Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.     

The socioecological complexity of ecological restoration in Mexico

Almost half of Mexican territory has been classified as environmentally degraded. The main response for the last 60 years has been reforestation to combat soil erosion and loss of forest cover, mostly carried out on private lands where negotiations with local stakeholders were critical. Despite four legal instruments referring to ecological restoration, no specific instrument that defines basic concepts, criteria and standards, required actions, or regulations to implement and evaluate ecological restoration exists. The Ministry of the Environment and Natural Resources is now solely in charge of restoration and only recently have external scientists been invited to be part of the process. Following important national and international events in Latin America and the Caribbean region, the First Mexican Symposium on Ecological Restoration was held in November, 2014. This historic event was the first action undertaken in Mexico to meet Objective 3 of the Global Strategy of Plant Conservation, coordinated in Mexico by the National Council for the Use and Knowledge of Biodiversity. Although mangrove ecosystems are the most endangered ecosystem type in Mexico, they were not well represented at the symposium. In contrast, several other ecosystem types, such as tropical dry forest and islands, have received increased attention. Overall, while the Symposium and above‐cited policy initiatives are important steps, Mexico needs to increase its institutional capacities and social organization of the rural sector with regard to ecological restoration. Better integration of social and natural scientists and increased participation of Mexico internationally is also needed.     

Morphometric variables related to metabolic profile in captive chimpanzees (Pan troglodytes)

Obesity is a risk factor for several diseases including type 2 diabetes and cardiovascular disease. The aim of this study was to compare the relationships of waist circumference and body weight with circulating markers of metabolic, cardiovascular, and hepatic function in chimpanzees (Pan troglodytes). After a 12-h fast, blood was collected from 39 adult captive chimpanzees for measurement of serum glucose, BUN, creatinine, albumin, cholesterol, ALT, AST, ALP, total and direct bilirubin, triglyceride, and insulin, and waist circumference and body weight were measured. Waist circumference was positively correlated with systolic and diastolic blood pressure, glucose, insulin resistance as estimated by the homeostatic model assessment method, and albumin in female chimpanzees and with triglyceride in female and male chimpanzees. Body weight was correlated significantly with systolic and diastolic blood pressure in female chimpanzees and triglyceride in male chimpanzees. Male chimpanzees were heavier and had lower diastolic blood pressure, greater creatinine, albumin, AST, ALP, total bilirubin, and direct bilirubin values than did female chimpanzees. The relationships between waist circumference and blood pressure and triglyceride are consistent with those reported in humans and other primate species. In conclusion, our study is the first work to demonstrate a relationship between waist circumference and metabolic risk factors in chimpanzees. Results demonstrated that waist circumference was associated with more metabolic risk factors than was body weight, particularly in female chimpanzees.     

Adaptive evolution of nontransgenic <Emphasis Type="Italic">Escherichia coli</Emphasis> KC01 for improved ethanol tolerance and homoethanol fermentation from xylose

Due to its excellent capability to ferment five-carbon sugars, Escherichia coli has been considered one of the platform organisms to be engineered for production of cellulosic ethanol. Nevertheless, genetically engineered ethanologenic E. coli lacks the essential trait of alcohol tolerance. Development of ethanol tolerance is required for cost-effective ethanol fermentation. In this study, we improved alcohol tolerance of a nontransgenic E. coli KC01 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) through adaptive evolution. During ~350 generations of adaptive evolution, a gradually increased concentration of ethanol was used as a selection pressure to enrich ethanol-tolerant mutants. The evolved mutant, E. coli SZ470, was able to grow anaerobically at 40 g l⁻¹ ethanol, a twofold improvement over parent KC01. When compared with KC01 for small-scale (500 ml) xylose (50 g l⁻¹) fermentation, SZ470 achieved 67% higher cell mass, 48% faster volumetric ethanol productivity, and 50% shorter time to complete fermentation with ethanol titer of 23.5 g l⁻¹ and yield of 94%. These results demonstrate that an industry-oriented nontransgenic E. coli strain could be developed through incremental improvements of desired traits by a combination of molecular biology and traditional microbiology techniques.     

Discovery and characterization of single nucleotide polymorphisms in Chinook salmon, Oncorhynchus tshawytscha

Molecular population genetics of non-model organisms has been dominated by the use of microsatellite loci over the last two decades. The availability of extensive genomic resources for many species is contributing to a transition to the use of single nucleotide polymorphisms (SNPs) for the study of many natural populations. Here we describe the discovery of a large number of SNPs in Chinook salmon, one of the world's most important fishery species, through large-scale Sanger sequencing of expressed sequence tag (EST) regions. More than 3 Mb of sequence was collected in a survey of variation in almost 132 kb of unique genic regions, from 225 separate ESTs, in a diverse ascertainment panel of 24 salmon. This survey yielded 117 TaqMan (5' nuclease) assays, almost all from separate ESTs, which were validated in population samples from five major stocks of salmon from the three largest basins on the Pacific coast of the contiguous United States: the Sacramento, Klamath and Columbia Rivers. The proportion of these loci that was variable in each of these stocks ranged from 86.3% to 90.6% and the mean minor allele frequency ranged from 0.194 to 0.236. There was substantial differentiation between populations with these markers, with a mean F(ST) estimate of 0.107, and values for individual loci ranging from 0 to 0.592. This substantial polymorphism and population-specific differentiation indicates that these markers will be broadly useful, including for both pedigree reconstruction and genetic stock identification applications.     

The relA homolog of Mycobacterium smegmatis affects cell appearance, viability, and gene expression

The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel(Msm). The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.     

Plant gamma-glutamyl hydrolases and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles

gamma-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low K(m) values (0.5-2 microm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with approximately 50 and approximately 10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16-60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack.     

Bactericidal effect of bovine lactoferrin, LFcin, LFampin and LFchimera on antibiotic-resistant Staphylococcus aureus and Escherichia coli

Increased prevalence of antibiotic-resistant bacteria has become a major threat to the health sector worldwide due to their virulence, limited therapeutic options and distribution in both hospital and community settings. Discovery and development of new agents to combat antibiotic-resistant bacteria is thus needed. This study therefore aimed to evaluate the ability of bovine lactoferrin (LF), peptides from two antimicrobial domains lactoferricin B (LFcin17-30) and lactoferrampin (LFampin265-284) and a chimeric construct (LFchimera) containing both peptides, as potential bactericidal agents against clinical isolates of antibiotic-resistant Staphylococcus aureus and Escherichia coli. Results in kinetics of growth show that LF chimera and peptides inhibited the growth of both bacterial species. By confocal microscopy and flow cytometry it was observed that LF and FITC-labeled peptides are able to interact with these bacteria and cause membrane permeabilization, as monitored by propidium iodide staining, these effects were decreased by preincubation with lipopolysaccharide in E. coli. By electron microscopy, a clear cellular damage was observed in bacteria after treatments with LFchimera and peptides, suggesting that interaction and membrane disruption are probably involved as a mechanism of action. In conclusion, results show that LFchimera, LF and peptides have potential as bactericidal agents in the antibiotic-resistant strains of S. aureus and E. coli and also the work strongly suggest that LFcin17-30 and LFampin265-284 acts synergistically with antibiotics against multidrug resistant EPEC and MRSA in vitro.