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The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities in relevant pathogens. The Gram-negative pathogen,Acinetobacter baumannii, poses an urgent threat by evading antibiotic treatment through both intrinsic and acquired mechanisms. Antibiotics kill bacteria by targeting essential gene products, but antibiotic-essential gene interactions have not been studied systematically inA. baumannii. Here, we use CRISPR interference (CRISPRi) to comprehensively phenotypeA. baumanniiessential genes. We show that certain essential genes are acutely sensitive to knockdown, providing a set of promising therapeutic targets. Screening our CRISPRi library against last-resort antibiotics revealed essential pathways that modulate beta-lactam resistance, an unexpected link between NADH dehydrogenase function and polymyxin killing, and the genetic basis for synergy between polymyxins and rifamycins. Our results demonstrate the power of systematic genetic approaches to identify weaknesses in Gram-negative pathogens and uncover antibiotic mechanisms that better inform combination therapies. Significance Acinetobacter baumanniiis a hospital-acquired pathogen that is resistant to common antibiotic treatments.A. baumanniiinfections, we need to identify promising therapeutic targets and effective antibiotic combinations. Here, we characterize genes critical forA. baumanniiviability and their interactions with antibiotics. We find that genes involved in proton gradient formation required for oxygen-dependent energy generation are central toA. baumanniiphysiology and represent appealing drug targets. We show that polymyxins interact with proton gradient genes, explaining how these antibiotics inhibit growth at sub-lethal concentrations and their efficacy in combination therapies. Our studies reveal antibiotic-gene interactions inA. baumanniithat can inform future therapies.

In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti–σ-factors. In Escherichia coli , Salmonella enterica , and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σ S -regulon by promoting the association of σ S with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σ S -RNAP in an open promoter complex with a σ S -regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σ S (σ S 2 ), the structure, along with p -benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β′-subunit that we call the β′-clamp-toe (β′CT). Deletion of the β′CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β′CT interaction. We conclude that Crl activates σ S -dependent transcription in part through stabilizing σ S -RNAP by tethering σ S 2 and the β′CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.

Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICEBs1 transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors Basic Protocol 3: Tn7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4: ICEBs1 transfer of Mobile‐CRISPRi to Bacillales Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3: Transformation of E. coli by electroporation Support Protocol 4: Transformation of CaCl2‐competent E. coli

Biofuels produced by microbial fermentation of plant feedstocks provide renewable and sustainable energy sources that have the potential to mitigate climate change and improve energy security. Engineered strains of the bacterium Z. mobilis can convert sugars extracted from plant feedstocks into next-generation biofuels like isobutanol; however, conversion by these strains remains inefficient due to key gaps in our knowledge about genes involved in metabolism and stress responses such as alcohol tolerance. Here, we develop CRISPRi as a tool to explore gene function in Z. mobilis. We characterize genes that are essential for growth, required to ferment sugar to ethanol, and involved in resistance to isobutanol. Our Z. mobilis CRISPRi system makes it straightforward to define gene function and can be applied to improve strain engineering and increase biofuel yields., Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields. IMPORTANCE Biofuels produced by microbial fermentation of plant feedstocks provide renewable and sustainable energy sources that have the potential to mitigate climate change and improve energy security. Engineered strains of the bacterium Z. mobilis can convert sugars extracted from plant feedstocks into next-generation biofuels like isobutanol; however, conversion by these strains remains inefficient due to key gaps in our knowledge about genes involved in metabolism and stress responses such as alcohol tolerance. Here, we develop CRISPRi as a tool to explore gene function in Z. mobilis. We characterize genes that are essential for growth, required to ferment sugar to ethanol, and involved in resistance to isobutanol. Our Z. mobilis CRISPRi system makes it straightforward to define gene function and can be applied to improve strain engineering and increase biofuel yields.

Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essentials. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes, or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.IMPORTANCEBiofuels produced by microbial fermentation of plant feedstocks provide renewable and sustainable energy sources that have the potential to mitigate climate change and improve energy security. Engineered strains of the bacterium Z. mobilis can convert sugars extracted from plant feedstocks into next generation biofuels such as isobutanol; however, conversion by these strains remains inefficient due to key gaps in our knowledge about genes involved in metabolism and stress responses such as alcohol tolerance. Here, we develop CRISPRi as a tool to characterize gene function in Z. mobilis. We identify genes that are essential for growth, required to ferment sugar to ethanol, and involved in resistance to alcohol. Our Z. mobilis CRISPRi system makes it straightforward to define gene function and can be applied to improve strain engineering and increase biofuel yields.

Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti–σ-factors. In Escherichia coli, Salmonella enterica, and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σ(S)-regulon by promoting the association of σ(S) with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σ(S)-RNAP in an open promoter complex with a σ(S)-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σ(S) (σ(S)(2)), the structure, along with p-benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β′-subunit that we call the β′-clamp-toe (β′CT). Deletion of the β′CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β′CT interaction. We conclude that Crl activates σ(S)-dependent transcription in part through stabilizing σ(S)-RNAP by tethering σ(S)(2) and the β′CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.

In bacteria, a primary σ factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ factors are negatively regulated by anti-σ factors. In Escherichia coli, Salmonella enterica, and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σS regulon by promoting the association of σS with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σS-RNAP in an open promoter complex with a σS regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σS (σS), the structure, along with p-benzoylphenylalanine crosslinking, reveals that Crl interacts with a structural element of the RNAP β’ subunit we call the β’-clamp-toe (β’CT). Deletion of the β’CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β’CT interaction. We conclude that Crl activates σS-dependent transcription in part through stabilizing σS-RNAP by tethering σS and the β’CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.Significance StatementIn bacteria, multiple σ factors can bind to a common core RNA polymerase (RNAP) to alter global transcriptional programs in response to environmental stresses. Many γ-proteobacteria, including the pathogens Yersinia pestis, Vibrio cholera, Escherichia coli, and Salmonella typhimurium, encode Crl, a transcription factor that activates σS-dependent genes. Many of these genes are involved in processes important for infection, such as biofilm formation. We determined a high-resolution cryo-electron microscopy structure of a Crl-σS-RNAP transcription initiation complex. The structure, combined with biochemical experiments, shows that Crl stabilizes σS-RNAP by tethering σS directly to the RNAP.

Sterols are essential eukaryotic lipids that are required for a variety of physiological roles. The diagenetic products of sterol lipids, sterane hydrocarbons, are preserved in ancient sedimentary rocks and are utilized as geological biomarkers, indicating the presence of both eukaryotes and oxic environments throughout Earth’s history. However, a few bacterial species are also known to produce sterols, bringing into question the significance of bacterial sterol synthesis for our interpretation of sterane biomarkers. Recent studies suggest that bacterial sterol synthesis may be distinct from what is observed in eukaryotes. In particular, phylogenomic analyses of sterol-producing bacteria have failed to identify homologs of several key eukaryotic sterol synthesis enzymes, most notably those required for demethylation at the C-4 position. In this study, we identified two genes of previously unknown function in the aerobic methanotrophic γ-Proteobacterium Methylococcus capsulatus that encode sterol demethylase proteins (Sdm). We show that a Rieske-type oxygenase (SdmA) and an NAD(P)-dependent reductase (SdmB) are responsible for converting 4,4-dimethylsterols to 4α-methylsterols. Identification of intermediate products synthesized during heterologous expression of SdmA-SdmB along with 13C-labeling studies support a sterol C-4 demethylation mechanism distinct from that of eukaryotes. SdmA-SdmB homologs were identified in several other sterol-producing bacterial genomes but not in any eukaryotic genomes, indicating that these proteins are unrelated to the eukaryotic C-4 sterol demethylase enzymes. These findings reveal a separate pathway for sterol synthesis exclusive to bacteria and show that demethylation of sterols evolved at least twice—once in bacteria and once in eukaryotes.

Tetrahymanol is a polycyclic triterpenoid lipid first discovered in the ciliate Tetrahymena pyriformis whose potential diagenetic product, gammacerane, is often used as a biomarker for water column stratification in ancient ecosystems. Bacteria are also a potential source of tetrahymanol, but neither the distribution of this lipid in extant bacteria nor the significance of bacterial tetrahymanol synthesis for interpreting gammacerane biosignatures is known. Here we couple comparative genomics with genetic and lipid analyses to link a protein of unknown function to tetrahymanol synthesis in bacteria. This tetrahymanol synthase (Ths) is found in a variety of bacterial genomes, including aerobic methanotrophs, nitrite-oxidizers, and sulfate-reducers, and in a subset of aquatic and terrestrial metagenomes. Thus, the potential to produce tetrahymanol is more widespread in the bacterial domain than previously thought. However, Ths is not encoded in any eukaryotic genomes, nor is it homologous to eukaryotic squalene-tetrahymanol cyclase, which catalyzes the cyclization of squalene directly to tetrahymanol. Rather, heterologous expression studies suggest that bacteria couple the cyclization of squalene to a hopene molecule by squalene-hopene cyclase with a subsequent Ths-dependent ring expansion to form tetrahymanol. Thus, bacteria and eukaryotes have evolved distinct biochemical mechanisms for producing tetrahymanol.

Bacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica , Crl positively regulates the σ S regulon by binding to σ S to promote its association with core RNAP. We recently characterized the determinants in σ S responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-σ S interaction in E. coli . We show that the P. mirabilis , Vibrio harveyi , and E. coli Crl homologs function similarly in E. coli , indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the interaction with σ S . The results of p -benzoylphenylalanine (BPA)-mediated UV cross-linking studies further support the model in which an evolutionarily conserved central cleft is the surface on Crl that binds to σ S . Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of Eσ S -dependent transcription in vivo and in vitro . Our study provides a physical basis for understanding the σ S -Crl interaction.

One of the major signalling pathways responsible for intercompartmental communication between the cell envelope and cytoplasm in Escherichia coli is mediated by the alternative sigma factor, sigmaE. sigmaE has been studied primarily for its role in response to the misfolding of outer membrane porins. This response is essentially reactionary; cells are stressed, porin folding is disrupted, and the response is activated. sigmaE can also be activated following starvation for a variety of nutrients by the alarmone ppGpp. This response is proactive, as sigmaE is activated in the absence of any obvious damage to the cell envelope sensed by the stress signalling pathway. Here we examine the mechanism of regulation of sigmaE by ppGpp. ppGpp has been proposed to activate at least two alternative sigma factors, sigmaN and sigmaS, indirectly by altering the competition for core RNA polymerase between the alternative sigma factors and the housekeeping sigma factor, sigma70. In vivo experiments with sigmaE are consistent with this model. However, ppGpp and its cofactor DksA can also activate transcription by EsigmaEin vitro, suggesting that the effects of ppGpp on sigmaE activity are both direct and indirect.

In spite of the extreme environmental conditions, deep-sea hydrothermal vents are home to a multitude of microbial species. But one ingredient was missing: terrestrial hot acid springs are inhabited by acidophiles, but although theory predicts the presence of acidic microhabitats in sulphide deposits at deep-seavents, until now all microbes isolated from these deposits have been neutrophiles, or at best acid tolerant. Now, at last, an extreme thermoacidophilic microbe has been isolated from a hydrothermal vent. It's not a bacterium, but a member of the Archaea DHVE2 (deep-sea hydrothermal vent Euryarchaeota 2) lineage. It grows at pHs between 3.3 and 5.8 and at temperatures of 55–75°C. It constitutes up to 15% of the archaeal population so may be the main player in the iron and sulphurcycles in these environments. Isolation and cultivation of an extreme thermoacidophilic archaeon from hydrothermal vents suggests that this organism may be important in the iron and sulphur cycles in these environments. Deep-sea hydrothermal vents are important in global biogeochemical cycles, providing biological oases at the sea floor that are supported by the thermal and chemical flux from the Earth's interior. As hot, acidic and reduced hydrothermal fluids mix with cold, alkaline and oxygenated sea water, minerals precipitate to form porous sulphide–sulphate deposits. These structures provide microhabitats for a diversity of prokaryotes that exploit the geochemical and physical gradients in this dynamic ecosystem1. It has been proposed that fluid pH in the actively venting sulphide structures is generally low (pH < 4.5)2, yet no extreme thermoacidophile has been isolated from vent deposits. Culture-independent surveys based on ribosomal RNA genes from deep-sea hydrothermal deposits have identified a widespread euryarchaeotal lineage, DHVE2 (deep-sea hydrothermal vent euryarchaeotic 2)3,4,5,6. Despite the ubiquity and apparent deep-sea endemism of DHVE2, cultivation of this group has been unsuccessful and thus its metabolism remains a mystery. Here we report the isolation and cultivation of a member of the DHVE2 group, which is an obligate thermoacidophilic sulphur- or iron-reducing heterotroph capable of growing from pH 3.3 to 5.8 and between 55 and 75 °C. In addition, we demonstrate that this isolate constitutes up to 15% of the archaeal population, providing evidence that thermoacidophiles may be key players in the sulphur and iron cycling at deep-sea vents.

A novel thermophilic, sulfur-oxidizing Gram-negative bacterium, designated strain SS-5T, was isolated from the Calcite Hot Springs in Yellowstone National Park, USA. The cells were motile rods (1·2–2·8 μm long and 0·6–0·8 μm wide). The new isolate was a facultative heterotroph capable of using elemental sulfur or thiosulfate as an electron donor and O2 (1–18 %; optimum 6 %, v/v) as an electron acceptor. Hydrogen did not support growth. The isolate grew autotrophically with CO2. In addition, strain SS-5T utilized various organic carbon sources such as yeast extract, tryptone, sugars, amino acids and organic acids. Growth was observed between 55 and 78 °C (optimum 70 °C; 3·5 h doubling time), pH 6·0 and 8·0 (optimum pH 7·5), and 0 and 0·6 % (w/v) NaCl (optimum 0 %). The G+C content of the genomic DNA was 32 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate was a member of the genus Sulfurihydrogenibium. On the basis of the physiological and molecular characteristics of the new isolate, we propose the name Sulfurihydrogenibium yellowstonense sp. nov. with SS-5T (=JCM 12773T=OCM 840T) as the type strain. In addition, emended descriptions of the genus Sulfurihydrogenibium, Sulfurihydrogenibium subterraneum and Sulfurihydrogenibium azorense are proposed.

Microorganisms associated with basalt core were compared to those suspended in groundwater pumped from the same well in the eastern Snake River Plain Aquifer (Idaho, USA). Two wells located at different distances from the source of a mixed-waste plume in the fractured basalt aquifer were examined. In the well more distal from the plume source, an array of dialysis chambers filled with either deionized water or crushed basalt was equilibrated to compare the microorganisms collected in this fashion with those from core and groundwater samples collected in a traditional manner from the same well. The samples were characterized to determine the total amount of biomass, presence of specific populations or physiological groups, and potential community functions. Microorganisms and their activities were nearly undetectable in core and groundwater collected from the well farthest from the plume source and substantially enriched in both core and groundwater from the well closest to the plume source. In both wells,...

A culture-independent molecular phylogenetic survey was carried out for a bacterial and archaeal community of a mineralized crust coating a sulphide spire, which was collected from the Edmond vent field (23 ° S, 69 ° E, 3300 m depth) on the Central Indian Ridge. Small-subunit rRNA genes (16S rDNA) were amplified from environmental DNA by PCR utilizing Bacteria-specific, and Archaea-specific 16S rDNA primers. PCR products were cloned and 26 bacterial and nine archaeal unique sequence types (phylotypes) were identified from 150 clones analysed by restriction fragment length polymorphism, representing eight and four distinct lineages, respectively. The majority (>90%) of the bacterial phylotypes group with the e -Proteobacteria and confirms the global prevalence of e -Proteobacteria in deep-sea hydrothermal environments. Among the e -Proteobacteria, >40% of the phylotypes were closely related to the recently isolated deep-sea vent thermophilic chemolithoautotrophic sulphur-reducer, Nautilia lithotrophica . A single bacterial sequence was nearly identical (99% similarity) to the thermophilic hydrogen-oxidizing Hydrogenobacter thermolithotrophum , and is the first report of Hydrogenobacter at deep-sea hydrothermal vents. A majority (97%) of the archaeal phylotypes grouped with the ‘Deep-sea Hydrothermal Vent Euryarchaeotal Group’, a phylogenetic lineage of uncultured Archaea that have only been reported from other deep-sea hydrothermal vents on the Mid-Atlantic Ridge, East Pacific Rise, Juan de Fuca Ridge, Isu‐Ogasawara Arc, Okinawa Trough and the Manus Basin. A single sequence was closely related to the hyperthermophilic sulphur-reducing Thermococcales frequently found in diverse deep-sea vent environments. Scanning electron micrographs of the mineralized crust reveal abundant filamentous, rod and coccoidal forms encased in sulphur and sulphide mineral precipitate, suggesting that the thermophilic chemolithoautorophs and sulphide-producing heterotrophs may influence the architecture and sulphur cycling of the sulphide spire.

Two thermophilic, strictly chemolithoautotrophic, microaerophilic, hydrogen-oxidizing members of the Bacteria designated strain EX-H1T and strain EX-H2T were isolated from two separate deep-sea hydrothermal vent sites at 9 degrees N 104 degrees W in the Pacific Ocean and Guaymas Basin. The motile 2-4-microm-long rods were Gram-negative and non-sporulating. The temperature range for growth was between 55 and 80 degrees C for EX- H1T (optimum at 73 degrees C) and 55-75 C for EX-H2T (optimum at 70 C). Both strains grew fastest at 2.5% (w/v) NaCl and at pH 6, although growth was observed from pH 4.7 to pH 7.5. EX-H1T and EX-H2T were able to use elemental sulfur, thiosulfate or hydrogen as an electron donor, and oxygen (2-3%, v/v) or nitrate as an electron acceptor. EX-H1T was also able to use elemental sulfur as an electron acceptor. EX-H1T and EX-H2T further differed in their genomic G+C content (38.5 and 37.4 mol%, respectively) and 16S rRNA sequences (4% difference). Maximum-likelihood analysis of the 16S rRNA phylogeny placed both isolates within the Aquificales as a distinct lineage and showed them to be only about 85% similar to Aquifex pyrophilus. On the basis of phenotypic and phylogenetic characteristics, it is proposed that EX-H1T and EX-H2T belong to a new genus within the Aquificales, namely Persephonella gen. nov. It is further proposed that EX-H1T be named Persephonella marina sp. nov., the type species of the genus, and that EX-H2T be named Persephonella guaymasensis sp. nov., a second species in this genus.

7 pages, 4 figures, supplementary data https://doi.org/10.1016/j.syapm.2014.04.002, The microbial diversity associated with actively venting deep-sea hydrothermal deposits is tightly connected to the geochemistry of the hydrothermal fluids. Although the dominant members of these deposits drive the structure of the microbial communities, it is less well understood whether the lower abundance groups are as closely connected to the geochemical milieu, or driven perhaps by biotic factors such as microbial community interactions. We used the natural geochemical gradients that exist in the back-arc basin, Eastern Lau Spreading Center and Valu-Fa Ridge (ELSC/VFR) in the Southwestern Pacific, to explore whether the chemolithotrophic Aquificales are influenced by geographical location, host-rock of the vent field or deposit type. Using a combination of cloning, DNA fingerprinting (DGGE) and enrichment culturing approaches, all genera of this order previously described at marine vents were detected, i.e., Desulfurobacterium, Thermovibrio, Aquifex, Hydrogenivirga, Persephonella and Hydrogenothermus. The comparison between clone libraries and DGGE showed similar patterns of distribution of different Aquificales whereas results differed for the enrichment cultures that were retrieved. However, the use of cultivation-based and -independent methods did provide complementary phylogenetic diversity overview of the Aquificales in these systems. Together, this survey revealed that the ELSC/VFR contains some of the largest diversity of Aquificales ever reported at a deep-sea vent area, that the diversity patterns are tied to the geography and geochemistry of the system, and that this geochemical diverse back-arc basin may harbor new members of the Aquificales, This research was supported by the United States National Science Foundation (OCE-0937404 and OCE-1235432 to A.-L.R.). I.F. was supported by a fellowship from the Spanish Ministry of Science and Education (MEC-Fulbright program)

Within the endemic invertebrate faunas of hydrothermal vents, five biogeographic provinces are recognized. Invertebrates at two Indian Ocean vent fields (Kairei and Edmond) belong to a sixth province, despite ecological settings and invertebrate-bacterial symbioses similar to those of both western Pacific and Atlantic vents. Most organisms found at these Indian Ocean vent fields have evolutionary affinities with western Pacific vent faunas, but a shrimp that ecologically dominates Indian Ocean vents closely resembles its Mid-Atlantic counterpart. These findings contribute to a global assessment of the biogeography of chemosynthetic faunas and indicate that the Indian Ocean vent community follows asymmetric assembly rules biased toward Pacific evolutionary alliances.

A cDNA clone encoding the human homolog of rat Jagged1 was isolated from normal human marrow. Analyses of human stromal cell lines indicate that this gene, designated hJagged1 , is expressed by marrow stromal cells typified by the cell line HS-27a, which supports the long-term maintenance of hematopoietic progenitor cells. G-CSF–induced differentiation of 32D cells expressing Notch1 was inhibited by coculturing with HS-27a. A peptide corresponding to the Delta/Serrate/LAG-2 domain of hJagged1 and supernatants from COS cells expressing a soluble form of the extracellular portion of hJagged1 were able to mimic this effect. These observations suggest that hJagged1 may function as a ligand for Notch1 and play a role in mediating cell fate decisions during hematopoiesis.

Alagille syndrome is an autosomal dominant disorder characterized by abnormal development of liver, heart, skeleton, eye, face and, less frequently, kidney. Analyses of many patients with cytogenetic deletions or rearrangements have mapped the gene to chromosome 20p12, although deletions are found in a relatively small proportion of patients (< 7%). We have mapped the human Jagged1 gene (JAG1), encoding a ligand for the developmentally important Notch transmembrane receptor, to the Alagille syndrome critical region within 20p12. The Notch intercellular signalling pathway has been shown to mediate cell fate decisions during development in invertebrates and vertebrates. We demonstrate four distinct coding mutations in JAG1 from four Alagille syndrome families, providing evidence that it is the causal gene for Alagille syndrome. All four mutations lie within conserved regions of the gene and cause translational frameshifts, resulting in gross alterations of the protein product Patients with cytogenetically detectable deletions including JAG1 have Alagille syndrome, supporting the hypothesis that haploinsufficiency for this gene is one of the mechanisms causing the Alagille syndrome phenotype.

Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other γ-proteobacteria, the transcription factor Crl stimulates σ(S)-dependent transcription during times of cellular stress by promoting the association of σ(S) with core RNA polymerase. The molecular basis for specific recognition of σ(S) by Crl, rather than the homologous and more abundant primary sigma factor σ(70), is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoyl-phenylalanine cross-linking in vitro to define the features in σ(S) responsible for specific recognition by Crl. We identify residues in σ(S) conserved domain 2 (σ(S)2) that are necessary and sufficient to allow recognition of σ(70) conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of σ(70). We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on σ(S). Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.

This is an author manuscript that has been accepted for publication in International Journal of Systematic and Evolutionary Microbiology, copyright Society for General Microbiology, but has not been copy-edited, formatted or proofed. Cite this article as appearing in International Journal of Systematic and Evolutionary Microbiology. This version of the manuscript may not be duplicated or reproduced, other than for personal use or within the rule of 'Fair Use of Copyrighted Materials' (section 17, Title 17, US Code), without permission from the copyright owner, Society for General Microbiology. The Society for General Microbiology disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by any other parties. The final copy-edited, published article, which is the version of record, can be found at http://mic.sgmjournals.org and is freely available without a subscription.; International audience; Three thermophilic, anaerobic, strictly chemolithoautotrophic, sulphur- and/or thiosulphate-reducing bacteria, designated SL17(T), SL19(T) and SL22(T), were isolated from deep-sea hydrothermal samples collected at 13 degrees N (East Pacific Rise), Guaymas Basin (Gulf of California) and 23 degrees N (Mid-Atlantic Ridge), respectively. These strains differed in their morphology, temperature range and optimum for growth, energy substrates and 16S rRNA gene sequences. The G+C content of the genomic DNA was 41 mol% (SL22(T)), 42 mol% (SL17(T)) and 46 mol% (SL19(T)). Comparative analysis of phenotypic and phylogenetic traits indicated that strains SL17(T) and SL22(T) represented two novel species of the genus Desulfurobacterium and that strain SL19(T) should be considered as a novel species of the genus Thermovibrio. The names Desulfurobacterium pacificum sp. nov. (type strain SL17(T)=DSM 15522(T)=JCM 12127(T)), Desulfurobacterium atlanticum sp. nov. (type strain SL22(T)=DSM 15668(T)=JCM 12129(T)) and Thermovibrio guaymasensis sp. nov. (type strain SL19(T)=DSM 15521(T)=JCM 12128(T)) are proposed for these organisms. Furthermore, phylogenetic data based on 16S rRNA gene sequence analyses correlated with the significant phenotypic differences between members of the lineage encompassing the genera Desulfurobacterium, Thermovibrio and Balnearium and that of the families Aquificaceae and Hydrogenothermaceae. It is therefore proposed that this lineage represents a new family, Desulfurobacteriaceae fam. nov., within the order Aquificales.

A continuous culture bioreactor was developed to enrich for nitrate and sulfate reducing thermophiles under in situ deep-sea pressures. The ultimate objective of this experimental design was to be able to study microbial activities at chemical and physical conditions relevant to seafloor hydrothermal vents. Sulfide, sulfate and oxide minerals from sampled seafloor vent-chimney structures [East Pacific Rise (9 degrees 46'N)] served as source mineral and microbial inoculum for enrichment culturing using nitrate and sulfate-enriched media at 70 and 90 degrees C and 250 bars. Changes in microbial diversity during the continuous reaction flow were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rRNA gene fragments. Time series changes in fluid chemistry were also monitored throughout the experiment to assess the feedback between mineral-fluid reaction and metabolic processes. Data indicate a shift from the dominance of epsilon Proteobacteria in the initial inoculum to the several Aquificales-like phylotypes in nitrate-reducing enrichment media and Thermodesulfobacteriales in the sulfate-reducing enrichment media. Methanogens were detected in the original sulfide sample and grew in selected sulfate-enriched experiments. Microbial interactions with anhydrite and pyrrhotite in the chimney material resulted in measurable changes in fluid chemistry despite a fluid residence time only 75 min in the reactor. Changes in temperature rather than source material resulted in greater differences in microbial enrichments and mediated geochemical reactions.

Three thermophilic strains of chemolithoautotrophic Fe(III)-reducers were isolated from mixed sediment and water samples (JW/KA-1 and JW/KA-2T: Calcite Spring, Yellowstone N.P., WY, USA; JW/JH-Fiji-2: Savusavu, Vanu Levu, Fiji). All were Gram stain positive rods (∼0.5 × 1.8 μm). Cells occurred singly or in V-shaped pairs, and they formed long chains in complex media. All utilized H2 to reduce amorphous iron (III) oxide/hydroxide to magnetite at temperatures from 50 to 75°C (opt. ∼73°C). Growth occurred within the pH60C range of 6.5-8.5 (opt. pH60C 7.1-7.3). Magnetite production by resting cells occurred at pH60C 5.5-10.3 (opt. 7.3). The iron (III) reduction rate was 1.3 μmol Fe(II) produced × h-1 × ml-1 in a culture with 3 × 107 cells, one of the highest rates reported. In the presence or absence of H2, JW/KA-2T did not utilize CO. The G + C content of the genomic DNA of the type strain is 52.7 ± 0.3 mol%. Strains JW/KA-1 and JW/KA-2T each contain two different 16S rRNA gene sequences. The 16S rRNA gene sequences from JW/KA-1, JW/KA-2T, or JW/JH-Fiji-2 possessed >99% similarity to each other but also 99% similarity to the 16S rRNA gene sequence from the anaerobic, thermophilic, hydrogenogenic CO-oxidizing bacterium 'Carboxydothermus restrictus' R1. DNA-DNA hybridization between strain JW/KA-2T and strain R1T yielded 35% similarity. Physiological characteristics and the 16S rRNA gene sequence analysis indicated that the strains represent two novel species and are placed into the novel genus Thermolithobacter within the phylum 'Firmicutes'. In addition, the levels of 16S rRNA gene sequence similarity between the lineage containing the Thermolithobacter and well-established members of the three existing classes of the 'Firmicutes' is less than 85%. Therefore, Thermolithobacter is proposed to constitute the first genus within a novel class of the 'Firmicutes', Thermolithobacteria. The Fe(III)-reducing Thermolithobacter ferrireducens gen. nov., sp. nov. is designated as the type species with strain JW/KA-2T (ATCC 700985T, DSM 13639 T) as its type strain. Strain R1T is the type strain for the hydrogenogenic, CO-oxidizing Thermolithobacter carboxydivorans sp. nov. (DSM 7242T, VKM 2359T).

The Aquificales are prevalent members of the microbial communities inhabiting many marine and terrestrial hydrothermal systems. Numerous new strains were obtained from deep-sea and terrestrial hydrothermal systems. In order to resolve the phylogenetic relationships within this group, three different phylogenetic datasets were used, namely the 16S rRNA gene, the intergenic transcribed spacer region between the 16S rRNA and 23S rRNA genes (ITS) and the gene coding for the ATP citrate lyase (aclB), a key enzyme in the reductive TCA cycle. The data were analyzed using neighbor-joining, parsimony and maximum likelihood. The resulting phylogenies appeared to be consistent between the three markers. The three genes confirmed the presence of isolates that merit further characterization and descriptions as new species and perhaps even new genera. The detailed phylogenetic interrelationships of these isolates are described here.

The genomic sequence of the human Jagged2 (JAG2) gene, which encodes a ligand for the Notch receptors, was determined. The 30-kb DNA sequence spanning the JAG2 gene contains 26 exons and a putative promoter region. Several potential binding sites for transcription factors, including NF-kappab, E47, E12, E2F, Ets-1, MyoD, and OCT-1, were found in the human JAG2 promoter region. The JAG2 gene was also mapped to the chromosomal region 14q32 using fluorescence in situ hybridization.

The first complete mammalian genomic sequence reported thus far in the Notch gene family, including a putative promoter region and 30 exons of the human NOTCH4 gene spanning 56.8 kb of DNA, were sequenced. The NOTCH4 locus contains a TATA-less promoter with two putative transcription initiation sites (Inr), three RBP-Jkappa sites, and two GATA recognition sites. Two cDNA isoforms, NOTCH4(L) and NOTCH4(S),were identified. Whereas the NOTCH4(S) isoform contains the entire coding sequence, the NOTCH4(L) isoform has two unspliced intronic sequences between exons 11 and 12 and exons 20 and 21 and a misspliced exon 6. Consistent with these results, two alternatively spliced isoforms of transcripts of approximately 9.3 and 6.7 kb were detected by Northern blot analysis. The predicted amino acid sequence of the NOTCH4 protein based on the NOTCH4(S) cDNA sequence contains 2003 amino acids and includes the predominant motifs of the Notch family: 29 epidermal growth factor (EGF)-like repeats, 3 Notch/lin-12 repeats, a transmembrane region, 6 cdc10/Ankyrin repeats, and a PEST domain.

The sequences and structures of RNase P RNAs of some Gram-positive bacteria, e.g. Bacillus subtilis, are very different than those of other bacteria. In order to expand our understanding of the structure and evolution of RNase P RNA in Gram-positive bacteria, gene sequences encoding RNase P RNAs from 10 additional species from this evolutionary group have been determined, doubling the number of sequences available for comparative analysis. The enlarged data set allows refinement of the secondary structure model of these unusual RNase P RNAs and the identification of potential tertiary interactions between P10.1 and L12, and between L5.1 and L15.1. The newly-obtained sequences suggest that RNase P RNA underwent an abrupt, dramatic restructuring in the ancestry of the low-G+C Gram-positive bacteria after the divergence of the branches leading to the ‘Clostridia and relatives’ and the remaining low-G+C Gram-positive species. The unusual structures of the RNase P RNAs of Mycoplasma hyopneumoniae and M.floccularre are apparently derived from RNAs with Bacillus-like structure rather than from intermediate, partially restructured ancestral RNAs. The structure of the RNase P RNA from the photosynthetic Heliobacillus mobilis supports the relationship of this specie with Bacillus and Staphylococcus rather than the ‘Clostridia and relatives’ as suggested by the sequences of their small-subunit ribosomal RNAs.