Chro­mo­some Or­ga­ni­za­tion the Pseudo­monas Way (1|2)

As an ex­cep­tion to our usual for­mat, this post comes in two parts. Part 1 in­tro­duces the dancers and gives an out­line of the chore­og­ra­phy, next week's Part 2 will lift the cur­tain and take a glimpse at the bal­let compa­ny's per­for­mance.

by Christoph

(Re-)Introducing the Pseudomon­ads   De­spite the some­what murky prove­nance of their name, pseudomon­ads are every­thing but "pseudo-" in terms of their meta­bolic ver­sa­til­ity: they are bac­terial om­ni­vores, het­erotrophs yet far from picky. Mem­bers of the fam­ily Pseudomon­adaceae (Gamma­pro­te­o­bac­teria) are ubiq­ui­tous and make up a re­spectable per­cent­age of the mi­crobes in any sam­ple taken from what­ever source, be it air, wa­ter, soil, plant or an­i­mal skin, plas­tic or metal sur­faces. No won­der they are also con­sid­ered quite fre­quently in this blog. As skilled chem­i­cal en­gineers, they can con­vert PET bot­tles to biodiesel and pro­mote ice nu­cle­ation at above-zero tem­pe­ratures on plant leaves. They grow aer­o­bi­cally and anaer­o­bi­cally, many are halo­tol­er­ant, and there are psy­chrophilic as well as ther­mophilic fam­ily mem­bers – in short, one would like to call them holophilic if that term were not al­ready taken by psy­chol­ogy. They speak 'quo­rum sens­ing' with each other and pro­lif­er­ate hap­pily as plank­tonic cells but are also cham­pi­ons of a life in bio­films. The lat­ter makes the ther­apy of per­sis­tent lung in­fec­tions by Pseudomonas aerug­i­nosa in pa­tients with cys­tic fi­bro­sis so de­mand­ing. And the main­te­nance of sewage pipes, to give a more mun­dane ex­am­ple. Luck­ily for mi­cro­bi­ol­o­gists, most of them are cul­ti­vat­able.

Fig­ure 1. Thin sec­tion trans­mis­sion elec­tron mi­crograph (TEM) of a P. aerug­i­nosa PAO1 cell grown in MOPS min­i­mal me­dium for 3 h. n nu­cleoid; c cy­to­plasm; i in­ner mem­brane; o outer mem­brane. Scale bar: 200 nm. Source

Pseudomonas genomes   Pseudomon­ads have by far the largest genomes among the known Gammaproteobacte­ria and – un­like many Al­phapro­teobac­te­ria with equally large genomes – they store most of their gene equip­ment in just one sin­gle, large, cir­cu­lar chro­mo­some, which in P. aerug­i­nosa PAO1 is 6.3 Mb in size and codes for ~5,600 genes. A re­cent re­view sum­ma­rizes what is presently known about how P. aerug­i­nosa or­ga­nizes its chromoso­me in the nu­cleoid to fit into the tiny – com­pared to the length of the DNA mol­e­cule – cell (see Fig. 1) and how it re-or­ga­nizes it dur­ing repli­ca­tion.

Reg­u­la­tion of chro­mo­some repli­ca­tion   P. aerug­i­nosa is one of the ex­cep­tional cases of bac­te­ria with two func­tional repli­ca­tion ori­gins, oriCs, for a sin­gle repli­con. Both are DnaA-de­pen­dent, yet only one, oriC1 lo­cated in the rpmH·dnaA in­ter­genic re­gion, can drive chro­mo­some repli­ca­tion alone (most likely be­cause the dele­tion of oriC1 si­mul­ta­ne­oulsy in­ac­ti­vated the dnaA gene). Both ori­gins are struc­turally very sim­i­lar but it is presently not known whether oriC2 lo­cated up­stream of gidA at a dis­tance of ~6 kb from oriC1 is used at all or only un­der un­known growth con­di­tions. In any case, fir­ing of both ori­gins al­ter­nately or even si­mul­ta­ne­ously would hardly un­balance the ori­gin-ter­mi­nus axis and thus cause prob­lems with cell cy­cle reg­u­la­tion: the dis­tance of ~6 kb be­tween the two oriCs seems neg­li­gi­ble as com­pared to the over­all ori­gin-ter­mi­nus dis­tance of ~3 Mb. We may have here the rare chance to wit­ness a long-term evo­lu­tion­ary experi­ment: whereas all mem­bers of the Pseudomonas fam­ily show this 'dual-oriC pat­tern', more dis­tant­ly re­lated 'cousins' have ap­par­ently cho­sen to re­tain only one of them, ei­ther next to gidA as in Cel­lvib­rio japon­i­cus (Pseudomon­adales) and Kang­iella ko­reen­sis (Oceanospir­il­lales), or up­stream of dnaA as in Ha­hella chejuen­sis (Oceanospir­il­lales) (my in sil­ico find­ings).

We know lit­tle about the reg­u­la­tion of ini­ti­a­tion of chro­mo­some repli­ca­tion in P. aerug­i­nosa. In E. coli, DiaA pro­motes higher-or­der com­plex for­ma­tion of the ini­tia­tor pro­tein DnaA bound to oriC prior to ini­ti­a­tion, while Hda – in con­cert with the β‑clamp sub­unit of the replica­tive DNA polyme­ra­se – in­ac­ti­vates DnaA shortly af­ter ini­ti­a­tion by stim­u­lat­ing DnaA's in­trin­sic AT­Pase ac­tiv­ity. Genes en­cod­ing ho­mologs of E. coli DiaA and Hda are present in the P. aerug­i­nosa genome but nei­ther pro­tein has been looked at ex­per­i­men­tally. By con­trast to E. coli, a func­tional equiv­a­lent of the Dam/SeqA sys­tem that lim­its ini­ti­a­tion to a nar­row time win­dow has not been found, nor is it known if its dnaA gene is sub­ject to au­toreg­u­la­tion. Like­wise, the ter­mi­na­tion step of chro­mo­some repli­ca­tion is only rudi­men­ta­r­ily un­der­stood in P. aerug­i­nosa. From the pres­ence of E. coli homo­logs for XerCD and FtsK, we may con­clude that in P. aerug­i­nosa as well FtsK translo­cates DNA across the sep­tum af­ter XerCD re­com­bi­nase dis­en­tan­gles the fully repli­cated chro­mo­somes at the dif site dur­ing the fi­nal stage of chro­mo­some seg­re­ga­tion (see also here for FtsK's func­tion). Clear­ly, un­der­stand­ing the reg­u­la­tion of repli­ca­tion in P. aerug­i­nosa de­mands fur­ther stud­ies.

Fig­ure 2. Schematic rep­re­sen­ta­tion of the small nu­­cleoid-as­so­ci­ated pro­teins and SMC. These pro­teins in­troduce DNA bends and also func­tion in 'bridg­ing' chro­mosomal loci lo­cated on sep­a­rate to­pological do­mains. Source

Chro­mo­some com­paction   P. aerug­i­nosa, as most other bac­te­ria, com­pacts its chro­mo­some is com­pacted by ne­gative su­per­coil­ing of the DNA through the com­bined ac­tions of topoi­so­merases and so-called nu­cleoid asso­ciated pro­teins (NAPs), in­clud­ing IHF, Fis, HU, and MvaT/ MvaU (pu­ta­tive E. coli H‑NS ho­mologs). Un­like clas­si­cal re­pres­sors, NAPs don't usu­ally bind to well-de­fined bin­ding sites in pro­moter re­gions of genes but bind to more re­laxed recog­ni­tion se­quences and par­tic­u­lar DNA struc­tures. When bound to DNA, NAPs in­duce bends and kinks and, by oligomer­iza­tion, lead to 'bridg­ing' of sep­a­rate neigh­bor­ing topo­log­i­cal do­mains (Fig. 2). Such do­mains are ap­prox­i­mately 10 kb in length and highly dy­namic, mean­ing that they form and dis­solve in re­sponse to on­going tran­scrip­tion and repli­ca­tion with­out hav­ing fixed po­si­tions.

In both eu­kary­otes and prokary­otes, chro­mo­some com­paction in­volves a set of SMC pro­teins, for 'Chro­mo­some Main­te­nance of Chro­mo­somes'. In a gig­gly mood, one would prob­a­bly com­pare the SMCs to chop­sticks for pick­ing up noo­dles from a bowl of Japan­ese Udon noo­dle soup. The first bac­te­r­ial SMC, MukB of E. coli, was de­tected in 1991 by Sota Hi­raga and its shape as a dimer isn't so far from that of a pair of chop­sticks (Fig. 3, A). The SMCs' job is in­deed to pick noo­dles: they 'clamp' to­gether the 10 kb topo­log­i­cal do­mains (Fig. 2) and can be, to­gether with their ac­ces­sory pro­teins, called con­densins.
 

Fig­ure 3. A B. sub­tilis SMC forms V‑shaped di­mers, whose two arms are con­nected by a fle­xible hinge. Af­ter as­so­ci­a­tion of the hinge re­gion with DNA, the two head do­mains can in­teract in an ATP-de­pen­dent man­ner, form­ing a ring­like struc­ture that em­braces seg­ments of dou­ble-stranded DNA. Engage­ment of the head do­mains from dif­fer­ent mole­cules re­sults in the for­mation of larger rings or more com­plex struc­tures, such as ro­settes and fil­a­ments. B Mo­del for the re­cruit­ment of SMC to the repli­cation ori­gin re­gion in B. sub­tilis. Spo0J (ParB) in­teracts with prox­i­mal parS sites and spreads into the flank­ing chro­mo­so­mal re­gions, giv­ing rise to large nu­cle­o­pro­tein fil­a­ments. SMC is specif­i­cally re­cruited to these com­plexes and then be­comes dis­tributed over the repli­ca­tion ori­gin re­gion by lat­eral dif­fu­sion. Source

Chro­mo­some par­ti­tion­ing   ad­di­tion to their func­tion in chro­mo­some com­paction, bac­te­r­ial SMCs par­tic­i­pate in chro­mo­some seg­re­ga­tion dur­ing repli­ca­tion in ways that are not yet fully un­der­stood. Un­like most other bac­teria, which rely on one set of SMC pro­teins ­– the B. sub­tilis SMC-type or the E. coli Muk­BEF-type – P. aerug­i­nosa PAO1 en­codes two con­densins, the B. sub­tilis-type SMC-Sc­pAB and the E. coli-type Mks­BEF. Both con­tribute to faith­ful chro­mo­some par­ti­tion­ing and their in­ac­ti­va­tion leads to for­ma­tion of anu­cle­ate cells. How­ever, Mks­BEF can com­ple­ment anu­cle­ate cell for­ma­tion in SMC-de­fi­­cient cells.

Many but not all bac­te­ria posses a ParABS sys­tem that or­ga­nizes and com­pacts the chro­mo­some at a higher di­men­sion – above the level of the 10 kb topo­log­i­cal do­mains – and ac­tively con­trols the move­ment of chromo­some seg­ments dur­ing repli­ca­tion. Present mod­els sug­gest that ATP-bound ParA oligomer­izes to fil­a­ments along the cell axis and loosely con­tacts the DNA. ParB dimers bind strongly to palin­dromic parS sites at the re­plication ori­gin re­gion. ParS-bound ParB stim­u­lates the in­trin­sic AT­Pase ac­tiv­ity of ParA upon con­tact with a fi­lament tip, caus­ing ParA to de-poly­mer­ize. By al­ter­nat­ing poly­mer­iza­tion-de­poly­mer­iza­tion steps of ParA, chromo­somal DNA teth­ered to ParB·parS is pulled to­wards and along the ParA fil­a­ment, thus spa­tially sep­a­rat­ing the repli­cat­ing sis­ter chro­mo­somes. All compo­nents of a func­tional ParABS sytem are en­coded in the P. aerug­i­nosa genome, and parA or parB mu­tants show se­vere growth de­fects, in­clud­ing aber­rant chro­mo­some seg­re­ga­tion and misloca­tion of oriC and ter. In­ter­est­ingly, four parS sites were found close to oriC and an­other four (of 10 al­to­gether) in an ori­gin do­main de­tected by Val­let-Gely and Boc­card (more de­tails to this in Part 2). So far only ob­served for B. sub­tilis and Strep­to­myces coeli­color, Spo0J/ParB can poly­mer­ize around its parS bind­ing sites form­ing large nu­cle­o­pro­tein com­plexes that then at­tract SMCs (Fig. 3, B). The ex­act na­ture of the 'com­mu­ni­ca­tion' be­tween the P. aerug­i­nosa SMCs and its ParABS sys­tem has yet to be de­ci­phered.

The above may sound like an ex­tended to-do list rather than a well es­tab­lished chore­og­ra­phy, but Part 2 will show that it was nev­er­the­less pos­si­ble to gain deeper in­sight into the bal­let of chromo­some or­ga­ni­za­tion in Pseudomonas aerug­i­nosa cells.

 

Ref­er­ences

Ry­benkov VV (2014). Main­te­nance of chro­mo­some struc­ture in Pseudomonas aerug­i­nosa. FEMS mi­cro­bi­ol­ogy let­ters, 356 (2), 154−165. PMID 24863732

Val­let-Gely I, Boc­card F (2013). Chro­mo­so­mal or­ga­ni­za­tion and seg­re­ga­tion in Pseudomonas aerug­i­nosa. PLoS ge­net­ics, 9 (5). PMID 23658532