2 The bacteria

Although free-living bacteria exist in huge numbers, relatively few species cause disease. The majority of these are well known and well studied; however, new pathogens continue to emerge and the significance of previously unrecognized infections becomes apparent. Good examples of the latter include infection with Legionella, the cause of Legionnaires’ disease, coronavirus-associated severe acute respiratory syndrome (SARS), and gastric ulcers associated with Helicobacter pylori infection.

Bacteria are single-celled prokaryotes, their DNA forming a long circular molecule, but not contained within a defined nucleus. Many are motile, using a unique pattern of flagella. The bacterial cell is surrounded by a complex cell wall and often a thick capsule. They reproduce by binary fission, often at very high rates, and show a wide range of metabolic patterns, both aerobic and anaerobic. Classification of bacteria uses both phenotypic and genotypic data. For clinical purposes, the phenotypic data are of most practical value, and rest on an understanding of bacterial structure and biology (see Fig. 32.15). Detailed summaries of members of the major bacterial groups are given in the Pathogen Parade (see online appendix).

Structure

Bacteria are ‘prokaryotes’ and have a characteristic cellular organization

The genetic information of bacteria is carried in a long, double-stranded (ds), circular molecule of DNA (Fig. 2.1). By analogy with eukaryotes (see Ch. 1), this can be termed a ‘chromosome’, but there are no introns; instead, the DNA comprises a continuous coding sequence of genes. The chromosome is not localized within a distinct nucleus; no nuclear membrane is present and the DNA is tightly coiled into a region known as the ‘nucleoid’. Genetic information in the cell may also be extrachromosomal, present as small circular self-replicating DNA molecules termed plasmids. The cytoplasm contains no organelles other than ribosomes for protein synthesis. Although ribosomal function is the same in both pro- and eukaryotic cells, organelle structure is different. Ribosomes are characterized as 70 S in prokaryotes and 80 S in eukaryotes (the ‘S’ unit relates to how a particle behaves when studied under extreme centrifugal force in an ultracentrifuge). The bacterial 70 S ribosome is specifically targeted by antimicrobials such as the aminoglycosides (see Ch. 33). Many of the metabolic functions performed in eukaryote cells by membrane-bound organelles such as mitochondria are carried out by the prokaryotic cell membrane. In all bacteria except mycoplasmas, the cell is surrounded by a complex cell wall. External to this wall may be capsules, flagella and pili. Knowledge of the cell wall and these external structures is important in diagnosis and pathogenicity and for understanding bacterial biology.

Figure 2.1 Diagrammatic structure of a generalized bacterium.

Bacteria are classified according to their cell wall as Gram-positive or Gram-negative

Gram staining is a basic microbiologic procedure for detection and identification of bacteria (see Ch. 32). The main structural component of the cell wall is a ‘peptidoglycan’ (mucopeptide or murein), a mixed polymer of hexose sugars (N-acetylglucosamine and N-acetylmuramic acid) and amino acids.

• In Gram-positive bacteria, the peptidoglycan forms a thick (20–80 nm) layer external to the cell membrane, and may contain other macromolecules.

• In Gram-negative species, the peptidoglycan layer is thin (5–10 nm) and is overlaid by an outer membrane, anchored to lipoprotein molecules in the peptidoglycan layer. The principal molecules of the outer membrane are lipopolysaccharides and lipoprotein (Fig. 2.2).

Figure 2.2 Construction of the cell walls of Gram-positive and Gram-negative bacteria.

The polysaccharides and charged amino acids in the peptidoglycan layer make it highly polar, providing the bacterium with a thick hydrophilic surface. This property allows Gram-positive organisms to resist the activity of bile in the intestine. Conversely, the layer is digested by lysozyme, an enzyme present in body secretions, which therefore has bactericidal properties. Synthesis of peptidoglycan is disrupted by beta-lactam and glycopeptides antibiotics (see Ch. 33).

In Gram-negative bacteria, the outer membrane is also hydrophilic, but the lipid components of the constituent molecules give hydrophobic properties as well. Entry of hydrophilic molecules such as sugars and amino acids is necessary for nutrition and is achieved through special channels or pores formed by proteins called ‘porins’. The lipopolysaccharide (LPS) in the membrane confers both antigenic properties (the ‘O antigens’ from the carbohydrate chains) and toxic properties (the ‘endotoxin’ from the lipid A component; see Ch. 17).

In the Gram-positive mycobacteria, the peptidoglycan layer has a different chemical basis for cross-linking to the lipoprotein layer, and the outer envelope contains a variety of complex lipids (mycolic acids). These create a waxy layer, which both alters the staining properties of these organisms (the so-called acid-fast bacteria) and gives considerable resistance to drying and other environmental factors. Mycobacterial cell wall components also have a pronounced adjuvant activity (i.e. they promote immunologic responsiveness).

External to the cell wall may be an additional capsule of high molecular weight polysaccharides (or amino acids in anthrax bacilli) that gives a slimy surface. This provides protection against phagocytosis by host cells and is important in determining virulence. With Streptococcus pneumoniae infection, only a few capsulated organisms can cause a fatal infection, but unencapsulated mutants cause no disease.

The cell wall is a major contributor to the ultimate shape of the organism, an important characteristic for bacterial identification. In general, bacterial shapes (Fig. 2.3) are categorized as either spherical (cocci), rods (bacilli) or helical (spirilla), although there are variations on these themes.

Figure 2.3 The three basic shapes of bacterial cells.

Many bacteria possess flagella

Flagella are long helical filaments extending from the cell surface, which enable bacteria to move in their environment. These may be restricted to the poles of the cell, singly (polar) or in tufts (lophotrichous), or distributed over the general surface of the cell (peritrichous). Bacterial flagella are quite different from eukaryote flagella, and the forces that result in movement are generated quite differently (being independent of adenosine triphosphate (ATP)). Motility allows positive and negative responses to chemical stimuli (chemotaxis). Flagella are built of protein components (flagellins), which are strongly antigenic. These antigens, the H antigens, are important targets of protective antibody responses.

Pili are another form of bacterial surface projection

Pili (fimbriae) are more rigid than flagella and function in attachment, either to other bacteria (the ‘sex’ pili) or to host cells (the ‘common’ pili). Adherence to host cells involves specific interactions between component molecules of the pili (adhesins) and molecules in host cell membranes. For example, the adhesins of Escherichia coli interact with fucose/mannose molecules on the surface of intestinal epithelial cells (see Ch. 22). The presence of many pili may help to prevent phagocytosis, reducing host resistance to bacterial infection. Although immunogenic, their antigens can be changed, allowing the bacteria to avoid immune recognition. The mechanism of ‘antigenic variation’ has been elucidated in the gonococci and is known to involve recombination of genes coding for ‘constant’ and ‘variable’ regions of pili molecules.

Nutrition

Bacteria obtain nutrients mainly by taking up small molecules across the cell wall

Bacteria take up small molecules such as amino acids, oligosaccharides and small peptides across the cell wall. Gram-negative species can also take up and use larger molecules after preliminary digestion in the periplasmic space. Uptake and transport of nutrients into the cytoplasm is achieved by the cell membrane using a variety of transport mechanisms, including facilitated diffusion which utilizes a carrier to move compounds to equalize their intra- and extracellular concentrations, and active transport where energy is expended to deliberately increase intracellular concentrations of a substrate. Oxidative metabolism (see below) also takes place at the membrane–cytoplasm interface.

Some species require only minimal nutrients in their environment, having considerable synthetic powers, whereas others have complex nutritional requirements. E. coli, for example, can be grown in media providing only glucose and inorganic salts; streptococci, on the other hand, will grow only in complex media providing them with many organic compounds. Nevertheless, all bacteria have similar general nutritional requirements for growth which are summarized in Table 2.1.

Table 2.1 Major nutritional requirements for bacterial growth

Element	Cell dry weight (%)	Major cellular role
Carbon	50	Molecular ‘building block’ obtained from organic compounds or CO₂
Oxygen	20	Molecular ‘building block’ obtained from organic compounds, O₂ or H₂O; O₂ is an electron acceptor in aerobic respiration
Nitrogen	14	Component of amino acids, nucleotides, nucleic acids and coenzymes obtained from organic compounds and inorganic sources such as NH4⁺
Hydrogen	8	Molecular ‘building block’ obtained from organic compounds, H₂O, or H₂; involved in respiration to produce energy
Phosphorus	3	Found in a variety of cellular components including nucleotides, nucleic acids, lipopolysaccharide (lps) and phospholipids; obtained from inorganic phosphates ()
Sulphur	1–2	Component of several amino acids and coenzymes; obtained from organic compounds and inorganic sources such as sulfates ()
Potassium	1–2	Important inorganic cation, enzyme cofactor, etc., obtained from inorganic sources

All pathogenic bacteria are heterotrophic

All bacteria obtain energy by oxidizing preformed organic molecules (carbohydrates, lipids and proteins) from their environment. Metabolism of these molecules yields ATP as an energy source. Metabolism may be aerobic, where the final electron acceptor is oxygen, or anaerobic, where the final acceptor may be an organic or inorganic molecule other than oxygen.

• In aerobic metabolism (i.e. aerobic respiration), complete utilization of an energy source such as glucose produces 38 molecules of ATP.

• Anaerobic metabolism utilizing an inorganic molecule other than oxygen as the final hydrogen acceptor (anaerobic respiration) is incomplete and produces fewer ATP molecules than aerobic respiration.

• Anaerobic metabolism utilizing an organic final hydrogen acceptor (fermentation) is much less efficient and produces only two molecules of ATP.

Anaerobic metabolism, while less efficient, can thus be used in the absence of oxygen when appropriate substrates are available, as they usually are in the host’s body. The requirement for oxygen in respiration may be ‘obligate’ or it may be ‘facultative’, some organisms being able to switch between aerobic and anaerobic metabolism. Those that use fermentation pathways often use the major product pyruvate in secondary fermentations by which additional energy can be generated. The interrelationship between these different metabolic pathways is illustrated in Figure 2.4.

Figure 2.4 Catabolic breakdown of glucose in relationship to final hydrogen acceptor.

The ability of bacteria to grow in the presence of atmospheric oxygen relates to their ability to enzymatically deal with potentially destructive intracellular reactive oxygen species (e.g. free radicals, anions containing oxygen, etc.) (Table 2.2). The interaction between these harmful compounds and detoxifying enzymes such as superoxide dismutase, peroxidase, and catalase is illustrated in Figure 2.5 (also see Ch. 9 and Box 9.2).

Table 2.2 Bacterial classification in response to environmental oxygen

Figure 2.5 Interaction between oxygen detoxifying enzymes.

Growth and division

The rate at which bacteria grow and divide depends in large part on the nutritional status of the environment. The growth and division of a single E. coli cell into identical ‘daughter cells’ may occur in as little as 20–30 min in rich laboratory media, whereas the same process is much slower (1–2 h) in a nutritionally depleted environment. Conversely, even in the best environment, other bacteria such as Mycobacterium tuberculosis may grow much more slowly, dividing every 24 h. When introduced into a new environment, bacterial growth follows a characteristic pattern depicted in Figure 2.6. After an initial period of adjustment (lag phase), cell division rapidly occurs, with the population doubling at a constant rate (generation time), for a period termed log or exponential phase. As nutrients are depleted and toxic products accumulate, cell growth slows to a stop (stationary phase) and eventually enters a phase of decline (death).

Figure 2.6 The bacterial growth curve. CFU, colony-forming units.

The septum is formed by an invagination of the cytoplasmic membrane and ingrowth of the peptidoglycan cell wall (and outer membrane in Gram-negative bacteria). Septation and DNA replication and genome segregation are not tightly coupled, but are sufficiently well coordinated to ensure that very few daughter cells do not have the correct complement of genomic DNA.

The mechanics of cell division result in reproducible cellular arrangements, when viewed by microscopic examination. For example, cocci dividing in one plane may appear chained (streptococci) or paired (diplococci), while division in multiple planes results in clusters (staphylococci). As with cell shape, these arrangements have served as an important characteristic for bacterial identification.

Bacterial growth and division are important targets for antimicrobial agents

Antimicrobials that target the processes involved in bacterial growth and division include:

• quinolones (ciprofloxacin and levofloxacin), which inhibit the unwinding of DNA by DNA gyrase during DNA replication

• the many inhibitors of peptidoglycan cell wall synthesis (e.g. beta-lactams such as the penicillins, cephalosporins and carbapenems, and glycopeptides such as vancomycin).

These are considered in more detail in Chapter 33.

Gene expression

Gene expression describes the processes involved in decoding the ‘genetic information’ contained within a gene to produce a functional protein or RNA molecule.

Most genes are transcribed into messenger RNA (mRNA)

The overwhelming majority of genes (e.g. up to 98% in E. coli) are transcribed into mRNA, which is then translated into proteins. Certain genes, however, are transcribed to produce ribosomal RNA species (5 S, 16 S, 23 S), which provide a scaffold for assembling ribosomal subunits; others are transcribed into transfer RNA (tRNA) molecules, which together with the ribosome participate in decoding mRNA into functional proteins.

Transcription

The DNA is copied by a DNA-dependent RNA polymerase to yield an RNA transcript. The polymerization reaction involves incorporation of ribonucleotides, which correctly base pair with the template DNA.

Transcription is initiated at promoters

Promoters are nucleotide sequences in DNA that can bind the RNA polymerase. The frequency of transcription initiation can be influenced by many factors, for example:

• the exact DNA sequence of the promoter site

• the overall topology (supercoiling) of the DNA

• the presence or absence of regulatory proteins that bind adjacent to and may overlap the promoter site.

Consequently, different promoters have widely different rates of transcriptional initiation (of up to 3000-fold). Their activities can be altered by regulatory proteins. Sigma factor (a component RNA polymerase) plays an important role in promoter recognition. The presence of several different sigma factors in bacteria enables sets of genes to be switched on simply by altering the level of expression of a particular sigma factor. This is particularly important in controlling the expression of genes involved in spore formation in Gram-positive bacteria.