The bldC developmental locus of Streptomyces coelicolor encodes a member of a family of small DNA-binding proteins related to the DNA-binding domains of the MerR family.

The bldC locus, required for formation of aerial hyphae in Streptomyces coelicolor, was localized by map-based cloning to the overlap between cosmids D17 and D25 of a minimal ordered library. Subcloning and sequencing showed that bldC encodes a member of a previously unrecognized family of small (58- to 78-residue) DNA-binding proteins, related to the DNA-binding domains of the MerR family of transcriptional activators. BldC family members are found in a wide range of gram-positive and gram-negative bacteria. Constructed {Delta}bldC mutants were defective in differentiation and antibiotic production. They failed to form an aerial mycelium on minimal medium and showed severe delays in aerial mycelium formation on rich medium. In addition, they failed to produce the polyketide antibiotic actinorhodin, and bldC was shown to be required for normal and sustained transcription of the pathway-specific activator gene actII-orf4. Although {Delta}bldC mutants produced the tripyrrole antibiotic undecylprodigiosin, transcripts of the pathway-specific activator gene (redD) were reduced to almost undetectable levels after 48 h in the bldC mutant, in contrast to the bldC+ parent strain in which redD transcription continued during aerial mycelium formation and sporulation. This suggests that bldC may be required for maintenance of redD transcription during differentiation. bldC is expressed from a single promoter. S1 nuclease protection assays and immunoblotting showed that bldC is constitutively expressed and that transcription of bldC does not depend on any of the other known bld genes. The bldC18 mutation that originally defined the locus causes a Y49C substitution that results in instability of the protein

DevA, a GntR-like transcriptional regulator required for development in streptomyces coelicolor

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

Characterization of Microbial Activity

The overall goal of this study is to investigate the phenomena that affect the fate and transport of radionuclides in the environment. The objective of this task, “Characterization of Microbial Activity”, is to develop a molecular biological method for the characterization of the microbial population indigenous to the Yucca Mountain Project site, with emphasis in detection and measurement of species or groups of microorganisms that could be involved in actinide and/or metal reduction, and subsurface transport. Subtasks consist of QA planning and preparation, and literature review. This task is part of a cooperative agreement between the UNLV Research Foundation and the U.S. Department of Energy (#DE-FC28-04RW12237) titled “Yucca Mountain Groundwater Characterization”

Characterization of microbial populations in the subsurface

This task is part of a cooperative agreement between the UNLV Research Foundation and the U.S. Department of Energy (#DE-FC28-04RW12237) titled “Yucca Mountain Groundwater Characterization”. The work was conducted in the Harry Reid Center for Environmental Studies, Microbiology Division of the University of Nevada, Las Vegas from October 1, 2004 to September 30, 2006. The overall goal of this research was to investigate the phenomena that affect the fate and transport of radionuclides in the environment. The purpose of this task (ORD-RF-01), “Characterization of Microbial Activity”, was to develop a molecular biological method for the characterization of the microbial population indigenous to the Yucca Mountain Project site, with emphasis in detection and measurement of species or groups of microorganisms that could be involved in actinide and/or metal reduction, and subsurface transport. To quantify and characterize the microbial populations, including microorganisms that may be viable but are not currently physiologically active, a molecular biological approach was utilized to amplify and detect microbial DNA present in the subsurface. This approach, termed polymerase chain reaction (PCR), results in the amplification of DNA sequences that are unique to the groups of microorganisms of interest. Quantitative PCR (QPCR) assays were developed and used for the measurement of subsurface microbial populations. The protocols were evaluated in laboratory tests involving representative microbial species and genera, and tested by assaying available subsurface samples previously collected from the Yucca Mountain Project site. Other subtasks included Quality Assurance (QA) planning and preparation, and a literature review. This work was subject to the Nevada System of Higher Education (NSHE) QA Program requirements

Translational control of the SigR-directed oxidative stress response in streptomyces via IF3-mediated repression of a noncanonical GTC start codon

The major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and posttranslational levels. Here we show that sigR has a highly unusual GTC start codon and that this leads to another level of SigR regulation, in which SigR translation is repressed by translation initiation factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overproduced relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the noncanonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper functioning of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Here we show that RsrA can be transcribed and translated independently of SigR, present evidence that RsrA is normally produced in excess of SigR, and describe the factors that determine SigR-RsrA stoichiometry.IMPORTANCE In all sigma factor-antisigma factor regulatory switches, the relative abundance of the two proteins is critical to the proper functioning of the system. Many sigma-antisigma operons are cotranscribed and translationally coupled, leading to a generic assumption that the sigma and antisigma factors are produced in a fixed 1:1 ratio. In the case of sigR-rsrA, we show instead that the antisigma factor is produced in excess over the sigma factor, providing a buffer to prevent spurious release of sigma activity. This excess arises in part because sigR has an extremely rare noncanonical GTC start codon, and as a result, SigR translation initiation is repressed by IF3. This finding highlights the potential significance of noncanonical start codons, very few of which have been characterized experimentally. It also emphasizes the limitations of predicting start codons using bioinformatic approaches, which rely heavily on the assumption that ATG, GTG, and TTG are the only permissible start codons

A connection between stress and development in the multicelular prokaryote Streptomyces coelicolor

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2–egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2–egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis

Defining the regulon of genes controlled by σE, a key regulator of the cell envelope stress response in Streptomyces coelicolor

The extracytoplasmic function (ECF) σ factor, σE is a key regulator of the cell envelope stress response in Streptomyces coelicolor. Although its role in maintaining cell wall integrity has been known for over a decade, a comprehensive analysis of the genes under its control has not been undertaken. Here, using a combination of chromatin immunoprecipitation‐sequencing (ChIP‐seq), microarray transcriptional profiling and bioinformatic analysis, we attempt to define the σE regulon. Approximately half of the genes identified encode proteins implicated in cell envelope function. 17 novel targets were validated by S1 nuclease mapping or in vitro transcription, establishing a σE binding consensus. Subsequently, we used bioinformatic analysis to look for conservation of the σE target promoters identified in S. coelicolor across 19 Streptomyces species. Key proteins under σE control across the genus include the actin homolog MreB, three penicillin‐binding proteins, two L,D‐transpeptidases, a LytR‐CpsA‐Psr‐family protein predicted to be involved in cell wall teichoic acid deposition, and a predicted MprF protein, which adds lysyl groups to phosphatidylglycerol to neutralize membrane surface charge. Taken together, these analyses provide biological insight into the σE‐mediated cell envelope stress response in the genus Streptomyces

Isolation and characterization of thermophilic, calcium-precipitating bacteria form calcite deposits at Yucca Mountain

Calcite deposits, composed of a mixture of calcium carbonate and silicon dioxide, were found in fractures and small cavities within the welded tuff of Yucca Mountain. This research investigation involves determining the presence of thermophilic, calciumprecipitating bacteria within these deposits. The possible existence of thermophilic bacteria may help to resolve the issue of whether these calcite deposits formed from precipitation of dissolved calcium carbonate in rain water transported from the overlying soil environment or as a result of upwelling of geothermally-heated waters transported from below the mountain. Evidence for microbially-influenced calcite precipitation in these deposits is indicated by the presence of moderately-thermophilic, calciumprecipitating bacteria. Growth of bacteria enriched from crushed calcite and calcite/tuff mixed samples collected from tunnels within Yucca Mountain indicate a tendency for thermophiles to be found in calcite deposits and mixed rock samples compared to tuff samples (no calcite) which lacked bacterial growth at temperatures of 50° C and higher. Calcite isolates growing at 60 and 70° C were identified as thermophiles, the most common identification being Bacillus stearothermophilus. SEM and electron dispersion spectroscopy (EDS) results showed that bacteria, isolated from Yucca Mountain calcite and calcite/tuff, produced calcite (CaCO3) when grown on calcium-enriched medium. This evidence indicates a possible warm water influence in the history of Yucca Mountain

Developmentally regulated volatiles geosmin and 2-methylisoborneol attract a soil arthropod to Streptomyces bacteria promoting spore dispersal

Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood1,2. Well-known examples are the soil-smelling terpenoids geosmin and 2-methylisoborneol (2-MIB)3,4, which humans and various animals sense at extremely low concentrations5,6. The conservation of geosmin biosynthesis genes among virtually all species of Streptomyces bacteria (and genes for the biosynthesis of 2-MIB in about 50%)7,8, suggests that the volatiles provide a selective advantage for these soil microbes. We show, in the present study, that these volatiles mediate interactions of apparent mutual benefit between streptomycetes and springtails (Collembola). In field experiments, springtails were attracted to odours emitted by Streptomyces colonies. Geosmin and 2-MIB in these odours induce electrophysiological responses in the antennae of the model springtail Folsomia candida, which is also attracted to both compounds. Moreover, the genes for geosmin and 2-MIB synthases are under the direct control of sporulation-specific transcription factors, constraining emission of the odorants to sporulating colonies. F. candida feeds on the Streptomyces colonies and disseminates spores both via faecal pellets and through adherence to its hydrophobic cuticle. The results indicate that geosmin and 2-MIB production is an integral part of the sporulation process, completing the Streptomyces life cycle by facilitating dispersal of spores by soil arthropods