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The ultrafast equilibrium fluctuations of the Fe(III)-NO complex of a single point mutation of Myoglobin (H64Q) have been studied using Fourier Transform 2D-IR spectroscopy. Comparison with data from wild type Myoglobin (wt-Mb) shows the presence of two conformational substates of the mutant haem pocket where only one exists in the wild type form. One of the substates of the mutant exhibits an almost identical NO stretching frequency and spectral diffusion dynamics to wt-Mb while the other is distinctly different in both respects. The remarkably contrasting dynamics are largely attributable to interactions between the NO ligand and a nearby distal side chain which provides a basis for understanding the roles of these side chains in other ferric haem proteins.

Filamentous fungi are arguably the most industrially important group of microorganisms. Production processes involving these simple eukaryotes are often highly aerobic in nature, which implies these cultures are routinely subject to oxidative stress. Despite this, little is known about how filamentous fungi cope with high levels of oxidative stress as experienced in fermenter systems. More surprisingly, much of our knowledge of oxidative stress responses in fungi comes from environmental or medical studies. Here, the current understanding of oxidative stress effects and cellular responses in filamentous fungi is critically discussed. In particular the role of alternative respiration is evaluated, and the contributions of the alternative oxidase and alternative dehydrogenases in defence against oxidative stress, and their profound influence on fungal metabolism is critically examined. Finally, the importance of further research which would underpin a less empirical approach to optimising fungal strains for the fermenter environment is emphasised.

Understanding morphological transformations is essential to elucidating the evolution and developmental biology of many organisms. The Gram-positive soil bacterium, Streptomyces coelicolor has a complex lifecycle which lends itself well to such studies. We recently identified a transcriptional regulator, devA, which is required for correct sporulation in this organism, with mutants forming short, mis-septate aerial hyphae. devA is highly conserved within the Streptomyces genus along with a duplicate copy, devE. Disruption of devE indicates this gene also plays a role in sporulation; however the phenotype of a devE mutant differs from a devA mutant, forming long un-septate aerial hyphae. Transcriptional analysis of devA and devE indicates that they are expressed at different stages of the lifecycle. This suggests that following duplication they have diverged in regulation and function. Analysis of fully sequenced actinomycete genomes shows that devA is found in a single copy in morphologically simpler actinobacteria, suggesting that duplication has lead to increased morphological complexity. Complementation studies with devA from Salinispora, which sporulates but does not form aerial hyphae, indicates the ancestral gene cannot complement devA or devE, suggesting neo-functionalisation has occurred. Analysis of the synonymous and non-synonymous nucleotide changes within the devA paralogues suggest subfunctionalisation has occurred as both copies have diverged from the ancestral sequences. Divergence is also asymmetric with a higher level of functional constraint observed in the DNA binding domain compared with the effector binding/oligomerisation domain, suggesting diversification in the substrate specificity of these paralogues has contributed to their evolution.

In this perspective we offer views on how microbial activities may change in response to the impact of human activities and an increasing human population. New knowledge is forthcoming in microbiology and immunology on the biology of emerging pathogens. With the exposure of human populations to changing environments, through climate change and human encroachment into previously unpopulated areas, there is likely to be increased incidences of diseases caused by existing, emerging and previously unrecognised pathogens.

One of the most abundant and widely distributed groups of Helix-turn-helix (HTH) transcription factors is the metabolite-responsive GntR family of regulators (>8500 members in the Pfam database; Jan 2009). These proteins contain a DNA-binding HTH domain at the N terminus of the protein and an effector-binding and/or oligomerisation domain at the C terminus, where upon on binding an effector molecule, a conformational change occurs in the protein which influences the DNA-binding properties of the regulator resulting in repression or activation of transcription. This review summarises what we know about the distribution, structure, function and classification of these regulators and suggests that they may have a future role in biotechnology.

Phylogenetic relationships and timings of major cladogenesis events are investigated in the Balearic Island lizards Podarcislilfordi and P.pityusensis using 2675bp of mitochondrial and nuclear DNA sequences. Partitioned Bayesian and Maximum Parsimony analyses provided a well-resolved phylogeny with high node-support values. Bayesian MCMC estimation of node dates was investigated by comparing means of posterior distributions from different subsets of the sequence against the most robust analysis which used multiple partitions and allowed for rate heterogeneity among branches under a rate-drift model. Evolutionary rates were systematically underestimated and thus divergence times overestimated when sequences containing lower numbers of variable sites were used (based on ingroup node constraints). The following analyses allowed the best recovery of node times under the constant-rate (i.e., perfect clock) model: (i) all cytochrome b sequence (partitioned by codon position), (ii) cytochrome b (codon position 3 alone), (iii) NADH dehydrogenase (subunits 1 and 2; partitioned by codon position), (iv) cytochrome b and NADH dehydrogenase sequence together (six gene-codon partitions), (v) all unpartitioned sequence, (vi) a full multipartition analysis (nine partitions). Of these, only (iv) and (vi) performed well under the rate-drift model. These findings have significant implications for dating of recent divergence times in other taxa. The earliest P.lilfordi cladogenesis event (divergence of Menorcan populations), occurred before the end of the Pliocene, some 2.6Ma. Subsequent events led to a West Mallorcan lineage (2.0Ma ago), followed 1.2Ma ago by divergence of populations from the southern part of the Cabrera archipelago from a widely-distributed group from north Cabrera, northern and southern Mallorcan islets. Divergence within P.pityusensis is more recent with the main Ibiza and Formentera clades sharing a common ancestor at about 1.0Ma ago. Climatic and sea level changes are likely to have initiated cladogenesis, with lineages making secondary contact during periodic landbridge formation. This oscillating cross-archipelago pattern in which ancient divergence is followed by repeated contact resembles that seen between East-West refugia populations from mainland Europe.

Most bacteria encode proteins for defence against infection by bacteriophages. The mechanisms that bring about phage defence are extremely diverse, suggesting frequent independent evolution of novel processes. Phage defence determinants are often plasmid or phage-encoded and many that are chromosomal show evidence of lateral transfer. Recent studies on restriction-modification (R-M) systems show that these genes are amongst the most rapidly evolving. Some bacteria have contingency genes that encode alternative target specificity determinants for Type I or Type III R-M systems, thus expanding the range of phages against which the host population is immune. The most counter-intuitive observation, however, is the prevalence of phase variation in many restriction systems, but recent arguments suggest that switching off expression of R-M systems can aid phage defence.

In the post-genomic area, the prediction of transcription factor regulons by position weight matrix-based programmes is a powerful approach to decipher biological pathways and to modelize regulatory networks in bacteria. The main difficulty once a regulon prediction is available is to estimate its reliability prior to start expensive experimental validations and therefore trying to find a way how to identify true positive hits from an endless list of potential target genes of a regulatory protein. Here we introduce PREDetector (Prokaryotic Regulatory Elements Detector), a tool developed for predicting regulons of DNA-binding proteins in bacterial genomes that, beside the automatic prediction, scoring and positioning of potential binding sites and their respective target genes in annotated bacterial genomes, it also provides an easy way to estimate the thresholds where to find reliable possible new target genes. PREDetector can be downloaded freely at http://www.montefiore.ulg.ac.be/~hiard/PreDetector/PreDetector.php.

Streptomyces coelicolor is the model organism for the actinobacteria, a group of high-GC Gram-positive bacteria with members that are notable both for their industrial importance as antibiotic producers and for their pathogenicity. The S. coelicolor genome encodes a subgroup of sensor kinases that is genetically linked to lipoprotein genes, at least one of which functions as an accessory protein to its co-translated kinase. Another member of this subgroup, MtrB, is widely conserved in the actinobacteria, along with its presumed cognate response regulator MtrA and the lipoprotein LpqB. Here, we postulate a possible role for LpqB in the MtrAB signal transduction pathway. We discuss what is known about this pathway in the actinobacteria and offer insights into why an essential response regulator does not necessarily need its cognate sensor kinase for activation.

Several processes have been described that could explain geographical variation and speciation within small islands, including fragmentation of populations through volcanic eruptions. Massive landslides, or debris avalanches, could cause similar effects. Here we analyse the potential impact of the 0.8 million-year-ago (Ma) Güimar valley debris avalanche on the phylogeography of the lizard Gallotia galloti on the Canary Island of Tenerife. Distributions of mitochondrial DNA lineages (based on cytochrome b sequences) were analysed on a 60-km southeastern coast transect centred on this area. Three main clades were detected, which can be divided into northern (one clade) and southern (two clades) groups that introgress across the valley. Maximum-likelihood estimates of migration rates (scaled for mutation rate) revealed highly asymmetric patterns, indicating that long-term gene flow into this region from both the northern and the southern populations greatly exceeded that in the opposite directions, consistent with recolonization of the area. The ancestral Tenerife node on the G. galloti tree is estimated at 0.80 Ma, matching closely with the geological estimate for the debris avalanche. Morphological variation (body dimensions and scalation) was also analysed and indicated a stepped cline in female scalation across the valley, although the patterns for male scalation and male and female body dimensions were not as clear. Together these findings provide support for the hypothesis that the debris avalanche has shaped the phylogeography of G. galloti and may even have been a primary cause of the within-island cladogenesis through population fragmentation and isolation. Current estimates of timing of island unification mean that the original hypothesis that within-island diversity is explained by the secondary contact of populations from the two ancient precursor islands of Teno and Anaga is less plausible for this and some other Tenerife species. Large-scale landslides have occurred on many volcanic islands, and so may have been instrumental in shaping within-island diversities.

The gram-positive filamentous bacterium Streptomyces coelicolor has a complex developmental cycle with three distinct phases: growth of the substrate mycelium, development of reproductive structures called aerial hyphae, and differentiation of these aerial filaments into long chains of exospores. During a transposon mutagenesis screen, we identified a novel gene (devA) required for proper development. The devA mutant produced only rare aerial hyphae, and those that were produced developed aberrant spore chains that were much shorter than wild-type chains and had misplaced septa. devA encodes a member of the GntR superfamily, a class of transcriptional regulators that typically respond to metabolite effector molecules. devA forms an operon with the downstream gene devB, which encodes a putative hydrolase that is also required for aerial mycelium formation on R5 medium. S1 nuclease protection analysis showed that transcription from the single devA promoter was temporally associated with vegetative growth, and enhanced green fluorescent protein transcriptional fusions showed that transcription was spatially confined to the substrate hyphae in the wild type. In contrast, devAB transcript levels were dramatically upregulated in a devA mutant and the devA promoter was also active in aerial hyphae and spores in this background, suggesting that DevA might negatively regulate its own production. This suggestion was confirmed by gel mobility shift assays that showed that DevA binds its own promoter region in vitro.

We report a system for the efficient removal of a marker flanked by two loxP sites in Streptomyces coelicolor, using a derivative of the temperate phage phiC31 that expresses Cre recombinase during a transient infection. As the test case for this recombinant phage (called Cre-phage), we present the construction of an in-frame deletion of a gene, pglW, required for phage growth limitation or Pgl in S.coelicolor. Cre-phage was also used for marker deletion in other strains of S.coelicolor.

Micromonospora echinospora differentiates in both submerged and surface cultures producing abundant dark spores after a period of vegetative mycelial growth. In submerged batch cultures, under either carbon or nitrogen limiting conditions, protease activity was found to coincide with sporulation indicating a relationship between proteolytic activity and differentiation in this organism. Further evidence for this link was provided from surface grown cultures wherein sporulation was inhibited by the serine protease inhibitors TLCK and TPCK. The association between proteolysis and differentiation apparent in this organism correlates with evidence of a similar phenomenon observed in the streptomycetes, suggesting that this may be a common response associated with differentiation in filamentous actinomycetes.

The heyday of continuous culture was in the 1960s, when its versatility and reproducibility were used to address fundamental problems in diverse microbiological fields such as biochemistry, ecology, genetics and physiology. The advent of molecular genetics in the 1970s and 1980s led to a decline in the popularity of continuous culture as a standard laboratory tool. The current trend of studying global proteomics, transcriptomics and metabolomics requires reproducible, reliable and biologically homogeneous datasets with which to approach a given problem. The use of continuous culture techniques can aid the acquisition of such data, and continuous cultures offer advantages over biologically heterogeneous batch cultures, where secondary growth and stress effects can often mask subtle physiological differences and trends. This review is intended to remind microbiologists of the value of continuous cultivation in a wide range of biological investigations, and describes some advantages and recent advances in applications of continuous culture in post-genomic studies.

The growth of Micromonospora echinospora was studied in high and low C/N ratio medium using both batch and continuous culture. Asparagine was consumed rapidly in batch cultures where it served as both a nitrogen and carbon source. Glucose consumption was low suggesting that asparagine functions as the major carbon source under these conditions. The effect of nutrient limitation on the accumulation of storage carbohydrate in batch culture revealed an intimate association between nitrogen limitation and the accumulation of carbonaceous reserves. This study revealed that glycogen constituted the major carbohydrate reserve associated with the onset of sporulation. Intracellular trehalose levels were found to be relatively low and may have been affected by the availability of carbon. Continuous culture studies revealed a correlation between glycogen accumulation and increasing growth rate. It was also found that elevated cellular ATP levels correlated with the increase in glycogen, and reduced glycolytic activity. At the higher growth rates cellular ATP levels were elevated and coincided with reduced activity of the key glycolytic enzyme, phosphofructokinase, suggesting that glycogen can act as a convenient energy reservoir when excess carbon flux dictates.

Streptomyces coelicolor is a Gram-positive soil bacterium that undergoes a complex developmental life cycle. The genome sequence of this organism was recently completed and has revealed the presence of over 60 sigma factors and a multitude of other transcriptional regulators, with a significant number of these being putative two-component signal transduction proteins. The authors have used the criteria established by Hoch and co-workers (Fabret et al., 1999, J Bacteriol 181, 1975-1983) to identify sensor kinase and response regulator genes encoded within the S. coelicolor genome. This analysis has revealed the presence of 84 sensor kinase genes, 67 of which lie adjacent to genes encoding response regulators. This strongly suggests that these paired genes encode two-component systems. In addition there are 13 orphan response regulators encoded in the genome, several of which have already been characterized and are implicated in development and antibiotic production, and 17 unpaired and as yet uncharacterized sensor kinases. This article attempts to infer useful information from sequence analysis and reviews what is currently known about the two-component systems, unpaired sensor kinases and orphan response regulators of S. coelicolor from both published reports and the authors' own unpublished data.

Aims: This study set out to investigate the effect of amino acids on the uptake of glucose by Micromonospora eichinospora (ATCC 15837)., Methods and Results: The specific rate of glucose uptake was found to be reduced when organic nitrogen components were present in the medium. Radioactive uptake studies revealed that the Km for glucose in this organism was 53 mm, indicating a low affinity for uptake compared with other actinomycete sugar transport systems. Individual amino acids negatively influenced the rate of glucose transport, suggesting a relationship between amino acid metabolism and glucose uptake in this organism. The sugar transport system was found to be an active process being inhibited by ionophores and KCN., Conclusions: The data suggest a direct link between amino acid metabolism and glucose uptake at the level of sugar transport., Significance and Impact of the Study: This study shows that the uptake of glucose, a major carbon source for many antibiotic fermentations, is significantly reduced in the presence of amino acids. This fact should inform the medium design and feeding regimes of fermentations involving similar actinomycetes.

The physiology of the actinomycete Micromonospora echinospora was examined during growth. Biphasic accumulation of glycogen occurred, initially during the early exponential growth phase, and again following the onset of sporulation at 120 h. Lipid levels increased during growth eventually representing 25% of the cell mass. A significant proportion of the lipid was found to be in the form of triacylglycerols, which were found to accumulate markedly during the sporulation phase. The disaccharide trehalose was also found to accumulate during growth with levels rising to 5% of the dry weight during the mycelial production phase, then remaining constant during sporulation. Antibiotic was produced transiently by the cultures over the period preceding sporulation.

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.