Importance of Type II Secretion for Survival of Legionella pneumophila in Tap Water and in Amoebae at Low Temperatures

Legionella pneumophila type II secretion mutants showed reduced survival in both tap water at 4 to 17°C and aquatic amoebae at 22 to 25°C. Wild-type supernatants stimulated the growth of these mutants, indicating that secreted factors promote low-temperature survival. There was a correlation between low-temperature survival and secretion function when 12 additional Legionella species were examined.     

Iron Superoxide Dismutase Protects against Chilling Damage in the Cyanobacterium Synechococcus species PCC7942

A strain of Synechococcus sp. PCC7942 lacking functional Fe superoxide dismutase (SOD), designated sodB⁻, was characterized by its growth rate, photosynthetic pigments, inhibition of photosynthetic electron transport activity, and total SOD activity at 0°C, 10°C, 17°C, and 27°C in moderate light. At 27°C, the sodB⁻ and wild-type strains had similar growth rates, chlorophyll and carotenoid contents, and cyclic photosynthetic electron transport activity. The sodB⁻ strain was more sensitive to chilling stress at 17°C than the wild type, indicating a role for FeSOD in protection against photooxidative damage during moderate chilling in light. However, both the wild-type and sodB⁻ strains exhibited similar chilling damage at 0°C and 10°C, indicating that the FeSOD does not provide protection against severe chilling stress in light. Total SOD activity was lower in the sodB⁻ strain than in the wild type at 17°C and 27°C. Total SOD activity decreased with decreasing temperature in both strains but more so in the wild type. Total SOD activity was equal in the two strains when assayed at 0°C.     

Identification of Proteins Involved in the Heat Stress Response of Bacillus cereus ATCC 14579

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42°C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50°C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42°C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis. These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus, which may result in increased survival in foods.     

Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42°C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30°C. Heat shock at 50°C reduced the viable cell population by 10³; however, pretreatment of the cells at 42°C made them more thermoresistant to exposure at 50°C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with ³⁵S upon heat shock at 42°C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.     

Levels of Endogenous Indole-3-acetic Acid in Achenes of Rosa rugosa during Dormancy Release and Germination

Indole-3-acetic acid (IAA) in highly purified extracts of rose achenes (Rosa rugosa var rubra) was quantified by means of ion-pair reversephase high performance liquid chromatography with spectrofluorimetric detection. Changes in IAA content were determined during a 14-week 4°C stratification, which leads to dormancy breakage, and during subsequent germination at 17°C. IAA was also determined in achenes stratified in parallel at 17°C, which does not induce release from dormancy. IAA decreased during the first 2 weeks of stratification both at 4°C and at 17°C. IAA remained low during the remaining 12 weeks of stratification at 4°C, whereas it continued to decrease in achenes kept at 17°C. An immediate increase in IAA during germination was followed by transients in the IAA level. The results suggest that IAA is without a regulating role in dormancy release although it seems to be involved in the germination process.     

Growth of Candida ingens on Supernatant from Anaerobically Fermented Pig Waste: Effects of Temperature and pH

Candida ingens, a pellicle-forming yeast utilizing volatile fatty acids, grew over a pH range of 4.1 to 6.0 on nonsterile supernatants from anaerobically fermented pig wastes; growth was inconsistent between pH 4.1 and 4.6. When ambient temperature above the pellicle was 21°C and the temperature of the medium was 29 to 32°C, a pH range of 4.8 to 5.0 gave yields of 1.90 to 3.31 g of dry matter per liter, and 0.059 to 0.065 mol of volatile fatty acids was utilized per liter. There was no advantage in utilization of volatile fatty acids and yield of dry matter in keeping the pH constant during a 24-h growth period. C. ingens grew at pH 4.8 and 5.0 when both ambient and medium temperatures were 30°C. When ambient temperature was 10°C, maximum yield and utilization of volatile fatty acids occurred at a medium temperature of 28 to 30°C.     

Effect of temperature conditioning on chilling injury of cucumber cotyledons: possible role of abscisic Acid and heat shock proteins

Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11-day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37°C for 6 hours followed by 4 days at 2.5 or 12.5°C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5°C, but not to 12.5°C, a nonchilling temperature. Exposure to 37°C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5°C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37°C and held at the nonchilling temperature of 12.5°C. There was no relationship between abscisic acid content following exposure to 12.5 or 37°C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42°C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42°C.     

Long-lived and short-lived heat-shock proteins in tobacco mesophyll protoplasts

We have studied modifications in the pattern of proteins synthesized by tobacco (Nicotiana tabacum var Maryland) mesophyll protoplasts when they are transferred from 25°C to 40°C. The synthesis of one group of proteins is practically unaffected by the heat shock. On the other hand, the synthesis of most other 25°C proteins is greatly reduced, while specific heat-shock proteins appear: 17 stable, neutral, major proteins, which are synthesized throughout the culture period at the higher temperature and which correspond to those observed in other organisms, and two basic proteins with a short lifetime and which are synthesized only during the first 2 hours of heat shock. We suggest that these latter proteins are regulatory peptides which intervene in the inhibition of 25°C syntheses.     

Concomitant changes in high temperature tolerance and heat-shock proteins in desert succulents

Raising the day/night air temperatures from 30°C/20°C to 50°C/40°C increases the high temperature tolerated by Agave deserti, Carnegiea gigantea, and Ferocactus acanthodes by 6°C to 8°C; the increase is about half completed in 3 days and fully completed in 10 days. A 25 to 27 kilodalton protein concomitantly accumulates for all three desert succulents upon transfer to 50°C/40°C, while accumulation of other heat “heat-shock” proteins is species specific. Some of the induced proteins are more abundant at 3 days, while others (including the 25-27 kilodalton protein) remain after completion of high temperature acclimation.     

Greening under High Light or Cold Temperature Affects the Level of Xanthophyll-Cycle Pigments, Early Light-Inducible Proteins, and Light-Harvesting Polypeptides in Wild-Type Barley and the Chlorina f2 Mutant

Etiolated seedlings of wild type and the chlorina f2 mutant of barley (Hordeum vulgare) were exposed to greening at either 5°C or 20°C and continuous illumination varying from 50 to 800 μmol m⁻² s⁻¹. Exposure to either moderate temperature and high light or low temperature and moderate light inhibited chlorophyll a and b accumulation in the wild type and in the f2 mutant. Continuous illumination under these greening conditions resulted in transient accumulations of zeaxanthin, concomitant transient decreases in violaxanthin, and fluctuations in the epoxidation state of the xanthophyll pool. Photoinhibition-induced xanthophyll-cycle activity was detectable after only 3 h of greening at 20°C and 250 μmol m⁻² s⁻¹. Immunoblot analyses of the accumulation of the 14-kD early light-inducible protein but not the major (Lhcb2) or minor (Lhcb5) light-harvesting polypeptides demonstrated transient kinetics similar to those observed for zeaxanthin accumulation during greening at either 5°C or 20°C for both the wild type and the f2 mutant. Furthermore, greening of the f2 mutant at either 5°C or 20°C indicated that Lhcb2 is not essential for the regulation of the xanthophyll cycle in barley. These results are consistent with the thesis that early light-inducible proteins may bind zeaxanthin as well as other xanthophylls and dissipate excess light energy to protect the developing photosynthetic apparatus from excess excitation. We discuss the role of energy balance and photosystem II excitation pressure in the regulation of the xanthophyll cycle during chloroplast biogenesis in wild-type barley and the f2 mutant.     

Corn mitochondrial protein synthesis in response to heat shock

Corn (Zea mays L., W23(N), OH43(N), and reciprocal single cross hybrid) seedling mitochondria respond to a 10°C temperature shift (27-37°C) by incorporating a greater amount of [³⁵S]methionine into acid-insoluble material than mitochondria incubated at the original growing temperature (27°C). This increase is in part manifested in the enhanced synthesis of a 52 kilodaltons protein. At both temperatures mitochondria of two inbreds and their reciprocal hybrids synthesize normal (N) cytoplasm proteins sensitive to chloramphenicol and insensitive to cyclohexamide treatment. The 52 kilodaltons protein is found in the supernatants of pelleted (15,000g, 5 min) mitochondria after heat shock. The role of this protein in the heat shock response is discussed in light of the implication of mitochondria as the primary cellular target to temperature stress.     

Photosynthetic and respiratory rates of two psychrophilic diatoms

The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3-10 and Nitzschia sp. K3-3 for cells grown at 0°C were 8 to 10 microliters O₂ evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10°C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0°C, and 6 to 10 for cells grown at 10°C. When cells grown at 0°C were immediately shifted to 10°C or cells grown at 10°C were shifted to 0°C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O₂ evolution showed much less immediate adaptation, especially on the up shift, 0° to 10°C. The chlorophyll a content of 0°C grown cells was about 0.5% of dry weight, in 10°C grown cells 1.3% (strain K3-10) and 2.2% (strain K3-3). In addition to a diminished chlorophyll a content in 0°C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10°C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0°C grown cells of Chaetoceros sp. strain K3-10 as compared to 10°C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0°C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins.     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Thermotolerance of isolated mitochondria associated with heat shock proteins

Mitochondria isolated from 2-day-old etiolated soybean (Glycine max) seedlings which had been subjected to various heat shock treatments, i.e. (A) 28°C (2 h), (B) 38°C (2 h), (C) 38°C (2 h)-42.5°C (0.5 h), and (D) 38°C (2 h)-42.5°C (0.5 h)-28°C (4 h), were monitored for O₂ uptake using an oxygen electrode. Mitochondria isolated after all four heat shock treatments were active in O₂ consumption at 28°C in response to succinate and ADP (derived P/O ratios were 1.6, 1.7, 1.3, and 1.3, respectively.) The mitochondria from all four treatments were also active in O₂ uptake at 42.5°C. However, only mitochondria isolated after treatment (C) were tightly coupling at 42.5°C (derived ADP/O ratio was about 1.4). Combined with our earlier findings on the subcellular localization of heat shock proteins, our present data demonstrate that association of heat shock proteins with mitochondria by treatment (C) enables them to phosphorylate at 42.5°C (i.e. they become thermotolerant). Isolated mitochondria from treatment (C) and treatment (A) were compared by electron microscopy. They appeared to be very similar and no significant ultrastructural differences were noted.     

Early and late heat shock proteins in wheats and other cereal species

Coleoptiles and roots of 3-day-old seedlings from five cereal species (Triticum aestivum L., T. durum Desf., Hordeum vulgare L., Secale cereale L., and Triticale) respond to heat shock at 40°C by synthesizing a new set of 13 strong bands (as revealed by one-dimensional sodium dodecyl sulfate gel electrophoresis) as well as some 20°C proteins. Heat shock proteins (HSPs) belong to three different size groups: high molecular mass HSPs in the 103 to 70 kilodalton range, intermediate molecular mass HSPs in the 62 to 32 kilodalton range, and low molecular mass HSPs about 17 to 16 kilodalton in size. At the beginning of the heat shock coleoptiles show a reduced ability to synthesize intermediate molecular mass HSPs but after 4 hours at 40°C they exhibit fully developed HSP patterns identical to that found in roots. Synthesis of early HSPs declines after 7 hours of treatment followed by the appearance of a new set of 12 protein bands (late HSPs) in the ranges 99 to 83, 69 to 35, and 15 to 14 kilodaltons. After 12 hours at 40°C, three other late HSPs of 89, 45, and 38 kilodalton are induced. The induction of late HSPs after 7 hours at 40°C appears to be associated with an enhancement of radioactive methionine incorporation into proteins.     

Abundances and Distributions of the Dominant nifH Phylotypes in the Northern Atlantic Ocean

Understanding the factors that influence the distribution and abundance of marine diazotrophs is important in order to assess their role in the oceanic nitrogen cycle. Environmental DNA samples from four cruises to the North Atlantic Ocean, covering a sampling area of 0°N to 42°N and 67°W to 13°W, were analyzed for the presence and amount of seven nifH phylotypes using real-time quantitative PCR and TaqMan probes. The cyanobacterial phylotypes dominated in abundance (94% of all nifH copies detected) and were the most widely distributed. The filamentous cyanobacterial type, which included both Trichodesmium and Katagnymene, was the most abundant (51%), followed by group A, an uncultured unicellular cyanobacterium (33%), and gamma A, an uncultured gammaproteobacterium (6%). Group B, unicellular cyanobacterium Crocosphaera, and group C Cyanothece-like phylotypes were not often detected (6.9% and 2.3%, respectively), but where present, could reach high concentrations. Gamma P, another uncultured gammaproteobacterium, was seldom detected (0.5%). Water temperature appeared to influence the distribution of many nifH phylotypes. Very high (up to 1 × 10⁶ copies liter⁻¹) nifH concentrations of group A were detected in the eastern basin (25 to 17°N, 27 to 30°W), where the temperature ranged from 20 to 23°C. The highest concentrations of filamentous phylotypes were measured between 25 and 30°C. The uncultured cluster III phylotype was uncommon (0.4%) and was associated with mean water temperatures of 18°C. Diazotroph abundance was highest in regions where modeled average dust deposition was between 1 and 2 g/m²/year.     

Effect of Temperature on Pratylenchus penetrans Development

Reproduction and development of Pratylenchus penetrans were studied on genetically transformed ladino clover roots. Solitary females developing on transformed roots in nutrient gellan gum medium (pH 5.5) deposited 1.2, 1.5, 1.6, 1.8, and 2.0 eggs per day at the respective temperatures of 17, 20, 25, 27, and 30 °C. The number of eggs deposited was highly correlated with temperature. A reduction in egg-laying rates at the start of hatching was observed at all temperatures. Juvenile mortality was higher at 17 °C (50.4%), 20 °C (50.3%), and 30 °C (58.4%) than at 25 °C (34.6%) and 27 °C (37.6%). Life-cycle (egg deposition to egg deposition) duration was 46, 38, 28, 26, and 22 days at the respective temperatures. The developmental zero degrees (°C) and the effective accumulative temperatures (degree-days) required for hatching, female emergence, and onset of oviposition (completion of one generation) of P. penetrans were estimated to be 2.7 and 200, 4.2 and 548, and 5.1 and 564, respectively. Pratylenchus penetrans reproduces over a wide range of temperatures.     

Temperature Effects on Development of Meloidogyne Enterolobii and M. Floridensis

Meloidogyne enterolobii and M. floridensis are virulent species that can overcome root-knot nematode resistance in economically important crops. Our objectives were to determine the effects of temperature on the infectivity of second-stage juveniles (J2) of these two species and determine differences in duration and thermal-time requirements (degree-days [DD]) to complete their developmental cycle. Florida isolates of M. enterolobii and M. floridensis were compared to M. incognita race 3. Tomato cv. BHN 589 seedlings following inoculation were placed in growth chambers set at constant temperatures of 25°C, and 30°C, and alternating temperatures of 30°C to 25°C (day–night). Root infection by the three nematode species was higher at 30°C than at 25°C, and intermediate at 30°C to 25°C, with 33%, 15%, and 24% infection rates, respectively. There was no difference, however, in the percentages of J2 that infected roots among species at each temperature. Developmental time from infective J2 to reproductive stage for the three species was shorter at 30°C than at 25°C, and 30°C to 25°C. The shortest time and DD to egg production for the three species were 13 days after inoculation (DAI) and 285.7 DD, respectively. During the experimental timeframe of 29 d, a single generation was completed at 30°C for all three species, whereas only M. floridensis completed a generation at 30°C to 25°C. The number of days and accumulated DD for completing the life cycle (from J2 to J2) were 23 d and 506.9 DD for M. enterolobii, and 25 d and 552.3 DD for M. floridensis and M. incognita, respectively. Exposure to lower (25°C) and intermediate temperatures (30°C to 25°C) decreased root penetration and slowed the developmental cycle of M. enterolobii and M. floridensis compared with 30°C.     

Effect of Turbulent-Flow Pasteurization on Survival of Mycobacterium avium subsp. paratuberculosis Added to Raw Milk

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72°C in duplicate trials. No strains survived at 72°C for 15 s; and only one strain survived at 69°C. Means of pooled D values (decimal reduction times) at 63 and 66°C were 15.0 ± 2.8 s (95% confidence interval) and 5.9 ± 0.7 s (95% confidence interval), respectively. The mean extrapolated D_72°C was <2.03 s. This was equivalent to a >7 log₁₀ kill at 72°C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6°C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72°C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log₁₀. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2° above 72°C.