Download Genome Sequencing and Informatics: New Tools

Plant Physiol. (1998) 117: 1129–1133
Update on Genomics
Genome Sequencing and Informatics: New Tools for
Biochemical Discoveries
Milton H. Saier, Jr.*
Department of Biology, University of California at San Diego, La Jolla, California 92093–0116
During the past 3 years, we have experienced a major
revolution in the biological sciences resulting from a tremendous flux of information generated by genomesequencing efforts. Our understanding of microorganisms,
the metabolic processes they catalyze, the genetic apparatuses encoding cellular proteinaceous constituents, and the
pathological conditions caused by these organisms has
greatly benefited from the availability of complete microbial genomic sequences. Many research institutes around
the world are now devoting their efforts solely to genome
sequencing and to analysis of the data produced. Dozens of
international conferences have been held with the primary
purpose of keeping the scientific community abreast of
recent developments. In this Update I will summarize some
of the exciting information reported at a recent conference
on microbial genomics.1
Compared with microbe genomics, plant genomics is
still in its infancy, since the sequencing of only the Arabidopsis genome is currently under way (Bevan et al., 1998).
However, within a few years we can expect that the
genomic sequences of at least two plants, Arabidopsis and
rice (Oryza sativa), will be available. Meanwhile, as of January 1998, 12 microbial genomes have already been sequenced and published. These include genomes of representative organisms from the three domains of life:
bacteria, archaea, and eukarya. The genomes of one eukaryote (the brewers’ yeast Saccharomyces cerevisiae) as well
as three archaea and eight bacteria have been completely
sequenced. By examining what has been learned from sequencing of microbial genomes, we can get some idea
about what kinds of information we can expect from plant
genome-sequencing efforts.
MICROBIAL GENOME SEQUENCING
Most of the bacterial genomes that have been sequenced
are small, of 2 Mbp or less. However, three large bacterial
genomes have been sequenced: those of the prototypic
* E-mail [email protected]; fax 1– 619 –534 –7108.
1
The conference, entitled “Microbial Genomics II,” was organized by Claire Fraser of the Institute for Genomic Research in the
United States and Bart Barrell of the Sanger Center in England, and
took place at Hilton Head, South Carolina, from January 31 to
February 3, 1998. The program and abstracts of the meeting are
presented in Microbial and Comparative Genomics (1998) 3: 1–96.
gram-negative bacterium Escherichia coli (Blattner et al.,
1997); the best-characterized gram-positive bacterium, Bacillus subtilis (Kunst et al., 1997); and a representative cyanobacterium, Synechocystis PCC6803 (Kaneko et al., 1996).
In addition, six microbial genomes have been completely
sequenced but not yet published. Pathogens with fully
sequenced genomes include Mycobacterium tuberculosis, the
causative agent of tuberculosis; Treponema pallidum, the
spirochete that causes syphilis; and Borrelia burgdorferi, another spirochete that causes Lyme disease. Two interesting
nonpathogens that have recently been sequenced are
Deinococcus radiodurans, the organism reported to be most
resistant to UV irradiation, and Aquifex aeolicus, a marine
hyperthermophile capable of growth at 95°C. Based on 16S
RNA analyses, A. aeolicus may represent the deepest lineage within the bacterial domain, and its genome may
therefore provide clues about primitive prokaryotic lifeforms that existed billions of years ago.
More than 50 microbial genomes are currently being
sequenced. These include the genomes of virtually every
major pathogen. Industrially important bacteria such as
Clostridium acetobutylicum, a principal producer of organic
solvents, are also being sequenced, as is the deep-sea
manganese-oxidizing bacterium Shewanella putrefaciens.
The sequence of the first animal genome, that of the worm
Caenorhabdeitis elegans, is expected to be completed in the
middle of this year (July, 1998). Although only 3% of the
human genome has been sequenced, this tiny fraction of
the human genome represents more DNA than that of all of
the completed microbial genomes sequenced to date.
Table I summarizes some of the most impressive
genome-sequencing efforts completed by the end of 1997.
The first organism to have its genome sequenced was
Haemophilus influenzae (Fleischmann et al., 1995). It has a
genome size of 1.8 Mbp encoding 1743 recognized genes.
One laboratory at the Institute for Genomic Research, involving about 40 people, completed the project in 1 year.
B. subtilis, with a genome of 4.2 Mbp, was sequenced by an
international consortium of 46 laboratories involving about
160 people in 5 years (Kunst et al., 1997).
S. cerevisiae possesses about 13 Mbp of DNA, and 12 Mbp
of this DNA has been fully sequenced. The remaining 1
Mbp consists of repetitive rDNA (encoding rRNA molecules) representing more than 100 repeats. This repetitive
Abbreviation: Mbp, megabase pair.
1129
Downloaded from on August 3, 2017 - Published by www.plantphysiol.org
Copyright © 1998 American Society of Plant Biologists. All rights reserved.
1130
Saier
Plant Physiol. Vol. 117, 1998
Table I. Representative genome-sequencing efforts
Organism
Genome Size
Genes
Laboratories
People
Years
1743
4100
5900
4300
1
46
96
1
40
160
640
17
1
5
6
10
Mbp
H. influenzae
B. subtilis
S. cerevisiae
E. coli
1.8
4.2
12
4.6
DNA was not sequenced as part of the yeast-sequencing
effort partly for technical reasons, and partly because it was
expected to yield little new information. Ninety-six laboratories and 640 people completed this project in 6 years
(Goffeau et al., 1997). The prototypical bacterium E. coli (4.6
Mbp) was sequenced by a single laboratory in an effort
involving 17 people, but the project took about 10 years
(Blattner et al., 1997). Because of recent technological advances, it is estimated that a microbial genome of about 2
Mbp can now be fully sequenced in 1 year by a single
laboratory of 6 people with an expenditure of less than $1
million.
GENOME SEQUENCING LEADS TO THE
IDENTIFICATION OF NEW PROTEINS
Tremendous benefits have resulted from the microbial
genome-sequencing efforts completed to date (Table II).
For example, important new proteins have been identified.
Owen White at the Institute for Genomic Research reported
that D. radiodurans is the first nonphotosynthetic organism
to be shown to possess the light-sensing protein phytochrome. In D. radiodurans this molecule may function to
regulate the synthesis of pigments that protect the organism from irradiation, and it also has a novel type of RecA
protein involved in DNA repair-related recombination.
Richard Roberts of New England Biolabs analyzed various genomes for DNA restriction-modification systems,
enzyme systems that protect organisms from the potentially detrimental effects of foreign DNA (such as that of
viruses). It was found that although B. subtilis and E. coli
possess 3 to 4 such restriction-modification systems, some
pathogenic bacteria have far more. Thus, H. influenzae has
7, Neisseria gonorrhoeae has 18, and Helicobacter pylori, which
is a causative agent of peptic ulcers, has 23. The physiological significance attributed to the possession of large
numbers of such systems in small-genome bacteria is a
point of debate.
Entirely new families of protein paralogs (families of
proteins arising by gene duplication within a single organism) have been revealed by genome sequencing. For examTable II. Benefits of genomics
New proteins identified
New protein families discovered
New pathways revealed
Total metabolic capabilities estimated
Virulence factors and mechanisms identified
Potential drug targets revealed
Gene transfer established
ple, Claire Fraser, Sherwood Casjens, and Steven Norris
reported that the genomes of the two pathogenic spirochetes T. pallidum and B. burgdorferi both exhibit large
families of species-specific paralogs of unknown function.
The same has been observed for methanogenic (methaneproducing) archaea. In the case of B. subtilis, a large family
of Bacillus-specific protein paralogs have been identified as
“Rap” phosphatases that release phosphate from phosphorylated aspartyl residues in response to regulatory proteins. These regulatory proteins control the initiation of
sporulation, a gram-positive bacterial cell-differentiation
process that leads to the generation of thick-walled, metabolically inert, environmentally resistant spores (Perego et
al., 1994, 1996).
DISCOVERY OF NEW METABOLIC PATHWAYS
Entirely new metabolic pathways have been revealed by
genome sequencing. Tyrrell Conway reported the discovery of a previously unrecognized pathway in E. coli for the
metabolism of the sugar acid idonate, a compound that had
not been known to be a substrate for the growth of this
bacterium (C. Bausch, N. Peekhaus, C. Utz, T. Blais, E.
Murray, T. Lowary, and T. Conway, unpublished data).
Additionally, analysis of several bacterial genomes showed
that many bacteria have all of the requisite enzymes of the
ribulose-monophosphate–hexulose-monophosphate pathway, a pathway for the interconversion of five- and sixcarbon sugars (Reizer et al., 1997). Such a pathway had
been previously demonstrated only in methanogenic bacteria. Finally, Owen White reported evidence that D. radiodurans has a novel pathway for the efficient repair of
double-stranded DNA breaks, a fact that explains the ability of this octaploid organism to repair more than 150
double-stranded DNA breaks in its genome without loss of
viability (Battista, 1997).
The availability of a complete genomic sequence allows
one to estimate the total metabolic capability of an organism. For example, knowledge of the complete gene complement permits estimation of the nutrients that can be
taken up by the cell. Such knowledge reflects the natural
lifestyle of the organism, and hence, ecological deductions
and inferences can be made regarding the use of the bacterium for purposes of bioremediation (Clayton et al., 1997;
Paulsen et al., 1998).
Based on the complement of genes encoded within microbial genomes, Peter Karp of the Pangea Corporation in
Oakland, CA, and his co-workers have created “EcoCyc”
for E. coli and “HinCyc” for H. influenzae, which are computerized displays of the complete metabolic pathways of
Downloaded from on August 3, 2017 - Published by www.plantphysiol.org
Copyright © 1998 American Society of Plant Biologists. All rights reserved.
Genome Sequencing and Informatics
Table III. Technological advances in genomics
Chips bearing probes for analysis of complete
organismal gene complements
Vectors for gene expression
Gene knockouts and reporter-gene fusions
Novel chips for sequencing
these two closely related organisms (Karp et al., 1996).
“EcoCyc,” which is available on the World Wide Web, is
now being expanded to include transport and regulatory
information as well as metabolic reactions.
Results reported at the Microbial Genomics II conference
clearly suggested that many obligate prokaryotic human
parasites have condensed and streamlined their genomes,
with the loss of regulatory functions and biosynthetic capabilities. Moreover, although strict human pathogens
such as Mycoplasma genitalium, T. pallidum, and B. burgdorferi have adapted to an anaerobic life style using glycolytic
sugar metabolism for their primary source of energy, as
reported by Claire Fraser, Chlamydia (a common sexually
transmitted disease agent [Peeling and Brunham, 1996])
and Rickettsia (the causative agent of Rocky Mountain spotted fever [Andersson and Andersson, 1997]) have adapted
to a strictly aerobic life style. These two bacteria have lost
their glycolytic enzymes and satisfy their energy needs by
metabolizing organic acids via the Krebs cycle and electron
flow, as reported by Richard Stephens and Siv Andersson,
respectively (unpublished results).
COMPARATIVE GENOMICS YIELDS NEW CLUES
ABOUT PATHOGENESIS AND HORIZONTAL
GENE TRANSFER
Virulence factors and novel mechanisms of pathogenesis
have been revealed as a result of the development of the
new discipline of comparative genomics. Thus, Fred Blattner, who recently sequenced a pathogenic E. coli strain and
compared it with the nonpathogenic E. coli K12 strain
commonly used for laboratory research, reported that the
former bacterium possesses 1.2 Mbp more DNA than the
latter, an approximately 20% increase in genomic size. This
additional genetic material codes for virulence factorbearing prophage, a virulence plasmid, and a “pathogenicity island” that allows the bacteria to secrete toxic proteins
directly from the bacterial cytoplasm into that of the host
animal cell (see Groisman and Ochman, 1996). In addition,
three new tRNAs, a eukaryotic-type Ser/Thr protein kinase, and a novel iron-transport system, all possibly important for pathogenicity, were revealed.
Potential drug targets were identified and studied. Lynn
Miesel reported on studies characterizing the targets of the
antituberculosis drug isoniazid. This drug appears to inhibit the growth of M. tuberculosis by blocking the function
of a biosynthetic enzyme that makes a cell-surface fatty
acid called mycolic acid. Isoniazid thereby disrupts the
outer protective layer of this fastidious bacterium, rendering it sensitive to host defense mechanisms.
Horizontal gene transfer between related gram-negative
bacteria could be established using the comparative
1131
genomics approach. Pathogenicity islands, encoding virulence factor genes and the apparatus for their transfer to
host cells, have now been identified in many bacteria (Groisman and Ochman, 1996). Fred Blattner noted that 30% of
the new DNA present in pathogenic E. coli, but lacking in
nonpathogenic E. coli, is shared by Yersinia pestis, the causative agent of plague.
GENOMICS, PROTEOMICS, AND BIOINFORMATICS
Tremendous technological advances are being made in
the related fields of genomics, proteomics, and bioinformatics (Tables III–V). In the area of genomics (the study of
an organism’s gene complement; Table III), novel chips
bearing oligonucleotide probes for analysis of the expression of the complete complement of genes encoded within
the genome of an organism have already been used and the
results published (DeRisi et al., 1997; Wodicka et al., 1997).
Such a chip costs about $200 and is good for a single
information-rich experiment. However, the machine that
reads the chip costs about $150,000.
Expression vectors, gene knockouts, and reporter-gene
constructs for all of the genes in an organism will soon be
available for at least two major experimental organisms,
the yeast S. cerevisiae, and the sporulating, gram-positive
bacterium B. subtilis. Our laboratory has recently taken
advantage of such technology to identify and characterize
a protein kinase that controls catabolite repression and
carbon metabolism in B. subtilis (Reizer et al., 1998). In
earlier studies we had purified the protein and determined
an N-terminal amino acid sequence, but the availability of
this sequence was insufficient to allow us to clone the gene.
When the complete genome of B. subtilis became available,
its identification became almost automatic. Thus, when
expression vectors, knockouts, and fusion constructs become available commercially, the academic molecular biologist will be largely obsolete.
In proteomics (the study of an organism’s proteins; Table
IV), technological advances are beginning to have an effect
on basic research. Tremendous advances have been made
in coupling two-dimensional gel analysis of the total protein complement of an organism to analysis by MS or to
N-terminal sequence analysis. Chips are being developed
for pico-quantity protein purification based on hydrophobic and ion-exchange chromatography as well as adsorption chromatography.
Rapid procedures for studying protein-protein interactions are being developed. For example, a yeast two-hybrid
system has recently been developed for the assay of whole
libraries of genes to determine which of the encoded proteins interact with high affinity with a specific test protein
(Williams et al., 1998). Additionally, novel chips for studying protein-ligand interactions are being developed.
Table IV. Technological advances in proteomics
Two-dimensional gel analysis coupled to MS
Chips for full proteome protein purification
Protein-protein interaction studies
Protein-ligand interaction studies
Downloaded from on August 3, 2017 - Published by www.plantphysiol.org
Copyright © 1998 American Society of Plant Biologists. All rights reserved.
1132
Saier
Finally, in the area of bioinformatics (the computational
analysis of genetic and biochemical data; Table V), novel
software is being developed for more refined homology
searches of the databases, for protein and nucleic acid
secondary and tertiary structural predictions, and for protein and DNA motif identification. Many of the programs
used routinely in academic laboratories for characterizing
families of proteins have been or are now being automated.
Such advances will be required to keep up with the exponentially increased volume of sequence data that will be
available in the near future.
Genome-sequence analyses have revealed that most of
the proteins encoded within the genome of a living organism belong to families of homologous proteins that share a
common evolutionary origin and are present in many dissimilar organisms. Some of these families are very large,
with hundreds of currently sequenced members (Pao et al.,
1998). Other families are small, with only a few currently
sequenced members (Saier, 1998). By constructing dendrograms (which show approximate relationships of the proteins to each other without providing numerical values for
the relative phylogenetic distances that separate them), or
instead by constructing phylogenetic trees (which not only
cluster the proteins according to their relative degrees of
sequence similarity, but also provide quantitative measures of their degrees of relatedness), one can estimate the
probability that any two members of a protein family will
prove to serve the same function. Phylogenetic trees thus
indicate relative degrees of sequence similarity and provide a reliable guide to biochemical function.
WHERE IS GENOMICS GOING?
Where is the new discipline of genomics taking us?
Directly into an exciting and ever-expanding new century
of scientific discovery. The current sequencing explosion
will lead to the development of novel disciplines such as
“comparative genomics” and “molecular archaeology” (Table VI). Because about 20% to 30% of each newly sequenced
genome consists of genes encoding proteins with no recognizable homologs in the current databases (Koonin et al.,
1997), a tremendous effort must be devoted to the functional identification of these “orphan” proteins. Novel regulatory constraints and virulence mechanisms will be revealed (Ewald, 1996). New kingdoms of currently
unculturable microbes (Bloomfield et al., 1998) are likely to
be revealed. Discoveries leading to tremendous expansion
Table V. Technological advances in informatics
Novel software for
More refined homology searches
Three-dimensional structural predictions
Motif identification
Repetitive sequence analysis
Automation for
Data searching
Multiple alignment generation
Phylogenetic-tree construction
Averaging similarity, hydropathy, amphipathicity
Plant Physiol. Vol. 117, 1998
Table VI. Microbial genomics: future directions
Sequencing explosion: genome sequencing will become the primary source of biological information
Comparative genomics: functional assignments in one
organism will be directly applicable to many others
Molecular archeology: sequence comparisons and
three-dimensional structural analyses will allow
deduction of evolutionary origins of most macromolecules
Functional identification: many new types of genes
must be characterized biochemically and physiologically
Regulatory constraints: identification of new genes
requires an understanding of how their expression
is regulated
Virulence mechanisms: novel factors and mechanisms
concerned with microbial pathogenesis will be
discovered
Unculturable organisms: microorganisms that cannot
be currently cultured in the laboratory will be
characterized
Industrial applications: industry will use microorganisms to an ever-increasing degree for the production of useful products
Novel technological advances: technologies will be
developed for understanding and using the genetic
information encoded within microbes
Unforeseen discoveries: many novel discoveries will
be made in almost every imaginable dimension,
with far-reaching consequences
of industrial applications will be made, and additional
novel technological advances will undoubtedly appear in
the not-too-distant future. But most importantly, genomics
will lead to discoveries currently unforeseen and nearly
unfathomable. Even 2 years ago we did not dream that
microbial genomes would exhibit the degree of plasticity
that has been revealed by genome sequencing, or that the
“minimal genome” would be so limited in scope. Living
organisms have solved the fundamental problems of life in
many diverse ways. It is an exciting time to be working in
the biological sciences, and this excitement is largely attributable to developments in genomics. The advances of the
future will be limited only by the imaginations of current
and future generations of molecular biologists.
Received April 5, 1998; accepted April 18, 1998.
Copyright Clearance Center: 0032–0889/98/117/1129/05.
LITERATURE CITED
Andersson JO, Andersson SGE (1997) Genomic rearrangements
during evolution of the obligate intracellular parasite Rickettsia
prowazekii as inferred from an analysis of 52015 bp nucleotide
sequence. Microbiology 143: 2783–2795
Battista JR (1997) Against the odds: the survival strategies of
Deinococcus radiodurans. Annu Rev Microbiol 51: 203–224
Bevan M, Bancroft I, Bent E, Love K, Goodman H, Dean C,
Bergkamp R, Dirkse W, Van Staveren M, Stiekema W, and
others (1998) Analysis of 1.9 Mb of contiguous sequence from
chromosome 4 of Arabidopsis thaliana. Nature 391: 485–488
Blattner FR, Plunkett G III, Bloch CA, Perna NT, Burland V,
Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayrew GF,
Downloaded from on August 3, 2017 - Published by www.plantphysiol.org
Copyright © 1998 American Society of Plant Biologists. All rights reserved.
Genome Sequencing and Informatics
and others (1997) The complete genome sequence of Escherichia
coli K-12. Science 277: 1453–1474
Bloomfield SF, Stewart GSAB, Dodd CER, Booth IR, Power
EGM (1998) The viable but non-culturable phenomenon explained? Microbiology 144: 1–2
Clayton RA, White O, Ketchum KA, Venter JC (1997) The first
genome from the third domain of life. Nature 387: 459–462
DeRisi JL, Iyer VR, Brown PO (1997) Exploring the metabolic and
genetic control of gene expression on a genomic scale. Science
278: 680–686
Ewald PW (1996) Guarding against the most dangerous emerging
pathogens: insights from evolutionary biology. Emerging Infect
Dis 2: 245–257
Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness
EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick
JM, and others (1995) Whole-genome random sequencing and
assembly of Haemophilus influenzae Rd. Science 269: 496–512
Goffeau A, Aert R, Agostini-Carbone ML, Ahmed A, Aigle M,
Alberghina L, Albermann K, Albers M, Aldea M, Alexandraki
D, and others (1997) The yeast genome directory. Nature
387(suppl): 1–105
Groisman EA, Ochman H (1996) Pathogenicity islands: bacterial
evolution in quantum leaps. Cell 87: 791–794
Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y,
Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, and others
(1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential
protein-coding regions. DNA Res 3: 109–136
Karp PD, Riley M, Paley SM, Pelligrini-Toole A (1996) EcoCyc:
an encyclopedia of Escherichia coli genes and metabolism. Nucleic Acids Res 24: 32–39
Koonin EV, Mushegian AR, Galperin MY, Walker DR (1997)
Comparison of archaeal and bacterial genomes: computer analysis of protein sequences predicts novel functions and suggests
a chimeric origin for the archaea. Mol Microbiol 25: 619–637
Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessieres P, Bolotin A, Borchert S, and
1133
others (1997) The complete genome sequence of the Grampositive bacterium Bacillus subtilis. Nature 390: 249–272
Pao SS, Paulsen IT, Saier MH Jr (1998) The major facilitator
superfamily. Microbiol Mol Biol Rev 62: 1–32
Paulsen IT, Sliwinski MK, Saier MH Jr (1998) Microbial genome
analyses: global comparisons of transport capabilities based on
phylogenies, bioenergetics and substrate specificities. J Mol Biol
277: 573–592
Peeling RW, Brunham RC (1996) Chlamydiae as pathogens: new
species and new issues. Emerging Infect Dis 2: 307–319
Perego M, Glaser P, Hoch JA (1996) Aspartyl-phosphate phosphatases deactivate the response regulator components of the
sporulation signal transduction system in Bacillus subtilis. Mol
Microbiol 19: 1151–1157
Perego M, Hanstein C, Welsh KM, Djavakhishvili T, Glaser P,
Hoch JA (1994) Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the
control of development in B. subtilis. Cell 79: 1047–1055
Reizer J, Hoischen C, Titgemeyer F, Rabus R, Stülke J, Rivolta C,
Karamata D, Saier MH Jr, Hillen W (1998) A novel protein
kinase that controls carbon catabolite repression in bacteria. Mol
Microbiol 27: 1157–1169
Reizer J, Reizer A, Saier MH Jr (1997) Is the ribulose monophosphate pathway widely distributed in bacteria? Microbiology 143:
2519–2520
Saier MH Jr (1998) Molecular phylogeny as a basis for the classification of transport proteins from bacteria, archaea and eukarya. In RK Poole, ed, Advances in Microbial Physiology Academic Press, San Diego, CA (in press)
Williams JM, Chen G-C, Zhu L, Rest RF (1998) Using the yeast
two-hybrid system to identify human epithelial cell proteins that
bind gonococcal Opa proteins: intracellular gonococci bind
pyruvate kinase via their Opa proteins and require host pyruvate for growth. Mol Microbiol 27: 171–186
Wodicka L, Dong H, Mittmann M, Ho MH, Lockhart DJ (1997)
Genome-wide expression monitoring in Saccharomyces cerevisiae.
Nature Biotechnol 15: 1359–1367
Downloaded from on August 3, 2017 - Published by www.plantphysiol.org
Copyright © 1998 American Society of Plant Biologists. All rights reserved.