Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved mariner-based transposon system with enhanced stability during propagation and versatile applications in mutagenesis. We used a low-copy-number plasmid as a transposon delivery vehicle, which affords a lower frequency of unintended recombination during vector construction and propagation in Escherichia coli. We generated a variety of transposons allowing for gene disruption or artificial overexpression, each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance the flexibility of future transposon modification and application in Bacillus and other organisms.

IMPORTANCE The stability of transposase-encoding vectors during cloning and propagation is crucial for the reliable application of transposons. Here, we increased the stability of the mariner delivery vehicle in E. coli. Moreover, the TnFLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased green fluorescent protein (GFP) fusions to report on protein translation and subcellular localization.

Dietary protein residue can result in microbial generation of various toxic metabolites in the gut, such as ammonia. A prebiotic is "a substrate that is selectively utilised by host microorganisms conferring a health benefit" (G. R. Gibson, R. Hutkins, M. E. Sanders, S. L. Prescott, et al., Nat Rev Gastroenterol Hepatol 14:491–502, 2017, https://doi.org/10.1038/nrgastro.2017.75). Prebiotics are carbohydrates that may have the potential to reverse the harmful effects of gut bacterial protein fermentation. Three-stage continuous colonic model systems were inoculated with fecal samples from omnivore and vegetarian volunteers. Casein (equivalent to 105 g protein consumption per day) was used within the systems as a protein source. Two different doses of inulin-type fructans (Synergy1) were later added (equivalent to 10 g per day in vivo and 15 g per day) to assess whether this influenced protein fermentation. Bacteria were enumerated by fluorescence in situ hybridization with flow cytometry. Metabolites from bacterial fermentation (short-chain fatty acid [SCFA], ammonia, phenol, indole, and p-cresol) were monitored to further analyze proteolysis and the prebiotic effect. A significantly higher number of bifidobacteria was observed with the addition of inulin together with reduction of Desulfovibrio spp. Furthermore, metabolites from protein fermentation, such as branched-chain fatty acids (BCFA) and ammonia, were significantly lowered with Synergy1. Production of p-cresol varied among donors, as we recognized four high producing models and two low producing models. Prebiotic addition reduced its production only in vegetarian high p-cresol producers.

IMPORTANCE Dietary protein levels are generally higher in Western populations than in the world average. We challenged three-stage continuous colonic model systems containing high protein levels and confirmed the production of potentially harmful metabolites from proteolysis, especially replicates of the transverse and distal colon. Fermentations of proteins with a prebiotic supplementation resulted in a change in the human gut microbiota and inhibited the production of some proteolytic metabolites. Moreover, we observed both bacterial and metabolic differences between fecal bacteria from omnivore donors and vegetarian donors. Proteins with prebiotic supplementation showed higher Bacteroides spp. and inhibited Clostridium cluster IX in omnivore models, while in vegetarian modes, Clostridium cluster IX was higher and Bacteroides spp. lower with high protein plus prebiotic supplementation. Synergy1 addition inhibited p-cresol production in vegetarian high p-cresol-producing models while the inhibitory effect was not seen in omnivore models.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C_16:0 and n-C_18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes. Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2. In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes.

IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

To better understand how associated microorganisms ("microbiota") influence organismal aging, we focused on the model organism Drosophila melanogaster. We conducted a metagenome-wide association (MGWA) as a screen to identify bacterial genes associated with variation in the D. melanogaster life span. The results of the MGWA predicted that bacterial cysteine and methionine metabolism genes influence fruit fly longevity. A mutant analysis, in which flies were inoculated with Escherichia coli strains bearing mutations in various methionine cycle genes, confirmed a role for some methionine cycle genes in extending or shortening fruit fly life span. Initially, we predicted these genes might influence longevity by mimicking or opposing methionine restriction, an established mechanism for life span extension in fruit flies. However, follow-up transcriptome sequencing (RNA-seq) and metabolomic experiments were generally inconsistent with this conclusion and instead implicated glucose and vitamin B₆ metabolism in these influences. We then tested if bacteria could influence life span through methionine restriction using a different set of bacterial strains. Flies reared with a bacterial strain that ectopically expressed bacterial transsulfuration genes and lowered the methionine content of the fly diet also extended female D. melanogaster life span. Taken together, the microbial influences shown here overlap with established host genetic mechanisms for aging and therefore suggest overlapping roles for host and microbial metabolism genes in organismal aging.

IMPORTANCE Associated microorganisms ("microbiota") are intimately connected to the behavior and physiology of their animal hosts, and defining the mechanisms of these interactions is an urgent imperative. This study focuses on how microorganisms influence the life span of a model host, the fruit fly Drosophila melanogaster. First, we performed a screen that suggested a strong influence of bacterial methionine metabolism on host life span. Follow-up analyses of gene expression and metabolite abundance identified stronger roles for vitamin B₆ and glucose than methionine metabolism among the tested mutants, possibly suggesting a more limited role for bacterial methionine metabolism genes in host life span effects. In a parallel set of experiments, we created a distinct bacterial strain that expressed life span-extending methionine metabolism genes and showed that this strain can extend fly life span. Therefore, this work identifies specific bacterial genes that influence host life span, including in ways that are consistent with the expectations of methionine restriction.

The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, lytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytB_E585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either lytB or producing the inactive LytB_E585A) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 μg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytB_E585A mutant formed biofilm more rapidly than lytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed.

IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

Deep-subsurface hot brines in northwest Poland, extracted through boreholes reaching 1.6 and 2.6 km below the ground surface, were microbiologically investigated using culture-independent and culture-dependent methods. The high-throughput sequencing of 16S rRNA gene amplicons showed a very low diversity of bacterial communities, which were dominated by phyla Proteobacteria and Firmicutes. Bacterial genera potentially involved in sulfur oxidation and nitrate reduction (Halothiobacillus and Methylobacterium) prevailed in both waters over the sulfate reducers ("Candidatus Desulforudis" and Desulfotomaculum). Only one archaeal taxon, affiliated with the order Thermoplasmatales, was detected in analyzed samples. Bacterial isolates obtained from these deep hot brines were closely related to Bacillus paralicheniformis based on the 16S rRNA sequence similarity. However, genomic and physiological analyses made for one of the isolates, Bacillus paralicheniformis strain TS6, revealed the existence of more diverse metabolic pathways than those of its moderate-temperature counterpart. These specific traits may be associated with the ecological adaptations to the extreme habitat, which suggest that some lineages of B. paralicheniformis are halothermophilic.

IMPORTANCE Deep-subsurface aquifers, buried thousands of meters down the Earth’s crust, belong to the most underexplored microbial habitats. Although a few studies revealed the existence of microbial life at the depths, the knowledge about the microbial life in the deep hydrosphere is still scarce due to the limited access to such environments. Studying the subsurface microbiome provides unique information on microbial diversity, community structure, and geomicrobiological processes occurring under extreme conditions of the deep subsurface. Our study shows that low-diversity microbial assemblages in subsurface hot brines were dominated by the bacteria involved in biogeochemical cycles of sulfur and nitrogen. Based on genomic and physiological analyses, we found that the Bacillus paralicheniformis isolate obtained from the brine under study differed from the mesophilic species in the presence of specific adaptations to harsh environmental conditions. We indicate that some lineages of B. paralicheniformis are halothermophilic, which was not previously reported.

Most freshwater bacterial communities are characterized by a few dominant taxa that are often ubiquitous across freshwater biomes worldwide. Our understanding of the genomic diversity within these taxonomic groups is limited to a subset of taxa. Here, we investigated the genomic diversity that enables Limnohabitans, a freshwater genus key in funneling carbon from primary producers to higher trophic levels, to achieve abundance and ubiquity. We reconstructed eight putative Limnohabitans metagenome-assembled genomes (MAGs) from stations located along broad environmental gradients existing in Lake Michigan, part of Earth’s largest surface freshwater system. De novo strain inference analysis resolved a total of 23 strains from these MAGs, which strongly partitioned into two habitat-specific clusters with cooccurring strains from different lineages. The largest number of strains belonged to the abundant LimB lineage, for which robust in situ strain delineation had not previously been achieved. Our data show that temperature and nutrient levels may be important environmental parameters associated with microdiversification within the Limnohabitans genus. In addition, strains predominant in low- and high-phosphorus conditions had larger genomic divergence than strains abundant under different temperatures. Comparative genomics and gene expression analysis yielded evidence for the ability of LimB populations to exhibit cellular motility and chemotaxis, a phenotype not yet associated with available Limnohabitans isolates. Our findings broaden historical marker gene-based surveys of Limnohabitans microdiversification and provide in situ evidence of genome diversity and its functional implications across freshwater gradients.

IMPORTANCE Limnohabitans is an important bacterial taxonomic group for cycling carbon in freshwater ecosystems worldwide. Here, we examined the genomic diversity of different Limnohabitans lineages. We focused on the LimB lineage of this genus, which is globally distributed and often abundant, and its abundance has shown to be largely invariant to environmental change. Our data show that the LimB lineage is actually comprised of multiple cooccurring populations for which the composition and genomic characteristics are associated with variations in temperature and nutrient levels. The gene expression profiles of this lineage suggest the importance of chemotaxis and motility, traits that had not yet been associated with the Limnohabitans genus, in adapting to environmental conditions.

Bacteria accumulate small, organic compounds called compatible solutes via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (encoded by ectABC-asp_ect) and glycine betaine (encoded by betIBA-proXWV), four betaine-carnitine-choline transporters (encoded by bccT1 to bccT4), and a second ProU transporter (encoded by proVWX). All of these systems are osmotically inducible with the exception of bccT2. Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABC-asp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBA-proXWV, bccT1, bccT3, bccT4, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed that expression of these systems is derepressed in the mutant at low salinity compared with the wild type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli green fluorescent protein (GFP) reporter assays, we demonstrated that CosR directly represses transcription of betIBA-proXWV, bccT3, and proVWX. Similar to Vibrio harveyi, we showed betIBA-proXWV was directly activated by the quorum-sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family, and bioinformatics analysis showed widespread distribution among Gammaproteobacteria in general. Incidentally, in Aliivibrio fischeri, Aliivibrio finisterrensis, Aliivibrio sifiae, and Aliivibrio wodanis, an unrelated MarR-type regulator gene named ectR was clustered with ectABC-asp, which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria.

IMPORTANCE Vibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gammaproteobacteria. Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.

Monoubiquitinated FANCI and FANCD2 constitute the ID complex, which forms a sliding clamp on DNA.

A chambered heart is common to all vertebrates, but reptiles show unparalleled variation in ventricular septation, ranging from almost absent in tuataras to full in crocodilians. Because mammals and birds evolved independently from reptile lineages, studies on reptile development may yield insight into the evolution and development of the full ventricular septum. Compared with reptiles, mammals and birds have evolved several other adaptations, including compact chamber walls and a specialized conduction system. These adaptations appear to have evolved from precursor structures that can be studied in present-day reptiles. The increase in the number of studies on reptile heart development has been greatly facilitated by sequencing of several genomes and the availability of good staging systems. Here, we place reptiles in their phylogenetic context with a focus on features that are primitive when compared with the homologous features of mammals. Further, an outline of major developmental events is given, and variation between reptile species is discussed.

Inflammasomes assemble in the cytosol of myeloid and epithelial cells on sensing of cellular stress and pathogen-associated molecular patterns and serve as scaffolds for recruitment and activation of inflammatory caspases. Inflammasomes play beneficial roles in host and immune responses against diverse pathogens but may also promote inflammatory tissue damage if uncontrolled. Gasdermin D (GSDMD) is a recently identified substrate of murine caspase-1 and caspase-11, and human caspases-1, -4, and -5 that mediates a regulated lytic cell death mode termed pyroptosis. Recent studies have identified pyroptosis as a critical inflammasome effector mechanism that controls inflammasome-dependent cytokine secretion and contributes to antimicrobial defense and inflammasome-mediated autoinflammatory diseases. Here, we review recent developments on inflammasome-associated effector functions with an emphasis on the emerging roles of gasdermin pores and pyroptosis.

Cell death is an invariant feature throughout our life span, starting with extensive scheduled cell death during morphogenesis and continuing with death under homeostasis in adult tissues. Additionally, cells become victims of accidental, unscheduled death following injury and infection. Cell death in each of these occasions triggers specific and specialized responses in the living cells that surround them or are attracted to the dying/dead cells. These responses sculpt tissues during morphogenesis, replenish lost cells in homeostasis to maintain tissue/system function, and repair damaged tissues after injury. Wherein lies the information that sets in motion the cascade of effector responses culminating in remodeling, renewal, or repair? Here, we attempt to provide a framework for thinking about cell death in terms of the specific effector responses that accompanies various modalities of cell death. We also propose an integrated threefold "cell death code" consisting of information intrinsic to the dying/dead cell, the surroundings of the dying cell, and the identity of the responder.

Adrian Tchaikovsky's latest novel The Doors of Eden rewrites Earth's evolutionary history, with highlights including fish that upload their minds to supercomputers and cats that rule over primates

A species of plant that grew about 400 million years ago (Early Devonian period) produced a spectrum of spore sizes, which is an essential innovation necessary for all advanced plant reproductive strategies, including seeds and flowers. The Devonian period is one of the most important time periods for the evolution of land plants. It witnessed [...]

Aurora kinase A (AURKA), AURKB and AURKC proteins regulate each other with surprising twists and turns in female mouse eggs, a finding that may play an

Synthetic biology is an emerging technology that shows promise for investigating some of the burning issues in biological research. It also has the potential to address some of the grand challenges facing society, such as climate change and energy security.

Cham, Switzerland : Springer, [2019]

Cham : Springer, c2019.

Cham : Springer, 2020.

[S.l.] : SPRINGER NATURE, 2020.

[Cham : Springer, 2019]

Singapore : Springer, 2019.

Cham : Springer, 2020.

Cham, Switzerland : Springer, [2019]

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Dewey Library - QH324.25.C66 2019

Hao, Da Cheng

Purich, Daniel L., author

Hayden Library - QH324.2.H358 2013

Hayden Library - QH324.2.L53 2014

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