Mechanisms involved in the increased sensitivity ofEscherichia coli to microcin 15m at 42°C

Escherichia coli cells show a markedly increased sensitivity to the antibiotic microcin 15m when briefly treated at 42°C as compared to the effect at 37°C. Furthermore, mutants resistant to the microcin at 37°C become sensitive at 42°C at microcin concentrations that are inactive at 37°C. This effect can be overcome byl-methionine. The mechanism involved seems to be based on an apparent inactivation of the homoserine-O-transsuccinylase activity. As previously established, this enzyme suffers a reversible partial inactivation when the cells are shifted to 42°C and the action of the microcin at this temperature seems to bring this process to a virtually irreversible stage. In mixed cultures of the microcin-producing strain and oneE. coli strain sensitive to the antibiotic, a much stronger growth inhibition of the latter strain has been observed at 42°C than at 37°C.     

Isolation and characterization of immunity temperature sensitive mutants of Enterobacter cloacae harbouring the cloacinogenic factor DF13

Summary Enterobacter cloacae cells, harbouring the cloacinogenic factor DF13 (Clo DF13) are immune to the cloacin they produce. We describe the isolation of eleven Enterobacter cloacae (Clo DF13) mutants, which are immune at 30°C, but lose their immunity at 42°C. The temperature sensitive immunity (Imm^ts) of these mutants appeared not to be transferable together with the Clo DF13 factor to non-cloacinogenic acceptor strains. Apparently host mutations are involved in the Imm^ts phenotype. Two different groups of Imm^ts mutants could be identified. Imm^tsC6 and Imm^tsC8, representatives of each group, have been compared with the parent strain. Imm^tsC6 as well as Imm^tsC8 is sensitive to crude cloacin at 42°C. Imm^ts mutants appeared to be also sensitive to cell components other than cloacin, indicating that the Imm^ts mutations may result in pleiotropic changes of cell properties.The Imm^tsC6 mutant is sensitive to deoxycholate and osmotic shock at 42°C. Spheroplasts of Imm^tsC6 cells incubated at 42°C are sensitive to DOC at 42°C and 30°C. The pleiotrophic changes of the Imm^tsC6 mutant may be attributed to a defect in the cell membrane.The Imm^tsC8, incubated at 42°C, is sensitive to deoxycholate, osmotic shock, ethylene-diaminetetraacetic acid, dyes, drugs and UV. Furthermore they form filaments. Imm^tsC8 spheroplasts are as sensitive to deoxycholate as the parent strain at 42°C. The pleiotropic changes in the phenotype of Imm^tsC8 are considered to be the result of a defect in the outer layers of the cell envelope, most likely the lipopolysaccharide layer.The possible relationship between the observed structural defects in the cell envelope of Imm^ts mutants and the phenomenon of immunity have been discussed.     

Response of antennal cold receptors of the catopid beetles,Speophyes lucidulus Delar. andCholera angustata Fab. to very slowly changing temperature

Summary The antennal cold cells in larval black-hair sensilla of the cave beetle,Speophyes lucidulus Delar., clearly respond to rates of temperature change 5 to 10 times lower than any tested on insect cold cells so far: often below 0.0005° C/s or 2° C/h. At a given ambient temperature between 11° C and 15° C, cold-cell impulse frequency was higher when temperature was falling at these rates than when it was rising at them in every one of the twelve cells examined. The mean differential sensitivity to the rate of change was -3340±1071 (imp/s)/(° C/s), in each case two to 5 times greater (sign ignored) than in any cold cell observed previously (Loftus 1969; Corbière-Tichané and Loftus 1983). The differential sensitivity to ambient temperature,-6.8 (imp/s)/° C, was statistically indistinguishable, on the other hand, from the earlier values forSpeophyes.Antennal cold cells of six first-stage larvae of another Catopid beetle,Choleva angustata Fab., displayed very similar responses to the same stimuli. Its mean differential sensitivities were -8.1+3.9 (imp/s)/° C to ambient temperature and-3790+2190 (imp/s)/(° C/s) to the rate of temperature change. UnlikeSpeophyes this beetle spends only part of its life cycle in caves.Abbreviations dT/dt rate of temperature change in °C/s - F impulse frequency in impulses/s (imp/s) - T temperature in ° C To Sylvie Deleurance, a helpful friend who dedicated much of her life to the study of cave insects     

Loss and restoration of viability of E. coli due to combinations of mutations affecting DNA polymerase I and repair activities

Summary We have found that the cells possessing the polA6 mutation affecting DNA polymerase I are unable to accept another mutation (uvr502) leading to UV-sensitivity. The introduction of the polA12 mutation determining the synthesis of a temperature sensitive DNA polymerase I into the uvr502 mutant results in the temperature sensitivity of colony forming ability of the double mutant. These data show that the uvr502 derivatives lacking DNA polymerase I are inviable. Reversions to temperature resistance in the population of the double mutant uvr502 polA12 may occur because of reverse mutations at one of the mutated sites or because of mutations suppressing DNA polymerase I deficiency but not UV- or MMS-sensitivity of revertants. DNA and protein synthesis in uvr502 polA12 cells continues after a shift to 45°C with rates almost indistinguishable from those in single mutants or wild type cells. No differences in DNA degradation were observed during incubation of single and double mutants at 45°C. The single strand molecular weight distribution of parent DNA from the double mutant as well as that from wild type cells is not affected by the shift to 45°C and 3 hours incubation at this temperature. We suggest that DNA polymerase I and/or the product altered by the uvr502 mutation are required for some step(s) of discontinuous DNA replication nonessential for the formation of acid insoluble DNA. The DNA polymerase I and the uvr gene product seem to be able to substitute for each other in accomplishing this process.     

Isolation and initial characterization of a Schizosaccharomyces pombe mutant exhibiting temperature-dependent radiation sensitivity due to a mutation in a previously unidentified rad locus

Summary We have isolated a mutant of the yeast Schizosaccharomyces pombe which exhibits sensitivity to UV light when grown at either 30° or 37°C, as compared to the parental wild-type strain. This increased sensitivity is more pronounced when cells are grown at 37°C. The mutant is also sensitive to 18 MeV electrons at the high temperature. Tetrad analysis of spores generated by crossing the mutant and a Rad⁺ strain revealed that sensitivity to both types of radiation cosegregate 2:2, relative to wild-type resistance, indicating that a single altered chromosomal locus is responsible for the radiation sensitivities observed. In addition, analysis of spores resulting from crosses between the mutant and all other known S. pombe rad mutants indicates that the temperature-dependent sensitivity described in this report is mediated by a mutation in a previously unidentified rad locus.     

Cold-sensitive expression of cytokinin habituation by tobacco pith cells in culture

Cloned, cytokinin-habituated tissues of Nicotiana tobacum L. cv. Havana 425 are able to grow in culture at 25° C without added cytokinin. These tissues vary in their expression of the habituated phenotype at 16° C. When cytokinin-requiring pith tissues are converted to the habituated state by 35° C treatment, all of the habituated cells are cold sensitive. After several transfers in culture, some of these habituated cells give rise to stable, cold resistant variants. Both phenotypes are inherited by individual cells. Cold sensitive clones at 16° C and non-habituated clones at 16° C as well as 25° C show the same dose response to the cytokinin, kinetin. This suggests that at the physiological level, cold sensitivity results from a decreased production of cell division factors rather than from a decreased affinity of cellular receptors for these factors.Abbreviations CDF Cell division factor(s) - NAA -naphthalencacetic acid     

Inactivation of Candida albicans by ultraviolet radiation

Summary Inactivation of Candida albicans by ultraviolet (uv) light is markedly dependent upon (a) the cell division stage and (b) the nutrition and growth temperatures of cells both before and after irradiation. Cells grown at 37°C after irradiation show lower survivals than those grown at 25°C. At either recovery temperature, cells which had been cultured before irradiation at 37°C are able to sustain less uv damage prior to inactivation than those cultured at 25°C. The radiosensitivities of budding and non-budding cells are the same when survivals are scored at 25°C; at low uv dosages, cells show slightly poorer recoveries on enriched medium than on minimal medium whereas at higher dosages, their recoveries on both kinds of media are equivalent. In contrast, at 37°C, uv treated non-budding cells are much more susceptible to inactivation than budding cells; non-budding cells also express much poorer recovery on enriched medium than on minimal medium at 37°C whereas budding cells survive equally well on either medium. Though non-budding cells grown for irradiation on minimal or enriched media exhibit the same radiosensitivites, budding cells grown for irradiation on enriched medium are more susceptible to inactivation at 37°C than those grown on minimal medium.The particularly poor recovery by irradiated non-budding cells at 37°C is correlated with their unique tendency to undergo a transitory filamentation when initiating growth at that temperature. Evidence is presented that neither the filamentous growth per se nor the temporary inhibition of cell division associated with filamentation causes the poor recovery. Furthermore, while irradiated non-budding cells at 37°C exhibit singular susceptibility to inhibition of recovery by metabolic antagonists which disturb protein synthesis, the course of their filamentous growth is not affected by such agents. It is concluded that recovery from irradiation and the instigation of cytokinesis by non-budding cells of C. albicans result from different metabolic processes which may be related through a common temperature sensitive step. C. albicans does not photoreactivate and observations on recovery by cells prevented from undergoing immediate postirradiation replication do not indicate the existence of a system for dark repair of DNA damage comparable to that occurring in bacteria. Difficulties attending a valid demonstration of DNA dark repair in yeasts are discussed.     

Cell division and the production of cells lacking nuclear bodies in a mutant of Salmonella typhimurium

Summary There are two distinct division phases when the temperature-sensitive DNA synthesis initiation mutant Salmonella typhimurium strain 11G is shifted from 25° to 38°. The first phase appears to represent segregation of the nuclear bodies formed at 38°. Division in this phase takes place at the normal size and produces mainly organisms with one nuclear body. It is dependent on the termination of the rounds of replication in operation at the time of the temperature shift and sensitive to low doses of penicillin. This division phase continues for 60–75 min and then after a short lag division restarts. At first the cells undergoing the second division phase are only slightly larger than normal but they soon grow into short filaments which bud off cells at both ends even if only one nuclear body is present. The cells budded off in this division phase are about 3 long on both broth and M M. They lack nuclear bodies but have a small amount of DNA which may be exclusively in the form of a large plasmid. This second division phase is also dependent on rounds of replication being allowed to terminate at 38° and is sensitive to low levels of penicillin. It is 80–90 min after the temperature shift before the second division phase starts and this lag is maintained even if rounds of replication have been completed prior to the temperature shift by amino acid starvation at 25°. The occurrence of this lag and the demonstration (using penicillin) of potential division sites at regular intervals along 11G filaments suggest that division is initiated some time before the actual division event.     

Escherichia coli mutants with temperature-sensitive synthesis of DNA

Summary Seven mutants of E. coli with temperature-sensitive synthesis of DNA have been isolated. Synthesis of RNA, protein and DNA precursors does not appear to be directly affected. The mutants can be divided into at least two groups on the basis of their pattern of DNA synthesis, their ability to support phage growth at 41° and their genetic mapping.Mutants of the first group are heterogeneous in their pattern of DNA synthesis at 40°. Some mutants cease DNA synthesis abruptly upon transfer to 40° and any residual DNA synthesis is barely detectable. In others there is substantial residual synthesis at 40°. All these Group 1 mutants are alike, however, in that they support the growth of phage T4 but not Lambda at 41°. Two mutants with barely detectable residual DNA synthesis carry DNA mutations which have been mapped by P1 transduction and show about 72% linkage to the malB locus. It has not yet proved possible to map accurately the mutants showing substantial residual synthesis, and the possibility that these mutations are in a different gene(s) has not been excluded.A single mutant has been placed in a second group. Like some Group 1 mutants it synthesizes substantial amounts of DNA at 40° before synthesis stops. However, unlike them it supports the growth of T4 and Lambda at 41°. The DNA mutation maps near the leu locus. Certain properties of this mutant are consistent with the idea that initiation of DNA synthesis is temperature-sensitive in this strain.Adapted from a dissertation presented in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported in part by U.S. Public Health Services Grant 5-TO1-GM00829 from the National Institute of General Medical Sciences and in part by U.S.P.H.S. research grant GM12524.     

Identification of optic lobe neurons of locusts by video films

The response characteristic of visual interneurons of the brain was studied in Locusta migratoria and Schistocerca gregaria. Alternating light and dark, moving dots, bars and striped patterns were used for stimulation (Fig. 3). These stimuli were recorded with a video system and replayed on TV-screens during the experiment to allow fast testing of the sensitivity of a neuron to different stimuli during the limited time of intracellular recording. Data were stored and analysed by computer. The neurons were anatomically identified by intracellular injection of Lucifer yellow. Neutral (non-visual) and several classes of spiking interneurons of the medulla and lobula sensitive to visual stimuli could be distinguished by anatomical and physiological characteristics (Figs. 1, 2). The visual cells respond either to light-on, or to light-off, flicker, moving small dots, bars or striped patterns (Figs. 2–6). One class is directionally sensitive to pattern movement either from back to front or into the reverse direction (horizontal cells; Figs. 7, 8) and may therefore be involved in optomotor flight control.Dedicated to Prof. Dr. Dietrich Burkhardt on the occasion of his 60th birthday     

Base pair substitution and frameshift mutagenesis induced by apurinic sites and two fluorene derivatives in a recA441 lexA (Def) strain

Summary One of the consequences of the induction of the Escherichia coli SOS system is the increased ability of the cells to perform mutagenesis. Induction of the SOS system is the result of derepression of a set of genes through a regulatory mechanism controlled by LexA and RecA. In response to an inducing signal, RecA is activated in a form that facilitates the proteolytic cleavage of LexA repressor. Previous works have shown that activated RecA plays a second role, i.e. it is required for the establishment of base pair substitution mutations promoted by UV irradiation. Using a forward mutatonal assay and recA441 lexA(Def) host bacteria, we show that the result can be extended not only to other mutagens promoting base pair substitution mutations (Apurinic sites, Ap sites and N-hydroxy-N-2-aminofluorene, N-OH-AF) but also mutagens promoting frameshift mutations (N-Acetoxy-N-2-acetylaminofluorene, N-AcO-AAF). In the recA441 lexA(Def) strain all the genes which are part of the lexA regulon, including recA itself, are expressed constitutively. The recA441 mutation allows RecA to acquire its activated form when the bacteria are grown at 42° C. We show that in such strains Ap sites or N-OH-AF induce a high level of mutations only when the bacteria are grown at 42° C. On the other hand, we show that N-AcO-AAF can promote mutations even at 30° C; the number of mutations being increased when the bacteria were grown at 42° C. Analysis of the mutants obtained at 30° C indicate that they belong to both type of mutations, UmuC-dependent or UmuC-independent. The much higher ability of N-AcO-AAF to induce RecA as compared to N-OH-AF strongly suggests that the former mutagen is able to induce at least partially the activated form of RexA441 even at 30°C in a strain which overproduces RecA, [lexA(Def)]. Furthermore, we show that the UmuC-independent type of mutagenesis induced by N-AcO-AAF depends on gene(s) that are part of the lexA regulon.     

Gonadal sex differentiation in Alligator mississippiensis,a species with temperature-dependent sex determination

Temperature of egg incubation determines sex in Alligator mississippiensis hatchlings. To define the timing and morphology of sexual differentiation, alligator gonads were examined histologically and ultrastructurally throughout embryogenesis. At the male-producing temperature (33° C), the onset of testis differentiation occurred in most embryos during developmental stages 21–22, when a number of somatic cells in the medulla of the gonad became enlarged, forming presumptive Sertoli cells. Some enlarged somatic cells were also observed at the female-producing temperature (30° C) during gonadogenesis, but they were less widespread than at 33° C. Ovarian differentiation at 30° C began slighlty later, during stage 22–23, and was characterised by proliferation of germs cells in the cortex of the gonad. Testis formation in alligators may depend upon presumptive Sertoli cells differentiating prior to a critical event in embryogenesis, such as germ cell proliferation and meiosis. If follows that ovary formation occurs if this requirement is not met, as at lower incubation temperatures.     

Changes in regulation of ribosome synthesis during different stages of the life cycle of Saccharomyces cerevisiae.

Summary Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34° C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34° C.     

Optic lobe projections of marginal ommatidia in Calliphora erythrocephala specialized for detecting polarized light

Summary The structure of ommatidia at the dorsal eye margin of the fly, Calliphora erythrocephala is specialized for the detection of the e-vector of polarized light. Marginal zone ommatidia are distinguished by R7/R8 receptor cells with large-diameter, short, untwisted rhabdomeres and long axons to the medulla. The arrangement of the R7 microvillar directions along the marginal zone is fan-shaped. Ommatidia lining the dorsal and frontal edge of the eye lack primary screening pigments and have foreshortened crystalline cones. The marginal ommatidia from each eye view a strip that is 5 °–20 ° contralateral to the fly's longitudinal axis and that coincides with the outer boundaries of the binocular overlap.Cobalt injection into the retina demonstrates that photoreceptor axons arising from marginal ommatidia define a special area of marginal neuropil in the second visual neuropil, the medulla. Small-field neurons arising from the marginal medulla area define, in turn, a special area of marginal neuropil in the two deepest visual neuropils, the lobula and the lobula plate. From these arise local assemblies of columnar neurons that relay the marginal zones of one optic lobe to equivalent areas of the opposite lobe and to midbrain regions from which arise descending neurons destined for the the thoracic ganglia.Optically, the marginal zone of the retina represents the lateral edge of a larger area of ommatidia involved in dorsofrontal binocular overlap. This binocularity area is also represented by special arrangements of columnar neurons, which map the binocularity area of one eye into the lobula beneath the opposite eye. Another type of binocularity neuron terminates in the midbrain.These neuronal arrangements suggest two novel features of the insect optic lobes and brain: (1) Marginal neurons that directly connect the left and right optic lobes imply that each lobe receives a common input from areas of the left and right eye, specialized for detecting the pattern of polarized light. (2) Information about the e-vector pattern of sky-light polarization may be integrated with binocular and monocular pathways at the level of descending neurons leading to thoracic motor neuropil.     

Radiation sensitivity of a mutant of Escherichia coli K-12 associated with DNA replication: Evidence for a new repair function

Summary The isolation and properties of a new radiation sensitive mutant of Escherichia coli K-12 are described which shows a correlation between radiation sensitivity and replication of irradiated DNA. The mutation, called rer, is located between argB and purD loci. The mutant, when grown in tryptone broth after irradiation, is sensitive to UV and -rays and incorporates little or no ³H-thymidine but in minimal glucose-salts medium both the radiation sensitivity and incorporation of ³H-thymidine remain identical to that of the parent strain. Studies with a temperature sensitive double mutant rer dnaC show that 1 hr incubation of irradiated cells at 42° C before their transfer to 30° C results in higher survival as compared to their incubation at 30° C only. It is suggested that rer controls the replication of irradiated DNA and thus regulates the coordination between replication and repair of DNA.     

Replication and maturation of phage P22 in a mutant of Salmonella typhimurium temperature sensitive in initiation of DNA replication.

Summary TB37 is a dna A-mutant of Salmonella typhimurium in which the initiation of DNA replication at the origin is stopped at 42°C. DNA synthesis in uninfected cells of this strain and in cells infected by phage P22 was followed by the pulse labelling technique. DNA replication ceases completely after about 50 minutes at the high temperature. After lytic infection with P22 (c2) at this time, DNA synthesis starts immediately and increases at a rate well comparable to the permissive control. Obviously the temperature sensitive function of the dnaA-product is dispensable for P22 DNA replication, especially for its initiation. This result is confirmed by the normal yield of phage particles under these conditions, provided that a late step in P22 maturation which naturally is temperature sensitive can proceed at low temperature. If TB37 is infected at 42°C with P22 wild type, an unexpected high rate of phage controlled DNA synthesis is observed. Preliminary results seem to indicate that the process of integration is a prerequisite for part of this synthesis.     

The dorsal eye of the dragonfly Sympetrum: specializations for prey detection against the blue sky

Dragonflies of the genus Sympetrum have compound eyes conspicuously divided into dorsal and ventral regions. Using anatomical, optical, electrophysiological, in-vivo photochemical and microspectrophotometrical methods, we have investigated the design and physiology of the dorsal part which is characterized by a pale yellow-orange screening pigment and extremely large facets. The upper part of the yellow dorsal region is a pronounced fovea with interommatidial angles approaching 0.3°, contrasting to the much larger values of 1.5°–2° in the rest of the eye. The dorsal eye part is exclusively sensitive to short wavelengths (below 520 nm). It contains predominantly blue-receptors with a sensitivity maximum at 420 nm, and a smaller amount of UV-receptors. The metarhodopsin of the blue-receptors absorbs maximally at 535 nm. The yellow screening pigment transmits longwavelength light (cut-on 580 nm), which increases the conversion rate from metarhodopsin to rhodopsin (see Fig. 11a). We demonstrate that because of the yellow pigment screen nearly all of the photopigment is in the rhodopsin state under natural conditions, thus maximizing sensitivity. Theoretical considerations show that the extremely long rhabdoms (1.1 mm) in the dorsal fovea are motivated for absorption reasons alone. A surprising consequence of the long rhabdoms is that the sensitivity gain, caused by pumping photopigment into the rhodopsin state, is small. To explain this puzzling fact we present arguments for a mechanism producing a gradient of rhodopsin concentration along the rhabdom, which would minimize saturation of transduction units, and hence improve the signal-to-noise ratio at high intensities. The latter is of special importance for the short integration time and high contrast sensitivity these animals need for spotting small prey at long distances.Abbreviations ERG electroretinogram - R rhodopsin - M metarhodopsin     

Antennal warm and cold receptors of the cave beetle,Speophyes lucidulus delar., in sensilla with a lamellated dendrite

Summary Electrophysiological examination of the 2 black-hair sensilla on the antennae of both larval stages of the cave beetle,Speophyes lucidulus, has revealed in each a pair of antagonistic thermal receptors (Fig. 1). Each sensillum was known to house the dendrites of 2 sensory cells which are associated with the extensively lamellated dendrite of a third (Corbière-Tichané 1971). One unit, a cold receptor, responds to temperature drops of 1 to 7 °C from initial temperatures between 9 and 14 °C with impulse frequencies up to 200 imp/s (Figs. 3, 4). Its antagonist, encountered less than 10% as often, is a warm receptor which responds with similar impulse frequencies to rapid rises in temperature from the same 9–14 °C (Figs. 3, 6). As indicated by the average gain of 24 imp/s for an increase of 1 °C in temperature drop, the cold unit appears almost twice as sensitive to sudden temperature change as the warm unit (14 (imp/s) °C). Examination of response scatter indicates that the average cold unit should on the basis of a single pair of responses be able to designate the greater of two temperature drops between 1 and 7 °C with 90% probability when they differ by 0.7 °C (Fig. 5). Though not yet definitive, evidence is accumulating that the third physiological unit is a dry air receptor.Abbreviations F impulse frequency in imp/s - Fc F as calculated - Fm F as measured - imp impulses - Pw partial pressure of water vapor in air - Ps saturation pressure of water vapor - r regression coefficient - T temperature - difference in Supported by the Deutsche Forschungsgemeinschaft, Sonder forschungsbereich 4, Projekt DThe authors wish to express their indebtedness to Dr. Renate Beinhauer, Faculty of Natural Sciences I — Mathematics, Univ. of Regensburg, for her help in applying statistical methods in determining resolving power.