Bacterial metabolism: catabolism and anabolism

Trace a single molecule of glucose into a bacterium that has oxygen. The sugar is taken across the membrane and enters glycolysis, a chain of ten reactions in the cytoplasm that needs no oxygen at all. Glycolysis splits the six-carbon sugar into two molecules of pyruvate, each of three carbons, and pays back a net profit of two ATP and two NADH; it is the only energy-yielding pathway that virtually every bacterium shares. Each pyruvate is then oxidised, losing one carbon as carbon dioxide and handing a pair of hydrogens to NAD⁺, and the remaining two-carbon fragment is attached to coenzyme A to form acetyl-CoA. Acetyl-CoA enters the Krebs cycle, also called the citric acid cycle, a ring of eight reactions that strips off more hydrogens and releases two molecules of carbon dioxide per turn, four for each glucose; each turn also makes one ATP equivalent, two per glucose. The hydrogens collected as NADH and FADH₂ are now presented to the electron transport chain, a set of protein complexes and mobile carriers — quinones, flavoproteins and cytochromes — embedded in the bacterial membrane. The chain passes electrons from one carrier to the next, and at each step a little energy is used to pump protons across the membrane. The protons then flow back through a rotary enzyme, ATP synthase, and that flow drives the addition of phosphate to ADP to make ATP, a process called chemiosmosis. Oxygen waits at the end of the chain as the final electron and hydrogen acceptor and combines with the spent hydrogens to form water. The bookkeeping is the point: respiration can yield about thirty to thirty-two ATP per glucose, against two for fermentation, and almost all of the extra comes from the oxygen step at the end.

Fermentation happens when a cell must get rid of hydrogen but has no external acceptor, so it uses an organic molecule that it has just made. The commonest donor is pyruvate, the end product of glycolysis, so the reaction that follows glycolysis is what names each fermenting organism. Lactic acid bacteria, such as Lactobacillus species in yoghurt and some streptococci, reduce pyruvate to lactic acid. Yeasts and a few bacteria convert pyruvate to ethanol and carbon dioxide, which is why bread dough rises and beer ferments. Members of the Enterobacteriaceae, including Escherichia coli, Salmonella and Klebsiella, carry out mixed-acid fermentation, producing several acids together with carbon dioxide and hydrogen gas. In every case the chemical purpose is the same: to regenerate NAD⁺ from NADH so that glycolysis can keep running, because fermentation makes no ATP of its own beyond the two molecules glycolysis already earned. In the laboratory this is a gift to identification. A tube of phenol red broth containing a sugar and an inverted Durham tube turns yellow when acid is made and traps a bubble when gas is produced, so four or five sugars tested side by side give a pattern — a biochemical fingerprint — that helps name the organism. The same products matter in food and in the body. Acid preserves yoghurt, cheese, sauerkraut and silage by making the environment too acid for spoilage organisms. In the colon, fermentation of dietary fibre by the microbiota yields short-chain fatty acids such as butyrate, the preferred fuel of the cells lining the colon, which helps keep that barrier healthy. When antibiotics wipe out those bacteria, or when a patient cannot digest lactose, the same chemistry produces bloating, cramps and diarrhoea. In a wound or an abscess, the acid and gas made by fermenting anaerobes also help to explain the smell of the pus.

An enzyme test works as an identification tool because the enzyme is inherited, not learned. The gene that codes for urease, catalase or oxidase is part of the organism’s chromosome and is passed, unchanged for practical purposes, from one generation to the next; the enzyme does not appear because of the patient, the antibiotic or the temperature of the ward. Some enzymes are constitutive, meaning the cell makes them all the time, while others are inducible, meaning they are produced only when their substrate is present — but even an inducible enzyme can be provoked in the laboratory by supplying the substrate, so the test remains usable. Identification therefore consists of asking a panel of questions and comparing the pattern of answers with a table. A catalase test drops hydrogen peroxide on a colony: bubbling means the organism makes catalase and can break peroxide into water and oxygen, a reaction that separates staphylococci from streptococci. An oxidase test asks whether the organism carries cytochrome oxidase in its electron transport chain, which helps separate Pseudomonas from the enterobacteria. A rapid urease test on a gastric biopsy gives an answer about Helicobacter pylori within an hour, long before culture would, and a urease-positive Proteus in a urine specimen explains both the alkaline urine and the struvite stones that can follow, because the ammonia produced raises the pH and precipitates magnesium ammonium phosphate. Commercial strips such as API 20E extend the same principle to twenty or more sugars and substrates at once, and modern laboratories add mass spectrometry of ribosomal proteins. The logic never changes: a stable, inherited enzyme catalyses a known reaction, and the product of that reaction is the organism’s signature.

Using oxygen as the final acceptor is energetically excellent but chemically dangerous, because oxygen and its partly reduced forms are reactive. During normal respiration a small fraction of the electrons escape and form superoxide radicals, hydrogen peroxide and hydroxyl radicals, all of which attack DNA, proteins and membrane lipids. Aerobes and facultative anaerobes survive this because they carry defensive enzymes: superoxide dismutase converts superoxide to hydrogen peroxide, and catalase or peroxidase then converts hydrogen peroxide to water and oxygen. The catalase test exploits exactly this defence. An organism that lacks these enzymes — an obligate or strict anaerobe — cannot tolerate oxygen at all and is killed by air; this is why obligate anaerobes live in the colon, in deep wounds, in abscesses and in the soil, and why a specimen for anaerobic culture must be protected from air and handled quickly. Other groups sit in between. Facultative anaerobes, such as Escherichia coli, prefer oxygen but can ferment when it is absent; microaerophiles need a little oxygen but are damaged by the full atmospheric concentration; aerotolerant anaerobes ignore oxygen because they never use it. The distinction is not academic. The gas in a wound, the smell of an abscess, the site of an infection and the choice of specimen all follow from whether the organism in front of you can use oxygen or is poisoned by it, and a nurse who understands that will not leave an anaerobic specimen sitting on a bench.