Growth-limiting Conditions Research Articles

Diarrheic toxins in field-sampled and cultivated Dinophysis spp. cells from southern Brazil

In southern Brazil, mixotrophic dinoflagellates belonging to the Dinophysis acuminata complex have recently been involved in diarrheic shellfish poisoning episodes through the production of lipophilic toxins such as okadaic acid (OA) and dinophysistoxin-1 (DTX-1). The present investigation used a combination of laboratory cultures and field surveys at three large estuarine systems in that region to examine toxin retention in Dinophysis spp. cells under optimum or growth-limiting conditions. This study represents the first successful culture of a Dinophysis isolate from the Atlantic South America region. Starved D. acuminata complex cells reached 5.6-fold higher cellular OA quotas (up to 18 pg cell−1) than Mesodinium rubrum-fed cultures 20 days after inoculation in the laboratory. Moreover, in field samples, light-limited cells at the bottom of a stratified water column were less abundant, yet 6.6- to 11-fold more toxic (up to 26.4 pg OA and 1.7 pg DTX-1 cell−1) than those located at the illuminated surface. This phenomenon of toxin retention by slow-dividing cells may partially explain the enormous variation in cell toxin quota found within Dinophysis spp. populations from a single location, and it may have serious implications for cell count-based monitoring program in bivalve aquaculture areas. In fact, only low to moderate OA levels were detected in the digestive glands of oysters Crassostrea spp. (up to 17.8 ng g−1) and the guts and livers of filter-feeding fish (44.7 ng g−1) during the present study, despite the relatively high Dinophysis cell densities (up to 19,500 cells L−1) found in the field.

PLANT SELECTION CRITERIA FOR LANDSCAPING IN GULBARGA

Plants are part and parcel of any urban landscape. Plants in urban areas serve many functions. They ar e preferred for climatic, environmental, visual, medicinal, functional and re ligious purposes. Planting design being a part of p rocess of landscaping, it needs to be based on site conditions. Gulbarga poses lim iting conditions for growth of plants in terms of h ot dry climate, soil type and water quality and physical environment. Plant selection becomes a major task in landscape process. Analyses of effects of these limiting conditions on plants are necessary to make planting successful. Plant selection criteria are worked ou t based on the limiting factors and adaptability of the plants to make landscaping successful.

The Transcriptional Landscape of Campylobacter jejuni under Iron Replete and Iron Limited Growth Conditions

The genome-wide Campylobacter jejuni transcriptional response under iron replete and iron limited conditions was characterized using RNA-seq. We have identified 111 novel C. jejuni 5’UTRs and mapped 377 co-transcribed genes into 230 transcriptional operons. In contrast to previous microarray results, the C. jejuni iron stimulon is less extensive than previously believed and consists of 77 iron activated genes and 50 iron repressed genes. As anticipated, the iron repressed genes are primarily those involved in iron acquisition or oxidative stress defense. Interestingly, these experiments have revealed that iron is an important modulator of flagellar biogenesis with almost all the components of the flagella found to be iron activated. Given that motility is a well-known C. jejuni colonization factor, this suggests that there is an important regulatory coupling of flagellar biogenesis and iron level in C. jejuni. In addition we have identified several consensus mutations in the C. jejuni NCTC11168 strain that are widespread in the Campylobacter research community and which may explain conflicting phenotypic reports for this strain. Comparative analysis of iron responsive genes with the known Fur regulon indicates that many iron responsive genes are not Fur responsive; suggesting that additional iron regulatory factors remain to be characterized in C. jejuni. Further analysis of the RNA-seq data identified multiple novel transcripts including 19 potential ncRNAs. The expression of selected ncRNAs was confirmed and quantified with qRT-PCR. The qRT-PCR results indicate that several of these novel transcripts are either Fur and/or iron responsive. The fact that several of these ncRNAs are iron responsive or Fur regulated suggests that they may perform regulatory roles in iron homeostasis.

Growth Limiting pH, Water Activity, and Temperature for Neurotoxigenic Strains of Clostridium butyricum

Some rare strains of Clostridium butyricum carry the gene encoding the botulinal type E neurotoxin and must be considered as possible hazards in certain types of food. The limiting growth conditions for C. butyricum were determined in peptone yeast glucose starch (PYGS) broth incubated anaerobically at 30°C for up to 42 days. The minimum pH values permitting growth depended on the acidulant and strain. Organic acids were more effective at inhibiting growth than HCl as expected. The lowest pH values at which growth of toxigenic and nontoxigenic strains of C. butyricum was observed in broth acidified with HCl were 4.1 and 4.2, respectively. In organic acids, however, the minimum pH varied between 4.4 and 5.1 depending on acid type and concentration. The minimum water activity for growth of toxigenic strains of C. butyricum was 0.96. The minimum growth temperatures of the toxigenic strains of C. butyricum (ca 10-11°C) were somewhat higher than for non-toxigenic ones (8°C). It was concluded that control of toxigenic C. butyricum in the food industry needs to allow for the greater pH tolerance of this species compared with proteolytic C. botulinum.

The Normalcy of Dormancy: Common Themes in Microbial Quiescence

All microorganisms are exposed to periodic stresses that inhibit growth. Many bacteria and fungi weather these periods by entering a hardy, nonreplicating state, often termed quiescence or dormancy. When this occurs during an infection, the resulting slowly growing pathogen is able to tolerate both immune insults and prolonged antibiotic exposure. While the stresses encountered in a free-living environment may differ from those imposed by host immunity, these growth-limiting conditions impose common pressures, and many of the corresponding microbial responses appear to be universal. In this review, we discuss the common features of these growth-limited states, which suggest new approaches for treating chronic infections such as tuberculosis.

Exploring two-stage fermentation strategy of polyhydroxyalkanoate production using Aeromonas hydrophila

With consideration of sustainable development, this study explored the fermentation strategy of cost-effective production of biodegradable polymer- polyhydroxyalanoates (PHAs) for feasibility of eco-friendly materials recycling during wastewater treatment. As prior studies showed that Aeromonas hydrophila NIU01 was a promising PHA-producing bacterium, this follow-up study tended to seek for optimal nutrient-supplementation strategy to stimulate maximal PHA accumulation of A. hydrophila NIU01 for cellular production. As maximal PHA production took place at growth-limiting conditions, two-stage fermentation was much more appropriate for practical applications compared to batch mode of operation. Moreover, this optimal two-stage operation strategy maximized cellular PHA production under nitrogen-limiting conditions at C/N molar ratio of 60/1. For materials recycling, this operation strategy could be applicable to simultaneous PHB production and wastewater decolorization using A. hydrophila.

Radiation and optical properties of Nannochloropsis oculata grown under different irradiances and spectra

This paper reports accurate measurements of the radiation characteristics and optical properties of Nannochloropsis oculata in the photosynthetically active radiation (PAR) region. These marine microalgae were grown in 2 cm thick culture bottles with vented caps exposed, on one side, to either white fluorescent light bulbs or red LEDs emitting at 630 nm. The illuminance varied from 2000 to 10,000 lux. The microalgae average equivalent diameter ranged from 2.52 to 2.63 μm. Their radiation characteristics and optical properties were statistically identical over most of the PAR region. Other N. oculata grown with 2 vol.% CO2 injection in 1cm thick flat bottles exposed to light from both sides reached a significantly larger mass concentration and featured lower pigment concentration and smaller absorption cross-sections. This was due to nutrient limited growth conditions. The refraction index was independent of illuminance, spectrum, and growth conditions and featured resonance at wavelengths corresponding to absorption peaks.

One-dimensional lateral growth of epitaxial islands on focused ion beam patterned substrates

Lateral growth of highly elongated SiGe islands in one dimension has been achieved by ex-situ substrate patterning using a focused ion beam (FIB) to create an array of surface grooves in the Si substrate. Growth of Si0.7Ge0.3 on this template results in preferential formation of strain-relieving islands next to the edges of the grooves under kinetically limited growth conditions. The length of the nanowire-like islands is limited only by the length of the patterned trench. Occasional bridging across the groove is also observed due to non-uniformity of the underlying topography and the strong preference for the formation of four-fold island structures in this system. The success of this method is dependent upon the initial Si buffer layer thickness and on the orientation of the grooves, which must be parallel to the crystallographic 〈110〉 directions. These results demonstrate that important crystallographic as well as kinetic factors must be considered for controlling the lateral growth of nanostructures on patterned substrates.

Localization of Methyl-Coenzyme M reductase as metabolic marker for diverse methanogenic Archaea.

Methyl-Coenzyme M reductase (MCR) as key enzyme for methanogenesis as well as for anaerobic oxidation of methane represents an important metabolic marker for both processes in microbial biofilms. Here, the potential of MCR-specific polyclonal antibodies as metabolic marker in various methanogenic Archaea is shown. For standard growth conditions in laboratory culture, the cytoplasmic localization of the enzyme in Methanothermobacter marburgensis, Methanothermobacter wolfei, Methanococcus maripaludis, Methanosarcina mazei, and in anaerobically methane-oxidizing biofilms is demonstrated. Under growth limiting conditions on nickel-depleted media, at low linear growth of cultures, a fraction of 50–70% of the enzyme was localized close to the cytoplasmic membrane, which implies “facultative” membrane association of the enzyme. This feature may be also useful for assessment of growth-limiting conditions in microbial biofilms.

Laccase production by Trametes versicolor under limited-growth conditions using dyes as inducers

Laccase production by pre-growth pellets of Trametes versicolor using two types of textile dyes as inducers was studied. By decoupling the enzyme production phase from the growth phase, it is possible to reduce the time and nutrients required for laccase production. At the glucose maintenance level, the effect of the nitrogen source and textile dye was analysed using response surface methodology. Ammonium chloride was used as the inorganic nitrogen source. Two types of dyes were tested: Grey Lanaset G (GLG), a metal complex dye mixture containing nitrogen; and Alizarin Red (AR), an anthraquinonic dye with no nitrogen in its chemical structure. GLG induces laccase production at a higher extent than AR. Despite the limiting conditions required for the production of laccase, enzyme production increases with increasing ammonium chloride. When AR, the N-free dye, was used as an inducer, the optimal supply of N for laccase production was 1.2 mg/(g dry cell weight·d) as ammonium chloride. The reuse of fungal pellets in the repeated-batch mode under maintenance conditions was found to be a good strategy for improving laccase production, as enzyme production increased to up to seven times the production of the first cycle. It was demonstrated that GLG can be used as an inducer and as an N source and, thus, it is possible to decolorize the dye and to induce laccase production at the same time without adding an extra N source.

Maf1, a general negative regulator of RNA polymerase III in yeast

tRNA synthesis by yeast RNA polymerase III (Pol III) is down-regulated under growth-limiting conditions. This control is mediated by Maf1, a global negative regulator of Pol III transcription. Conserved from yeast to man, Maf1 was originally discovered in Saccharomyces cerevisiae by a genetic approach. Details regarding the molecular basis of Pol III repression by Maf1 are now emerging from the recently reported structural and biochemical data on Pol III and Maf1. The phosphorylation status of Maf1 determines its nuclear localization and interaction with the Pol III complex and several Maf1 kinases have been identified to be involved in Pol III control. Moreover, Maf1 indirectly affects tRNA maturation and decay. Here I discuss the current understanding of the mechanisms that oversee the Maf1-mediated regulation of Pol III activity and the role of Maf1 in the control of tRNA biosynthesis in yeast. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Visualization of capsular polysaccharide induction in Acidithiobacillus ferrooxidans

Extracellular Polymeric Substances (EPS) are of fundamental importance for attachment to metal sulfides, biofilm formation and leaching efficiency of Acidithiobacillus ferrooxidans. In this work we have visualized the capsular polysaccharides (CPS) of A. ferrooxidans ATCC 23270 using the fluorescently-labeled lectin Concanavalin A (ConA) and confocal laser scanning microscopy (CLSM). Ferrous iron-grown cells exhibited low amounts of CPS. A strong induction of CPS production was observed for planktonic and mineral-attached cells 24h after transfer to pyrite (FeS2). Additionally, CPS production was inducible in the presence of metal sulfide degradation products, even when cells had no direct contact to metal sulfide surfaces. Despite being an autotrophic bacterium, it has been found that sugars, presumably serving as polysaccharide building blocks stimulate CPS production in A. ferrooxidans. A strong induction of CPS was found after growth of cells on floating filters in ferrous iron medium supplemented with 2.5mg/mL glucose or galactose, while the presence of 0.25mg/mL casaminoacids did not show effects on induction of CPS. A similar phenotype was also found after growth under phosphate (Pi) limited growth conditions. Our results strongly suggest that CPS expression is inducible by several factors, including bacterial contact with metal sulfide degradation products, while direct mineral contact seems not to be a prerequisite. The molecular mechanisms regulating expression of genes encoding CPS biosynthesis and export systems in A. ferrooxidans remain to be elucidated.

The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (A|| = 20 mT). Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s) for Cu(II) ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I) and low Cu(II) affinity properties.

Agronomic Considerations for Sweet Sorghum Biofuel Production in the South-Central USA

Sweet sorghum (Sorghum bicolor (L.) Moench) is currently recognized throughout the world as a highly promising biomass energy crop. Production systems and management practices for sweet sorghum have not been fully developed for the USA, although sporadic research efforts during recent decades have provided some insights into production of sweet sorghum primarily for fermentable sugar production. Field plot experiments were conducted at sites across Louisiana to assess biomass and sugar yield responses to N fertilizer, plant density, and selected cultivars. Although linear increases in stem biomass production and fermentable sugar yield were obtained with increasing N fertilizer rate under irrigated conditions, most of the increase was from the initial 45 kg N ha−1 increment. Nitrogen fertilization increased stem biomass production but not fermentable sugar yield in some non-irrigated environments. Increased plant density contributed to fermentable sugar yield only under growth-limiting conditions, particularly under limited soil moisture. Location effects indicate that sweet sorghum may not be suitable for some sub-optimal cropland and pasture environments in Louisiana. During the primary growing season, cultivar did not affect fermentable sugar yields, although Dale was consistently high in sugar concentration during this period. Nitrogen fertilizer increased fermentable sugar yields only when moisture was not limiting. Overall results indicate that in environments where soil moisture limits plant growth, sugar yield responses are likely from increased plant density and not from increased N fertilization.

Thermoinhibition Uncovers a Role for Strigolactones in Arabidopsis Seed Germination

Strigolactones are host factors that stimulate seed germination of parasitic plant species such as Striga and Orobanche. This hormone is also important in shoot branching architecture and photomorphogenic development. Strigolactone biosynthetic and signaling mutants in model systems, unlike parasitic plants, only show seed germination phenotypes under limited growth condition. To understand the roles of strigolactones in seed germination, it is necessary to develop a tractable experimental system using model plants such as Arabidopsis. Here, we report that thermoinhibition, which involves exposing seeds to high temperatures, uncovers a clear role for strigolactones in promoting Arabidopsis seed germination. Both strigolactone biosynthetic and signaling mutants showed increased sensitivity to seed thermoinhibition. The synthetic strigolactone GR24 rescued germination of thermoinbibited biosynthetic mutant seeds but not a signaling mutant. Hormone analysis revealed that strigolactones alleviate thermoinhibition by modulating levels of the two plant hormones, GA and ABA. We also showed that GR24 was able to counteract secondary dormancy in Arabidopsis ecotype Columbia (Col) and Cape Verde island (Cvi). Systematic hormone analysis of germinating Striga helmonthica seeds suggested a common mechanism between the parasitic and non-parasitic seeds with respect to how hormones regulate germination. Thus, our simple assay system using Arabidopsis thermoinhibition allows comparisons to determine similarities and differences between parasitic plants and model experimental systems for the use of strigolactones.

Growth and accumulation dynamics of poly(3‐hydroxyalkanoate) (PHA) in Pseudomonas putida GPo1 cultivated in continuous culture under transient feed conditions

It has been shown that Pseudomonas putida GPo1 is able to grow in continuous culture simultaneously limited by ammonium (N source) and octanoate (C source), and concomitantly accumulate poly([R]-3-hydroxyalkanoate) (PHA). Under such growth conditions the material properties of PHA can be fine-tuned if a second PHA precursor substrate is supplied. To determine the range of dual carbon and nitrogen (C, N)-limited growth conditions, tedious chemostat experiments need to be carried out for each carbon source separately. To determine the growth regime, the C/N ratio of the feed (f) to a chemostat was changed in a stepwise manner at a constant dilution rate of 0.3/h. Dual-(C, N)-limited growth was observed between C(f) /N(f) ≤ 6.4 g/g and C(f) /N(f) >9.5 g/g. In the following, we analyzed alternative approaches, using continuous medium gradients at the same dilution rate, that do not require time consuming establishments of steady states. Different dynamic approaches were selected in which the C(f) /N(f) ratio was changed continuously through a convex increase of C(f) , a convex increase of N(f) , or a linear decrease of C(f) (gradients 1, 2, and 3, respectively). In these experiments, the dual-(C, N)-limited growth regime was between 7.2 and 11.0 g/g for gradient 1, 4.3 and 6.9 g/g for gradient 2, and 5.1 and 8.9 g/g for gradient 3. A mathematical equation was developed that compensated a time delay of the gradient that was caused by the wash-in/wash-out effects of the medium feed.

Effects of phosphorus availability and genetic variation of leaf terpene content and emission rate in Pinus pinaster seedlings susceptible and resistant to the pine weevil, Hylobius abietis

We studied the effects of phosphorus fertilisation on foliar terpene concentrations and foliar volatile terpene emission rates in six half-sib families of Pinus pinaster Ait. seedlings. Half of the seedlings were resistant to attack of the pine weevil Hylobius abietis L., a generalist phloem feeder, and the remaining seedlings were susceptible to this insect. We hypothesised that P stress could modify the terpene concentration in the needles and thus lead to altered terpene emission patterns relevant to plant-insect signalling. The total concentration and emission rate ranged between 5732 and 13,995 μg·g(-1) DW and between 2 and 22 μg·g(-1) DW·h(-1), respectively. Storage and emission were dominated by the isomers α- and β-pinene (77.2% and 84.2% of the total terpene amount amassed and released, respectively). In both resistant and susceptible families, P stress caused an increase of 31% in foliar terpene concentration with an associated 5-fold decrease in terpene emission rates. A higher terpene content in the leaves implies that the 'excess carbon', available under limiting growth conditions (P scarcity), is allocated to terpene production. Sensitive families showed a greater increase in terpene emission rates with increasing P concentrations, which could explain their susceptibility to H. abietis.

Microbial community engineering for biopolymer production from glycerol

In this work, the potential of using microbial community engineering for production of polyhydroxyalkanoates (PHA) from glycerol was explored. Crude glycerol is a by-product of the biofuel (biodiesel and bioethanol) industry and potentially a good substrate for bioplastic production. A PHA-producing microbial community was enriched based on cultivation in a feast-famine regime as successfully applied before for fatty acids-based biopolymer production. A glycerol-fed sequencing batch reactor operated at a 2-day liquid and biomass residence time and with feast-famine cycles of 24 h was used to enrich a mixed community of PHA producers. In a subsequent fed-batch PHA production step under growth-limiting conditions, the enriched mixed community produced PHA up to a dry weight content of 80 wt.%. The conversion efficiency of substrate to PHA on electron basis was 53%. Since glycerol is entering the metabolic pathways of the cell in the glycolytic pathway, it was anticipated that besides PHA, polyglucose could be formed as storage polymer as well. Indeed, polyglucose was produced in low amounts (∼ 10 wt.%). The results indicated that the feast-famine-based enrichment strategy was comparably successful to obtain a microbial community compared to fatty acids-based enrichment described before.

HP-23: Co-ordinated Regulation of Gene Expression in H. Pylori in Response to Low pH and Iron Limiting Growth Conditions

HP-23: Co-ordinated Regulation of Gene Expression in H. Pylori in Response to Low pH and Iron Limiting Growth Conditions

HP-23: Co-ordinated Regulation of Gene Expression in H. Pylori in Response to Low pH and Iron Limiting Growth Conditions

AbstractEstablishing a successful infection within the gastric environment and its varied and changing niches requires adaptation achieved through regulation of bacterial gene expression. In the gastric environment in addition to the low pH Helicobacter pylori is required to adapt to conditions of iron limitation. In this study, we used cDNA microarrays to identify genes whose expression was altered when the bacterium was grown under differing conditions of pH (5 versus 7) and iron limitation. cDNA microarray analyses following exposure to different growth conditions were carried out for three H. pylori strains 26695 (gastritis-associated), J99 (ulcer-associated) and AG-1 (atrophic gastritis-associated). Genes were identified whose expression was acid pH-dependent in all the strains: 68 genes were up-regulated, these included urease-related genes (ureA, ureB, ureI, UreF), amidase (aimE) and genes involved in flagellar synthesis (flgB, fliM, flgE). Additionally, up-regulation of transport-related genes and genes encoding binding proteins (glnQ, glnH, exbD) were noted. Furthermore, 37 genes were down-regulated from different functional groups (e.g., outer membrane proteins and chaperones). At pH 7, iron limitation mainly induced motility-associated genes, whereas at pH5, iron depletion although inducing down-regulation of 12 genes, led to the increased expression of 21 genes including those coding for carbonic anhydrase and proteins involved in metal ion transport, instead of genes coding for urease and amidase enzymes. Genes encoding virulence factors (e.g., cagA and vacA) and proteins involved in motility were over-expressed under all the conditions of stress. Collectively, as effectors of the H. pylori stress response, this transcriptome analysis implicating up-regulation of various metal responsive regulators and metal transport-related genes, in addition to induction of ammonia-producing enzymes and virulence factors (CagA and VacA), emphasizes the link between the bacterial response to acidity, metal metabolism and virulence. Moreover, this response was similar in H. pylori strains associated with different pathologies.