Involvement of a Novel Class C Beta-Lactamase in the Transglutaminase Mediated Cross-Linking Cascade of Streptomyces mobaraensis DSM 40847

Streptomyces mobaraensis DSM 40847 secretes transglutaminase that cross-links proteins via γ-glutamyl-ε-lysine isopeptide bonds. Characterized substrates are inhibitory proteins acting against various serine, cysteine and metalloproteases. In the present study, the bacterial secretome was examined to uncover additional transglutaminase substrates. Fractional ethanol precipitation of the exported proteins at various times of culture growth, electrophoresis of the precipitated proteins, and sequencing of a 39 kDa protein by mass spectrometry revealed the novel beta-lactamase Sml-1. As indicated by biotinylated probes, Sml-1, produced in E. coli, exhibits glutamine and lysine residues accessible for transglutaminase. The chromogenic cephalosporin analogue, nitrocefin, was hydrolyzed by Sml-1 with low velocity. The obtained K_m and k_cat values of the recombinant enzyme were 94.3±1.8 μM and 0.39±0.03 s^-1, respectively. Penicillin G and ampicillin proved to be weak inhibitors of nitrocefin hydrolysis (K_i of 0.1 mM and 0.18 mM). Negligible influence of metals on β-lactamase activity ruled out that Sml-1 is a Zn²⁺-dependent class B beta-lactamase. Rather, sequence motifs such as SITK, YSN, and HDG forming the active core in a hypothetical structure may be typical for class C beta-lactamases. Based on the results, we assume that the novel transglutaminase substrate ensures undisturbed growth of aerial hyphae in Streptomyces mobaraensis by trapping and inactivating hostile beta-lactam antibiotics.     

Salt Intake and Health Risk in Climate Change Vulnerable Coastal Bangladesh: What Role Do Beliefs and Practices Play?

BackgroundHigh salt consumption is an important risk factor of elevated blood pressure. In Bangladesh about 20 million people are at high risk of hypertension due to climate change induced saline intrusion in water. The objective of this study is to assess beliefs, perceptions, and practices associated with salt consumption in coastal Bangladesh.MethodsThe study was conducted in Chakaria, Bangladesh between April-June 2011. It was a cross sectional mixed method study. For the qualitative study 6 focus group discussions, 8 key informant interviews, 60 free listing exercises, 20 ranking exercises and 10 observations were conducted. 400 adults were randomly selected for quantitative survey. For analysis we used SPSS for quantitative data, and Anthropac and Nvivo for qualitative data.ResultsSalt was described as an essential component of food with strong cultural and religious roots. People described both health benefits and risks related to salt intake. The overall risk perception regarding excessive salt consumption was low and respondents believed that the cooking process can render the salt harmless. Respondents were aware that salt is added in many foods even if they do not taste salty but did not recognize that salt can occur naturally in both foods and water.ConclusionsIn the study community people had low awareness of the risks associated with excess salt consumption and salt reduction strategies were not high in their agenda. The easy access to and low cost of salt as well as unrecognised presence of salt in drinking water has created an environment conducive to excess salt consumption. It is important to design general messages related to salt reduction and test tailored strategies especially for those at high risk of hypertension.     

ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking

COPI‐coated vesicles mediate retrograde membrane traffic from the cis‐Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER‐resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V‐dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI?Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.     

DNA damage induced by the anticodon nuclease from a Pichia acaciae killer strain is linked to ribonucleotide reductase depletion

Virus like element (VLE) encoded killer toxins of Pichia acaciae and Kluyveromyces lactis kill target cells through anticodon nuclease (ACNase) activity directed against tRNA^Gln and tRNA^Glu respectively. Not only does tRNA cleavage disable translation, it also affects DNA integrity as well. Consistent with DNA damage, which is involved in toxicity, target cells' mutation frequencies are elevated upon ACNase exposure, suggesting a link between translational integrity and genome surveillance. Here, we analysed whether ACNase action impedes the periodically and highly expressed S‐phase specific ribonucleotide reductase (RNR) and proved that RNR expression is severely affected by PaT. Because RNR catalyses the rate‐limiting step in dNTP synthesis, mutants affected in dNTP synthesis were scrutinized with respect to ACNase action. Mutations elevating cellular dNTPs antagonized the action of both the above ACNases, whereas mutations lowering dNTPs aggravated toxicity. Consistently, prevention of tRNA cleavage in elp3 or trm9 mutants, which both affect the wobble uridine modification of the target tRNA, suppressed the toxin hypersensitivity of a dNTP synthesis mutant. Moreover, dNTP synthesis defects exacerbated the PaT ACNase sensitivity of cells defective in homologous recombination, proving that dNTP depletion is responsible for subsequent DNA damage.     

Nutrient induced fluorescence transients (NIFTs) provide a rapid measure of P and C (co-)limitation in a green alga

Nutrient Induced Fluorescence Transients (NIFTs) have been shown to be a possible way of testing for the limiting nutrient in algal populations. In this study we tested the hypothesis that NIFTs can be used to detect a (co-)limitation for inorganic phosphorus (Pi) and CO₂ in the green alga Chlamydomonas acidophila and that the magnitude of the NIFTs can be related to cellular P:C ratios. We show a co-limitation response for Pi and CO₂ via traditional nutrient enrichment experiments in natural phytoplankton populations dominated by C. acidophila. We measured NIFT responses after a Pi- or a CO₂-spike in C. acidophila batch cultures at various stages of Pi and inorganic C limitation. Significant NIFTs were observed in response to spikes in both nutrients. The NIFT response to a Pi-spike showed a strong negative correlation with cellular P:C ratio that was pronounced below 3 mmol P: mol C (equivalent to 0.2 pg P cell^–1). Both cellular P and C content influenced the extent of the Pi-NIFT response. The NIFT response to a CO₂-spike correlated to low CO₂ culturing conditions and also had a negative correlation with cellular P content. A secondary response within the Pi-NIFT response was related to the CO₂ concentration and potentially reflected co-limitation. In conclusion, NIFTs provided a quick and reliable method to detect the growth-limiting nutrient in an extremophile green alga, under Pi-, CO_2- and Pi/CO₂ (co-)limited growth conditions.