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1.  Bacillus cereus efflux protein BC3310 – a multidrug transporter of the unknown major facilitator family, UMF-2 
Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the “unknown major facilitator family-2” (UMF-2). BC3310 was shown to be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in Escherichia coli DH5α ΔacrAB. A conserved aspartate residue (D105) in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide efflux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF-2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria.
PMCID: PMC4601019  PMID: 26528249
MFS; drug resistance; efflux protein; Bacillus cereus; UMF-2
2.  Genetically and Phenotypically Distinct Pseudomonas aeruginosa Cystic Fibrosis Isolates Share a Core Proteomic Signature 
PLoS ONE  2015;10(10):e0138527.
The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and compared them with each other and with the model strain PAO1. Phenotypic analysis of CF isolates showed significant variability in colonization and virulence-related traits suggesting different strategies for adaptation to the CF lung. Genomic analysis indicated these strains shared a large set of core genes with the standard laboratory strain PAO1, and identified the genetic basis for some of the observed phenotypic differences. Proteomics revealed that in a conventional laboratory medium PAO1 expressed 827 proteins that were absent in the CF isolates while the CF isolates shared a distinctive signature set of 703 proteins not detected in PAO1. PAO1 expressed many transporters for the uptake of organic nutrients and relatively few biosynthetic pathways. Conversely, the CF isolates expressed a narrower range of transporters and a broader set of metabolic pathways for the biosynthesis of amino acids, carbohydrates, nucleotides and polyamines. The proteomic data suggests that in a common laboratory medium PAO1 may transport a diverse set of “ready-made” nutrients from the rich medium, whereas the CF isolates may only utilize a limited number of nutrients from the medium relying mainly on their own metabolism for synthesis of essential nutrients. These variations indicate significant differences between the metabolism and physiology of P. aeruginosa CF isolates and PAO1 that cannot be detected at the genome level alone. The widening gap between the increasing genomic data and the lack of phenotypic data means that researchers are increasingly reliant on extrapolating from genomic comparisons using experimentally characterized model organisms such as PAO1. While comparative genomics can provide valuable information, our data suggests that such extrapolations may be fraught with peril.
PMCID: PMC4592193  PMID: 26431321
3.  Insights on virulence from the complete genome of Staphylococcus capitis 
Staphylococcus capitis is an opportunistic pathogen of the coagulase negative staphylococci (CoNS). Functional genomic studies of S. capitis have thus far been limited by a lack of available complete genome sequences. Here, we determined the closed S. capitis genome and methylome using Single Molecule Real Time (SMRT) sequencing. The strain, AYP1020, harbors a single circular chromosome of 2.44 Mb encoding 2304 predicted proteins, which is the smallest of all complete staphylococcal genomes sequenced to date. AYP1020 harbors two large mobile genetic elements; a plasmid designated pAYP1020 (59.6 Kb) and a prophage, ΦAYP1020 (48.5 Kb). Methylome analysis identified significant adenine methylation across the genome involving two distinct methylation motifs (1972 putative 6-methyladenine (m6A) residues identified). Putative adenine methyltransferases were also identified. Comparative analysis of AYP1020 and the closely related CoNS, S. epidermidis RP62a, revealed a host of virulence factors that likely contribute to S. capitis pathogenicity, most notably genes important for biofilm formation and a suite of phenol soluble modulins (PSMs); the expression/production of these factors were corroborated by functional assays. The complete S. capitis genome will aid future studies on the evolution and pathogenesis of the coagulase negative staphylococci.
PMCID: PMC4585213  PMID: 26441910
coagulase-negative staphylococci; CoNS; SMRT sequencing; genomics; methylation
4.  Ecology and Evolution of the Human Microbiota: Fire, Farming and Antibiotics 
Genes  2015;6(3):841-857.
Human activities significantly affect all ecosystems on the planet, including the assemblages that comprise our own microbiota. Over the last five million years, various evolutionary and ecological drivers have altered the composition of the human microbiota, including the use of fire, the invention of agriculture, and the increasing availability of processed foods after the Industrial Revolution. However, no factor has had a faster or more direct effect than antimicrobial agents. Biocides, disinfectants and antibiotics select for individual cells that carry resistance genes, immediately reducing both overall microbial diversity and within-species genetic diversity. Treated individuals may never recover their original diversity, and repeated treatments lead to a series of genetic bottlenecks. The sequential introduction of diverse antimicrobial agents has selected for increasingly complex DNA elements that carry multiple resistance genes, and has fostered their spread through the human microbiota. Practices that interfere with microbial colonization, such as sanitation, Caesarian births and bottle-feeding, exacerbate the effects of antimicrobials, generating species-poor and less resilient microbial assemblages in the developed world. More and more evidence is accumulating that these perturbations to our internal ecosystems lie at the heart of many diseases whose frequency has shown a dramatic increase over the last half century.
PMCID: PMC4584332  PMID: 26371047
integron; evolution; mercury; disinfectant; resistance; antibiotic; Anthropocene; dysbiosis
5.  Pseudomonas aeruginosa inhibits the growth of Scedosporium aurantiacum, an opportunistic fungal pathogen isolated from the lungs of cystic fibrosis patients 
The filamentous fungus Scedosporium aurantiacum and the bacterium Pseudomonas aeruginosa are opportunistic pathogens isolated from lungs of the cystic fibrosis (CF) patients. P. aeruginosa has been known to suppress the growth of a number of CF related fungi such as Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. However, the interactions between P. aeruginosa and S. aurantiacum have not been investigated in depth. Hence we assessed the effect of P. aeruginosa reference strain PAO1 and two clinical isolates PASS1 and PASS2 on the growth of two clinical S. aurantiacum isolates WM 06.482 and WM 08.202 using solid plate assays and liquid cultures, in a synthetic medium mimicking the nutrient condition in the CF sputum. Solid plate assays showed a clear inhibition of growth of both S. aurantiacum strains when cultured with P. aeruginosa strains PASS1 and PAO1. The inhibitory effect was confirmed by confocal microscopy. In addition to using chemical fluorescent stains, strains tagged with yfp (P. aeruginosa PASS1) and mCherry (S. aurantiacum WM 06.482) were created to facilitate detailed microscopic observations on strain interaction. To our knowledge, this is the first study describing successful genetic transformation of S. aurantiacum. Inhibition of growth was observed only in co-cultures of P. aeruginosa and S. aurantiacum; the cell fractions obtained from independent bacterial monocultures failed to initiate a response against the fungus. In the liquid co-cultures, biofilm forming P. aeruginosa strains PASS1 and PAO1 displayed higher inhibition of fungal growth when compared to PASS2. No change was observed in the inhibition pattern when direct cell contact between the bacterial and fungal strains was prevented using a separation membrane suggesting the involvement of extracellular metabolites in the fungal inhibition. However, one of the most commonly described bacterial virulence factors, pyocyanin, had no effect against either of the S. aurantiacum strains. This study shows that P. aeruginosa has a substantial inhibitory effect on the growth of the recently described CF fungal pathogen S. aurantiacum. The findings also highlighted that P. aeruginosa biofilm formation is important but not crucial for inhibiting the growth of S. aurantiacum in a lung- mimicking environment.
PMCID: PMC4547459  PMID: 26379643
co-culture; S. aurantiacum; P. aeruginosa; interactions; growth inhibition; phenazines; SCFM; biofilms
6.  An ace up their sleeve: a transcriptomic approach exposes the AceI efflux protein of Acinetobacter baumannii and reveals the drug efflux potential hidden in many microbial pathogens 
The era of antibiotics as a cure-all for bacterial infections appears to be coming to an end. The emergence of multidrug resistance in many hospital-associated pathogens has resulted in “superbugs” that are effectively untreatable. Multidrug efflux pumps are well known mediators of bacterial drug resistance. Genome sequencing efforts have highlighted an abundance of putative efflux pump genes in bacteria. However, it is not clear how many of these pumps play a role in antimicrobial resistance. Efflux pump genes that participate in drug resistance can be under tight regulatory control and expressed only in response to substrates. Consequently, changes in gene expression following antimicrobial shock may be used to identify efflux pumps that mediate antimicrobial resistance. Using this approach we have characterized several novel efflux pumps in bacteria. In one example we recently identified the Acinetobacter chlorhexidine efflux protein (AceI) efflux pump in Acinetobacter. AceI is a prototype for a novel family of multidrug efflux pumps conserved in many proteobacterial lineages. The discovery of this family raises the possibility that additional undiscovered intrinsic resistance proteins may be encoded in the core genomes of pathogenic bacteria.
PMCID: PMC4406071  PMID: 25954261
multidrug efflux systems; bacterial transmembrane pair; adaptive resistance; bacterial drug resistance transcriptomics
7.  A novel family of integrases associated with prophages and genomic islands integrated within the tRNA-dihydrouridine synthase A (dusA) gene 
Nucleic Acids Research  2015;43(9):4547-4557.
Genomic islands play a key role in prokaryotic genome plasticity. Genomic islands integrate into chromosomal loci such as transfer RNA genes and protein coding genes, whilst retaining various cargo genes that potentially bestow novel functions on the host organism. A gene encoding a putative integrase was identified at a single site within the 5′ end of the dusA gene in the genomes of over 200 bacteria. This integrase was discovered to be a component of numerous genomic islands, which appear to share a target site within the dusA gene. dusA encodes the tRNA-dihydrouridine synthase A enzyme, which catalyses the post-transcriptional reduction of uridine to dihydrouridine in tRNA. Genomic islands encoding homologous dusA-associated integrases were found at a much lower frequency within the related dusB and dusC genes, and non-dus genes. Excision of these dusA-associated islands from the chromosome as circularized intermediates was confirmed by polymerase chain reaction. Analysis of the dusA-associated islands indicated that they were highly diverse, with the integrase gene representing the only universal common feature.
PMCID: PMC4482086  PMID: 25883135
8.  Phenotypic Profiling of Scedosporium aurantiacum, an Opportunistic Pathogen Colonizing Human Lungs 
PLoS ONE  2015;10(3):e0122354.
Genotyping studies of Australian Scedosporium isolates have revealed the strong prevalence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibrosis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using a Galleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (saccharose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two different temperature conditions tested (28 and 37°C). Phenotype microarray data were integrated with the whole-genome sequence data of S. aurantiacum to reconstruct a pathway map for the metabolism of selected substrates to further elucidate differences between the strains.
PMCID: PMC4374879  PMID: 25811884
9.  The Rsm regulon of plant growth-promoting Pseudomonas fluorescens SS101: role of small RNAs in regulation of lipopeptide biosynthesis 
Microbial Biotechnology  2014;8(2):296-310.
The rhizobacterium Pseudomonas fluorescens SS101 inhibits growth of oomycete and fungal pathogens, and induces resistance in plants against pathogens and insects. To unravel regulatory pathways of secondary metabolite production in SS101, we conducted a genome-wide search for sRNAs and performed transcriptomic analyses to identify genes associated with the Rsm (repressor of secondary metabolites) regulon. In silico analysis led to the identification of 16 putative sRNAs in the SS101 genome. In frame deletion of the sRNAs rsmY and rsmZ showed that the Rsm system regulates the biosynthesis of the lipopeptide massetolide A and involves the two repressor proteins RsmA and RsmE, with the LuxR-type transcriptional regulator MassAR as their most likely target. Transcriptome analyses of the rsmYZ mutant further revealed that genes associated with iron acquisition, motility and chemotaxis were significantly upregulated, whereas genes of the type VI secretion system were downregulated. Comparative transcriptomic analyses showed that most, but not all, of the genes controlled by RsmY/RsmZ are also controlled by the GacS/GacA two-component system. We conclude that the Rsm regulon of P. fluorescens SS101 plays a critical role in the regulation of lipopeptide biosynthesis and controls the expression of other genes involved in motility, competition and survival in the plant rhizosphere.
PMCID: PMC4353343  PMID: 25488342
10.  Defining the microbial effluxome in the content of the host-microbiome interaction 
PMCID: PMC4333769  PMID: 25745401
efflux transporters; multidrug resistance; microbiome; host-pathogen interaction; effluxome
11.  Draft Genome of Australian Environmental Strain WM 09.24 of the Opportunistic Human Pathogen Scedosporium aurantiacum 
Genome Announcements  2015;3(1):e01526-14.
We report here the first genome assembly and annotation of the human-pathogenic fungus Scedosporium aurantiacum, with a predicted 10,525 genes, and 11,661 transcripts. The strain WM 09.24 was isolated from the environment at Circular Quay, Sydney, New South Wales, Australia.
PMCID: PMC4333655  PMID: 25676755
12.  Homologs of the Acinetobacter baumannii AceI Transporter Represent a New Family of Bacterial Multidrug Efflux Systems 
mBio  2015;6(1):e01982-14.
Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins.
Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug efflux systems which are very common in Proteobacteria: the proteobacterial antimicrobial compound efflux (PACE) family. This is the first new family of multidrug efflux pumps to be described in 15 years.
PMCID: PMC4337561  PMID: 25670776
13.  The EcoCyc Database 
EcoSal Plus  2014;6(1):10.1128/ecosalplus.ESP-0009-2013.
EcoCyc is a bioinformatics database available at that describes the genome and the biochemical machinery of Escherichia coli K-12 MG1655. The long-term goal of the project is to describe the complete molecular catalog of the E. coli cell, as well as the functions of each of its molecular parts, to facilitate a system-level understanding of E. coli. EcoCyc is an electronic reference source for E. coli biologists, and for biologists who work with related microorganisms. The database includes information pages on each E. coli gene, metabolite, reaction, operon, and metabolic pathway. The database also includes information on E. coli gene essentiality, and on nutrient conditions that do or do not support the growth of E. coli. The web site and downloadable software contain tools for analysis of high-throughput datasets. In addition, a steady-state metabolic flux model is generated from each new version of EcoCyc. The model can predict metabolic flux rates, nutrient uptake rates, and growth rates for different gene knockouts and nutrient conditions. This chapter provides a detailed description of the data content of EcoCyc, and of the procedures by which this content is generated.
PMCID: PMC4243172  PMID: 26442933
14.  Comparative analysis of surface-exposed virulence factors of Acinetobacter baumannii 
BMC Genomics  2014;15(1):1020.
Acinetobacter baumannii is a significant hospital pathogen, particularly due to the dissemination of highly multidrug resistant isolates. Genome data have revealed that A. baumannii is highly genetically diverse, which correlates with major variations seen at the phenotypic level. Thus far, comparative genomic studies have been aimed at identifying resistance determinants in A. baumannii. In this study, we extend and expand on these analyses to gain greater insight into the virulence factors across eight A. baumannii strains which are clonally, temporally and geographically distinct, and includes an isolate considered non-pathogenic and a community-acquired A. baumannii.
We have identified a large number of genes in the A. baumannii genomes that are known to play a role in virulence in other pathogens, such as the recently studied proline-alanine-alanine-arginine (PAAR)-repeat domains of the type VI secretion systems. Not surprising, many virulence candidates appear to be part of the A. baumannii core genome of virulent isolates but were often found to be insertionally disrupted in the avirulent A. baumannii strain SDF. Our study also reveals that many known or putative virulence determinants are restricted to specific clonal lineages, which suggests that these virulence determinants may be crucial for the success of these widespread common clones. It has previously been suggested that the high level of intrinsic and adaptive resistance has enabled the widespread presence of A. baumannii in the hospital environment. This appears to have facilitated the expansion of its repertoire of virulence traits, as in general, the nosocomial strains in this study possess more virulence genes compared to the community-acquired isolate.
Major genetic variation in known or putative virulence factors was seen across the eight strains included in this study, suggesting that virulence mechanisms are complex and multifaceted in A. baumannii. Overall, these analyses increase our understanding of A. baumannii pathogenicity and will assist in future studies determining the significance of virulence factors within clonal lineages and/or across the species.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1020) contains supplementary material, which is available to authorized users.
PMCID: PMC4256060  PMID: 25422040
Genomics; Virulome; Type VI secretion systems; Membrane
15.  Draft Genome Sequence of Bacillus alcalophilus AV1934, a Classic Alkaliphile Isolated from Human Feces in 1934 
Genome Announcements  2014;2(6):e01175-14.
Bacillus alcalophilus AV1934, isolated from human feces, was described in 1934 before microbiome studies and recent indications of novel potassium ion coupling to motility in this extremophile. Here, we report draft sequences that will facilitate an examination of whether that coupling is part of a larger cycle of potassium ion-coupled transporters.
PMCID: PMC4241669  PMID: 25395643
16.  Structure of a short-chain dehydrogenase/reductase (SDR) within a genomic island from a clinical strain of Acinetobacter baumannii  
The structure of a short-chain dehydrogenase encoded within genomic islands of A. baumannii strains has been solved to 2.4 Å resolution. This classical SDR incorporates a flexible helical subdomain. The NADP-binding site and catalytic side chains are identified.
Over 15% of the genome of an Australian clinical isolate of Acinetobacter baumannii occurs within genomic islands. An uncharacterized protein encoded within one island feature common to this and other International Clone II strains has been studied by X-ray crystallography. The 2.4 Å resolution structure of SDR-WM99c reveals it to be a new member of the classical short-chain dehydrogenase/reductase (SDR) superfamily. The enzyme contains a nucleotide-binding domain and, like many other SDRs, is tetrameric in form. The active site contains a catalytic tetrad (Asn117, Ser146, Tyr159 and Lys163) and water molecules occupying the presumed NADP cofactor-binding pocket. An adjacent cleft is capped by a relatively mobile helical subdomain, which is well positioned to control substrate access.
PMCID: PMC4188072  PMID: 25286932
opportunistic pathogen; Acinetobacter baumannii WM99c; nosocomial strain; multidrug resistance; Rossmann fold
18.  Addition of Escherichia coli K-12 Growth Observation and Gene Essentiality Data to the EcoCyc Database 
Journal of Bacteriology  2014;196(5):982-988.
The sets of compounds that can support growth of an organism are defined by the presence of transporters and metabolic pathways that convert nutrient sources into cellular components and energy for growth. A collection of known nutrient sources can therefore serve both as an impetus for investigating new metabolic pathways and transporters and as a reference for computational modeling of known metabolic pathways. To establish such a collection for Escherichia coli K-12, we have integrated data on the growth or nongrowth of E. coli K-12 obtained from published observations using a variety of individual media and from high-throughput phenotype microarrays into the EcoCyc database. The assembled collection revealed a substantial number of discrepancies between the high-throughput data sets, which we investigated where possible using low-throughput growth assays on soft agar and in liquid culture. We also integrated six data sets describing 16,119 observations of the growth of single-gene knockout mutants of E. coli K-12 into EcoCyc, which are relevant to antimicrobial drug design, provide clues regarding the roles of genes of unknown function, and are useful for validating metabolic models. To make this information easily accessible to EcoCyc users, we developed software for capturing, querying, and visualizing cellular growth assays and gene essentiality data.
PMCID: PMC3957686  PMID: 24363340
19.  SecDF as Part of the Sec-Translocase Facilitates Efficient Secretion of Bacillus cereus Toxins and Cell Wall-Associated Proteins 
PLoS ONE  2014;9(8):e103326.
The aim of this study was to explore the role of SecDF in protein secretion in Bacillus cereus ATCC 14579 by in-depth characterization of a markerless secDF knock out mutant. Deletion of secDF resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the ΔsecDF mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of secDF indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen B. cereus, and further supports the notion that B. cereus enterotoxins are secreted by the Sec-system.
PMCID: PMC4118872  PMID: 25083861
20.  A genome-scale metabolic flux model of Escherichia coli K–12 derived from the EcoCyc database 
BMC Systems Biology  2014;8:79.
Constraint-based models of Escherichia coli metabolic flux have played a key role in computational studies of cellular metabolism at the genome scale. We sought to develop a next-generation constraint-based E. coli model that achieved improved phenotypic prediction accuracy while being frequently updated and easy to use. We also sought to compare model predictions with experimental data to highlight open questions in E. coli biology.
We present EcoCyc–18.0–GEM, a genome-scale model of the E. coli K–12 MG1655 metabolic network. The model is automatically generated from the current state of EcoCyc using the MetaFlux software, enabling the release of multiple model updates per year. EcoCyc–18.0–GEM encompasses 1445 genes, 2286 unique metabolic reactions, and 1453 unique metabolites. We demonstrate a three-part validation of the model that breaks new ground in breadth and accuracy: (i) Comparison of simulated growth in aerobic and anaerobic glucose culture with experimental results from chemostat culture and simulation results from the E. coli modeling literature. (ii) Essentiality prediction for the 1445 genes represented in the model, in which EcoCyc–18.0–GEM achieves an improved accuracy of 95.2% in predicting the growth phenotype of experimental gene knockouts. (iii) Nutrient utilization predictions under 431 different media conditions, for which the model achieves an overall accuracy of 80.7%. The model’s derivation from EcoCyc enables query and visualization via the EcoCyc website, facilitating model reuse and validation by inspection. We present an extensive investigation of disagreements between EcoCyc–18.0–GEM predictions and experimental data to highlight areas of interest to E. coli modelers and experimentalists, including 70 incorrect predictions of gene essentiality on glucose, 80 incorrect predictions of gene essentiality on glycerol, and 83 incorrect predictions of nutrient utilization.
Significant advantages can be derived from the combination of model organism databases and flux balance modeling represented by MetaFlux. Interpretation of the EcoCyc database as a flux balance model results in a highly accurate metabolic model and provides a rigorous consistency check for information stored in the database.
PMCID: PMC4086706  PMID: 24974895
Escherichia coli; Flux balance analysis; Constraint-based modeling; Metabolic network reconstruction; Metabolic modeling; Genome-scale model; Gene essentiality; Systems biology; EcoCyc; Pathway Tools
21.  Genome Sequence of the Neurotoxigenic Clostridium butyricum Strain 5521 
Genome Announcements  2014;2(3):e00632-14.
Clostridium strains from six phylogenetic groups, C. botulinum groups I to IV, C. baratii, and C. butyricum, display the capacity to produce botulinum neurotoxin. Here, we present the genome sequence of a C. butyricum isolate, the neurotoxigenic strain 5521, which encodes the type E botulinum neurotoxin.
PMCID: PMC4073117  PMID: 24970833
22.  Maintenance of essential amino acid synthesis pathways in the Blattabacterium cuenoti symbiont of a wood-feeding cockroach 
Biology Letters  2013;9(3):20121153.
In addition to harbouring intestinal symbionts, some animal species also possess intracellular symbiotic microbes. The relative contributions of gut-resident and intracellular symbionts to host metabolism, and how they coevolve are not well understood. Cockroaches and the termite Mastotermes darwiniensis present a unique opportunity to examine the evolution of spatially separated symbionts, as they harbour gut symbionts and the intracellular symbiont Blattabacterium cuenoti. The genomes of B. cuenoti from M. darwiniensis and the social wood-feeding cockroach Cryptocercus punctulatus are each missing most of the pathways for the synthesis of essential amino acids found in the genomes of relatives from non-wood-feeding hosts. Hypotheses to explain this pathway degradation include: (i) feeding on microbes present in rotting wood by ancestral hosts; (ii) the evolution of high-fidelity transfer of gut microbes via social behaviour. To test these hypotheses, we sequenced the B. cuenoti genome of a third wood-feeding species, the phylogenetically distant and non-social Panesthia angustipennis. We show that host wood-feeding does not necessarily lead to degradation of essential amino acid synthesis pathways in B. cuenoti, and argue that ancestral high-fidelity transfer of gut microbes best explains their loss in strains from M. darwiniensis and C. punctulatus.
PMCID: PMC3645023  PMID: 23515978
mutualism; termite; genome degradation
23.  Life in the dark: metagenomic evidence that a microbial slime community is driven by inorganic nitrogen metabolism 
The ISME Journal  2013;7(6):1227-1236.
Beneath Australia's large, dry Nullarbor Plain lies an extensive underwater cave system, where dense microbial communities known as ‘slime curtains' are found. These communities exist in isolation from photosynthetically derived carbon and are presumed to be chemoautotrophic. Earlier work found high levels of nitrite and nitrate in the cave waters and a high relative abundance of Nitrospirae in bacterial 16S rRNA clone libraries. This suggested that these communities may be supported by nitrite oxidation, however, details of the inorganic nitrogen cycling in these communities remained unclear. Here we report analysis of 16S rRNA amplicon and metagenomic sequence data from the Weebubbie cave slime curtain community. The microbial community is comprised of a diverse assortment of bacterial and archaeal genera, including an abundant population of Thaumarchaeota. Sufficient thaumarchaeotal sequence was recovered to enable a partial genome sequence to be assembled, which showed considerable synteny with the corresponding regions in the genome of the autotrophic ammonia oxidiser Nitrosopumilus maritimus SCM1. This partial genome sequence, contained regions with high sequence identity to the ammonia mono-oxygenase operon and carbon fixing 3-hydroxypropionate/4-hydroxybutyrate cycle genes of N. maritimus SCM1. Additionally, the community, as a whole, included genes encoding key enzymes for inorganic nitrogen transformations, including nitrification and denitrification. We propose that the Weebubbie slime curtain community represents a distinctive microbial ecosystem, in which primary productivity is due to the combined activity of archaeal ammonia-oxidisers and bacterial nitrite oxidisers.
PMCID: PMC3660674  PMID: 23426011
ammonia oxidising archaea (AOA); chemolithotrophy; metagenomics; microbial community; nitrogen cycling; Nullarbor caves
24.  pA506, a Conjugative Plasmid of the Plant Epiphyte Pseudomonas fluorescens A506 
Applied and Environmental Microbiology  2013;79(17):5272-5282.
Conjugative plasmids are known to facilitate the acquisition and dispersal of genes contributing to the fitness of Pseudomonas spp. Here, we report the characterization of pA506, the 57-kb conjugative plasmid of Pseudomonas fluorescens A506, a plant epiphyte used in the United States for the biological control of fire blight disease of pear and apple. Twenty-nine of the 67 open reading frames (ORFs) of pA506 have putative functions in conjugation, including a type IV secretion system related to that of MOBP6 family plasmids and a gene cluster for type IV pili. We demonstrate that pA506 is self-transmissible via conjugation between A506 and strains of Pseudomonas spp. or the Enterobacteriaceae. The origin of vegetative replication (oriV) of pA506 is typical of those in pPT23A family plasmids, which are present in many pathovars of Pseudomonas syringae, but pA506 lacks repA, a defining locus for pPT23A plasmids, and has a novel partitioning region. We selected a plasmid-cured derivative of A506 and compared it to the wild type to identify plasmid-encoded phenotypes. pA506 conferred UV resistance, presumably due to the plasmid-borne rulAB genes, but did not influence epiphytic fitness of A506 on pear or apple blossoms in the field. pA506 does not appear to confer resistance to antibiotics or other toxic elements. Based on the conjugative nature of pA506 and the large number of its genes that are shared with plasmids from diverse groups of environmental bacteria, the plasmid is likely to serve as a vehicle for genetic exchange between A506 and its coinhabitants on plant surfaces.
PMCID: PMC3753976  PMID: 23811504
25.  H-NS Plays a Role in Expression of Acinetobacter baumannii Virulence Features 
Infection and Immunity  2013;81(7):2574-2583.
Acinetobacter baumannii has become a major problem in the clinical setting with the prevalence of infections caused by multidrug-resistant strains on the increase. Nevertheless, only a limited number of molecular mechanisms involved in the success of A. baumannii as a human pathogen have been described. In this study, we examined the virulence features of a hypermotile derivative of A. baumannii strain ATCC 17978, which was found to display enhanced adherence to human pneumocytes and elevated levels of lethality toward Caenorhabditis elegans nematodes. Analysis of cellular lipids revealed modifications to the fatty acid composition, providing a possible explanation for the observed changes in hydrophobicity and subsequent alteration in adherence and motility. Comparison of the genome sequences of the hypermotile variant and parental strain revealed that an insertion sequence had disrupted an hns-like gene in the variant. This gene encodes a homologue of the histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes seen in the autotransporter Ata, a type VI secretion system, and a type I pilus cluster. Interestingly, isolation and analysis of a second independent hypermotile ATCC 17978 variant revealed a mutation to a residue within the DNA binding region of H-NS. Taken together, these mutants indicate that the phenotypic and transcriptomic differences seen are due to loss of regulatory control effected by H-NS.
PMCID: PMC3697591  PMID: 23649094

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