Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product.

The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M(r) of 76,725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M(r) of the mature form is 75,000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676-1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.     

QTL Mapping of Agronomic Waterlogging Tolerance Using Recombinant Inbred Lines Derived from Tropical Maize (Zea mays L) Germplasm

Waterlogging is an important abiotic stress constraint that causes significant yield losses in maize grown throughout south and south-east Asia due to erratic rainfall patterns. The most economic option to offset the damage caused by waterlogging is to genetically incorporate tolerance in cultivars that are grown widely in the target agro-ecologies. We assessed the genetic variation in a population of recombinant inbred lines (RILs) derived from crossing a waterlogging tolerant line (CAWL-46-3-1) to an elite but sensitive line (CML311-2-1-3) and observed significant range of variation for grain yield (GY) under waterlogging stress along with a number of other secondary traits such as brace roots (BR), chlorophyll content (SPAD), % stem and root lodging (S&RL) among the RILs. Significant positive correlation of GY with BR and SPAD and negative correlation with S&RL indicated the potential use of these secondary traits in selection indices under waterlogged conditions. RILs were genotyped with 331 polymorphic single nucleotide polymorphism (SNP) markers using KASP (Kompetitive Allele Specific PCR) Platform. QTL mapping revealed five QTL on chromosomes 1, 3, 5, 7 and 10, which together explained approximately 30% of phenotypic variance for GY based on evaluation of RIL families under waterlogged conditions, with effects ranging from 520 to 640 kg/ha for individual genomic regions. 13 QTL were identified for various secondary traits associated with waterlogging tolerance, each individually explaining from 3 to 14% of phenotypic variance. Of the 22 candidate genes with known functional domains identified within the physical intervals delimited by the flanking markers of the QTL influencing GY and other secondary traits, six have previously been demonstrated to be associated with anaerobic responses in either maize or other model species. A pair of flanking SNP markers has been identified for each of the QTL and high throughput marker assays were developed to facilitate rapid introgression of waterlogging tolerance in tropical maize breeding programs.     

Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug‐resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug‐resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug‐resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A‐site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem‐loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug‐resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug‐resistant tuberculosis disease.     

Travellers as sentinels: Assaying the worldwide distribution of polymorphisms associated with artemisinin combination therapy resistance in Plasmodium falciparum using malaria cases imported into Scotland

There is growing evidence that Plasmodium falciparum parasites in southeastern Asia have developed resistance to artemisinin combination therapy. The resistance phenotype has recently been shown to be associated with four single nucleotide polymorphisms in the parasite’s genome. We assessed the prevalence of two of these single nucleotide polymorphisms in P. falciparum parasites imported into Scotland between 2009 and 2012, and in additional field samples from six countries in southeastern Asia. We analysed 28 samples from 11 African countries, and 25 samples from nine countries in Asia/southeastern Asia/Oceania. Single nucleotide polymorphisms associated with artemisinin combination therapy resistance were not observed outside Thailand and Cambodia.     

Assay of activity of a soil deaminase

Summary A simple method of assaying activity of a soil deaminase is described. The substrate used in the assay, 1,2-diamino-4-nitrobenzene, is red in colour, intensity decreasing with deamination of the benzene ring. This colour changes was used as the basis for a new assay in which substrate was incubated in a buffered soil slurry for 20 h, after which, the amount of substrate deaminated was determined. The method is simple and precise and, unlike existing soil deaminase assays, does not rely on measurement of the NH ₄ ⁺ product. After demonstration of enzyme kinetics, the assay was used to assess differences in deaminase activities in soils of differing crop-cover and N-status.     

Methods for Determining the Uncertainty of Population Estimates Derived from Satellite Imagery and Limited Survey Data: A Case Study of Bo City,Sierra Leone

This study demonstrates the use of bootstrap methods to estimate the total population of urban and periurban areas using satellite imagery and limited survey data. We conducted complete household surveys in 20 neighborhoods in the city of Bo, Sierra Leone, which collectively were home to 25,954 persons living in 1,979 residential structures. For five of those twenty sections, we quantized the rooftop areas of structures extracted from satellite images. We used bootstrap statistical methods to estimate the total population of the pooled sections, including the associated uncertainty intervals, as a function of sample size. Evaluations based either on rooftop area per person or on the mean number of occupants per residence both converged on the true population size. We demonstrate with this simulation that demographic surveys of a relatively small proportion of residences can provide a foundation for accurately estimating the total population in conjunction with aerial photographs.     

Chemosensory responses of Aporcelaimellus nivalis (Nematoda: Dorylaimida) towards kairomones emitted by prey nematodes belonging to different trophic groups

Abstract

Observations were made on the attraction of Aporcelaimellus nivalis towards kairomones/attractants emitted by prey nematodes belonging to different trophic categories, viz., saprophagous, epidermal, migratory semi-endodermal, predatory nematodes, virus vectors and cortical feeders. Aporcelaimellus nivalis responded positively and significantly to prey kairomones, but showed variation in their individual behaviour. Predators are most attracted towards epidermal feeders and least attracted to virus vectors. The differential responses of A. nivalis towards different prey were attributed to the inert behaviour of predators, their preference for a particular species of prey, chemical composition, concentration, quality, quantity of prey attractant, formation of minimum perceptible attraction gradient of prey and minimum response threshold of predators. Various factors such as prey density, period of prey incubation, starvation of predators, temperature, agar concentration, agar thickness and distance of predators from the source of attraction (prey) govern chemosensory responses of predator. Aporcelaimellus nivalis maximum response was towards Hirschmanniella oryzae, when tested as 10 day starved predators in agar plates containing 2 mm thick layer of 1% water-agar with 200 prey individuals previously incubated for 16 h at 30°C. Prey kairomones were most attractive when A. nivalis were tested from a distance of 2 and 3 cm.     

Inhibition of Mushroom Formation and Induction of Glycerol Release—Ecological Strategies of Burkholderia terrae BS001 to Create a Hospitable Niche at the Fungus Lyophyllum sp. Strain Karsten

We investigated the ecological strategies exerted by the soil bacterium Burkholderia terrae BS001 at the hyphae of the soil saprotrophic fungus Lyophyllum sp. strain Karsten. Recently, this bacterium has been reported to form biofilms around, and to comigrate with, growing hyphae of Lyophyllum sp. strain Karsten. In addition, it was found to be able to utilize fungal metabolites. Here, we extend this work to shed some light on the interactions between the bacterial and fungal partner which allow ecological success for the former. In standing liquid microcosms inoculated with Lyophyllum sp. strain Karsten, we detected, upon prolonged incubation, the formation of a mycelial mat at the liquid–air interface. From this mat, primordia were formed after 4–6 weeks, which eventually resulted in mushrooms. However, upon addition of strain BS001 to the bulk liquid, mushroom formation from the fungal mat was clearly inhibited, as evidenced by (1) the formation of significantly lower numbers of primordia and (2) a delay of the onset of primordia formation. Moreover and importantly, the presence of strain BS001 caused the fungus to secrete large amounts of exudates at the mycelial mat, whereas such exudation was absent from control (uninoculated) or Escherichia coli K12- or Variovorax paradoxus BS64-inoculated microcosms. In the exudates, glycerol was the main carbonaceous component, and this compound could be easily utilized by strain BS001. Thus, in different experimental set-ups with the fungal partner, strain BS001 was shown to grow in the fungal exudates on the mat. The two fungal-interactive phenotypes were specific for B. terrae strain BS001, as the other bacteria used in our study, i.e. E. coli K12 and V. paradoxus BS64, did not exhibit any of these phenomena.     

Synthesis and antibacterial activity of novel phosphonium salts on the basis of pyridoxine

A series of 13 phosphonium salts on the basis of pyridoxine derivatives were synthesized and their antibacterial activity against clinically relevant strains was tested in vitro. All compounds were almost inactive against gram-negative bacteria and exhibited structure-dependent activity against gram-positive bacteria. A crucial role of ketal protection group in phosphonium salts for their antibacterial properties was demonstrated. Among synthesized compounds 5,6-bis[triphenylphosphonio(methyl)]-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine dichloride (compound 20) was found to be the most effective towards Staphylococcus aureus and Staphylococcus epidermidis strains (MIC 5 μg/ml). The mechanism of antibacterial activity of this compound probably involves cell penetration and interaction with genomic and plasmid DNA.