the repeating-unit O-antigen polysaccharide (panel A). The LPS forms the surface of the outer membrane, whose inner layer is composed of phospholipids. The thin peptidoglycan (also called murein) of Gram-negative species is located in the periplasm between the inner and outer membrane.
In contrast, the cell wall of Gram-positive species lacks LPS and an outer membrane (panel B). The thick peptidoglycan is composed of multiple, cross–linked layers. A substantial amount of the Gram-positive cell wall is made up of another anionic polymer called wall teichoic acid (WTA, or TA) and lipoteichoic acid (LTA), both of which contain repeat units of ribitol or glycerol phosphate linked to amino sugars, sometimes to amino acids, such as D-alanine, and sometimes to ternary amines, such as phosphorylcholine. TA is covalently bonded to the peptidoglycan, whereas LTA is linked to a lipid anchor in the cellular membrane.
Other bacteria have cell walls with compositions that are a variation on one of these two types plus a few different components. For instance, the cell wall of mycobacteria, including Mycobacterium tuberculosis (the causative agent of tuberculosis), is similar to Gram-negative bacteria, but mycobacteria have a thick outer membrane comprised of two leaflets: an inner leaflet composed of arabinogalactan and mycolic acid (hence the name “mycobacteria”) and an outer leaflet composed of phosphoglycolipids (panel C). Further details can be found in The Physiology and Biochemistry of Prokaryotes, 4th Edition by David White, James Drummond, and Clay Fuqua (Oxford University Press, 2011).
It should be noted that the differences in bacterial surfaces and their components between Gram-positive and Gram-negative bacteria often dictate how they interact with their environments and, in particular, how they interact with their hosts. Importantly, the host immune system responds quite differently to these components. The properties conferred by these components necessitate that hosts have alternative strategies to recognize and combat Gram-negative versus Gram-positive pathogens. The presence of the outer membrane in Gram negatives also creates an extra challenge for the bacteria to export molecules on its surface and to secrete its toxins and other virulence factors into the external medium.
A different type of question is why different people respond differently to the same bacterium. In some cases, the human response can range all the way from a virtual lack of symptoms in some individuals to severe illness and death in others. Taking this issue one step further, prior exposure to one microbe can impact the nature and degree of response to subsequent exposure to another microbe. Indeed, it is now well-established that maternal exposure to pathogens or their components (e.g., LPS) modulates the immune competence and immune response of offspring in species ranging from insects to plants to birds and mammals. Clearly, it would be helpful to understand this range of reactions so that people who are most susceptible to an infectious agent could be quickly identified and given priority in treatment.
These and similar practical problems with controlling bacterial infections have driven a new interest in the interaction between bacteria and the human body at the molecular level. Fortunately, a cornucopia of new molecular tools and paradigms has become available that have made it possible to explore the host-pathogen interaction in a detailed way. It has even become more feasible to investigate infections that involve more than one species of bacteria or infections in which the bacterial pathogen acts in an area of the body, such as the mouth or small intestine, where there are many other bacteria that may influence the course of the disease.
The New Age of Genomics
Tremendous breakthroughs in DNA-sequencing technologies and bioinformatics now allow scientists to rapidly sequence, analyze, and study entire bacterial genomes (also known as genomics). There are now more than 13,000 complete genome sequences for bacteria, about half of which are from medically relevant bacteria, and there are over 175,000 incomplete or ongoing bacterial sequencing projects. These numbers and the pace of discovery are staggering considering that only a handful of complete bacterial genomes were available in the year 2000. At one time, it would have made sense to provide a list of available genome sequences, but additions to this list are coming so fast that the best solution is to provide the address of a website that keeps track of genomes that have been or are being sequenced: https://gold.jgi.doe.gov/.
Once a genome sequence becomes available, scientists examine the open reading frames (orfs) (i.e., the putative genes) one by one to try to assign each gene a name and function. In some cases, this process of genome annotation is easy, because the gene and its expressed protein product have already been characterized. In other cases, tentative identification of a gene is made on the basis of similarities to known genes or proteins present in other organisms that have been deposited into public repositories for DNA and protein sequences—DNA databases and protein databases, respectively. These automated assignments are useful but should be treated with some degree of caution, as many are based on relatively poor sequence matches. The best way to approach DNA sequence data is to realize that the function of a gene based on its sequence similarity to known genes is only a hypothesis that needs to be confirmed by more rigorous testing. A sobering fact is that even in the case of well-studied bacteria, such as E. coli and Salmonella, at least one-third of the genes in their genomes have no similarity to any known genes. A challenging job for future scientists is to determine the biological role of these genes of unknown function.
The way in which DNA sequence information can reveal surprising things about an organism is illustrated by the genome sequence of Borrelia burgdorferi, the spirochete that causes Lyme disease. Scientists noted that no genes corresponding to the usual iron-containing proteins normally found in bacteria were present in the genome of this organism. This suggested a radical hypothesis: that B. burgdorferi copes with the problem of low iron concentrations in the mammalian host by not using iron at all. Instead, its proteins use other metals that are abundant in humans, such as manganese. Scientists who were trained in an era in which every article on iron utilization by bacteria started by describing that all bacteria require iron were startled by this suggestion. Biochemical analyses confirmed, however, that indeed B. burgdorferi apparently does live without the need for iron, thus solving one problem most other pathogens have to confront: how to obtain iron in a host whose iron sequestration mechanisms keep the supply of available iron very low. As can be seen from this example, genome sequences not only provide valuable insights into unique bacterial metabolic processes, but also are excellent hypothesis-generating tools for understanding virulence mechanisms.
Along with the availability of complete genome sequences has come new technology enabling the high-throughput sequencing of an organism’s total RNA (RNA-seq, also called whole transcriptome shotgun sequencing). This technology has provided the means for scientists to measure the expression of thousands of genes in a single experiment. If the number of bacteria is high enough in a body site of a colonized or infected animal, RNA isolated from bacteria growing under these in vivo conditions can be obtained and analyzed by RNA-seq to assess the expression profile (i.e., the transcriptome) of different genes in the animal. Comparison of this expression profile with that obtained from bacteria grown outside of the host body has led, in turn, to the identification of genes that are only expressed during an infection and that might contribute to virulence in the host.
Another form of genomic analysis being used to detect and identify unknown pathogenic bacteria takes advantage of the fact that ribosomal RNA (rRNA) genes contain highly conserved regions of sequence separated by more variable regions. PCR primers that target conserved regions of the rRNA genes are used to amplify these genes from genomic DNA extracted from tissue suspected to contain an infectious organism. The PCR-amplified DNA is called an amplicon. Of course, if there are no bacteria present or if the level of bacterial DNA is too low, no PCR amplicon will be obtained; however, if an amplicon is obtained, its sequence can be determined and compared to the thousands of rRNA gene sequences now available in the DNA databases.
The variable regions of the 16S rRNA gene are particularly valuable in helping determine what known microbe is most similar to the one found in
