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Roles of different ecological classes of algal exometabolites in regulating microbial community composition are not well understood. Here, we identify exometabolites from the model diatom Phaeodactylum tricornutum and demonstrate their potential to influence bacterial abundances. We profiled exometabolites across a time course of axenic algal growth using liquid chromatography-tandem mass spectrometry. We then investigated growth of 12 bacterial isolates on individual-identified exometabolites. Lastly, we compared responses of a P. tricornutum-adapted enrichment community to additions of two contrasting metabolites: selective growth substrate 4-hydroxybenzoic acid and putative signaling/facilitator molecule lumichrome. We identified 50 P. tricornutum metabolites and found distinct temporal accumulation patterns. Two exometabolites (of 12 tested) supported growth of distinct subsets of bacterial isolates. While algal exudates and algal presence drove similar changes in community composition compared with controls, exogenous 4-hydroxybenzoic acid addition promoted increased abundances of taxa that utilized it in isolation, and also revealed the importance of factors relating to algal presence in regulating community composition. This work demonstrates that secretion of selective bacterial growth substrates represents one mechanism by which algal exometabolites can influence bacterial community composition and illustrates how the algal exometabolome has the potential to modulate bacterial communities as a function of algal growth.

Dissolved exometabolites mediate algal interactions in aquatic ecosystems, but microalgal exometabolomes remain understudied. We conducted an untargeted metabolomic analysis of nonpolar exometabolites exuded from four phylogenetically and ecologically diverse eukaryotic microalgal strains grown in the laboratory, freshwater Chlamydomonas reinhardtii, brackish Desmodesmus sp., marine Phaeodactylum tricornutum, and marine Microchloropsis salina, to identify released metabolites based on relative enrichment in the exometabolomes compared to cell pellet metabolomes. Exudates from the different taxa were distinct, but we did not observe clear phylogenetic patterns. We used feature-based molecular networking to explore the identities of these metabolites, revealing several distinct di- and tripeptides secreted by each of the algae, lumichrome, a compound that is known to be involved in plant growth and bacterial quorum sensing, and novel prostaglandin-like compounds. We further investigated the impacts of exogenous additions of eight compounds selected based on exometabolome enrichment on algal growth. Of these compounds, five (lumichrome, 5'-S-methyl-5'-thioadenosine, 17-phenyl trinor prostaglandin A2, dodecanedioic acid, and aleuritic acid) impacted growth in at least one of the algal cultures. Two of these compounds (dodecanedioic acid and aleuritic acid) produced contrasting results, increasing growth in some algae and decreasing growth in others. Together, our results reveal new groups of microalgal exometabolites, some of which could alter algal growth when provided exogenously, suggesting potential roles in allelopathy and algal interactions. IMPORTANCE Microalgae are responsible for nearly half of primary production on earth and play an important role in global biogeochemical cycling as well as in a range of industrial applications. Algal exometabolites are important mediators of algal-algal and algal-bacterial interactions that ultimately affect algal growth and physiology. In this study, we characterize exometabolomes across marine and freshwater algae to gain insights into the diverse metabolites they release into their environments ("exudates"). We observe that while phylogeny can play a role in exometabolome content, environmental conditions or habitat origin (freshwater versus marine) are also important. We also find that several of these compounds can influence algal growth (as measured by chlorophyll production) when provided exogenously, highlighting the importance of characterization of these novel compounds and their role in microalgal ecophysiology.

Plant cell walls contain a renewable, nearly limitless supply of sugar that could be used to support microbial production of commodity chemicals and biofuels. Imidazolium ionic liquid (IIL) solvents are among the best reagents for gaining access to the sugars in this otherwise recalcitrant biomass. However, the sugars from IIL-treated biomass are inevitably contaminated with residual IILs that inhibit growth in bacteria and yeast, blocking biochemical production by these organisms. IIL toxicity is, therefore, a critical roadblock in many industrial biosynthetic pathways. Although several IIL-tolerant (IILT) bacterial and yeast isolates have been identified in nature, few genetic mechanisms have been identified. In this study, we identified two IILTBacillus isolates as well as a spontaneous IILTEscherichia coli lab strain that are tolerant to high levels of two widely used IILs. We demonstrate that all three IILT strains contain one or more pumps of the small multidrug resistance (SMR) family, and two of these strains contain mutations that affect an adjacent regulatory guanidine riboswitch. Furthermore, we show that the regulation of E. colisugE by the guanidine II riboswitch can be exploited to promote IIL tolerance by the simple addition of guanidine to the medium. Our results demonstrate the critical role that transporter genes play in IIL tolerance in their native bacterial hosts. The study presented here is another step in engineering IIL tolerance into industrial strains toward overcoming this key gap in biofuels and industrial biochemical production processes.IMPORTANCE This study identifies bacteria that are tolerant to ionic liquid solvents used in the production of biofuels and industrial biochemicals. For industrial microbiology, it is essential to find less-harmful reagents and microbes that are resistant to their cytotoxic effects. We identified a family of small multidrug resistance efflux transporters, which are responsible for the tolerance of these strains. We also found that this resistance can be caused by mutations in the sequences of guanidine-specific riboswitches that regulate these efflux pumps. Extending this knowledge, we demonstrated that guanidine itself can promote ionic liquid tolerance. Our findings will inform genetic engineering strategies that improve conversion of cellulosic sugars into biofuels and biochemicals in processes where low concentrations of ionic liquids surpass bacterial tolerance.

In the original version of this Article, an incorrect URL was provided in the Data Availability Statement regarding the deposition of plasmids listed in Supplementary Table 4. The correct URL is https://public-registry.jbei.org/folders/378 . This error has been corrected in both the PDF and HTML versions of the Article.

Tightly regulated promoters are essential for numerous biological applications, where strong inducibility, portability, and scalability are desirable. Current systems are often incompatible with large-scale fermentations due to high inducer costs and strict media requirements. Here, we describe the bottom-up engineering of 'Jungle Express', an expression system that enables efficient gene regulation in diverse proteobacteria. This system is guided by EilR, a multidrug-binding repressor with high affinity to its optimized operator and cationic dyes that act as powerful inducers at negligible costs. In E. coli, the engineered promoters exhibit minimal basal transcription and are inducible over four orders of magnitude by 1 µM crystal violet, reaching expression levels exceeding those of the strongest current bacterial systems. Further, we provide molecular insights into specific interactions of EilR with its operator and with two inducers. The versatility of Jungle Express opens the way for tightly controlled and efficient gene expression that is not restricted to host organism, substrate, or scale.

Imidazolium ionic liquids (IILs) have a range of biotechnological applications, including as pretreatment solvents that extract cellulose from plant biomass for microbial fermentation into sustainable bioenergy. However, residual levels of IILs, such as 1-ethyl-3-methylimidazolium chloride ([C2C1im]Cl), are toxic to biofuel-producing microbes, including the yeast Saccharomyces cerevisiae. S. cerevisiae strains isolated from diverse ecological niches differ in genomic sequence and in phenotypes potentially beneficial for industrial applications, including tolerance to inhibitory compounds present in hydrolyzed plant feedstocks. We evaluated >100 genome-sequenced S. cerevisiae strains for tolerance to [C2C1im]Cl and identified one strain with exceptional tolerance. By screening a library of genomic DNA fragments from the [C2C1im]Cl-tolerant strain for improved IIL tolerance, we identified SGE1, which encodes a plasma membrane multidrug efflux pump, and a previously uncharacterized gene that we named ionic liquid tolerance 1 (ILT1), which encodes a predicted membrane protein. Analyses of SGE1 sequences from our panel of S. cerevisiae strains together with growth phenotypes implicated two single nucleotide polymorphisms (SNPs) that associated with IIL tolerance and sensitivity. We confirmed these phenotypic effects by transferring the SGE1 SNPs into a [C2C1im]Cl-sensitive yeast strain using CRISPR/Cas9 genome editing. Further studies indicated that these SNPs affect Sge1 protein stability and cell surface localization, influencing the amount of toxic IILs that cells can pump out of the cytoplasm. Our results highlight the general potential for discovering useful biotechnological functions from untapped natural sequence variation and provide functional insight into emergent SGE1 alleles with reduced capacities to protect against IIL toxicity.

Chemical and physical pretreatment of biomass is a critical step in the conversion of lignocellulose to biofuels and bioproducts. Ionic liquid (IL) pretreatment has attracted significant attention due to the unique ability of certain ILs to solubilize some or all components of the plant cell wall. However, these ILs inhibit not only the enzyme activities but also the growth and productivity of microorganisms used in downstream hydrolysis and fermentation processes. While pretreated biomass can be washed to remove residual IL and reduce inhibition, extensive washing is costly and not feasible in large-scale processes. IL-tolerant microorganisms and microbial communities have been discovered from environmental samples and studies begun to elucidate mechanisms of IL tolerance. The discovery of IL tolerance in environmental microbial communities and individual microbes has lead to the proposal of molecular mechanisms of resistance. In this article, we review recent progress on discovering IL-tolerant microorganisms, identifying metabolic pathways and mechanisms of tolerance, and engineering microorganisms for IL tolerance. Research in these areas will yield new approaches to overcome inhibition in lignocellulosic biomass bioconversion processes and increase opportunities for the use of ILs in biomass pretreatment.

Although it is becoming clear that many microbial primary producers can also play a role as organic consumers, we know very little about the metabolic regulation of photoautotroph organic matter consumption. Cyanobacteria in phototrophic biofilms can reuse extracellular organic carbon, but the metabolic drivers of extracellular processes are surprisingly complex. We investigated the metabolic foundations of organic matter reuse by comparing exoproteome composition and incorporation of (13)C-labeled and (15)N-labeled cyanobacterial extracellular organic matter (EOM) in a unicyanobacterial biofilm incubated using different light regimes. In the light and the dark, cyanobacterial direct organic C assimilation accounted for 32% and 43%, respectively, of all organic C assimilation in the community. Under photosynthesis conditions, we measured increased excretion of extracellular polymeric substances (EPS) and proteins involved in micronutrient transport, suggesting that requirements for micronutrients may drive EOM assimilation during daylight hours. This interpretation was supported by photosynthesis inhibition experiments, in which cyanobacteria incorporated N-rich EOM-derived material. In contrast, under dark, C-starved conditions, cyanobacteria incorporated C-rich EOM-derived organic matter, decreased excretion of EPS, and showed an increased abundance of degradative exoproteins, demonstrating the use of the extracellular domain for C storage. Sequence-structure modeling of one of these exoproteins predicted a specific hydrolytic activity that was subsequently detected, confirming increased EOM degradation in the dark. Associated heterotrophic bacteria increased in abundance and upregulated transport proteins under dark relative to light conditions. Taken together, our results indicate that biofilm cyanobacteria are successful competitors for organic C and N and that cyanobacterial nutrient and energy requirements control the use of EOM. Cyanobacteria are globally distributed primary producers, and the fate of their fixed C influences microbial biogeochemical cycling. This fate is complicated by cyanobacterial degradation and assimilation of organic matter, but because cyanobacteria are assumed to be poor competitors for organic matter consumption, regulation of this process is not well tested. In mats and biofilms, this is especially relevant because cyanobacteria produce an extensive organic extracellular matrix, providing the community with a rich source of nutrients. Light is a well-known regulator of cyanobacterial metabolism, so we characterized the effects of light availability on the incorporation of organic matter. Using stable isotope tracing at the single-cell level, we quantified photoautotroph assimilation under different metabolic conditions and integrated the results with proteomics to elucidate metabolic status. We found that cyanobacteria effectively compete for organic matter in the light and the dark and that nutrient requirements and community interactions contribute to cycling of extracellular organic matter.

The aims of this study were to identify thermophilic microbial communities that degrade green waste in the presence of the ionic liquids (IL) tetrabutylphosphonium chloride and tributylethylphosphonium diethylphosphate and examine preservation methods for IL-tolerant communities. High-solids incubations with stepwise increases in IL concentration were conducted to enrich for thermophilic IL-tolerant communities that decomposed green waste. 16S rRNA sequencing of enriched communities revealed microorganisms capable of tolerating high levels of IL. Cryogenic preservation of enriched communities reduced the IL tolerance of the community and decreased the relative abundance of IL-tolerant organisms. The use of cryoprotectants did not have an effect on microbial activity on green waste of the stored community. A successful approach was developed to enrich communities that decompose green waste in thermophilic high-solids environments in the presence of IL. Alternative community storage and revival methods are necessary for maintenance and recovery of IL-tolerant communities. The enriched communities provide a targeted source of enzymes for the bioconversion of IL-pretreated green waste for conversion to biofuels.

Cyanobacterial organic matter excretion is crucial to carbon cycling in many microbial communities, but the nature and bioavailability of this C depend on unknown physiological functions. Cyanobacteria-dominated hypersaline laminated mats are a useful model ecosystem for the study of C flow in complex communities, as they use photosynthesis to sustain a more or less closed system. Although such mats have a large C reservoir in the extracellular polymeric substances (EPSs), the production and degradation of organic carbon is not well defined. To identify extracellular processes in cyanobacterial mats, we examined mats collected from Elkhorn Slough (ES) at Monterey Bay, California, for glycosyl and protein composition of the EPS. We found a prevalence of simple glucose polysaccharides containing either α or β (1,4) linkages, indicating distinct sources of glucose with differing enzymatic accessibility. Using proteomics, we identified cyanobacterial extracellular enzymes, and also detected activities that indicate a capacity for EPS degradation. In a less complex system, we characterized the EPS of a cyanobacterial isolate from ES, ESFC-1, and found the extracellular composition of biofilms produced by this unicyanobacterial culture were similar to that of natural mats. By tracing isotopically labeled EPS into single cells of ESFC-1, we demonstrated rapid incorporation of extracellular-derived carbon. Taken together, these results indicate cyanobacteria reuse excess organic carbon, constituting a dynamic pool of extracellular resources in these mats.

The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR', PydfO', and PydfA', were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA' and PmarR' based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA' and PmarR' in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR'-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR' can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinniaelegans. Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.

During microbial evolution, genome rearrangement increases with increasing sequence divergence. If the relationship between synteny and sequence divergence can be modeled, gene clusters in genomes of distantly related organisms exhibiting anomalous synteny can be identified and used to infer functional conservation. We applied the phylogenetic pairwise comparison method to establish and model a strong correlation between synteny and sequence divergence in all 634 available Archaeal and Bacterial genomes from the NCBI database and four newly assembled genomes of uncultivated Archaea from an acid mine drainage (AMD) community. In parallel, we established and modeled the trend between synteny and functional relatedness in the 118 genomes available in the STRING database. By combining these models, we developed a gene functional annotation method that weights evolutionary distance to estimate the probability of functional associations of syntenous proteins between genome pairs. The method was applied to the hypothetical proteins and poorly annotated genes in newly assembled acid mine drainage Archaeal genomes to add or improve gene annotations. This is the first method to assign possible functions to poorly annotated genes through quantification of the probability of gene functional relationships based on synteny at a significant evolutionary distance, and has the potential for broad application.

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth ∼20×). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types (∼94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination.

Objectives: Morphologic anomalies of the feline gallbladder (GB) have been previously reported in the literature. These morphologic variants are frequently encountered on routine abdominal ultrasound examination. The aim of this study was to provide an ultrasonographic classification system of these variants and document the overall incidence in the feline population. Methods: A prospective, descriptive study was undertaken; cats that had an abdominal ultrasound examination that included at least one sagittal and transverse plane image of the GB were included. GB shape was evaluated and categorized based on a classification scheme of morphologic variants modified from the human literature. Septated (S), bilobed (B1, B2, B3), duplex (D) and complex (C) categories were described. Results: Of 516 cats included in the study, 389 had normal GB morphology, while 127 had anomalous GB morphology. The overall incidence rate of anomalous GB morphology was 24.61%. When examined by morphologic type, the septated (S) morphology had an incidence of 9.69%. A bilobed (B) morphology was the most commonly observed classification; incidence was 14.35% within our population; incidence of B1, B2 and B3 subtypes were 2.91%, 6.98% and 4.46%, respectively. Duplex GBs only made up 0.39% of the total population. The incidence of complex (C) morphologies was 0.19%. Conclusions and relevance: The incidence of GB morphologic anomalies was higher in our population than previously reported. Identification of these anomalies on routine ultrasound evaluation is common; numerous different morphologies can be identified and a standardized classification scheme is proposed. Complete evaluation of morphology can be challenging, particularly with regard to cystic duct anatomy. Clinical significance is uncertain and future studies are warranted to determine the relationship between morphologic variants and hepatobiliary disease. [ABSTRACT FROM AUTHOR]

In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes (1). Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation (2). However, improperly repaired DSBs can cause meiotic arrest or mutation (3,4); thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse (5) and SPO11 and its accessory factors remain abundant long after most DSB formation ceases (1), implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency., SPO11 creates DSBs via a covalent protein-DNA intermediate that is endonucleolytically cleaved to release SPO11 attached to a short oligonucleotide, freeing DSB ends for further processing and recombination (5) (Fig. [...]

Results indicate that activation or the endonucleolytic activity of the N-terminal domain of XPF elicit the 5' and 3' incisions at the site of a psoralen interstrand cross-link, mediated by a structure-specific endonuclease formed by the binding of ERCC1 to XPF.

Nuclear Overhauser effect spectroscopy demonstrates that N-terminal residues 837-844 of the human DNA ligase IIIalpha BRCA1 carboxy-terminus domain seem to undergo interconversion between helix and random coil conformations. The solution structure of ligase, residues 837-922, reveals a beta2beta1beta3beta4 parallel sheet with two alpha-helix bundle attached to the sheet.

The biochemical role of the human DNA repair protein XPF has been studied by expressing and purifying the independent 104 kDa recombinant XPF protein from Escherichia coli. The XPF/ERCC1 protein complex is responsible for making one of the necessary incisions in eliminating a broad range of DNA adducts during nucleotide excision repair. Results indicate that XPF is an endonuclease and can bind DNA even when the ERCC1 subunit is not present. It has an N-terminal catalytic domain based on the endonuclease activity of a stable 70 kDa proteolysis fragment of XPF.

A study was conducted to examine the chemical composition of extracellular polymeric substances (EPS) extracted from two natural microbial pellicle biofilms growing on acid mine drainage (AMD) solutions. The results indicated that the biochemical composition of the EPS from the acidic biofilms is dependent on maturity and is regulated by the microbial communities as well as the local geochemical environment.

The analysis of near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmnd Mine, Iron Mountain, Ca, acid mine drainage biofilm is presented. Both the bacterial groups are found to have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers and multiple pathways for osmotic protection.

The characterization of a novel, soluble cytochrome with an unusual visible signature at 579 nm (Cyt(579)) is described. The results suggested that Cyt(579) plays an important role as an electron transfer protein to shuttle electrons derived from Fe(II) oxidation to support critical metabolical functions in the acidophilic microbial community.