Intracellular glucose 6-phosphate content in Streptomyces coelicolor upon environmental changes in a defined medium

A new, chemically defined medium providing dispersed growth and high biomass formation and a method for quantitative extraction of intracellular metabolites was used to investigate the cellular response of Streptomyces coelicolor A3(2) during growth and upon changes in nutrient utilization. Fast changes of the glucose 6-phosphate content precisely signaled transitions in the flow of carbon sources. The results indicate that intracellular pool sizes may be used to detect early nutrient limitations in view of the onset of antibiotic production. Additionally the results disclose characteristics of the regulation of maltose and glutamic acid uptake and degradation in S. coelicolor A3(2).     

Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.     

Resonance Raman spectroscopy of xanthophylls in pigment mutant thylakoid membranes of pea

Low-temperature resonance Raman spectroscopy was used to study the changes in the molecular structure and configuration of the major xanthophylls in thylakoid membranes isolated from mutants of pea with modified pigment content and altered structural organization of their pigment-protein complexes. The Raman spectra contained four known groups of bands, nu(1)-nu(4), which could be assigned to originate mainly from the long wavelength absorbing lutein and neoxanthin upon 514.5 nm and at 488 nm excitations, respectively. The overall configuration of these bound xanthophyll molecules in the mutants appeared to be similar to the wild type, and the configuration in the wild type was almost identical with that in the isolated main chlorophyll a/b light harvesting protein complex of photosystem II (LHCII). Significant differences were found mainly in the region of nu(4) (around 960 cm(-1)), which suggest that the macroorganization of PS II-LHCII supercomplexes and/or of the LHCII-only domains are modified in the mutants compared to the wild type.     

Orcein staining and the study of the effect of chemicals on chromosomes

Summary Plant materials of both dicotyledonous and monocotyledonous groups were subjected to treatment with alloxan followed by coumarin, phenols, oxyquinoline etc. for 3 to 4 hours.Root tips were then heated in an orcein-acid mixture for 10 to 40 seconds and studied after mounting in 1% orcein. Clear metaphase plates were obtained after 10 seconds of heating, whereas after 20 and 30 seconds considerable erosion and fragmentation respectively were recorded. Complete loss of stainability resulted after heating for 40 seconds.Fragmentation is caused only by orcein under heat conditions. This is demonstrated by control series checking all variables.Pretreatment with the chemicals stated is of absolute necessity for fragmentation. It is suggested that this pretreatment causes depolymerisation of DNA and lability of nucleoprotein linkage at certain segments from which the DNA becomes detached during subsequent heating.Of all the chemicals tested for pretreatment effects, marked positive results were obtained with alloxan (1 hour) followed by coumarin (2 hours at 16–20 ° C.). Phenols also gave positive results. The other chemicals tried need slightly longer time of treatment for fragment induction.     

Bonding, postpartum dysphoria, and social ties

Since the late 1970s, disruptions and “failure” of maternal-infant bonding have been causally linked to postpartum depression. Part I of this paper examines the grounds for this connection while tracing the ramifications of bonding theory (Klaus and Kennell 1976) through obstetrics, pediatrics, and psychiatry, as well as in the (mis)representations of it in the popular media. This discussion resolves into a view of maternal attachment as a long-term development progressively established through intensive mother-infant interaction. The forms of this interaction are phylogenetically determined, albeit culturally and personally mediated. Flowing from this premise, Part II of the paper casts postpartum depression as an adaptive response to threat (from whatever cause) to adequate mothering, and develops an argument for the evolutionary role of enacted social ties in the establishment of maternal responsiveness.     

A rapid method for the determination of microbial biomass by dry weight using a moisture analyser with an infrared heating source and an analytical balance

Aims: Microbial biomass is an important biotechnological parameter. The traditional method for its determination involves an oven‐drying step and equilibration to room temperature before weighing, and it is tedious and time consuming. This work studied the utilisation of a moisture analyser consisting of an efficient infrared‐heating module and an analytical balance for the determination of microbial biomass by dry weight. Methods and Results: The method duration depended on the sample volume and was between 7 and 40 min for sample volumes of 1–10 ml. The method precision depended on the total dry weight analysed – 10 mg of total dry weight being sufficient to achieve coefficients of variation of 5% or less. Comparison with the conventional oven method provided a correlation coefficient r² of 0·99. The recovery of an internal standard ranged between 94·2 and 106·4% with a precision of 1·39–4·53%CV. Conclusions: Validation revealed sufficient method accuracy, precision and robustness and was successfully applied to the study of yeast and bacterial growth kinetics. Techniques are discussed that allow for increased method precision at low biomass concentrations, and equations are provided to estimate required drying time and method precision based on sample volume and total sample dry weight, respectively. Significance and Impact of the Study: This work presents a rapid method for the determination of microbial biomass, allowing for the timely implementation of biomass‐based information in biotechnological and laboratory protocols.     

Four chromosome replication origins in the archaeon Pyrobaculum calidifontis

Replication origins were mapped in hyperthermophilic crenarchaea, using high‐throughput sequencing‐based marker frequency analysis. We confirm previous origin mapping in Sulfolobus acidocaldarius, and demonstrate that the single chromosome of Pyrobaculum calidifontis contains four replication origins, the highest number detected in a prokaryotic organism. The relative positions of the origins in both organisms coincided with regions enriched in highly conserved (core) archaeal genes. We show that core gene distribution provides a useful tool for origin identification in archaea, and predict multiple replication origins in a range of species. One of the P. calidifontis origins was mapped in detail, and electrophoretic mobility shift assays demonstrated binding of the Cdc6/Orc1 replication initiator protein to a repeated sequence element, denoted Orb‐1, within the origin. The high‐throughput sequencing approach also allowed for an annotation update of both genomes, resulting in the restoration of open reading frames encoding proteins involved in, e.g., sugar, nitrate and energy metabolism, as well as in glycosylation and DNA repair.     

Novel Proteorhodopsin variants from the Mediterranean and Red Seas

Proteorhodopsins, ubiquitous retinylidene photoactive proton pumps, were recently found in the widespread uncultured SAR86 bacterial group in oceanic surface waters. To survey proteorhodopsin diversity, new degenerate sets of proteorhodopsin primers were designed based on a genomic proteorhodopsin gene sequence originating from an Antarctic fosmid library. New proteorhodopsin variants were identified in Red Sea samples that were most similar to the original green-light absorbing proteorhodopsins found in Monterey Bay California. Unlike green-absorbing proteorhodopsins however, these new variants contained a glutamine residue at position 105, the same site recently shown to control spectral tuning in naturally occurring proteorhodopsins. Different proteorhodopsin variants were also found in the Mediterranean Sea. These proteorhodopsins formed new and distinctive proteorhodopsin groups. Phylogenetic analyses show that some of the new variants were very different from previously characterized proteorhodopsins, and formed the deepest branching groups found so far among marine proteorhodopsins. The existence of these varied proteorhodopsin sequences suggests that this class of proteins has undergone substantial evolution. These variants could represent functionally divergent paralogous genes, derived from the same or similar species, or orthologous proteorhodopsins that are distributed amongst divergent planktonic microbial taxa.