Friday, October 22, 2010

Reorganization of Part III

I have lots of bits of text and ideas for the final section of the review, but before I write any more I need to sort out their organization.

Distinguish (here or earlier) between the signal-transduction mechanism and the signals themselves, and between these and the output (the changed phenotype and its consequences for survival and growth).

1.  Summarize the results of the survey:

Great diversity in regulatory mechanism, diversity in signals, core set of induced genes plus wide diversity of other induced genes.  Regulatory information is available for only a few species.  Where we do have information for close relatives we usually see differences, and at greater evolutionary distances the regulatory mechanisms appear unrelated.  Also wide variation in the non-core genes in the regulon (both number and functions).

2.  Practical applications of this information:

Does the current knowledge of how competence is regulated suggest better ways of inducing competence in lab cultures?  The standard 'competence rituals' for the model organisms were developed by trial and error, long before we understood their regulation.  Are they unnecessarily troublesome?  Can new methods increase transformation efficiencies/frequencies?  Use of mutations and plasmids that increase competence?

Does this knowledge suggest ways of inducing competence in bacteria where it is apparently unregulated (Neisseria sp.), or where little is known about regulation (Helicobacter?)?  Look at this in phylogenetic context?  Or is regulation so evolutionarily variable that even comparisons within families are not very predictive?

Does it suggest better ways of testing for competence in bacteria not known to be transformable? For example, does the regulation by cAMP and CRP shown in H. influenzae and postulated (better word) in related species suggests that other members of the Past, Ent (?) and Vib families that induction/stimulation by cAMP should be checked in other members of these families.  Perhaps it should also be tested in other species where cAMP and CRP have regulatory roles, such as throughout the gammaproteobacteria.  Such analysis has been recently done for Streptococci Halvorstein 2010 Mol Micb 78:541).

3. What are the broad questions about the role(s) of competence in bacteria? 

Explain the controversy about function (nutrients, repair, recombination).  Keep this whole section and the next quite short; save the detailed exposition for the other review.  (OK to initially write the details here and then move much of them to the other article.)

What are the predictions of the different hypotheses?  If uptake is selected because incoming DNA provides templates for DNA repair, competence should be regulated by the same damage signals that induce recA, or that induce the RecA-regulated SOS response.  If uptake is selected because incoming DNA provides nucleotides and other nutrients, competence should be regulated by nucleotide pools and/or by processes that sense availability of sources of C, N and P.  If uptake is selected because incoming DNA sometimes carries beneficial new alleles that replace inferior alleles in the chromosome by recombination, then competence should be regulated by ... what? ... I've suggested that under this selection competence might be expected to be a 'when all else fails' response, induced when the cell's other stress responses have been mobilized but have failed to solve the problem.  How strictly this test (what test? the test of whether the other stress responses have worked?) is applied would probably depend on how costly DNA uptake and recombination were, considering both the physiological costs/risks of DNA uptake and the genetic costs of recombining in alleles that reduce fitness.

'Stress' is difficult to quantitate.  We can measure the decrease in growth rate or numbers of viable cells caused by a macroscopic perturbation of culture conditions.  But often our best evidence of the importance of disruptive events is the presence of evolved mechanisms to prevent them or mitigate their effects.  For example, the presence of a gene for photolyase is evidence that pyrimidine dimers is not only a tol that lets us measure the effect of unrepaired damage on viability, but evidence that this effect has been important over evolutionary time.  The logic is straightforward for processes whose benefits and costs are direct,  but where the effects are indirect the inference is always on shaky ground, with interpretations compromised by our guesses of what might matter to the bacteria.

Lack of nutrients is often considered as a form of stress.  Difficulty of drawing a line between nutrient signals and signals of other kinds of stress?  Some signals are clearly nutritional - PTS sugars, but even cAMP has subtle complications in bacteria other than Ent and relatives.  Same for phosphate and nitrogen limitation?  Signals of nucleotide depletion?  Purine syn regulated by guanine and hypoxanthine, pyrimidine by post-transcrptional effects (?), other effects on transcription likely.  Secreted autoinducers ('quorum sensing') integrate both local cell density and the physical properties of the microenvironment, such that may be activated by dense populations in well mixed cultures or by single cells in confined spaces. And the term 'stress' is usually used very loosely; it is rarely well enough defined that hypotheses can be rigorously tested.  Somebody (John Roth?) suggested that rapid growth in rich medium might be more stressful than slow growth in minimal medium - maybe any condition is stressful if the doubling time is less than the time needed to replicate the chromosome.

The direct and indirect costs of genetic processes are rarely considered explicitly, but evaluating them is essential to a true understanding of function.  When cells become competent energy and materials must be diverted from other processes (e.g. growth) to synthesize and assemble the DNA uptake machinery.  Once in place, the machinery may interfere with membrane integrity or with other membrane transport processes.  The energy costs of the uptake process remain unknown, but, at a minimum they include a cost of pilus retraction of about ???? per bp.  Transport across the inner membrane???  There are also genetic (and perhaps DNA damage) costs.  The obvious one is the cost of replacing well functioning alleles with worse ones, likely to be overrepresented in environmental DNA.  The infrequent non-homologous recombination events will almost always be deleterious.  Any process that disrupts the integrity of double-stranded DNA is likely to increase the risk of DNA damage - we have no measure of this.

4.  What does the survey of regulation tell us about function?

What do we learn from the breadth of 'competence' regulons (the 'effectors')?  Are there any consistencies in the genes that don't directly contribute to DNA uptake or transformation?  Do competence regulons overlap with other global regulons?  For which species do we have microarray (or other) surveys of what genes are regulated?  (Not only H. influenzae, B. subtilis, S. pneumoniae, right?)  Do genes that enhance transformation but don't contribute to uptake have specific other functions?

What do we learn from surveying the signals that induce competence?  Given the diversity, are there any unifying features of the regulation of competence?  Is there always a nutrient component?  A 'stress' component?  What would be the most important experiments to do now?

Do the steps by which competence is regulated tell us anything beyond what we learn from the nature of the inducing signals?  Certainly quorum sensing does, but what about other cascades?

What can we conclude about how the regulation has evolved?  Which features of regulation have been conserved in related species?  Which have been particularly labile?

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