Pharmacogenomics of in vitro response of the NCI-60 cancer cell line panel to Indian natural products.

BACKGROUND: Indian natural products have been anecdotally used for cancer treatment but with limited efficacy. To better understand their mechanism, we examined the publicly available data for the activity of Indian natural products in the NCI-60 cell line panel. METHODS: We examined associations of molecular genomic features in the well-characterized NCI-60 cancer cell line panel with in vitro response to treatment with 75 compounds derived from Indian plant-based natural products. We analyzed expression measures for annotated transcripts, lncRNAs, and miRNAs, and protein-changing single nucleotide variants in cancer-related genes. We also examined the similarities between cancer cell line response to Indian natural products and response to reference anti-tumor compounds recorded in a U.S. National Cancer Institute (NCI) Developmental Therapeutics Program database. RESULTS: Hierarchical clustering based on cell line response measures identified clustering of Phyllanthus and cucurbitacin products with known anti-tumor agents with anti-mitotic mechanisms of action. Curcumin and curcuminoids mostly clustered together. We found associations of response to Indian natural products with expression of multiple genes, notably including SLC7A11 involved in solute transport and ATAD3A and ATAD3B encoding mitochondrial ATPase proteins, as well as significant associations with functional single nucleotide variants, including BRAF V600E. CONCLUSION: These findings suggest potential mechanisms of action and novel associations of in vitro response with gene expression and some cancer-related mutations that increase our understanding of these Indian natural products.

Group testing can improve the cost-efficiency of prospective-retrospective biomarker studies.

BACKGROUND: Cancer treatment is increasingly dependent on biomarkers for prognostication and treatment selection. Potential biomarkers are frequently evaluated in prospective-retrospective studies in which biomarkers are measured retrospectively on archived specimens after completion of prospective clinical trials. In light of the high costs of some assays, random sampling designs have been proposed that measure biomarkers for a random sub-sample of subjects selected on the basis of observed outcome and possibly other variables. Compared with a standard design that measures biomarkers on all subjects, a random sampling design can be cost-efficient in the sense of reducing the cost of the study substantially while achieving a reasonable level of precision. METHODS: For a biomarker that indicates the presence of some molecular alteration (e.g., mutation in a gene), we explore the use of a group testing strategy, which involves physically pooling specimens across subjects and assaying pooled samples for the presence of the molecular alteration of interest, for further improvement in cost-efficiency beyond random sampling. We propose simple and general approaches to estimating the prognostic and predictive values of biomarkers with group testing, and conduct simulation studies to validate the proposed estimation procedures and to assess the cost-efficiency of the group testing design in comparison to the standard and random sampling designs. RESULTS: Simulation results show that the proposed estimation procedures perform well in realistic settings and that a group testing design can have considerably higher cost-efficiency than a random sampling design. CONCLUSIONS: Group testing can be used to improve the cost-efficiency of biomarker studies.

Correlation of gene expression and associated mutation profiles of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT with chemosensitivity of cancer cell lines to drug treatment.

BACKGROUND: The APOBEC gene family of cytidine deaminases plays important roles in DNA repair and mRNA editing. In many cancers, APOBEC3B increases the mutation load, generating clusters of closely spaced, single-strand-specific DNA substitutions with a characteristic hypermutation signature. Some studies also suggested a possible involvement of APOBEC3A, REV1, UNG, and FHIT in molecular processes affecting APOBEC mutagenesis. It is important to understand how mutagenic processes linked to the activity of these genes may affect sensitivity of cancer cells to treatment. RESULTS: We used information from the Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer resources to examine associations of the prevalence of APOBEC-like motifs and mutational loads with expression of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT and with cell line chemosensitivity to 255 antitumor drugs. Among the five genes, APOBEC3B expression levels were bimodally distributed, whereas expression of APOBEC3A, REV1, UNG, and FHIT was unimodally distributed. The majority of the cell lines had low levels of APOBEC3A expression. The strongest correlations of gene expression levels with mutational loads or with measures of prevalence of APOBEC-like motif counts and kataegis clusters were observed for REV1, UNG, and APOBEC3A. Sensitivity or resistance of cell lines to JQ1, palbociclib, bicalutamide, 17-AAG, TAE684, MEK inhibitors refametinib, PD-0325901, and trametinib and a number of other agents was correlated with candidate gene expression levels or with abundance of APOBEC-like motif clusters in specific cancers or across cancer types. CONCLUSIONS: We observed correlations of expression levels of the five candidate genes in cell line models with sensitivity to cancer drug treatment. We also noted suggestive correlations between measures of abundance of APOBEC-like sequence motifs with drug sensitivity in small samples of cell lines from individual cancer categories, which require further validation in larger datasets. Molecular mechanisms underlying the links between the activities of the products of each of the five genes, the resulting mutagenic processes, and sensitivity to each category of antitumor agents require further investigation.

Epigenetic analysis of neurocognitive development at 1 year of age in a community-based pregnancy cohort.

Multiple studies show that molecular genetic changes and epigenetic modifications affect the risk of cognitive disability or impairment. However, the role of epigenetic variation in cognitive development of neurotypical young children remains largely unknown. Using data from a prospective, community-based study of mother-infant pairs, we investigated the association of DNA methylation patterns in neonatal umbilical cord blood with cognitive and language development at 1 year of age. No CpG loci achieved genome-wide significance, although a small number of weakly suggestive associations with Bayley-III Receptive Communication scales were noted. While umbilical cord blood is a convenient resource for genetic analyses of birth outcomes, our results do not provide conclusive evidence that its use for DNA methylation profiling yields epigenetic markers that are directly related to postnatal neurocognitive outcomes at 1 year of age.

Association of changes in expression of HDAC and SIRT genes after drug treatment with cancer cell line sensitivity to kinase inhibitors.

Histone deacetylases (HDACs) and sirtuins (SIRTs) are important epigenetic regulators of cancer pathways. There is a limited understanding of how transcriptional regulation of their genes is affected by chemotherapeutic agents, and how such transcriptional changes affect tumour sensitivity to drug treatment. We investigated the concerted transcriptional response of HDAC and SIRT genes to 15 approved antitumor agents in the NCI-60 cancer cell line panel. Antitumor agents with diverse mechanisms of action induced upregulation or downregulation of multiple HDAC and SIRT genes. HDAC5 was upregulated by dasatinib and erlotinib in the majority of the cell lines. Tumour cell line sensitivity to kinase inhibitors was associated with upregulation of HDAC5, HDAC1, and several SIRT genes. We confirmed changes in HDAC and SIRT expression in independent datasets. We also experimentally validated the upregulation of HDAC5 mRNA and protein expression by dasatinib in the highly sensitive IGROV1 cell line. HDAC5 was not upregulated in the UACC-257 cell line resistant to dasatinib. The effects of cancer drug treatment on expression of HDAC and SIRT genes may influence chemosensitivity and may need to be considered during chemotherapy.

Phylogenetic classification of diverse LysR-type transcriptional regulators of a model prokaryote Geobacter sulfurreducens.

The protein family of LysR-type transcriptional regulators (LTTRs) is highly abundant among prokaryotes. We analyzed 10,145 non-redundant microbial sequences with homology to eight LysR family regulators of a model prokaryote, Geobacter sulfurreducens, and employed phylogenetic tree inference for LTTR classification. We also analyzed the arrangement of genome clusters containing G. sulfurreducens LTTR genes and searched for LTTR regulatory motifs, suggesting likely regulatory targets of G. sulfurreducens LTTRs. This is the first study to date providing a detailed classification of LTTRs in the deltaproteobacterial family Geobacteraceae.

Newborn umbilical cord blood DNA methylation and gene expression levels exhibit limited association with birth weight.

Most cases of fetal growth retardation are unexplained. These newborns are at high risk of serious illness or death in the neonatal period and exhibit significantly increased risk of specific chronic illnesses later in life. While there are several hypotheses to explain the well-established association between low birth weight and later risk of disease, the true etiology is unknown. To search for molecular patterns that may explain the biological basis for reduced fetal growth in a clinically normal cohort, and possibly provide clues for the lifelong increased risk of disease, we surveyed genome-wide DNA methylation and gene expression patterns in the umbilical cord blood of newborns born in Shelby County, TN. While we did not find genome-wide significant associations of birth weight with either leukocytic gene expression or DNA methylation, we did find suggestive associations in several genes with known effects on pre- or postnatal growth and health. As with previous molecular epidemiological studies of birth weight, we did not sample the most biologically relevant tissues in the newborn. However, our discovery of biologically plausible associations in a peripheral tissue suggests that further studies of tissues key to fetal growth regulation are warranted.

Increased copy number of imprinted genes in the chromosomal region 20q11-q13.32 is associated with resistance to antitumor agents in cancer cell lines.

BACKGROUND: Parent of origin-specific allelic expression of imprinted genes is epigenetically controlled. In cancer, imprinted genes undergo both genomic and epigenomic alterations, including frequent copy number changes. We investigated whether copy number loss or gain of imprinted genes in cancer cell lines is associated with response to chemotherapy treatment. RESULTS: We analyzed 198 human imprinted genes including protein-coding genes and noncoding RNA genes using data from tumor cell lines from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer datasets. We examined whether copy number of the imprinted genes in 35 different genome locations was associated with response to cancer drug treatment. We also analyzed associations of pretreatment expression and DNA methylation of imprinted genes with drug response. Higher copy number of BLCAP, GNAS, NNAT, GNAS-AS1, HM13, MIR296, MIR298, and PSIMCT-1 in the chromosomal region 20q11-q13.32 was associated with resistance to multiple antitumor agents. Increased expression of BLCAP and HM13 was also associated with drug resistance, whereas higher methylation of gene regions of BLCAP, NNAT, SGK2, and GNAS was associated with drug sensitivity. While expression and methylation of imprinted genes in several other chromosomal regions was also associated with drug response and many imprinted genes in different chromosomal locations showed a considerable copy number variation, only imprinted genes at 20q11-q13.32 had a consistent association of their copy number with drug response. Copy number values among the imprinted genes in the 20q11-q13.32 region were strongly correlated. They were also correlated with the copy number of cancer-related non-imprinted genes MYBL2, AURKA, and ZNF217 in that chromosomal region. Expression of genes at 20q11-q13.32 was associated with ex vivo drug response in primary tumor samples from the Beat AML 1.0 acute myeloid leukemia patient cohort. Association of the increased copy number of the 20q11-q13.32 region with drug resistance may be complex and could involve multiple genes. CONCLUSIONS: Copy number of imprinted and non-imprinted genes in the chromosomal region 20q11-q13.32 was associated with cancer drug resistance. The genes in this chromosomal region may have a modulating effect on tumor response to chemotherapy.

Parental ages and levels of DNA methylation in the newborn are correlated.

BACKGROUND: Changes in DNA methylation patterns with age frequently have been observed and implicated in the normal aging process and its associated increasing risk of disease, particularly cancer. Additionally, the offspring of older parents are at significantly increased risk of cancer, diabetes, and neurodevelopmental disorders. Only a proportion of these increased risks among the children of older parents can be attributed to nondisjunction and chromosomal rearrangements. RESULTS: Using a genome-wide survey of 27,578 CpG dinucleotides in a cohort of 168 newborns, we examined the relationship between DNA methylation in newborns and a variety of parental and newborn traits. We found that methylation levels of 144 CpGs belonging to 142 genes were significantly correlated with maternal age. A weaker correlation was observed with paternal age. Among these genes, processes related to cancer were over-represented, as were functions related to neurological regulation, glucose/carbohydrate metabolism, nucleocytoplasmic transport, and transcriptional regulation. CpGs exhibiting gender differences in methylation were overwhelmingly located on the X chromosome, although a small subset of autosomal CpGs were found in genes previously shown to exhibit gender-specific differences in methylation levels. CONCLUSIONS: These results indicate that there are differences in CpG methylation levels at birth that are related to parental age and that could influence disease risk in childhood and throughout life.

Racial differences in gene-specific DNA methylation levels are present at birth.

BACKGROUND: DNA methylation patterns differ among children and adults and play an unambiguous role in several disease processes, particularly cancers. The origin of these differences is inadequately understood, and this is a question of specific relevance to childhood and adult cancer. METHODS: DNA methylation levels at 26,485 autosomal CpGs were assayed in 201 newborns (107 African American and 94 Caucasian). Nonparametric analyses were performed to examine the relation between these methylation levels and maternal parity, maternal age, newborn gestational age, newborn gender, and newborn race. To identify the possible influences of confounding, stratification was performed by a second and third variable. For genes containing CpGs with significant differences in DNA methylation levels between races, analyses were performed to identify highly represented gene ontological terms and functional pathways. RESULTS: 13.7% (3623) of the autosomal CpGs exhibited significantly different levels of DNA methylation between African Americans and Caucasians; 2% of autosomal CpGs had significantly different DNA methylation levels between male and female newborns. Cancer pathways, including four (pancreatic, prostate, bladder, and melanoma) with substantial differences in incidence between the races, were highly represented among the genes containing significant race-divergent CpGs. CONCLUSIONS: At birth, there are significantly different DNA methylation levels between African Americans and Caucasians at a subset of CpG dinucleotides. It is possible that some of the epigenetic precursors to cancer exist at birth and that these differences partially explain the different incidence rates of specific cancers between the races.

Association of expression of epigenetic molecular factors with DNA methylation and sensitivity to chemotherapeutic agents in cancer cell lines.

BACKGROUND: Altered DNA methylation patterns play important roles in cancer development and progression. We examined whether expression levels of genes directly or indirectly involved in DNA methylation and demethylation may be associated with response of cancer cell lines to chemotherapy treatment with a variety of antitumor agents. RESULTS: We analyzed 72 genes encoding epigenetic factors directly or indirectly involved in DNA methylation and demethylation processes. We examined association of their pretreatment expression levels with methylation beta-values of individual DNA methylation probes, DNA methylation averaged within gene regions, and average epigenome-wide methylation levels. We analyzed data from 645 cancer cell lines and 23 cancer types from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer datasets. We observed numerous correlations between expression of genes encoding epigenetic factors and response to chemotherapeutic agents. Expression of genes encoding a variety of epigenetic factors, including KDM2B, DNMT1, EHMT2, SETDB1, EZH2, APOBEC3G, and other genes, was correlated with response to multiple agents. DNA methylation of numerous target probes and gene regions was associated with expression of multiple genes encoding epigenetic factors, underscoring complex regulation of epigenome methylation by multiple intersecting molecular pathways. The genes whose expression was associated with methylation of multiple epigenome targets encode DNA methyltransferases, TET DNA methylcytosine dioxygenases, the methylated DNA-binding protein ZBTB38, KDM2B, SETDB1, and other molecular factors which are involved in diverse epigenetic processes affecting DNA methylation. While baseline DNA methylation of numerous epigenome targets was correlated with cell line response to antitumor agents, the complex relationships between the overlapping effects of each epigenetic factor on methylation of specific targets and the importance of such influences in tumor response to individual agents require further investigation. CONCLUSIONS: Expression of multiple genes encoding epigenetic factors is associated with drug response and with DNA methylation of numerous epigenome targets that may affect response to therapeutic agents. Our findings suggest complex and interconnected pathways regulating DNA methylation in the epigenome, which may both directly and indirectly affect response to chemotherapy.

Molecular genomic features associated with in vitro response of the NCI-60 cancer cell line panel to natural products.

Natural products remain a significant source of anticancer chemotherapeutics. The search for targeted drugs for cancer treatment includes consideration of natural products, which may provide new opportunities for antitumor cytotoxicity as single agents or in combination therapy. We examined the association of molecular genomic features in the well-characterized NCI-60 cancer cell line panel with in vitro response to treatment with 1302 small molecules which included natural products, semisynthetic natural product derivatives, and synthetic compounds based on a natural product pharmacophore from the Developmental Therapeutics Program of the US National Cancer Institute's database. These compounds were obtained from a variety of plant, marine, and microbial species. Molecular information utilized for the analysis included expression measures for 23059 annotated transcripts, lncRNAs, and miRNAs, and data on protein-changing single nucleotide variants in 211 cancer-related genes. We found associations of expression of multiple genes including SLFN11, CYP2J2, EPHX1, GPC1, ELF3, and MGMT involved in DNA damage repair, NOTCH family members, ABC and SLC transporters, and both mutations in tyrosine kinases and BRAF V600E with NCI-60 responses to specific categories of natural products. Hierarchical clustering identified groups of natural products, which correlated with a specific mechanism of action. Specifically, several natural product clusters were associated with SLFN11 gene expression, suggesting that potential action of these compounds may involve DNA damage. The associations between gene expression or genome alterations of functionally relevant genes with the response of cancer cells to natural products provide new information about potential mechanisms of action of these identified clusters of compounds with potentially similar biological effects. This information will assist in future drug discovery and in design of new targeted cancer chemotherapy agents.

Association of birth weight with polymorphisms in the IGF2, H19, and IGF2R genes.

There is a substantial genetic component for birth weight variation. We tested 18 single nucleotide polymorphisms (SNPs) in the IGF2, H19, and IGF2R genes for associations with birth weight variation in 342 mother-newborn pairs (birth weight 2.1-4.7 kg at term) and 527 parent-newborn trios (birth weight 2.1-5.1 kg) across three localities. SNPs in the IGF2R (rs8191754; maternal genotype), IGF2 (rs3741205; newborn genotype), and 5' region of the H19 (rs2067051, rs2251375, and rs4929984) genes were associated with birth weight. Detailed analyses to distinguish direct maternal, direct newborn, and parent of origin effects for the most strongly associated H19 SNP (rs4929984) determined that the association of maternal genotype with newborn birth weight was due to parent of origin effects not direct maternal effects. That SNP is located near the CTCF binding sites that influence expression of the maternally imprinted IGF2 and paternally imprinted H19 locus, and there are statistically significant and independent opposite effects of the same rs4929984 allele, depending on the parent from which it was inherited.

Computational analysis of the evolution of 1-deoxy-D-xylulose-5-phosphate Reductoisomerase, an important enzyme in plant terpene biosynthesis.

Isoprenoids are a highly diverse and important group of natural compounds. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) catalyzes a key regulatory step in the non-mevalonate isoprenoid biosynthetic pathway in eubacteria and in plant plastids. For example, in Artemisia annua DXR participates in regulation of the biosynthesis of artemisinin, an important antimalarial drug. We performed phylogenetic analysis using DXR protein sequences from a model prokaryote, Escherichia coli, a picoplanktonic alga, Ostreococcus lucimarinus, and higher plants. The functional domain of DXR was conserved, allowing molecular evolutionary comparisons of both prokaryotic and eukaryotic sequences of DXR. Despite this conservation, for some plant species such as Campthoteca acuminata and Arabidopsis thaliana, phylogenetic relationships of their lineages were consistently violated. Our analysis revealed that plant DXR has an N-terminal transit domain that is likely bipartite, consisting of a chloroplast transit peptide (cTP) and a lumen transit peptide (lTP). Several features observed in the lTP suggest that, while DXR is targeted to the chloroplast, it is localized to the thylakoid lumen. These features include a twin arginine motif, a hydrophobic region, and a proline-rich region. The transit peptide also showed putative motifs for a 14-3-3 binding site with a chaperone phosphorylation site at Thr.

Bioinformatics Tools and Resources for Cancer Immunotherapy Study.

In recent years, cancer immunotherapy has emerged as a highly promising approach to treat patients with cancer, as the patient's own immune system is harnessed to attack cancer cells. However, the application of these approaches is still limited to a minority of patients with cancer and it is difficult to predict which patients will derive the greatest clinical benefit.One of the challenges faced by the biomedical community in the search of more effective biomarkers is the fact that translational research efforts involve collecting and accessing data at many different levels: from the type of material examined (e.g., cell line, animal models, clinical samples) to multiple data type (e.g., pharmacodynamic markers, genetic sequencing data) to the scale of a study (e.g., small preclinical study, moderate retrospective study on stored specimen sets, clinical trials with large cohorts).This chapter reviews several publicly available bioinformatics tools and data resources for high throughput molecular analyses applied to a range of data types, including those generated from microarray, whole-exome sequencing (WES), RNA-seq, DNA copy number, and DNA methylation assays, that are extensively used for integrative multidimensional data analysis and visualization.

Epigenome-wide DNA methylation analysis of small cell lung cancer cell lines suggests potential chemotherapy targets.

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. SCLC progression and treatment resistance involve epigenetic processes. However, links between SCLC DNA methylation and drug response remain unclear. We performed an epigenome-wide study of 66 human SCLC cell lines using the Illumina Infinium MethylationEPIC BeadChip array. Correlations of SCLC DNA methylation and gene expression with in vitro response to 526 antitumor agents were examined. RESULTS: We found multiple significant correlations between DNA methylation and chemosensitivity. A potentially important association was observed for TREX1, which encodes the 3' exonuclease I that serves as a STING antagonist in the regulation of a cytosolic DNA-sensing pathway. Increased methylation and low expression of TREX1 were associated with the sensitivity to Aurora kinase inhibitors AZD-1152, SCH-1473759, SNS-314, and TAK-901; the CDK inhibitor R-547; the Vertex ATR inhibitor Cpd 45; and the mitotic spindle disruptor vinorelbine. Compared with cell lines of other cancer types, TREX1 had low mRNA expression and increased upstream region methylation in SCLC, suggesting a possible relationship with SCLC sensitivity to Aurora kinase inhibitors. We also identified multiple additional correlations indicative of potential mechanisms of chemosensitivity. Methylation of the 3'UTR of CEP350 and MLPH, involved in centrosome machinery and microtubule tracking, respectively, was associated with response to Aurora kinase inhibitors and other agents. EPAS1 methylation was associated with response to Aurora kinase inhibitors, a PLK-1 inhibitor and a Bcl-2 inhibitor. KDM1A methylation was associated with PLK-1 inhibitors and a KSP inhibitor. Increased promoter methylation of SLFN11 was correlated with resistance to DNA damaging agents, as a result of low or no SLFN11 expression. The 5' UTR of the epigenetic modifier EZH2 was associated with response to Aurora kinase inhibitors and a FGFR inhibitor. Methylation and expression of YAP1 were correlated with response to an mTOR inhibitor. Among non-neuroendocrine markers, EPHA2 was associated with response to Aurora kinase inhibitors and a PLK-1 inhibitor and CD151 with Bcl-2 inhibitors. CONCLUSIONS: Multiple associations indicate potential epigenetic mechanisms affecting SCLC response to chemotherapy and suggest targets for combination therapies. While many correlations were not specific to SCLC lineages, several lineage markers were associated with specific agents.

SCLC-CellMiner: A Resource for Small Cell Lung Cancer Cell Line Genomics and Pharmacology Based on Genomic Signatures.

CellMiner-SCLC (https://discover.nci.nih.gov/SclcCellMinerCDB/) integrates drug sensitivity and genomic data, including high-resolution methylome and transcriptome from 118 patient-derived small cell lung cancer (SCLC) cell lines, providing a resource for research into this "recalcitrant cancer." We demonstrate the reproducibility and stability of data from multiple sources and validate the SCLC consensus nomenclature on the basis of expression of master transcription factors NEUROD1, ASCL1, POU2F3, and YAP1. Our analyses reveal transcription networks linking SCLC subtypes with MYC and its paralogs and the NOTCH and HIPPO pathways. SCLC subsets express specific surface markers, providing potential opportunities for antibody-based targeted therapies. YAP1-driven SCLCs are notable for differential expression of the NOTCH pathway, epithelial-mesenchymal transition (EMT), and antigen-presenting machinery (APM) genes and sensitivity to mTOR and AKT inhibitors. These analyses provide insights into SCLC biology and a framework for future investigations into subtype-specific SCLC vulnerabilities.

Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens.

BACKGROUND: The role of the RNA polymerase sigma factor RpoN in regulation of gene expression in Geobacter sulfurreducens was investigated to better understand transcriptional regulatory networks as part of an effort to develop regulatory modules for genome-scale in silico models, which can predict the physiological responses of Geobacter species during groundwater bioremediation or electricity production. RESULTS: An rpoN deletion mutant could not be obtained under all conditions tested. In order to investigate the regulon of the G. sulfurreducens RpoN, an RpoN over-expression strain was made in which an extra copy of the rpoN gene was under the control of a taclac promoter. Combining both the microarray transcriptome analysis and the computational prediction revealed that the G. sulfurreducens RpoN controls genes involved in a wide range of cellular functions. Most importantly, RpoN controls the expression of the dcuB gene encoding the fumarate/succinate exchanger, which is essential for cell growth with fumarate as the terminal electron acceptor in G. sulfurreducens. RpoN also controls genes, which encode enzymes for both pathways of ammonia assimilation that is predicted to be essential under all growth conditions in G. sulfurreducens. Other genes that were identified as part of the RpoN regulon using either the computational prediction or the microarray transcriptome analysis included genes involved in flagella biosynthesis, pili biosynthesis and genes involved in central metabolism enzymes and cytochromes involved in extracellular electron transfer to Fe(III), which are known to be important for growth in subsurface environment or electricity production in microbial fuel cells. The consensus sequence for the predicted RpoN-regulated promoter elements is TTGGCACGGTTTTTGCT. CONCLUSION: The G. sulfurreducens RpoN is an essential sigma factor and a global regulator involved in a complex transcriptional network controlling a variety of cellular processes.

Diversity of promoter elements in a Geobacter sulfurreducens mutant adapted to disruption in electron transfer.

The delta-proteobacterium, Geobacter sulfurreducens, can obtain energy by coupling the oxidation of organic matter to the reduction of insoluble Fe(III) or the anode of a microbial fuel cell. Because Fe(III) oxide or the anode surface, in contrast to oxygen, nitrate, or sulfate, is not soluble nor can it be reduced readily, Geobacter species have developed mechanisms which allow electrons to be delivered across outer membrane to the cell surface. OmcB is an outer-membrane c-type cytochrome important for G. sulfurreducens Fe(III) respiration. In the absence of OmcB, cells lost the ability to reduce soluble or insoluble Fe(III). However, the omcB deletion mutant can slowly adapt to growth on soluble Fe(III) over prolonged incubation in the medium with acetate as the electron donor. We discuss available information about predicted or experimentally validated promoters and transcription regulatory sites identified upstream of operons with transcriptional expression significantly changed in the adapted omcB mutant. DNA sequences of upstream regions of coregulated operons in the adapted mutant are divergent, suggesting the presence of recognition sites for different transcriptional regulators and indicating that adaptation of the omcB mutant to growth on soluble Fe(III) has shifted the relevant expression networks involved to a more diverse molecular basis.

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens.

BACKGROUND: The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences. RESULTS: The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second putative succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recently in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes. CONCLUSION: The genomic evidence suggests that metabolism, physiology and regulation of gene expression in G. metallireducens may be dramatically different from other Geobacteraceae.