Free-living bacteria need genetic information to synthesize proteins for executing vital functions.

Most bacteria have a single chromosome with DNA that is about 2Mbp (mega base pairs) long (1Mbp=1X106 base pairs), but the DNA content of different species varies from 0.58 to greater than 9 Mbp of DNA. In contrast to the linear chromosomes found in eukaryotic cells, the strains of bacteria initially studied were found to have single, covalently closed, circular chromosomes. The circularity of the bacterial chromosome was elegantly demonstrated by electron microscopy in both Gram negative bacteria (such as Escherichia coli) and Gram positive bacteria (such as Bacillus subtilis). Bacterial plasmids were also shown to be circular. In fact, the experiments were so beautiful and the evidence was so convincing that the idea that bacterial chromosomes are circular and eukaryotic chromosomes are linear was quickly accepted as a definitive distinction between prokaryotic and eukaryotic cells. However, like most other distinctions between prokaryotic and eukaryotic cells, it is now clear that this dichotomy is incorrect. Not all bacteria have a single circular chromosome: some bacteria have multiple circular chromosomes, and many bacteria have linear SBT&HS

Bacterial Chromosome

chromosomes and linear plasmids.

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 Some bacteria have multiple chromosomes. The first convincing evidence that some bacteria have multiple chromosomes came from studies on Rhodobacter sphaeroides. Both molecular and genetic studies clearly demonstrated that R. sphaeroides has two large circular chromosomes. One of the chromosomes is 3.0 Mb and the other is 0.9 Mb. Leptospira also has two chromosomes of 4.4 and 4.6 Mbp. The largest bacterial genome yet analysed is that of Myxococcus xanthus, with 9.2Mbp. However, the best studied organism in nature is E. coli, which has a 4.6-Mbp chromosome with 4288 genes for proteins, seven operons for ribosomal ribonucleic acids (RNAs), and 86 genes for transfer RNAs. Some bacteria have linear chromosomes also. The first published evidence for linear chromosomes was in 1979, but because the techniques used at that time were limited and because the dogma that all bacterial chromosomes are circular was so entrenched, few people believed that linear chromosomes and plasmids occured in bacteria until 1989. Borrelia have linear chromosomes and most strains contain both linear and circular plasmids; most of the bacteria in the genus Streptomyces have linear chromosomes and plasmids and some have circular plasmids as well. In addition, in some cases there may be a dynamic equlibrium SBT&HS

between linear and circular forms of a DNA molecule. There is some evidence that linearization may be due to integration of a linear phage genome into the circular DNA molecule. On a macroscopic scale, bacterial chromosomes are either circular or linear. Circular chromosomes are most common, at least among the beststudied bacteria. However, the causative agent of Lyme disease, Borrelia burgdorphei, has a 2-Mb linear chromosome plus 12 different linear plasmids. Eukaryotic chromosomes are invariably linear, and they have two ends, each a telomere, and a organized region, the centromere which allows the chromosome to attach to cellular machinery that moves it to the proper place during cell division. The ends of linear DNA molecules (called telomeres) pose two problems that do not apply to circular DNA molecules. First, since free double-stranded DNA ends are very sensitive to degradation by intracellular nucleases, there must be a mechanism to protect the ends. Second, the ends of linear DNA molecules must have a special mechanism for DNA replication. These problems are solved by features of the telomeres. Two different types of telomeres have been observed in bacteria: hairpin telomeres and invertron telomeres. SBT&HS

 Linear DNA molecules in bacteria are protected by both types of telomeres: palindromic hairpin loops are protected by the lack of free double-stranded ends, and invertron telomeres are protected by proteins that bind to the 5'-ends.

Both of these mechanisms are also used by some phage, eukaryotic viruses, and eukaryotic plasmids. Telomeres also solve the problem of DNA replication differently. Invertron telomeres have a protein covalently attached to the 5' ends of the DNA molecule (called the 5'-terminal protein or TP). DNA polymerase interacts with the TP at the telomere and catalyzes the formation of a covalent bond between the TP and a dNTP. The dNTP bound to the TP has a free 3'-OH group which acts as the primer for chain elongation. Replication of hairpin telomeres is less well understood. Apparently multiple hairpin sequences can pair to form concatemers that are replication intermediates. SBT&HS

 One critical facet of chromosome structure is that DNA is a plectonemic helix, which means that two helical strands entwine about each other. For duplex DNA the two antiparallel strands are interwound once for every 10 bp. Because of this wound configuration, biochemical transactions that involve strand separation require chromosome movement (spin) about DNAs long axis. The processes ofDNA replication, recombination and transcription all require DNA rotation, and during DNA synthesis the rotation speed approaches 6000 rpm.

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 Chromosomal DNA molecules are very long and thin, so DNA must fold many times to fit within the confines of a bacterial cell. How does DNA twisting transpire without chromosomes getting tangled up? The problem was important enough that Watson and Crick considered a paranemic helix, which is a pair of coils that lay side by side without interwinding. Strands of a paranemic helix can separate without rotation. None the less DNA is plectonemic, and keeping chromosomes untangled requires a special class of enzymes called topoisomerases. In the dimensions of B-form DNA, E. coli is a sphere that is 6 kbp long (8 nm) and 4 kbp wide (2 nm), so the 4.6-Mbp chromosome must SBT&HS

be folded many times to fit within a cell. Negative supercoiling forces DNA into an interwound configuration. First, the DNA molecule doubles back on itself so that length is halved relative to that of the linear form. Second, supercoiled branches are dynamic so that opposing DNA sites in the interwound network are constantly changing. A protein bound to DNA in one supercoiling domain interacts more than 100 times more frequently with other proteins in the same domain than it does with proteins bound to a different domain. When DNA is liberated from cells by breaking the peptidoglycan coat, chromosomes form bundled loops that represent domains. Such preparations (called nucleoids) behave as discrete bodies. Many reactions ofthe chromosome require the formation of intricate DNAprotein machines to replicate, transcribe or recombine DNA at specific sequences. A group of chromosome-associated proteins assists in the formation of these complexes by shaping DNA. These proteins are sometimes described as histone-like, although they share no structural similarity with the eukaryotic histones. Chromosome-associated proteins include HU, H-NS, intregation host factor (IHF) and factor for inversion stimulation (FIS). HU is encoded by two genes, hupA and hupB, which are closely related to each other and to the genes encoding the two IHF SBT&HS

subunits. HU is the most abundant doublestranded DNA-binding protein in E. coli, although it also binds RNA. Although HU shows a preference for structurally bendable sequences, it binds DNA relatively nonspecifically, forming complexes that stabilize negative supercoils in double-stranded DNA. H-NS is a second relatively nonspecific DNA-binding protein that forms dimers, tetramers and possibly higher order associations when bound to DNA. Like HU, H-NS constrains negative supercoils. H-NS binds bendable DNA, which include AT-rich sequences often found near bacterial promoters; H-NS also modulates the transcription of numerous operons in E. coli and of lysogenic phages. FIS protein forms homodimers and binds DNA at specific sites. FIS was discovered nearly simultaneously in two related site-specific recombination systems: the Hin inversion reaction of Salmonella typhimurium and the Gin inversion reaction ofphage Mu. FIS concentration rises in early log phase when the protein has regulatory roles in replication initiation at oriC and transcription of ribosomal RNA operons, and then FIS abundance declines in stationary phase. IHF protein is composed oftwo subunits (encoded by the himA and hip genes) that are about 100 amino acids long and which make SBT&HS

stable heterodimers. IHF protein binds to a consensus sequence that was first discovered in phage , where it stimulates insertional and excisional recombination in vitro by a factor of 104. IHF bends DNA severely (more than 90); such bends are critical for site specific recombination in , for repression and activation of phage Mu, and for repressionactivation in a number of cellular operons, including ilv, oxyR and nifA.

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Supercoiling

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The diverse group of prokaryotes share even more common features with the eukaryotes. The circular genomes of mitochondrial and chloroplast are a notable exception to the rule that eukaryotic chromosomes are linear. However, this nicely fit into the dichotomy that eukaryotic chromosomes are linear and bacterial chromosomes are circular because these organelles seem to have evolved from entrapped bacteria. Other examples include the presence of introns, and poly-A tails on mRNA. This genus includes B. burgdorferi, the causative agent of Lyme disease. 1.Streptomyces make a wide variety of useful antibiotics, including streptomycin. 2.For example, linear DNA was precipitated in the most commonly used procedures for purifying bacterial plasmids, and the procedures for purifying chromosomal DNA relied upon the differential binding of ethidium bromide to "sheared DNA fragments" compared to circular DNA. 3.It is not intuitively obvious how the ends of a linear DNA molecule could be completely replicated. All known DNA polymerases require a pre-existing primer for initiation of DNA replication. The primer is usually a short RNA molecule with a free 3'-OH group that can be extended by DNA polymerase. If a linear DNA molecule was primed at one end, DNA synthesis could continue to the other end. However, once the primer is removed, the DNA corresponding to the primer could not be replicated. The telomers at the end of chromosomes of most eukaryotic cells are replicated by a different mechanism: most telomeres are short GC-rich repeats that are added in a 5' to 3' direction by the enzyme telomerase

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