In glucose-limited continuous culture the minimum substrate concentration for growth,smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii,an environmentally abundant bacterium

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters μ_max and K_s were determined for growth with glucose. For both strains, μ_max was determined in batch culture after different precultivation conditions. In the case of C. heintzii, μ_max was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h⁻¹ C. heintzii grew immediately with a μ_max of 0.17±0.03 h⁻¹. After five transfers in batch culture, μ_max had increased only slightly to 0.18±0.03 h⁻¹. A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D=0.075 h⁻¹ into glucose-excess batch medium E. coli grew only after an acceleration phase of ∼3.5 h with a μ_max of 0.52 h⁻¹. After 120 generations and several transfers into fresh medium, μ_max had increased to 0.80±0.03 h⁻¹. For long-term adapted chemostat-cultivated cells, a K_s for glucose of 15 μg l⁻¹ for C. heintzii, and of 35 μg l⁻¹ for E. coli, respectively, was determined in ¹⁴C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h⁻¹. Using the determined pure culture parameter values for K_s and μ_max, it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s_min). The values estimated for s_min were dependent on growth rate; at D=0.05 h⁻¹, it was 12.6 and 0 μg l⁻¹ for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h⁻¹, s_min for E. coli had to be raised to 34.9 μg l⁻¹ whereas s_min for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K_s value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s_min.     

A test for plasticity in sperm motility activation in response to osmotic environment in an anuran amphibian

Evolutionary theory predicts that selection will favor phenotypic plasticity in sperm traits that maximize fertilization success in dynamic fertilization environments. In species with external fertilization, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but evidence for osmotic‐induced sperm plasticity is limited to euryhaline fish and marine invertebrates. Whether this capacity extends to freshwater taxa remains unknown. Here, we provide the first test for plasticity in sperm‐motility activation in response to osmotic environment in an anuran amphibian. Male common eastern froglets (Crinia signifera) were acclimated to either low (0 mOsmol kg⁻¹) or high (50 mOsmol kg⁻¹) environmental osmolality, and using a split‐sample experimental design, sperm were activated across a range of osmolality treatments (0, 25, 50, 75, 100, and 200 ± 2 mOsmol kg⁻¹). Unexpectedly, there was no detectable shift in the optimal osmolality for sperm‐motility activation after approximately 13 weeks of acclimation (a period reflecting the duration of the winter breeding season). However, in both the low and high acclimation treatments, the optimal osmolality for sperm‐motility activation mirrored the osmolality at the natural breeding site, indicating a phenotypic match to the local environment. Previously it has been shown that C. signifera display among‐population covariation between environmental osmolality and sperm performance. Coupled with this finding, the results of the present study suggest that inter‐population differences reflect genetic divergence and local adaptation. We discuss the need for experimental tests of osmotic‐induced sperm plasticity in more freshwater taxa to better understand the environmental and evolutionary contexts favoring adaptive plasticity in sperm‐motility activation.     

In vitro antioxidant capacity and free radical scavenging evaluation of active metabolite constituents of Newbouldia laevis ethanolic leaf extract

Background

The aim of the present study was to evaluate the in vitro antioxidant and free radical scavenging capacity of bioactive metabolites present in Newbouldia laevis leaf extract.

Results

Chromatographic and spectrophotometric methods were used in the study and modified where necessary in the study. Bioactivity of the extract was determined at 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 400 μg/ml concentrations expressed in % inhibition. The yield of the ethanolic leaf extract of N.laevis was 30.3 g (9.93%). Evaluation of bioactive metabolic constituents gave high levels of ascorbic acid (515.53 ± 12 IU/100 g [25.7 mg/100 g]), vitamin E (26.46 ± 1.08 IU/100 g), saponins (6.2 ± 0.10), alkaloids (2.20 ± 0.03), cardiac glycosides(1.48 ± 0.22), amino acids and steroids (8.01 ± 0.04) measured in mg/100 g dry weight; moderate levels of vitamin A (188.28 ± 6.19 IU/100 g), tannins (0.09 ± 0.30), terpenoids (3.42 ± 0.67); low level of flavonoids (1.01 ± 0.34 mg/100 g) and absence of cyanogenic glycosides, carboxylic acids and aldehydes/ketones. The extracts percentage inhibition of DPPH, hydroxyl radical (OH^.), superoxide anion (O₂^.-), iron chelating, nitric oxide radical (NO), peroxynitrite (ONOO⁻), singlet oxygen (¹O₂), hypochlorous acid (HOCl), lipid peroxidation (LPO) and FRAP showed a concentration-dependent antioxidant activity with no significant difference with the controls. Though, IC₅₀ of the extract showed significant difference only in singlet oxygen (¹O₂) and iron chelating activity when compared with the controls.

Conclusions

The extract is a potential source of antioxidants/free radical scavengers having important metabolites which maybe linked to its ethno-medicinal use.     

Anion inhibition studies of the Zn(II)-bound ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii

Burkholderia territorii, a Gram-negative bacterium, encodes for the ι-class carbonic anhydrase (CA, EC 4.2.1.1) BteCAι, which was recently characterised. It acts as a good catalyst for the hydration of CO₂ to bicarbonate and protons, with a k_cat value of 3.0 × 10⁵ s⁻¹ and k_cat/K_M value of 3.9 × 10⁷ M⁻¹ s⁻¹. No inhibition data on this new class of enzymes are available to date. We report here an anion and small molecules inhibition study of BteCAι, which we prove to be a zinc(II)- and not manganese(II)-containing enzyme, as reported for diatom ι-CAs. The best inhibitors were sulphamic acid, stannate, phenylarsonic acid, phenylboronic acid and sulfamide (K_I values of 6.2–94 µM), whereas diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors (K_I values of 0.71–0.94 mM). The halides (except iodide), thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed K_I values in the range of 3.1–9.3 mM.     

Prevalence of Histidine Decarboxylase Genes of Gram-Positive Bacteria in Surströmming as Revealed by qPCR

Histamine is a degradation product of the bacterial decarboxylation of the amino acid histidine; such activity is determined by histidine decarboxylase encoded by a gene cluster, carried by some Gram-positive bacteria, that includes the hdcA gene. In this study, the presence of the hdcA gene in ready-to-eat surströmming samples collected from three producers based in Sweden was directly assessed via qPCR analysis for the very first time. Samples from producer A showed hdcA average gene abundance of 6.67 ± 0.13 Log cells/gene copies g⁻¹; in samples from producer B the average value attested at 5.56 ± 0.06 Log cells/gene copies g⁻¹, whereas for samples of producer C hdcA average gene abundance attested at 5.30 ± 0.08 Log cells/gene copies g⁻¹. ANOVA showed a significantly higher average hdcA gene copy number in samples from producer A, whereas no significant differences were seen between average values of hdcA gene copy numbers detected in samples from producer B and C. The hdcA gene copies detected in the present study could give an estimation of the load of potential histamine-producing bacteria in surströmming.     

Corals shed bacteria as a potential mechanism of resilience to organic matter enrichment

Understanding the mechanisms of resilience of coral reefs to anthropogenic stressors is a critical step toward mitigating their current global decline. Coral–bacteria associations are fundamental to reef health and disease, but direct observations of these interactions remain largely unexplored. Here, we use novel technology, high-speed laser scanning confocal microscopy on live coral (Pocillopora damicornis), to test the hypothesis that corals exert control over the abundance of their associated bacterial communities by releasing (‘shedding'') bacteria from their surface, and that this mechanism can counteract bacterial growth stimulated by organic inputs. We also test the hypothesis that the coral pathogen Vibrio coralliilyticus can evade such a defense mechanism. This first report of direct observation with high-speed confocal microscopy of living coral and its associated bacterial community revealed a layer (3.3–146.8 μm thick) on the coral surface where bacteria were concentrated. The results of two independent experiments showed that the bacterial abundance in this layer was not sensitive to enrichment (5 mg l⁻¹ peptone), and that coral fragments exposed to enrichment released significantly more bacteria from their surfaces than control corals (P<0.01; 35.9±1.4 × 10⁵ cells cm⁻² coral versus 1.3±0.5 × 10⁵ cells cm⁻² coral). Our results provide direct support to the hypothesis that shedding bacteria may be an important mechanism by which coral-associated bacterial abundances are regulated under organic matter stress. Additionally, the novel ability to watch this ecological behavior in real-time at the microscale opens an unexplored avenue for mechanistic studies of coral–microbe interactions.     

The basis of antagonistic pleiotropy in hfq mutations that have opposite effects on fitness at slow and fast growth rates

Mutations beneficial in one environment may cause costs in different environments, resulting in antagonistic pleiotropy. Here, we describe a novel form of antagonistic pleiotropy that operates even within the same environment, where benefits and deleterious effects exhibit themselves at different growth rates. The fitness of hfq mutations in Escherichia coli affecting the RNA chaperone involved in small-RNA regulation is remarkably sensitive to growth rate. E. coli populations evolving in chemostats under nutrient limitation acquired beneficial mutations in hfq during slow growth (0.1 h⁻¹) but not in populations growing sixfold faster. Four identified hfq alleles from parallel populations were beneficial at 0.1 h⁻¹ and deleterious at 0.6 h⁻¹. The hfq mutations were beneficial, deleterious or neutral at an intermediate growth rate (0.5 h⁻¹) and one changed from beneficial to deleterious within a 36 min difference in doubling time. The benefit of hfq mutations was due to the greater transport of limiting nutrient, which diminished at higher growth rates. The deleterious effects of hfq mutations at 0.6 h⁻¹ were less clear, with decreased viability a contributing factor. The results demonstrate distinct pleiotropy characteristics in the alleles of the same gene, probably because the altered residues in Hfq affected the regulation of expression of different genes in distinct ways. In addition, these results point to a source of variation in experimental measurement of the selective advantage of a mutation; estimates of fitness need to consider variation in growth rate impacting on the magnitude of the benefit of mutations and on their fitness distributions.     

Effect of amino acids and amines on the activity of the recombinant ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii

We here report a study on the activation of the ι-class bacterial CA from Burkholderia territorii (BteCAι). This protein was recently characterised as a zinc-dependent enzyme that shows a significant catalytic activity (k_cat 3.0 × 10⁵ s⁻¹) for the physiological reaction of CO₂ hydration to bicarbonate and protons. Some amino acids and amines, among which some proteinogenic derivatives as well as histamine, dopamine and serotonin, showed efficient activating properties towards BteCAι, with activation constants in the range 3.9–13.3 µM. L-Phe, L-Asn, L-Glu, and some pyridyl-alkylamines, showed a weaker activating effect towards BteCAι, with K_A values ranging between 18.4 µM and 45.6 µM. Nowadays, no information is available on active site architecture, metal ion coordination and catalytic mechanism of members of the ι-group of CAs, and this study represents another contribution towards a better understanding of this still uncharacterised class of enzymes.     

Association of TNF-alpha,IL-6 and IL-1beta gene polymorphisms with polycystic ovary syndrome: a meta-analysis

Background

Several studies on the association of TNF-alpha (−308 G/A), IL-6 (−174 G/C) and IL-1beta (−511 C/T) polymorphisms with polycystic ovary syndrome (PCOS) risk have reported conflicting results. The aim of the present study was to assess these associations by meta-analysis.

Results

A total of 14 eligible articles (1665 cases/1687 controls) were included in this meta-analysis. The results suggested that there was no obvious association between the TNF-alpha (−308 G/A) polymorphism and PCOS in the overall population or subgroup analysis by ethnicity, Hardy–Weinberg equilibrium (HWE) in controls, genotyping method, PCOS diagnosis criteria, and study sample size. Also, no obvious association was found between the TNF-alpha (−308 G/A) polymorphism and obesity in patients with PCOS (body mass index [BMI] ≥ 25 kg/m² vs. BMI < 25 kg/m²). Regarding the IL-6 (−174 G/C) polymorphism, also no association was found in the overall population in heterozygote comparison, dominant model, and recessive model. Even though an allelic model (odds ratio [OR] = 0.63, 95% confidence interval [CI] = 0.41–0.96) and a homozygote comparison (OR = 0.52, 95% CI = 0.30–0.93) showed that the IL-6 (−174 G/C) polymorphism was marginally associated with PCOS. Further subgroup analysis suggested that the effect size was not significant among HWE in controls (sample size ≤ 200) and genotyping method of pyrosequencing under all genetic models. Similarly, there was no association between the IL-1beta (−511 C/T) polymorphism and PCOS in the overall population or subgroup analysis under all genetic models. Furthermore, no significant association was found between the IL-1beta (−511 C/T) polymorphism and several clinical and biochemical parameters in patients with PCOS.

Conclusions

The results of this meta-analysis suggest that the TNF-alpha (−308 G/A), IL-6 (−174 G/C), and IL-1beta (−511 C/T) polymorphisms may not be associated with PCOS risk. However, further case–control studies with larger sample sizes are needed to confirm our results.

Electronic supplementary material

The online version of this article (doi:10.1186/s12863-015-0165-4) contains supplementary material, which is available to authorized users.     

Evaluation of the mutagenicity and antimutagenicity of Ziziphus joazeiro Mart. bark in the micronucleus assay

The aim of this study was to evaluate the mutagenicity (clastogenicity/aneugenicity) of a glycolic extract of Ziziphus joazeiro bark (GEZJ) by the micronucleus assay in mice bone marrow. Antimutagenic activity was also assessed using treatments associated with GEZJ and doxorubicin (DXR). Mice were evaluated 24–48 h after exposure to positive (N-nitroso-N-ethylurea, NEU - 50 mg.kg⁻¹ and DXR - 5 mg.kg⁻¹) and negative (150 mM NaCl) controls, as well as treatment with GEZJ (0.5–2 g.kg⁻¹), GEZJ (2 g.kg⁻¹) + NEU and GEZJ (2 g.kg⁻¹) + DXR. There were no significant differences in the frequencies of micronucleated polychromatic erythrocytes in mice treated with GEJZ and GEJZ + DXR compared to the negative controls, indicating that GEZJ was not mutagenic. Analysis of the polychromatic:normochromatic erythrocyte ratio revealed significant differences in the responses to doses of 0.5 g.kg⁻¹ and 1–2 g.kg⁻¹ and the positive control (NEU). These results indicated no systemic toxicity and moderate toxicity at lower and higher doses of GEZJ. The lack of mutagenicity and systemic toxicity in the antimutagenic assays, especially for treatment with GEZJ + DXR, suggested that phytochemical compounds in Z. joazeiro bark attenuated DXR-induced mutagenicity and the moderate systemic toxicity of a high dose of Z. joazeiro bark (2 g.kg⁻¹). Further studies on the genotoxicity of Z. joazeiro extracts are necessary to establish the possible health risk in humans and to determine the potential as a chemopreventive agent for therapeutic use.     

Improving key enzyme activities and quality of rice under various methods of zinc application

Zinc (Zn) is an important micronutrient for the physiology of plants. It is poorly available to the plants in soil solution. A pot experiment was conducted to evaluate effectiveness of various Zn application methods on key enzyme activities and protein content of two contrasting rice genotypes viz., PD16 (Zn efficient) and NDR359 (Zn inefficient). The treatments were, control (0 mg Zn kg⁻¹ soil), soil application (5 mg Zn kg⁻¹ soil), foliar application (0.5 % ZnSO₄ + 0.25 % lime at 30, 60 and 90 days after transplanting), soil (5 mg Zn kg⁻¹ soil) + foliar application of 0.5 % ZnSO₄ + 0.25 % lime at 30, 60 and 90 days after transplanting. Among all the methods tested soil+foliar application of Zn fertilizers was found most effective in increasing superoxide dismutase (SOD) and carbonic anhydrase (CA) activities as well as chlorophyll and protein content in both the rice varieties. NDR359, showed higher enzyme activities and more chlorophyll content in leaves than PD16, when Zn was applied either through foliar spray alone or in soil along with foliar application. Regarding the protein content in grains, PD16 showed higher protein content than NDR359, thus showed better translocation of Zn from leaves to grains.     

Butyric acid fermentation from pretreated and hydrolysed wheat straw by an adapted Clostridium tyrobutyricum strain

Butyric acid is a valuable building-block for the production of chemicals and materials and nowadays it is produced exclusively from petroleum. The aim of this study was to develop a suitable and robust strain of Clostridium tyrobutyricum that produces butyric acid at a high yield and selectivity from lignocellulosic biomasses. Pretreated (by wet explosion) and enzymatically hydrolysed wheat straw (PHWS), rich in C6 and C5 sugars (71.6 and 55.4 g l⁻¹ of glucose and xylose respectively), was used as substrate. After one year of serial selections, an adapted strain of C. tyrobutyricum was developed. The adapted strain was able to grow in 80% (v v⁻¹) PHWS without addition of yeast extract compared with an initial tolerance to less than 10% PHWS and was able to ferment both glucose and xylose. It is noticeable that the adapted C. tyrobutyricum strain was characterized by a high yield and selectivity to butyric acid. Specifically, the butyric acid yield at 60–80% PHWS lie between 0.37 and 0.46 g g⁻¹ of sugar, while the selectivity for butyric acid was as high as 0.9–1.0 g g⁻¹ of acid. Moreover, the strain exhibited a robust response in regards to growth and product profile at pH 6 and 7.     

An efficient plant regeneration system through callus for Pseudarthria viscida (L.) Wright and Arn., a rare ethnomedicinal herb

The purpose of this study was to develop a protocol to induce high frequency of callus and subsequent plantlet regeneration for Pseudarthria viscida; an important medicinal plant. The cotyledonary node and young leaf pieces (1 × 0.5 cm, length × breadth) were used as explants for callus induction and subsequent shoot regeneration and adventitious roots induction from the shoots. The best results were achieved on the following media: (1) 96 % callus induction from cotyledonary node explants on MS medium supplemented with 1.5 mgl⁻¹ 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgl⁻¹ 1-naphthalene acetic acid (NAA), (2) 97 % shoot regeneration from cotyledonary node derived calli with an average of 44.9 shoots per explant on MS medium fortified with 3.0 mgl⁻¹ N₆-benzylaminopurine (BA) and 1 mgl⁻¹ NAA,37 (3) 98 % rooting with an average number of 3.3 roots per shoot on MS medium containing indole-3-butyric acid (IBA) or NAA (0.5–4 mgl⁻¹) after 45 days. The plantlets were transferred to the field after acclimatization. Of the 40 plantlets transplanted to the soil, 29 survived (72.5 %).     

Pan-genome dynamics of Pseudomonas gene complements enriched across hexachlorocyclohexane dumpsite

Background

Phylogenetic heterogeneity across Pseudomonas genus is complemented by its diverse genome architecture enriched by accessory genetic elements (plasmids, transposons, and integrons) conferring resistance across this genus. Here, we sequenced a stress tolerant genotype i.e. Pseudomonas sp. strain RL isolated from a hexachlorocyclohexane (HCH) contaminated pond (45 mg of total HCH g⁻¹ sediment) and further compared its gene repertoire with 17 reference ecotypes belonging to P. stutzeri, P. mendocina, P. aeruginosa, P. psychrotolerans and P. denitrificans, representing metabolically diverse ecosystems (i.e. marine, clinical, and soil/sludge). Metagenomic data from HCH contaminated pond sediment and similar HCH contaminated sites were further used to analyze the pan-genome dynamics of Pseudomonas genotypes enriched across increasing HCH gradient.

Results

Although strain RL demonstrated clear species demarcation (ANI ≤ 80.03%) from the rest of its phylogenetic relatives, it was found to be closest to P. stutzeri clade which was further complemented functionally. Comparative functional analysis elucidated strain specific enrichment of metabolic pathways like α-linoleic acid degradation and carbazole degradation in Pseudomonas sp. strain RL and P. stutzeri XLDN-R, respectively. Composition based methods (%codon bias and %G + C difference) further highlighted the significance of horizontal gene transfer (HGT) in evolution of nitrogen metabolism, two-component system (TCS) and methionine metabolism across the Pseudomonas genomes used in this study. An intact mobile class-I integron (3,552 bp) with a captured gene cassette encoding for dihydrofolate reductase (dhfra1) was detected in strain RL, distinctly demarcated from other integron harboring species (i.e. P. aeruginosa, P. stutzeri, and P. putida). Mobility of this integron was confirmed by its association with Tnp21-like transposon (95% identity) suggesting stress specific mobilization across HCH contaminated sites. Metagenomics data from pond sediment and recently surveyed HCH adulterated soils revealed the in situ enrichment of integron associated transposase gene (TnpA6100) across increasing HCH contamination (0.7 to 450 mg HCH g⁻¹ of soil).

Conclusions

Unlocking the potential of comparative genomics supplemented with metagenomics, we have attempted to resolve the environment and strain specific demarcations across 18 Pseudomonas gene complements. Pan-genome analyses of these strains indicate at astoundingly diverse metabolic strategies and provide genetic basis for the cosmopolitan existence of this taxon.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1488-2) contains supplementary material, which is available to authorized users.