The bacterial chromosome

Bacteria represent the vast majority of biological diversity found on Earth. In this review, we focus on selected aspects of their genetic material, those providing insight into structural, functional, dynamic, and evolutionary aspects of their genomes. Bacterial chromosomes are far more dynamic than previously realized, and dozens of mechanisms giving rise to genomic plasticity are now understood. Maturation of the genomics era has provided the tools for unraveling the interwoven details of DNA structure/function relationships that provide a basis for organismal diversity. Some of the most throughly understood processes that underlie the dynamics of genomic structure and function in prokaryotes are examined.     

Organization of transfer ribonucleic acid genes in the Escherichia coli chromosome.

The arrangement of transfer ribonucleic acid (RNA) genes in the chromosome of Escherichia coli K-12 (C600) was examined with the techniques of restriction endonuclease digestion and Southern blotting. The number and size of restriction fragments containing transfer or ribosomal RNA sequences or both were estimated by a variety of restriction endonucleases, including EcoRI, BglI, SmaI, SalI, BamHI, and PstI. EcoRI liberated a minimum of 27 fragments which hybridized to transfer RNA and 16 which hybridized to ribosomal RNA. Enzymes which did not cut within the ribosomal RNA operons (PstI and BamHI) liberated 16 and 13 fragments, respectively, which hybridized to transfer RNA. Five PstI and six BamHi fragments also hybridized to ribosomal RNA, suggesting that there may be at least 11 chromosomal locations distinct from ribosomal RNA operons which encode transfer RNA genes. In addition, our data indicated that several transfer RNA genes may be very close to the 5' proximal ends of certain ribosomal RNA operons and close to the 3' distal ends of all seven ribosomal RNA operons. Similar studies have been carried out with 22 purified species of transfer RNA, and we report here the number and size of EcoRI restriction fragments which hybridize to these transfer RNA species.     

Effect of chromosome location on bacterial mutation rates.

In previous comparisons of enterobacterial sequences, synonymous substitution rates were higher in genes closer to the replication terminus, suggesting that mutation rates increase with distance from the replication origin. In order to directly test for the effects of chromosomal location on the rates of point mutations, we assayed the reversion rates of two lacZ alleles inserted at four positions in the Salmonella enterica chromosome. Mutation rates at an intermediate locus were significantly higher than those at loci nearer to and farther from the replication origin. The higher reversion rates at this locus were neither the result of an overall increase in mutation rates produced by the insertion at this location nor a function of the mutations' immediate neighbors, but rather a regional effect. At all loci, regardless of chromosome location, T.A --> G.C transversions were more frequent than A.T --> G.C transitions during the exponential phase.     

Upheaval in the bacterial nucleoid. An active chromosome segregation mechanism

Recent advances have completely overturned the classical view of chromosome segregation in bacteria. Far from being a passive process involving gradual separation of the chromosomes, an active, possibly mitotic-like machinery is now known to exist. Soon after the initiation of DNA replication, the newly replicated copies of the oriC region, behaving rather like eukaryotic centromeres, move rapidly apart towards opposite poles of the cell. They then determine the positions that will be taken up by the newly formed sister nucleoids when DNA replication has been completed. Thus, the gradual expansion of the diffuse nucleoid camouflages an underlying active mechanism. Several genes involved in chromosome segregation in bacteria have now been defined; their possible functions are discussed.     

DNA motifs that sculpt the bacterial chromosome

During the bacterial cell cycle, the processes of chromosome replication, DNA segregation, DNA repair and cell division are coordinated by precisely defined events. Tremendous progress has been made in recent years in identifying the mechanisms that underlie these processes. A striking feature common to these processes is that non-coding DNA motifs play a central part, thus 'sculpting' the bacterial chromosome. Here, we review the roles of these motifs in the mechanisms that ensure faithful transmission of genetic information to daughter cells. We show how their chromosomal distribution is crucial for their function and how it can be analysed quantitatively. Finally, the potential roles of these motifs in bacterial chromosome evolution are discussed.     

Implication of gene distribution in the bacterial chromosome for the bacterial cell factory

As bacterial genome sequences accumulate, more and more pieces of data suggest that there is a significant correlation between the distribution of genes along the chromosome and the physical architecture of the cell, suggesting that the map of the cell is in the chromosome. Considering sequences and experimental data indicative of cell compartmentalisation, mRNA folding and turnover, as well as known structural features of protein and membrane complexes, we show that preliminary in silico analysis of whole genome sequences strongly substantiates this hypothesis. If there is a correlation between the genome sequence and the cell architecture, it must derive from some selection pressure in the organisms growing in the wild. As a consequence, the underlying constraints should be optimised in genetically modified organisms if one is to expect high product yields. Consequences in terms of gene expression for biotechnology are straightforward: knocking genes out and in genomes should not be randomly performed, but should follow the rules of chromosome organisation.     

Plasmids that mobilize bacterial chromosome

Cma can now be accepted as a common property of plasmids rather than one limited to a few, intensively studied examples. The mechanisms by which plasmids mobilize chromosome, in particular the role of plasmid integration into the bacterial chromosome and the part played by insertion sequences, are areas to which future interest should be directed. Solutions to these problems will be instructive in our understanding of plasmid and bacterial chromosome structure and function. The role of the IncP-1 plasmids can be expected to be central to this future work.The wide variety of bacterial genera for which conjugational analysis, mapping, and recombinant isolation are now available has extended microbial genetics into new areas. These advances are promising for the solution of a variety of practical problems in medicine, agriculture, and environmental science in the foreseeable future.     

The structure and function of the bacterial chromosome

Advances in microscopic and cell biological techniques have considerably improved our understanding of bacterial chromosome organization and dynamics. The nucleoid was formerly perceived to be an amorphous entity divided into ill-defined domains of supercoiling that are randomly deposited in the cell. Recent work, however, has demonstrated a remarkable degree of spatial organization. A highly ordered chromosome structure, established while DNA replication and partitioning are in progress, is maintained and propagated during growth. Duplication of the chromosome and partitioning of the newly generated daughter strands are interwoven processes driven by the dynamic interplay between the synthesis, segregation and condensation of DNA. These events are intimately coupled with the bacterial cell cycle and exhibit a previously unanticipated complexity reminiscent of eukaryotic systems.     

A bacterial artificial chromosome based physical map of the Ustilago maydis genome.

Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and co-digested with HaeIII to reduce fragments to 50-750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P < or = 1 x 10(-6) to 1 x 10(-24)). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P < or = 1 x 10(-7) to 1 x 10 (-12). The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P < or = 1 x 10(-7). The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18-20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.     

Supercoiling and map stability in the bacterial chromosome

A major goal of comparative genomics is an understanding of the forces which control gene order. This assumes that gene order is important, a supposition backed by the existence of genomic colinearity between many related species. In the bacterial chromosome, a polarity in the order of genes has been suggested, influenced by distance and orientation relative to the origin of DNA replication. We propose a model of the bacterial chromosome in which gene order is maintained by the adaptation of gene expression to local superhelical context. This force acts not directly at the genomic level but rather at the local gene level. A full understanding of gene-order conservation must therefore come from the bottom up. Correspondence to: R.L. Charlebois     

Characterization of a human chromosome 22 enriched bacterial artificial chromosome sublibrary

Selection of chromosomal sublibraries from total human genomic libraries is critical for chromosome-based physical mapping approaches. We have previously reported a method of screening total human genomic library using flow sorted chromosomal DNA as a hybridization probe and selection of a human chromosome 22-enriched sublibrary from a total human bacterial artificial chromosome (BAC) library (Nucleic Acids Res 1995; 23: 1838–1839). We describe here further details of the method of construction as well as characterization of the chromosome 22-enriched sublibrary thus constructed. Nearly 40% of the BAC clones that have been mapped by fluorescence in situ hybridization (FISH) analysis were localized to chromosome 22. By screening the sublibrary using chromosome 22-specific hybridization probes, we estimated that the sublibrary represents at least 2.5 × coverage of chromosome 22. This is in good agreement with the results from FISH mapping experiments. FISH map data also indicate that chromosome 22-specific BACs in the sublibrary represent all the subregions of chromosome 22.     

Bacteriophage P1 site-specific recombination. II. Recombination between loxP and the bacterial chromosome

Lipoprotein crystals of hagfish yolk platelets (Myxine glutinosa L.) are, by electron diffraction of embedded specimens, monoclinic (a > 19.8 nm, b > 8.9 nm, c > 9.0 nm, β ~ 105 °; space group C2: 2 dimers per cell). Using sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the four major protein bands in Myxine (molecular weights 30,000; 44,000: 80,000 and 130,000) correspond, with small differences, to similar bands of Xenopus yolk lipoproteins. The symmetric lipovitellin-phosvitin dimer in cyclostomes is unique and probably reflects the lack of diversification of homologous vertebrate protein species.