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A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2-35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region. [ABSTRACT FROM AUTHOR]

The hok / sok locus has been shown to enhance the growth of bacteria in adverse growth conditions such as high temperature, low starting-culture densities and antibiotic treatment. This is in addition to their well-established plasmid-stabilization effect via post-segregational killing of plasmid-free daughter cells. It delays the onset of growth by prolonging the lag phase of bacterial culture, and increases the rate of exponential growth when growth eventually begins. This enables the cells adapt to the prevailing growth conditions and enhance their survival in stressful conditions. These effects functionally complement defective SOS response mechanism, and appear analogous to the growth effects of FtsZ in the SOS pathway. In this study, the role of FtsZ in the hok/ sok -induced changes in bacterial growth and cell division was investigated. Morphologic studies of early growth-phase cultures and cells growing under temperature stress showed elongated cells typical of FtsZ inhibition/deficiency. Both ftsZ silencing and over-expression produced comparable growth effects in control cells, and altered the growth changes observed otherwise in the hok / sok + cells. These changes were diminished in SOS-deficient strain containing mutant FtsZ. The involvement of FtsZ in the hok / sok -induced growth changes may be exploited as drug target in host bacteria, which often propagate antibiotic resistance elements. [ABSTRACT FROM AUTHOR]

The sfk1 (suppressor of four kinase) gene has been mainly studied in Saccharomyces cerevisiae, where it was shown to be involved in growth and thermal stress resistance. This gene is widely conserved within the phylum Ascomycota. Despite this, to date sfk1 has not been studied in any filamentous fungus. Previously, we found that the orthologous of sfk1 was differentially expressed in a strain of Penicillium roqueforti with an altered phenotype. In this work, we have performed a functional characterization of this gene by using RNAi-silencing technology. The silencing of sfk1 in P. roqueforti resulted in decreased apical growth and the promotion of conidial germination, but interesting, it had no effect on conidiation. In addition, the attenuation of the sfk1 expression sensitized the fungus to osmotic stress, but not to thermal stress. RNAmediated gene-silencing of sfk1 also affected cell wall integrity in the fungus. Finally, the silencing of sfk1 depleted the production of the main secondary metabolites of P. roqueforti, namely roquefortine C, andrastin A, and mycophenolic acid. To the best of our knowledge this is the first study of the sfk1 gene in filamentous fungi. [ABSTRACT FROM AUTHOR]

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat-stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat-induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat-induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered. [ABSTRACT FROM AUTHOR]

Heat resistance of microorganisms can be affected by different influencing factors. Although, the effect of heating rates has been scarcely explored by the scientific community, recent researches have unraveled its important effect on the thermal resistance of different species of vegetative bacteria. Typically heating rates described in the literature ranged from 1 to 20°C/min but the impact of much higher heating rates is unclear. The aim of this research was to explore the effect of different heating rates, such as those currently achieved in the heat exchangers used in the food industry, on the heat resistance of Escherichia coli. A pilot plant tubular heat exchanger and a thermoresistometer Mastia were used for this purpose. Results showed that fast heating rates had a deep impact on the thermal resistance of E. coli. Heating rates between 20 and 50°C/min were achieved in the heat exchanger, which were much slower than those around 20°C/s achieved in the thermoresistometer. In all cases, these high heating rates led to higher inactivation than expected: in the heat exchanger, for all the experiments performed, when the observed inactivation had reached about seven log cycles, the predictions estimated about 1 log cycle of inactivation; in the thermoresistometer these differences between observed and predicted values were even more than 10 times higher, from 4.07 log cycles observed to 0.34 predicted at a flow rate of 70mL/min and a maximum heating rate of 14.7°C/s. A quantification of the impact of the heating rates on the level of inactivation achieved was established. These results point out the important effect that the heating rate has on the thermal resistance of E. coli, with high heating rates resulting in an additional sensitization to heat and therefore an effective food safety strategy in terms of food processing. [ABSTRACT FROM AUTHOR]

The symbiotic association of corals and unicellular algae of the genus Symbiodinium in the southern Persian/Arabian Gulf (PAG) display an exceptional heat tolerance, enduring summer peak temperatures of up to 36 °C. As yet, it is not clear whether this resilience is related to the presence of specific symbiont types that are exclusively found in this region. Therefore, we used molecular markers to identify the symbiotic algae of three Porites species along >1000 km of coastline in the PAG and the Gulf of Oman and found that a recently described species, Symbiodinium thermophilum, is integral to coral survival in the southern PAG, the world's hottest sea. Despite the geographic isolation of the PAG, we discovered that representatives of the S. thermophilum group can also be found in the adjacent Gulf of Oman providing a potential source of thermotolerant symbionts that might facilitate the adaptation of Indian Ocean populations to the higher water temperatures expected for the future. However, corals from the PAG associated with S. thermophilum show strong local adaptation not only to high temperatures but also to the exceptionally high salinity of their habitat. We show that their superior heat tolerance can be lost when these corals are exposed to reduced salinity levels common for oceanic environments elsewhere. Consequently, the salinity prevailing in most reefs outside the PAG might represent a distribution barrier for extreme temperature-tolerant coral/Symbiodinium associations from the PAG. [ABSTRACT FROM AUTHOR]

The combined effect of heat treatment and electro-activated solution (EAS) on the heat resistance of spores of Clostridium sporogenes and Geobacillus stearothermophilus was assessed under various heating and exposure time combinations. The acid and neutral EAS showed the highest inhibitory activity, indicating that these solutions may be considered as strong sporicidal disinfectants. These EAS were able to cause a reduction of ≥6 log of spores of C. sporogenes at 60 °C in only 1 min of exposition. For G . stearothermophilus spores, a reduction of 4.5 log was observed at 60 °C in 1 min, while in 5 min, ≥7 log CFU/ml reduction was observed. Inoculated puree of pea and corn were used as a food matrix for the determination of the heat resistance of these spores during the treatments in glass capillaries. The inactivation kinetics of the spores was studied in an oil bath. Combined treatment by EAS and temperature demonstrated a significant decrease in the heat resistance of C . sporogenes . The D 100°C in pea puree with NaCl solution was 66.86 min while with acid and neutral EAS it was reduced down to 3.97 and 2.19 min, respectively. The spore of G . stearothermophilus displayed higher heat resistance as confirmed by other similar studies. Its D 130°C in pea puree showed a decrease from 1.45 min in NaCl solution down to 1.30 and 0.93 min for acid and neutral EAS, respectively. The differences between the spores of these species are attributable to their different sensitivities with respect to pH, Redox potential and oxygen. [ABSTRACT FROM AUTHOR]

Heat shock factor 1 (HSF-1) is activated by heat stress and induces the expression of heat shock proteins. However, the role of HSF-1 in thermotolerance remains unclear. We previously reported that heat stress reversibly reduces thrashing movement in Caenorhabditis elegans . In this study, we analyzed the function of HSF-1 on thermotolerance by monitoring thrashing movement. hsf-1 RNAi suppressed the restoration of thrashing reduced by heat stress. In contrast, hsf-1 knockdown cancelled prevention of movement reduction in insulin/IGF-1-like growth factor 1 receptor ( daf-2 ) mutant, but didn't suppress thrashing restoration in daf-2 mutant. In addition, hsf-1 RNAi accelerated the reduction of thrashing in heat-shocked wild-type C. elegans . And, daf-16 KO didn't accelerate the reduction of thrashing by heat stress. Taken together, these results suggest that HSF-1 prevents the reduction of thrashing caused by heat shock. [ABSTRACT FROM AUTHOR]

A comprehensive monitoring program has been performed at an industrial bioleaching heap at the Escondida mine in Chile since 2006, in order to study possible changes to the indigenous microbial population. In the initial stage of the bioleaching heap operation, the microbial population was dominated by mesophilic microorganisms. When the height of the heap increased, the microbial population changed from predominantly mesophilic to thermotolerant and moderate thermophilic microorganisms. The results of molecular analyses indicated that under these conditions Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans are the most abundant microorganisms. Those results obtained show that the highest ferrous iron oxidation activity at the third lift occurs at the same operational temperature window as that of the thermotolerant and moderate thermophiles. An increase in the expression of genes (including rfa F1) associated with cell envelope biogenesis was indicative of the active growth of L. ferriphilum . We conclude that the increasing heap height and the consequent increase in temperature within the heap triggered a change in the microbial community from mesophilic to moderate thermophilic microorganisms. Further, this shift had a relevant impact on the metallurgical performance of the industrial bioheap process at Escondida mine. [ABSTRACT FROM AUTHOR]

Thermal acclimation of soil organic matter (SOM) decomposition is frequently observed and has often been attributed to substrate depletion under warming, but other mechanisms, such as changes in microbial community structure and functioning, have received less attention. In order to determine whether shifts in microbial community structure and functioning are involved in thermal acclimation of SOM decomposition, a laboratory incubation experiment was conducted using an artificial forest soil. Samples were first subjected to different temperatures of 5, 15, and 25 °C during a 72-day pre-incubation period and then half of the microcosms from each pre-incubation temperature were incubated at 5 or 25 °C for a period of 11 days. Substantial thermal acclimation of SOM decomposition was observed, with the SOM decomposition in soils pre-incubated at higher temperatures being less sensitive to temperature. Along with the reduced temperature sensitivity in response to warming, significant changes in microbial community PLFAs, microbial biomass carbon (MBC), and the potential activities of 11 enzymes were also observed. Nevertheless, shifts in microbial community PLFAs and particular enzyme activities provided the most explanatory power for the decreased temperature sensitivity with warming, as revealed by a multivariate regression analysis. The microbial community structure shifts were mainly manifested as an increase in the relative abundance of Gram-positive bacteria and decreases in the relative abundances of Gram-negative bacteria and fungi. Microbial communities pre-incubated under lower temperatures experienced greater shifts in their structure. Substrate depletion did not occur in this short-term incubation experiment, since neither total organic carbon (TOC) nor dissolved organic carbon (DOC) decreased with increasing temperature. Our results suggest that shifts in microbial community structure and functioning may underlie the thermal acclimation of SOM decomposition and should be taken into account when predicting the response of soil CO2 efflux to global warming. [ABSTRACT FROM AUTHOR]

Abstract: The study characterized the influences of various combinations of process and product parameters namely, heating temperature (53, 55, 57.5, 60, 62°C), pH (2.0, 3.0, 4.5, 6.0, 7.0), and soluble solids (SS) (1.4, 15, 35, 55, 69°Brix) on the thermal inactivation of non-adapted and acid-adapted E. coli O157:H7 (HCIPH 96055) in a defined liquid heating medium (LHM). Acid adaptation was conducted by propagating cells in a gradually acidifying nutrient broth medium, supplemented with 1% glucose. The D values of non-adapted cells ranged from 1.43s (0.02min) to 304.89s (5.08min). Acid-adapted cells had D values that ranged from 1.33s (0.02min) to 2628.57s (43.81min). Adaptation did not always result in more resistant cells as indicated by the Log (D adapted/D non-adapted) values calculated in all combinations tested, with values ranging from −1.10 to 1.40. The linear effects of temperature and pH, and the joint effects of pH and SS significantly influenced the thermal resistance of non-adapted cells. Only the linear and quadratic effects of both pH and SS significantly influenced the D values of acid-adapted cells. Generally, the D values of acid-adapted cells decreased at SS greater than 55 °Brix, suggesting the possible cancelation of thermal cross protection by acid habituation at such SS levels. The relatively wide ranges of LHM pH and SS values tested in the study allowed for better examination of the effects of these factors on the thermal death of the pathogen. The results established in this work may be used in the evaluation, control and improvement of safety of juice products; and of other liquid foods with physicochemical properties that fall within the ranges tested in this work. [Copyright &y& Elsevier]

The Japanese government legally restricts entry of the bacterial fruit blotch disease of cucurbits caused by Acidovorax citrulli. However, the disease has occurred several times in Japan caused by infestations of melon, watermelon and wax gourd seeds with the pathogen. We prepared seeds infested with this pathogen for melon, cucumber, squash, wax gourd and bottle gourd and treated the seeds with dry heat for disinfection from the pathogen, varying the temperature and duration. Percentages of seed infested with the pathogen were determined after the dry heat treatments. The healthy or infested seeds (after the dry heat treatments) were sown in soil and the germination percentage evaluated. Complete disinfection of seeds of melon, cucumber and small-seeded squash (and without preventing germination) was achieved with some dry heat treatments, with 85°C for 3-5 day being preferable. The large-seeded squash, wax gourd and bottle gourd were sensitive to dry heat, additionally harsh conditions of ≥90°C and 7 day at 85°C were needed for complete disinfection. Thus, there were no feasible conditions for seed disinfection without affecting germination for the large-seeded crops. [ABSTRACT FROM AUTHOR]

Abstract: Free-living bacteria constantly monitor their ambient temperature. Drastic deviations elicit immediate protective responses known as cold shock or heat shock response. Many mammalian pathogens use temperature surveillance systems to recognize the successful invasion of a host by its body temperature, usually 37°C. Translation of temperature-responsive genes can be modulated by RNA thermometers (RNATs). RNATs form complex structures primarily in the 5′-untranslated region of their transcripts. Most RNATs block the ribosome binding site at low temperatures. Translation is induced at increasing temperature by melting of the RNA structure. The analysis of such temperature-dependent RNA elements calls for adequate test systems that function in the appropriate temperature range. Here, we summarize previously established reporter gene systems based on the classical β-galactosidase LacZ, the heat-stable β-galactosidase BgaB and the green fluorescent protein GFP. We validate these systems by testing known RNATs and describe the construction and application of an optimized bgaB system. Finally, two novel RNA thermometer candidates from Escherichia coli and Salmonella will be presented. [Copyright &y& Elsevier]

We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene (sharing 48% protein sequence identity to AldO) was identified, cloned and expressed in Escherichia coli. Following 6xHis tag purification, characterization revealed the protein to be a covalent flavoprotein of 47 kDa with a remarkably similar reactivity and substrate specificity to that of AldO. A steady-state kinetic analysis with a number of different polyol substrates revealed lower catalytic rates but slightly altered substrate specificity when compared to AldO. Thermostability measurements revealed that the novel AldO is a highly thermostable enzyme with an unfolding temperature of 84 °C and an activity half-life at 75 °C of 112 min, prompting the name HotAldO. Inspired by earlier studies, we attempted a straightforward, exploratory approach to improve the thermostability of AldO by replacing residues with high B-factors with corresponding residues from HotAldO. None of these mutations resulted in a more thermostable oxidase; a fact that was corroborated by in silico analysis. [ABSTRACT FROM AUTHOR]

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1 and PhaC2). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1 to evaluate the potential of the resulting protein as a 'thermostable LPE'. The mutated PhaC1 [PhaC1(STQK)] showed high thermal stability in synthesizing P(LA- co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding). [ABSTRACT FROM AUTHOR]

Our results revealed that a 7-log reduction of Salmonella can be achieved by exposing fresh chicken litter for 80.5 to 100.8, 78.4 to 93.1, and 44.1 to 63 min at 70, 75, and 80°C, respectively, depending on initial moisture contents. However, the aged chicken litter requires more heat treatment. [ABSTRACT FROM AUTHOR]

Abstract: Until recently, ohmic heating was commonly thought to kill microorganisms through a thermal effect. However a growing body of evidence suggests that non-thermal effects may occur. Our aim was to determine the kinetics of inactivation of Geobacillus stearothermophilus spores (ATCC 7953) under ohmic and conventional heating using a specially constructed test chamber with capillary sized cells to eliminate potential sources of error and ensure that identical thermal histories were experienced both by conventionally and ohmically heated samples. Ohmic treatments at frequencies of 60Hz and 10kHz were compared with conventional heating at 121, 125 and 130°C for four different holding times. Both ohmic treatments showed a general trend of accelerated spore inactivation. It is hypothesized that vibration of polar dipicolinic acid molecules (DPA) and spore proteins to electric fields at high temperature conditions may result in the accelerated inactivation. [Copyright &y& Elsevier]

Abstract: This study reports on the influence of heat and hydrogen peroxide combination on the inactivation kinetics of two heat resistant molds: Neosartorya fischeri and Paecilomyces variotii. Spores of different ages (1 and 4 months) of these molds were prepared and D-values (the time required at certain temperature/hydrogen peroxide combination to inactivate 90% of the mold ascospores) were determined using thermal death tubes. D-values found for P. variotii ranged from 1.2 to 25.1 s after exposure to different combinations of heat (40 or 60 °C) and hydrogen peroxide (35 or 40% w/w) while for N. fischeri they varied from 2.7 to 14.3 s after exposure to the same hydrogen peroxide concentrations and higher temperatures (60 or 70 °C). The influence of temperature and hydrogen peroxide concentration on the d-values varied with the genus of mold and their ages. A synergistic effect of heat and hydrogen peroxide in reducing D-values of Paecilomyces variotti and N. fischeri has been observed. In addition to strict control of temperature, time and hydrogen concentration, hygienic storage and handling of laminated paperboard material must be considered to reduce the probability of package’s contamination. All these measures together will ensure package’s sterility that is imperative for the effectiveness of aseptic processing and consequently to ensure the microbiological stability of processed foods during shelf-life. [Copyright &y& Elsevier]

Abstract: The objective of this study was to investigate the effect of nisin alone and in combination with heat (63 °C/5 min) on the inactivation of Listeria innocua in white cheese. Nisin was added at different concentrations (500, 1000, and 1500 IU ml−1) to pasteurized milk before curd formation. The curd was soaked for 24 h in 10% solution of brine containing ca 106 CFU ml−1 of a cocktail mixture of three strains of L. innocua. Part of the nisin treated samples were heat treated at 63 °C/5 min. Total mesophilic count (TMC), L. innocua survivors and changes in the pH of white cheese were monitored each 2 d for a period of 12 d of storage at 4 or 10 °C. Nisin at 500 IU ml−1 did not diminish TMC in white cheese compared to the control. The combination of heat and nisin (1000 or 1500 IU ml−1) exhibited a bacteriostatic effect on TMC throughout the storage period at 4 or 10 °C. Nisin at 500 IU ml−1 had a marginal inhibitory activity against L. innocua. However, nisin at 1000 and 1500 IU ml−1l resulted in a more than 2 log10 reduction in L. innocua count and the effect was more prominent at 10 °C. In comparison, the combination of nisin (1000 or1500 IU ml−1) and heat treatment exhibited a synergistic inhibitory activity against L. innocua, where a complete elimination of the organism was accrued after 6 and 8 d of storage at 10 and 4 °C. Therefore, nisin and heat combination could be used as a prudent hurdle to preclude the growth of Listeria in white cheese, especially under the condition of abused refrigeration conditions. [Copyright &y& Elsevier]

Abstract: Nine Lactococcus lactis c2 phages propagated on different hosts were screened for thermal resistance in skimmed milk. Pronounced variations in thermal resistance were found. Three phages displayed high sensitivity towards heat resulting in >8 log reductions after 70 °C for 5 min, whereas the most thermal resistant phages required 80 °C for 5 min to obtain the same reduction. Inactivation kinetics were determined for a thermo-sensitive and a thermo-resistant phage at 60–70 °C and 65–78 °C, respectively, using a submerged-coil system with extremely short heating-up times. Inactivation followed first-order kinetics with correlation coefficients of 0.96–0.99. D70-values of 12 s and 16.6 min were calculated for the most sensitive and resistant phage, respectively. Release of phage DNA from capsids, and disintegration of phage heads and tails were among the first morphological changes observed for moderately thermal inactivated lysates (15% phage inactivation) of the heat tolerant phage P635. [Copyright &y& Elsevier]

Abstract: Cronobacter sakazakii is an emerging foodborne pathogen that has been implicated in severe forms of meningitis, septicemia or necrotizing colitis in pre-term neonates. Although illness outbreaks (primarily associated with powdered infant formula, PIF) caused by this pathogen are rare, the case-fatality rate may reach 50%. Successful treatment of C. sakazakii infection is reliant upon clinical use of antibiotics (AB) such as ampicillin. Recent reports showed increased resistance of C. sakazakii to broad-spectrum antibiotics. The objective of this study was to evaluate the effect of extreme pH (3.5 for 30min or 11.25 for 5min), cold (4°C for 24h), heat (55°C for 5min), and desiccation (cells were dried at 40°C for 2h and held at 21°C for 4 d) stresses on susceptibility of five isolated strains of C. sakazakii to streptomycin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, tilmicosin, florfenicol, ampicillin, amoxicillin, vancomycin, ciprofloxacin and enrofloxacin. All unstressed strains of C. sakazakii were sensitive to streptomycin, gentamycin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and amoxicillin, but were moderately resistant or resistant to the rest. Exposing cells to alkaline or acidic stress did not change their sensitivity toward streptomycin, gentamycin, kanamycin or ciprofloxacin, but their resistance toward the other AB was increased. Cells stressed by desiccation showed increased sensitivity toward streptomycin, gentamicin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and doxycycline, but showed resistance toward the others. Cold-stressed cells were more sensitive to streptomycin, gentamicin, kanamycin, and ciprofloxacin compared with heat-stressed cells, but both heat and cold-stressed cells showed increased resistance toward all the other AB. Results obtained will help in understanding the effect of environmental stresses during processing on C. sakazakii susceptibility to AB. [Copyright &y& Elsevier]

Soil denitrification is a major source of nitrous oxide emission that causes ozone depletion and global warming. Low soil pH influences the relative amount of NO produced and consumed by denitrification. Furthermore, denitrification is strongly inhibited in pure cultures of denitrifying microorganisms below pH 5. Soils, however, have been shown to denitrify at pH values as low as pH 3. Here we used a continuous bioreactor to investigate the possibility of significant denitrification at low pH under controlled conditions with soil microorganisms and naturally available electron donors. Significant NO and NO reduction were observed for 3 months without the addition of any external electron donor. Batch incubations with the enriched biomass showed that low pH as well as low electron donor availability promoted the relative abundance of NO as denitrification end-product. Molecular analysis of the enriched biomass revealed that a Rhodanobacter-like bacterium dominated the community in 16S rRNA gene libraries as well as in FISH microscopy during the highest denitrification activity in the reactor. We conclude that denitrification at pH 4 with natural electron donors is possible and that a Rhodanobacter species may be one of the microorganisms involved in acidic denitrification in soils. [ABSTRACT FROM AUTHOR]

Steam soil disinfestation is now being reconsidered in open field and greenhouse horticulture for its efficiency in controlling or even eradicating soil borne pathogens, nematodes and weed seeds, while ensuring low ecological impact. Due to the high energy and labour costs of this treatment, technical solutions able to increase efficiency and to reduce workload are needed and are being sought. This paper presents the design, development and testing of two steam supply systems for soil disinfestation. Since soil conditions, in terms of texture and humidity, play an important role and significantly influence the efficacy of the treatment, determining the thermal properties of the soil and influencing steam diffusion, the equipment design followed an extensive laboratory experimentation phase during which the effect of soil parameters on heat efficiency was assessed. In a companion paper (), an innovative implement, based on the coupling of the two devices presented in this paper, has been built, connected to a caterpillar track machine, equipped with a steam generator and an automatic guidance system, and used in open field tests. [ABSTRACT FROM AUTHOR]

Abstract: Gene regulation in trypanosomatids occurs mainly by post-transcriptional mechanisms modulating mRNA stability and translation. We have investigated heat shock protein (HSP) 70 gene regulation in Trypanosoma cruzi, the causal agent of Chagas’ disease. The HSP70 mRNA’s half-life increases after heat shock, and the stabilization is dependent on protein synthesis. In a cell-free RNA decay assay, a U-rich region in the 3′ untranslated region (UTR) is a target for degradation, which is reduced when in the presence of protein extracts from heat shocked cells. In a transfected reporter gene assay, both the 5′- and 3′-UTRs confer temperature-dependent regulation. Both UTRs must be present to increase mRNA stability at 37°C, indicating that the 5′- and 3′-UTRs act cooperatively to stabilize HSP70 mRNA during heat shock. We conclude that HSP70 5′- and 3′-UTRs regulate mRNA stability during heat shock in T. cruzi. [ABSTRACT FROM AUTHOR]

Abstract: In previous work, we reported on the identification and purification of a multi-enzyme complex (MEC) from Bacillus licheniformis SVD1. The predominant activity within the MEC was xylanase activity and this study examined the effect of various environmental parameters such as pH, temperature, substrate concentration and compounds such as Mg2+, Mn2+, Fe2+, Zn2+, Ca2+, EDTA, SDS, xylose, xylobiose and ethanol on complexed xylanase activity. The pH optimum was found to be between pH 6.0 and 7.0 and the temperature optimum at 55°C. High levels of residual activity were present over a broad range of pH values. Enhancement of complexed xylanase activity was found in the presence of Mg2+ at 2mM and 10mM, while Ca2+ displayed a slight activation at 2mM but inhibition at 10mM. Mn2+, Fe2+, Zn2+, EDTA and SDS all displayed an inhibitory effect on complexed xylanase activity, with the greatest inhibition found in the presence of Mn2+. Xylose and xylobiose were found to enhance complexed xylanase activity up to 50%, which has not been reported in literature previously. Ethanol was found to inhibit complexed xylanase activity in a competitive manner, but 58% residual activity was still present at concentrations of 50g/l ethanol. Complexed xylanases from B. licheniformis SVD1, being uninhibited by products of degradation and only mildly inhibited by ethanol, would be suitable for use in biotechnological applications such as bioethanol production. [ABSTRACT FROM AUTHOR]

Abstract: Entrapped bacteria are used in several applications including food and beverage production, antibiotic production, and wastewater treatment. To date in order to determine the viability of entrapped bacteria, they have to be de-entrapped from the matrix first. However, the cell de-entrapment procedures such as matrix dissolution by acid or heating at high temperatures, may affect the viability of the cells. In this study, the uses of two quantitative approaches for in situ viability estimation of calcium alginate and phosphorylated poly(vinyl) alcohol (PPVA) entrapped Escherichia coli were investigated. Bioreducible tetrazolium salt (XTT) and adenosine triphosphate (ATP) based assays were used to determine microbial viability without the dissolution of the matrices (spherical beads). The data from both assays showed linearity and strong correlations between the viability signals and number of beads in which each bead contained a similar number of live cells. An application of XTT assay on the PPVA entrapped bacterial beads was an exception to these results. Effects of the acid and heat dissolution de-entrapment procedures on cell viability were also evaluated by using both assays and a traditional plate count method. The heating process showed the greatest reduction in bacterial viability when compared to the other de-entrapment procedures. The ATP assay is a more sensitive and less time consuming approach for viability estimation when compared to the XTT assay and traditional plate count method. Both XTT and ATP assays have potential for use in quantifying the viability of entrapped bacteria. [ABSTRACT FROM AUTHOR]

Porphyra haitanensis is one of the most economical nutritive marine algae; however, its production and quality are significantly jeopardized by high temperatures. Selection of heat-resistant strains will greatly reduce the economic risks and benefit to the nori industry. Three previously isolated and improved strains with a high yield were screened at 28°C and identified, of which one strain, ZS-1, showed significantly improved heat tolerance. Upon further characterizing of the cultures of the ZS-1 strain and the wild-type ( WT) strain at 28°C and 30°C, the ZS-1 conchospore germlings survived at rates of 69.9% and 59.6%, while the WT conchospore germlings survived at significantly lower rates of 15.9% and 6.7%, respectively, over a period of 15 days. Furthermore, ZS-1 conchospore germlings divided at significantly higher rates of 100% and 88.6% compared to the WT conchospore germlings with 90.4% and 63.8%, respectively. When the 35-day-old conchospore germlings were transferred from the optimal temperature of 24°C to higher temperatures of 28°C and 30°C, the ZS-1 blades sustained growth over a 25-day period without decay and increase of blade lengths with a factor of 18.5 and 10.3 times, respectively. The blade lengths of the WT germlings only increased by a factor of 1.7 and 0.9 times and began to decay after being cultured for 15 days at 28°C and 30°C. At 24, 28, and 30°C, the ZS-1 blades grew 3.4, 8.6, and 8.0 times faster than those of the WT. Evidently, ZS-1 is a fast-growing and heat-resistant strain compared to the WT strain and may offer an alternative for the nori industry. [ABSTRACT FROM AUTHOR]

Abstract: Heat resistance of spores is affected by many factors such as temperature, pH, water activity (aw) and others. Previous studies have reported that free fatty acids can affect the germination and growth of bacterial spores. In this study, we investigated the influence of free fatty acids in heating medium or in recovery medium on the heat resistance of spores of Bacillus cereus NTCC 11145 and Clostridium sporogenes Pasteur 79.3. Four free fatty acids were studied: palmitic, palmitoleic, stearic and oleic acids. During thermal treatments, the impact of these FFA in heating media was generally low, but the presence of free fatty acids in the recovery medium highly decreases bacterial spore apparent heat resistance, particularly with unsaturated fatty acids. A mathematical model was developed to describe and quantify the influence of free fatty acids in recovery media on the D-values. The z′FFA parameter values which quantify the impact of free fatty acids were determined. The variation of this parameter value according to the free fatty acid type was compared with MIC value variation given in the literature. The model enables the decrease in D-values in the presence of free fatty acids to be estimated. The high concentrations of free fatty acids in liver or canned duck may explain the microbial stability with low sterilization values applied. [Copyright &y& Elsevier]

Abstract: This study shows the behaviour of Listeria monocytogenes under heat and Pulsed Electric Fields (PEF) treatments, as well as the influence of sigB in bacterial resistance and recovery. Absence of the sigB gene resulted in a decreased heat tolerance, showing that σB influences L. monocytogenes heat survival. Heat treatments at 60°C (40s) caused sublethal membrane injuries in 99.99% of survivors. The repair of heat damage required energy production and lipid, protein, and RNA synthesis, and it lasted 6h. Furthermore, deletion of sigB did not affect the heat injuries repair. PEF resistance at pH 4.0 and 7.0 was not influenced by sigB. Sublethal damage after PEF treatments was only detected when PEF-treated cells had previously been heat-shocked (45°C/1h). The membrane repair only required energy production, and it was independent of sigB. Although both heat and PEF treatments have an effect on cellular membrane, the repair of the sublethal damages suggests different membrane targets, and thus we propose a different mechanism of inactivation by these food preservation technologies. [Copyright &y& Elsevier]

Abstract: The ability of Staphylococcus aureus to develop stress resistance responses was investigated. Exponential growth phase cells of S. aureus CECT 4459 were exposed to sublethal conditions (acid and alkaline pH, hydrogen peroxide, and heat) and then the acquisition of resistance to acid (pH 2.5), alkali (pH 12.0), hydrogen peroxide (50mM), and heat (58°C) was determined. Conditions resulting in the maximum development of homologous resistance (tolerance to the same stress), while preventing lethal effects in the population, were pH 4.5 (2h), pH 9.5 (30min), 0.05mM H2O2 (30min), and 45°C (2h). Under these adaptation conditions, times for the first decimal reduction (TFDC) to a lethal treatment at acid pH, alkaline pH, hydrogen peroxide, and heat were increased by a factor of 1.6, 2, 2, and 6, respectively. The presence of chloramphenicol or rifampicin in the adaptation medium completely abolished the increase in homologous resistance to acid pH and to hydrogen peroxide. By contrast, the development of homologous resistance to alkaline pH resulted independently of the presence of either chloramphenicol or rifampicin. S. aureus heat resistance increased in the presence of the inhibitors during the heat shock, but only partially. In some cases, the exposure to a given stress induced cross-protection against other agents. Protective combinations of sublethal stress and lethal agents were: acid pH–heat, acid pH–hydrogen peroxide, alkaline pH–hydrogen peroxide, heat–acid pH, and heat–hydrogen peroxide. These combinations of agents applied sequentially should be avoided in food-processing environments. [Copyright &y& Elsevier]

In a novel approach to separate the co-occurring freshwater cyanobacteria Microcystis and Synechoccous , published ecological characteristics are used to manipulate temperature and nutrient concentrations to successfully establish a unialgal Microcystis strain. The simple protocol has implications for future cyanobacterial culturing approaches and the establishment of new cyanobacteria strains. [ABSTRACT FROM AUTHOR]

Abstract: Food allergy is epidemic and prompts investigation to reduce allergic predisposition. It was hypothesized that heat-killed Escherichia coli injected intramuscularly (im) with or without interferon gamma (IFN-γ), reduces neonatal susceptibility to experimental egg allergy. Two litters of Yorkshire pigs were assigned to three intramuscular treatment groups (four/group): control (PBS), heat-killed E. coli with or without IFN-γ-expressing plasmid. Pigs were sensitized to ovomucoid (Ovm) by intraperitoneal injection with cholera toxin. To assess induction of allergy, pigs were fed egg white in yoghurt and assigned scores for allergic signs. Significantly fewer pigs developed allergy and passive cutaneous anaphylaxis in E. coli and E. coli +IFN-γ vs control groups. E. coli-treated pigs also had significantly lower frequency of mean clinical scores. E. coli and E. coli +IFN-γ groups did not differ. Serum antibody associated with IgG (H & L), IgG1, IgG2 or IgE all correlated but did not differ by treatment groups. Thus, treatment of neonatal pigs with heat-killed E. coli by im injection reduced susceptibility to allergic sensitization with Ovm. Inclusion of the type-1 cytokine, IFN-γ, had no additional effect. Results indicate a method for prophylaxis of allergy and suggest support for the “hygiene hypothesis”. [Copyright &y& Elsevier]

Salmonella typhimurium is a potentially intracellular pathogen and is responsible for thousands of reported cases of acute gastroenteritis and diarrhea each year. Although many successful physiological and genetic approaches have been taken to conclude the key virulence determinants encoded by this organism, the total number of uncharacterized reading frames observed within the S. typhimurium genome suggests that many virulence factors remain to be discovered. This study was conducted to evaluate the role of heat induced phage shock protein A (PspA), in the pathogenicity of S. typhimurium. The stress proteins detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identified specifically by immunoblotting with polyclonal antibody against PspA. PspA was produced in response to heat stress at 45°C and it was over-expressed at 65°C. At this temperature, the stressed bacterial cells producing PspA were more virulent (16 folds greater) to female 6–8 week-old Balb/c mice. Correspondency between decrease in LD50 and increase in PspA production during heat stress and lower pathogenicity in non-producing cells that emerged during stress at 55°C represents PspA as an important virulence factor in heat stressed S. typhimurium. [ABSTRACT FROM AUTHOR]

Abstract: Innovative methods less invasive for man and environment are needed to solve the problem of weeds, pests and diseases in cropping systems. Soil solarization is one practice that involves passively heating soil covered by a thin clear plastic film for several weeks during the periods of high solar radiation before the crop is planted. Soil solarization treatments were repeated to verify their long-term effectiveness on soil microbial biomass and its respiration activities, in addition to yield response and root-knot nematodes control in tomato and melon crops following soil heating. Experiments in greenhouse were carried out in Southern Italy (40°24′ N, 16°28′ E) during the summer of 1998, 1999 and 2000 on soil infested by the root-knot nematode Meloidogyne javanica. Crop yields and fruit quality of all vegetable crops were positively affected by repeating solarization for two and three years, compared to an untreated soil and soil treated once in 1998. Solarization was effective in controlling root-knot nematode damage, but solarization affected the soil biota populations and their activities as a consequence of repeated treatments. [Copyright &y& Elsevier]

Abstract: In a two year field study, the effect of varying intensities of sub-lethal heating on the efficiency of Brassica amendments in controlling viable populations of Macrophomina phaseolina and Fusarium oxysporum f sp. cumini was determined in an arid region of India. After 30d of dry summer exposure of pathogen infested soil, incorporation of mustard residues and oil cake (0.18% and 0.04% w/w) and then applying one irrigation caused significant reduction by 75.3–81.3% in viable counts of M. phaseolina that causes dry root rot of legumes and by 93.9% in counts of F.o. f. sp. cumini causing wilt of cumin (Cuminum cyminum L.) at 0–15 and 16–30cm depths. Increasing duration of summer exposure to 60d improved the reductions in viable propagules of M. phaseolina by 83.6–90.4% and in F.o. f. sp. cumini by 78.2–94.8% at same soil depths. At certain heat levels, reduction in viable population of Fusarium due to amendments and irrigation was greater than that recorded in Macrophomina. Significantly low levels of reduction in pathogenic propagules of Macrophomina (63.9–71.4%) and Fusarium (48.0–57.2%) under shade compared to unshaded conditions indicated that mild heating did not cause discernible weakening effect. In second season also, 89.2–91.5% and 78.5–95.8% reduction in counts of Macrophomina and Fusarium, respectively was achieved by the application of amendments after 60d of summer exposure at 0–30cm soil depth. These results suggested a new approach to improve the control of soil-borne plant pathogens in hot arid regions by combining prolonged sub-lethal heating, effective naturally available on-farm wastes as soil amendments and one summer irrigation. [Copyright &y& Elsevier]

Abstract: Enterobacter sakazakii is an emerging opportunistic pathogen associated with life-threatening illnesses in infants, with infant formula serving as the principal mode of transmission. In the present study, C. sakazakii (formely E. sakazakii) BCRC 13988 was subjected to various heat shock treatments (42–48°C for 5–15min). Its subsequent survival at 51°C and the leakage of intracellular materials was investigated. It was found that 47°C was the maximum growth temperature of the test organism. In addition, heat shock enhanced the thermal tolerance of C. sakazakii BCRC 13988. Within heat shock temperatures between 42 and 47°C, the thermal tolerance enhancing effect increased as the length or temperature of the heat shock treatment was increased. However, increasing the heat shock temperature to 48°C reduced the thermal tolerance enhancing effect. Among the various heat shocked cells examined, the 47°C–15min-heat shocked C. sakazakii exhibited the highest thermal tolerance. Moreover, electron micrograph analysis showed that heat shock treatment caused damage and disruption in C. sakazakii cells. There was a significant increase (P <0.05) in the leakage of nucleic acid and protein in the supernatant of the heat shocked cell suspension that increased as the temperature and duration of heat shock increased. [Copyright &y& Elsevier]

Abstract: Acidification of foods with organic acids, either by fermentation or by intentional addition, is an important and common mechanism for controlling foodborne pathogens in a diversity of food products. The objective of this work was to study thermal inactivation of Listeria innocua, an acid tolerant microorganism, at 52.5, 60.0 and 65.0 °C, at different pH values (4.5, 6.0 and 7.5), using three types of acid (lactic, acetic and hydrochloric) and three different plating media (Tryptic Soy Agar with 0.6% yeast extract—TSAYE; TSAYE plus 5% NaCl—TSAYE+5%NaCl; and Palcam Agar with selective supplement—Palcam Agar), according to a 34 factorial experimental design. Survival data experimentally obtained were fitted with a Gompertz-inspired model and kinetic parameters (shoulder, maximum inactivation rate—k max, and tail) were estimated for all conditions considered. The influence of temperature, pH, type of acid and enumeration media on kinetic parameters was assessed. Results showed that, with the exception of the type of acid, all the remaining factors and their combinations significantly affected the shoulder period and k max. In relation to tail, temperature and recovery media were the affectable factors. It was concluded that the survival of this bacteria is higher when combining low temperature with neutral pH, and when TSAYE is the enumeration medium. Bigelow-inspired models were successfully developed and describe accurately the temperature and pH effects on the kinetic parameters. [Copyright &y& Elsevier]

Abstract: Campylobacter jejuni is a Gram-negative, fragile, spiral bacterium, known worldwide to be a major cause of acute human enteritis. Like many other food-borne bacteria, campylobacters must be able to survive under diverse conditions both inside the host and in the environment. Understanding stress response mechanisms provides information necessary for improving food processing and strategies that enhance food safety as well as clarifying the pathogenesis of campylobacteriosis. We investigated the relation between stress response to starvation and pathogenic potential in C. jejuni. Starvation changed the morphology and physiology of C. jejuni cells. However, the lower metabolic activity of 5-h-starved culture was not a dormant state, but probably a viable but non-culturable (VBNC) form of the cells, since starved C. jejuni induced heat stress resistance. The health hazard potential of starved cells is still unclear. We showed that, in spite of starvation, C. jejuni survived in vitro within Caco-2 enterocites up to 4 days and caused systemic campylobacteriosis in vivo in a mouse model. However, bacterial numbers in investigated organs were significantly lower and the infection was resolved sooner. Our results show that nutrient insufficiency is responsible for C. jejuni transformation, influencing but not abolishing its survival and virulence properties while in the VBNC state. [Copyright &y& Elsevier]

We examined short- and long-term desiccation tolerance of 31 strains of thermophilic and hyperthermophilic Archaea and thermophilic phylogenetically deep-branching Bacteria. Seventeen organisms showed a significant high ability to withstand desiccation. The desiccation tolerance turned out to be species-specific and was influenced by several parameters such as storage temperature, pH, substrate or presence of oxygen. All organisms showed a higher survival rate at low storage temperatures (−20°C or below) than at room temperature. Anaerobic and microaerophilic strains are influenced negatively in their survival by the presence of oxygen during desiccation and storage. The desiccation tolerance of Sulfolobales strains is co-influenced by the pH and the substrate of the pre-culture. The distribution of desiccation tolerance in the phylogenetic tree of life is not domain specific. Surprisingly, there are dramatic differences in desiccation tolerance among organisms from the same order and even from closely related strains of the same genus. Our results show that tolerance of vegetative cells to desiccation is a common phenomenon of thermophilic and hyperthermophilic microorganisms although they originated from quite different non-arid habitats like boiling acidic springs or black smoker chimneys. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a foodborne pathogen that has the ability to survive relatively high temperatures compared with other nonsporulating foodborne pathogens. This study was performed to determine whether L. monocytogenes strains with relatively high heat resistances are adequately inactivated in a high-temperature, short-time pasteurization process (72°C for 15 s). To obtain heat-resistant strains, 48 strains were exposed to 55°C for up to 3 h. The energy of activation constant and inactivation constant of strains that survived best (strains 1E and NV8) were subsequently determined in a continuous-flow-through system. Strain Scott A was taken along as a reference. The 3 strains were cultured in whole milk and in brain heart infusion broth at 30 and 7°C. Strains 1E and NV8 were significantly more heat resistant than was strain Scott A after growth in brain heart infusion broth at 30°C and after growth in milk at 7°C. From the inactivation parameters, it was calculated that exposure to high-temperature, short-time pasteurization (72°C for 15 s) will result in 12.1-, 14.2-, and 87.5-1og reductions for the strains 1E, NV8, and Scott A, respectively. These results demonstrate that industrial pasteurization conditions suffice to inactivate the most heat-resistant L. monocytogenes strains tested in this study. [ABSTRACT FROM AUTHOR]

Aims: To compare conventional plate counting and indirect conductimetry as techniques for ranking the resistance of Salmonella spp. to processing stressors. Methods and Results: Forty Salmonella isolates were subjected to three separate stressors used in food processing; irradiation, heat and high hydrostatic pressure (HHP). Total viable counts (TVC) using conventional plate counts and time to detection (TTD) using indirect conductimetry were determined. A significant negative correlation between TVC and TTD was seen with irradiation ( P < 0·01) and heat ( P < 0·05) but not HHP. Conclusions: For a group of salmonellas, indirect conductimetry can rapidly determine a ranking of isolate sensitivity to irradiation and heat. However, for HHP, the results indicated that conventional plate counting alone cannot be used to determine sensitivity. Significance and Impact of the Study: The resistance of micro-organisms to processing systems must be ranked to allow the selection of appropriate isolates for process validation. TTD measurements allow rapid screening of salmonellas to rank isolates for resistance to irradiation and heat stress. However, following HHP, the TVC of survivors is independent of the time required for growth to a set cell density and therefore it cannot be used as the sole measure of relative stress resistance. [ABSTRACT FROM AUTHOR]

Desiccation-tolerant cells must either protect their cellular components from desiccation-induced damage and/or repair it upon rewetting. Subcellular damage to the anhydrobiotic cyanobacterium Chroococcidiopsis sp. CCMEE 029 stored in the desiccated state for 4 years was evaluated at the single-cell level using fluorescent DNA strand breakage labelling, membrane integrity and potential related molecular probes, oxidant-sensing fluorochrome and redox dye. Covalent modifications of dried genomes were assessed by testing their suitability as PCR template. Results suggest that desiccation survivors avoid/and or limit genome fragmentation and genome covalent modifications, preserve intact plasma membranes and phycobiliprotein autofluorescence, exhibit spatially-reduced ROS accumulation and dehydrogenase activity upon rewetting. Damaged cells undergo genome fragmentation, loss of plasma membrane potential and integrity, phycobiliprotein bleaching, whole-cell ROS accumulation and lack respiratory activity upon rewetting. The co-occurrence of live and dead cells within dried aggregates of Chroococcidiopsis confirms that desiccation resistance is not a simple process and that subtle modifications to the cellular milieu are required to dry without dying. It rises also intriguing questions about the triggers of dead cells in response to drying. The capability of desiccation survivors to avoid and/or reduce subcellular damage, shows that protection mechanisms are relevant in the desiccation tolerance of this cyanobacterium. [ABSTRACT FROM AUTHOR]

Thirty out of 8,000 different colony morphotypes isolated from soil samples of Isla de los Estados were selected based on their ability to produce antimicrobials. The significant influence of culture media and incubation temperature on antimicrobial production was proved, being LB medium and 8°C the conditions of choice. Most of the psychrotolerant isolates were phylogenetically related to Serratia proteamaculans (96.4–97.9%) while the psychrophilic isolated 8H1 was closely related to Pseudomonas sp. (90–94% similarity). Produced antimicrobials showed a promising wide spectrum of activity both against gram-positive and gram-negative pathogenic bacteria. They were suspected to be microcin-like compounds (Mw <2,000 Da) and showed a marked tolerance to heat (1 h in boiling water bath) and pH-treatments (1–12). Antimicrobial compounds also showed to partially keep their activity even after overnight freezing at −20 and −80°C and displayed a negative net charge at pH 8.0, a common feature of class II microcins. [ABSTRACT FROM AUTHOR]

The composition of fatty acids in 12 strains of the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus was analyzed by gas chromatography–mass spectrometry. Major FAs found in the profiles included i-15:0, i-17:0, ai-15:0, i-16:0, 16:0, ai-17:0, together with some minor components. Branched FAs were predominant, forming more than 80% of all FAs measured. Fast atom bombardment-mass spectrometry was used for analysis of unusual glycophospholipids, i.e., acylglycosylcardiolipins from genera Geobacillus and Alicyclobacillus and 1-(hydroxy(2-( O-acylglycosyl-oxy)hexadecyloxy)phosphoryloxy) hexadecan-2-yl esters of C15–C17 acids from genera Thermus and Meiothermus. Cloning and preliminary sequence analysis of 16S rDNA showed that these isolates belong to the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus. [ABSTRACT FROM AUTHOR]

Minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. Studies have shown that the MCM complex from the thermoacidophilic euryarchaeon Thermoplasma acidophilum (TaMCM) has some properties not reported in other archaeal MCM helicases. Here, the biochemical properties of the TaMCM are studied. The protein binds single-stranded DNA, has DNA-dependent ATPase activity and ATP-dependent 3′ → 5′ helicase activity. The optimal helicase conditions with regard to temperature, pH and salinity are similar to the intracellular conditions in T. acidophilum. It is also found that about 1,000 molecules of TaMCM are present per actively growing cell. [ABSTRACT FROM AUTHOR]

The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are compared. [ABSTRACT FROM AUTHOR]