Why, oh, why, can't life be less complicated ?

by Christoph

 

It could all be so easy! Take a bacterial cell, say, a single E. coli  cell with all its ~4,300 genes nicely strung like pearls on the necklace of its single circular chromosome. Present this E. coli  cell with something to eat and it starts growing and, while growing, replicates its chromosome starting at oriC, the unique replication origin. When the cell has reached twice its original size and now con­tains two chromosomes, the cell divides by binary fission, giving rise to two monoploid daughter cells of equal size. That's neat, orderly, and experimentally observed for planktonic E. coli  cells grown in minimal medium with a poor carbon source (glycerol) at 30°C. Alas, life is more complica­ted...
 

Figure 1. 'Circles in the Sand'. Source. Front­pa­ge: S. elongatus  chromosomes were tagged + observed in vivo using a fluorescent re­pres­sor-operator system (lacO/LacI-GFP). LacO arrays were integrated near the origin of repli­cation (NS1), which appear as foci (green) in cells (red) when imaged using wide field fluo­res­cence mi­croscopy. Source

Bacteria can live a solitary planktonic lifestyle but most prefer not to. Wildtype Bacillus subtilis  cells, for example, tend to stick together after division and grow in longer chains as do many Cyanobacteria. And the cells in these chains of siblings communicate with each other, of cour­se! Think of the long filaments of Nostoc  or Anabaena  in which one cell in a chain is "persuaded" to take the job of nitrogen acquisition as a so-called heterocyst (see a pic­tu­re here). Other bacteria usually come in "packages" of four cells, like Deinococcus, or two cells, like Neisseria go­norrhoeae (see a picture here). And, finally, most known bacteria are capable of forming biofilm communities, ei­ther among siblings or in a "multi-species enterprise" as in dental plaque. To form and maintain biofilms, the in­dividual cells communicate heavily with each other – so­metimes with fatal consequences – and reciprocally in­flu­ence their cell cycles.
 

So, living a "single-celled life" is more of a textbook abstraction than reality for bacteria. But what about the paradigmatic single circular chromosome with one replication origin (Figure 1) ? Recent­ly, E. coli  was engineered to have a linear chromosome and the cells were, by all means, absolute­ly fine. Linear chromosomes are known from the Spirochaetes, for example from Borrelia burg­dorf­eri, the causative agent of Lyme disease, and from Streptomyces  species that can switch be­tween linear and circular forms of their (single) chromosomes. When it comes to chro­mo­somal replication origins, bacteria mostly have just one oriC  (not counting the repressed phage origins in lysogens, of course, or oriTs of chromosomally integrated conjugative plasmids). A no­table ex­cept­ion is found in the Gammaproteobacteria where not only Pseudomonas aeruginosa  but all 90+ sequenced Pseudomonaceae have two closely spaced oriCs that may actually "count as one", de­pending on which one fires first in the cell cycle, thus preventing the second oriC  from firing. Simi­larly in the Oceanospirillales, sister family of the pseudomonads, you can spot genomes with two closely spaced oriCs, or one oriC  at either of the two possible locations (the author's unpublished observation).
 

The flexibility of bacterial genome organization does not end here! The Vibrionaceae, sister family of the Enterobacteraceae (E. coli  and its cousins), are the best known examples for bacteria having their genomes distributed over two (circular) chromosomes of different sizes, yet this is also found among various plant-associated and phototrophic Alphaproteobacteria. Only recently, however, vi­brios were found that have skipped the "2 chromosome rule" by fusing them into one large chro­mo­some. Also, a clinical isolate of Vibrio cholerae O1 El Tor apparently went this route too. Ex­cept­ions do obviously not stop with the exceptions. Note that the vibrios with their two chromosomes are, nevertheless, monoploid sensu stricto.
 

Figure 2. A Chromosomes in cells visualized through Lacl-GFP in an NSl-lacO background showed two different spatial arrangements. The chromosomes are either aligned along the major axis of the cell (I) or randomly localized (II). B Time-lapse imaging of single cells re­veal­ed a transient alignment of the chromosomes (4 h, yellow arrows) approximately 3 h before the cell entered cytokinesis. Source

Yet there are oligoploid bacteria. Deinococcus radiodu­rans  comes to mind here, which does not simply gets along as a cell quadruple (=pack-of-four) but with each individual cell harboring two chromosomes (and some plasmids). The numbers for chromosome 1 per nucleoid were found to range between 4 and 10 (the numbers for chromosome 2 were not measured in this study). Ther­mus thermophilus HB8, from the same bacterial phylum, has 4 – 5 copies of each of its two chromosomes per cell.
 

Another striking example for bacterial oligoploidy was found in the cyanobacterium Synechococ­cus elongatus PCC 7942. By tagging the one-chromosome genome with an array of lacO binding sites for a LacI repressor-GFP fusion close to the (single) replication origin, oriC, Chen et al.  could show by fluorescence microscopy (lighting up the GFP) that newborn S. elongatus  cells have, on average, 4 nucleoids, a number that increased to ~8 prior to cell division (see frontpage picture and Figure 2A). Replication of individual chro­mo­so­mes occurred randomly over the cell cycle for one chromosome at a time (which certainly helps to prevent chromosome entangling), and the in­creasing cell lengths during the growth phase cor­related well with number of nucleoids per cell. Bi­nary fission-type cell divisions left daughter cells with mostly four nucleoids yet unequal divisions were also found (similar to a log-normal distri­bu­tion). What had not been observed so far in other oligoploid bacteria was the transient nucleoid alignment along the cellular length axis approxi­ma­te­ly 3 h before cell division (Figure 2B). The mo­lecular composition of this "mitotic apparatus" is presently unknown, as is the regulation of its transient formation and decay.
 

(Click to enlarge )
Figure 3. Overview of the me­thod for quantifying ploidy levels. Results from a qPCR experiment are shown including, as tem­plates, serial dilutions of the standard fragment (red curves), the cytoplasmic extract (blue cur­ves) and the cytoplasmic extract with added in­ternal standard (green curves). The horizontal line used for the quantification of the Ct values is also included as well as the resulting standard curve. Source

Jörg Soppa and his coworkers at Goethe University Frank­furt, Germany, have devised a particularly sensitive meth­od to determine bacterial ploidy. Briefly, Genomic DNA is used as a template in a conventional PCR reaction to am­plify a fragment of about 1 kb length. A dilution series of this fragment is prepared and used for Real Time PCR (qPCR) analysis. A fragment of about 300 bp, internal to the standard fragment, is amplified. The results are used to generate a standard curve. To determine the genome copy number, cells are lysed and a dilution series of the resulting cell extract is analyzed by qPCR in parallel to the standards. The results allow calculating the number of genome copies in the cell extract and, in combination with the cell density of the culture, the ploidy level (see diagram in Figure 3). By applying this method they found a full spectrum of mono-, oligo (<10), and polyploidies (>10): 1 for Wolinella succinogenes (Epsilonproteobac­te­ria) 1 – 8 for slowly growing Azotobacter vinelandii (Gam­maproteobacteria, Pseudomonadaceae), 2 for Neisseria lactamica (Betaproteobacteria) and Caulobacter crescen­tus (Alphaproteobacteria), 3 for Neisseria gonorrhoe, 4 for Neisseria meningitides and Desulfovibrio vulgaris (Deltaproteobacteria), 9 – 17 for Desulfovibrio gigas de­pending on growth conditions, 20 for Pseudomonas pu­tida, 30 for fast growing A. vinelandii, and a whopping 120 for Buchnera (the latter number was highly variable and depended on the host's developmental stage). Pro­bably the most intriguing of their observations was the fluctuation of ploidy numbers with growth stage and growth conditions. Also, ploidies were not fixed for one species. Rather, it varied among strains of a species, and also among closely related genera. From the present status of the "ploidy census" it appears that monoploid bacteria are actually not the majority, and the cases of Deino­coccus or Synechococcus elongatus not the usual exceptions of the rule. Who would have thought?
 

Bacterial ploidy is far from being just *interesting* for genome nerds. On-site sampling of bac­te­ri­al communities has become a standard tool in microbial ecology, and "whole sample seque­nc­ing" a means to evaluate, independent of cultivation, species diversity and abundance for any imagi­na­ble habitat. It is easily conceivable, though, that the percentage distribution of species in a sample as calculated from the numbers of "sequence reads" would be considerably skewed if ploidies of the sampled species were not accounted for. Back to square one: why can't life be less complicated?