Mutants: Enhanced Tolerance or Sensitivity to Temperature and pH Ranges

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Mutants: enhanced tolerance or sensitivity to temperature and pH ranges

Microorganisms have optimal environmental conditions under which they grow best. Classification of microorganisms in terms of growth rate dependence on temperature includes the thermopiles, the mesophiles and psychrophiles. Similarly, while most organisms grow well in neutral pH conditions, some organisms grow well under acidic conditions, while others can grow under alkaline conditions. The mechanism by which such control exists is being studied in detail. This will overcome the need to obtain mutants by a slow and unsure process of acclimatization.

When some organisms are subjected to high temperatures, they respond by synthesizing a group of proteins that help to stabilize the internal cellular environment. These, called heat shock proteins, are present in both prokaryotes and eukaryotes . Heat stress specifically induces the transcription of genes encoding these proteins. Comparisons of amino acid sequences of these proteins from the bacteria Escherichia coli and the fruit fly Drosophila show that they are 40%50% identical. This is remarkable considering the length of evolutionary time separating the two organisms.

Fungi are able to sense extracellular pH and alter the expression of genes. Some fungi secrete acids during growth making their environment particularly acidic. A strain of Asperigillus nidulans encodes a regulatory protein that activates transcription of genes during growth under alkaline conditions and prevents transcription of genes expressed in acidic conditions. A number of other genes originally found by analysis of mutants have been identified as mediating pH regulation, and some of these have been cloned. Improved understanding of pH sensing and regulation of gene expression will play an important role in gene manipulation for biotechnology .

The pH of the external growth medium has been shown to regulate gene expression in several enteric bacteria like Vibrio cholerae. Some of the acid-shock genes in Salmonella may turn out to assist its growth, possibly by preventing lysosomal acidification. Interestingly, acid also induces virulence in the plant pathogen (harmful microorganism) Agrobacterium tumefaciens.

Study of pH-regulated genes is slowly leading to knowledge about pH homeostasis, an important capability of many enteric bacteria by which they maintain intracellular pH. Furthermore, it is felt that pH interacts in important ways with other environmental and metabolic pathways involving anaerobiosis, sodium (Na+) and potassium (K+) levels, DNA repair, and amino acid degradation. Two different kinds of inducible pH homeostasis mechanisms that have been demonstrated are acid tolerance and the sodium-proton antiporter NhaA. Both cases are complex, involving several different stimuli and gene loci.

Salmonella typhimurium (the bacteria responsible for typhoid fever ) that grows in moderately acid medium (pH 5.56.0) induces genes whose products enable cells to retain viability (ability to live) under more extreme acid conditions (below pH 4) where growth is not possible. Close to 100% of acid-tolerant (or acid-adapted) cells can recover from extreme-acid exposure and grow at neutral pH. The inducible survival mechanism is called acid tolerance response. The retention of viability by acid-tolerant cells correlates with improved pH homeostasis at low external pH represents inducible pH homeostasis.

Cells detect external alkalization with the help of a mechanism known as the alkaline signal transduction system. Under such environmental conditions, an inducible system for internal pH homeostasis works in E. coli. The so-called sodium-proton antiporter gene NhaA is induced at high external pH in the presence of high sodium. The NhaA antiporter acts to acidify the cytoplasm through proton/sodium exchange. This allows the microorganism to survive above its normal pH range. As B. alkalophilus may have as many as three sodium-proton antiporters, it is felt that the number of antiporters may relate to the alkalophilicity of a species.

The search for extremophiles has intensified recently. Standard enzymes stop working when exposed to heat or other extreme conditions, so manufacturers that rely on them must often take special steps to protect (stabilize) the proteins during reactions or storage. By remaining active when other enzymes would fail, enzymes from extremophiles (extremozymes) can potentially eliminate the need for those added steps, thereby increasing efficiency and reducing costs in many applications.

Many routes are being followed to use the capacity that such extremophiles possess. First, the direct use of these natural mutants to grow and produce the useful products. Also, it is possible with recombinant DNA technology to isolate genes from such organisms that grow under unusual conditions and clone them on to a fast growing organism. For example, an enzyme alpha-amylase is required to function at high temperature for the hydrolysis of starch to glucose. The gene for the enzyme was isolated from Bacillus stearothermophilus, an organism that is grows naturally at 194°F (90°C), and cloned into another suitable organism. Finally, attempts are being made to stabilize the proteins themselves by adding some groups (e.g., disulfide bonds) that prevent its easy denaturation. This process is called protein engineering.

Conventional mutagenesis and selection schemes can be used in an attempt to create and perpetuate a mutant form of a gene that encodes a protein with the desired properties. However, the number of mutant proteins that are possible after alteration of individual nucleotides within a structural gene by this method is extremely large. This type of mutagenesis also could lead to significant decrease in the activity of the enzyme. By using set techniques that specifically change amino-acids encoded by a cloned gene, proteins with properties that are better than those obtained from the naturally occurring strain can be obtained. Unfortunately, it is not possible to know in advance which particular amino acid or short sequence of amino acids will contribute to particular changes in physical, chemical, or kinetic properties. A particular property of a protein, for example, will be influenced by amino acids quite far apart in the linear chain as a consequence of the folding of the protein, which may bring them into close proximity. The amino acid sequences that would bring about change in physical properties of the protein can be obtained after characterization of the three dimensional structure of purified and crystallized protein using x-ray crystallography and other analytical procedures. Many approaches are being tried to bring about this type of "directed mutagenesis" once the specific nucleotide that needs to be altered is known.

See also Bacterial adaptation; Evolutionary origin of bacteria and viruses; Microbial genetics; Mutations and mutagenesis