Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

Effect of oxygen and air flow on xylanolytic and cellulolytic activity from bacterial cultures grown on bagasse pith

Summary The xylanolytic and cellulolytic activity fromCellulomanas were reduced by high O₂ concentrations in continous culture as well as by an air flow passed trough the samples, suggesting an inhibition or inactivation of enzymes in such conditions.     

Influence of immunizing dose and presence or absence of adult worms on the development of resistance to Nematospiroides dubius challenge infections of mice

H-2 congenic strains expressing resistant (H-2q, H-2f) or susceptible (H-2k) haplotypes were compared for their ability to resist challenge infection with N. dubius following a 6- or 14-day ivermectin-abbreviated immunizing infection. B10.BR mice (H-2k) were considerably more resistant to infection when the priming interval was shortened from 14 to 6 days. B10.Q (H-2q) and B10.M (H-2f) mice resisted challenge regardless of which immunization regimen was used. The influence of parasite numbers on the response to challenge was studied by comparing infections in resistant DBA/1 (H-2q) and susceptible CBA/J (H-2k) mice that differ at both H-2 and non-H-2 genes. DBA/1 mice, immunized with 50 or 150 L3 of N. dubius for 14 days, resisted challenge, whereas mice receiving 300 worms did not. In contrast, CBA/J mice failed to resist challenge at all priming doses tested. When the immunizing infection was shortened from 14 to 6 days, DBA/1 mice resisted challenge regardless of priming dose and CBA/J mice resisted challenge only when the highest dose of 300 worms was used for priming. The data suggest that susceptible strains of mice may be preferentially immunosuppressed, particularly at low infective doses, and that suppression is associated with adult worms present in the lumen of the small intestine.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

The LOX1 Gene of Arabidopsis Is Temporally and Spatially Regulated in Germinating Seedlings

We examined the temporal and spatial expression patterns of the LOX1 gene during the development of Arabidopsis thaliana seedlings. Measurements of steady-state LOX1 mRNA levels indicated that this gene is transiently expressed during germination. LOX1 mRNA was not detected in seed that had imbibed (T0) but reached a maximum level by 1 d in both light- and dark-grown seedlings. The induction of the LOX1 gene was not light dependent; however, mRNA levels were 4-fold greater in light-grown seedlings. Immunoblot analysis of lipoxygenase protein levels and measurements of enzyme activity suggested that the induction of the LOX1 gene resulted in the production of functional lipoxygenase enzyme. Lipoxygenase protein was not present in dry seed or seed that had imbibed, but was first detected by immunoblot analysis after 1 and 2 d of growth in the light and dark, respectively. In both cases, lipoxygenase protein levels remained high for 2 d and then declined. Lipoxygenase activity paralleled the changes in protein levels. In situ hybridization studies revealed that the LOX1 gene is transiently expressed in the epidermis and the aleurone layer during germination. LOX1 mRNA levels were particularly high in the epidermis of the radicle and the adaxial side of the cotyledons. These results suggest that the LOX1 gene product is produced specifically during early germination and plays a role in the functioning of the epidermis.     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5’-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate. The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments. Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and “holey” native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane. The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.     

Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties

Bacterial biofilms are communities of bacteria entangled in a self‐produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid‐polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum‐of‐the‐parts ¹³C solid‐state nuclear magnetic resonance (NMR) analysis to define the curli‐to‐pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well‐studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid‐air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic‐associated) and the strain itself.     

Isolation and Characterization of Mercury Resistant Bacillus sp. from Soils with an Extensive History as Substrates for Mercury Extraction in Mexico

Metal phytoextraction assisted by bacteria plays an important role in bioremediation systems. In this work, mercury-resistant bacterial strains were isolated from soils with high levels of mercury (San Joaquin, Queretaro State, Mexico) and identified as Bacillus sp. based on the 16S rDNA gene sequence analysis. The bacterial strains were found to exhibit different multiple mercury-resistance and carbon source utilization characteristics. The mercury reduction ability was tested through a volatilization assay. The bacterial isolates were also evaluated for their ability to promote growth and mercury uptake in tomato plants. In a roll towel assay, the maximum vigor index of tomato plants was obtained with the inoculation of Bacillus sp. A2, A12, B11, B15 and C1, while in a pot assay, the maximum vigor index was obtained with the inoculation of Bacillus sp. A6, A7 and B20, compared with un-inoculated controls in the presence of HgCl₂. Maximum Hg accumulation in the roots and shoots of tomato plants was obtained only with Bacillus sp. A7 in the roll towel assay, whereas in the pot assay, maximum accumulation was obtained with Bacillus sp. A12 compared with un-inoculated controls. Our results show that mercury accumulation in tissue is enhanced by these plant growth promoting bacterial strains, which recommends their possible use as microbe-assisted phytoremediation systems in mercury-polluted soils.