Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates.

In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs most commonly through the addition of fumarate to a subterminal carbon, producing an alkylsuccinate. Alkylsuccinate synthases are complex, multi-subunit enzymes that utilize a catalytic glycyl radical and require a partner, activating enzyme for hydrogen abstraction. While many genes encoding putative alkylsuccinate synthases have been identified, primarily from nitrate- and sulfate-reducing bacteria, few have been characterized and none have been reported to be functionally expressed in a heterologous host. Here, we describe the functional expression of the (1-methylalkyl)succinate synthase (Mas) system from Azoarcus sp. strain HxN1 in recombinant Escherichia coli. Mass spectrometry confirms anaerobic biosynthesis of the expected products of fumarate addition to hexane, butane, and propane. Maximum production of (1-methylpentyl)succinate is observed when masC, masD, masE, masB, and masG are all present on the expression plasmid; omitting masC reduces production by 66% while omitting any other gene eliminates production. Meanwhile, deleting iscR (encoding the repressor of the E. coli iron-sulfur cluster operon) improves product titer, as does performing the biotransformation at reduced temperature (18°C), both suggesting alkylsuccinate biosynthesis is largely limited by functional expression of this enzyme system.

Current Development and Application of Anaerobic Glycolytic Enzymes in Urothelial Cancer.

Urothelial cancer is a malignant tumor with metastatic ability and high mortality. Malignant tumors of the urinary system include upper tract urothelial cancer and bladder cancer. In addition to typical genetic alterations and epigenetic modifications, metabolism-related events also occur in urothelial cancer. This metabolic reprogramming includes aberrant expression levels of genes, metabolites, and associated networks and pathways. In this review, we summarize the dysfunctions of glycolytic enzymes in urothelial cancer and discuss the relevant phenotype and signal transduction. Moreover, we describe potential prognostic factors and risks to the survival of clinical cancer patients. More importantly, based on several available databases, we explore relationships between glycolytic enzymes and genetic changes or drug responses in urothelial cancer cells. Current advances in glycolysis-based inhibitors and their combinations are also discussed. Combining all of the evidence, we indicate their potential value for further research in basic science and clinical applications.

Comparative genomic analysis reveals metabolic flexibility of Woesearchaeota.

The archaeal phylum Woesearchaeota, within the DPANN superphylum, includes phylogenetically diverse microorganisms that inhabit various environments. Their biology is poorly understood due to the lack of cultured isolates. Here, we analyze datasets of Woesearchaeota 16S rRNA gene sequences and metagenome-assembled genomes to infer global distribution patterns, ecological preferences and metabolic capabilities. Phylogenomic analyses indicate that the phylum can be classified into ten subgroups, termed A-J. While a symbiotic lifestyle is predicted for most, some members of subgroup J might be host-independent. The genomes of several Woesearchaeota, including subgroup J, encode putative [FeFe] hydrogenases (known to be important for fermentation in other organisms), suggesting that these archaea might be anaerobic fermentative heterotrophs.

Host adaptation in gut Firmicutes is associated with sporulation loss and altered transmission cycle.

BACKGROUND: Human-to-human transmission of symbiotic, anaerobic bacteria is a fundamental evolutionary adaptation essential for membership of the human gut microbiota. However, despite its importance, the genomic and biological adaptations underpinning symbiont transmission remain poorly understood. The Firmicutes are a dominant phylum within the intestinal microbiota that are capable of producing resistant endospores that maintain viability within the environment and germinate within the intestine to facilitate transmission. However, the impact of host transmission on the evolutionary and adaptive processes within the intestinal microbiota remains unknown. RESULTS: We analyze 1358 genomes of Firmicutes bacteria derived from host and environment-associated habitats. Characterization of genomes as spore-forming based on the presence of sporulation-predictive genes reveals multiple losses of sporulation in many distinct lineages. Loss of sporulation in gut Firmicutes is associated with features of host-adaptation such as genome reduction and specialized metabolic capabilities. Consistent with these data, analysis of 9966 gut metagenomes from adults around the world demonstrates that bacteria now incapable of sporulation are more abundant within individuals but less prevalent in the human population compared to spore-forming bacteria. CONCLUSIONS: Our results suggest host adaptation in gut Firmicutes is an evolutionary trade-off between transmission range and colonization abundance. We reveal host transmission as an underappreciated process that shapes the evolution, assembly, and functions of gut Firmicutes.

Cellulosome Localization Patterns Vary across Life Stages of Anaerobic Fungi.

Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores are powerful biomass-degrading organisms that enhance their degradative ability through the formation of cellulosomes, multienzyme complexes that synergistically colocalize enzymes to extract sugars from recalcitrant plant matter. However, a functional understanding of how fungal cellulosomes are deployed in vivo to orchestrate plant matter degradation is lacking, as is knowledge of how cellulosome production and function vary throughout the morphologically diverse life cycle of anaerobic fungi. In this work, we generated antibodies against three major fungal cellulosome protein domains, a dockerin, scaffoldin, and glycoside hydrolase (GH) 48 protein, and used them in conjunction with helium ion and immunofluorescence microscopy to characterize cellulosome localization patterns throughout the life cycle of Piromyces finnis when grown on simple sugars and complex cellulosic carbon sources. Our analyses reveal that fungal cellulosomes are cell-localized entities specifically targeted to the rhizoids of mature fungal cells and bodies of zoospores. Examination of cellulosome localization patterns across life stages also revealed that cellulosome production is independent of growth substrate in zoospores but repressed by simple sugars in mature cells. This suggests that further exploration of gene regulation patterns in zoospores is needed and can inform potential strategies for derepressing cellulosome expression and boosting hydrolytic enzyme yields from fungal cultures. Collectively, these findings underscore how life cycle-dependent cell morphology and regulation of cellulosome production impact biomass degradation by anaerobic fungi, insights that will benefit ongoing efforts to develop these organisms and their cellulosomes into platforms for converting waste biomass into valuable bioproducts. IMPORTANCE Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses. We developed and used imaging tools to observe cellulosome spatial localization patterns across life stages of the anaerobic fungus Piromyces finnis under different environmental conditions. The resulting spatial details of how anaerobic fungi orchestrate biomass degradation and uncovered relationships between life cycle progression and regulation of cellulosome production will benefit ongoing efforts to develop anaerobic fungi and their cellulosomes into useful biomass-upgrading platforms.

Profiling temporal dynamics of acetogenic communities in anaerobic digesters using next-generation sequencing and T-RFLP.

Acetogens play a key role in anaerobic degradation of organic material and in maintaining biogas process efficiency. Profiling this community and its temporal changes can help evaluate process stability and function, especially under disturbance/stress conditions, and avoid complete process failure. The formyltetrahydrofolate synthetase (FTHFS) gene can be used as a marker for acetogenic community profiling in diverse environments. In this study, we developed a new high-throughput FTHFS gene sequencing method for acetogenic community profiling and compared it with conventional terminal restriction fragment length polymorphism of the FTHFS gene, 16S rRNA gene-based profiling of the whole bacterial community, and indirect analysis via 16S rRNA profiling of the FTHFS gene-harbouring community. Analyses and method comparisons were made using samples from two laboratory-scale biogas processes, one operated under stable control and one exposed to controlled overloading disturbance. Comparative analysis revealed satisfactory detection of the bacterial community and its changes for all methods, but with some differences in resolution and taxonomic identification. FTHFS gene sequencing was found to be the most suitable and reliable method to study acetogenic communities. These results pave the way for community profiling in various biogas processes and in other environments where the dynamics of acetogenic bacteria have not been well studied.

Metagenomic insights into mixotrophic denitrification facilitated nitrogen removal in a full-scale A2/O wastewater treatment plant.

Wastewater treatment plants (WWTPs) are important for pollutant removal from wastewater, elimination of point discharges of nutrients into the environment and water resource protection. The anaerobic/anoxic/oxic (A2/O) process is widely used in WWTPs for nitrogen removal, but the requirement for additional organics to ensure a suitable nitrogen removal efficiency makes this process costly and energy consuming. In this study, we report mixotrophic denitrification at a low COD (chemical oxygen demand)/TN (total nitrogen) ratio in a full-scale A2/O WWTP with relatively high sulfate in the inlet. Nitrogen and sulfur species analysis in different units of this A2/O WWTP showed that the internal sulfur cycle of sulfate reduction and reoxidation occurred and that the reduced sulfur species might contribute to denitrification. Microbial community analysis revealed that Thiobacillus, an autotrophic sulfur-oxidizing denitrifier, dominated the activated sludge bacterial community. Metagenomics data also supported the potential of sulfur-based denitrification when high levels of denitrification occurred, and sulfur oxidation and sulfate reduction genes coexisted in the activated sludge. Although most of the denitrification genes were affiliated with heterotrophic denitrifiers with high abundance, the narG and napA genes were mainly associated with autotrophic sulfur-oxidizing denitrifiers. The functional genes related to nitrogen removal were actively expressed even in the unit containing relatively highly reduced sulfur species, indicating that the mixotrophic denitrification process in A2/O could overcome not only a shortage of carbon sources but also the inhibition by reduced sulfur of nitrification and denitrification. Our results indicate that a mixotrophic denitrification process could be developed in full-scale WWTPs and reduce the requirement for additional carbon sources, which could endow WWTPs with more flexible and adaptable nitrogen removal.

Pseudomonas aeruginosa T6SS-mediated molybdate transport contributes to bacterial competition during anaerobiosis.

Type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria and functions as a versatile protein export machinery that translocates effectors into eukaryotic or prokaryotic target cells. Growing evidence indicates that T6SS can deliver several effectors to promote bacterial survival in harmful environments through metal ion acquisition. Here, we report that the Pseudomonas aeruginosa H2-T6SS mediates molybdate (MoO42-) acquisition by secretion of a molybdate-binding protein, ModA. The expression of H2-T6SS genes is activated by the master regulator Anr and anaerobiosis. We also identified a ModA-binding protein, IcmP, an insulin-cleaving metalloproteinase outer membrane protein. The T6SS-ModA-IcmP system provides P. aeruginosa with a growth advantage in bacterial competition under anaerobic conditions and plays an important role in bacterial virulence. Overall, this study clarifies the role of T6SS in secretion of an anion-binding protein, emphasizing the fundamental importance of this bacterium using T6SS-mediated molybdate uptake to adapt to complex environmental conditions.

Anaerobic gut fungi are an untapped reservoir of natural products.

Anaerobic fungi (class Neocallimastigomycetes) thrive as low-abundance members of the herbivore digestive tract. The genomes of anaerobic gut fungi are poorly characterized and have not been extensively mined for the biosynthetic enzymes of natural products such as antibiotics. Here, we investigate the potential of anaerobic gut fungi to synthesize natural products that could regulate membership within the gut microbiome. Complementary 'omics' approaches were combined to catalog the natural products of anaerobic gut fungi from four different representative species: Anaeromyces robustus (Arobustus), Caecomyces churrovis (Cchurrovis), Neocallimastix californiae (Ncaliforniae), and Piromyces finnis (Pfinnis). In total, 146 genes were identified that encode biosynthetic enzymes for diverse types of natural products, including nonribosomal peptide synthetases and polyketide synthases. In addition, N. californiae and C. churrovis genomes encoded seven putative bacteriocins, a class of antimicrobial peptides typically produced by bacteria. During standard laboratory growth on plant biomass or soluble substrates, 26% of total core biosynthetic genes in all four strains were transcribed. Across all four fungal strains, 30% of total biosynthetic gene products were detected via proteomics when grown on cellobiose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of fungal supernatants detected 72 likely natural products from A. robustus alone. A compound produced by all four strains of anaerobic fungi was putatively identified as the polyketide-related styrylpyrone baumin. Molecular networking quantified similarities between tandem mass spectrometry (MS/MS) spectra among these fungi, enabling three groups of natural products to be identified that are unique to anaerobic fungi. Overall, these results support the finding that anaerobic gut fungi synthesize natural products, which could be harnessed as a source of antimicrobials, therapeutics, and other bioactive compounds.

Inactivation of genes in oxidative respiration and iron acquisition pathways in pediatric clinical isolates of Small colony variant Enterobacteriaceae.

Isolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and can fundamentally change the outcome of the associated infections. Bacterial SCVs often emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The basis of SCV emergence in vivo is not well understood in Gram-negative bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of NCV and SCV co-isolates of Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei. We confirmed SCV emergence was attributed to limited genomic mutations: 4 single nucleotide variants in the E. coli SCV, 5 in C. freundii, and 8 in E. hormaechei. In addition, a 10.2 kb chromosomal segment containing 11 genes was deleted in the E. hormaechei SCV isolate. Each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved in iron capture. Chemical and genetic rescue confirmed defects in heme biosynthesis for E. coli and C. freundii and lipoic acid biosynthesis in E. hormaachei were responsible for the SCV phenotype. Prototrophic growth in all 3 SCV Enterobacteriaceae species was unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo.

Illumina sequencing of 16S rRNA genes reveals a unique microbial community in three anaerobic sludge digesters of Dubai.

Understanding the microbial communities in anaerobic digesters, especially bacteria and archaea, is key to its better operation and regulation. Microbial communities in the anaerobic digesters of the Gulf region where climatic conditions and other factors may impact the incoming feed are not documented. Therefore, Archaeal and Bacterial communities of three full-scale anaerobic digesters, namely AD1, AD3, and AD5 of the Jebel Ali Sewage water Treatment Plant (JASTP) were analyzed by Illumina sequencing of 16S rRNA genes. Among bacteria, the most abundant genus was fermentative bacteria Acetobacteroides (Blvii28). Other predominant bacterial genera in the digesters included thermophilic bacteria (Fervidobacterium and Coprothermobacter) and halophilic bacteria like Haloterrigena and Sediminibacter. This can be correlated with the climatic condition in Dubai, where the bacteria in the incoming feed may be thermophilic or halophilic as much of the water used in the country is desalinated seawater. The predominant Archaea include mainly the members of the phyla Euryarchaeota and Crenarchaeota belonging to the genus Methanocorpusculum, Metallosphaera, Methanocella, and Methanococcus. The highest population of Methanocorpusculum (more than 50% of total Archaea), and other hydrogenotrophic archaea, is in agreement with the high population of bacterial genera Acetobacteroides (Blvii28) and Fervidobacterium, capable of fermenting organic substrates into acetate and H2. Coprothermobacter, which is known to improve protein degradation by establishing syntrophy with hydrogenotrophic archaea, is also one of the digesters' dominant genera. The results suggest that the microbial community in three full-scale anaerobic digesters is different. To best of our knowledge this is the first detailed report from the UAE.

An epigenetic pathway in rice connects genetic variation to anaerobic germination and seedling establishment.

Rice production is shifting from transplanting seedlings to direct sowing of seeds. Following heavy rains, directly sown seeds may need to germinate under anaerobic environments, but most rice (Oryza sativa) genotypes cannot survive these conditions. To identify the genetic architecture of complex traits, we quantified percentage anaerobic germination (AG) in 2,700 (wet-season) and 1,500 (dry-season) sequenced rice genotypes and performed genome-wide association studies (GWAS) using 693,502 single nucleotide polymorphisms. This was followed by post-GWAS analysis with a generalized SNP-to-gene set analysis, meta-analysis, and network analysis. We determined that percentage AG is intermediate-to-high among indica subpopulations, and AG is a polygenic trait associated with transcription factors linked to ethylene responses or genes involved in metabolic processes that are known to be associated with AG. Our post-GWAS analysis identified several genes involved in a wide variety of metabolic processes. We subsequently performed functional analysis focused on the small RNA and methylation pathways. We selected CLASSY 1 (CLSY1), a gene involved in the RNA-directed DNA methylation (RdDm) pathway, for further analyses under AG and found several lines of evidence that CLSY1 influences AG. We propose that the RdDm pathway plays a role in rice responses to water status during germination and seedling establishment developmental stages.

The Mastigamoeba balamuthi Genome and the Nature of the Free-Living Ancestor of Entamoeba.

The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living Mastigamoeba balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).

Comparative Genomics Provides Insights into the Taxonomy of Azoarcus and Reveals Separate Origins of Nif Genes in the Proposed Azoarcus and Aromatoleum Genera.

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The "plant-associated" group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes.

Complete Genomes of the Anaerobic Degradation Specialists Aromatoleum petrolei ToN1T and Aromatoleum bremense PbN1T.

The betaproteobacterial genus Aromatoleum comprises facultative denitrifiers specialized in the anaerobic degradation of recalcitrant organic compounds (aromatic and terpenoid). This study reports on the complete and manually annotated genomes of Ar. petrolei ToN1T (5.41 Mbp) and Ar. bremense PbN1T (4.38 Mbp), which cover the phylogenetic breadth of the genus Aromatoleum together with previously genome sequenced Ar. aromaticum EbN1T [Rabus et al., Arch Microbiol. 2005 Jan;183(1):27-36]. The gene clusters for the anaerobic degradation of aromatic and terpenoid (strain ToN1T only) compounds are scattered across the genomes of strains ToN1T and PbN1T. The richness in mobile genetic elements is shared with other Aromatoleum spp., substantiating that horizontal gene transfer should have been a major driver in shaping the genomes of this genus. The composite catabolic network of strains ToN1T and PbN1T comprises 88 proteins, the coding genes of which occupy 86.1 and 76.4 kbp (1.59 and 1.75%) of the respective genome. The strain-specific gene clusters for anaerobic degradation of ethyl-/propylbenzene (strain PbN1T) and toluene/monoterpenes (strain ToN1T) share high similarity with their counterparts in Ar. aromaticum strains EbN1T and pCyN1, respectively. Glucose is degraded via the ED-pathway in strain ToN1T, while gluconeogenesis proceeds via the reverse EMP-pathway in strains ToN1T, PbN1T, and EbN1T. The diazotrophic, endophytic lifestyle of closest related genus Azoarcus is known to be associated with nitrogenase and type-6 secretion system (T6SS). By contrast, strains ToN1T, PbN1T, and EbN1T lack nif genes for nitrogenase (including cofactor synthesis and enzyme maturation). Moreover, strains PbN1T and EbN1T do not possess tss genes for T6SS, while strain ToN1T does and facultative endophytic "Aromatoleum" sp. CIB is known to even have both. These findings underpin the functional heterogeneity among Aromatoleum members, correlating with the high plasticity of their genomes.

The hypoxia adaptation of small mammals to plateau and underground burrow conditions.

Oxygen is one of the important substances for the survival of most life systems on the earth, and plateau and underground burrow systems are two typical hypoxic environments. Small mammals living in hypoxic environments have evolved different adaptation strategies, which include increased oxygen delivery, metabolic regulation of physiological responses and other physiological responses that change tissue oxygen utilization. Multi-omics predictions have also shown that these animals have evolved different adaptations to extreme environments. In particular, vascular endothelial growth factor (VEGF) and erythropoietin (EPO), which have specific functions in the control of O2 delivery, have evolved adaptively in small mammals in hypoxic environments. Naked mole-rats and blind mole-rats are typical hypoxic model animals as they have some resistance to cancer. This review primarily summarizes the main living environment of hypoxia tolerant small mammals, as well as the changes of phenotype, physiochemical characteristics and gene expression mode of their long-term living in hypoxia environment.

Heme biosynthesis in prokaryotes.

The cyclic tetrapyrrole heme is used as a prosthetic group in a broad variety of different proteins in almost all organisms. Often, it is essential for vital biochemical processes such as aerobic and anaerobic respiration as well as photosynthesis. In Nature, heme is made from the common tetrapyrrole precursor 5-aminolevulinic acid, and for a long time it was assumed that heme is biosynthesized by a single, common pathway in all organisms. However, although this is indeed the case in eukaryotes, heme biosynthesis is more diverse in the prokaryotic world, where two additional pathways exist. The final elucidation of the two 'alternative' heme biosynthesis routes operating in some bacteria and archaea was achieved within the last decade. This review summarizes the three different heme biosynthesis pathways with a special emphasis on the two 'new' prokaryotic routes.

Salmonella enterica Serovars Dublin and Enteritidis Comparative Proteomics Reveals Differential Expression of Proteins Involved in Stress Resistance, Virulence, and Anaerobic Metabolism.

The Enteritidis and Dublin serovars of Salmonella enterica are phylogenetically closely related yet differ significantly in host range and virulence. S Enteritidis is a broad-host-range serovar that commonly causes self-limited gastroenteritis in humans, whereas S Dublin is a cattle-adapted serovar that can infect humans, often resulting in invasive extraintestinal disease. The mechanism underlying the higher invasiveness of S Dublin remains undetermined. In this work, we quantitatively compared the proteomes of clinical isolates of each serovar grown under gut-mimicking conditions. Compared to S Enteritidis, the S Dublin proteome was enriched in proteins linked to response to several stress conditions, such as those encountered during host infection, as well as to virulence. The S Enteritidis proteome contained several proteins related to central anaerobic metabolism pathways that were undetected in S Dublin. In contrast to what has been observed in other extraintestinal serovars, most of the coding genes for these pathways are not degraded in S Dublin. Thus, we provide evidence that S Dublin metabolic functions may be much more affected than previously reported based on genomic studies. Single and double null mutants in stress response proteins Dps, YciF, and YgaU demonstrate their relevance to S Dublin invasiveness in a murine model of invasive salmonellosis. All in all, this work provides a basis for understanding interserovar differences in invasiveness and niche adaptation, underscoring the relevance of using proteomic approaches to complement genomic studies.

Fate of antibiotic resistant E. coli and antibiotic resistance genes during full scale conventional and advanced anaerobic digestion of sewage sludge.

Antibiotic resistant bacteria (ARB) and their genes (ARGs) have become recognised as significant emerging environmental pollutants. ARB and ARGs in sewage sludge can be transmitted back to humans via the food chain when sludge is recycled to agricultural land, making sludge treatment key to control the release of ARB and ARGs to the environment. This study investigated the fate of antibiotic resistant Escherichia coli and a large set of antibiotic resistance genes (ARGs) during full scale anaerobic digestion (AD) of sewage sludge at two U.K. wastewater treatment plants and evaluated the impact of thermal hydrolysis (TH) pre-treatment on their abundance and diversity. Absolute abundance of 13 ARGs and the Class I integron gene intI1 was calculated using single gene quantitative (q) PCR. High through-put qPCR analysis was also used to determine the relative abundance of 370 ARGs and mobile genetic elements (MGEs). Results revealed that TH reduced the absolute abundance of all ARGs tested and intI1 by 10-12,000 fold. After subsequent AD, a rebound effect was seen in many ARGs. The fate of ARGs during AD without pre-treatment was variable. Relative abundance of most ARGs and MGEs decreased or fluctuated, with the exception of macrolide resistance genes, which were enriched at both plants, and tetracyline and glycopeptide resistance genes which were enriched in the plant employing TH. Diversity of ARGs and MGEs decreased in both plants during sludge treatment. Principal coordinates analysis revealed that ARGs are clearly distinguished according to treatment step, whereas MGEs in digested sludge cluster according to site. This study provides a comprehensive within-digestor analysis of the fate of ARGs, MGEs and antibiotic resistant E. coli and highlights the effectiveness of AD, particularly when TH is used as a pre-treatment, at reducing the abundance of most ARGs and MGEs in sludgeand preventing their release into the environment.

Differential proteomic analysis by SWATH-MS unravels the most dominant mechanisms underlying yeast adaptation to non-optimal temperatures under anaerobic conditions.

Elucidation of temperature tolerance mechanisms in yeast is essential for enhancing cellular robustness of strains, providing more economically and sustainable processes. We investigated the differential responses of three distinct Saccharomyces cerevisiae strains, an industrial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and an industrial bioethanol strain, Ethanol Red, grown at sub- and supra-optimal temperatures under chemostat conditions. We employed anaerobic conditions, mimicking the industrial processes. The proteomic profile of these strains in all conditions was performed by sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS), allowing the quantification of 997 proteins, data available via ProteomeXchange (PXD016567). Our analysis demonstrated that temperature responses differ between the strains; however, we also found some common responsive proteins, revealing that the response to temperature involves general stress and specific mechanisms. Overall, sub-optimal temperature conditions involved a higher remodeling of the proteome. The proteomic data evidenced that the cold response involves strong repression of translation-related proteins as well as induction of amino acid metabolism, together with components related to protein folding and degradation while, the high temperature response mainly recruits amino acid metabolism. Our study provides a global and thorough insight into how growth temperature affects the yeast proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance.