Identity and functional characterisation of the transporter supporting the Na+-dependent high-affinity NO3− uptake in Zostera marina L.

Zostera marina is a seagrass, a group of angiosperms that evolved from land to live submerged in seawater, an environment of high salinity, alkaline pH and usually very low NO3−. In 2000, we reported the first physiological evidence for the Na+-dependent high-affinity NO3− uptake in this plant. Now, to determine the molecular identity of this process, we searched for NO3− transporters common to other vascular plants encoded in Z. marina's genome. We cloned two candidates, ZosmaNPF6.3 and ZosmaNRT2 with its partner protein ZosmaNAR2. ZosmaNAR2 expression levels increase up to 4.5-fold in Z. marina leaves under NO3−-deficiency, while ZosmaNRT2 and ZosmaNPF6.3 expressions were low and unaffected by NO3−. NO3− transport capacity, kinetic properties and H+ or Na+-dependence were examined by heterologous expression in the Hansenula polymorpha high-affinity NO3− transporter gene disrupted strain (∆ynt1). ZosmaNPF6.3 functions as a H+-dependent NO3− transporter, without functionality at alkaline pH and apparent dual kinetics (KM = 11.1 µM at NO3− concentrations below 50 µM). ZosmaNRT2 transports NO3− in a H+-independent but Na+-dependent manner (KM = 1 mM Na+), with low NO3− affinity (KM = 30 µM). When ZosmaNRT2 and ZosmaNAR2 are co-expressed, a Na+-dependent high-affinity NO3− transport occurs (KM = 5.7 µM NO3−), mimicking the in vivo value. These results are discussed in the physiological context, providing evidence that ZosmaNRT2 is a Na+-dependent high-affinity NO3− transporter, the first of its kind to be functionally characterised in a vascular plant, that requires ZosmaNAR2 to achieve the necessary high-affinity for nitrate uptake from seawater.Research Funds of Malaga University, Grant/Award Number: 0837002020 B4‐2021‐08; Andalusia Regional Government, Grant/Award Number: GLOCOMA‐FEDER‐UCA 18‐107243; Funding for open access charge: Universidad de Málaga / CBUA

Effectiveness of Fosfomycin for the Treatment of Multidrug-Resistant Escherichia coli Bacteremic Urinary Tract Infections

IMPORTANCE The consumption of broad-spectrum drugs has increased as a consequence of the spread of multidrug-resistant (MDR) Escherichia coli. Finding alternatives for these infections is critical, for which some neglected drugs may be an option. OBJECTIVE To determine whether fosfomycin is noninferior to ceftriaxone or meropenem in the targeted treatment of bacteremic urinary tract infections (bUTIs) due to MDR E coli. DESIGN, SETTING, AND PARTICIPANTS This multicenter, randomized, pragmatic, open clinical trial was conducted at 22 Spanish hospitals from June 2014 to December 2018. Eligible participants were adult patients with bacteremic urinary tract infections due to MDR E coli; 161 of 1578 screened patients were randomized and followed up for 60 days. Data were analyzed in May 2021. INTERVENTIONS Patients were randomized 1 to 1 to receive intravenous fosfomycin disodium at 4 g every 6 hours (70 participants) or a comparator (ceftriaxone or meropenem if resistant; 73 participants) with the option to switch to oral fosfomycin trometamol for the fosfomycin group or an active oral drug or pa renteral ertapenem for the comparator group after 4 days. MAIN OUTCOMES AND MEASURES The primary outcome was clinical and microbiological cure (CMC) 5 to 7 days after finalization of treatment; a noninferiority margin of 7% was considered. RESULTS Among 143 patients in the modified intention-to-treat population (median [IQR] age, 72 [62-81] years; 73 [51.0%] women), 48 of 70 patients (68.6%) treated with fosfomycin and 57 of 73 patients (78.1%) treated with comparators reached CMC (risk difference, -9.4 percentage points; 1-sided 95% CI, -21.5 to infinity percentage points; P = .10). While clinical or microbiological failure occurred among 10 patients (14.3%) treated with fosfomycin and 14 patients (19.7%) treated with comparators (risk difference, -5.4 percentage points; 1-sided 95% CI. -infinity to 4.9; percentage points; P = .19), an increased rate of adverse event-related discontinuations occurred with fosfomycin vs comparators (6 discontinuations [8.5%] vs 0 discontinuations; P = .006). In an exploratory analysis among a subset of 38 patients who underwent rectal colonization studies, patients treated with fosfomycin acquired a new ceftriaxone-resistant or meropenem-resistant gram-negative bacteria at a decreased rate compared with patients treated with comparators (0 of 21 patients vs 4 of 17 patients [23.5%]; 1-sided P = .01). CONCLUSIONS AND RELEVANCE This study found that fosfomycin did not demonstrate noninferiority to comparators as targeted treatment of bUTI from MDR E coli; this was due to an increased rate of adverse event-related discontinuations. This finding suggests that fosfomycin may be considered for selected patients with these infections

Newly discovered seed dispersal system of Juniperus cedrus questions the pristine nature of the high elevation scrub of El Teide (Tenerife, Canary Islands)

As a working hypothesis, we examined evidence for the former presence of a climacic woodland of Juniperus cedrus above the pine forest in the high elevation area of Tenerife (Canary Islands), which would indicate that the current dominant vegetation (endemic Spartocytisus supranubius scrub) may not be pristine. The main causes of the great regression of this woodland were caused by human activities (timber harvesting, herbivory by goats, and fires). The main support for this hypothesis is the survival of a presumably relict seed dispersal system of the endangered endemic J. cedrus, which relies mainly on the wintering thrush Turdus torquatus. The fact that genetic factors are directly involved in the control of bird migration routes strongly supports the idea that this interaction could be remnant of an older system, probably more widespread in the past. To test this hypothesis, we propose that a paleoecological approach could reconstruct the vegetation dynamics in the Teide National Park (Tenerife) and the past presence of this seed disperser migratory thrush. The analysis of plant microfossils in sediments (e.g., pollen, spores, phytoliths, coprolites, and charcoal) would allow us to evaluate whether the current vegetation is the same as that which naturally existed in the past, and assess the impact of the anthropogenic and natural factors to which it has been subjected during history. The results of these analyses will be useful for future management policies and practices aimed at restoring the pristine landscape and biotic interactions of the Teide National Park. To our knowledge, the case presented in this contribution, based on the high dependence of the seed dispersal of an endemic tree (J. cedrus) on a migratory bird, is the only reported in the context of oceanic islands.Ministerio de Ciencia e InnovaciónGobierno de las Islas CanariasOrganismo Autónomo de Parques Nacionale

One‐step, PCR‐mediated, gene disruption in the yeast Hansenula polymorpha

Previous evidence based on the experience of our laboratory showed that one‐step gene disruption in the yeast Hansenula polymorpha is not straightforward. A systematic study of several factors which could affect gene disruption frequency was carried out. We found that the more critical factor affecting one‐step gene disruption in H. polymorpha is the length of the target gene region flanking the marker gene. Target gene regions of about 1 kb flanking the marker gene were necessary to obtain a disruption frequency of about 50%. However, the gene marker, either homologous or heterologous, the locus and the strain examined did not significantly affect the frequency of disruption; the highest disruption frequency obtained for the YNR1 gene was in the strain HMI39, using the Saccharomyces cerevisiae URA3 gene as a marker. Since long regions flanking the gene marker do not allow the easy PCR‐mediated strategies, developed for S. cerevisiae, to obtain constructs to disrupt a given gene in H. polymorpha, an alternative PCR strategy was developed.This work was supported by Grant PB 97-1484from Dirección General de Investigación Científica y Técnica (DGICYT) to JMS.Peer reviewe

Evidence for multiple nitrate uptake systems in the yeast Hansenula polymorpha

Hansenula polymorpha mutants disrupted in the high-affinity nitrate transporter gene (YNT1) are still able to grow in nitrate. To detect the nitrate transporter(s) responsible for this growth a strain containing disruption of the nitrate assimilation gene cluster and expressing nitrate reductase gene (YNR1) under the control of H. polymorpha MOX1 (methanol oxidase) promoter was used (FM31 strain). In this strain nitrate taken up is transformed into nitrite by nitrate reductase and excreted to the medium where it is easily detected. Nitrate uptake which is neither induced by nitrate nor repressed by reduced nitrogen sources was detected in the FM31 strain. Likewise, nitrate uptake detected in the strain FM31 is independent of both Ynt1p and Yna1p and is not affected by ammonium, glutamine or chlorate. The inhibition of nitrite extrusion by extracellular nitrite suggests that the nitrate uptake system shown in the FM31 strain could also be involved in nitrite uptake.This work was supported by grants from the Dirección General de Investigación Científica y Técnica (P97-1484) and Gobierno de Canarias (PI1998) to J.M.S. G.P. and F.M. had fellowships from the Gobierno de Canarias.Peer reviewe

Tobacco Nia2 cDNA functionally complements a Hansenula polymorpha yeast mutant lacking nitrate reductase. A new expression system for the study of plant proteins involved in nitrate assimilation

An integrative expression vector based on promoter and terminator transcriptional sequences from the Hansenula polymorpha nitrate reductase gene (YNR1) has been developed to express nitrate assimilation plant genes in the nitrate assimilatory yeast H. polymorpha. Using this vector a plant nitrate reductase cDNA (tobacco Nia2) was expressed for the first time in a nitrate assimilatory yeast. The heterologous nitrate reductase produced retained its biochemical and physiological properties such as its NADH-dependent nitrate reductase activity, and allowed growth in nitrate containing media in a strain lacking endogenous nitrate reductase activity. In the transgenic strain, maximum tobacco nitrate reductase activity was about 70% of that presented in the wild-type. On the other hand, the disappearance of nitrate reductase activity correlated with that of the enzyme protein in response to the addition of ammonium to the medium and took place more rapidly in the transgenic strain than in the wild-type. Nitrate reductase activity of the recombinant strain assayed in the presence of Mg2+ was about 30% of that observed when assayed with EDTA. This result, together with a decreased growth rate in nitrate, suggests that tobacco nitrate reductase could be partially inactivated in H. polymorpha by phosphorylation and binding of 14-3-3-like proteins. These results show that H. polymorpha is a useful yeast heterologous expression system for studying plant proteins involved in nitrate assimilation.This work was supported by grants from the Ministerio de Ciencia y Tecnología (BMC2001-3719) and Gobierno de Canarias (PI2001/050) to J.M.S. G.P. and F.M. were recipients of predoctoral fellowships from the Gobierno de Canarias; P.T. and F.N. from Ministerio de Educación, Cultura y Deportes (Spain).Peer reviewe