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European Biopharmaceutical Review
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Prophages are common and often uncontrable elements in bacterial
strains, with effects ranging from increased host fi tness to
potentially fatal infections. These factors warrant careful
consideration before selecting lysogenic bacteria as an expression
system for protein manufacture.
Over the course of their evolution, viruses have developed several
traits. Among these is virulence. This occurs after a cell is infected;
the virus produces the maximum amount of progeny, usually killing the
cell in the process. Another trait is the establishment of a provirus,
the only difference being that a fraction of infected cells do not
produce and release viruses, but instead viral genetic material is
integrated with the cell chromosome, and may resume its development
cycle later, usually triggered by some environmental signals. In the
world of bacteria, both strategies are commonly used by bacteriophages –
the viruses which attack them. Dormant bacteriophages are called
prophages.
Are Prophages Common?
In the past, prophages were considered as a moderately frequent
occurence in the natural environment, their abundance revealed by
intensive sequencing of different bacterial strains. Many of them,
especially pathogenic strains, appeared to have many different prophages
in their genomes – some more than 20. Prophage content may form as much
as 10-20 per cent of the whole genetic content of a given strain (1).
Due to relative lack of knowledge about bacteriophages, many prophages
may go unnoticed during sequencing projects and subsequent analysis of
obtained results. Until now scientists have not fully understood their
effect on the bacterial strain itself, on its environmental fi tness and
interactions with other bacteria and bacteriophages, or on its
interaction with higher organisms. However, there are a lot of things we
already know. The altered abilities of bacterial strains bearing
prophages are important not only from the point of view of health
hazards, but also from the point of view of production safety when
bacteria are used to produce pharmaceutical or biotechnology products.
The Good
In general, the additional burden of prophage genetic material present
in chromosomes should force bacteria to collect more resources before
the chromosome can be replicated and so should prolong the process of
chromosome replication, where the prophage is physically integrated with
the chromosome. Contrary to these assumptions, some prophages have been
observed to have a positive impact on host fi tness. This is
particularly apparent in conditions of carbon starvation, and these
conditions are frequently used in the process of biopharmaceutical
molecule production (2). For a long time it was not understood how
prophages compensated their host for the additional costs of their
presence − essential to ensuring their survival and the success of this
evolutionary strategy. The explanation is lysogenic conversion genes
and, in some cases, modulation of the gluconeogenesis pathway (3).
Lysogenic conversion genes usually bring some entirely new
characteristics to the host strain, which can help the host cell to be
more competitive in certain conditions. Modulation of the
gluconeogenesis pathway allows for a more economic use of carbon sources
− crucial when carbon source is a growthlimiting factor. On this
principle, bacteria lysogenic with phage may show better growth and
production characteristsics in all types of biotechnology processes, as
conversion of various carbon sources, most common of which is glucose,
into the fi nal product, is the basis of this business.
Another positive factor resulting from the presence of prophage(s) in a
given strain is that they often encode powerful phage exclusion systems,
which may prevent infection of the strain with the much more dangerous
phages. From the point of view of production safety, this defence is
very important, as infecting bacteriophages can contaminate a facility,
paralysing productiveness. Prophages are also a useful tool for
molecular biology and biotechnology. They allow for inserting a gene of
interest into a bacterial chromosome, which enables strict control of
the gene copy number, stable gene maintenance and, when inserted into
the right place, may also provide control over gene expression
triggering. The most frequently used prophage in biotechnology is DE3,
which allows for controlled expression of T7 RNA polymerase. This is a
very convenient system enabling high protein overexpression with minimal
burden for a cell before the expression of T7 RNA polymerase gene is
triggered (4). Prophages also provide effective and easy-to-use
molecular tools, such as Red recombination system from phage lambda,
which is commonly used for bacterial strain engineering.
The Bad
Unfortunately, the presence of prophage in bacterial cells also has
drawbacks. One of the most important is that the same mechanism that
allows prophages to increase their host fi tness also help them to
convert harmless bacteria into lethal pathogens. The association of some
prophages with pathogenicity of different bacterial strains was fi rst
noticed some time ago: since then, a growing number of diseases has been
discovered to be associated with the strains which were lysogenised by
phages bearing major pathogenicity genes (5). One example is diphtheria,
caused by Corynebacterium diphteriae being lysogenised by
prophage β. The strain bearing prophage is able to cause disease, while
prophage triggers extremely effective toxin production and can kill an
infected person. Another well known example is cholera, caused by
saprofi tic Vibrio cholerae – quite common bacterium found in
waterassociated environments. It is lysogenisation by CTXφ, which turn
this harmless bacteria into a real killer.
Prophages also play an important role in emerging diseases. One such example is enterohaemorragic Escherichia coli,
causing food-borne outbreaks with potential fatalities. Thus, the
presence of lysogenic conversion genes in a production host, especially
when biopharmaceuticals are to be produced, should raise questions
regarding their impact on product safety and possible cross reactions in
patients organisms, since few purifi cation processes can deliver a
truly homogenous protein solution. A good example is bacteriophage
lambda and its derivatives, which carry the immune evasion gene bor. The
gene product modifi es the complement system disabling the classical
complement activation pathway, and is associated with the cell wall,
leading to manufactures attempting to minimise the risk of co-purifi
cation of bor with expressed protein in most circumstances.
Prophages are also dangerous in biotechnology. When induced, they may
cause a lysis of the bacterial cells. Synchronised induction can result
in unexpected and very rapid destruction of the whole bacterial culture,
regardless of size. In many cases the massive induction is caused by
factors triggering an SOS response, including diverse DNA-damaging
factors and conditions. The problem is that overexpression of some
proteins may also trigger an SOS response, so there is a possibility
that after beginning protein production one will obtain a phage lysate
instead of the protein of interest. This should of course be revealed by
an initial study; however, since fermentation conditions may differ
slightly from batch to batch, the risk of inducing prophage may still
exist, even if well designed preliminary tests excluded such
possibility. To make the situation worse, there is a large group of
prophages whose induction is SOS-independent, which means that we
usually do not know which stimuli can effi ciently induce them. An
example of such phage is P2-like phage Wφ, which is present in E coli W. Due to the fact that no one knows what condition may trigger massive
prophage induction in such cases, it is much harder to prevent it and,
when it does occur, it is also harder to understand the root cause of
fermentation failure.
The Ugly
Some characteristics of prophages don’t necessarily have a negative
impact on all biopharmaceutical production, but can still be problematic
in some cases. They quite often go unnoticed and due to their sneaky
nature may cause long-term problems in facility environment. The fi rst
of these is spontaneous induction. Even when conditions which trigger
prophage induction are avoided, in the case of many prophages, a small
fraction of a cell's prophage will be induced anyway. The degree of
induction strongly depends on the prophage itself and cultivation
conditions used, but in many cases it is possible to detect using
standard methods. Due to homoimmunity of lysogenised cells to the same
type of phage, this is not a signifi cant problem, at least as long as
various strains are not used for production in the same facility. When a
facility runs multiple projects, a higher degree of care must be used
in order to avoid cross-contamination and phage-caused outbreak or
lysogenisation of other strains. There are several documented cases of
horizontal spread of prophage in laboratory environment (6).
Due to bacteriophages’ abilities to perform similar processes as the
host cell, but usually in a different and effective way, there are some
potential problems encountered when lysogenic strains are used for
production. One of the characteristics that may affect the strain itself
is the very potent recombination system found in the majority of
bacteriophages. This system allows them to evolve very quickly and to
adapt to the changing environment. One side effect of its activation may
be the decreased stability of host cells. To prevent problems with host
stability, proper methods of cell bank storage and propagation as well
as suitable tests should be employed. Another possible effect is
decreased stability of plasmids used for product expression, especially
during over-expression of a gene from the afore-mentioned plasmid. This
may be due to several factors. The most evident will be improved growth
rate of host cells, which minimise the effort necessary to express the
gene by mutating or deleting the gene or regulatory elements responsible
for the product formation. This enables them to grow much faster, and
in effect, they may constitute an important fraction of bacterial
culture.
Another factor may be the fluctuation of repressor concentration in
cells with strong over-expression of cloned gene. Repressor in prophage
prevents its induction, and thus prevents expression of the vast
majority of genes, excluding lysogenic conversion genes and repressor
gene itself. When the protein synthesis system is saturated with the
protein of interest, the production of repressor may be less effective,
which may cause the repression to become ‘leaky’. This can lead to
expression of small quantities of prophage genes, including
recombination genes, which in turn may increase the probability of
recombination occurrence in the host. One effect may be an increased
frequency of loss of ability to overproduce the given protein.
Conclusion
Should prophages be eliminated from production strains? As in many
similar cases, the answer is ‘it depends’. In general, using a lysogenic
strain can be a good idea when high process effi ciency, especially for
the production of secondary metabolites, is required. In the case of
unusual hosts, fi nding a prophage even after strain sequencing may be
quite tricky, and the removal may not be possible or economically
justifi able (1). Additional protection against some virulent phages,
which some prophages provide for the host strain, may be very valuable,
especially in large scale fermentation where a proper degree of
sterility is much harder to obtain than a smaller volume fermentation
usually used for protein production. In protein production strains of E coli,
prophage DE3 provides an effi cient, well known expresion system
successfully used for decades and tested with many different products.
Despite its well documented contribution to protein expression, it is
highly recommended to test this with the protein of interest in the
early stages of a project under real fermentation conditions in order to
avoid problems, which may be caused by extensive induction of defective
DE3 prophage, that still carries full repertoire of functions required
for cell lysis.
Another aspect is the presence of lysogenic conversion genes, especially
when they encode toxins or immune system modulating proteins. They may
be problematic, especially in the pharmaceutical production of proteins.
This does not usually constitute a great risk as purifi cation
procedures should be effi cient enough to eliminate them; however, when
constructing a new protein production process, one should consider
removal or inactivation of such genes.
These potential problems do not exclude lysogenic bacteria as proper
hosts for the production of either proteins or secondary metabolites.
What is required when using such hosts is a higher degree of care during
construction of the production process, well developed control tests
and procedures, and last but not least, proper training of the personnel
involved in production.
References
1. Srividhya KV, Alaguraj V, Poornima G, Kumar D, Singh GP,
Raghavenderan L, Mohan Katta AVSK, Mehta P and Krishnaswamy S, Identifi
cation of Prophages in Bacterial Genomes by Dinucleotide Relative
Abundance Difference, PLoS ONE 2(11): e1193, 2007
2. Dykhuizen D, Campbell JH and Rolfe BG, The infl uences of a lambda prophage on the growth rate of Escherichia coli, Microbios 23(92): pp99-113, 1978
3. Chen Y, Golding I, Sawai S, Guo L and Cox EC, Population
Fitness and the Regulation of Escherichia coli Genes by Bacterial
Viruses, PLoS Biol 3(7): e229, 2005
4. Studier FW and Moffatt BA, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J Mol Biol, 189(1): pp113-130, 1986
5. Los M, Kuzio J, McConnell MR, Kropinski AM, Wegrzyn G and
Christie GE, Lysogenic conversion in bacteria of importance to the food
industry, in Sabour PM and Griffi ths MW (eds) Bacteriophages In the Control of Food- and Waterborne Pathogens, pp157-198, 2010
6. Rotman E, Amado L and Kuzminov A, Unauthorized Horizontal
Spread in the Laboratory Environment: The Tactics of Lula, a Temperate
Lambdoid Bacteriophage of Escherichia coli, PLoS ONE 5(6): e11106, 2010.
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