Two temperature-sensitive photosystem II mutants of pea

Two nuclear gene mutants of pea, chlorotica-887 and chlorina-5756, are temperature-sensitive in the development of photosystem II activity. Low temperature flourescence emission spectra of leaves show that the peak at 697 nm from the reaction center of photosystem II is present when the mutants have been grown at 18°C, but absent when they have been grown at 30°C. For leaves of chlorina-5756 grown at 18°C the relative size of the peak at 697 nm is reduced compared to that of leaves of the wild type or chlorotica-887 grown at this temperature. Flourescence induction curves of leaves from wild type plants and chlorotica-887 grown at 18°C possess two steps, while those of leaves from chlorina-5756 grown at 18°C or 30°C and chlorotica-887 grown at 30°C show at fast rise to the maximal level of fluorescence. Measurements on chloroplasts isolated from the mutants indicated that the photosystem I activity per g leaf material is comparable for plants grown at 18°C and plants grown at 30°C. In contrast, no photosystem II activity was detected when the mutants had been grown at 30°C. It is suggested that these mutants are affected in a component required for the assembly of functional photosystem II complexes.     

A new platform for ultra-high density Staphylococcus aureus transposon libraries

Background

Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. High throughput, parallel-sequencing methods for analyzing transposon mutant libraries have the potential to revolutionize studies of S. aureus, but the genetic tools to take advantage of the power of next generation sequencing have not been fully developed.

Results

Here we report a phage-based transposition system to make ultra-high density transposon libraries for genome-wide analysis of mutant fitness in any Φ11-transducible S. aureus strain. The high efficiency of the delivery system has made it possible to multiplex transposon cassettes containing different regulatory elements in order to make libraries in which genes are over- or under-expressed as well as deleted. By incorporating transposon-specific barcodes into the cassettes, we can evaluate how null mutations and changes in gene expression levels affect fitness in a single sequencing data set. Demonstrating the power of the system, we have prepared a library containing more than 690,000 unique insertions. Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress. We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures.

Conclusions

The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1361-3) contains supplementary material, which is available to authorized users.     

Heat-induced changes of chlorophyll fluorescence in intact leaves correlated with damage of the photosynthetic apparatus

Methods were developed to measure chlorophyll fluorescence yield of intact leaf tissue during heat treatment under varying conditions of light intensity and photosynthetic activity. Fluorescence yield of a dark-adapted leaf increases by 2- to 3-fold with an increase of temperature into the region where heat-damage occurs. The temperatures of the fluorescence transition correlate well with the temperatures where quantum yield of CO₂ fixation is irreversibly depressed. Fluorescence-temperature (F-T) curves allow ranking of different species according to their heat sensitivity. Within a single species acclimation to different growth temperatures is reflected by shifts of the transition temperatures in the F-T curves. When F-T curves are recorded in the steady light states at increasing light intensities, substantial shifts (up to 6°C) of transition temperatures to higher values are observed. Quantum yield measurements of CO₂ fixation confirm that hight-light conditions protect from heat-damage. It is suggested that chlorophyll acts as an intrinsic fluorescence probe of the thylakoid membrane and responds to the same changes which cause irreversible denaturation of photosynthetic enzymes.C.I.W.-D.P.B. Publication No. 594     

Mutation of the gene for the second-largest subunit of RNA polymerase I prolongs the period length of the circadian conidiation rhythm in Neurospora crassa

The period length of the circadian conidiation rhythm was examined in a mutant strain of Neurospora crassa, un-18, that is temperature sensitive for mycelial growth. The un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation rhythm. Below 22° C, the un-18 mutation did not affect the period length, but at temperatures between 22° C and 32° C, the period length of the un-18 mutant was ∼2 h longer than that of the wild-type strain. The un-18 ⁺ gene was cloned and was found to encode the second-largest subunit of RNA polymerase I, which is involved in the synthesis of rRNA. These results indicate that a defect in ribosome synthesis, which must result in a lower rate of protein synthesis, lengthens the period of the circadian conidiation rhythm in Neurospora. Received: 17 October 1997 / Accepted: 26 April 1998     

Mitochondrial calcium flux in temperature-sensitive mutants of Drosophila

The mechanisms of mitochondrial calcium flux in normal and temperature-sensitive mutants of $\text{Drosophila}$ were investigated. Adult mitochondria from all stocks, when analysed with an oxygen electrode, gave respiration rates which exhibited normal responses to adenosine diphosphate and uncoupling agents but no stimulation by calcium. In contrast, calcium stimulates the respiration rate of normal larval mitochondria particularly those of the second instar. This is not evident in second instar mitochondria from the temperature sensitive mutants. There is a rapid accumulation of mitochondrial calcium during normal larval ontogenesis. The levels in temperature-sensitive second instar mitochondria are higher than those of any of the normal larval stages. Adult mitochondria in all cases contain very low levels of calcium. The amount of calcium taken up by mitochondria of second instar temperature-sensitive mutants is lower than that of normal. This may reflect the higher endogenous levels already present in the former. The implications of these variations in calcium metabolism for the behavioural defects of the temperature sensitive mutants is discussed.     

Mitochondrial calcium flux in temperature-sensitive mutants of

The mechanisms of mitochondrial calcium flux in normal and temperature-sensitive mutants of were investigated. Adult mitochondria from all stocks, when analysed with an oxygen electrode, gave respiration rates which exhibited normal responses to adenosine diphosphate and uncoupling agents but no stimulation by calcium. In contrast, calcium stimulates the respiration rate of normal larval mitochondria particularly those of the second instar. This is not evident in second instar mitochondria from the temperature sensitive mutants. There is a rapid accumulation of mitochondrial calcium during normal larval ontogenesis. The levels in temperature-sensitive second instar mitochondria are higher than those of any of the normal larval stages. Adult mitochondria in all cases contain very low levels of calcium. The amount of calcium taken up by mitochondria of second instar temperature-sensitive mutants is lower than that of normal. This may reflect the higher endogenous levels already present in the former. The implications of these variations in calcium metabolism for the behavioural defects of the temperature sensitive mutants is discussed.