binds to a ligand, its RNA can take on a form that either impedes the translation of the mRNA (translational riboswitch)—in which case the entire mRNA is synthesized but its message is silenced—or stops the transcription of the genes downstream of it (transcriptional riboswitch), which leads to the synthesis of a very short RNA. If the riboswitch does not bind to the ligand, the RNA is transcribed in its entirety and is also completely translated. These riboswitch ligands vary greatly in nature, from S-adenosylmethionine (SAM) to vitamin B1 or B12 to transfer RNA or metals such as magnesium.
Riboswitches not only regulate mRNAs as explained above, they can also regulate noncoding RNA. There is such a case in the foodborne pathogen Listeria monocytogenes, in which a vitamin B12 riboswitch controls an antisense noncoding RNA for the regulator of a series of genes. These genes encode enzymes that process propanediol, a compound present in the intestine that is produced by the fermentation of certain sugars by commensal bacteria. These enzymes require vitamin B12 to function as follows: (i) in the presence of B12, the riboswitch leads to the synthesis of a short RNA while allowing synthesis of the regulator protein PocR, and PocR then activates the synthesis of genes under its control; (ii) in the absence of vitamin B12, the riboswitch is configured such that a long form of antisense noncoding RNA is produced, which hybridizes to the mRNA that codes for PocR, stopping its production. Thus the PocR activator is not produced unless conditions are favorable, that is, if proteins encoded by the genes that it regulates can be activated by vitamin B12.
Figure 7. Schematic representation of the chromosome region encoding PocR. In the absence of vitamin B12 (left), the long transcript AspocR hybridizes with the transcript for pocR, which is then destroyed, preventing the synthesis of PocR. In the presence of B12, the pocR messenger RNA allows synthesis of the protein PocR.
Another example of a nonclassical riboswitch is a different vitamin B12 riboswitch found in L. monocytogenes and Enterococcus faecalis, both of which cause intestinal infections. This riboswitch controls a noncoding RNA that can sequester a regulator protein that activates the eut genes. eut genes code for proteins involved in the utilization of ethanolamine, a compound found in abundance in the intestine. The riboswitch works as follows: (i) if vitamin B12 is present, a short form of RNA is produced, a form that does not sequester the regulator protein, which is then free to activate the expression of the eut genes; (ii) if vitamin B12 is absent, a long form of noncoding RNA is produced that sequesters the regulator protein, which is thus unable to activate the eut genes.
This complex alternative process is crucial to the survival of pathogenic intestinal bacteria. Pathogens can use ethanolamine, but only when vitamin B12 is present. Since eut genes are not present in commensal bacteria, they provide pathogens a significant advantage over commensal bacteria.
RNAIII in Staphylococcus aureus
The RNAIII of S. aureus is regulated by quorum sensing, which means it is expressed once the bacterial population reaches a certain threshold. RNAIII controls the expression of a certain number of virulence factors. It impedes the translation of proteins, such as protein A, expressed on the surface of the bacterium or secreted during the beginning of an infection. It also impedes the translation of transcription regulators such as RotA. However, it activates the expression of the toxin known as alpha-hemolysin (Hla) by acting as an antisense that allows the corresponding RNA to be translated. Additionally, RNAIII codes for the small Hld protein, another toxin of 26 amino acids. The 514-nucleotide-long RNAIII in S. aureus is a very active molecule and thus can regulate many facets of bacterial physiology over the course of an infection.
The excludon
Some RNAs function both as antisense and as messenger. They are encoded in recently discovered regions of bacterial chromosomes called excludons. These regions were originally detected in the Listeria genome but were then found to exist in various other bacteria. Excludons are made up of two DNA regions encoding genes or operons that are oriented in opposition to one another on the bacterial chromosome. They encode a long RNA (up to 6,000 nucleotides) that is antisense to one of the regions. The first part of this RNA functions as an actual antisense that has a negative effect on the genetic expression of the gene or operon located on the strand opposite to the one that codes the RNA. But the second part of the RNA can act as an mRNA (Fig. 8).
Figure 8. Example of an excludon. Once the transcription beginning at P2 is expressed and generates a long transcript, the operon on the right becomes less expressed.
CHAPTER 4
From the CRISPR Defense System to the CRISPR/ Cas9 Method for Modifying Genomes
In nature, bacteria need to defend themselves constantly, particularly against bacteriophages (or phages), the viruses that specifically attack bacteria. A phage generally attaches itself to a bacterium, injects its DNA into it, and subverts the bacterium’s mechanisms of replication, transcription, and translation in order to replicate itself. The phage DNA reproduces its own DNA, transcribes it into RNA, and produces phage proteins that accumulate to generate new phages and eventually cause the bacterial cell to explode (or lyse), releasing hundreds of new bacteriophages. Phages continually infect bacteria everywhere—in soil, in water, and even in our own intestinal microbiota (Fig. 9). Bacteriophage families are numerous and vary widely in their form, size, composition, and the bacteria they target.
To begin their attack, bacteriophages need a site of attachment, a particular component on the surface of a bacterium. This site of attachment is specific for each virus and the bacteria that it can infect.
Infections of bacteria by phages are of great concern, particularly in the dairy industry, which uses certain bacteria, for example Streptococcus thermophilus, to make yogurt and cheese. S. thermophilus transforms the lactose in milk into lactic acid. Additionally, each different bacterial strain contributes its own unique taste and texture to the yogurt, which must remain consistent to ensure a reliable product and successful sales. If a bacterial strain disappears as the result of a bacteriophage infection, the economic consequences for the manufacturer can be disastrous.
Figure 9. Bacteriophages infecting an Escherichia coli bacterium.
One of the great discoveries of this decade is that bacteria have an immune system called CRISPR, for clustered regularly interspaced short palindromic repeats, meaning small, regularly spaced, palindromic (the sequence reads the same from either end) repeats. CRISPR regions in the chromosomes allow bacteria to recognize predators, particularly previously encountered phages, and to destroy them. CRISPR regions protect and essentially “vaccinate” bacteria against bacteriophages.
In fact, it has been shown that bacteria
