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1.  Genome Sequence of SCB34, a Sequence Type 131 Multidrug-Resistant Escherichia coli Isolate Causing Neonatal Early-Onset Sepsis 
Genome Announcements  2014;2(3):e00514-14.
SCB34 is a sequence type 131, highly invasive, multidrug-resistant Escherichia coli isolate that produced neonatal bacteremia. Whole-genome sequencing was performed using a 250-bp library on the Illumina MiSeq platform; 5,910,264 reads were assembled de novo using the A5 assembly pipeline. The total contig length was 5,227,742 bp; the RAST server was used for annotation.
doi:10.1128/genomeA.00514-14
PMCID: PMC4056292  PMID: 24926049
2.  Homologous Recombination in E3 Genes of Human Adenovirus Species D 
Journal of Virology  2013;87(22):12481-12488.
Genes within the E3 transcription unit of human adenoviruses modulate host immune responses to infection. A comprehensive genomics and bioinformatics analysis of the E3 transcription unit for 38 viruses within human adenovirus species D (HAdV-D) revealed distinct and surprising patterns of homologous recombination. Homologous recombination was identified in open reading frames for E3 CR1α, CR1β, and CR1γ, similar to that previously observed with genes encoding the three major structural capsid proteins, the penton base, hexon, and fiber.
doi:10.1128/JVI.01927-13
PMCID: PMC3807923  PMID: 24027303
3.  Control of RNA Stability by NrrF, an Iron-Regulated Small RNA in Neisseria gonorrhoeae 
Journal of Bacteriology  2013;195(22):5166-5173.
Regulation of gene expression by small noncoding RNAs (sRNAs) plays a critical role in bacterial response to physiological stresses. NrrF, a trans-acting sRNA in Neisseria meningitidis and Neisseria gonorrhoeae, has been shown in the meningococcus to control indirectly, in response to iron (Fe) availability, the transcription of genes encoding subunits of succinate dehydrogenase, a Fe-requiring enzyme. Given that in other organisms, sRNAs target multiple mRNAs to control gene expression, we used a global approach to examine the role of NrrF in controlling gonococcal transcription. Three strains, including N. gonorrhoeae FA1090, an nrrF deletion mutant, and a complemented derivative, were examined using a custom CombiMatrix microarray to assess the role of this sRNA in controlling gene expression in response to Fe availability. In the absence of NrrF, the mRNA half-lives for 12 genes under Fe-depleted growth conditions were longer than those in FA1090. The 12 genes controlled by NrrF encoded proteins with biological functions including energy metabolism, oxidative stress, antibiotic resistance, and amino acid synthesis, as well as hypothetical proteins and a regulatory protein whose functions are not fully understood.
doi:10.1128/JB.00839-13
PMCID: PMC3811587  PMID: 24039262
4.  Molecular evolution of human adenoviruses 
Scientific Reports  2013;3:1812.
The recent emergence of highly virulent human adenoviruses (HAdVs) with new tissue tropisms underscores the need to determine their ontogeny. Here we report complete high quality genome sequences and analyses for all the previously unsequenced HAdV serotypes (n = 20) within HAdV species D. Analysis of nucleotide sequence variability for these in conjunction with another 40 HAdV prototypes, comprising all seven HAdV species, confirmed the uniquely hypervariable regions within species. The mutation rate among HAdV-Ds was low when compared to other HAdV species. Homologous recombination was identified in at least two of five examined hypervariable regions for every virus, suggesting the evolution of HAdV-Ds has been highly dependent on homologous recombination. Patterns of alternating GC and AT rich motifs correlated well with hypervariable region recombination sites across the HAdV-D genomes, suggesting foci of DNA instability lead to formulaic patterns of homologous recombination and confer agility to adenovirus evolution.
doi:10.1038/srep01812
PMCID: PMC3648800  PMID: 23657240
5.  Predicting the Next Eye Pathogen: Analysis of a Novel Adenovirus 
mBio  2013;4(2):e00595-12.
ABSTRACT
For DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis of in silico and laboratory approaches revealed a potential ocular tropism for the new virus. In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses.
IMPORTANCE
The ongoing dance between a virus and its host distinctly shapes how the virus evolves. While human adenoviruses typically cause mild infections, recent reports have described newly characterized adenoviruses that cause severe, sometimes fatal human infections. Here, we report a systems biology approach to show how evolution has affected the disease potential of a recently identified novel human adenovirus. A comprehensive understanding of viral evolution and pathogenicity is essential to our capacity to foretell the potential impact on human disease for new and emerging viruses.
doi:10.1128/mBio.00595-12
PMCID: PMC3622935  PMID: 23572555
6.  Genome Sequence of the Fish Pathogen Flavobacterium columnare ATCC 49512 
Journal of Bacteriology  2012;194(10):2763-2764.
Flavobacterium columnare is a Gram-negative, rod-shaped, motile, and highly prevalent fish pathogen causing columnaris disease in freshwater fish worldwide. Here, we present the complete genome sequence of F. columnare strain ATCC 49512.
doi:10.1128/JB.00281-12
PMCID: PMC3347163  PMID: 22535941
7.  Analysis of Human Adenovirus Type 19 Associated with Epidemic Keratoconjunctivitis and its Reclassification as Adenovirus Type 64 
Purpose.
Human adenovirus species D type 19 (HAdV-D19) has been associated with epidemic keratoconjunctivitis (EKC), a highly inflammatory infection of the ocular surface. Confusion exists regarding the origins of HAdV-D19. The prototype virus (HAdV-D19p) does not cause EKC, while a virus identified later with the identical serologic determinant is a significant ocular pathogen.
Methods.
High throughput genome sequencing and bioinformatics analysis were performed on HAdV-D19p and three HAdV-D19 EKC strains, and compared to the previously sequenced clinical isolate, HAdV-D19 (C) and HAdV-D37. Corneas of C57BL/6J mice were injected with HAdV-D19p, HAdV-D19 (C), or virus-free buffer, and inflammation assessed by clinical examination, flow cytometry, and cytokine ELISA. Confocal microscopy and real-time PCR of infected corneal cell cultures were used to test viral entry.
Results.
HAdV-D19 (C) and the other clinical EKC isolates showed nearly 100% sequence identity. EKC strains diverged from HAdV-D19p in the penton base, E3, and fiber transcription units. Simplot analysis showed recombination between EKC-associated HAdV-D19 with HAdV-D37, HAdV-D22, and HAdV-D19p, the latter contributing only the hexon gene, the principal serum neutralization determinant. HAdV-D19p induced stromal keratitis in the C57BL/6J mouse, but failed to infect productively human corneal epithelial cells. These data led to retyping of the clinical EKC isolates with a HAdV-D19 hexon gene as HAdV-D64.
Conclusions.
HAdV-D19 associated with EKC (HAdV-D64) originated from a recombination between HAdV-D19p, HAdV-D37, and HAdV-D22, and was mischaracterized because of a shared hexon gene. HAdV-D19p is not infectious for corneal epithelial cells, thus explaining the lack of any association with keratitis.
Zhou and colleagues study the viral genomes of clinical isolates of human adenovirus type 19, a common cause of epidemic keratoconjunctivitis, and show that the virus is really a naturally occurring recombinant of type 19, type 37 and type 22, with only the hexon gene derived from type 19.
doi:10.1167/iovs.12-9656
PMCID: PMC3367469  PMID: 22467570
8.  Divergence of the SigB regulon and pathogenesis of the Bacillus cereus sensu lato group 
BMC Genomics  2012;13:564.
Background
The Bacillus cereus sensu lato group currently includes seven species (B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus) that recent phylogenetic and phylogenomic analyses suggest are likely a single species, despite their varied phenotypes. Although horizontal gene transfer and insertion-deletion events are clearly important for promoting divergence among these genomes, recent studies have demonstrated that a major basis for phenotypic diversity in these organisms may be differential regulation of the highly similar gene content shared by these organisms. To explore this hypothesis, we used an in silico approach to evaluate the relationship of pathogenic potential and the divergence of the SigB-dependent general stress response within the B. cereus sensu lato group, since SigB has been demonstrated to support pathogenesis in Bacillus, Listeria and Staphylococcus species.
Results
During the divergence of these organisms from a common “SigB-less” ancestor, the placement of SigB promoters at varied locations in the B. cereus sensu lato genomes predict alternative structures for the SigB regulon in different organisms. Predicted promoter changes suggesting differential transcriptional control of a common gene pool predominate over evidence of indels or horizontal gene transfer for explaining SigB regulon divergence.
Conclusions
Four lineages of the SigB regulon have arisen that encompass different gene contents and suggest different strategies for supporting pathogenesis. This is consistent with the hypothesis that divergence within the B. cereus sensu lato group rests in part on alternative strategies for regulation of a common gene pool.
doi:10.1186/1471-2164-13-564
PMCID: PMC3485630  PMID: 23088190
Microbial evolution; Generalized stress response; SigB sigma factor
9.  Overreliance on the Hexon Gene, Leading to Misclassification of Human Adenoviruses 
Journal of Virology  2012;86(8):4693-4695.
The genome of human adenovirus (HAdV) D30 was sequenced in depth. Sequence assembly and analysis revealed two distinct viral sequences with identical hexon genes, which were the same as the one previously reported for HAdV-D30. However, one of the two viruses was found to be a recombinant of HAdV-D29. Exclusive reliance on serum neutralization can lead to mischaracterization of adenoviruses and miss coinfections. Whole-genome sequencing remains the gold standard for proper classification of HAdVs.
doi:10.1128/JVI.06969-11
PMCID: PMC3318657  PMID: 22301156
10.  The E3 CR1-gamma gene in human adenoviruses associated with epidemic keratoconjunctivitis 
Virus research  2011;160(1-2):120-127.
Human adenovirus species D type 37 (HAdV-D37) is an important etiologic agent of epidemic keratoconjunctivitis. Annotation of the whole genome revealed an open reading frame (ORF) in the E3 transcription unit predicted to encode a 31.6 kDa protein. This ORF, also known as CR1-γ, is predicted to be an integral membrane protein containing N-terminal signal sequence, luminal, transmembrane, and cytoplasmic domains. HAdV-D19 (C), another viral pathogen causing epidemic keratoconjunctivitis, contains an ORF 100% identical to its HAdV-D37 homologue but only 66% identical to other HAdV-D homologues. Kinetics of RNA expression and confirmation of splicing to the adenovirus tripartite leader sequence suggest a role for the protein product of CR1-γin the late stages of the viral replication cycle. Confocal microscopy is consistent with expression in the cytoplasm. Sequence analysis reveals a hypervariable luminal domain and a conserved cytoplasmic domain. The luminal domain is predicted to contain multiple N-glycosylation sites. The cytoplasmic domain contains a putative protein kinase C phosphorylation site and potential YXXϕ and dileucine (LL) motifs suggesting a potential role in modification of host proteins.
doi:10.1016/j.virusres.2011.05.022
PMCID: PMC3163711  PMID: 21683743
Human adenovirus type 37; CR1-gamma; 31.6K; E3 transcription unit; epidemic keratoconjunctivitis
11.  MpeR Regulates the mtr Efflux Locus in Neisseria gonorrhoeae and Modulates Antimicrobial Resistance by an Iron-Responsive Mechanism 
Previous studies have shown that the MpeR transcriptional regulator produced by Neisseria gonorrhoeae represses the expression of mtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation of mpeR has been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of the mtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that the mtrR gene, which encodes a direct repressor (MtrR) of mtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of the mtrCDE efflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression of mtrR was direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of the mtr efflux system and modulate levels of gonococcal susceptibility to efflux pump substrates.
doi:10.1128/AAC.06112-11
PMCID: PMC3294918  PMID: 22214775
12.  Molecular evolution of human species D adenoviruses 
Adenoviruses are medium-sized double stranded DNA viruses that infect vertebrates. Human adenoviruses cause an array of diseases. Currently there are 56 human adenovirus types recognized and characterized within seven species (A-G). Of those types, a majority belongs to species D. In this review, the genomic conservation and diversity are examined amongst human adenoviruses within species D, particularly in contrast to other human adenovirus species. Specifically, homologous recombination is presented as a driving force for the molecular evolution of human adenoviruses and the emergence of new adenovirus pathogens.
doi:10.1016/j.meegid.2011.04.031
PMCID: PMC3139803  PMID: 21570490
human adenovirus; HAdV-D; Recombination
13.  Five Genome Sequences of Subspecies B1 Human Adenoviruses Associated with Acute Respiratory Disease 
Journal of Virology  2012;86(1):635-636.
Five genomes of human subspecies B1 adenoviruses isolated from cases of acute respiratory disease have been sequenced and archived for reference. These include representatives of two prevalent genomic variants of HAdV-7, i.e., HAdV-7h and HAdV-7d2. The other three are HAdV-3/16, HAdV-16 strain E26, and HAdV-3+7 strain Takeuchi. All are recombinant genomes. Genomics and bioinformatics provide detailed views into the genetic makeup of these pathogens and insight into their molecular evolution. Retrospective characterization of particularly problematic older pathogens such as HAdV-7h (1987) and intriguing isolates such as HAdV-3+7 strain Takeuchi (1958) may provide clues to their phenotypes and serology and may suggest protocols for prevention and treatment.
doi:10.1128/JVI.06593-11
PMCID: PMC3255920  PMID: 22158846
14.  Complete Genome Sequence of Human Adenovirus Prototype 17 
Journal of Virology  2011;85(21):11540-11541.
As one of the first five human adenoviruses (HAdVs) to be sequenced, type 17 was important as a reference tool for comparative genomics of recently isolated HAdV pathogens in species D. HAdV-D17 was the first species D adenovirus to be sequenced and was deposited in GenBank in 1999. These genome data were not of high quality, and a redetermination of the same stock virus provides corrected data; among the differences are a length of 35,139 bp versus 35,100 bp in the original, and 160 mismatches to the original genome were found. Annotation of the coding sequences reveals 39 as opposed to 8, a finding which is important for phylogenomic studies.
doi:10.1128/JVI.06051-11
PMCID: PMC3194981  PMID: 21980031
15.  Computational Analysis of Two Species C Human Adenoviruses Provides Evidence of a Novel Virus▿‡ 
Journal of Clinical Microbiology  2011;49(10):3482-3490.
Human adenovirus C (HAdV-C) species are a common cause of respiratory infections and can occasionally produce severe clinical manifestations. A deeper understanding of the variation and evolution in species HAdV-C is especially important since these viruses, including HAdV-C6, are used as gene delivery vectors for human gene therapy and in other biotechnological applications. Here, the full-genome analysis of the prototype HAdV-C6 and a recently identified virus provisionally termed HAdV-C57 are reported. Although the genomes of all species HAdV-C members are very similar to each other, the E3 region, hexon and fiber (ten proteins total) present a wide range of identity values at the amino acid level. Studies of these viruses in comparison to the other three HAdV-C prototypes (1, 2, and 5) comprise a comprehensive analysis of the diversity and conservation within HAdV-C species. HAdV-C6 contains a recombination event within the constant region of the hexon gene. HAdV-C57 is a recombinant virus with a fiber gene nearly identical to HAdV-C6 and a unique hexon distinguished by its loop 2 motif.
doi:10.1128/JCM.00156-11
PMCID: PMC3187342  PMID: 21849694
16.  Computational and Serologic Analysis of Novel and Known Viruses in Species Human Adenovirus D in Which Serology and Genomics Do Not Correlate 
PLoS ONE  2012;7(3):e33212.
In November of 2007 a human adenovirus (HAdV) was isolated from a bronchoalveolar lavage (BAL) sample recovered from a biopsy of an AIDS patient who presented with fever, cough, tachycardia, and expiratory wheezes. To better understand the isolated virus, the genome was sequenced and analyzed using bioinformatic and phylogenomic analysis. The results suggest that this novel virus, which is provisionally named HAdV-D59, may have been created from multiple recombination events. Specifically, the penton, hexon, and fiber genes have high nucleotide identity to HAdV-D19C, HAdV-D25, and HAdV-D56, respectively. Serological results demonstrated that HAdV-D59 has a neutralization profile that is similar yet not identical to that of HAdV-D25. Furthermore, we observed a two-fold difference between the ability of HAdV-D15 and HAdV-D25 to be neutralized by reciprocal antiserum indicating that the two hexon proteins may be more similar in epitopic conformation than previously assumed. In contrast, hexon loops 1 and 2 of HAdV-D15 and HAdV-D25 share 79.13 and 92.56 percent nucleotide identity, respectively. These data suggest that serology and genomics do not always correlate.
doi:10.1371/journal.pone.0033212
PMCID: PMC3302849  PMID: 22427992
17.  Computational analysis and identification of an emergent human adenovirus pathogen implicated in a respiratory fatality 
Virology  2010;409(2):141-147.
Adenoviral infections are typically acute, self-limiting, and not associated with death. However, we present the genomic and bioinformatics analysis of a novel recombinant human adenovirus (HAdV-D56) isolated in France that caused a rare neonatal fatality, and keratoconjunctivitis in three health care workers who cared for the neonate. Whole genome alignments revealed the expected diversity in the penton base, hexon, E3, and fiber coding regions, and provided evidence for extensive recombination. Bootscan analysis confirmed recombination between HAdV-D9, HAdV-D26, HAdV-D15, and HAdV-D29 in the penton base and hexon proteins, centered around hypervariable loops within the putative proteins. Protein structure analysis of the fiber coding region revealed similarity with HAdV-D8, HAdV-D9, and HAdV-D53, possibly accounting for the ocular tropism of the virus. Based on these data, this virus appears to be a new HAdV-D type (HAdV-D56), underscoring the importance of recombination events in human adenovirus evolution and the emergence of new adenovirus pathogens.
doi:10.1016/j.virol.2010.10.020
PMCID: PMC3006489  PMID: 21056888
Adenovirus; viral genomics; viral recombination; viral evolution; viral bioinformatics
18.  Sequence Variation in the Herpes Simplex Virus US1 Ocular Virulence Determinant 
The HSV-1 US1 gene encodes an ocular virulence determinant that is highly modified after translation. To better determine sequence variability in the protein, the gene was sequenced in six ocular isolates, and bioinformatics analysis was carried out. The data will be useful for designing structure-function studies.
Purpose.
The herpes simplex virus type 1 (HSV-1) US1 gene encodes host-range and ocular virulence determinants. Mutations in US1 affecting virulence are known in strain OD4, but the genomic variation across several strains is not known. The goal was to determine the degree of sequence variation in the gene from several ocular HSV isolates.
Methods.
The US1 gene from six ocular HSV-1 isolates, as well as strains KOS and F, were sequenced, and bioinformatics analyses were applied to the data.
Results.
Strains 17, F, CJ394, and CJ311 had identical amino acid sequences. With the other strains, most of the variability was concentrated in the amino-terminal third of the protein. MEME analysis identified a 63-residue core sequence (motif 1) present in all α-herpesvirus US1 homologs that were located in a region identified as structured. Ten amino acids were absolutely conserved in all the α-herpesvirus US1 homologs and were all located in the central core. Consensus-binding motifs for cyclin-dependent kinases and pocket proteins were also identified.
Conclusions.
These results suggest that significant sequence variation exists in the US1 gene, that the α22 protein contains a conserved central core region with structurally variable regions at the amino- and carboxyl termini, that 10 amino acids are conserved in α-herpes US1 homologs, and that additional host proteins may interact with the HSV-1 US1 and US1.5 proteins. This information will be valuable in designing further studies on structure-function relationships and on the role these play in host-range determination and keratitis.
doi:10.1167/iovs.10-7032
PMCID: PMC3175936  PMID: 21519032
19.  Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analyses 
BMC Genomics  2011;12:570.
Background
Pneumonia and myocarditis are the most commonly reported diseases due to Histophilus somni, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in H. somni using traditional methods. Analyses of the genome sequences of several Pasteurellaceae species have provided insights into their biology and evolution. In view of the economic and ecological importance of H. somni, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the Pasteurellaceae.
Results
The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, several H. somni genes that may encode proteins involved in virulence (e.g., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor.
Conclusions
Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two H. somni strains.
doi:10.1186/1471-2164-12-570
PMCID: PMC3339403  PMID: 22111657
20.  Genome sequence of Haemophilus parasuis strain 29755 
Standards in Genomic Sciences  2011;5(1):61-68.
Haemophilus parasuis is a member of the family Pasteurellaceae and is the etiologic agent of Glässer’s disease in pigs, a systemic syndrome associated with only a subset of isolates. The genetic basis for virulence and systemic spread of particular H. parasuis isolates is currently unknown. Strain 29755 is an invasive isolate that has long been used in the study of Glässer’s disease. Accordingly, the genome sequence of strain 29755 is of considerable importance to investigators endeavoring to understand the molecular pathogenesis of H. parasuis. Here we describe the features of the 2,224,137 bp draft genome sequence of strain 29755 generated from 454-FLX pyrosequencing. These data comprise the first publicly available genome sequence for this bacterium.
doi:10.4056/sigs.2245029
PMCID: PMC3236040  PMID: 22180811
Haemophilus parasuis; Glässer’s disease; swine
21.  Genetic Analysis of a Novel Human Adenovirus with a Serologically Unique Hexon and a Recombinant Fiber Gene 
PLoS ONE  2011;6(9):e24491.
In February of 1996 a human adenovirus (formerly known as Ad-Cor-96-487) was isolated from the stool of an AIDS patient who presented with severe chronic diarrhea. To characterize this apparently novel pathogen of potential public health significance, the complete genome of this adenovirus was sequenced to elucidate its origin. Bioinformatic and phylogenetic analyses of this genome demonstrate that this virus, heretofore referred to as HAdV-D58, contains a novel hexon gene as well as a recombinant fiber gene. In addition, serological analysis demonstrated that HAdV-D58 has a different neutralization profile than all previously characterized HAdVs. Bootscan analysis of the HAdV-D58 fiber gene strongly suggests one recombination event.
doi:10.1371/journal.pone.0024491
PMCID: PMC3168504  PMID: 21915339
22.  Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group 
BMC Genomics  2011;12:430.
Background
The Bacillus cereus sensu lato group consists of six species (B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. weihenstephanensis). While classical microbial taxonomy proposed these organisms as distinct species, newer molecular phylogenies and comparative genome sequencing suggests that these organisms should be classified as a single species (thus, we will refer to these organisms collectively as the Bc species-group). How do we account for the underlying similarity of these phenotypically diverse microbes? It has been established for some time that the most rapidly evolving and evolutionarily flexible portions of the bacterial genome are regulatory sequences and transcriptional networks. Other studies have suggested that the sigma factor gene family of these organisms has diverged and expanded significantly relative to their ancestors; sigma factors are those portions of the bacterial transcriptional apparatus that control RNA polymerase recognition for promoter selection. Thus, examining sigma factor divergence in these organisms would concurrently examine both regulatory sequences and transcriptional networks important for divergence. We began this examination by comparison to the sigma factor gene set of B. subtilis.
Results
Phylogenetic analysis of the Bc species-group utilizing 157 single-copy genes of the family Bacillaceae suggests that several taxonomic revisions of the genus Bacillus should be considered. Within the Bc species-group there is little indication that the currently recognized species form related sub-groupings, suggesting that they are members of the same species. The sigma factor gene family encoded by the Bc species-group appears to be the result of a dynamic gene-duplication and gene-loss process that in previous analyses underestimated the true heterogeneity of the sigma factor content in the Bc species-group.
Conclusions
Expansion of the sigma factor gene family appears to have preferentially occurred within the extracytoplasmic function (ECF) sigma factor genes, while the primary alternative (PA) sigma factor genes are, in general, highly conserved with those found in B. subtilis. Divergence of the sigma-controlled transcriptional regulons among various members of the Bc species-group likely has a major role in explaining the diversity of phenotypic characteristics seen in members of the Bc species-group.
doi:10.1186/1471-2164-12-430
PMCID: PMC3171730  PMID: 21864360
23.  Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon▿ † 
Journal of Bacteriology  2009;192(1):77-85.
To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies—temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)—to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.
doi:10.1128/JB.00741-09
PMCID: PMC2798271  PMID: 19854902
24.  Characterization and Comparative Analysis of the Genes Encoding Haemophilus parasuis Outer Membrane Proteins P2 and P5▿ † 
Journal of Bacteriology  2009;191(19):5988-6002.
Haemophilus parasuis is a swine pathogen of significant industry concern, but little is known about how the organism causes disease. A related human pathogen, Haemophilus influenzae, has been better studied, and many of its virulence factors have been identified. Two of these, outer membrane proteins P2 and P5, are known to have important virulence properties. The goals of this study were to identify, analyze, and compare the genetic relatedness of orthologous genes encoding P2 and P5 proteins in a diverse group of 35 H. parasuis strains. Genes encoding P2 and P5 proteins were detected in all H. parasuis strains evaluated. The predicted amino acid sequences for both P2 and P5 proteins exhibit considerable heterogeneity, particularly in regions corresponding to predicted extracellular loops. Twenty-five variants of P2 and 17 variants of P5 were identified. The P2 proteins of seven strains were predicted to contain a highly conserved additional extracellular loop compared to the remaining strains and to H. influenzae P2. Antigenic-site predictions coincided with predicted extracellular loop regions of both P2 and P5. Neighbor-joining trees constructed using P2 and P5 sequences predicted divergent evolutionary histories distinct from those predicted by a multilocus sequence typing phylogeny based on partial sequencing of seven housekeeping genes. Real-time reverse transcription-PCR indicated that both genes are expressed in all of the strains.
doi:10.1128/JB.00469-09
PMCID: PMC2747881  PMID: 19633080
25.  Computational Analysis of Human Adenovirus Type 22 Provides Evidence for Recombination among Species D Human Adenoviruses in the Penton Base Gene ▿  
Journal of Virology  2009;83(17):8980-8985.
Recombination in human adenoviruses (HAdV) may confer virulence upon an otherwise nonvirulent strain. The genome sequence of species D HAdV type 22 (HAdV-D22) revealed evidence for recombination with HAdV-D19 and HAdV-D37 within the capsid penton base gene. Bootscan analysis demonstrated that recombination sites within the penton base gene frame the coding sequences for the two external hypervariable loops in the protein. A similar pattern of recombination was evident within other HAdV-D types but not other HAdV species. Further study of recombination among HAdVs is needed to better predict possible recombination events among wild-type viruses and adenoviral gene therapy vectors.
doi:10.1128/JVI.00786-09
PMCID: PMC2738150  PMID: 19553309

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