Probing regulon of ArcA in Shewanella oneidensis MR-1 by integrated genomic analyses

<p>Abstract</p> <p>Background</p> <p>The Arc two-component system is a global regulator controlling many genes involved in aerobic/anaerobic respiration and fermentative metabolism in <it>Escherichia coli</it>. <it>Shewanella oneidensis </it>MR-1 contains a gene encoding a putative ArcA homolog with ~81% amino acid sequence identity to the <it>E. coli </it>ArcA protein but not a full-length <it>arcB </it>gene.</p> <p>Results</p> <p>To understand the role of ArcA in <it>S. oneidensis</it>, an <it>arcA </it>deletion strain was constructed and subjected to both physiological characterization and microarray analysis. Compared to the wild-type MR-1, the mutant exhibited impaired aerobic growth and a defect in utilizing DMSO in the absence of O₂. Microarray analyses on cells grown aerobically and anaerobically on fumarate revealed that expression of 1009 genes was significantly affected (<it>p </it>< 0.05) by the mutation. In contrast to <it>E. coli </it>ArcA, the protein appears to be dispensable in regulation of the TCA cycle in <it>S. oneidensis</it>. To further determine genes regulated by the Arc system, an ArcA recognition weight matrix from DNA-binding data and bioinformatics analysis was generated and used to produce an ArcA sequence affinity map. By combining both techniques, we identified an ArcA regulon of at least 50 operons, of which only 6 were found to be directly controlled by ArcA in <it>E. coli</it>.</p> <p>Conclusion</p> <p>These results indicate that the Arc system in <it>S. oneidensis </it>differs from that in <it>E. coli </it>substantially in terms of its physiological function and regulon while their binding motif are strikingly similar.</p

Genetic and Molecular Characterization of Flagellar Assembly in Shewanella oneidensis

Shewanella oneidensis is a highly motile organism by virtue of a polar flagellum. Unlike most flagellated bacteria, it contains only one major chromosome segment encoding the components of the flagellum with the exception of the motor proteins. In this region, three genes encode flagellinsaccording to the original genome annotation. However, we find that only flaA and flaB encode functional filament subunits. Although these two genesare under the control of different promoters, they are actively transcribed and subsequently translated, producing a considerable number of flagellin proteins. Additionally, both flagellins are able to interact with their chaperon FliS and are subjected to feedback regulation. Furthermore, FlaA and FlaB are glycosylated by a pathwayinvolving a major glycosylating enzyme,PseB, in spite of the lack of the majority of theconsensus glycosylation sites. In conclusion, flagellar assembly in S. oneidensis has novel features despite the conservation of homologous genes across taxa

Oxidized OxyR Up-Regulates ahpCF Expression to Suppress Plating Defects of oxyR- and Catalase-Deficient Strains

It is well established that in bacteria, such as Escherichia coli, OxyR is a transcriptional regulator that mediates the response to H2O2 by activating the OxyR regulon, which consists of many genes that play vital roles in oxidative stress resistance. In Shewanella, OxyR regulates, however, in both reduced and oxidized states, the production of H2O2 scavengers, including major catalase KatB and NADH peroxidase AhpCF. Here we showed that the oxyR mutant carried a plating defect manifested as division arresting, a phenotype that can be completely suppressed by an OxyR variant constitutively existing in oxidized form (OxyRL197P). This effect of OxyRL197P could not be solely attributed to the increment in KatB production, since the suppression was also observed in the absence of KatB. Although expression of peroxidase CcpA was greatly activated by OxyRL197P, the contribution of the protein in alleviating plating defect was negligible. We eventually identified AhpCF as the critical factor, when produced at substantially elevated levels by OxyRL197P, to protect the cell from H2O2 attack. Our data indicate that AhpCF is a particularly important peroxidase in oxidative stress resistance in Shewanella, not only playing a compensatory role for catalase, but also by itself providing sufficient protection from killing of H2O2 generated abiotically

Constructing gene co-expression networks and predicting functions of unknown genes by random matrix theory

<p>Abstract</p> <p>Background</p> <p>Large-scale sequencing of entire genomes has ushered in a new age in biology. One of the next grand challenges is to dissect the cellular networks consisting of many individual functional modules. Defining co-expression networks without ambiguity based on genome-wide microarray data is difficult and current methods are not robust and consistent with different data sets. This is particularly problematic for little understood organisms since not much existing biological knowledge can be exploited for determining the threshold to differentiate true correlation from random noise. Random matrix theory (RMT), which has been widely and successfully used in physics, is a powerful approach to distinguish system-specific, non-random properties embedded in complex systems from random noise. Here, we have hypothesized that the universal predictions of RMT are also applicable to biological systems and the correlation threshold can be determined by characterizing the correlation matrix of microarray profiles using random matrix theory.</p> <p>Results</p> <p>Application of random matrix theory to microarray data of <it>S. oneidensis</it>, <it>E. coli</it>, yeast, <it>A. thaliana</it>, <it>Drosophila</it>, mouse and human indicates that there is a sharp transition of nearest neighbour spacing distribution (NNSD) of correlation matrix after gradually removing certain elements insider the matrix. Testing on an <it>in silico </it>modular model has demonstrated that this transition can be used to determine the correlation threshold for revealing modular co-expression networks. The co-expression network derived from yeast cell cycling microarray data is supported by gene annotation. The topological properties of the resulting co-expression network agree well with the general properties of biological networks. Computational evaluations have showed that RMT approach is sensitive and robust. Furthermore, evaluation on sampled expression data of an <it>in silico </it>modular gene system has showed that under-sampled expressions do not affect the recovery of gene co-expression network. Moreover, the cellular roles of 215 functionally unknown genes from yeast, <it>E. coli </it>and <it>S. oneidensis </it>are predicted by the gene co-expression networks using guilt-by-association principle, many of which are supported by existing information or our experimental verification, further demonstrating the reliability of this approach for gene function prediction.</p> <p>Conclusion</p> <p>Our rigorous analysis of gene expression microarray profiles using RMT has showed that the transition of NNSD of correlation matrix of microarray profile provides a profound theoretical criterion to determine the correlation threshold for identifying gene co-expression networks.</p

A high-throughput percentage-of-binding strategy to measure binding energies in DNA–protein interactions: application to genome-scale site discovery

Quantifying the binding energy in DNA–protein interactions is of critical importance to understand transcriptional regulation. Based on a simple computational model, this study describes a high-throughput percentage-of-binding strategy to measure the binding energy in DNA–protein interactions between the Shewanella oneidensis ArcA two-component transcription factor protein and a systematic set of mutants in an ArcA-P (phosphorylated ArcA) binding site. The binding energies corresponding to each of the 4 nt at each position in the 15-bp binding site were used to construct a position-specific energy matrix (PEM) that allowed a reliable prediction of ArcA-P binding sites not only in Shewanella but also in related bacterial genomes

Physiological Roles of ArcA, Crp, and EtrA and Their Interactive Control on Aerobic and Anaerobic Respiration in Shewanella oneidensis

In the genome of Shewanella oneidensis, genes encoding the global regulators ArcA, Crp, and EtrA have been identified. All these proteins deviate from their counterparts in E. coli significantly in terms of functionality and regulon. It is worth investigating the involvement and relationship of these global regulators in aerobic and anaerobic respiration in S. oneidensis. In this study, the impact of the transcriptional factors ArcA, Crp, and EtrA on aerobic and anaerobic respiration in S. oneidensis were assessed. While all these proteins appeared to be functional in vivo, the importance of individual proteins in these two major biological processes differed. The ArcA transcriptional factor was critical in aerobic respiration while the Crp protein was indispensible in anaerobic respiration. Using a newly developed reporter system, it was found that expression of arcA and etrA was not influenced by growth conditions but transcription of crp was induced by removal of oxygen. An analysis of the impact of each protein on transcription of the others revealed that Crp expression was independent of the other factors whereas ArcA repressed both etrA and its own transcription while EtrA also repressed arcA transcription. Transcriptional levels of arcA in the wild type, crp, and etrA strains under either aerobic or anaerobic conditions were further validated by quantitative immunoblotting with a polyclonal antibody against ArcA. This extensive survey demonstrated that all these three global regulators are functional in S. oneidensis. In addition, the reporter system constructed in this study will facilitate in vivo transcriptional analysis of targeted promoters

Functional Assessment of EnvZ/OmpR Two-Component System in Shewanella oneidensis

EnvZ and OmpR constitute the bacterial two-component signal transduction system known to mediate osmotic stress response in a number of Gram-negative bacteria. In an effort to understand the mechanism through which Shewanella oneidensis senses and responds to environmental osmolarity changes, structure of the ompR-envZ operon was determined with Northern blotting assay and roles of the EnvZ/OmpR two-component system in response to various stresses were investigated with mutational analysis, quantitative reverse transcriptase PCR (qRT-PCR), and phenotype microarrays. Results from the mutational analysis and qRT-PCR suggested that the EnvZ/OmpR system contributed to osmotic stress response of S. oneidensis and very likely engaged a similar strategy employed by E. coli, which involved reciprocal regulation of two major porin coding genes. Additionally, the ompR-envZ system was also found related to cell motility. We further showed that the ompR-envZ dependent regulation of porin genes and motility resided almost completely on ompR and only partially on envZ, indicating additional mechanisms for OmpR phosphorylation. In contrast to E. coli lacking ompR-envZ, however, growth of S. oneidensis did not show a significant dependence on ompR-envZ even under osmotic stress. Further analysis with phenotype microarrays revealed that the S. oneidensis strains lacking a complete ompR-envZ system displayed hypersensitivities to a number of agents, especially in alkaline environment. Taken together, our results suggest that the function of the ompR-envZ system in S. oneidensis, although still connected with osmoregulation, has diverged considerably from that of E. coli. Additional mechanism must exist to support growth of S. oneidensis under osmotic stress

Characterization of starch structures isolated from the grains of waxy, sweet, and hybrid sorghum (Sorghum bicolor L. Moench)

In this study, starches were isolated from inbred (sweet and waxy) and hybrid (sweet and waxy) sorghum grains. Structural and property differences between (inbred and hybrid) sweet and waxy sorghum starches were evaluated and discussed. The intermediate fraction and amylose content present in hybrid sweet starch were lower than those in inbred sweet starch, while the opposite trend occurred with waxy starch. Furthermore, there was a higher A chain (30.93–35.73% waxy, 13.73–31.81% sweet) and lower B2 + B3 chain (18.04–16.56% waxy, 24.07–17.43% sweet) of amylopectin in hybrid sorghum starch. X-ray diffraction (XRD) and Fourier transform infrared reflection measurements affirm the relative crystalline and ordered structures of both varieties as follows: inbred waxy &gt; hybrid waxy &gt; hybrid sweet &gt; inbred sweet. Small angle X-ray scattering and 13C CP/MAS nuclear magnetic resonance proved that the amylopectin content of waxy starch was positively correlated with lamellar ordering. In contrast, an opposite trend was observed in sweet sorghum starch due to its long B2 + B3 chain content. Furthermore, the relationship between starch granule structure and function was also concluded. These findings could provide a basic theory for the accurate application of existing sorghum varieties precisely