Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast.

Biosensors are valuable and versatile tools in synthetic biology that are used to modulate gene expression in response to a wide range of stimuli. Ligand responsive transcription factors are a class of biosensor that can be used to couple intracellular metabolite concentration with gene expression to enable dynamic regulation and high-throughput metabolite producer screening. We have established the Saccharomyces cerevisiae WAR1 transcriptional regulator and PDR12 promoter as an organic acid biosensor that can be used to detect varying levels of para-hydroxybenzoic acid (PHBA) production from the shikimate pathway and output green fluorescent protein (GFP) expression in response. The dynamic range of GFP expression in response to PHBA was dramatically increased by engineering positive-feedback expression of the WAR1 transcriptional regulator from its target PDR12 promoter. In addition, the noise in GFP expression at the population-level was controlled by normalising GFP fluorescence to constitutively expressed mCherry fluorescence within each cell. These biosensor modifications increased the high-throughput screening efficiency of yeast cells engineered to produce PHBA by 5,000-fold, enabling accurate fluorescence activated cell sorting isolation of producer cells that were mixed at a ratio of 1 in 10,000 with non-producers. Positive-feedback, ratiometric transcriptional regulator expression is likely applicable to many other transcription-factor/promoter pairs used in synthetic biology and metabolic engineering for both dynamic regulation and high-throughput screening applications

Tiny microbes with a big impact: The role of cyanobacteria and their metabolites in shaping our future

© 2016 by the authors; licensee MDPI. Cyanobacteria are among the first microorganisms to have inhabited the Earth. Throughout the last few billion years, they have played a major role in shaping the Earth as the planet we live in, and they continue to play a significant role in our everyday lives. Besides being an essential source of atmospheric oxygen, marine cyanobacteria are prolific secondary metabolite producers, often despite the exceptionally small genomes. Secondary metabolites produced by these organisms are diverse and complex; these include compounds, such as pigments and fluorescent dyes, as well as biologically-active compounds with a particular interest for the pharmaceutical industry. Cyanobacteria are currently regarded as an important source of nutrients and biofuels and form an integral part of novel innovative energy-efficient designs. Being autotrophic organisms, cyanobacteria are well suited for large-scale biotechnological applications due to the low requirements for organic nutrients. Recent advances in molecular biology techniques have considerably enhanced the potential for industries to optimize the production of cyanobacteria secondary metabolites with desired functions. This manuscript reviews the environmental role of marine cyanobacteria with a particular focus on their secondary metabolites and discusses current and future developments in both the production of desired cyanobacterial metabolites and their potential uses in future innovative projects

Physical enrichment of transposon mutants from saturation mutant libraries using the TraDISort approach

Transposon-insertion sequencing methods are finding their way into the molecular toolbox of many fields of microbiology. These methods can identify the genomic locations and density of transposon insertions in saturated transposon mutant libraries and can be used to make inferences on gene function. For example, where no insertions or very few insertions are identified within a gene in a mutant library grown under permissive conditions, the gene may be essential. Furthermore, where mutations are enriched or lost in a gene after passaging the library through a selective process, the gene is likely to be involved in the process. Typically, a fitness based selection such as a stress condition is used in these experiments and the processed sequencing data is used to identify genes required for fitness under the selection. Our research team recently expanded the utility of the transposon directed insertion sequencing (TraDIS) method by applying a physical separation of a transposon mutant library mediated by fluorescence activated cell sorting, rather than a fitness-based selection. This approach, which we have named “TraDISort” is significant because it allows the study of phenotypes that are not linked to cell survival. The TraDISort approach has a broad range of future applications, in drug development, metabolic engineering and in studies of basic bacterial cell physiology

Cell size, genome size, and maximum growth rate are near-independent dimensions of ecological variation across bacteria and archaea.

Among bacteria and archaea, maximum relative growth rate, cell diameter, and genome size are widely regarded as important influences on ecological strategy. Via the most extensive data compilation so far for these traits across all clades and habitats, we ask whether they are correlated and if so how. Overall, we found little correlation among them, indicating they should be considered as independent dimensions of ecological variation. Nor was correlation evident within particular habitat types. A weak nonlinearity (6% of variance) was found whereby high maximum growth rates (temperature-adjusted) tended to occur in the midrange of cell diameters. Species identified in the literature as oligotrophs or copiotrophs were clearly separated on the dimension of maximum growth rate, but not on the dimensions of genome size or cell diameter

Homologs of the Acinetobacter baumannii AceI Transporter Represent a New Family of Bacterial Multidrug Efflux Systems

Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. IMPORTANCE Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug efflux systems which are very common in Proteobacteria: the proteobacterial antimicrobial compound efflux (PACE) family. This is the first new family of multidrug efflux pumps to be described in 15 years

A Sample-to-Sequence Protocol for Genus Targeted Transcriptomic Profiling: Application to Marine Synechococcus.

Recent studies using whole community metagenomic and metatranscriptomic approaches are revealing important new insights into the functional potential and activity of natural marine microbial communities. Here, we complement these approaches by describing a complete ocean sample-to-sequence protocol, specifically designed to target a single bacterial genus for purposes of both DNA and RNA profiling using fluorescence activated cell sorting (FACS). The importance of defining and understanding the effects of a sampling protocol are critical if we are to gain meaningful data from environmental surveys. Rigorous pipeline trials with a cultured isolate, Synechococcus sp. BL107 demonstrate that water filtration has a well-defined but limited impact on the transcriptomic profile of this organism, whilst sample storage and multiple rounds of cell sorting have almost no effect on the resulting RNA sequence profile. Attractively, the means to replicate the sampling strategy is within the budget and expertise of most researchers

Functional characterisation of substrate-binding proteins to address nutrient uptake in marine picocyanobacteria.

Marine cyanobacteria are key primary producers, contributing significantly to the microbial food web and biogeochemical cycles by releasing and importing many essential nutrients cycled through the environment. A subgroup of these, the picocyanobacteria (Synechococcus and Prochlorococcus), have colonised almost all marine ecosystems, covering a range of distinct light and temperature conditions, and nutrient profiles. The intra-clade diversities displayed by this monophyletic branch of cyanobacteria is indicative of their success across a broad range of environments. Part of this diversity is due to nutrient acquisition mechanisms, such as the use of high-affinity ATP-binding cassette (ABC) transporters to competitively acquire nutrients, particularly in oligotrophic (nutrient scarce) marine environments. The specificity of nutrient uptake in ABC transporters is primarily determined by the peripheral substrate-binding protein (SBP), a receptor protein that mediates ligand recognition and initiates translocation into the cell. The recent availability of large numbers of sequenced picocyanobacterial genomes indicates both Synechococcus and Prochlorococcus apportion >50% of their transport capacity to ABC transport systems. However, the low degree of sequence homology among the SBP family limits the reliability of functional assignments using sequence annotation and prediction tools. This review highlights the use of known SBP structural representatives for the uptake of key nutrient classes by cyanobacteria to compare with predicted SBP functionalities within sequenced marine picocyanobacteria genomes. This review shows the broad range of conserved biochemical functions of picocyanobacteria and the range of novel and hypothetical ABC transport systems that require further functional characterisation

Unicellular cyanobacteria are important components of phytoplankton communities in Australia's northern oceanic ecoregions

© 2019 Moore, Huang, Ostrowski, Mazard, Kumar, Gamage, Brown, Messer, Seymour and Paulsen. The tropical marine environments of northern Australia encompasses a diverse range of geomorphological and oceanographic conditions and high levels of productivity and nitrogen fixation. However, efforts to characterize phytoplankton assemblages in these waters have been restricted to studies using microscopic and pigment analyses, leading to the current consensus that this region is dominated by large diatoms, dinoflagellates, and the marine cyanobacterium Trichodesmium. During an oceanographic transect from the Arafura Sea through the Torres Strait to the Coral Sea, we characterized prokaryotic and eukaryotic phytoplankton communities in surface waters using a combination of flow cytometry and Illumina based 16S and 18S ribosomal RNA amplicon sequencing. Similar to observations in other marine regions around Australian, phytoplankton assemblages throughout this entire region were rich in unicellular picocyanobacterial primary producers while picoeukaryotic phytoplankton formed a consistent, though smaller proportion of the photosynthetic biomass. Major taxonomic groups displayed distinct biogeographic patterns linked to oceanographic and nutrient conditions. Unicellular picocyanobacteria dominated in both flow cytometric abundance and carbon biomass, with members of the Synechococcus genus dominating in the shallower Arafura Sea and Torres Strait where chlorophyll a was relatively higher (averaging 0.4 ± 0.2 mg m-3), and Prochlorococcus dominating in the oligotrophic Coral Sea where chlorophyll a averaged 0.13 ± 0.07 mg m-3. Consistent with previous microscopic and pigment-based observations, we found from sequence analysis that a variety of diatoms (Bacillariophyceae) exhibited high relative abundance in the Arafura Sea and Torres Strait, while dinoflagellates (Dinophyceae) and prymnesiophytes (Prymnesiophyceae) were more abundant in the Coral Sea. Ordination analysis identified temperature, nutrient concentrations and water depth as key drivers of the region's assemblage composition. This is the first molecular and flow cytometric survey of the abundance and diversity of both prokaryotic and picoeukaryotic phytoplankton in this region, and points to the need to include the picocyanobacterial populations as an essential oceanic variable for sustained monitoring in order to better understand the health of these important coastal waters as global oceans change

Genomic and phenotypic analyses of diverse non-clinical Acinetobacter baumannii strains reveals strain-specific virulence and resistance capacity.

Acinetobacter baumannii is a critically important pathogen known for its widespread antibiotic resistance and ability to persist in hospital-associated environments. Whilst the majority of A. baumannii infections are hospital-acquired, infections from outside the hospital have been reported with high mortality. Despite this, little is known about the natural environmental reservoir(s) of A. baumannii and the virulence potential underlying non-clinical strains. Here, we report the complete genome sequences of six diverse strains isolated from environments such as river, soil, and industrial sites around the world. Phylogenetic analyses showed that four of these strains were unrelated to representative nosocomial strains and do not share a monophyletic origin, whereas two had sequence types belonging to the global clone lineages GC1 and GC2. Further, the majority of these strains harboured genes linked to virulence and stress protection in nosocomial strains. These genotypic properties correlated well with in vitro virulence phenotypic assays testing resistance to abiotic stresses, serum survival, and capsule formation. Virulence potential was confirmed in vivo, with most environmental strains able to effectively kill Galleria mellonella greater wax moth larvae. Using phenomic arrays and antibiotic resistance profiling, environmental and nosocomial strains were shown to have similar substrate utilisation patterns although environmental strains were distinctly more sensitive to antibiotics. Taken together, these features of environmental A. baumannii strains suggest the existence of a strain-specific distinct gene pools for niche specific adaptation. Furthermore, environmental strains appear to be equally virulent as contemporary nosocomial strains but remain largely antibiotic sensitive