Dawn M. Klingeman

Global Transcriptome Analysis of Shewanella oneidensis MR-1 Exposed to Different Terminal Electron Acceptors

To gain insight into the complex structure of the energy-generating networks in the dissimilatory metal reducer Shewanella oneidensis MR-1, global mRNA patterns were examined in cells exposed to a wide range of metal and non-metal electron acceptors. Gene expression patterns were similar irrespective of which metal ion was used as electron acceptor, with 60% of the differentially expressed genes showing similar induction or repression relative to fumarate-respiring conditions. Several groups of genes exhibited elevated expression levels in the presence of metals, including those encoding putative multidrug efflux transporters, detoxification proteins, extracytoplasmic sigma factors and PAS-domain regulators. Only one of the 42 predicted c-type cytochromes in MR-1, SO3300, displayed significantly elevated transcript levels across all metal-reducing conditions. Genes encoding decaheme cytochromes MtrC and MtrA that were previously linked to the reduction of different forms of Fe(III) and Mn(IV), exhibited only slight decreases in relative mRNA abundances under metal-reducing conditions. In contrast, specific transcriptome responses were displayed to individual non-metal electron acceptors resulting in the identification of unique groups of nitrate-, thiosulfate- and TMAO-induced genes including previously uncharacterized multi-cytochrome gene clusters. Collectively, the gene expression results reflect the fundamental differences between metal and non-metal respiratory pathways of S. oneidensis MR-1, where the coordinate induction of detoxification and stress response genes play a key role in adaptation of this organism under metal-reducing conditions. Moreover, the relative paucity and/or the constitutive nature of genes involved in electron transfer to metals is likely due to the low-specificity and the opportunistic nature of the metal-reducing electron transport pathways.

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

Twenty-One Genome Sequences from Pseudomonas Species and 19 Genome Sequences from Diverse Bacteria Isolated from the Rhizosphere and Endosphere of Populus deltoides

To aid in the investigation of the Populus deltoides microbiome, we generated draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium, and Variovorax were generated.

Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae

The application of systems biology tools holds promise for rational industrial microbial strain development. Here, we characterize a Zymomonas mobilis mutant (AcR) demonstrating sodium acetate tolerance that has potential importance in biofuel development. The genome changes associated with AcR are determined using microarray comparative genome sequencing (CGS) and 454-pyrosequencing. Sanger sequencing analysis is employed to validate genomic differences and to investigate CGS and 454-pyrosequencing limitations. Transcriptomics, genetic data and growth studies indicate that over-expression of the sodium-proton antiporter gene nhaA confers the elevated AcR sodium acetate tolerance phenotype. nhaA over-expression mostly confers enhanced sodium (Na+) tolerance and not acetate (Ac-) tolerance, unless both ions are present in sufficient quantities. NaAc is more inhibitory than potassium and ammonium acetate for Z. mobilis and the combination of elevated Na+ and Ac- ions exerts a synergistic inhibitory effect for strain ZM4. A structural model for the NhaA sodium-proton antiporter is constructed to provide mechanistic insights. We demonstrate that Saccharomyces cerevisiae sodium-proton antiporter genes also contribute to sodium acetate, potassium acetate, and ammonium acetate tolerances. The present combination of classical and systems biology tools is a paradigm for accelerated industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies.

Improved genome annotation for Zymomonas mobilis

893 respective quality scores and the details of the software and parameters used in study are available at our website (Supplementary Table 1). We have also sequenced the genome of an acetate-tolerant strain derived from Z. mobilis ZM4 ATCC31821 that was selected in another geographically separated laboratory7 and report 454 pyrosequencing and Sanger sequencing and peptide support for our changes to the ZM4 chromosome (Supplementary Table 1). In addition, the entire ZM4 pyrosequencing data set has been deposited in the National Center for Biotechnology Information (NCBI) shortread archive database (Study SRP000908). We processed the updated sequence data using the automated Oak Ridge National Laboratory (ORNL) microbial genome annotation pipeline. Finally, we examined the gene models predicted in the original GenBank annotation, the TIGR reannotation and our new reannotation and updated the ZM4 annotation in a final manual curation step. The final curation was performed in conjunction with a defined set of criteria (available with reannotation) and several proteomics data sets that showed peptide support for more than half of the theoretical proteome. An overview of the extensive changes made to the ZM4 chromosome based upon mass-spectrometry proteomics and pyrosequencing data and six illustrative examples are presented (Table 1 and Supplementary Fig. 1, respectively). We have converted 61 pseudogenes in the original annotation into 43 full-length coding sequences, which include predicted genes with important metabolic and physiological functions (e.g., GenBank acc. nos. for tRNA synthetases ZMO0460, ZMO0843, ZMO0845, ZMO1508, ZMO1878 and flagella gene fliF, ZMO0633) (Supplementary Table 2). Several of the updated chromosomal nucleotides are consistent with earlier ZM4 fosmid DNA sequence data (e.g., GenBank acc. no. AAG29859) and we have peptide support for 6 of our 37 newly predicted chromosomal genes (Supplementary Table 3). We did not identify peptides corresponding to any of the putative genes that we deleted. A comprehensive comparison on a gene-by-gene basis is presented in Supplementary Table 4. We have provided our analysis to the authors of the primary genome annotation and they are in the process of updating their GenBank submission. Plasmid DNA was also identified in our 454-pyrosequencing data, which was the financial sustainability of biomedical innovation in the private sector. This in turn will help secure the future of these areas against any further crises.

Evaluation and validation of de novo and hybrid assembly techniques to derive high-quality genome sequences

Motivation: To assess the potential of different types of sequence data combined with de novo and hybrid assembly approaches to improve existing draft genome sequences. Results: Illumina, 454 and PacBio sequencing technologies were used to generate de novo and hybrid genome assemblies for four different bacteria, which were assessed for quality using summary statistics (e.g. number of contigs, N50) and in silico evaluation tools. Differences in predictions of multiple copies of rDNA operons for each respective bacterium were evaluated by PCR and Sanger sequencing, and then the validated results were applied as an additional criterion to rank assemblies. In general, assemblies using longer PacBio reads were better able to resolve repetitive regions. In this study, the combination of Illumina and PacBio sequence data assembled through the ALLPATHS-LG algorithm gave the best summary statistics and most accurate rDNA operon number predictions. This study will aid others looking to improve existing draft genome assemblies. Availability and implementation: All assembly tools except CLC Genomics Workbench are freely available under GNU General Public License. Contact: brownsd@ornl.gov Supplementary information: Supplementary data are available at Bioinformatics online.

Dramatic performance of Clostridium thermocellum explained by its wide range of cellulase modalities

The multi–length scale nature of its glycoside hydrolase system explains the remarkable ability demonstrated by Clostridium thermocellum. Clostridium thermocellum is the most efficient microorganism for solubilizing lignocellulosic biomass known to date. Its high cellulose digestion capability is attributed to efficient cellulases consisting of both a free-enzyme system and a tethered cellulosomal system wherein carbohydrate active enzymes (CAZymes) are organized by primary and secondary scaffoldin proteins to generate large protein complexes attached to the bacterial cell wall. This study demonstrates that C. thermocellum also uses a type of cellulosomal system not bound to the bacterial cell wall, called the “cell-free” cellulosomal system. The cell-free cellulosome complex can be seen as a “long range cellulosome” because it can diffuse away from the cell and degrade polysaccharide substrates remotely from the bacterial cell. The contribution of these two types of cellulosomal systems in C. thermocellum was elucidated by characterization of mutants with different combinations of scaffoldin gene deletions. The primary scaffoldin, CipA, was found to play the most important role in cellulose degradation by C. thermocellum, whereas the secondary scaffoldins have less important roles. Additionally, the distinct and efficient mode of action of the C. thermocellum exoproteome, wherein the cellulosomes splay or divide biomass particles, changes when either the primary or secondary scaffolds are removed, showing that the intact wild-type cellulosomal system is necessary for this essential mode of action. This new transcriptional and proteomic evidence shows that a functional primary scaffoldin plays a more important role compared to secondary scaffoldins in the proper regulation of CAZyme genes, cellodextrin transport, and other cellular functions.

Complete Genome Sequence of the Cellulolytic Thermophile Caldicellulosiruptor obsidiansis OB47T

Caldicellulosiruptor obsidiansis OB47(T) (ATCC BAA-2073, JCM 16842) is an extremely thermophilic, anaerobic bacterium capable of hydrolyzing plant-derived polymers through the expression of multidomain/multifunctional hydrolases. The complete genome sequence reveals a diverse set of carbohydrate-active enzymes and provides further insight into lignocellulosic biomass hydrolysis at high temperatures.

Knock-out of SO1377 gene, which encodes the member of a conserved hypothetical bacterial protein family COG2268, results in alteration of iron metabolism, increased spontaneous mutation and hydrogen peroxide sensitivity in Shewanella oneidensis MR-1

BackgroundShewanella oneidensis MR-1 is a facultative, gram-negative bacterium capable of coupling the oxidation of organic carbon to a wide range of electron acceptors such as oxygen, nitrate and metals, and has potential for bioremediation of heavy metal contaminated sites. The complete 5-Mb genome of S. oneidensis MR-1 was sequenced and standard sequence-comparison methods revealed approximately 42% of the MR-1 genome encodes proteins of unknown function. Defining the functions of hypothetical proteins is a great challenge and may need a systems approach. In this study, by using integrated approaches including whole genomic microarray and proteomics, we examined knockout effects of the gene encoding SO1377 (gi24372955), a member of the conserved, hypothetical, bacterial protein family COG2268 (C lusters of O rthologous G roup) in bacterium Shewanella oneidensis MR-1, under various physiological conditions.ResultsCompared with the wild-type strain, growth assays showed that the deletion mutant had a decreased growth rate when cultured aerobically, but not affected under anaerobic conditions. Whole-genome expression (RNA and protein) profiles revealed numerous gene and protein expression changes relative to the wild-type control, including some involved in iron metabolism, oxidative damage protection and respiratory electron transfer, e. g. complex IV of the respiration chain. Although total intracellular iron levels remained unchanged, whole-cell electron paramagnetic resonance (EPR) demonstrated that the level of free iron in mutant cells was 3 times less than that of the wild-type strain. Siderophore excretion in the mutant also decreased in iron-depleted medium. The mutant was more sensitive to hydrogen peroxide and gave rise to 100 times more colonies resistant to gentamicin or kanamycin.ConclusionOur results showed that the knock-out of SO1377 gene had pleiotropic effects and suggested that SO1377 may play a role in iron homeostasis and oxidative damage protection in S. oneidensis MR-1.

Draft Genome Sequences for Clostridium thermocellum Wild-Type Strain YS and Derived Cellulose Adhesion-Defective Mutant Strain AD2

Clostridium thermocellum wild-type strain YS is an anaerobic, thermophilic, cellulolytic bacterium capable of directly converting cellulosic substrates into ethanol. Strain YS and a derived cellulose adhesion-defective mutant strain, AD2, played pivotal roles in describing the original cellulosome concept. We present their draft genome sequences.