Microorganisms around the globe have adapted to colonise environments over a broad range of temperature, from sub-zero to higher than 100°C, although no individual species can exist over a range greater than about 45°C. No particular species cannot exist outside of its temperature limits.
This property is being tested by the current trends in climate change. For instance, sea-ice plankton have been less able to perform phototrophic primary production due to the global reduction in polar sea ice. Since there is a greater propensity for microbial life in low-temperature environments, it is vitally important to understand the effects of rising temperatures on these and other psychrophiles.
It has already been established that cold-adapted microorganisms tend to grow faster at temperatures higher than their normal growth environment, in contrast to other thermal classes which grow quickest at their typical environmental temperature (Topt). This has been explained in terms of the kinetic effects on the cellular reactions of the psychrophiles.
However, research covering the temperature effects on microorganisms has been limited, with the majority of studies comparing two temperatures only. Many of the studies have been carried out on the Antarctic species Methanococcoides burtonii, which is regarded as a model cold-adapted species.
In a new study, the changes in the proteome of M. burtonii over its complete growth range have been studied by a research team based in Australia in an attempt to define the cellular process that are influenced by temperature, especially at the extremes of its range.
Isotope labelling quantitates proteins over temperature range
Ricardo Cavicchioli and colleagues from the University of New South Wales, Sydney, with Haluk Ertan also affiliated to Istanbul University, conducted a quantitative proteomics comparison for the microorganism grown at seven different temperatures.
They began at the lower extreme, -2°C (Tmin), continued at 1°C which is the temperature of its natural environment, and extended to 4, 10 and 16°C. Finally, they also studied the microorganism at 23°C (Topt) and its maximum temperature of 28°C (Tmax).
The length of time required for the lab cultures to grow to a particular degree (to an optical density at 620 nm of 0.25) increased as the temperature decreased, ranging from 20 days at Topt to 170 days at 1°C. The cultures at Tmax took 80 days.
Growth at Tmin was extremely slow. It could not be measured by optical density due to the formation of dense aggregates but took 720 days to provide sufficient biomass to provide the minimum amount of protein required for subsequent study.
The proteins from the soluble (enriched for cytoplasmic proteins), insoluble (membrane and membrane-associated proteins and macromolecular complexes) and cell supernatant (secreted proteins) fractions were harvested from five replicate cultures at each temperature. Then they were labelled using the 8-plex iTRAQ isotope labelling system.
Following enzymatic digestion, the peptides formed were analysed by two-dimensional LC coupled to a tandem mass spectrometer. The data were searched against a M. burtonii database for protein identification.
Three distinct physiological states defined by temperature
A total of 714 proteins were found to be common to all of the cultures, representing 29.4% of the 2431 protein-encoding genes. From this pool, the abundances of 336 proteins were changed by 1.2-fold or more compared with those at Topt, which was chosen as the reference.
The underlying trends of the protein abundances were deciphered using principal components analysis which revealed three distinct physiological states depending on the growth temperature.
The state of cold stress existed at -2°C, which showed the largest number of differentially expressed proteins, 64 being absent from the supernatant fraction compared with higher temperatures.
Cells growing at 23 and 28°C (Topt and Tmax, respectively) displayed signs of heat stress. However, there was also a marked similarity between the Tmin and Tmax profiles, suggesting that temperature stress, whether hot or cold, produced similar effects.
In between, the profiles at 1, 4, 10 and 16°C were reflective of cold adaptation, as opposed to cold stress, with those at 4 and 10°C being most similar. Not surprisingly, the profile at 1°C had some similarity to that at -2°C.
The researchers went on to discuss at length the proteins changes associated with each of the three growth states. For example, at -2°C, the levels of proteins associated with oxidative stress and electron transfer were increased, including catalase and flavoproteins.
These proteins were mostly different to those oxidative stress proteins that were elevated at 23 and 28°C, reflecting different causes of stress. Protein chaperones, which aid protein refolding, were also raised at these higher temperatures but were down-regulated at lower temperatures.
Further studies on more psychrophiles would be of interest to see if they too are subjected to oxidative stress at their thermal extremes of existence.
This is the first time that a microorganism has been studied across its whole growth range by quantitative proteomics. The results illustrate the value of the multiplexed labelling approach combined with principal components analysis, which should be applicable to other microorganisms.
Related links
Environmental Microbiology 2011 (Article in Press): "Defining the response of a microorganism to temperatures that span its complete growth temperature range (-2°C to 28°C) using multiplex quantitative proteomics"
Source: Proteomics and Genomics
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