Genome Sequence and Comparative Pathogenic Determinants of Multidrug Resistant Uropathogenic Escherichia coli O25b:H4, A Clinical Isolate from Saudi Arabia

Essam J. Alyamani, Anamil M. Khiyami, Rayan Y. Booq, Fayez S. Bahwerth, Benjamin Vaisvil, Daniel P. Schmitt and Vinayak Kapatral

Escherichia coli serotype O25b:H4 is involved in human urinary tract infections. In this study, we sequenced and analyzed E. coli O25b:H4 isolated from a patient suffering from recurring UTI infections in an intensive care unit at Hera General Hospital in Makkah, Saudi Arabia. We aimed to determine the virulence genes for pathogenesis and drug resistance of this isolate compared to other E. coli strains. We sequenced and analyzed the E. coli O25b:H4 Saudi strain clinical isolate using next generation sequencing. Using the ERGO genome analysis platform, we performed annotations and identified virulence and antibiotic resistance determinants of this clinical isolate. The E. coli O25b:H4 genome was assembled into four contigs representing a total chromosome size of 5.28 Mb, and three contigs were identified, including a 130.9 kb (virulence plasmid) contig bearing the bla-CTX gene and 32 kb and 29 kb contigs. In comparing this genome to other uropathogenic E. coli genomes, we identified unique drug resistance and pathogenicity factors. In this work, whole-genome sequencing and targeted comparative analysis of a clinical isolate of uropathogenic Escherichia coli O25b:H4 was performed. This strain encodes virulence genes linked with extraintestinal pathogenic E. coli (ExPEC) that are expressed constitutively in E. coli ST131. We identified the genes responsible for pathogenesis and drug resistance and performed comparative analyses of the virulence and antibiotic resistance determinants with those of other E. coli UPEC isolates. This is the first report of genome sequencing and analysis of a UPEC strain from Saudi Arabia.

Published Nov 5. 2016. DOI: 10.22207/JPAM.10.4.01

Transcriptional Profiling the 150 kb Linear Megaplasmid of Borrelia turicatae Suggests a Role in Vector Colonization and Initiating Mammalian Infection

Hannah K. Wilder, Sandra J. Raffel, Alan G. Barbour, Stephen F. Porcella, Daniel E. Sturdevant, Benjamin Vaisvil, Vinayak Kapatral, Daniel P. Schmitt, Tom G. Schwan, Job E. Lopez

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3’ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.

Published: February 4, 2016 DOI: 10.1371/journal.pone.0147707

The genome of Shigella dysenteriae strain Sd1617 comparison to representative strains in evaluating pathogenesis

Ajchara A. Vongsawan , Vinayak Kapatral , Benjamin Vaisvil , Henry Burd , Oralak Serichantalergs , Malabi M. Venkatesan , Carl J. Mason

We sequenced and analyzed Shigella dysenteriae strain Sd1617 serotype 1 that is widely used as model strain for vaccine design, trials and research. A combination of next-generation sequencing platforms and assembly yielded two contigs representing a chromosome size of 4.34 Mb and the large virulence plasmid of 177 kb. This genome sequence is compared with other Shigella genomes in order to understand gene complexity and pathogenic factors.

FEMS Microbiology Letters, 362, 2015, fnv011 doi: 10.1093/femsle/fnv011

Complete Genome Sequence of Flavobacterium psychrophilum Strain CSF259-93, Used To Select Rainbow Trout for Increased Genetic Resistance against Bacterial Cold Water Disease.

Wiens GD, LaPatra SE, Welch TJ, Rexroad C 3rd, Call DR, Cain KD, LaFrentz BR, Vaisvil B, Schmitt DP, Kapatral V.

The genome sequence of Flavobacterium psychrophilum strain CSF259-93, isolated from rainbow trout (Oncorhynchus mykiss), consists of a single circular genome of 2,900,735 bp and 2,701 predicted open reading frames (ORFs). Strain CSF259-93 has been used to select a line of rainbow trout with increased genetic resistance against bacterial cold water disease.

Genome Announc. 2014 Sep 18;2(5). pii: e00889-14. doi: 10.1128/genomeA.00889-14.

Draft Genome Sequence of a New Homofermentative, Lactic AcidProducing Enterococcus faecalis Isolate, CBRD01

Lew P. Christopher, Vinayak Kapatral, Benjamin Vaisvil, Ginger Emel,b and Linda C. DeVeauxc

We report here the draft genome sequence of the novel homofermentative Enterococcus faecalis isolate CBRD01, which is capable of high lactic acid productivity and yields, with minimal nutritional requirements. The genome is 2.8 Mbp, with 37% G+C, and contains genes for two lactate dehydrogenase (LDH) enzymes found in related organisms.

Genome Announc. 2014 Mar-Apr; 2(2): e00147-14.
Published online 2014 Mar 27. doi:  10.1128/genomeA.00147-14

Metabolic Network Analysis-Based Identification of Antimicrobial Drug Targets in Category A Bioterrorism Agents

Yong-Yeol Ahn, Deok-Sun Lee, Henry Burd, William Blank, Vinayak Kapatral 

The 2001 anthrax mail attacks in the United States demonstrated the potential threat of bioterrorism, hence driving the need to develop sophisticated treatment and diagnostic protocols to counter biological warfare. Here, by performing flux balance analyses on the fully-annotated metabolic networks of multiple, whole genome-sequenced bacterial strains, we have identified a large number of metabolic enzymes as potential drug targets for each of the three Category A-designated bioterrorism agents including Bacillus anthracis, Francisella tularensis and Yersinia pestis. Nine metabolic enzymes- belonging to the coenzyme A, folate, phosphatidyl-ethanolamine and nucleic acid pathways common to all strains across the three distinct genera were identified as targets. Antimicrobial agents against some of these enzymes are available. Thus, a combination of cross species-specific antibiotics and common antimicrobials against shared targets may represent a useful combinatorial therapeutic approach against all Category A bioterrorism agents.

Published: January 15, 2014DOI: 10.1371/journal.pone.0085195

Complete genome sequence of Yersinia enterocolitica subsp. palearctica serogroup O:3.

Batzilla J, Höper D, Antonenka U, Heesemann J, Rakin A.

We report here the first finished and annotated genome sequence of a representative of the most epidemiologically successful Yersinia group, Y. enterocolitica subsp. palearctica strain Y11, serotype O:3, biotype 4. This strain is a certified type strain of the German DSMZ collection (DSM no. 13030; Yersinia enterocolitica subsp. palearctica) that was isolated from the stool of a human patient (H. Neubauer, S. Aleksic, A. Hensel, E. J. Finke, and H. Meyer. Int. J. Med. Microbiol. 290:61-64, 2000).

J Bacteriol. 2011 Apr;193(8):2067. doi: 10.1128/JB.01484-10. Epub 2011 Feb 4.

Blueprint for antimicrobial hit discovery targeting metabolic networks.

Shen Y, Liu J, Estiu G, Isin B, Ahn YY, Lee DS, Barabási AL, Kapatral V, Wiest O, Oltvai ZN.

Advances in genome analysis, network biology, and computational chemistry have the potential to revolutionize drug discovery by combining system-level identification of drug targets with the atomistic modeling of small molecules capable of modulating their activity. To demonstrate the effectiveness of such a discovery pipeline, we deduced common antibiotic targets in Escherichia coli and Staphylococcus aureus by identifying shared tissue-specific or uniformly essential metabolic reactions in their metabolic networks. We then predicted through virtual screening dozens of potential inhibitors for several enzymes of these reactions and showed experimentally that a subset of these inhibited both enzyme activities in vitro and bacterial cell viability. This blueprint is applicable for any sequenced organism with high-quality metabolic reconstruction and suggests a general strategy for strain-specific antiinfective therapy.

Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1082-7. doi: 10.1073/pnas.0909181107. Epub 2010 Jan 5.

Comparative Genome-Scale Metabolic Reconstruction and Flux Balance Analysis of Multiple Staphylococcus aureus Genomes Identify Novel Antimicrobial Drug Targets

Deok-Sun Lee, Henry Burd, Jiangxia Liu, Eivind Almaas, Olaf Wiest, Albert-László Barabási, Zoltán N. Oltvai, and  Vinayak Kapatral

Mortality due to multidrug-resistant Staphylococcus aureus infection is predicted to surpass that of human immunodeficiency virus/AIDS in the United States. Despite the various treatment options for S. aureusinfections, it remains a major hospital- and community-acquired opportunistic pathogen. With the emergence of multidrug-resistant S. aureus strains, there is an urgent need for the discovery of new antimicrobial drug targets in the organism. To this end, we reconstructed the metabolic networks of multidrug-resistant S. aureus strains using genome annotation, functional-pathway analysis, and comparative genomic approaches, followed by flux balance analysis-based in silico single and double gene deletion experiments. We identified 70 single enzymes and 54 pairs of enzymes whose corresponding metabolic reactions are predicted to be unconditionally essential for growth. Of these, 44 single enzymes and 10 enzyme pairs proved to be common to all 13 S. aureus strains, including many that had not been previously identified as being essential for growth by gene deletion experiments in S. aureus. We thus conclude that metabolic reconstruction and in silico analyses of multiple strains of the same bacterial species provide a novel approach for potential antibiotic target identification.

J Bacteriol. 2009 Jun; 191(12): 4015–4024.
Published online 2009 Apr 17. doi:  10.1128/JB.01743-08

Host cell-free growth of the Q fever bacterium Coxiella burnetii.

Omsland A1, Cockrell DC, Howe D, Fischer ER, Virtaneva K, Sturdevant DE, Porcella SF, Heinzen RA.

The inability to propagate obligate intracellular pathogens under axenic (host cell-free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen that replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organism's metabolic requirements using expression microarrays, genomic reconstruction, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (approximately 3 log(10)) of C. burnetii in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious for Vero cells and exhibited developmental forms characteristic of in vivo grown organisms. Axenic cultivation of C. burnetii will facilitate studies of the organism's pathogenesis and genetics and aid development of Q fever preventatives such as an effective subunit vaccine. Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellular pathogens.

Proc Natl Acad Sci U S A. 2009 Mar 17;106(11):4430-4. doi: 10.1073/pnas.0812074106. Epub 2009 Feb 25.

Comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus Coxiella.

Beare PA1, Unsworth N, Andoh M, Voth DE, Omsland A, Gilk SD, Williams KP, Sobral BW, Kupko JJ 3rd, Porcella SF, Samuel JE, Heinzen RA.

Genetically distinct isolates of Coxiella burnetii, the cause of human Q fever, display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. Moreover, correlations between C. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting that isolates have distinct virulence characteristics. To provide a more-complete understanding of C. burnetii's genetic diversity, evolution, and pathogenic potential, we deciphered the whole-genome sequences of the K (Q154) and G (Q212) human chronic endocarditis isolates and the naturally attenuated Dugway (5J108-111) rodent isolate. Cross-genome comparisons that included the previously sequenced Nine Mile (NM) reference isolate (RSA493) revealed both novel gene content and disparate collections of pseudogenes that may contribute to isolate virulence and other phenotypes. While C. burnetii genomes are highly syntenous, recombination between abundant insertion sequence (IS) elements has resulted in genome plasticity manifested as chromosomal rearrangement of syntenic blocks and DNA insertions/deletions. The numerous IS elements, genomic rearrangements, and pseudogenes of C. burnetii isolates are consistent with genome structures of other bacterial pathogens that have recently emerged from nonpathogens with expanded niches. The observation that the attenuated Dugway isolate has the largest genome with the fewest pseudogenes and IS elements suggests that this isolate's lineage is at an earlier stage of pathoadaptation than the NM, K, and G lineages.

Infect Immun. 2009 Feb;77(2):642-56. doi: 10.1128/IAI.01141-08. Epub 2008 Dec 1.

Genome sequence of the fish pathogen Renibacterium salmoninarum suggests reductive evolution away from an environmental Arthrobacter ancestor.

Wiens GD, Rockey DD, Wu Z, Chang J, Levy R, Crane S, Chen DS, Capri GR, Burnett JR, Sudheesh PS, Schipma MJ, Burd H, Bhattacharyya A, Rhodes LD, Kaul R, Strom MS.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

J Bacteriol. 2008 Nov;190(21):6970-82. 
doi: 10.1128/JB.00721-08. Epub 2008 Aug 22.

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.