Growth, lipid accumulation, and fatty acid composition in obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts

Obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts were cultured in a carbon-rich medium at different temperatures to investigate whether growth parameters, lipid accumulation, and fatty acid (FA) composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to a lower temperature decreased their specific growth rate, but did not affect their biomass yield (Y_X/S). Obligate and facultative psychrophiles exhibited the highest Y_X/S. Acclimation to lower temperature decreased the lipid yield (Y_L/X) in mesophilic yeasts, but did not affect Y_L/X in facultative psychrophilic ones. Similar Y_L/X were found in both groups of psychrophiles, suggesting that lipid accumulation is not a distinctive characteristic of adaptation to permanently cold environments. The unsaturation of FAs was one major adaptive feature of the yeasts colonizing permanently cold ecosystems. Remarkable amounts of α-linolenic acid were found in obligate psychrophiles at the expense of linoleic acid, whereas it was scarce or absent in all the other strains. Increased unsaturation of FAs was also a general acclimatory response of facultative psychrophiles to a lower temperature. These results improve the knowledge of the responses enabling psychrophilic yeasts to cope with the cold and may be of support for potential biotechnological exploitation of these strains.     

Effects of temperature on the growth of psychrophilic bacteria from glaciers.

Growth of five strains of psychrophilic bacteria (four Arthrobacter and one Pseudomonas) isolated from glacial deposits was studied at different temperatures. Three strains were facultative psychrophiles, having an optimum temperature for growth at about 25-28 degrees C and a maximum at about 32-34 degrees C. The two Arthrobacter glacialis strains were found to be obligate psychrophiles with an optimum at 13-15 degrees C and a maximum at 18 degrees C. Arrhenius plots showed that A. glacialis could compete with the facultative psychrophilic bacteria only at 0 degrees C, that is, the temperature of its natural environment. The psychrophilic Arthrobacter species studied here are more resistant to thermal stress than are marine psychrophilic bacteria. For Arthrobacter, in contrast to Pseudomonas, temperatures above the optimum induced formation of filaments and abnormal cells. The culture turbidity increased 10 to 30 times, whereas viable count tended to decrease. The thermal block seems to prevent cell wall synthesis and septation, but at a different step for each species.     

Temperature range variants of Bacillus megaterium

Facultatively and obligately thermophilic variants were isolated from 3 out of 12 tested mesophilic Bacillus megaterium strains. The variants occurred at a frequency of 10^-8–10^-9. The ability to grow at elevated temperatures was cured by means of treatment with acridine orange. Stable revertants were isolated from facultatively and obligately thermophilic variants. An unknown type of megacin was produced by the facultative thermophiles. This megacin attacked mesophilic and obligately thermophilic strains. The thermophiles displayed a few divergent taxonomic characteristics but a close relationship between the strains was indicated by the megacin spectrum and sensitivity to phage. Arrhenius plots revealed that the strains could be considered as temperature range variants and that the temperature characteristic increased with growth at a higher temperature range. The case for a plasmid involvement in the phenomenon is discussed.Abbreviations M Mesophilic - Fp facultatively psychrophilic - Ft facultatively thermophilic - Ot obligately thermophilic     

The Bacteria in an Antarctic Peat

S ummary : Of a total of 119 strains of bacteria from 3 depths in an acid peat on Signy Island, S. Orkney, 52% belonged to the genus Brevibacterium. Twelve other genera were recorded of which numerically the most important were Arthrobacter, Cellulomonas, Kurthia and Micrococcus. 62% of the collection were psychrophilic, but only 4 strains were obligate psychrophiles. No pattern could be established for the various genera from different depths. The fine structure of an obligately psychrophilic pleomorphic rod from the peat is illustrated and discussed.     

Membrane lipid composition of obligately and facultatively alkalophilic strains of Bacillus spp.

The membrane lipids from two obligately and two facultatively alkalophilic strains of Bacillus spp. were characterized in a comparative study that included B. subtilis. Preparations of membrane lipids were made from pH 10.5-grown cells of all of the alkalophiles and from pH 7.5- or 7.0-grown cells of the two facultative strains and B. subtilis. The two obligate alkalophiles contained high ratios of membrane lipid to membrane protein, and the lipid fraction contained a high proportion of neutral lipid. These characteristics are probably not prerequisites for growth at very high pH since one or another of the facultative strains failed to show these properties at high pH. All of the alkalophiles contained appreciable amounts of squalene and C40 isoprenoids. Among the polar lipids, the alkalophiles all contained high concentrations of anionic phospholipids, including phosphatidylglycerol and especially large amounts of cardiolipin; phosphatidylethanolamine was the other major phospholipid. Small amounts of bis(monoacylglycero)phosphate were found in most, but not all, of the alkalophile preparations. Glycolipids and phosphoglycolipids were absent. The fatty acid composition of the total phospholipid and individual fractions revealed two features that distinguished between the obligate and facultative strains. Membranes from the obligately alkalophilic species contained a high concentration of branched-chain fatty acids, comparable to that in membranes from B. subtilis, as well as a relatively high content of unsaturated fatty acids. By contrast, the facultatively alkalophilic strains contained almost no unsaturated fatty acids and a lower concentration of branched-chain fatty acids than either the obligate alkalophiles or B. subtilis.     

Enzymatic profiles of 11 barophilic bacteria under in situ conditions: evidence for pressure modulation of phenotype.

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm = 101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.     

Enzymatic profiles of 11 barophilic bacteria under in situ conditions: evidence for pressure modulation of phenotype

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm = 101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.     

Psychrophilic Microorganisms from Areas Associated with the Viking Spacecraft

Microorganisms capable of growth at 7 C were enumerated and isolated from soil samples from the manufacture and assembly areas of the Viking spacecraft. Populations ranging from 4.2 X 10(3) to 7.7 X 10(6)/g of soil were isolated from the 15 soil samples examined. Temperature requirements were determined, and those growing at 3 C, but not at 32 C, were designated as obligate psychrophiles in this investigation. Populations of soil bacteria, including aerobic sporeformers, ranging from 1.5 X 10(2) to 9.8 X 10(5)/g were capable of growth at 3 C, but not at 32 C. Bacterial isolates were identified to major generic groups. No psychrophilic sporeformers were isolated from soil from the manufacture area, but psychrophilic sporeformers ranged from 0 to 6.1 X 10(3)/g from soil from the assembly area.     

Studies on psychrophilic bacteria in two lakes of different trophy

The number of bacteria capable of growth at low temperatures in two lakes was found to be subject to considerable variation throughout the year although the changes were not correlated with changes in ambient temperature. A correlation was, however, observed between temperature and ratio of number of psychrophiles to "total" count. All of the psychrophilic bacteria isolated from the studied lakes were able to grow at temperatures below 0 degrees C. Microorganisms isolated from eutrophic lakes were characterized by more pronounced stenothermy than those isolated from lake with low tropic level. These bacteria represented a type resembling a type resembling obligatory or ,,true" psychrophiles. Most of the isolates belonged to the genus Pseudomonas and to the Flavobacterium--Cytophaga group.     

Community Size and Metabolic Rates of Psychrophilic Sulfate-Reducing Bacteria in Arctic Marine Sediments

The numbers of sulfate reducers in two Arctic sediments with in situ temperatures of 2.6 and −1.7°C were determined. Most-probable-number counts were higher at 10°C than at 20°C, indicating the predominance of a psychrophilic community. Mean specific sulfate reduction rates of 19 isolated psychrophiles were compared to corresponding rates of 9 marine, mesophilic sulfate-reducing bacteria. The results indicate that, as a physiological adaptation to the permanently cold Arctic environment, psychrophilic sulfate reducers have considerably higher specific metabolic rates than their mesophilic counterparts at similarly low temperatures.     

Effect of Abrupt Temperature Shift on the Growth of Mesophilic and Psychrophilic Yeasts

Exponentially growing cultures of mesophilic and psychrophilic yeasts were subjected to abrupt changes in temperature. Temperature shifts made within the range in which the temperature characteristic, mu, is relatively constant (moderate temperatures) immediately induced growth at the normal exponential rate for the new temperature. Prior incubation at temperatures defined as moderate enabled some yeasts to grow for a few generations at temperatures higher than their normal maximal temperature for growth. Shifts made to or from temperatures above or below those in the moderate temperature range resulted in growth rates that were intermediate between the normal steady-state rates for the initial and final temperatures. A period of transient growth rate at the new temperature outside the moderate temperature range seems to be required before normal steady-state growth rates can be attained after such temperature shifts. The psychrophiles gave transient growth rates only below 10 C, whereas the mesophiles gave transient rates below 20 C. However, the psychrophiles cannot be distinguished from the mesophiles on the basis of the temperature characteristic, mu, which was found to be about 12,000 cal/mole for both types.     

Identity and physiology of a new psychrophilic eukaryotic green alga, <Emphasis Type="Italic">Chlorella</Emphasis> sp., strain BI,isolated from a transitory pond near Bratina Island,Antarctica

Permanently low temperature environments are one of the most abundant microbial habitats on earth. As in most ecosystems, photosynthetic organisms drive primary production in low temperature food webs. Many of these phototrophic microorganisms are psychrophilic; however, functioning of the photosynthetic processes of these enigmatic psychrophiles (the "photopsychrophiles") in cold environments is not well understood. Here we describe a new chlorophyte isolated from a low temperature pond, on the Ross Ice Shelf near Bratina Island, Antarctica. Phylogenetic and morphological analyses place this strain in the Chlorella clade, and we have named this new chlorophyte Chlorella BI. Chlorella BI is a psychrophilic species, exhibiting optimum temperature for growth at around 10 degrees C. However, psychrophily in the Antarctic Chlorella was not linked to high levels of membrane-associated poly-unsaturated fatty acids. Unlike the model Antarctic lake alga, Chlamydomonas raudensis UWO241, Chlorella BI has retained the ability for dynamic short term adjustment of light energy distribution between photosystem II (PS II) and photosystem I (PS I). In addition, Chlorella BI can grow under a variety of trophic modes, including heterotrophic growth in the dark. Thus, this newly isolated photopsychrophile has retained a higher versatility in response to environmental change than other well studied cold-adapted chlorophytes.     

A continuous culture study of an obligately psychrophilic Pseudomonas species

Summary The optimum temperatures for growth and respiration of an obligately psychrophilic Pseudomonas spec. were 14°C and 23°C, respectively. The maximum temperature for growth was between 19 and 20°C. When cells were grown in a chemostat with lactate as the growth-limiting substrate at a specific growth rate of 0.05 hr^-1 over a temperature range of 5–19°C, it was found that RNA concentration was lowest at 14°C. At lower temperatures the cells compensated the decrease of reaction rates by increasing the concentration of RNA and of respiratory enzymes. A temperature raise above 14°C also increased cellular RNA, which probably counteracted an impairment of protein synthesis. Above 18°C the RNA increase ceased, resulting in a rapid decrease of protein synthesis, until between 19 and 20°C growth ceased entirely. Cells grown at 14°C showed a linear increase of RNA content and values with growth rate, when this was varied from 0.025 to the maximum value of 0.2 hr^-1.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.     

Thermally adaptive tradeoffs in closely related marine bacterial strains

Time series studies have shown that some bacterial taxa occur only at specific times of the year while others are ubiquitous in spite of seasonal shifts in environmental variables. Here, we ask if these ubiquitous clades are generalists that grow over a wide range of environmental conditions, or clusters of strain‐level environmental specialists. To answer this question, vibrio strains isolated at a coastal time series were phylogenetically and physiologically characterized revealing three dominant strategies within the vibrio: mesophiles, psychrophiles and apparently generalist broad thermal range clades. Thermal performance curves from laboratory growth rate experiments help explain field observations of relative abundances: the mesophilic clade grows optimally at temperatures 16°C higher than the psychrophilic clade. Strains in the broad thermal range clade all have similar optimal growth temperatures but also exhibit temperature‐related tradeoffs with faster growth rates for warm temperature strains and broader growth ranges for strains from cool temperatures. Moreover, the mechanisms of thermal adaptation apparently differ based on evolutionary time scales: shifts in the temperature of maximal growth occur between deeply branching clades but thermal performance curve shape changes on shorter time scales. Thus, apparently ubiquitous clades are likely not generalists, but contain subclusters with distinct environmental preferences.     

Genomic signatures of obligate host dependence in the luminous bacterial symbiont of a vertebrate

The majority of bacteria engaged in bioluminescent symbiosis are environmentally acquired and facultatively symbiotic. A few enigmatic bioluminescent symbionts have not been successfully cultured, which has led to speculation that they may be obligately dependent on their hosts. Here, we report the draft genome of the uncultured luminous symbiont of an anomalopid flashlight fish, ‘Candidatus Photodesmus katoptron’. The genome of the anomalopid symbiont is reduced by 80% compared with close relatives and lacks almost all genes necessary for amino acid synthesis and for metabolism of energy sources other than glucose, supporting obligate dependence on the host for growth. ‘Candidatus Photodesmus katoptron’ is the first described obligate mutualistic symbiont of a vertebrate. Unlike most other obligate mutualists, the anomalopid symbiont genome has retained complete pathways for chemotaxis and motility as well as most genes involved in cell wall production, consistent with the hypothesis that these bacteria may be transmitted environmentally during an extra‐host phase.     

Manipulations of AMP metabolic genes increase growth rate and cold tolerance in Escherichia coli: implications for psychrophilic evolution

Diverse organisms have adapted to thrive at low temperatures (i.e., <20 °C, termed psychrophiles), colonizing the majority of earth's biosphere. In contrast with mesophiles (20-40 °C thermal range), all observed psychrophiles increase intracellular adenosine 5'-triphosphate concentrations as temperatures decline; this phenomenon has been described as an important compensatory mechanism to deal with decreases in thermal energy and molecular motion. We considered purine metabolic pathways in class gammaproteobacteria (n = 115) to investigate metabolic and evolutionary bases of this process. A survey of the KEGG database indicated that psychrophilic purine metabolic pathways tend to be enriched with de novo adenosine 5'-monophosphate (AMP) synthetic enzymes, whereas mesophiles tend to be enriched with AMP degradative enzymes. Function of the observed psychrophilic pathway structure was tested by engineering the mesophilic gammaproteobacterium Escherichia coli to reflect psychrophilic purine metabolism, specifically by expressing adenylosuccinate synthetase (purA) from the psychrophilic gammaproteobacterium, Psychrobacter cryohalolentis, in an AMP nucleosidase (amn)-deficient background. Modified E. coli was capable of growing up to ～70% faster at low temperatures and became up to ～10-fold more cold tolerant relative to wild type. These findings highlight potentially important transitional steps in psychrophilic evolution.     

Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria)

Bacterial strains affiliated to the phylogenetically shallow subcluster C (PnecC) of the Polynucleobacter cluster, which is characterized by a minimal 16S rRNA gene sequence similarity of approximately 98.5%, have been reported to occur as obligate endosymbionts of ciliates (Euplotes spp.), as well as to occur as free-living cells in the pelagic zone of freshwater habitats. We investigated if these two groups of closely related bacteria represent strains fundamentally differing in lifestyle, or if they simply represent different stages of a facultative endosymbiotic lifestyle. The phylogenetic analysis of 16S rRNA gene and 16S-23S ITS sequences of five endosymbiont strains from two different Euplotes species and 40 pure culture strains demonstrated host-species-specific clustering of the endosymbiont sequences within the PnecC subcluster. The sequences of the endosymbionts showed characteristics indicating an obligate endosymbiotic lifestyle. Cultivation experiments revealed fundamental differences in physiological adaptations, and determination of the genome sizes indicated a slight size reduction in endosymbiotic strains. We conclude that the two groups of PnecC bacteria represent obligately free-living and obligately endosymbiotic strains, respectively, and do not represent different stages of the same complex life cycle. These closely related strains occupy completely separated ecological niches. To our best knowledge, this is the closest phylogenetic relationship between obligate endosymbionts and obligately free-living bacteria ever revealed.     

Relationship of critical temperature to macromolecular synthesis and growth yield in Psychrobacter cryopegella

Most microorganisms isolated from low-temperature environments (below 4 degrees C) are eury-, not steno-, psychrophiles. While psychrophiles maximize or maintain growth yield at low temperatures to compensate for low growth rate, the mechanisms involved remain unknown, as does the strategy used by eurypsychrophiles to survive wide ranges of temperatures that include subzero temperatures. Our studies involve the eurypsychrophilic bacterium Psychrobacter cryopegella, which was isolated from a briny water lens within Siberian permafrost, where the temperature is -12 degrees C. P. cryopegella is capable of reproducing from -10 to 28 degrees C, with its maximum growth rate at 22 degrees C. We examined the temperature dependence of growth rate, growth yield, and macromolecular (DNA, RNA, and protein) synthesis rates for P. cryopegella. Below 22 degrees C, the growth of P. cryopegella was separated into two domains at the critical temperature (T(critical) = 4 degrees C). RNA, protein, and DNA synthesis rates decreased exponentially with decreasing temperatures. Only the temperature dependence of the DNA synthesis rate changed at T(critical). When normalized to growth rate, RNA and protein synthesis reached a minimum at T(critical), while DNA synthesis remained constant over the entire temperature range. Growth yield peaked at about T(critical) and declined rapidly as temperature decreased further. Similar to some stenopsychrophiles, P. cryopegella maximized growth yield at low temperatures and did so by streamlining growth processes at T(critical). Identifying the specific processes which result in T(critical) will be vital to understanding both low-temperature growth and growth over a wide range of temperatures.