Dale_Molecular Genetics of Bacteria 4th ed

Apart from marine vibrios, related systems have been identified in over 50 Gramnegative bacterial species.

Gram-positive bacteria also regulate a variety of processes in response to cell density, using small secreted peptides (Figure 3.26), rather than the acyl homoserine lactones used by Gram-negative bacteria. Initially, a polypeptide signal precursor is synthesized which is later cleaved to yield a small peptide (8–20 amino acids). When the external concentration of this peptide signal reaches a threshold concentration, an HPK of a two-component system (see above) detects the signal and activates a RR which then stimulates transcription of the target genes. A marked contrast with the Gram-negative system is that these peptides do not diffuse across the cell membrane – they are specifically secreted and the cells respond to the extracellular concentration via a signal transduction mechanism.

Quorum sensing facilitates multicellular cooperation in bacteria. Myxobacteria (such as Myxococcus xanthus) are predatory bacteria which obtain nutrients through the lysis of other bacteria, by secreting lytic extracellular enzymes. The attack of a single myxobacterial cell would be ineffective since the digestive enzymes and any released nutrients would be rapidly diluted. As a consequence, myxobacteria use a ‘wolf-pack’ hunting strategy, in which quorum sensing co-

Signal peptides

P

Peptide

Response regulator

Precursor protein

P Target

gene(s)

Precursor protein gene

Figure 3.26 Quorum sensing: Gram-positive bacteria. Quorum sensing in Gram-posi- tive bacteria is typically mediated by small peptides which are transported across the membrane by a specific peptide transporter. These peptides are not taken up by the target cells, but are recognized by a membrane receptor which transmits the signal to the interior of the cell (see also two-component regulation)

ordinates the formation of a cooperatively feeding swarm of individual cells. We will look at other aspects of communication in M. xanthus in Chapter 9.

Similarly, the plant pathogen Erwinia carotovora uses a homoserine lactone quorum sensing system to ensure that digestive enzymes, which attack plant structures, are only produced if there are sufficient cell numbers to mount a concerted and effective attack on the plant tissue. As another example, we will see in Chapter 6 that some mechanisms involved in the transfer of genetic information between bacteria are also dependent on quorum sensing and only work at high cell density.

3.3 Translational control

3.3.1 Ribosome binding

After the mRNA has been produced, the next step is the attachment of ribosomes and the translation of the sequence into protein. This stage seems to play a comparatively minor role in the natural control of gene expression in bacteria. This is not really surprising as it would be rather wasteful to produce large amounts of mRNA that are not required for translation. Translational control can however become important with genetically-engineered bacteria, when very high levels of transcription of a specific gene have been achieved.

Binding of the ribosomes to the mRNA occurs at specific ribosome binding sites (see Chapter 1) up to seven bases upstream from the AUG translation initiation codon. In bacteria, this sequence determines where the ribosomes will bind to the mRNA and thus determines which AUG codon is used for the initiation of translation.

The actual sequence of a ribosome binding site (RBS) and its distance from the start codon can vary somewhat, so it is to be expected that there will be weak and strong ribosome binding sites, just as there are weak and strong promoters. However, the sequence of an RBS does not seem to affect the level of translation – it either works or it does not. But the distance separating the ribosome binding site from the initiation codon can have a powerful effect on gene expression.

In polycistronic operons, the ribosome may, after translating the first gene, dissociate from the mRNA. The next gene then must have a site to which ribosomes can attach in order for it to be translated. The efficiency of this can vary which may result in the subsequent genes being translated less effectively. This effect, known as polarity, is an alternative to the attenuation mechanism referred to earlier for achieving different levels of expression of genes within an operon.

Polarity is particularly marked if there is a nonsense mutation causing premature termination of translation. Such a mutation may prevent translation of the

best characterized examples come from the regulation of the mobility of insertion sequences (see Chapter 7).

3.3. 2 Codon usage

The effectiveness of translation can also be influenced by the nature of the codons used throughout the gene. Most amino acids can be coded for by more than one codon. In some cases, the codons are effectively equivalent, since the same tRNA will recognize both equally well (see Chapter 1). But in many cases, a different tRNA species is responsible for recognition of the different codons and some of these tRNA species are known to be present in the cell at quite low levels. A gene that contains many codons that require these ‘rare’ tRNA molecules will then be expected to suffer delays in translation that may affect the amount of end-product formed.

Some indirect evidence of this codon usage effect comes from the study of gene sequences of E. coli, which shows that highly expressed genes have a high degree of ‘codon bias’, i.e. they have a marked preference for codons that can be recognized by the common tRNA species. Genes that are expressed at moderate or low levels do not on the whole show such a codon bias and they may contain several codons that require relatively rare tRNAs. Codon usage appears to be more important for highly expressed genes.

3.3.3 Stringent response

Ribosomal protein synthesis provides a specific example of translational control. The production of these proteins needs to be coordinated so that equal amounts of each are made. This is achieved, in part, by autogenous control of translation, i.e. the accumulation of a ribosomal protein will repress the translation of the corresponding mRNA. This also provides a way of relating ribosomal protein production to the amount of rRNA. If there is a shortage of rRNA, free ribosomal proteins will start to accumulate and will shut down further translation of the relevant mRNAs. In this way, the cell is able to respond to changes in growth conditions. When the cells are short of nutrients, they need fewer ribosomes. This can be achieved by reducing rRNA production, with a consequent cessation of ribosomal protein production. This is one aspect of what is known as the stringent response.

The stringent response is triggered by amino acid starvation, which leads to the presence of uncharged tRNA occupying the A site of the ribosome. A protein known as the stringent factor is involved in the conversion of GTP to two unusual nucleotides, ppGpp (guanosine-50-diphosphate-30-diphosphate) and pppGpp (guanosine-50-triphosphate-30-diphosphate). These nucleotides prevent

transcription of the rRNA operons; the production of ribosomal proteins will then be reduced as a consequence of the reduced amount of rRNA available.

3.3.4 Regulatory RNA

In addition to proteins acting as transcriptional regulators, in recent years regulatory RNA molecules (often denoted riboregulators) that regulate gene expression at the post-transcriptional level have been discovered. In many cases this involves the production of ‘antisense’ RNA. If a region of a gene, particularly the region including the ribosome binding site and translation initiation point, is transcribed in the opposite direction, an RNA molecule will be produced that is complementary to the mRNA. This molecule can hybridize to the mRNA, and thus block the binding of ribosomes and the initiation of translation. Interactions of this kind are known to be involved in the regulation of some bacterial genes such as ompF, which codes for a porin in the outer membrane. Translation of ompF mRNA is regulated by a short antisense RNA called MicF. Under stress conditions, the amount of MicF increases, and consequently the production of OmpF is decreased.

Typically, antisense RNA binds to mRNA made from the opposite DNA strand and is therefore specific for a single gene. However, some regulatory RNAs regulate multiple targets, often in different ways and with different consequences. For example, DsrA is an 87-kb RNA which regulates translation of two different genes, hns (which codes for the H-NS protein, a silencer of expression of a wide range of genes) and rpoS (coding for the stationary-phase sigma factor sS, see above). DsrA binds to the two ends of the hns mRNA, preventing translation. In contrast, its binding to rpoS mRNA prevents formation of a stem–loop structure that otherwise obscures the ribosome binding site; DsrA thus stimulates translation of rpoS. We will encounter other facets of antisense RNA activity when considering the control of plasmid replication in Chapter 5 and for genetic manipulation (Chapter 9).

3.3.5 Phase variation

Whilst the expression of most bacterial genes is controlled at the level of transcription and the amplitude of the response can be graded, a number of genes are controlled in an ‘all or nothing’ manner. The high frequency switching of gene expression between an ON or OFF state is called phase variation and is another important mechanism for regulating gene expression in bacteria. This will be discussed in detail in Chapter 7.

Injection of phage DNA

Replication of DNA

Infection

Synthesis of

phage particles

Lytic cycle

Lysis

Packaging of DNA into phage heads

Figure 4.1 Lytic growth of bacteriophages

indicator cells) and the phage suspended in soft agar poured as an overlay on the top of an agar plate, rather than spread on the surface. This gives a threedimensional plaque, which is easier to see.

Some bacteriophages, such as (lambda), do not always enter the lytic cycle described above. These are temperate phages and can establish a more or less stable relationship with the host cell, known as lysogeny. In this state, almost all the phage genes are completely repressed, as is replication of the nucleic acid, so no phage particles are produced until the lysogenic state breaks down again. Temperate bacteriophages will usually produce plaques in an agar overlay with a suitable bacterial indicator since many of the infected cells will lyse rather than become lysogenic. However, since those cells that enter the lysogenic state will continue to grow (and are resistant to further infection), the plaques will be turbid rather than the clear plaques seen with a virulent phage (i.e. a phage that is unable to establish lysogeny). Lysogeny is covered in more detail later in this chapter.

Many phages have been characterized to a greater or lesser extent; the main examples used in this chapter are all viruses that infect E. coli. These come in a variety of shapes and sizes as shown in Figure 4.2, including the small, simple

φX174

MS2

λ

(lambda)

T4

M13

Figure 4.2 Morphology of selected bacteriophages. Note that the filamentous structure of M13 is longer than can be represented on this scale

structures of fX174 and MS2, more complex structures with identifiable heads and tails ( , T4) and filamentous structures such as M13.

The phage particle consists of a nucleic acid molecule contained within a protein coat. In the simpler phages, such as MS2, the coat contains a number of copies of a single major protein which essentially polymerize spontaneously. The manner in which they associate defines both the shape and the size of the phage particle. In principle, filamentous structures can also be produced by spontaneous polymerization of protein subunits – but this is not sufficient to define the length of the filament. A common way of ensuring that filaments of a defined length are produced is to use a template, around which the protein polymerizes. As we will see later on, with filamentous phages such as M13 the phage DNA provides the template.

The assembly of M13, where the proteins making the phage coat polymerize around the DNA, is a marked contrast to the larger phages such as lambda and T4, where the DNA is packaged into pre-formed empty phage heads and therefore the size of the phage head is defined by the proteins of which it is composed. The size and complexity of these structures necessitates a different approach to their production – spontaneous polymerization would be too inefficient and the structures generally unstable until they are complete. Assembly of these phage

Figure 4.3 Scaffolded assembly. Assembly of complex phages is assisted by scaffolding proteins. These are removed once assembly is complete

heads therefore requires the temporary presence of a number of additional proteins, which are removed before the final maturation of the phage (see Figure 4.3). These components which assist with assembly but are not present in the final particle are referred to as scaffolding proteins.

Bacteriophages also differ in the nature of their nucleic acid content. Phages fX174 and M13 contain circular single-stranded DNA, MS2 has an RNA genome, while and T4 particles contain double-stranded DNA. Each of these phages is worth considering in more detail as they provide examples of fundamental molecular processes.

4.1 Single-stranded DNA bacteriophages

4.1.1 fX174

The phage fX174 is an icosahedral phage that contains a circular single-stranded DNA molecule of 5386 nucleotides. It codes for 11 proteins, each of which has been identified. Adding together the size of all those proteins comes to 2330 amino acids, which would require 6990 nucleotides (3 2330) – substantially more than the total length of the genome. How this is achieved becomes apparent from inspection of the genome sequence of the virus (Figure 4.4).

Firstly the genes are very tightly packed – there is very little non-coding sequence in the genome. In most cases, the end of one gene is directly adjacent to (or slightly overlaps with) the start of the next. Secondly, one of the proteins (A*) corresponds to the C-terminal region of protein A, due to the use of an