Regulation of podJ Expression during the Caulobacter crescentus Cell Cycle

The polar organelle development gene, podJ, is expressed during the swarmer-to-stalked cell transition of the Caulobacter crescentus cell cycle. Mutants with insertions that inactivate the podJ gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally. In contrast, hyperexpression of podJ results in a lethal cell division defect. Nucleotide sequence analysis of the podJ promoter region revealed a binding site for the global response regulator, CtrA. Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the podJ promoter. Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site. Thus, although the level of podJ promoter activity is dependent on the CtrA binding site, the temporal control of podJ promoter expression is dependent on other factors.     

A high-density molecular map for ryegrass (Lolium perenne) using AFLP markers

AFLP markers have been successfully employed for the development of a high-density linkage map of ryegrass (Lolium perenne L.) using a progeny set of 95 plants from a testcross involving a doubled-haploid tester. This genetic map covered 930 cM in seven linkage groups and was based on 463 amplified fragment length polymorphism (AFLP) markers using 17 primer pairs, three isozymes and five EST markers. The average density of markers was approximately 1 per 2.0 cM. However, strong clustering of AFLP markers was observed at putative centromeric regions. Around these regions, 272 markers covered about 137 cM whereas the remaining 199 markers covered approximately 793 cM. Most genetic distances between consecutive pairs of markers were smaller than 20 cM except for five gaps on groups A, C, D, F and G. A skeletal map with a uniform distribution of markers can be extracted from this high-density map, and can be applied to detect and map QTLs. We report here the application of AFLP markers to genome mapping, in Lolium as a prelude to quantitative trait locus (QTL) identification for diverse agronomic traits in ryegrass and for marker-assisted plant breeding. Received: 4 November 1998 / Accepted:15 March 1999     

Altered Virulence of Vaccine Strains of Measles Virus after Prolonged Replication in Human Tissue

To understand the molecular determinants of measles virus (MV) virulence, we have used the SCID-hu thymus/liver xenograft model (SCID-hu thy/liv) in which in vivo MV virulence phenotypes are faithfully duplicated. Stromal epithelial and monocytic cells are infected by MV in thymus implants, and virulent strains induce massive thymocyte apoptosis, although thymocytes are not infected. To determine whether passage of an avirulent vaccine strain in human tissue increases virulence, we studied a virus isolated from thymic tissue 90 days after infection with the vaccine strain Moraten (pMor-1) and a virus isolated from an immunodeficient child with progressive vaccine-induced disease (Hu2). These viruses were compared to a minimally passaged wild-type Edmonston strain (Ed-wt) and the vaccine strain Moraten. pMor-1, Hu2, and Ed-wt displayed virulent phenotypes in thymic implants, with high levels of virus being detected by 3 days after infection (10(5.2), 10(2.8), and 10(3. 4), respectively) and maximal levels being detected between 7 and 14 days after infection. In contrast, Moraten required over 14 days to grow to detectable levels. pMor-1 produced the highest levels of virus throughout infection, suggesting thymic adaptation of this strain. Similar to other virulent strains, Ed-wt, Hu2, and pMor-1 caused a decrease in the number of viable thymocytes as assessed by trypan blue exclusion and fluorescence-activated cell sorter analysis. Thymic architecture was also disrupted by these strains. Sequence analysis of the hemagglutinin (H) and matrix (M) genes showed no common changes in Hu2 and pMor-1. M sequences were identical in pMor-1 and Mor and varied in H at amino acid 469 (threonine to alanine), a position near the base of propeller 4 in the propeller blade/stem model of H structure. Further study will provide insights into the determinants of virulence.     

Requirements for RNA Replication of a Poliovirus Replicon by Coxsackievirus B3 RNA Polymerase

A chimeric poliovirus type 1 (PV1) genome was constructed in which the 3D RNA polymerase (3D(pol)) coding sequences were replaced with those from coxsackievirus B3 (CVB3). No infectious virus was produced from HeLa cells transfected with the chimeric RNA. Processing of the PV1 capsid protein precursor was incomplete, presumably due to inefficient recognition of the P1 protein substrate by the chimeric 3CD proteinase containing CVB3 3D sequences. The ability of the chimeric RNA to replicate in the absence of capsid formation was measured after replacement of the P1 region with a luciferase reporter gene. No RNA synthesis was detected, despite efficient production of enzymatically active 3D(pol) from the 3D portion of the chimeric 3CD. The chimeric 3CD protein was unable to efficiently bind to the cloverleaf-like structure (CL) at the 5' end of PV1 RNA, which has been demonstrated previously to be required for viral RNA synthesis. The CVB3 3CD protein bound the PV1 CL as well as PV1 3CD. An additional chimeric PV1 RNA that contained CVB3 3CD sequences also failed to produce virus after transfection. Since processing of PV1 capsid protein precursors by the CVB3 3CD was again incomplete, a luciferase-containing replicon was also analyzed for RNA replication. The 3CD chimera replicated at 33 degrees C, but not at 37 degrees C. Replacement of the PV1 5'-terminal CL with that of CVB3 did not rescue the temperature-sensitive phenotype. Thus, there is an essential interaction(s) between 3CD and other viral P2 or P3 protein products required for efficient RNA replication which is not fully achieved between proteins from the two different members of the same virus genus.     

Role of Volatile Fatty Acids in Development of the Cecal Microflora in Broiler Chickens during Growth

It is known that volatile fatty acids can inhibit growth of species of the family Enterobacteriaceae in vitro. However, whether these volatile fatty acids affect bacterial populations in the ceca of chickens is unknown. Therefore, a study was conducted to investigate if changes in volatile fatty acids in ceca of broiler chickens during growth affect bacterial populations. Results showed that members of the Enterobacteriaceae and enterococci are present in large numbers in 3-day-old broilers and start to decrease when broilers grow older. Lactobacilli are present in large numbers as well in 3-day-old broilers, but they remain stable during the growth of broilers. Acetate, butyrate, and propionate increase from undetectable levels in 1-day-old broilers to high concentrations in 15-day-old broilers, after which they stabilize. Significant negative correlations could be calculated between numbers of Enterobacteriaceae and concentrations of undissociated acetate, propionate, and butyrate. Furthermore, pure cultures of Enterobacteriaceae isolated from the ceca were grown in the presence of volatile fatty acids. Growth rates and maximal optical density decreased when these strains grew in the presence of increasing volatile fatty acid concentrations. It is concluded that volatile fatty acids are responsible for the reduction in numbers of Enterobacteriaceae in the ceca of broiler chickens during growth.     

Carbon and nutrient losses during manure storage under traditional and improved practices in smallholder crop-livestock systems—evidence from Kenya

In the absence of mineral fertiliser, animal manure may be the only nutrient resource available to smallholder farmers in Africa, and manure is often the main input of C to the soil when crop residues are removed from the fields. Assessments of C and nutrient balances and cycling within agroecosystems or of greenhouse gas emissions often assume average C and nutrient mass fractions in manure, disregarding the impact that manure storage may have on C and nutrient losses from the system. To quantify such losses, in order to refine our models of C and nutrient cycling in smallholder (crop-livestock) farming systems, an experiment was conducted reproducing farmers’ practices: heaps vs. pits of a mix of cattle manure and maize stover (2:3 v/v) stored in the open air during 6 months. Heaps stored under a simple roof were also evaluated as an affordable improvement of the storage conditions. The results were used to derive empirical models and graphs for the estimation of C and nutrient losses. Heaps and pits were turned every month, weighed, and sampled to determine organic matter, total and mineral N, P and K mass fractions. Soils beneath heaps/pits were sampled to measure mineral N to a depth of 1 m, and leaching tube tests in the laboratory were used to estimate P leaching from manure. After 6 months, ca. 70% remained of the initial dry mass of manure stored in pits, but only half of or less of the manure stored in heaps. The stored manure lost 45% of its C in the open air and 69% under roof. The efficiencies of nutrient retention during storage varied between 24–38% for total N, 34–38% for P and 18–34% for K, with the heaps under a roof having greater efficiencies of retention of N and K. Laboratory tests indicated that up to 25% of the P contained in fresh manure could be lost by leaching. Results suggest that reducing the period of storage by, for example, more frequent application and incorporation of manure into the soil may have a larger impact on retaining C and nutrient within the farm system than improving storage conditions.     

Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.     

Behavior of Yersinia enterocolitica in the Presence of the Bacterivorous Acanthamoeba castellanii

Free-living protozoa play an important role in the ecology and epidemiology of human-pathogenic bacteria. In the present study, the interaction between Yersinia enterocolitica, an important food-borne pathogen, and the free-living amoeba Acanthamoeba castellanii was studied. Several cocultivation assays were set up to assess the resistance of Y. enterocolitica to A. castellanii predation and the impact of environmental factors and bacterial strain-specific characteristics. Results showed that all Y. enterocolitica strains persist in association with A. castellanii for at least 14 days, and associations with A. castellanii enhanced survival of Yersinia under nutrient-rich conditions at 25°C and under nutrient-poor conditions at 37°C. Amoebae cultivated in the supernatant of one Yersinia strain showed temperature- and time-dependent permeabilization. Intraprotozoan survival of Y. enterocolitica depended on nutrient availability and temperature, with up to 2.8 log CFU/ml bacteria displaying intracellular survival at 7°C for at least 4 days in nutrient-rich medium. Transmission electron microscopy was performed to locate the Yersinia cells inside the amoebae. As Yersinia and Acanthamoeba share similar ecological niches, this interaction identifies a role of free-living protozoa in the ecology and epidemiology of Y. enterocolitica.