arabinose dehydrogenase, d-arabinoate dehydratase, 2-keto-3-deoxy-d-arabinoate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase (3).In this study, we present a comprehensive analysis of the complete d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. This halophilic archaeon was chosen because it exerts several suitable properties for the analyses. For example, it can be cultivated on synthetic media with sugars, e.g. xylose, an advantage for in vivo labeling studies in growing cultures. Furthermore, a shotgun DNA microarray of H. volcanii is available (7) allowing the identification of xylose-inducible genes, and H. volcanii is one of the few archaea for which an efficient protocol was recently described to generate in-frame deletion mutants.Accordingly, the d-xylose degradation pathway was elucidated following in vivo labeling experiments with [¹³C]xylose, DNA microarray analyses, and the characterization of enzymes involved and their encoding genes. The functional involvement of genes and enzymes was proven by constructing corresponding in-frame deletion mutants and their analysis by selective growth experiments on xylose versus glucose. The data show that d-xylose was exclusively degraded to α-ketoglutarate involving xylose dehydrogenase, a novel xylonate dehydratase, 2-keto-3-deoxyxylonate dehydratase, and α-ketoglutarate semialdehyde dehydrogenase.     

Microbial degradation of street dust polycyclic aromatic hydrocarbons in microcosms simulating diffuse pollution of urban soil

Diffuse pollution with polycyclic aromatic hydrocarbons (PAHs) of topsoil in urban regions has caused increasing concerns in recent years. We simulated diffuse pollution of soil in microcosms by spiking sandy topsoil (A-horizon) and coarse, mineral subsoil (C-horizon) with street dust (PM63) isolated from municipal street sweepings from central Copenhagen. The microbial communities adapted to PAH degradation in microcosms spiked with street dust in both A-horizon and C-horizon soils, in spite of low PAH-concentrations. The increased potential for PAH degradation was demonstrated on several levels: by slowly diminishing PAH-concentrations, increased mineralization of 14C-PAHs, increasing numbers of PAH degraders and increased prevalence of nah and pdo1 PAH degradation genes, i.e. the microbial communities quickly adapted to PAH degradation. Three- and 4-ring PAHs from the street dust were biodegraded to some extent (10-20%), but 5- and 6-ring PAHs were not biodegraded in spite of frequent soil mixing and high PAH degradation potentials. In addition to biodegradation, leaching of 2-, 3- and 4-ring PAHs from the A-horizon to the C-horizon seems to reduce PAH-levels in surface soil. Over time, levels of 2-, 3- and 4-ring PAHs in surface soil may reach equilibrium between input and the combination of biodegradation and leaching. However, levels of the environmentally critical 5- and 6-ring PAHs will probably continue to rise. We presume that sorption to black carbon particles is responsible for the persistence and low bioaccessibility of 5- and 6-ring PAHs in diffusely polluted soil.     

Quality assessment of autografting by probability evaluation: model estimation by clinical end-points in newly diagnosed multiple myeloma patients

BACKGROUND: Pre-transplant clinical evaluation of autografting is an important step in predicting post-transplant support, complications and safety. Today, unfavorable outcomes such as early death or graft failure are rare, making them unsuitable for quality assessment of supportive autografting. However, end-points constructed from frequently occurring clinical events may estimate clinically relevant prognostic models. METHODS: The present retrospective analysis was based on two consecutive clinical trials in the Nordic area including up to 640 newly diagnosed multiple myeloma patients. RESULTS: In the model, the efficacy (time on antibiotics and use of transfusions) was influenced by pre-transplant variables, including sex, nationality, serum creatinine, hemoglobin, disease stage at diagnosis, response following induction therapy, length of priming and average graft CD34+ cell number per day of harvest. The toxicity end-point (time to blood cell recovery) was influenced by nationality, marrow plasma cell percentage, serum creatinine, M-component isotype, response to induction therapy, length of priming and graft CD34+ cell number. The safety (early disease recurrence or death) was influenced by serum creatinine, hemoglobin, treatment response and CD34+ cell number. DISCUSSION: In conclusion, the model illustrates that intervention strategies in quality assessment of autografting may benefit from probability estimates of graded clinical end-points.     

Freezing at -800 degrees C distorts the DNA composition of bacterial communities in intestinal samples

Terminal-restriction fragment length polymorphism (T-RFLP) was used to evaluate how to store intestinal specimens for bacterial community analysis. Bacterial communities are increasingly often described by means of DNA-based methods and it is common practice to store intestinal or faecal specimens either at -20 degrees C or -80 degrees C. In this study, samples of intestines from five different pigs were stored at -80 degrees C and -20 degrees C, respectively and a thawing and freezing procedure was carried out three times for each intestinal per pig per temperature. The cumulative sum of the T-RFLP peak heights (T-RF intensities) decreased as the temperature decreased. The composition of the bacterial community changed when stored at -80 degrees C compared to the samples stored at -20 degrees C. Thus it is recommended from this study that samples of intestinal content are stored at -20 degrees C before use for bacterial community analysis, instead of the current practice at -80 degrees C.     

Characterization of the Caenorhabditis elegans G protein-coupled serotonin receptors

Serotonin (5-HT) regulates a wide range of behaviors in Caenorhabditis elegans, including egg laying, male mating, locomotion and pharyngeal pumping. So far, four serotonin receptors have been described in the nematode C. elegans, three of which are G protein-coupled receptors (GPCR), (SER-1, SER-4 and SER-7), and one is an ion channel (MOD-1). By searching the C. elegans genome for additional 5-HT GPCR genes, we identified five further genes which encode putative 5-HT receptors, based on sequence similarities to 5-HT receptors from other species. Using loss-of-function mutants and RNAi, we performed a systematic study of the role of the eight GPCR genes in serotonin-modulated behaviors of C. elegans (F59C12.2, Y22D7AR.13, K02F2.6, C09B7.1, M03F4.3, F16D3.7, T02E9.3, C24A8.1). We also examined their expression patterns. Finally, we tested whether the most likely candidate receptors were able to modulate adenylate cyclase activity in transfected cells in a 5-HT-dependent manner. This paper is the first comprehensive study of G protein-coupled serotonin receptors of C. elegans. It provides a direct comparison of the expression patterns and functional roles for 5-HT receptors in C. elegans.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A spatial genetic structure and effects of relatedness on mate choice in a wild bird population

Inbreeding depression, as commonly found in natural populations, should favour the evolution of inbreeding avoidance mechanisms. If natal dispersal, the first and probably most effective mechanism, does not lead to a complete separation of males and females from a common origin, a small-scale genetic population structure may result and other mechanisms to avoid inbreeding may exist. We studied the genetic population structure and individual mating patterns in blue tits (Parus caeruleus). The population showed a local genetic structure in two out of four years: genetic relatedness between individuals (estimated from microsatellite markers) decreased with distance. This pattern was mainly caused by immigrants to the study area; these, if paired with fellow immigrants, were more related than expected by chance. Since blue tits did not avoid inbreeding with their social partner, we examined if individuals preferred less related partners at later stages of the mate choice process. We found no evidence that females or males avoided inbreeding through extra-pair copulations or through mate desertion and postbreeding dispersal. Although the small-scale genetic population structure suggests that blue tits could use a simple rule of thumb to select less related mates, females did not generally prefer more distantly breeding extra-pair partners. However, the proportion of young fathered by an extra-pair male in mixed paternity broods depended on the genetic relatedness with the female. This suggests that there is a fertilization bias towards less related copulation partners and that blue tits are able to reduce the costs of inbreeding through a postcopulatory process.     

The influence of root-zone temperature on growth of Betula pendula Roth.

We have examined shoot and root growth and the concentration of carbohydrates in seedlings of a northern (67°N) and a southern (61°N) ecotype of Betula pendula Roth. cultivated at root-zone temperatures of 2, 6, 12 and 17°C. Three hydroponic experiments were conducted in controlled environments. We used three different pretreatments before seedlings were subjected to the experimental temperature treatments. Actively growing seedlings that were acclimated to the hydroponic solution for 3 weeks at a root temperature of 17°C, continued to grow at all the experimental temperatures, with an expected increase in growth from 2 to 17°C. However, if we started with ecodormant cold stored plants or used seedlings grown actively in perlite, no growth was observed at 2°C and only minor growth was found at 6°C. The highest root temperature always produced the best growth. The concentration of nonstructural carbohydrates was higher in seedlings grown at 2°C than at 17°C, and this is probably due to extensive incorporation of carbohydrates into cell walls and other structural elements at 17°C. We found no evidence for differences between the two ecotypes in root growth, in timing of bud burst, but shoot growth terminated in the northern ecotype in the first experiment because the natural photoperiod was below the critical value. Our study highlights the importance of post-transplantation stress (planting check) related to root growth, and that root threshold temperatures may change according to the way plants are pretreated.