Experimental determination and system level analysis of essential genes in Escherichia coli MG1655.

Gerdes SY, Scholle MD, Campbell JW, Balázsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabási AL, Oltvai ZN, Osterman AL.

Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.

J Bacteriol. 2003 Oct;185(19):5673-84.

Comparative Genomics of the Vitamin B12 Metabolism and Regulation in Prokaryotes

Dmitry A. Rodionov, Alexey G. Vitreschak, Andrey A. Mironov, Mikhail S. Gelfand

Using comparative analysis of genes, operons, and regulatory elements, we describe the cobalamin (vitamin B12) biosynthetic pathway in available prokaryotic genomes. Here we found a highly conserved RNA secondary structure, the regulatory B12 element, which is widely distributed in the upstream regions of cobalamin biosynthetic/transport genes in eubacteria. In addition, the binding signal (CBL-box) for a hypothetical B12 regulator was identified in some archaea. A search for B12 elements and CBL-boxes and positional analysis identified a large number of new candidate B12-regulated genes in various prokaryotes. Among newly assigned functions associated with the cobalamin biosynthesis, there are several new types of cobalt transporters, ChlI and ChlD subunits of the CobN-dependent cobaltochelatase complex, cobalt reductase BluB, adenosyltransferase PduO, several new proteins linked to the lower ligand assembly pathway, l-threonine kinase PduX, and a large number of other hypothetical proteins. Most missing genes detected within the cobalamin biosynthetic pathways of various bacteria were identified as nonorthologous substitutes. The variable parts of the cobalamin metabolism appear to be the cobalt transport and insertion, the CobG/CbiG- and CobF/CbiD-catalyzed reactions, and the lower ligand synthesis pathway. The most interesting result of analysis of B12 elements is that B12-independent isozymes of the methionine synthase and ribonucleotide reductase are regulated by B12 elements in bacteria that have both B12-dependent and B12-independent isozymes. Moreover, B12 regulons of various bacteria are thought to include enzymes from known B12-dependent or alternative pathways.

Genome Analysis of F. nucleatum sub spp vincentii and Its Comparison With the Genome of F. nucleatum ATCC 25586

Vinayak Kapatral, Natalia Ivanova, Iain Anderson, Gary Reznik, Anamitra Bhattacharyya, Warren L. Gardner, Natalia Mikhailova, Alla Lapidus, Niels Larsen, Mark D'Souza, Theresa Walunas, Robert Haselkorn, Ross Overbeek, and Nikos Kyrpides

We present the draft genome sequence and its analysis for Fusobacterium nucleatum sub spp. vincentii (FNV), and compare that genome with F. nucleatum ATCC 25586 (FN). A total of 441 FNV open reading frames (ORFs) with no orthologs in FN have been identified. Of these, 118 ORFs have no known function and are unique to FNV, whereas 323 ORFs have functional orthologs in other organisms. In addition to the excretion of butyrate, H2S and ammonia-like FN, FNV has the additional capability to excrete lactate and aminobutyrate. Unlike FN, FNV is likely to incorporate galactopyranose, galacturonate, and sialic acid into its O-antigen. It appears to transport ferrous iron by an anaerobic ferrous transporter. Genes for eukaryotic type serine/threonine kinase and phosphatase, transpeptidase E-transglycosylase Pbp1A are found in FNV but not in FN. Unique ABC transporters, cryptic phages, and three types of restriction-modification systems have been identified in FNV. ORFs for ethanolamine utilization, thermostable carboxypeptidase, γ glutamyl-transpeptidase, and deblocking aminopeptidases are absent from FNV. FNV, like FN, lacks the classical catalase-peroxidase system, but thioredoxin/glutaredoxin enzymes might alleviate oxidative stress. Genes for resistance to antibiotics such as acriflavin, bacitracin, bleomycin, daunorubicin, florfenicol, and other general multidrug resistance are present. These capabilities allow Fusobacteria to survive in a mixed culture in the mouth.

Genome Res. 2003 Jun; 13(6a): 1180–1189.
doi:  10.1101/gr.566003

Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis.

Ivanova N, Sorokin A, Anderson I, Galleron N, Candelon B, Kapatral V,
Bhattacharyya A, Reznik G, Mikhailova N, Lapidus A, Chu L, Mazur M, Goltsman E,
Larsen N, D'Souza M, Walunas T, Grechkin Y, Pusch G, Haselkorn R, Fonstein M,
Ehrlich SD, Overbeek R, Kyrpides N.

Bacillus cereus is an opportunistic pathogen causing food poisoning manifested by
diarrhoeal or emetic syndromes. It is closely related to the animal and human
pathogen Bacillus anthracis and the insect pathogen Bacillus thuringiensis, the
former being used as a biological weapon and the latter as a pesticide. B.
anthracis and B. thuringiensis are readily distinguished from B. cereus by the
presence of plasmid-borne specific toxins (B. anthracis and B. thuringiensis) and
capsule (B. anthracis). But phylogenetic studies based on the analysis of
chromosomal genes bring controversial results, and it is unclear whether B.
cereus, B. anthracis and B. thuringiensis are varieties of the same species or
different species. Here we report the sequencing and analysis of the type strain
B. cereus ATCC 14579. The complete genome sequence of B. cereus ATCC 14579
together with the gapped genome of B. anthracis A2012 enables us to perform
comparative analysis, and hence to identify the genes that are conserved between
B. cereus and B. anthracis, and the genes that are unique for each species. We
use the former to clarify the phylogeny of the cereus group, and the latter to
determine plasmid-independent species-specific markers.

Nature. 2003 May 1;423(6935):87-91.

Missing genes in metabolic pathways: a comparative genomics approach.

Osterman A, Overbeek R.

The new techniques of genome context analysis--chromosomal gene clustering, protein fusions, occurrence profiles and shared regulatory sites--infer functional coupling between genes. In combination with metabolic reconstructions, these techniques can dramatically accelerate the pace of gene discovery.

Curr Opin Chem Biol. 2003 Apr;7(2):238-51.

The ERGO genome analysis and discovery system.

Overbeek R, Larsen N, Walunas T, D'Souza M, Pusch G, Selkov E Jr, Liolios K, Joukov V, Kaznadzey D, Anderson I, Bhattacharyya A, Burd H, Gardner W, Hanke P, Kapatral V, Mikhailova N, Vasieva O, Osterman A, Vonstein V, Fonstein M, Ivanova N, Kyrpides N.

The ERGO (http://ergo.integratedgenomics.com/ERGO/) genome analysis and discovery suite is an integration of biological data from genomics, biochemistry, high-throughput expression profiling, genetics and peer-reviewed journals to achieve a comprehensive analysis of genes and genomes. Far beyond any conventional systems that facilitate functional assignments, ERGO combines pattern-based analysis with comparative genomics by visualizing genes within the context of regulation, expression profiling, phylogenetic clusters, fusion events, networked cellular pathways and chromosomal neighborhoods of other functionally related genes. The result of this multifaceted approach is to provide an extensively curated database of the largest available integration of genomes, with a vast collection of reconstructed cellular pathways spanning all domains of life. Although access to ERGO is provided only under subscription, it is already widely used by the academic community. The current version of the system integrates 500 genomes from all domains of life in various levels of completion, 403 of which are available for subscription.

Nucleic Acids Res. 2003 Jan 1;31(1):164-71.

FlhD/FlhC is a regulator of anaerobic respiration and the Entner-Doudoroff pathway through induction of the methyl-accepting chemotaxis protein Aer.

Prüss BM, Campbell JW, Van Dyk TK, Zhu C, Kogan Y, Matsumura P.

The regulation by two transcriptional activators of flagellar expression (FlhD and FlhC) and the chemotaxis methyl-accepting protein Aer was studied with glass slide DNA microarrays. An flhD::Kan insertion and an aer deletion were independently introduced into two Escherichia coli K-12 strains, and the effects upon gene regulation were investigated. Altogether, the flhD::Kan insertion altered the expression of 29 operons of known function. Among them was Aer, which in turn regulated a subset of these operons, namely, the ones involved in anaerobic respiration and the Entner-Doudoroff pathway. In addition, FlhD/FlhC repressed enzymes involved in aerobic respiration and regulated many other metabolic enzymes and transporters in an Aer-independent manner. Expression of 12 genes of uncharacterized function was also affected. FlhD increased gltBD, gcvTHP, and ompT expression. The regulation of half of these genes was subsequently confirmed with reporter gene fusions, enzyme assays, and real-time PCR. Growth phenotypes of flhD and flhC mutants were determined with Phenotype MicroArrays and correlated with gene expression.

J Bacteriol. 2003 Jan; 185(2): 534–543.
doi:  10.1128/JB.185.2.534-543.2003

The ERGO genome analysis and discovery system.

Overbeek R, Larsen N, Walunas T, D'Souza M, Pusch G, Selkov E Jr, Liolios K, Joukov V, Kaznadzey D, Anderson I, Bhattacharyya A, Burd H, Gardner W, Hanke P, Kapatral V, Mikhailova N, Vasieva O, Osterman A, Vonstein V, Fonstein M, Ivanova N, Kyrpides N.

The ERGO (http://ergo.integratedgenomics.com/ERGO/) genome analysis and discovery suite is an integration of biological data from genomics, biochemistry, high-throughput expression profiling, genetics and peer-reviewed journals to achieve a comprehensive analysis of genes and genomes. Far beyond any conventional systems that facilitate functional assignments, ERGO combines pattern-based analysis with comparative genomics by visualizing genes within the context of regulation, expression profiling, phylogenetic clusters, fusion events, networked cellular pathways and chromosomal neighborhoods of other functionally related genes. The result of this multifaceted approach is to provide an extensively curated database of the largest available integration of genomes, with a vast collection of reconstructed cellular pathways spanning all domains of life. Although access to ERGO is provided only under subscription, it is already widely used by the academic community. The current version of the system integrates 500 genomes from all domains of life in various levels of completion, 403 of which are available for subscription.

Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesis.

Kurnasov OV, Polanuyer BM, Ananta S, Sloutsky R, Tam A, Gerdes SY, Osterman AL.

NAD is an indispensable redox cofactor in all organisms. Most of the genes required for NAD biosynthesis in various species are known. Ribosylnicotinamide kinase (RNK) was among the few unknown (missing) genes involved with NAD salvage and recycling pathways. Using a comparative genome analysis involving reconstruction of NAD metabolism from genomic data, we predicted and experimentally verified that bacterial RNK is encoded within the 3' region of the nadR gene. Based on these results and previous data, the full-size multifunctional NadR protein (as in Escherichia coli) is composed of (i) an N-terminal DNA-binding domain involved in the transcriptional regulation of NAD biosynthesis, (ii) a central nicotinamide mononucleotide adenylyltransferase (NMNAT) domain, and (iii) a C-terminal RNK domain. The RNK and NMNAT enzymatic activities of recombinant NadR proteins from Salmonella enterica serovar Typhimurium and Haemophilus influenzae were quantitatively characterized. We propose a model for the complete salvage pathway from exogenous N-ribosylnicotinamide to NAD which involves the concerted action of the PnuC transporter and NRK, followed by the NMNAT activity of the NadR protein. Both the pnuC and nadR genes were proven to be essential for the growth and survival of H. influenzae, thus implicating them as potential narrow-spectrum drug targets.

J Bacteriol. 2002 Dec;184(24):6906-17.

Bioinformatics classification and functional analysis of PhoH homologs.

Kazakov AE, Vassieva O, Gelfand MS, Osterman A, Overbeek R.

PhoH protein is a putative ATPase belonging to the phosphate regulon in Escherichia coli. EC-PhoH homologs are present in different organisms, but it is not clear if they are functionally related, besides nothing is known about their regulation. To distinguish true functional orthologs of EC-PhoH in different classes of bacteria and to identify their functional role in bacterial metabolic network we performed phylogenetic analysis of these proteins and comparative study of position and regulation of the related genes. Three groups of proteins were identified. Proteins of the first group (BS-PhoH orthologs) are present in most of bacteria and are proposed to be functionally linked to phospholipid metabolism and RNA modification. Proteins of the second group (BS-YlaK orthologs) are present in most of aerobes and Actinobacterial YlaK orthologs are shown to be members of a fatty acid beta-oxidation regulons. EC-PhoH orthologs are classified in a third group, specific for Enterobacteria. Functional role of PhoH homologs in the lipid and RNA metabolism and proposed interrelation of PhoH paralogs in one organism are discussed.

In Silico Biol. 2003;3(1-2):3-15. Epub 2002 Dec 30

Genes for the cytoskeletal protein tubulin in the bacterial genus Prosthecobacter.

Jenkins C, Samudrala R, Anderson I, Hedlund BP, Petroni G, Michailova N, Pinel N, Overbeek R, Rosati G, Staley JT.

Tubulins, the protein constituents of the microtubule cytoskeleton, are present in all known eukaryotes but have never been found in the Bacteria or Archaea. Here we report the presence of two tubulin-like genes [bacterial tubulin a (btuba) and bacterial tubulin b (btubb)] in bacteria of the genus Prosthecobacter (Division Verrucomicrobia). In this study, we investigated the organization and expression of these genes and conducted a comparative analysis of the bacterial and eukaryotic protein sequences, focusing on their phylogeny and 3D structures. The btuba and btubb genes are arranged as adjacent loci within the genome along with a kinesin light chain gene homolog. RT-PCR experiments indicate that these three genes are cotranscribed, and a probable promoter was identified upstream of btuba. On the basis of comparative modeling data, we predict that the Prosthecobacter tubulins are monomeric, unlike eukaryotic alpha and beta tubulins, which form dimers and are therefore unlikely to form microtubule-like structures. Phylogenetic analyses indicate that the Prosthecobacter tubulins are quite divergent and do not support recent horizontal transfer of the genes from a eukaryote. The discovery of genes for tubulin in a bacterial genus may offer new insights into the evolution of the cytoskeleton.

Proc Natl Acad Sci U S A. 2002 Dec 24; 99(26): 17049–17054.
Published online 2002 Dec 16. 
doi:  10.1073/pnas.012516899

Draft Sequencing and Comparative Genomics of Xylella fastidiosa Strains Reveal Novel Biological Insights.

Anamitra Bhattacharyya, Stephanie Stilwagen, Gary Reznik, Helene Feil, William S. Feil, Iain Anderson, Axel Bernal, Mark D'Souza, Natalia Ivanova, Vinayak Kapatral, Niels Larsen, Tamara Los, Athanasios Lykidis, Eugene Selkov, Jr., Theresa L. Walunas, Alexander Purcell, Rob A. Edwards, Trevor Hawkins, Robert Haselkorn, Ross Overbeek, Nikos C. Kyrpides, and Paul F. Predki

Draft sequencing is a rapid and efficient method for determining the near-complete sequence of microbial genomes. Here we report a comparative analysis of one complete and two draft genome sequences of the phytopathogenic bacterium, Xylella fastidiosa, which causes serious disease in plants, including citrus, almond, and oleander. We present highlights of an in silico analysis based on a comparison of reconstructions of core biological subsystems. Cellular pathway reconstructions have been used to identify a small number of genes, which are likely to reside within the draft genomes but are not captured in the draft assembly. These represented only a small fraction of all genes and were predominantly large and small ribosomal subunit protein components. By using this approach, some of the inherent limitations of draft sequence can be significantly reduced. Despite the incomplete nature of the draft genomes, it is possible to identify several phage-related genes, which appear to be absent from the draft genomes and not the result of insufficient sequence sampling. This region may therefore identify potential host-specific functions. Based on this first functional reconstruction of a phytopathogenic microbe, we spotlight an unusual respiration machinery as a potential target for biological control. We also predicted and developed a new defined growth medium for Xylella.

Genome Res. 2002 Oct; 12(10): 1556–1563.
doi:  10.1101/gr.370702

The genome of Brucella melitensis.

DelVecchio VG, Kapatral V, Elzer P, Patra G, Mujer CV.

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3,198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other alpha-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.

Vet Microbiol. 2002 Dec 20;90(1-4):587-92.
doi:10.1016/S0378-1135(02)00238-9

Whole-genome comparative analysis of three phytopathogenic Xylella fastidiosa strains.

Bhattacharyya A, Stilwagen S, Ivanova N, D'Souza M, Bernal A, Lykidis A, Kapatral V, Anderson I, Larsen N, Los T, Reznik G, Selkov E Jr, Walunas TL, Feil H, Feil WS, Purcell A, Lassez JL, Hawkins TL, Haselkorn R, Overbeek R, Predki PF, Kyrpides NC.

Xylella fastidiosa (Xf) causes wilt disease in plants and is responsible for major economic and crop losses globally. Owing to the public importance of this phytopathogen we embarked on a comparative analysis of the complete genome of Xf pv citrus and the partial genomes of two recently sequenced strains of this species: Xf pv almond and Xf pv oleander, which cause leaf scorch in almond and oleander plants, respectively. We report a reanalysis of the previously sequenced Xf 9a5c (CVC, citrus) strain and the two "gapped" Xf genomes revealing ORFs encoding critical functions in pathogenicity and conjugative transfer. Second, a detailed whole-genome functional comparison was based on the three sequenced Xf strains, identifying the unique genes present in each strain, in addition to those shared between strains. Third, an "in silico" cellular reconstruction of these organisms was made, based on a comparison of their core functional subsystems that led to a characterization of their conjugative transfer machinery, identification of potential differences in their adhesion mechanisms, and highlighting of the absence of a classical quorum-sensing mechanism. This study demonstrates the effectiveness of comparative analysis strategies in the interpretation of genomes that are closely related.

Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12403-8. Epub 2002 Aug 30.

From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways.

Gerdes SY, Scholle MD, D'Souza M, Bernal A, Baev MV, Farrell M, Kurnasov OV, Daugherty MD, Mseeh F, Polanuyer BM, Campbell JW, Anantha S, Shatalin KY, Chowdhury SA, Fonstein MY, Osterman AL.

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability of E. coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E. coli genes nadD, coaD, and ribF), are discussed in detail.

J Bacteriol. 2002 Aug;184(16):4555-72.

Microarray analysis of gene expression during bacteriophage T4 infection.

Luke K, Radek A, Liu X, Campbell J, Uzan M, Haselkorn R, Kogan Y.

Genomic microarrays were used to examine the complex temporal program of gene expression exhibited by bacteriophage T4 during the course of development. The microarray data confirm the existence of distinct early, middle, and late transcriptional classes during the bacteriophage replicative cycle. This approach allows assignment of previously uncharacterized genes to specific temporal classes. The genomic expression data verify many promoter assignments and predict the existence of previously unidentified promoters.

Virology. 2002 Aug 1;299(2):182-91.

Discovering lactic acid bacteria by genomics.

Klaenhammer T, Altermann E, Arigoni F, Bolotin A, Breidt F, Broadbent J, Cano R, Chaillou S, Deutscher J, Gasson M, van de Guchte M, Guzzo J, Hartke A, Hawkins T, Hols P, Hutkins R, Kleerebezem M, Kok J, Kuipers O, Lubbers M, Maguin E, McKay L, Mills D, Nauta A, Overbeek R, Pel H, Pridmore D, Saier M, van Sinderen D, Sorokin A, Steele J, O'Sullivan D, de Vos W, Weimer B, Zagorec M, Siezen R.

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, environmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram-positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.

Exact mapping of prokaryotic gene starts.

Baytaluk MV, Gelfand MS, Mironov AA.

It is known that while the programs used to find genes in prokaryotic genomes reliably map protein-coding regions, they often fail in the exact determination of gene starts. This problem is further aggravated by sequencing errors, most notably insertions and deletions leading to frame-shifts. Therefore, the exact mapping of gene starts and identification of frame-shifts are important problems of the computer-assisted functional analysis of newly sequenced genomes. Here we review methods of gene recognition and describe a new algorithm for correction of gene starts and identification of frame-shifts in prokaryotic genomes. The algorithm is based on the comparison of nucleotide and protein sequences of homologous genes from related organisms, using the assumption that the rate of evolutionary changes in protein-coding regions is lower than that in non-coding regions. A dynamic programming algorithm is used to align protein sequences obtained by formal translation of genomic nucleotide sequences. The possibility of frame-shifts is taken into account. The algorithm was tested on several groups of related organisms: gamma-proteobacteria, the Bacillus/Clostridium group, and three Pyrococcus genomes. The testing demonstrated that, dependent or a genome, 1-10 per cent of genes have incorrect starts or contain frame-shifts. The algorithm is implemented in the program package Orthologator-GeneCorrector.

Brief Bioinform. 2002 Jun;3(2):181-94.

Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics.

Daugherty M, Polanuyer B, Farrell M, Scholle M, Lykidis A, de Crécy-Lagard V, Osterman A.

The biosynthesis of CoA from pantothenic acid (vitamin B5) is an essential universal pathway in prokaryotes and eukaryotes. The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown. Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC ), phosphopantothenoylcysteine decarboxylase (EC ), phosphopantetheine adenylyltransferase (EC ), and dephospho-CoA kinase (EC ). Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis. The individual human enzymes were overexpressed in E. coli and purified, and the corresponding activities were experimentally verified. In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes. Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized. This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.

J Biol Chem. 2002 Jun 14;277(24):21431-9. Epub 2002 Mar 28.

Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586.

Kapatral V, Anderson I, Ivanova N, Reznik G, Los T, Lykidis A, Bhattacharyya A, Bartman A, Gardner W, Grechkin G, Zhu L, Vasieva O, Chu L, Kogan Y, Chaga O, Goltsman E, Bernal A, Larsen N, D'Souza M, Walunas T, Pusch G, Haselkorn R, Fonstein M, Kyrpides N, Overbeek R.

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H(2)S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth.

J Bacteriol. 2002 Apr;184(7):2005-18.