Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Publisher Summary The chapter introduces the mitogen-activated protein (MAP) kinase (MAPK) module. The identification of MAP kinase pathways exemplifies the power of combining biochemical and genetic approaches to molecular problems. The chapter discusses the mammalian MAPK pathways—ERKl/2 and MKKl/2 pathways—and stress-activated protein kinase pathways. The regulation of MAPK pathways by protein phosphatases is discussed in the chapter describing in detail about dual specificity phosphatases, serinenhreonine phosphatases, and protein tyrosine phosphatases. The chapter explores the cellular substrates of MAP kinases, wherein it discusses about protein kinase substrates for MAPKS, nuclear transcription factors, signaling components, and cytoskeletal proteins. Responses to MAPK pathways, regulation of cell growth and transformation, and regulation of cell differentiation and development have also been summarized in the chapter. The chapter describes the yeast MAPK pathways of saccharomyces cerevisiae (Budding Yeast) and Schizosaccharomyces pombe (Fission Yeast). The chapter provides the description of the intracellular targeting and spatial regulation of MAPK pathway components, signaling complexes, and the nuclear translocation of MAPK and MKK. Eukaryotic MAPK cascades provide excellent examples of signal transduction mechanisms that embody key principles common to many, if not all, signaling pathways. Many fundamental questions remain for future studies to investigate the mechanisms by which these pathways are regulated as well as the cellular responses that they control.

Signal transduction through MAP kinase cascades.

Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times ( 2 S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments.

Recent Advances in Petroleum Microbiology

The cellular stress response is a universal mechanism of extraordinary physiological/pathophysiological significance. It represents a defense reaction of cells to damage that environmental forces inflict on macromolecules. Many aspects of the cellular stress response are not stressor specific because cells monitor stress based on macromolecular damage without regard to the type of stress that causes such damage. Cellular mechanisms activated by DNA damage and protein damage are interconnected and share common elements. Other cellular responses directed at re-establishing homeostasis are stressor specific and often activated in parallel to the cellular stress response. All organisms have stress proteins, and universally conserved stress proteins can be regarded as the minimal stress proteome. Functional analysis of the minimal stress proteome yields information about key aspects of the cellular stress response, including physiological mechanisms of sensing membrane lipid, protein, and DNA damage; redox sensing and regulation; cell cycle control; macromolecular stabilization/repair; and control of energy metabolism. In addition, cells can quantify stress and activate a death program (apoptosis) when tolerance limits are exceeded.

Molecular and evolutionary basis of the cellular stress response

The extracellular signal-regulated kinase (ERK) cascade is a central pathway that transmits signals from many extracellular agents to regulate cellular processes such as proliferation, differentiation and cell cycle progression. The signaling via the ERK cascade is mediated by sequential phosphorylation and activation of protein kinases in the different tiers of the cascade. Although the main core phosphorylation chain of the cascade includes Raf kinases, MEK1/2, ERK1/2 (ERKs) and RSKs, other alternatively spliced forms and distinct components exist in the different tiers, and participate in ERK signaling under specific conditions. These components enhance the complexity of the ERK cascade and thereby, enable the wide variety of functions that are regulated by it. Another factor that is important for the dissemination of ERKs' signals is the multiplicity of the cascade's substrates, which include transcription factors, protein kinases and phosphatases, cytoskeletal elements, regulators of apoptosis, and a variety of other signaling-related molecules. About 160 substrates have already been discovered for ERKs, and the list of these substrates, as well as the function and mechanism of activation of representative substrates, are described in the current review. Many of these substrates are localized in the nucleus, and seem to participate in the regulation of transcription upon stimulation. However, other substrates are found in the cytosol as well as in other cellular organelles, and those are responsible for processes such as translation, mitosis and apoptosis. Understanding of these processes may provide a full picture of the distinct, and even opposing cellular processes that are regulated by the ERK cascade.

The extracellular signal-regulated kinase: Multiple substrates regulate diverse cellular functions

Many regulons controlled by alternative c factors, including cs and c32, are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of cs ...

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Regulation of σ factor competition by the alarmone ppGpp

The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta-cleavage pathway enzyme is discussed.

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Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600.

Alternative bacterial sigma factors bind the catalytic core RNA polymerase to confer promoter selectivity on the holoenzyme. The different holoenzymes are thus programmed to recognize the distinct promoter classes in the genome to allow coordinated activation of discrete sets of genes needed for adaptive responses. To form the holoenzymes, the different sigma factors must be available to compete for their common substrate (core RNA polymerase). This review highlights (a) the roles of antisigma factors in controlling the availability of alternative sigma factors and (b) the involvement of diverse regulatory molecules that promote the use of alternative sigma factors through subversion of the domineering housekeeping σ(70). The latter include the nucleotide alarmone ppGpp and small proteins (DksA, Rsd, and Crl), which directly target the transcriptional machinery to mediate their effects.

Regulation of Alternative Sigma Factor Use

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid located dmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-known meta-cleavage pathway for catechol, and include the recently discovered meta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of these meta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to the meta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations between meta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.

Genetics and biochemistry of phenol degradation by Pseudomonas sp. CF600

Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmid-encoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequence and the polypeptide products of this 5.5-kb region. A combination of deletion analysis, expression of subfragments in tac expression vectors, and identification of polypeptide products in maxicells was used to demonstrate that the polypeptides observed are produced from the six open reading frames identified in the sequence. Expression of phenol hydroxylase activity in a laboratory Pseudomonas strain allows growth on phenol, owing to expression of this enzyme and the chromosomally encoded ortho-cleavage pathway. This system, in conjunction with six plasmids that each expressed all but one of the polypeptides, was used to demonstrate that all six polypeptides are required for growth on phenol.

Victoria Shingler

Papers

Regulation of σ factor competition by the alarmone ppGpp

Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600.

Regulation of Alternative Sigma Factor Use

Genetics and biochemistry of phenol degradation by Pseudomonas sp. CF600

Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.