Bacterial Biochemistry And Metabolism
Microbial metabolism consists of the biochemical reactions bacteria use to break down organic compounds as well as those they use to synthesize new bacterial parts from the resulting carbon skeletons. Energy for the new constructions is generated during the metabolic breakdown of the substrate. The occurrence of all biochemical reactions in the cell depends on the presence and activity of specific enzymes. Thus metabolism can be regulated in the cell either by regulating the production of an enzyme itself (a genetic type of regulation, in which production of the enzyme can be induced or suppressed by molecules present in the cell) or by regulating the activity of the enzyme (via feedback inhibition, in which the products of the enzymatic reaction or a succeeding enzymatic reaction inhibit the activity of the enzyme). Bacteria vary widely in their ability to use various compounds as substrates and in the end products
generated. A variety of biochemical pathways exis for substrate breakdown in the microbial world, and the particular pathway used determines the end product and final pH of the medium (Figure 1-9). Microbiologists use these metabolic differences as phenotypic markers in the identification of bacteria. Diagnostic schemes analyze each unknown microorganism for (1) utilization of a variety of substrates as a carbon source, (2) production of specific end products from various substrates, and (3) production of an acid or alkaline pH in the test medium. Thus knowledge of the biochemistry and metabolism of bacteria is important in the clinical laborator
Fermentation and Respiration
Bacteria use biochemical pathways to catabolize (break down) carbohydrates and produce energy by
two mechanisms-fermentation and respiration (commonly referred to as oxidation). Fermentation is an anaerobic process carried out by both obligate and facultative anaerobes. In fermentation the electron acceptor is an organic compound. Fermentation is less efficient in energy generation than respiration (oxidation) because the beginning substrate is not completely reduced; therefore all the energy in the substrate is not released. When fermentation occurs,a mixture of end products (e.g., lactate, butyrate,ethanol, and acetoin) accumulates in the medium.Analysis of these end products is particularly useful for the identification of anaerobic bacteria. End-product determination is also used in the Voges-Proskauer (VP) and methyl red tests, two important diagnostic tests used in the identification of the Enterobacteriaceae.([he term fermentation is often used loosely in the
diagnostic microbiology laboratory to indicate any type of utilization-fermentative or oxidative-of a
carbohydrate-sugar-with the resulting production of an acid pH.)
Respiration is an efficient energy-generating process in which molecular oxygen is the final electron acceptor. Obligate aerobes and facultative anaerobes carry out aerobic respiration, in which oxygen (02) is the final electron acceptor. Certain anaerobes can carry out anaerobic respiration, in which inorganic forms of oxygen, such as nitrate and sulfate, act as the final electron acceptors.
Biochemical Pathways from Glucose to Pyruvic Acid
The starting carbohydrate for bacterial fermentations or oxidations is glucose. When bacteria use
other sugars as a carbon source, they first convert the sugar to glucose, which is then processed by
one of three pathways. These pathways are designed to generate pyruvic acid, a key three-carbon intermediate. The three major biochemical pathways bacteria use to break down glucose to pyruvic acid are (1) the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (Figure 1-10), (2) the pentose phosphate pathway (Figure 1-11), and (3) the Entner-Doudoroff pathway (see Figure 1-11). Pyruvate can then be further processed either fermentatively or oxidatively. The three major metabolic pathways and their key characteristics are described in Box 1-1.
Anaerobic Utilization of Pyruvic Acid (Fermentation)
Pyruvic acid is a key metabolic intermediate. Bacteria process pyruvic acid further using a variety of fermentation pathways. Each pathway yields different end products, which can be analyzed and used as phenotypic markers (see Figure 1-9). Some of the fermentation pathways used by the microbes that inhabit the human body are as follows:
Alcoholic fermentation: The major end product is ethanol. This is the pathway used by yeasts when
they ferment glucose to produce ethanol.
Homolactic fermentation: The end product is almost exclusively lactic acid. All members of the Streptococcus genus and many members of the Lactobacillus genus ferment pyruvate using this
pathway. Heterolactic fermentation: Some lactobacilli use this mixed fermentation pathway, of which, in addition to lactic acid, the end products include carbon dioxide, alcohols, formic acid, and acetic acid.
Propionic acid fermentation: Propionic acid is the major end product of fermentations carried out by Propionibacterium acnes and some anaerobic non-spore-forming gram-positive bacilli. Mixed acid fermentation: Members of the genera Escherichia, Salmonella, and Shigella within the Enterobacteriaceae use this pathway for sugar fermentation and produce a number of acids as end products-lactic, acetic, succinic, and formic acids. The strong acid produced is the basis for the positive reaction on the methyl red test exhibited
by these organisms Butanediol fermentation: Members of the genera Klebsiella, Enterobacter, and Serratia within the Enterobacteriaceae use this pathway for sugar fermentation. The end products are acetoin (acetyl methyl carbinol) and 2,3-butanediol. Detection of acetoin is the basis for the positive VP reaction characteristic of these microorganisms. Little acid is produced by this pathway. Thus organisms that have a positive VP reaction usually have a negative reaction on the methyl red test, and vice versa. Butyric acid fermentation: Certain obligate anaerobes, including many Clostridium species, Fusobacterium, and Eubacterium, produce butyric acid as their primary end product along with acetic acid, carbon dioxide, and hydrogen
Aerobic Utilization of Pyruvate (Oxidation)
The most important pathway for the complete oxidation of a substrate under aerobic conditions is the Krebs or TCA (tricarboxylic acid) cycle. In this cycle, pyruvate is oxidized, carbon skeletons for biosynthetic reactions are created, and the electrons donated by pyruvate are passed through an electron transport chain and used to generate energy in the form of ATP. This cycle results in the production of acid and the evolution of carbon dioxide (Figure 1-12).
Carbohydrate Utilization and Lactose Fermentation
The ability of microorganisms to use various “sugars” (carbohydrates) for growth is an integral part of most diagnostic identification schemes. The fermentation of the sugar is usually detected by acid production and a concomitant change of color resulting from a pH indicator present in the culture medium. In general,bacteria ferment glucose preferentially over other sugars, and therefore glucose must not be present if the ability to ferment another sugar is being tested One of the important steps in classifying members of the Enterobacteriaceae family is the determination of the microorganism’s ability to ferment lactose. These bacteria are classified as either lactose fermenters or lactose nonfermenters. Lactose is a disaccharide consisting of one molecule of glucose and one molecule of galactose linked together by a galactoside bond. Two steps are involved in the utilization of lactose by a bacterium. The first step requires an enzyme, I)-galactoside permease, for the transport of lactose across the cell wall into the bacterial cytoplasm. The second step occurs inside the cell and requires the enzyme ~-galactosidase to break the galactoside bond, releasing glucose, which can then be fermented. Thus all organisms that can ferment
lactose can also ferment glucose