Chloride dependence of endospore germination in Halobacillus halophilus

The ion requirement for germination and outgrowth of endospores from the moderately halophilic salt marsh bacterium Halobacillus halophilus was studied. Germination and outgrowth of endospores plated onto nutrient broth was dependent on the salt concentration in the artificial seawater used as the source of ions. Maximal germination and outgrowth were observed when double-concentrated artificial seawater was used. Replacement of chloride salts in the artificial seawater by other salts resulted in a complete loss of germination and outgrowth that was restored upon addition of chloride. To analyze the role of chloride more directly and quantitatively, a defined growth medium was used in which the artificial seawater was substituted by a solution of magnesium sulfate and sodium chloride. Spore germination and outgrowth were strictly dependent on the chloride concentration; maximal germination and outgrowth were observed at ≈ 1.3 M Cl^–. Chloride could be substituted by bromide, but not by sulfate or nitrate. Microscopic examinations of single spores clearly showed that germination is the chloride-dependent step. This first report on chloride dependence of spore germination in any endospore-forming bacterium adds another function to chloride in H. halophilus apart from its being essential for the physiology of the vegetative cell. Received: 21 May 1999 / Accepted: 26 July 1999     

Chloride dependence of glycine betaine transport in Halobacillus halophilus

Growth of Halobacillus halophilus is strictly chloride-dependent but the physiological basis for the chloride dependence remains to be elucidated. To address the function of Cl(-) in H. halophilus, a physiological study was performed. It was found that uptake of the compatible solute glycine betaine under isoosmotic conditions was stimulated by increasing salt concentrations. Uptake of glycine betaine required both, Na(+) and Cl(-). Cl(-) could be substituted by nitrate and bromide, but not by sulfate. Glycine betaine transport was optimal at around 0.7 M Cl(-). Cells responded to an osmotic upshock by accumulating glycine betaine, but only in the presence of chloride. These studies revealed the first chloride-dependent glycine betaine transporter in a prokaryote.     

Motility and flagellum synthesis in Halobacillus halophilus are chloride dependent

The motility of Halobacillus halophilus as observed on swarm agar plates was strictly dependent on the chloride concentration. Cl(-) was apparently not used as the coupling ion for flagellar rotation. Cells grown in the absence of chloride were devoid of flagella, but flagellation was restored upon the addition of chloride. These experiments indicate that chloride is involved in synthesis of flagella in H. halophilus.     

Growth phase-dependent switch in osmolyte strategy in a moderate halophile: ectoine is a minor osmolyte but major stationary phase solute in Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus switches its osmolyte strategy with the salinity in its environment by the production of different compatible solutes. Ectoine is produced predominantly at very high salinities, along with proline. Interestingly, ectoine production is growth phase dependent which led to a more than 1000-fold change in the ectoine : proline ratio from 0.04 in exponential to 27.4 in late stationary phase cultures. The genes encoding the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC . They form an operon that is expressed in a salinity-dependent manner with low-level expression below 1.5 M NaCl but 10-fold and 23-fold increased expression at 2.5 and 3.0 M NaCl respectively. The temporal expression of genes involved in osmoresponse is different with gdh / gln and pro genes being first, followed by ect genes. Chloride had no effect on expression of ect genes, but stimulated cellular EctC synthesis as well as ectoine production. These data demonstrate, for the first time, a growth-phase dependent switch in osmolyte strategy in a moderate halophile and, additionally, represent another piece of the chloride regulon of H. halophilus .     

Biochemical and molecular characterization of the biosynthesis of glutamine and glutamate, two major compatible solutes in the moderately halophilic bacterium Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes.     

Hyperspectral and Physiological Analyses of Coral-Algal Interactions

Space limitation leads to competition between benthic, sessile organisms on coral reefs. As a primary example, reef-building corals are in direct contact with each other and many different species and functional groups of algae. Here we characterize interactions between three coral genera and three algal functional groups using a combination of hyperspectral imaging and oxygen microprofiling. We also performed in situ interaction transects to quantify the relative occurrence of these interaction on coral reefs. These studies were conducted in the Southern Line Islands, home to some of the most remote and near-pristine reefs in the world. Our goal was to determine if different types of coral-coral and coral-algal interactions were characterized by unique fine-scale physiological signatures. This is the first report using hyperspectral imaging for characterization of marine benthic organisms at the micron scale and proved to be a valuable tool for discriminating among different photosynthetic organisms. Consistent patterns emerged in physiology across different types of competitive interactions. In cases where corals were in direct contact with turf or macroalgae, there was a zone of hypoxia and altered pigmentation on the coral. In contrast, interaction zones between corals and crustose coralline algae (CCA) were not hypoxic and the coral tissue was consistent across the colony. Our results suggest that at least two main characteristic coral interaction phenotypes exist: 1) hypoxia and coral tissue disruption, seen with interactions between corals and fleshy turf and/or some species of macroalgae, and 2) no hypoxia or tissue disruption, seen with interactions between corals and some species of CCA. Hyperspectral imaging in combination with oxygen profiling provided useful information on competitive interactions between benthic reef organisms, and demonstrated that some turf and fleshy macroalgae can be a constant source of stress for corals, while CCA are not.     

Development of a genetic system for the moderately halophilic bacterium Halobacillus halophilus: generation and characterization of mutants defect in the production of the compatible solute proline

A procedure for markerless mutagenesis gene deletions was developed for the moderately halophilic model strain Halobacillus halophilus. Gene transfer was achieved by protoplast fusion and allelic replacement by a two-step procedure. In the first step the non-replicating plasmid integrated over the upstream or the downstream region of the target gene or operon into the chromosome to obtain single-crossover mutants. When cells were grown under non-selective conditions a second homologous recombination happened (segregation). This resulted in either the wild-type or the mutated allele. The method was used to delete the proHJA operon from H. halophilus. The mutant still produced proline and thus was not proline auxotroph but it completely lost the ability to produce proline as a compatible solute. However, growth was not impaired and the loss of the solute proline was compensated for by an increase in glutamate, glutamine and ectoine concentration. Expressions of the genes encoding the biosynthesis enzymes of theses solutes were upregulated and the activity of the key enzyme in glutamine biosynthesis, the glutamine synthetase, was increased. A model for the proline biosynthesis in the ΔproHJA mutant is discussed.     

Optimisation of Potassium Chloride Nutrition for Proper Growth,Physiological Development and Bioactive Component Production in Prunella vulgaris L

Prunella vulgaris L. is an important medicinal plant with a variety of pharmacological activities, but limited information is available about its response to potassium chloride (KCl) supplementation. P. vulgaris seedlings were cultured in media with four different KCl levels (0, 1.00, 6.00 and 40.00 mM). Characteristics relating to the growth, foliar potassium, water and chlorophyll content, photosynthesis, transpiration, nitrogen metabolism, bioactive constituent concentrations and yield were determined after three months. The appropriate KCl concentration was 6.00 mM to result in the highest values for dry weight, shoot height, spica and root weight, spica length and number in P. vulgaris. The optimum KCl concentration resulted in a maximum net photosynthetic rate (Pn) that could be associated with the highest chlorophyll content and fully open stomata conductance. A supply of surplus KCl resulted in a higher concentration of foliar potassium and negatively correlated with the biomass. Plants that were treated with the appropriate KCl level showed a greater capacity for nitrate assimilation. The Pn was significantly and positively correlated with nitrate reductase (NR) and glutamine synthetase (GS) activities and was positively correlated with leaf-soluble protein and free amino acid (FAA) contents. Both KCl starvation (0 mM) and high KCl (40.00 mM) led to water loss through a high transpiration rate and low water absorption, respectively, and resulted in increased concentrations of ursolic acid (UA), oleanolic acid (OA) and flavonoids, with the exception of rosmarinic acid (RA). Moreover, the optimum concentration of KCl significantly increased the yields of RA, UA, OA and flavonoids. Our findings suggested that significantly higher plant biomass; chlorophyll content; Pn; stronger nitrogen anabolism; lower RA, UA, OA and flavonoid accumulation; and greater RA, UA, OA and flavonoid yields in P. vulgaris could be expected in the presence of the appropriate KCl concentration (6.00 mM).     

Quantitative Analyses of Cell Division in Plants

At the molecular level regulatory interactions between cell cycle genes are being uncovered rapidly, but less progress is made in unravelling how these molecular events regulate growth processes at the level of cells and of the whole organism. The main obstacle is the absence of a set of analytical tools that are powerful enough to determine pertinent parameters and, at the same time, relatively easy to use by non-specialized laboratories. Appropriate methodology to obtain this type of data has been pioneered in the first half of the last century and is now commonly defined as ‘kinematic analysis’. Unfortunately, the laborious nature of these analyses and the relatively complex numerical methods used, have limited their use to only a handful of specialized research groups. In this article we attempt to present an accessible entry to this methodology, particularly in terms of the mathematical framework. We start describing the simplest possible system, i.e., a virtually homogenous cell suspension culture. Then, we outline the analysis of dicotyledonous leaves, root tips, monocotyledonous leaves, and finally shoot apical meristems. For each of these systems we discuss the details of the calculation of cell division parameters such as cell cycle duration, size of the meristem and number of cells contained in it, which enables answering fundamental questions about the relative contribution of differences in cell production and cell size to variation in growth. In addition, we discuss the assumptions and limitations of these and alternative methodologies with the aim to facilitate the choice of appropriate analyses depending on the specific research question.     

Physiological and Transcriptome Analyses of Transgenic FGF21 Immature Rice Seeds

Russian Journal of Plant Physiology - In addition to its role as one of the largest staple food crops in the world, rice (Oryza sativa L.) currently serves as an important bioreactor in molecular...     

Evolutionary Analyses of Morphological and Physiological Plasticity in Thermally Variable Environments

SYNOPSIS. Morphological and physiological plasticity is oftenthought to represent an adaptive response to variable environments.However, determining whether a given pattern of plasticity isin fact adaptive is analytically challenging, as is evaluatingthe degree of and limits to adaptive plasticity. Here we describea general methodological framework for studying the evolutionof plastic responses. This framework synthesizes recent analyticaladvances from both evolutionary ecology and functional biology,and it does so by integrating field experiments, functionaland physiological analyses, environmental data, and geneticstudies of plasticity. We argue that studies of plasticity inresponse to the thermal environment may be particularly valuablein understanding the role of environmental variation in theevolution of plasticity: not only can thermally-relevant traitsoften be mechanistically and physiologically linked to the thermalenvironment, but also the variability and predictability ofthe thermal environment itself can be quantified on ecologicallyrelevant time scales. We illustrate this approach by reviewinga case study of seasonal plasticity in the extent of wing melanizationin Western White Butterflies (Pontia occidentalis). This reviewdemonstrates that 1) wing melanin plasticity is heritable, 2)plasticity does increase fitness in nature, but the effect variesbetween seasons and between years, 3) selection on existingvariation in the magnitude of plasticity favors increased plasticityin one melanin trait that affects thermoregulation, but 4) themarked unpredictability of short-term (within-season) weatherpatterns substantially limits the capacity of plasticity tomatch optimal wing phenotypes to the weather conditions actuallyexperienced. We complement the above case study with a casualreview of selected aspects of thermal acclimation responses.The magnitude of thermal acclimation ("flexibility") is demonstrablymodest rather than fully compensatory. The magnitude of geneticvariation (crucial to evolutionary responses to selection) inthermal acclimation responses has been investigated in onlya few species to date. In conclusion, we suggest that an understandingof selection and evolution of thermal acclimation will be enhancedby experimental examinations of mechanistic links between traitsand environments, of the physiological bases and functionalconsequences of acclimation, of patterns of environmental variabilityand predictability, of the fitness consequences of acclimationin nature, and of potential genetic constraints.     

Evaluating Dependence Among Mule Deer Siblings in Fetal and Neonatal Survival Analyses

Abstract: The assumption of independent sample units is potentially violated in survival analyses where siblings comprise a high proportion of the sample. Violation of the independence assumption causes sample data to be overdispersed relative to a binomial model, which leads to underestimates of sampling variances. A variance inflation factor, c, is therefore required to obtain appropriate estimates of variances. We evaluated overdispersion in fetal and neonatal mule deer (Odocoileus hemionus) datasets where more than half of the sample units were comprised of siblings. We developed a likelihood function for estimating fetal survival when the fates of some fetuses are unknown, and we used several variations of the binomial model to estimate neonatal survival. We compared theoretical variance estimates obtained from these analyses with empirical variance estimates obtained from data-bootstrap analyses to estimate the overdispersion parameter, c. Our estimates of c for fetal survival ranged from 0.678 to 1.118, which indicate little to no evidence of overdispersion. For neonatal survival, 3 different models indicated that ĉ ranged from 1.1 to 1.4 and averaged 1.24–1.26, providing evidence of limited overdispersion (i.e., limited sibling dependence). Our results indicate that fates of sibling mule deer fetuses and neonates may often be independent even though they have the same dam. Predation tends to act independently on sibling neonates because of dam-neonate behavioral adaptations. The effect of maternal characteristics on sibling fate dependence is less straightforward and may vary by circumstance. We recommend that future neonatal survival studies incorporate additional sampling intensity to accommodate modest overdispersion (i.e., ĉ = 1.25), which would facilitate a corresponding ĉ adjustment in a model selection analysis using quasi-likelihood without a reduction in power. Our computational approach could be used to evaluate sample unit dependence in other studies where fates of individually marked siblings are monitored.