Begin Part II (the survey of competence regulation)

(Put elsewhere the discussion of how role of T4P in DNA uptake means we often can't separate T4P regulation from competence regulation.  In Part I?  Is there any T4P regulation independent of 'competence' regulation?)

An introductory paragraph:  Should we relate regulation to the old framework of 'early' and 'late' competence genes?  Is this still useful? Also for each species the traditional competence rituals used in the lab (if they exist), and the levels of competence achieved (with whatever DNA) and fraction of cells that are competent.  For many species this is all that's known.

Also explain that the species are considered here in phylogenetic context (not just gram+ vs gram-).  Refer to the detailed survey by Johnsborg et al (2007 review).  Should this start with H. influenzae and other Paseurellaceae?  Yes, as this provides a good framework for the Vibrio information (better than Vibrio first).  So have a couple of paragraphs about H. influenzae, then one about other Pasteurellaceae. 

Our survey begins with the Gamma proteobacteria.  Ref for phylogeny Williams et al 2010 (http://jb.asm.org/cgi/content/full/192/9/2305).  Competence is common and well studied in several families, and regulation appears to have been conserved in the clade containing the families Pasteurellaceae, Enterobacteraceae and Vibrionacea (ref Cameron 2006).  We begin with H. influenzae because its regulation is the best known.

Pasteurellaceae:  Haemophilus influenzae:  Competence is traditionally induced by transfer of exponentially growing cells from rich medium to a starvation medium lacking nucleotides and essential cofactors.  This gives high transformation frequencies (10-3-10-2 with chromosomal DNA), with most cells in the culture competent.



H. influenzae has a compact competence regulon, consisting of 25 genes under the control of promoters activated by the regulatory proteins CRP and Sxy (called TfoX in some species).  Most of these genes have been directly or indirectly implicated in DNA uptake, and several more act on DNA intracellularly; only a few (how many?) have unknown functions and none (?) have known functions unconnected to competence or transformation.  The first step in competence induction is transcription of sxy, which requires both the transcription factor CRP and elevated levels of its cofactor cyclic AMP.  As in E. coli, production of cAMP occurs when the phosphotransferase system lacks preferred sugars to transport.  However this transcription is not enough, as effective translation of the sxy transcript requires additional signals, thought to derive from depletion of purine nucleotide pools.  Sxy translation is regulated by secondary structure (all homologs produce mRNAs with long untranslated leaders).  Once both Sxy and active CRP are present, they together activate transcription of genes with the CRP-S promoter motif; these genes comprise the competence regulon.  The mode of action of Sxy is not known, but is thought to involve direct contact with CRP (Sinha 2009).  Similar CRP-S regulons are also present in other Pasteurellaceae and in the Enterobacteraceae and Vibrionaceae (see below).
(CRP thus acts both early and later...)


Other Pasteurellaceae:  Similar mechanisms regulate competence in other Pasteurellaceae (ref Maughan chapter). Although only some Pasteurellacea are known to be naturally competent in the laboratory, all sequenced genomes have the same competence genes as H. influenzae, with CRP-S motifs in their promoters.  Evidence that these regulated genes are needed for competence, or that the regulation uses the same mechanism? A. pleuro competence needs Sxy (Janine?), so does Act. act (Bhattacharjee 2007).  Evidence that cAMP stimulates competence?  Any evidence that regulation might be different?


Competence in the Enterobacteraceae?  (There are no Enterobacteria in the Johnsborg list.) Enterobacterial genomes contain homologs of many of the H. influenzae competence genes, and the complete genomes analyzed in detail appear to contain CRP-S regulons like that of H. influenzae.  However there are no reports of natural transformation for any of the Enterobacteriaceae.  One interpretation is that cells use these genes to take up DNA, but that the DNA is quickly degraded and rarely or never recombines with the chromosome. (Figure of gammaproteobacterial competence genes and phylogeny?)

(Nominalization alert)  Although several protocols have been described that allow plasmid transformation of E. coli without the standard permeabilization treatments (cold divalent cations, electroporation), these do not require any of the genes specific to natural competence.  (One exception is our finding that low-calcium transformation requires sxy...) 

Artificial overexpression of E. coli sxy does induce the CRP-S regulon genes but its toxicity precluded investigation of competence.  Comprehensive screens for culture conditions that induce the CRP-S-regulated ppdD gene (encoding the type IV pilin) have been unsuccessful.  In this paragraph describe Finkel's demonstration that E. coli can use DNA as food, dependent on the CRP-S genes.

Vibrionaceae:  cholerae, parahemolyticus, fischeri, vulnificus, others?  Marine Vibrios JH Paul old papers.  Regulated by Sxy (cholerae, fischeri, others?), which is regulated by chitin, which Vibrios break down as major nutrient source (minimal inducer for V. cholerae is dimer of GlcNac - the chitin subunit).  V. cholerae has two Sxy homologs - one is required for competence, and overexpression of it bypasses the need for chitin.  No function has been assigned to the other Sxy homolog, which is regulated by a cyclic-di-GMP-sensitive 'GEMM' riboswitch (Kulshina 2009) (Sudarsan 2008).  Expression of the competence-regulating sxy gene is subject to  translational regulation by dimers of GlcNac (Yamamoto ref).  Yamamoto et al. overlooked a very strong CRP-N site 50 nt upstream of the 35 promoter element; this site suggests that V. cholerae competence may be regulated by cAMP and CRP as in H. influenzae.  Consistent with this, Meibom et al. found that addition of glucose prevents competence induction by chitin; induction by cAMP has not been reported.  As in H. influenzae and E. coli, the activity of V. cholerae CRP is controlled by its phosphotransferase system (Karatan & Watnick 2009 http://mmbr.asm.org/cgi/content/full/73/2/310)

Read Meibom 2005 paper - they did microarrays!

The quorum-sensing regulator HapR gene also controls V. cholerae competence (Meibom et al), and cultures become more competent as density increased (but this is a weird and not very compelling experiment).  Two sequenced V. cholerae strains that  Meibom et al. found unable to develop competence are known to carry frameshift mutations in hapR, (relevance to variation in natural populations?).

Pseudomonadaceae:  Moraxella catarrhalis (Stutzmann et al. FEMS Micb. Letters 2006).  Transformation on plates for 5 hr, frequencies v. high (>10% with chromosomal insertion!).  No transformation when cells were in stationary phase before plating.

Acinetobacter:   (Averhoff and Graf 2008)  (I can't find any other refs about regulation.)  Wackernagel papers (see email)?  Palmen 1994.

Pseudomonas:  Have competence genes (Cameron 2006) but not sxy homolog. Competence regulation has been studied in P. stutzeri (Lalucat et al.  good review MMBR 2006).  Induced by transition to stationary phase and by nutritional downshifts.  Nothing known about regulatory genes?  Transformable strains common, but 10% of strains are nontransformable.  I can't find anything about natural transformation methods in P. aeruginosa or any other pseudomonas species.  Bob Hancock says there aren't any for P. aeruginosa.  Paul Rainey also says they aren't, except stutzeri (Spiers et al. Microbiology 2000).  But see Carlson et al. 1982 (JB 153:93-99)  which says also several close relatives of stutzeri, all best at transition to stationary phase.  See email from Wackernagel.

Azotobacter:  (Ref Page Can J. Micro 1983).  Competence is induced by a nutritional downshift into an iron-limited nitrogen-free medium. cAMP also induces.  TFs are high, 10-3 - 10-2. No recent work.

Xanthomonadaceae:  Xanthomonas:  1957 paper (Corey and Starr, J. Bact?) demonstrating efficient transformation of X. phaseoli in broth by a chromosomal StrR marker .  TF ~10^-2.  Cited in 1985 as 'early reports claiming transformation...'), but no confirmations.

Legionellaceae: Legionella pneumophila (Sexton and Vogel J. Bact 2004) two genes that repress competence!  TF increases with cell density, max ~ 10^-2 at OD ~ 1.0, then falls (with a plasmid-borne homologous marker). Knocking out its only exoRNase induces competence! (nutrient nucleotides???Charpentier et al. JB 2008)


Nov. 24 additions:  Yesterday I went over the Johnsborg review with the Wu et al. phylogenetic tree in hand.  Cardiobacterium (Cardiobacteriaceae) is the only other Gammaproteobacteria reported to be competent.  This is based on a single report from 1985 (Tonjum et al, behind a paywall), with no regulation information available.  I'm emailing her to get a copy.  Also Dichelobacter?