Showing papers in "Advances in Microbial Physiology in 2002"

The extracytoplasmic function (ECF) sigma factors.

Abstract: Bacterial sigma (sigma) factors are an essential component of RNA polymerase and determine promoter selectivity. The substitution of one sigma factor for another can redirect some or all of the RNA polymerase in a cell to activate the transcription of genes that would otherwise be silent. As a class, alternative sigma factors play key roles in coordinating gene transcription during various stress responses and during morphological development. The extracytoplasmic function (ECF) sigma factors are small regulatory proteins that are quite divergent in sequence relative to most other sigma factors. Many bacteria, particularly those with more complex genomes, contain multiple ECF sigma factors and these regulators often outnumber all other types of sigma factor combined. Examples include Bacillus subtilis (7 ECF sigma factors), Mycobacterium tuberculosis (10), Caulobacter crescentus (13), Pseudomonas aeruginosa (approximately 19), and Streptomyces coelicolor (approximately 50). The roles and mechanisms of regulation for these various ECF sigma factors are largely unknown, but significant progress has been made in selected systems. As a general trend, most ECF sigma factors are cotranscribed with one or more negative regulators. Often, these include a transmembrane protein functioning as an anti-sigma factor that binds, and inhibits, the cognate sigma factor. Upon receiving a stimulus from the environment, the sigma factor is released and can bind to RNA polymerase to stimulate transcription. In many ways, these anti-sigma:sigma pairs are analogous to the more familiar two-component regulatory systems consisting of a transmembrane histidine protein kinase and a DNA-binding response regulator. Both are mechanisms of coordinating a cytoplasmic transcriptional response to signals perceived by protein domains external to the cell membrane. Here, I review current knowledge of some of the better characterized ECF sigma factors, discuss the variety of experimental approaches that have proven productive in defining the roles of ECF sigma factors, and present some unifying themes that are beginning to emerge as more systems are studied.

The physiology and collective recalcitrance of microbial biofilm communities

Abstract: Microbial biofilms impinge upon all aspects of our lives. Whilst much of this impact is positive, there are many areas in which the presence and activities of biofilms are regarded as problematic and in need of control. It is in this respect that biofilms reveal their recalcitrance towards many of the long-established antibiotics, and industrial and medical treatment strategies. The nature of the resistance of biofilms, in spite of much research, remains an enigma. Whilst it is recognized that reaction--diffusion limitation properties of the biofilm matrix towards the majority of treatment agents will impede access, this cannot be the sole explanation of the observed resistance. Rather, it will delay the death of cells within the community to various extents. Similarly, it is recognized that biofilm communities are phenotypically heterogeneous and that their eradication will reflect the susceptibility of the most resistant phenotype. The nutrient and gaseous gradients that generate this heterogeneity will, however, be destroyed as a result of antimicrobial treatments and cause the phenotype of the survivors to alter from slow-growing resistant cells to fast-growing susceptible ones. Accordingly both explanations can only delay death of the community. In order to explain more fully the long-term recalcitrance of biofilms towards such a wide variety of biocidal agents, more radical hypotheses must be considered. Amongst these are that multidrug efflux pumps could be up-regulated on expression of a biofilm phenotype. Whilst this is an appealing and simple explanation, because of its ability to explain the breadth of agents to which biofilms are resistant, recent work has suggested that this is not the case. Alternative hypotheses attempt to explain the diversity of agents by invoking a common cause of death for which singular resistance mechanisms could be applied. It is therefore suggested that an altruistic majority of sublethally damaged cells in a population commit suicide (apoptosis), thereby providing some protection to the survivors. A proportion of cells (persisters) is suggested to be defective, or repressed, in their suicide response, and survive. The persisters thereby benefit from the self-sacrifice of their compatriots and maintain the gene pool. A second explanation of the presence of persisters is that the general stress response, well known to include the adoption of a viable, nonculturable state of quiescence, is up-regulated in small pockets of the biofilm community, where nutrients are particularly scarce. Such quiescent cells noted for their resistance towards the metabolically acting biocides would potentially have their dormancy broken after treatment by the replenished supply of nutrients caused by the death of the majority. A more recent hypothesis suggests that extracellular signals, 'alarmones', released from killed cells might prime recipients into a state of resistance. Thus, in biofilm communities deep lying cells might be alerted into a resistant state by the premature death of peripheral cells. It is equally possible that 'alarmones', in this context, are merely the post-treatment 'wake-up' call to a previously quiescent subset of cells. In this review, we attempt to provide a holistic view of the potential mechanisms by which biofilms express resistance. Since these mechanisms are multifaceted then their impact upon resistance will be considered against the context of biofilm formation, growth and maturation.

How oxygen damages microbes: oxygen tolerance and obligate anaerobiosis

Abstract: The orbital structure of molecular oxygen constrains it to accept electrons one at a time, and its unfavourable univalent reduction potential ensures that it can do so only with low-potential redox partners. In E. coli, this restriction prevents oxygen from oxidizing structural molecules. Instead, it primarily oxidizes reduced flavins, a reaction that is harmful only in that it generates superoxide and hydrogen peroxide as products. These species are stronger oxidants than is oxygen itself. They can oxidize dehydratase iron-sulphur clusters and sulphydryls, respectively, and thereby inactivate enzymes that are dependent upon these functional groups. Hydrogen peroxide also oxidizes free iron, generating hydroxyl radicals. Because hydroxyl radicals react with virtually any biomolecules they encounter, their reactivity is broadly dissipated, and only their reactions with DNA are known to have an important physiological impact. E. coli elaborates scavenging and repair systems to minimize the impact of this adventitious chemistry; mutants that lack these defences grow poorly in aerobic habitats. Some of the growth deficits of these mutants cannot be easily ascribed to sulphydryl, cluster, or DNA damage, indicating that important aspects of oxidative stress still lack a biochemical explanation. Obligate anaerobes cannot tolerate oxygen because they utilize metabolic schemes built around enzymes that react with oxidants. The reliance upon low-potential flavoproteins for anaerobic respiration probably causes substantial superoxide and hydrogen peroxide to be produced when anaerobes are exposed to air. These species then generate damage of the same type that they produce in aerotolerant bacteria. However, obligate anaerobes also utilize several classes of dioxygen-sensitive enzymes that are not needed by aerobes. These enzymes are used for processes that help maintain the redox balance during anaerobic fermentations. They catalyse reactions that are chemically difficult, and the reaction mechanisms require the solvent exposure of radicals or low-potential metal clusters that can react rapidly with oxygen. Recent work has uncovered adaptive strategies by which obligate anaerobes seek to minimize the damage done by superoxide and hydrogen peroxide. Their failure to divest themselves of enzymes that can be directly damaged by molecular oxygen suggests that evolution has not yet provided economical options to them.

Global adjustment of microbial physiology during free radical stress.

Abstract: Oxidation can damage all biological macromolecules, and the survival of a cell therefore depends on its ability to control the level of oxidants. Microbes possess an astonishing variety of antioxidant defences, ranging from small, oxidant-scavenging molecules to self-regulating, homeostatic gene networks. Most often these antioxidant defences are activated by exposure to specific classes of oxidants. Interestingly, the isolation of pleiotropic mutations that impair or exacerbate the expression of subsets of oxidant-responsive genes led to the identification of global regulators. In a few, well-characterized cases, these regulators can transduce oxidative damage into gene regulation. Recently, the application of genomic tools to study the antioxidant responses of E. coli has both confirmed previous observations and provided evidence for a wealth of putative new anti-oxidant functions. Here, we review the remarkable diversity of antioxidant defence mechanisms, with emphasis on signal transduction by global regulator proteins and the corresponding genetic networks that protect the microbial cell against oxidative stress.

Biochemistry, regulation and genomics of haem biosynthesis in prokaryotes.

Abstract: Haems are involved in many cellular processes in prokaryotes and eukaryotes The biosynthetic pathway leading to haem formation is, with few exceptions, well-conserved, and is controlled in accordance with cellular function Here, we review the biosynthesis of haem and its regulation in prokaryotes In addition, we focus on a modification of haem for cytochrome c biogenesis, a complex process that entails both transport between cellular compartments and a specific thioether linkage between the haem moiety and the apoprotein Finally, a whole genome analysis from 63 prokaryotes indicates intriguing exceptions to the universality of the haem biosynthetic pathway and helps define new frontiers for future study

Drug resistance in yeasts--an emerging scenario.

Abstract: In view of the increasing threat posed by fungal infections in immunocompromised patients and due to the non-availability of effective treatments, it has become imperative to find novel antifungals and vigorously search for new drug targets. Fungal pathogens acquire resistance to drugs (antifungals), a well-established phenomenon termed multidrug resistance (MDR), which hampers effective treatment strategies. The MDR phenomenon is spread throughout the evolutionary scale. Accordingly, a host of responsible genes have been identified in the genetically tractable budding yeast Saccharomyces cerevisiae, as well as in a pathogenic yeast Candida albicans. Studies so far suggest that, while antifungal resistance is the culmination of multiple factors, there may be a unifying mechanism of drug resistance in these pathogens. ABC (ATP binding cassette) and MFS (major facilitator superfamily) drug transporters belonging to two different superfamilies, are the most prominent contributors to MDR in yeasts. Considering the abundance of the drug transporters and their wider specificity, it is believed that these drug transporters may not exclusively export drugs in fungi. It has become apparent that the drug transporters of the ABC superfamily of S. cerevisiae and C. albicans are multifunctional proteins, which mediate important physiological functions. This review summarizes current research on the molecular mechanisms underlying drug resistance, the emerging regulatory circuits of MDR genes, and the physiological relevance of drug transporters.

Microarray analysis of bacterial pathogenicity.

Abstract: The DNA microarray, a surface that contains an ordered arrangement of each identified open reading frame of a sequenced genome, is the engine of functional genomics. Its output, the expression profile, provides a genome wide snap-shot of the transcriptome. Refined by array-specific statistical instruments and data-mined by clustering algorithms and metabolic pathway databases, the expression profile discloses, at the transcriptional level, how the microbe adapts to new conditions of growth - the regulatory networks that govern the adaptive response and the metabolic and biosynthetic pathways that effect the new phenotype. Adaptation to host microenvironments underlies the capacity of infectious agents to persist in and damage host tissues. While monitoring the whole genome transcriptional response of bacterial pathogens within infected tissues has not been achieved, it is likely that the complex, tissue-specific response is but the sum of individual responses of the bacteria to specific physicochemical features that characterize the host milieu. These are amenable to experimentation in vitro and whole-genome expression studies of this kind have defined the transcriptional response to iron starvation, low oxygen, acid pH, quorum-sensing pheromones and reactive oxygen intermediates. These have disclosed new information about even well-studied processes and provide a portrait of the adapting bacterium as a ‘system’, rather than the product of a few genes or even a few regulons. Amongst the regulated genes that compose this adaptive system are transcription factors. Expression profiling experiments of transcription factor mutants delineate the corresponding regulatory cascade. The genetic basis for pathogenicity can also be studied by using microarray-based comparative genomics to characterize and quantify the extent of genetic variability within natural populations at the gene level of resolution. Also identified are differences between pathogen and commensal that point to possible virulence determinants or disclose evolutionary history. The host vigorously engages the pathogen; expression studies using host genome microarrays and bacterially infected cell cultures show that the initial host reaction is dominated by the innate immune response. However, within the complex expression profile of the host cell are components mediated by pathogen-specific determinants. In the future, the combined use of bacterial and host microarrays to study the same infected tissue will reveal the dialogue between pathogen and host in a gene-by-gene and site- and time-specific manner. Translating this conversation will not be easy and will probably require a combination of powerful bioinformatic tools and traditional experimental approaches - and considerable effort and time.