0 INSTITUT PASTEUF&LSEVIER Paris 1998

Bull. Inst. Pasteur 1998, 96, 25-33

REVIEW

Molecular evolution in bacteria : genome size, cell size, restriction-modification

and recognition

J.T. Trevors

Laboratory of Microbial Technology, Department of Environmental Biology, University of Guelph, Guelph, Ontario (Canada) NlG 2WI

The ability of bacteria to alter their genome sizes and the order of their genes, yet maintain a relatively constant genome, provides a mechanism for diversity and evolu- tion in bacteria. Moreover, bacteria may have evolved by increasing their genome sizes and rearranging gene orders with the assistance of restriction endonucleases cleaving foreign DNA and providing a diverse pool of DNA sequences for genetic recombination. This review examines some of these evolutionary aspects of bacteria including molecu- lar recognition of biomolecules.

Introduction

The bacterial cell’s micrometre dimensions provide a stable configuration for cellular metab- olism and replication of DNA. It is also generally believed that bacteria were the initial living organisms [l]. These microorganisms are still the oldest inhabitants on the Earth [l]. Moreover, their ability to grow and divide under a multitude of diverse environmental conditions reveals their capability to adapt to and alter the biosphere. Organisms at the cellular and biochemical level (DNA, RNA, proteins) are fundamentally similar even if the organisms are distantly related [l]. How- ever, multicellular organisms exhibit a variety of diverse properties which are still not understood.

During early evolution, the primitive atmos- phere of the Barth lacked 0,. It is therefore pos- tulated that the first bacteria were anaerobes. As 0, accumulated in the biosphere due to photo- synthesis, some anaerobic metabolic pathways in

bacteria were supplemented with cytochromes to link metabolism to 0,. This type of integrated metabolism was necessary for prokaryotic molec- ular evolution and optimization.

It is difficult to envision early prokaryotic molecular evolution in the absence of enzyme catalysis. It has been suggested that metabolic pathways appeared earlier in evolutionary time than the enzymes that catalyse the pathways [2]. Bishop et al. [2] reported on the non-enzymatic transamination (e.g., alanine + phenylpyruvic acid -+ phenylalanine ; glutamic acid + pyruvic acid 4 alanine ; alanine + alpha ketoglutaric acid 4 glutamic acid; phenylalanine + alpha ketoglu- taric acid -+ glutamic acid) between amino acids and keto acids without the need for a pyridoxal coenzyme. This direct transamination process may have been the beginning of complex amino acid metabolism in the absence of enzymes on the primitive Earth [2]. The role of enzymes in evolution may have been an evolutionary

Submitted September 14, 1997, accepted November 11, 1997.

26 J.T. TREVORS

development following non-enzymatic amino acid metabolism. Over time, non-enzymatic self- assembly was replaced with enzymatic assembly, replication of genomes and the formation of cells, cell growth and division. Figure 1 contains some possible major steps in the assembly of primitive prokaryotic genomes, bacterial diversification and bacterial colonization of the biosphere. Fig- ure 2 summarizes some factors that would have a

UNIVERSAL CONSTRUCTION KIT (ELEMENTS IN PERIODIC

TABLE) BIOMOLECULES RECOGNIZE EACH - OTHER

I

SELF-ASSEMBLY OF GENETIC MATERIAL

PRlMlTlVE REPLlCATlON OF GENETlC MATERIAL -

PRE-GENOME

EVOLVING GENOMES AND CEU-(S)

MOLECULAR OPTlMlZATlON -

I

BACTERIAL DIVERSIFICATION

I BACTERIAL COLONlZATlON OF

BIOSPHERE

Fig. 1. Some possible major steps in the assembly of primitive prokaryotic genomes.

significant influence on early evolving bacterial genomes and cells.

The bacterial genome (all genes present in the microorganism) is capable of change, while remaining relatively constant. These opposing states provide the essential mechanisms for some change and also the maintenance of an almost constant bacterial genome. Bacterial genome sizes within a genus remain somewhat constant, with the potential for minor changes in gene order. However, one way for bacterial cells to alter their genome sizes is to lose or acquire one or more plasmids.

Previous research by Trevors [3] on bacterial evolution and genome sizes revealed that genome sizes were not normally distributed about the arithmetic means of 3,812 kilobase pairs (kbp) for Gram-negative bacteria and 3,115 kbp for Gram-positive bacteria. It was also noteworthy that the genome sizes for Pseudomonas aerugi- now ranged from 4,100 to 5,400, 5,460 to 5,900 and from 2,200 to 2,800 kbp depending on the strains analysed [3]. In addition, differences in average genome sizes can change depending on the Gram-negative and Gram-positive genomes used in the size calculations and the presence or absence of plasmid( especially megaplasmids such as in Rhizobium spp.

Bacterial genomes range from about 1,000 to 9,000 kbp [3, 41. This range is a function of the evolutionary diversity of different bacterial spe- cies. The genome sizes of both Escherichia coli and Salmonella typhimurium (both Gram-nega- tive microorganisms) are about 4 million base- pairs. This is equivalent to a molecular weight of 3 x log daltons and a length of about 1.5mm. A better estimate of the E. coli genome is 4,500 kbp, with other bacterial genomes within a factor of about two of this value [4]. By summing sizes of DNA restriction fragments, the size of E. coli K12 EMG2 was determined to be 4,700 kbp (equivalent to 3.1 x log daltons - 3.1 megadal- tons [S]. The chromosome of E. coli and S. typhi-

be = base pairs. W = kilobase pairs.

R-M = restriction-modification.

MOLECULAR BACTERIAL EVOLUTION 27

EXTERNAL ENVIRONMENT

BlLLlONS OF YEAFIS- SUFFICIENT TIME

- temperature cycling - initially anoxic conditions - essential elements or

compounds lacking - extreme pH values

Initially, lnlstable Initially, cells would not

encapsulation of - pj - “nds;wcell;itision

be able to replicate

evolving genome genettc material and

- small genome size - small evolving cell size - metabolism not integrated - limitedgenetransfer - enzyme catalysis not present or

negligible - limited energy sources for cells - limited uptake of elements and

Fig. 2. Some factors influencing evolving bacterial genomes and cells.

murium also have common properties - a single, continuous, negatively supercoiled molecule with folding, to yield a nucleoid structure with bends at specific DNA sequences [6].

There is still a paucity of information on genome sizes in bacteria, even though methods are available to study this. Genthner et al. [7] used high-pressure liquid chromatography to determine the molecular mass of bacterial genomic DNA and plasmid copy number. A hydroxylapatite column capable of separating single-stranded DNA, double-stranded DNA and plasmid DNA was used for the analysis. DNA with an A260 to 48o ratio of 1.8 to 2.0 was con- sidered pure. The genome size of E. coli B was estimated to be 2,940 kbp. This value is smaller than the 4,500-kbp value described earlier for

E. coli. The plasmid copy number for pBR322 was estimated to be 29. The copy number was calculated by multiplying the ratio of the amount of plasmid to chromosomal DNA by the ratio of the Mr (molecular mass) values of the chromo- some and plasmid [7].

The genomes of P. aeruginosa strains (Gram- negative) have been estimated to range from 4,400 to 5,400kbp [S]. Some rhizobia (Gram- negative) cyanobacteria have genome sizes of up to 8,000kbp [5]. The chromosome of Bacillus subtilis was reported to be 2,000 kbp in size [9]. However, Anagnostopoulos er al. [lo] reported that endonuclease restriction of the B. subtilis 168 genome with SfiI and Not1 yielded a genome size of 4,188kbp. The latter value may be more accurate. This value of 4,188 kbp is

28 J.T. TREVORS

slightly smaller than the E. coli chromosome (4,700 kbp).

It is possible that bacteria with large genomes are the result of doubling of chromosome sizes during evolution. In addition, most bacterial chro- mosomes have likely been stable over millions to billions of years. Some DNA is cryptic (defined by Young [ll] as a phenotypically silent DNA sequence normally not expressed, but capable of activation as a rare event in some members of a population by mutation, recombination insertion of a DNA element or other genetic mechanisms) in bacteria. One or more DNA mutational changes have the potential to make the gene(s) functional.

Plasmids (double-stranded, circular DNA molecules that replicate independently of chro- mosomes) [12, 13, 141 are a significant pool of diverse genetic information. Plasmids vary in size from as small as 1 kbp to greater than 400kbp [12]. A 400-kbp plasmid may represent 10 % or more of a bacterial genome depending on the size of the host bacterial chromosome. Plasmids are virtually ubiquitous in genera like Pseudomonas [15, 161. However, the frequency of plasmid occurrence varies in particular species. Natural bacterial populations of Pseudomonas contain plasmids of diverse sizes that help to determine similar or different phenotypes. This in vivo genetic engineering by the bacteria themselves has allowed for changes in genome size. Plas- mids assist in this by promoting transfer of genetic information between bacteria belonging to the same as well as different taxonomic groups. It is also noteworthy that plasmids have different copy numbers (defined as the ratio of the number of moles of plasmid DNA to the number of mole equivalents of chromosomal DNA [17].

Bacterial cells maintain relative constancy in their genome while at the same time changing by genetic recombination, acquiring and losing plas- mids and transposons and undergoing transduc- tion and deletion/insertion of DNA that can pro- duce mutations. This creates a situation that gives rise to variants and diversity in bacterial popula- tions while maintaining the microorganism [5,6]. Related bacteria have similar genome sizes with

negligible variation in G+C (guanine+cytosine) content in different parts of the genome [5, 61. In addition, bacteria exhibit a wide G+C content - from about 23 to about 72% [S, 61.

During evolution, genetic information for unused metabolic systems was likely lost [18]. Stouthamer and Kooijman [lS] proposed that major metabolic differences between phylogenet- ically related bacteria may be explained by a loss of metabolic genes to maintain a small genome size and a suitable cell-surface to cell-volume ratio. This was explained on the basis of growth of individual microbial cells until replication of the genome is completed. Bacterial cells with a large genome size are larger at cell division [18], when distribution of cellular material from the parental cell to the two new cells at division (cell segregation) occurs [19]. Stouthamer and Kooij- man [18] suggested that this is a less favourable cell-surface to volume ratio, which has a negative influence on bacterial population growth rates. This implies that a smaller bacterial genome size has a selective advantage [18]. When nutrition- ally poor conditions are present, cells will not increase in volume during replication of DNA. Therefore, loss of part of the genome is of no advantage nor is loss of unused genes. This may lead to an average genome size [20].

It is possible that division of the genome into units occurred during the evolution of metabolic functions in microorganisms [20]. This is illus- trated by the enzymatic versatility of Pseudomo- nus spp. [15, 161, which often contain catabolic plasmids that encode for genes necessary for metabolism of a variety of organic compounds such as toxic xenobiotics, defined as completely synthetic chemical compounds not naturally occurring on Earth.

Genome mapping

A common method for chromosome and genome mapping is pulsed-field agarose gel elec- trophoresis. For example, if a genome with a ran- dom distribution of the 4 nucleotides was digested with restriction enzymes that recognized 4, 6, or 8 base pairs (bp), the resulting number of

MOLECULAR BACTERIAL EVOLUTION 29

fragments would be 256, 4,100 and 64,000 bp, respectively [21]. Since percent G+C and A+T ratios are not random, and the genome is species- dependent, the average fragment length is not a simple function of the base-recognition length. This aspect is important, as the percent G+C con- tent of bacteria varies from about 25 to 75 % or slightly higher.

DNA concentration in soil bacteria

Soil is the largest reservoir of diverse micro- organisms in the biosphere. Bakken and Olsen [22] reported that the DNA concentration of indige- nous soil bacteria ranged from 1.6 to 2.4 fg/cell. They suggested that dwarf cells in soil (capable of passing through a 0.40~p,rn membrane filter and often viable but non-culmrable) had an intact genome necessary for bacterial cell division. This research demonstrated the relative constancy of the bacterial genome in soil bacteria. In addition, gene order in the chromosome is very similar for related bacterial species that have been geograph- ically isolated, which have probably replicated their genomes billions of times and diverged from common ancestors for unknown lengths of time.

Restriction-modification (R-M) and genome size

Genome size during bacterial molecular evolu- tion may have been controlled, in part, by restric- tion-modification (R-M) systems. Endonuclease enzymes and methylase enzymes are catalytically active at the same DNA sequences [23]. Each endonuclease restriction enzyme recognizes a specific DNA sequence and catalyses cleavage of the DNA unless the sequence is methylated by a modification enzyme [23]. DNA in bacteria hav- ing an R-M system will be methylated, and are not suitable as a substrate for restriction [23]. The normal substrate for restriction enzymes is for- eign DNA sequences entering cells as bacterio- phages, or plasmid and chromosomal DNA enter- ing competent bacterial cells via transformation. Since R-M systems are widespread in prokar-

yotes, this suggests an important evolutionary role in these microorganisms. R-M systems are also not essential for bacterial growth, DNA syn- thesis, recombination or DNA repair [23]. Price and Bickle [23] suggested that protection of the host bacterial cell by R-M is analogous to an immune system in higher eukaryotes. The R-M system maintains a particular genome size and order while cleaving incoming foreign DNA and making it available for genetic recombination in these same cells.

Type I restriction enzymes consist of three non-identical subunits, with ATP (adenosine tri- phosphate) hydrolysis required for activity. Between 1 in 100 and 1 in 10,000 phages escape restriction. This system is therefore not efficient in the protection of cells from incoming phages. Type I restriction enzymes require specific DNA sites for recognition, while DNA cleavage is ran- dom with respect to sites. The type I restriction enzyme cleaves once, with no enzyme turnover.

Type II restriction enzymes are a single poly- peptide that recognizes a DNA sequence and then usually cuts within the sequence, as well as some- times outside the recognition DNA sequence, but at a fixed distance. Magnesium (Mg*‘) is the required cofactor, and the restriction efficiency is virtually 100%. For example, only 1 in 10” infecting phages is not restricted [23].

Type III R-M systems likely have a purpose similar to type II systems. Type III R-M restric- tion enzymes are composed of two different sub- units and require ATP, which is not hydrolysed during the catalytic reaction. They are efficient at restricting foreign DNA. The fact that bacterial cells have three different systems implies that evolving bacteria would have a virtual limitless supply of DNA sequences for use in genetic recombination and therefore in evolution.

Any DNA that escapes restriction is poten- tially capable of interacting with DNA in recipi- ent cells to bring the DNA into the chromosome by homologous recombination. This event requires that the homology of incoming and resi- dent DNA be relatively high. Several R-M systems are also encoded on conjugative plas- mids, which means that genes can be transferred from donor to recipient cells and assist in the

30 J.T. TREVORS

flow of these R-M systems throughout a micro- bial population. However, some phages have anti- restriction systems to escape restriction [23]. Moreover, some methylase enzymes that methyl- ate cytosine may have had a role in evolution, as cytosine methylation is itself mutagenic [23].

Point mutations, caused by addition/deletion of one or more DNA bases, occur in bacteria. However, when bases are added to or removed from a gene sequence in multiples of 3, the effect on the identity of adjacent amino acids is nil. The mutational event is simply addition or removal of one or several amino acids at the mutational site. It has been reported that after exposure to stress- ful conditions, many strains of Streptomyces exhibit as much as 900-kb DNA deletions and tandem amplification of selected chromosomal DNA sequences that range from 10 to 50% of the total DNA [4].

Bacterial genomes and cell sizes

Bacterial cells are large because they carry out a diverse range of integrated metabolic functions and are capable of cell division. Microbial cells must be large enough to maintain a stable struc- ture that ensures proper cell functions, which in turn ensure that metabolic and replication activ- ities are not in a state of constant fluctuation.

There are between 3,000 and 6,000 different molecules in a single bacterial cell, and about 40,500,000,000 total molecules per cell - 40,000,000,000 of these being H,O. Moreover, the transport rates of nutrients into the cells are in part a function of the surface area of the cells. Bacterial cells are large because they have evolved elaborate metabolic and replicative func- tions that need to be contained in a stable cyto- plasmic environment by membranes and a cell wall: they are the correct size for their diverse functions. It is not a matter of whether they are small or large, but a matter of evolutionary opti- mization over billions of years for the environ- ments in which they carry out their activities.

There is a paucity of information on bacterial genome sizes and whether they have any rela- tionship to cell size and morphology. For exam-

ple, ZVeisseriu gonorrhoeae is a small cocci, 0.6- 1.0 pm in diameter, with a genome of 1,800 kbp. Bacillus megaterium has a genome size of 4,670 kbp, and is a large rod ranging from 1.2- 1.5 pm in width and from 2-5 pm in length. These bacteria have different morpholo- gies, cell sizes and genome sizes. Staphylococ- cus aureus has a spherical cell morphology from 0.5 to 1.0 pm in diameter. Its genome size is 2,900 kbp. Streptococcus spp. also have rela- tively small genome sizes (1,700 and 2,300 kbp). As more information is forthcoming on bacterial genome size, it will be possible to statistically analyse data on genome size and determine whether it has a relationship to cell size/morphology.

Molecular recognition

The principal function of configured biomol- ecules in cells is to recognize other molecules. An excellent example is enzyme-substrate- product recognition during enzyme catalysis. Biomolecules also adopt specific configurations that require the minimal amount of free energy. Bacterial cells have evolved and optimized themselves to the necessary cell and genome size.

The evolutionary trend would be to optimize the sizes of cells so as to optimize their functions and eventually evolve specialized cells for differ- ent purposes. This would be central to eukaryotic evolution. For example, in cell division and the production, storage and use of chemical energy, the genetic code and present mechanisms of chemical energy conversion are optimized for life as we know it. The genetic code is simple, as it requires little information to encode the message; this is an excellent example of molecular optimi- zation.

An additional aspect that requires examination is pairing or recognition of biomolecules in pro- karyotic evolution. One primary function of pair- ing is that it allows one biomolecule to recognize another biomolecule. The DNA double helix is an excellent example of pairing of bases. The exact pairing also permits a mechanism for replicating

MOLECULAR BACTERIAL EVOLUTION 31

the genetic code. Other examples are enzyme- substrate recognition, duplicated genes, and paired donor and recipient cells in bacterial con- jugation. Once a macromolecule is produced, its primary function is to recognize other molecules, such as in the case of enzymes and their respec- tive substrates. This pairing or recognition also occurs rapidly - in the magnitude of lo6 per second - in some enzymatic reactions.

Pairing or recognition was important in early bacterial evolution, as it may have provided a means for efficient recognition of biomolecules and later, in prokaryotic evolution, for the rapid and protected transfer of DNA by conjugation between mating pairs of cells. Recognition between biomolecules was likely central to the initial self-assembly of primitive genomes, mem- branes and finally, cells capable of growth and division. Without recognition, primitive genomes and cells could not have assembled even in the presence of enzyme catalysis.

Recognition at the molecular level would also be influenced by the environment in which the recognition events were occurring and the con- centrations and types of molecules present. In a turbulent, changing environment, recognition may have been difficult due to constant changes or chaos in the environment. However, a pro- tected molecular environment such as the sur- face of minerals or clays may have provided the location upon which self-assembly of life began and proceeded [24] under elevated temperature conditions of cooling and warming and wetting and drying (interfaces between land and water) [25-301. Therefore, the first genomes and bacte- ria could have been thermotolerant or thermo- philes. However, the first genetic material was likely not DNA but most likely some precursor of RNA. It may have also been self-catalytic, and this assisted in its self-assembly.

Before RNA, there may have been numerous molecules involved in self-assembly and replica- tion [31]. It is not known if these non-living, unknown molecules were ancient, self-assem- bling primitive genomes that preceded RNA and DNA, or if RNA and DNA were formed indepen- dently of these ancient genomes without any con- nection to them.

It is known that primitive cells evolved and optimized. The transition from a random, unor- ganized lifeless Earth to the present situation, where the Earth is virtually covered with nucleic acids and diverse and complex species, required numerous molecular changes and the integration of metabolic reactions over billions of years [27, 321.

It is known that during molecular evolution, events like enzyme catalysis, membrane assem- bly, metabolism, production and storage of chem- ical energy by cells and DNA and RNA assembly were occurring on a molecular level. One com- mon ingredient in an all-encompassing theory may be time. The expanding universe at a cosmic level occurs over vast amounts of time. Molecular evolution also required time to arrive at a self- replicating, self-maintaining, living organism such as a common universal prokaryotic ancestor, and the subsequent evolution of bacterial popula- tions throughout the biosphere [33]. Collectively, microbes are the most versatile of living organ- isms, and they have shaped and help to defiF the limits of life in the biosphere over billions of years [34].

Key-words: Genome, Prokaryote, Phylogene- sis ; Genome and cell size, Restriction-modifica- tion, Molecular recognition ; Review.

Acknowledgements

This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) operating grants program.

fivolution molCculaire chez les bactiries : taille gknomique, taille cell&tire,

restriction-modification et reconnaissance

11 est admis que les cellules primitives ont tvoluC et atteint leur optimum. La transition entre un monde sans vie, non organid, alkatoire, et‘ la situa- tion prksente, 0iJ la terre est virtuellement couverte par les acides nuclkiques et diffkrentes espkes com- plexes, implique de nombreuses modifications au niveau molCculaire et l’integration de rkactions mktaboliques sur des milliards d’annt5es.

J.T. TREVORS

La capacite des batteries de modifier la taille de leur genome et l’ordre de leurs genes, tout en main- tenant un gtnome relativement constant, represente le mecanisme de l’evolution et de la diversid chez les bacdries. De plus, les batteries ont peut-&tre tvolu& en augmentant la taille de leur genome et en reamenageant l’ordre de leurs genes a l’aide d’endonucleases de restriction clivant 1’ADN etranger et produisant un ensemble diversifie de sequences d’ADN pour les recombinaisons gtnetiques.

Les cellules bacteriennes maintiennent leur gtnome relativement constant alors que paralle- lement elles changent par recombinaison genttique, acquierent et/au perdent des plasmides et des trans- posons, et subissent la transduction et la deletion/ insertion d’ADN pouvant produire des muta- tions. Cela crte une situation qui favorise le sur- gissement de variants et la diversite des populations bacttriennes tout en maintenant le microorganisme d’origine.

Au tours de l’evolution, l’information gtnetique pour des systemes mttaboliques non utilises a cer- tainement 6tC perdue. 11 est possible que les differences metaboliques majeures entre les batteries phylogCnCtiquement apparenttes peuvent s’expliquer par une perte des genes metaboliques afin de maintenir une faible taille genomique et un rapport adequoit entre la surface et le volume cellu- lakes.

Cette revue examine quelques-uns des aspects de l’evolution des bacttkies.

Mats-cl&s : Genome, Procaryote, Phylogenese ; Taille genomique et cellulaire, Restriction-modifica- tion, Reconnaissance molCculaire; Revue.

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