Comparative genome analysis and genome-guided physiological analysis of Roseobacter litoralis

Daniela Kalhoefer, Sebastian Thole, Sonja Voget, Rüdiger Lehmann, Heiko Liesegang, Antje Wollher, Rolf Daniel, Meinhard Simon & Thorsten Brinkhoff

Background

Roseobacter litoralis OCh149, the type species of the genus, and Roseobacter denitrificans OCh114 were the first described organisms of the Roseobacter clade, an ecologically important group of marine bacteria. Both species were isolated from seaweed and are able to perform aerobic anoxygenic photosynthesis.

Results

The genome of R. litoralis OCh149 contains one circular chromosome of 4,505,211 bp and three plasmids of 93,578 bp (pRLO149_94), 83,129 bp (pRLO149_83) and 63,532 bp (pRLO149_63). Of the 4537 genes predicted for R. litoralis, 1122 (24.7%) are not present in the genome of R. denitrificans. Many of the unique genes of R. litoralis are located in genomic islands and on plasmids. On pRLO149_83 several potential heavy metal resistance genes are encoded which are not present in the genome of R. denitrificans. The comparison of the heavy metal tolerance of the two organisms showed an increased zinc tolerance of R. litoralis. In contrast to R. denitrificans, the photosynthesis genes of R. litoralis are plasmid encoded. The activity of the photosynthetic apparatus was confirmed by respiration rate measurements, indicating a growth-phase dependent response to light. Comparative genomics with other members of the Roseobacter clade revealed several genomic regions that were only conserved in the two Roseobacter species. One of those regions encodes a variety of genes that might play a role in host association of the organisms. The catabolism of different carbon and nitrogen sources was predicted from the genome and combined with experimental data. In several cases, e.g. the degradation of some algal osmolytes and sugars, the genome-derived predictions of the metabolic pathways in R. litoralis differed from the phenotype.

Conclusions

The genomic differences between the two Roseobacter species are mainly due to lateral gene transfer and genomic rearrangements. Plasmid pRLO149_83 contains predominantly recently acquired genetic material whereas pRLO149_94 was probably translocated from the chromosome. Plasmid pRLO149_63 and one plasmid of R. denitrifcans (pTB2) seem to have a common ancestor and are important for cell envelope biosynthesis. Several new mechanisms of substrate degradation were indicated from the combination of experimental and genomic data. The photosynthetic activity of R. litoralis is probably regulated by nutrient availability.

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.

The genome of Syntrophus aciditrophicus: Life at the thermodynamic limit of microbial growth

Michael J. McInerney, Lars Rohlin, Housna Mouttaki, UnMi Kim, Rebecca S. Krupp, Luis Rios-Hernandez, Jessica Sieber, Christopher G. Struchtemeyer, Anamitra Bhattacharyya, John W. Campbell, and Robert P. Gunsalus

Biochemically, the syntrophic bacteria constitute the missing link in our understanding of anaerobic flow of carbon in the biosphere. The completed genome sequence of Syntrophus aciditrophicus SB, a model fatty acid- and aromatic acid-degrading syntrophic bacterium, provides a glimpse of the composition and architecture of the electron transfer and energy-transducing systems needed to exist on marginal energy economies of a syntrophic lifestyle. The genome contains 3,179,300 base pairs and 3,169 genes where 1,618 genes were assigned putative functions. Metabolic reconstruction of the gene inventory revealed that most biosynthetic pathways of a typical Gram-negative microbe were present. A distinctive feature of syntrophic metabolism is the need for reverse electron transport; the presence of a unique Rnf-type ion-translocating electron transfer complex, menaquinone, and membrane-bound Fe-S proteins with associated heterodisulfide reductase domains suggests mechanisms to accomplish this task. Previously undescribed approaches to degrade fatty and aromatic acids, including multiple AMP-forming CoA ligases and acyl-CoA synthetases seem to be present as ways to form and dissipate ion gradients by using a sodium-based energy strategy. Thus, S. aciditrophicus, although nutritionally self-sufficient, seems to be a syntrophic specialist with limited fermentative and respiratory metabolism. Genomic analysis confirms the S. aciditrophicus metabolic and regulatory commitment to a nonconventional mode of life compared with our prevailing understanding of microbiology.

Published online 2007 Apr 18. doi:  10.1073/pnas.0610456104

The cyanobacterial genome core and the origin of photosynthesis.

Armen Y. Mulkidjanian, Eugene V. Koonin, Kira S. Makarova, Sergey L. Mekhedov, Alexander Sorokin, Yuri I. Wolf, Alexis Dufresne, Frédéric Partensky, Henry Burd, Denis Kaznadzey, Robert Haselkorn, and Michael Y. Galperin

Comparative analysis of 15 complete cyanobacterial genome sequences, including “near minimal” genomes of five strains of Prochlorococcus spp., revealed 1,054 protein families [core cyanobacterial clusters of orthologous groups of proteins (core CyOGs)] encoded in at least 14 of them. The majority of the core CyOGs are involved in central cellular functions that are shared with other bacteria; 50 core CyOGs are specific for cyanobacteria, whereas 84 are exclusively shared by cyanobacteria and plants and/or other plastid-carrying eukaryotes, such as diatoms or apicomplexans. The latter group includes 35 families of uncharacterized proteins, which could also be involved in photosynthesis. Only a few components of cyanobacterial photosynthetic machinery are represented in the genomes of the anoxygenic phototrophic bacteria Chlorobium tepidum, Rhodopseudomonas palustris, Chloroflexus aurantiacus, or Heliobacillus mobilis. These observations, coupled with recent geological data on the properties of the ancient phototrophs, suggest that photosynthesis originated in the cyanobacterial lineage under the selective pressures of UV light and depletion of electron donors. We propose that the first phototrophs were anaerobic ancestors of cyanobacteria (“procyanobacteria”) that conducted anoxygenic photosynthesis using a photosystem I-like reaction center, somewhat similar to the heterocysts of modern filamentous cyanobacteria. From procyanobacteria, photosynthesis spread to other phyla by way of lateral gene transfer.

Proc Natl Acad Sci U S A. 2006 Aug 29; 103(35): 13126–13131.
Published online 2006 Aug 21. doi:  10.1073/pnas.0605709103

Growth of Escherichia coli MG1655 on LB medium: determining metabolic strategy with transcriptional microarrays.

Baev MV, Baev D, Radek AJ, Campbell JW.

Expression profiles of genes related to stress responses, substrate assimilation, acetate metabolism, and biosynthesis were obtained by monitoring growth of Escherichia coli MG1655 in Luria-Bertani (LB) medium with transcriptional microarrays. Superimposing gene expression profiles on a plot of specific growth rate demonstrates that the cells pass through four distinct physiological states during fermentation before entering stationary phase. Each of these states can be characterized by specific patterns of substrate utilization and cellular biosynthesis corresponding to the nutrient status of the medium. These data allow the growth phases of the classical microbial growth curve to be redefined in terms of the physiological states and environmental changes commonly occurring during bacterial growth in batch culture on LB medium.

Appl Microbiol Biotechnol. 2006 Jul;71(3):323-8. Epub 2006 Apr 28

Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of sugars, alcohols, and organic acids with transcriptional microarrays.

Baev MV, Baev D, Radek AJ, Campbell JW.

Microorganisms respond to environmental changes by reprogramming their metabolism primarily through altered patterns of gene expression. DNA microarrays provide a tool for exploiting microorganisms as living sensors of their environment. The potential of DNA microarrays to reflect availability of nutrient components during fermentations on complex media was examined by monitoring global gene expression throughout batch cultivation of Escherichia coli MG1655 on Luria-Bertani (LB) medium. Gene expression profiles group into pathways that clearly demonstrate the metabolic changes occurring in the course of fermentation. Functional analysis of the gene expression related to metabolism of sugars, alcohols, and organic acids revealed that E. coli growing on LB medium switches from a sequential mode of substrate utilization to the simultaneous one in the course of the growth. Maltose and maltodextrins are the first of these substrates to support growth. Utilization of these nutrients associated with the highest growth rate of the culture was followed by simultaneous induction of enzymes involved in assimilation of a large group of other carbon sources including D-mannose, melibiose, D-galactose, L-fucose, L-rhamnose, D-mannitol, amino sugars, trehalose, L-arabinose, glycerol, and lactate. Availability of these nutrients to the cells was monitored by induction of corresponding transport and/or catabolic systems specific for each of the compounds.

Appl Microbiol Biotechnol. 2006 Jul;71(3):310-6. Epub 2006 Apr 21.

The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution.

van de Guchte M, Penaud S, Grimaldi C, Barbe V, Bryson K, Nicolas P, Robert C,
Oztas S, Mangenot S, Couloux A, Loux V, Dervyn R, Bossy R, Bolotin A, Batto JM,
Walunas T, Gibrat JF, Bessières P, Weissenbach J, Ehrlich SD, Maguin E.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of
the group of lactic acid-producing bacteria, mainly known for its worldwide
application in yogurt production. The genome sequence of this bacterium has been
determined and shows the signs of ongoing specialization, with a substantial
number of pseudogenes and incomplete metabolic pathways and relatively few
regulatory functions. Several unique features of the L. bulgaricus genome support
the hypothesis that the genome is in a phase of rapid evolution. (i)
Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may
indicate that the L. bulgaricus genome has known a recent phase of important size
reduction, in agreement with the observed high frequency of gene inactivation and
elimination; (ii) a much higher GC content at codon position 3 than expected on
the basis of the overall GC content suggests that the composition of the genome
is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp
inverted repeat in the replication termination region, an extremely rare feature
in bacterial genomes, may be interpreted as a transient stage in genome
evolution. The results indicate the adaptation of L. bulgaricus from a
plant-associated habitat to the stable protein and lactose-rich milk environment
through the loss of superfluous functions and protocooperation with Streptococcus
thermophilus.

Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):9274-9. Epub 2006 Jun 5.

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2.

Gabriel DW, Allen C, Schell M, Denny TP, Greenberg JT, Duan YP, Flores-Cruz Z, Huang Q, Clifford JM, Presting G, González ET, Reddy J, Elphinstone J, Swanson J, Yao J, Mulholland V, Liu L, Farmerie W, Patnaikuni M, Balogh B, Norman D, Alvarez A, Castillo JA, Jones J, Saddler G, Walunas T, Zhukov A, Mikhailova N.

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Mol Plant Microbe Interact. 2006 Jan;19(1):69-79.
http://dx.doi.org/10.1094/MPMI-19-0069

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 data mining of lactic acid bacteria: the impact of bioinformatics.

Siezen RJ, van Enckevort FH, Kleerebezem M, Teusink B.

Lactic acid bacteria (LAB) have been widely used in food fermentations and, more
recently, as probiotics in health-promoting food products. Genome sequencing and
functional genomics studies of a variety of LAB are now rapidly providing
insights into their diversity and evolution and revealing the molecular basis for
important traits such as flavor formation, sugar metabolism, stress response,
adaptation and interactions. Bioinformatics plays a key role in handling,
integrating and analyzing the flood of 'omics' data being generated.
Reconstruction of metabolic potential using bioinformatics tools and databases,
followed by targeted experimental verification and exploration of the metabolic
and regulatory network properties, are the present challenges that should lead to
improved exploitation of these versatile food bacteria.

Curr Opin Biotechnol. 2004 Apr;15(2):105-15.

Genome of Methanocaldococcus (methanococcus) jannaschii.

Graham DE, Kyrpides N, Anderson IJ, Overbeek R, Whitman WB.

Methanocaldococcus (Methanococcus) jannaschii strain JAL-1 is a hyperthermophilic methanogenic archaeon that was isolated from surface material collected at a “white smoker” chimney at a depth of 2600 m in the East Pacific Rise near the western coast of Mexico. Cells are irregular cocci possessing polar bundles of flagella. The cell envelope is composed of a cytoplasmic membrane and a protein surface layer. Similar isolates have been obtained from hydrothermally active sediments in the Guaymas Basin and the Mid-Atlantic Ridge, and related species have been found at other marine hydrothermal vents. Because these hyperthermophilic species are very different from the mesophilic methanococci, they have been reclassified into a new family, Methanocaldococcaceae, and two new genera, Methanocaldococcus and Methanotorris. The characteristics of the source material for these isolates suggest that they possess adaptations for growth at high temperature and pressure as well as moderate salinity.

Methods Enzymol. 2001;330:40-123. doi:10.1016/S0076-6879(01)30370-1

Aerobic tryptophan degradation pathway in bacteria: novel kynurenine formamidase.

Kurnasov O1, Jablonski L, Polanuyer B, Dorrestein P, Begley T, Osterman A.

While a variety of chemical transformations related to the aerobic degradation of L-tryptophan (kynurenine pathway), and most of the genes and corresponding enzymes involved therein have been predominantly characterized in eukaryotes, relatively little was known about this pathway in bacteria. Using genome comparative analysis techniques we have predicted the existence of the three-step pathway of aerobic L-tryptophan degradation to anthranilate (anthranilate pathway) in several bacteria. Based on the chromosomal gene clustering analysis, we have identified a previously unknown gene encoding for kynurenine formamidase (EC 3.5.1.19) involved with the second step of the anthranilate pathway. This functional prediction was experimentally verified by cloning, expression and enzymatic characterization of recombinant kynurenine formamidase orthologs from Bacillus cereus, Pseudomonas aeruginosa and Ralstonia metallidurans. Experimental verification of the inferred anthranilate pathway was achieved by functional expression in Escherichia coli of the R. metallidurans putative kynBAU operon encoding three required enzymes: tryptophan 2,3-dioxygenase (gene kynA), kynurenine formamidase (gene kynB), and kynureninase (gene kynU). Our data provide the first experimental evidence of the connection between these genes (only one of which, kynU, was previously characterized) and L-tryptophan aerobic degradation pathway in bacteria.

FEMS Microbiol Lett. 2003 Oct 24;227(2):219-27.