DNA Microarray and Proteomic Analyses of the RpoS Regulon in Geobacter sulfurreducens

Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa Cuernavaca, Morelos 62210, Mexico.
Journal of Bacteriology (Impact Factor: 2.81). 05/2006; 188(8):2792-800. DOI: 10.1128/JB.188.8.2792-2800.2006
Source: PubMed


The regulon of the sigma factor RpoS was defined in Geobacter sulfurreducens by using a combination of DNA microarray expression profiles and proteomics. An rpoS mutant was examined under steady-state conditions with acetate as an electron donor and fumarate as an electron acceptor
and with additional transcriptional profiling using Fe(III) as an electron acceptor. Expression analysis revealed that RpoS
acts as both a positive and negative regulator. Many of the RpoS-dependent genes determined play roles in energy metabolism,
including the tricarboxylic acid cycle, signal transduction, transport, protein synthesis and degradation, and amino acid
metabolism and transport. As expected, RpoS activated genes involved in oxidative stress resistance and adaptation to nutrient
limitation. Transcription of the cytochrome c oxidase operon, necessary for G. sulfurreducens growth using oxygen as an electron acceptor, and expression of at least 13 c-type cytochromes, including one previously shown to participate in Fe(III) reduction (MacA), were RpoS dependent. Analysis
of a subset of the rpoS mutant proteome indicated that 15 major protein species showed reproducible differences in abundance relative to those of
the wild-type strain. Protein identification using mass spectrometry indicated that the expression of seven of these proteins
correlated with the microarray data. Collectively, these results indicate that RpoS exerts global effects on G. sulfurreducens physiology and that RpoS is vital to G. sulfurreducens survival under conditions typically encountered in its native subsurface environments.


Available from: Tripti Khare
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    • "This was reflected by increased abundances of enzymes related to the utilization of alternative electron acceptors, signal transduction and protection against oxidative stress (Fig. 5). In G. sulfurreducens, some of these homologous proteins were also found to belong to the regulon of sigma RpoS, a master regulator of the general stress response [29]. Such physiology is advantageous for Geobactereaceae to survive in natural habitats with energy limitation , heavy metals, or oxidative stress. "
    [Show abstract] [Hide abstract] ABSTRACT: The strict anaerobe Geobacter metallireducens was cultivated in retentostats under acetate and acetate plus benzoate limitation in the presence of Fe(III) citrate in order to investigate its physiology under close to natural conditions. Growth rates below 0.003 h−1 were achieved in the course of cultivation. A nano-liquid chromatography–tandem mass spectrometry-based proteomic approach (nano-LC–MS/MS) with subsequent label-free quantification was performed on proteins extracted from cells sampled at different time points during retentostat cultivation. Proteins detected at low (0.002 h−1) and high (0.06 h−1) growth rates were compared between corresponding growth conditions (acetate or acetate plus benzoate). Carbon limitation significantly increased the abundances of several catabolic proteins involved in the degradation of substrates not present in the medium (ethanol, butyrate, fatty acids, and aromatic compounds). Growth rate-specific physiology was reflected in the changed abundances of energy-, chemotaxis-, oxidative stress-, and transport-related proteins. Mimicking natural conditions by extremely slow bacterial growth allowed to show how G. metallireducens optimized its physiology in order to survive in its natural habitats, since it was prepared to consume several carbon sources simultaneously and to withstand various environmental stresses.
    Full-text · Article · Jun 2014 · Systematic and Applied Microbiology
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    • "microarray techniques) since mRNAs are not inherently part of the ECM, and not all proteins that contribute to the formation of skeletal elements are produced in the vicinity of these elements. In addition, mRNA abundance has been shown to correlate poorly to the protein content [7,8,9], and does not take into account the wide variety of post-translational modifications which are critical to protein functions [10]. This makes proteomics an essential tool for characterizing the composition of the skeletal ECM. "
    [Show abstract] [Hide abstract] ABSTRACT: The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular proteins, including most of the bone and cartilage specific proteins previously reported in mammalian species. By comparing these extracellular proteins at larval, juvenile, and adult developmental stages, 123 proteins were found that differed significantly in abundance during development. Proteins with a reported function in bone formation increased in abundance during zebrafish development, while analysis of the cartilage matrix revealed major compositional changes during development. The protein list includes ligands and inhibitors of various signaling pathways implicated in skeletogenesis such as the Int/Wingless as well as the insulin-like growth factor signaling pathways. This first proteomic analysis of zebrafish skeletal development reveals that the zebrafish skeleton is comparable with the skeleton of other vertebrate species including mammals. In addition, our study reveals 6 novel proteins that have never been related to vertebrate skeletogenesis and shows a surprisingly large number of differences in the cartilage and bone proteome between the head, axis and caudal fin regions. Our study provides the first systematic assessment of bone and cartilage protein composition in an entire vertebrate at different stages of development.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "RpoS regulated genes and proteins were identified in Geobacter sulfurreducens and Burkholderia pseudomallei (Nunez et al., 2006; Osiriphun et al., 2009). As expected, most of the RpoS regulated genes and proteins are related to stress responses, but many of the RpoS regulated genes and proteins are involved in the expression of diverse physiological traits, such as general metabolism, transcription, translation, cell motility and secretions (Nunez et al., 2006; Osiriphun et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: The stationary-phase sigma factor, RpoS, influences the expression of factors important in survival of Pseudomonas chlororaphis O6 in the rhizosphere. A partial proteomic profile of a rpoS mutant in P. chlororaphis O6 was conducted to identify proteins under RpoS regulation. Five of 14 differentially regulated proteins had unknown roles. Changes in levels of proteins in P. chlororaphis O6 rpoS mutant were associated with iron metabolism, and protection against oxidative stress. The P. chlororaphis O6 rpoS mutant showed increased production of a pyoverdine-like siderophore, indole acetic acid, and altered isozyme patterns for peroxidase, catalase and superoxide dismutase. Consequently, sensitivity to hydrogen peroxide exposure increased in the P. chlororaphis O6 rpoS mutant, compared with the wild type. Taken together, RpoS exerted regulatory control over factors important for the habitat of P. chlororaphis O6 in soil and on root surfaces. The properties of several of the proteins in the RpoS regulon are currently unknown.
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