Genome sequence analysis of the emerging human pathogenic acetic acid bacterium Granulibacter bethesdensis.

Greenberg DE, Porcella SF, Zelazny AM, Virtaneva K, Sturdevant DE, Kupko JJ 3rd, Barbian KD, Babar A, Dorward DW, Holland SM.

Chronic granulomatous disease (CGD) is an inherited immune deficiency characterized by increased susceptibility to infection with Staphylococcus, certain gram-negative bacteria, and fungi. Granulibacter bethesdensis, a newly described genus and species within the family Acetobacteraceae, was recently isolated from four CGD patients residing in geographically distinct locales who presented with fever and lymphadenitis. We sequenced the genome of the reference strain of Granulibacter bethesdensis, which was isolated from lymph nodes of the original patient. The genome contains 2,708,355 base pairs in a single circular chromosome, in which 2,437 putative open reading frames (ORFs) were identified, 1,470 of which share sequence similarity with ORFs in the nonpathogenic but related Gluconobacter oxydans genome. Included in the 967 ORFs that are unique to G. bethesdensis are ORFs potentially important for virulence, adherence, DNA uptake, and methanol utilization. GC% values and best BLAST analysis suggested that some of these unique ORFs were recently acquired. Comparison of G. bethesdensis to other known CGD pathogens demonstrated conservation of some putative virulence factors, suggesting possible common mechanisms involved in pathogenesis in CGD. Genotyping of the four patient isolates by use of a custom microarray demonstrated genome-wide variations in regions encoding DNA uptake systems and transcriptional regulators and in hypothetical ORFs. G. bethesdensis is a genetically diverse emerging human pathogen that may have recently acquired virulence factors new to this family of organisms.

J Bacteriol. 2007 Dec;189(23):8727-36. Epub 2007 Sep 7.

Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis.

Anderson I, Sorokin A, Kapatral V, Reznik G, Bhattacharya A, Mikhailova N, Burd H, Joukov V, Kaznadzey D, Walunas T, Markd'Souza, Larsen N, Pusch G, Liolios K, Grechkin Y, Lapidus A, Goltsman E, Chu L, Fonstein M, Ehrlich SD, Overbeek R, Kyrpides N, Ivanova N.

Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp. israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.

FEMS Microbiol Lett. 2005 Sep 15;250(2):175-84.
DOI: http://dx.doi.org/10.1016/j.femsle.2005.07.008

The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode.

Foster J, Ganatra M, Kamal I, Ware J, Makarova K, Ivanova N, Bhattacharyya A, Kapatral V, Kumar S, Posfai J, Vincze T, Ingram J, Moran L, Lapidus A, Omelchenko M, Kyrpides N, Ghedin E, Wang S, Goltsman E, Joukov V, Ostrovskaya O, Tsukerman K, Mazur M, Comb D, Koonin E, Slatko B.

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.

PLoS Biol. 2005 Apr; 3(4): e121.
Published online 2005 Mar 29. doi:  10.1371/journal.pbio.0030121

Gene array analysis of Yersinia enterocolitica FlhD and FlhC: regulation of enzymes affecting synthesis and degradation of carbamoylphosphate.

Kapatral V, Campbell JW, Minnich SA, Thomson NR, Matsumura P, Prüss BM.

This paper focuses on global gene regulation by FlhD/FlhC in enteric bacteria. Even though Yersinia enterocolitica FlhD/FlhC can complement an Escherichia coli flhDC mutant for motility, it is not known if the Y. enterocolitica FlhD/FlhC complex has an effect on metabolism similar to E. coli. To study metabolic gene regulation, a partial Yersinia enterocolitica 8081c microarray was constructed and the expression patterns of wild-type cells were compared to an flhDC mutant strain at 25 and 37 degrees C. The overlap between the E. coli and Y. enterocolitica FlhD/FlhC regulated genes was 25 %. Genes that were regulated at least fivefold by FlhD/FlhC in Y. enterocolitica are genes encoding urocanate hydratase (hutU), imidazolone propionase (hutI), carbamoylphosphate synthetase (carAB) and aspartate carbamoyltransferase (pyrBI). These enzymes are part of a pathway that is involved in the degradation of L-histidine to L-glutamate and eventually leads into purine/pyrimidine biosynthesis via carbamoylphosphate and carbamoylaspartate. A number of other genes were regulated at a lower rate. In two additional experiments, the expression of wild-type cells grown at 4 or 25 degrees C was compared to the same strain grown at 37 degrees C. The expression of the flagella master operon flhD was not affected by temperature, whereas the flagella-specific sigma factor fliA was highly expressed at 25 degrees C and reduced at 4 and 37 degrees C. Several other flagella genes, all of which are under the control of FliA, exhibited a similar temperature profile. These data are consistent with the hypothesis that temperature regulation of flagella genes might be mediated by the flagella-specific sigma factor FliA and not the flagella master regulator FlhD/FlhC.

Microbiology. 2004 Jul;150(Pt 7):2289-300.

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.

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

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.

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

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.

The genome sequence of the facultative intracellular pathogen Brucella melitensis.

DelVecchio VG, Kapatral V, Redkar RJ, Patra G, Mujer C, Los T, Ivanova N,
Anderson I, Bhattacharyya A, Lykidis A, Reznik G, Jablonski L, Larsen N, D'Souza
M, Bernal A, Mazur M, Goltsman E, Selkov E, Elzer PH, Hagius S, O'Callaghan D,
Letesson JJ, Haselkorn R, Kyrpides N, Overbeek R.

Brucella melitensis is a facultative intracellular bacterial pathogen that causes
abortion in goats and sheep and Malta fever in humans. The genome of B.
melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed
over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197
ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned
functions. The origins of replication of the two chromosomes are similar to those
of other alpha-proteobacteria. Housekeeping genes, including those involved in
DNA replication, transcription, translation, core metabolism, and cell wall
biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion
systems are absent, but genes encoding sec-dependent, sec-independent, and
flagella-specific type III, type IV, and type V secretion systems as well as
adhesins, invasins, and hemolysins were identified. Several features of the B.
melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.

Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):443-8. Epub 2001 Dec 26.