by Christoph
Figure 1.1 Movie by Christina Savage PhD (@Dr_Savage_Sci), with permission. Source. Frontispiece: Detail from Plate XXIV by Antoni van Leeuwenhoek. Animalcule in "fig G" is probably a spirochaete. Source
Christina Savage, postdoc in Kumaran Ramamurthi's lab at the NIH, recently posted on Twitter the movie you see on the right with the remark:"And we just got our darkfield objectives in today! This was taken with my camera looking down the eye-piece, so not great quality. But I just love watching them!" I replied:"everytime I have the rare pleasure to see these slender spirals 'live', i.e. buzzing around, I can't suppress the notion that their shape correlates so neatly with them having a linear chromosome. stupid, i know. correlations can be so misleading!" Texting on Twitter is not proper writing, obviously, and the 280‑character limit of tweets contributes to churning out cryptic sentences, sometimes. So, let me explain... and this explanation will come in two parts: here I will go into the outright confusing genome organization of Borrelia burgdorferi, which are starring in the microscopic movie, and in the the second part I will consider their extravagant periplasmic(!) flagella. There will be no "loose ends" left in either part, I promise.
Borrelia burgdorferi was identified in 1982 as the causative agent of Lyme disease (considered earlier in these pages here and here.) Together with Treponema pallidum, which causes syphilis in humans, and various Leptospira species known to cause leptospirosis, Borrelia belongs to the phylum Spirochaetes, whose members are Gram-negative and characterized by elongated, wave-like or helical ("corkscrew") cell morphologies, and endoflagella. Antoni van Leeuwenhoek was probably the first to draw a "portrait" of a spirochaete after he had scraped samples from his teeth for microscopy (see frontispiece.) There are free-living spirochaetes and ubiquitous "inhabitants", that is, ectosymbionts of all kinds of animals, marine or terrestrial. At least one spirochaete related to Treponema was identified as endosymbiont of a termit gut protist.
Figure 1.2 A Cumulative GC-skew plot for the sequence: Borrelia burgdorferi B31 [NC_001318.1], with stepsize: 909 and windowsize: 909. GC-skew minimum at pos. 456820. By the author using Genskew. B SIDD prediction (superhelical-stress induced duplex destabilization) for the putative replication origin of B. burgdorferi B31. Orange: low neg. superhelicity (σ=0.004), pink: strong neg. superhelicity (σ=0.006). DnaA boxes 5'-TTWTNCACA are shown with the half circle indicating their direction (all predicted DnaA boxes have the 5'-TGTGNAWAA reverse direction). Arrowhead points to the most likely point of DNA unwinding during replication initiation (pos. 458180). By the author
The genome of B. burgdorferi B31 was completely sequenced in 1997, the third ever completely sequenced bacterial genome. The authors found the genome composed of "a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs" (which, with 38 authors, gives a decent ratio of ~2 replicons per author.) Together, plasmids and the chromosome make for a rather small genome with limited coding capacity, akin to the streamlined genomes of free living bacteria like Prochlorococcus or the reduced genomes of endosymbionts. And indeed, many biosynthetic pathways are missing from the Borrelia genome.
The authors detected inverted repeats and closed hairpins at both chromosome ends and speculated that replication of the linear chromosome may start there like, for example, in poxvirus. For bi-directional replication, they considered an alternative "...because bacterial chromosomal replication origins are usually near dnaA, it is intriguing to note that this gene lies almost exactly at the center of the linear B. burgdorferi chromosome." Another hint for the localization of a replication origin "...near dnaA" came from a 'genometric' method developed by Steven Salzberg, one of the co-authors: certain 8-mer oligonucleotide sequences are preferentially found on the leading strand of replication resulting in a "strand switch" close to the actual replication origin. The most useful application of this genometric method is the so-called GC‑skew, that is, the observation that leading strands contain on average more G than C residues (calculated as (G − C)/(G + C) for a given sequence window and easily accumulated for adjacent windows over an entire chromosome.)
For the B. burgdorferi linear chromosome, one "switch point" of the GC-skew, the GC-skew minimum, is at position 456820 (Figure 1.2A), at a distance of 1.36 kb of the position where DnaA‑dependent replication is initiated (according to my unpublished prediction from 2006.) This oriC structure is rather conventional as it comprises 1. an intergenic region with counter‑clockwise transcription of the left-flanking gene (here: dnaN); 2. a superhelicity-responsive DNA‑unwinding element (DUE), and an array of DnaA-binding sites ('DnaA boxes'); 3. importantly, the DUE-proximal DnaA box at a distance of ~2 helical turns is in reverse orientation (5'‑TGTGNAWAA) (Figure 1.2B). (An aside. On the scale of a chromosome of 910 kp in length, a difference of 1.36 kb between the GC-skew minimum and the unwinding point in the (predicted) DUE is negligible. But in other bacterial chromosomes this difference can range from < 1 kb to ~20 kb, which clearly argues that the GC-skew can be used to pinpoint "the origin region", but does not detect "the origin" per se.)
By nascent strand analysis, Picardeau et al. (1999) had "mapped the replication initiation site of the B. burgdorferi chromosome to an ~2 kb region containing dnaA, dnaN and gyrB, which are also adjacent to the origin in other prokaryotes" and replication starts "most probably in the 240 bp sequence between dnaA and dnaN." Case solved: the linear B. burgdorferi chromosome replicates bidirectionally from a replication origin positioned right in its middle. But what happens with replication at the ends, after the replisomes have run through the inverted repeats and the hairpins? Not an 'end replication problem' here, but...
Figure 1.3 Hairpin telomere generation by protelomerase TelA of A. tumefaciens C58 (1 circular and 1 linear chromosome). A Schematic diagram showing how a bacterial linear chromosome wih hairpin telomeres is replicated. The last step is resolution of concatenated chromosomes at the inverted repeat junctions, which is carried out by protelomerase (telomere resolvase). B A model for the hairpin telomere formation by the protelomerase TelA, based on the crystal structures presented in this study. A key feature of the reaction mechanism of TelA revealed by this study is the "re‑folding intermediate"conformation that is stabilized by a set of transient protein–DNA and DNA–DNA interactions. Source
This riddle was solved for phage N15, which has a linear genome with hairpin-ends, and for the 2.19 Mb large linear chromosome 2 of Agrobacterium tumefaciens C58. In short, a gyrase-type protein called protelomerase (or telomere resolvase) binds as dimer to the inverted repeats, each monomer then cuts one strand of one of the repeats and − instead of wrapping the DNA strands around each other as gyrases do − re-folds the inverted repeat and seals the nick, thus re-creating the hairpin (see Figure 1.3). Et voilà, tout est prêt. Almost! I did not come back yet to the stunning zoo of circular and linear plasmids in the B. burgdorferi genome, which are often lost during cultivation in the lab. Here, Christine Jacobs-Wagner, an expert in all things Borrelia, contributed an important detail in the short thread on Twitter:"Some B. burgdorferi plasmids can be difficult to lose, even in culture. The circular plasmid cp26 cannot be lost because it carries the gene resT encoding the telomere resolvase, which is essential for cellular replication." No "loose ends" for Borrelia's linear chromosome anymore, fine.
Borrelia demonstrates that it is possible for a bacterium to live comfortably with a linear chromosome. But does it cope better with this than with a circular chromosome? Turning this question on its head, Cui et al. (2007) asked how E. coli fares with a linearized chromosome. Merry summarized their work in her 2008 post 'Some Like it Linear':"...they borrowed some tricks from the lambdoid coliphage N15, a temperate dsDNA phage whose prophage exists and replicates as a 46 kb linear plasmid. When tested in L broth at 37°C, the modified E. coli were indistinguishable from wild-type in terms of growth rate, nucleoid appearance, and overall pattern of transcription – provided that the linearization occurred at or near the chromosome replication terminus." As an aside, the "linear" E. coli could also do without the dif site close to the terminus that their "circular" siblings need to resolve the chromosome dimers that inevitably arise during replication (the FtsK translocase together with the XerCD resolvase monomerise dimeric chromosomes at dif). In conclusion, it doesn't make much of a dif-ference for a bacterium whether its chromosome(s) is linear or circular. Unless said bacterium is a Streptomyces sp., but then that would be the topic for a separate post.
Footnote
B. burgdorferi cells "are long in length and thin in diameter" (exact quote) with "dimensions of 10–20 µm and approximately 0.3 µm, respectively." A back-to-the-envelope calculation gives a length of ~310 µm for a fully stretched-out linear Borrelia chromosome. Thus my above mentioned tweet about a correlation of cell-length and a linear chromosome was badly derailed (which I was aware of, but some associations are persistent.) All bacteria, Borrelia included, have SMC-like proteins for chromosome compaction via looping, and up to a dozen nucleoid associated proteins (NAPs) that cover and compact their chromosomes and eventually act as transcription factors. One such NAP, EbfC, was shown by Jutras et al. (2012) to bind on average every 1,000 bp to Borrelia chromosomal (and plasmid) DNA, and as GFP fusion "lights up" a compacted nucleoid that spans the entire cell length in fluorescent micrographs.
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