Targeting the role of a key conserved motif for substrate selection and catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.     

Evaluation of genetic isolation within an island flora reveals unusually widespread local adaptation and supports sympatric speciation

It is now recognized that speciation can proceed even when divergent natural selection is opposed by gene flow. Understanding the extent to which environmental gradients and geographical distance can limit gene flow within species can shed light on the relative roles of selection and dispersal limitation during the early stages of population divergence and speciation. On the remote Lord Howe Island (Australia), ecological speciation with gene flow is thought to have taken place in several plant genera. The aim of this study was to establish the contributions of isolation by environment (IBE) and isolation by community (IBC) to the genetic structure of 19 plant species, from a number of distantly related families, which have been subjected to similar environmental pressures over comparable time scales. We applied an individual-based, multivariate, model averaging approach to quantify IBE and IBC, while controlling for isolation by distance (IBD). Our analyses demonstrated that all species experienced some degree of ecologically driven isolation, whereas only 12 of 19 species were subjected to IBD. The prevalence of IBE within these plant species indicates that divergent selection in plants frequently produces local adaptation and supports hypotheses that ecological divergence can drive speciation in sympatry.     

Emergent Global Patterns of Ecosystem Structure and Function from a Mechanistic General Ecosystem Model

Anthropogenic activities are causing widespread degradation of ecosystems worldwide, threatening the ecosystem services upon which all human life depends. Improved understanding of this degradation is urgently needed to improve avoidance and mitigation measures. One tool to assist these efforts is predictive models of ecosystem structure and function that are mechanistic: based on fundamental ecological principles. Here we present the first mechanistic General Ecosystem Model (GEM) of ecosystem structure and function that is both global and applies in all terrestrial and marine environments. Functional forms and parameter values were derived from the theoretical and empirical literature where possible. Simulations of the fate of all organisms with body masses between 10 µg and 150,000 kg (a range of 14 orders of magnitude) across the globe led to emergent properties at individual (e.g., growth rate), community (e.g., biomass turnover rates), ecosystem (e.g., trophic pyramids), and macroecological scales (e.g., global patterns of trophic structure) that are in general agreement with current data and theory. These properties emerged from our encoding of the biology of, and interactions among, individual organisms without any direct constraints on the properties themselves. Our results indicate that ecologists have gathered sufficient information to begin to build realistic, global, and mechanistic models of ecosystems, capable of predicting a diverse range of ecosystem properties and their response to human pressures.     

The crystal structure of pyrimidine/thiamin biosynthesis precursor-like domain-containing protein CAE31940 from proteobacterium Bordetella bronchiseptica RB50, and evolutionary insight into the NMT1/THI5 family

We report a 2.0 Å structure of the CAE31940 protein, a proteobacterial NMT1/THI5-like domain-containing protein. We also discuss the primary and tertiary structure similarity with its homologs. The highly conserved FGGXMP motif was identified in CAE31940, which corresponds to the GCCCX motif located in the vicinity of the active center characteristic for THi5-like proteins found in yeast. This suggests that the FGGXMP motif may be a unique hallmark of proteobacterial NMT1/THI5-like proteins.     

Diverse lineages of pathogenic Leptospira species are widespread in the environment in Puerto Rico,USA

BackgroundLeptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology.Methodology/Principal findingsWe collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro.We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup.Conclusions/SignificanceDiverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics.     

Hymenolepis diminuta: nuclear magnetic resonance analysis of the excretory products resulting from the metabolism of D-[13C6]glucose

Excretory products of the tapeworm Hymenolepis diminuta, fed D-[13C6]glucose in vitro for 90 min, were studied using 1H and 13C nuclear magnetic resonance spectroscopy. Signals due to lactate, succinate, acetate, and alanine were identified in the spectra. Several differently labeled species were present for these metabolites; the variations of higher concentration were a consequence of metabolic factors while those of lower concentration could be accounted for by residual 12C in the glucose. The two major labeled lactates, U-13C and 2,3-13C2, were in the ratio 2:1, respectively, and the three major labeled succinates, 1,2,2'-13C3,2,2'-13C2, and U-13C, were present in the ratio 20:10:3, respectively. The different species of labeled end products are related to the overall glucose metabolism of H. diminuta.     

A polydisperse linear random coil model for the quaternary structure of pig colonic mucin

The distribution of molecular weights for polymeric colonic mucus glycoprotein or ``mucin' isolated and solubilised in the presence of protease inhibitors from pig colons is shown to be considerably greater than its ``subunit' (thiol reduction product) and papain digested forms using the technique of size-exclusion chromatography coupled to multi-angle laser light scattering, and confirmed by sedimentation equilibrium measurements. The conformation of this mucin is probed by examining the molecular weight – intrinsic viscosity relationship in terms of the Mark-Houwink-Kuhn-Sakurada analysis for its polymeric (or ``whole'), reduced and papain-digested forms: an exponent ``a' of (1.1±0.1) is obtained indicating a linear random coil conformation consistent with other mucins. Size-exclusion chromatography coupled to multi-angle laser light scattering is shown to provide a relatively simple complementary technique to sedimentation equilibrium for the molecular weight distribution analysis of polydisperse materials. Received: 29 November 1996 / Accepted: 2 December 1996     

Biotelemetry monitoring of translocated Crocodylus niloticus in Lake Ngezi, Zimbabwe

Radiotelemetry was used from June 1986 through February 1987 to observe the movements of four Nile crocodiles, Crocodylus niloticus Laurenti, introduced into a resident population at Lake Ngezi, Zimbabwe. The use of surgically-implanted transmitters was the first application of this technique on wild Nile crocodiles. A subadult female (194 cm TL, 35 kg) and two males (256 cm, 81 kg; 348 cm, 260 kg) wandered throughout the area. A large female (297 cm, 149 kg) moved > 12 km from her point of release to Ngezi River and nested; she guarded the nest site through 21 January 1987 when she moved the hatchlings to a nearby creche. A juvenile crocodile (120 cm TL, 8 kg) captured in Lake Ngezi was also monitored from June 1986 to February 1987. It traversed some 15 km of the shoreline of Lake Ngezi and moved as much as 10 km overnight. Evidence is presented that the translocated crocodiles, excluding the nesting female, exhibited exploratory behaviour throughout the nine-month observation period, and did not establish home ranges per se.