Novel Alphaproteobacteria transcribe genes for nitric oxide transformation at high levels in a marine oxygen-deficient zone.

Marine oxygen-deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs is still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase (nod) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribed nod genes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria, rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribe nod, but at lower relative abundance than UBA11136 in the upper ODZ. The nod-transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here named ptd) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs.IMPORTANCEIn marine oxygen-deficient zones (ODZs), microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the world's largest ODZ, but the identity of the microbes transforming nitric oxide remains unknown. Here, we show that highly transcribed nitric oxide dismutase (nod) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribe nod at lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria.

In situ community transcriptomics illuminates CO2-fixation potentials and supporting roles of phagotrophy and proton pump in plankton in a subtropical marginal sea.

Lineage-wise physiological activities of plankton communities in the ocean are important but challenging to characterize. Here, we conducted whole-assemblage metatranscriptomic profiling at continental shelf and slope sites in the South China Sea to investigate carbon fixation potential in different lineages. RuBisCO expression, the proxy of Calvin carbon fixation (CCF) potential, was mainly contributed by Bacillariophyta, Chlorophyta, Cyanobacteria, and Haptophyta, which was differentially affected by environmental factors among lineages. CCF potential exhibited positive or negative correlations with phagotrophy gene expression, suggesting phagotrophy possibly enhances or complements CCF. Our data also reveal significant non-Calvin carbon fixation (NCF) potential, as indicated by the active expression of genes in all five currently recognized NCF pathways, mainly contributed by Flavobacteriales, Alteromonadales, and Oceanospirillales. Furthermore, in Flavobacteriales, Alteromonadales, Pelagibacterales, and Rhodobacterales, NCF potential was positively correlated with proton-pump rhodopsin (PPR) expression, suggesting that NCF might be energetically supported by PPR. The novel insights into the lineage-differential potential of carbon fixation, widespread mixotrophy, and PPR as an energy source for NCF lay a methodological and informational foundation for further research to understand carbon fixation and the trophic landscape in the ocean.IMPORTANCEMarine plankton plays an important role in global carbon cycling and climate regulation. Phytoplankton and cyanobacteria fix CO2 to produce organic compounds using solar energy and mainly by the Calvin cycle, whereas autotrophic bacteria and archaea may fix CO2 by non-Calvin cycle carbon fixation pathways. How active individual lineages are in carbon fixation and mixotrophy, and what energy source bacteria may employ in non-Calvin carbon fixation, in a natural plankton assemblage are poorly understood and underexplored. Using metatranscriptomics, we studied carbon fixation in marine plankton with lineage resolution in tropical marginal shelf and slope areas. Based on the sequencing results, we characterized the carbon fixation potential of different lineages and assessed Calvin- and non-Calvin- carbon fixation activities and energy sources. Data revealed a high number of unigenes (4.4 million), lineage-dependent differential potentials of Calvin carbon fixation and responses to environmental conditions, major contributors of non-Calvin carbon fixation, and their potential energy source.

Cycloalkane degradation by an uncultivated novel genus of Gammaproteobacteria derived from China's marginal seas.

The consumption of cycloalkanes is prevalent in low-temperature marine environments, likely influenced by psychrophilic microorganisms. Despite their significance, the primary active species responsible for marine cycloalkane degradation remain largely unidentified due to cultivation challenges. In this study, we provide compelling evidence indicating that the uncultured genus C1-B045 of Gammaproteobacteria is a pivotal participant in cycloalkane decomposition within China's marginal seas. Notably, the relative abundance of C1-B045 surged from 15.9% in the methylcyclohexane (MCH)-consuming starter culture to as high as 97.5% in MCH-utilizing extinction cultures following successive dilution-to-extinction and incubation cycles. We used stable isotope probing, Raman-activated gravity-driven encapsulation, and 16 S rRNA gene sequencing to link cycloalkane-metabolizing phenotype to genotype at the single-cell level. By annotating key enzymes (e.g., alkane monooxygenase, cyclohexanone monooxygenase, and 6-hexanolactone hydrolase) involved in MCH metabolism within C1-B045's representative metagenome-assembled genome, we developed a putative MCH degradation pathway.

A novel soluble di-iron monooxygenase from the soil bacterium Solimonas soli.

Soluble di-iron monooxygenase (SDIMO) enzymes enable insertion of oxygen into diverse substrates and play significant roles in biogeochemistry, bioremediation and biocatalysis. An unusual SDIMO was detected in an earlier study in the genome of the soil organism Solimonas soli, but was not characterized. Here, we show that the S. soli SDIMO is part of a new clade, which we define as 'Group 7'; these share a conserved gene organization with alkene monooxygenases but have only low amino acid identity. The S. soli genes (named zmoABCD) could be functionally expressed in Pseudomonas putida KT2440 but not in Escherichia coli TOP10. The recombinants made epoxides from C2 C8 alkenes, preferring small linear alkenes (e.g. propene), but also epoxidating branched, carboxylated and chlorinated substrates. Enzymatic epoxidation of acrylic acid was observed for the first time. ZmoABCD oxidised the organochlorine pollutants vinyl chloride (VC) and cis-1,2-dichloroethene (cDCE), with the release of inorganic chloride from VC but not cDCE. The original host bacterium S. soli could not grow on any alkenes tested but grew well on phenol and n-octane. Further work is needed to link ZmoABCD and the other Group 7 SDIMOs to specific physiological and ecological roles.

Diaphorin, a polyketide produced by a bacterial endosymbiont of the Asian citrus psyllid, adversely affects the in vitro gene expression with ribosomes from Escherichia coli and Bacillus subtilis.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria), an obligate mutualist of an important agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera). Our previous study demonstrated that diaphorin, at physiological concentrations in D. citri, inhibits the growth and cell division of Bacillus subtilis (Firmicutes) but promotes the growth and metabolic activity of Escherichia coli (Gammaproteobacteria). This unique property of diaphorin may aid microbial mutualism in D. citri, potentially affecting the transmission of "Candidatus Liberibacter spp." (Alphaproteobacteria), the pathogens of the most destructive citrus disease Huanglongbing. Moreover, this property may be exploited to promote microbes' efficiency in producing industrial materials. However, the mechanism underlying this activity is unknown. Diaphorin belongs to the family of pederin-type compounds, which inhibit protein synthesis in eukaryotes by binding to eukaryotic ribosomes. Therefore, as a first step to assess diaphorin's direct influence on bacterial gene expression, this study examined the effect of diaphorin on the in vitro translation using ribosomes of B. subtilis and E. coli, quantifying the production of the green fluorescent protein. The results showed that the gene expression involving B. subtilis and E. coli ribosomes along with five millimolar diaphorin was 29.6% and 13.1%, respectively, less active than the control. This suggests that the diaphorin's adverse effects on B. subtilis are attributed to, at least partly, its inhibitory effects on gene expression. Moreover, as ingredients of the translation system were common other than ribosomes, the greater inhibitory effects observed with the B. subtilis ribosome imply that the ribosome is among the potential targets of diaphorin. On the other hand, the results also imply that diaphorin's positive effects on E. coli are due to targets other than the core machinery of transcription and translation. This study demonstrated for the first time that a pederin congener affects bacterial gene expression.

Acetylcysteine increases sensitivity of ceftazidime-avibactam-resistant enterobacterales with different enzymatic resistance to ceftazidime-avibactam in vitro and in vivo.

BACKGROUND: Ceftazidime-avibactam (CZA) improves treatment outcomes for infections caused by carbapenem-resistant organisms, but has led to serious bacterial resistance. Acetylcysteine (NAC) is an approved medication that protects the respiratory tract through antioxidant and anti-inflammatory effects. RESULTS: This study found that NAC combined with CZA effectively inhibits the growth of CZA-resistant clinical Enterobacterales strains. The CZA/NAC combination inhibits biofilm formation in vitro and decreases bacterial burden in a mouse thigh infection model. The combination is biocompatible and primarily increases cell membrane permeability to cause bacterial death. CONCLUSIONS: These findings prove that the CZA/NAC combination has potential as a treatment for CZA-resistant Enterobacterales infections.

Proterozoic Acquisition of Archaeal Genes for Extracellular Electron Transfer: A Metabolic Adaptation of Aerobic Ammonia-Oxidizing Bacteria to Oxygen Limitation.

Many aerobic microbes can utilize alternative electron acceptors under oxygen-limited conditions. In some cases, this is mediated by extracellular electron transfer (or EET), wherein electrons are transferred to extracellular oxidants such as iron oxide and manganese oxide minerals. Here, we show that an ammonia-oxidizer previously known to be strictly aerobic, Nitrosomonas communis, may have been able to utilize a poised electrode to maintain metabolic activity in anoxic conditions. The presence and activity of multiheme cytochromes in N. communis further suggest a capacity for EET. Molecular clock analysis shows that the ancestors of ß-proteobacterial ammonia oxidizers appeared after Earth's atmospheric oxygenation when the oxygen levels were >10-4pO2 (present atmospheric level [PAL]), consistent with aerobic origins. Equally important, phylogenetic reconciliations of gene and species trees show that the multiheme c-type EET proteins in Nitrosomonas and Nitrosospira lineages were likely acquired by gene transfer from γ-proteobacteria when the oxygen levels were between 0.1 and 1 pO2 (PAL). These results suggest that ß-proteobacterial EET evolved during the Proterozoic when oxygen limitation was widespread, but oxidized minerals were abundant.

The mRNA binding-mediated self-regulatory function of small heat shock protein IbpA in γ-proteobacteria is conferred by a conserved arginine.

Bacterial small heat shock proteins, such as inclusion body-associated protein A (IbpA) and IbpB, coaggregate with denatured proteins and recruit other chaperones for the processing of aggregates thereby assisting in protein refolding. In addition, as a recently revealed uncommon feature, Escherichia coli IbpA self-represses its own translation through interaction with the 5'-untranslated region of the ibpA mRNA, enabling IbpA to act as a mediator of negative feedback regulation. Although IbpA also suppresses the expression of IbpB, IbpB does not have this self-repression activity despite the two Ibps being highly homologous. In this study, we demonstrate that the self-repression function of IbpA is conserved in other γ-proteobacterial IbpAs. Moreover, we show a cationic residue-rich region in the α-crystallin domain of IbpA, which is not conserved in IbpB, is critical for the self-suppression activity. Notably, we found arginine 93 (R93) located within the α-crystallin domain is an essential residue that cannot be replaced by any of the other 19 amino acids including lysine. We observed that IbpA-R93 mutants completely lost the interaction with the 5' untranslated region of the ibpA mRNA, but retained almost all chaperone activity and were able to sequester denatured proteins. Taken together, we propose the conserved Arg93-mediated translational control of IbpA through RNA binding would be beneficial for a rapid and massive supply of the chaperone on demand.

Identification and characterization of a novel high-activity amylosucrase from Salinispirillum sp. LH10-3-1.

In this study, a novel high-activity amylosucrase from Salinispirillum sp. LH10-3-1 (SaAS) was identified and characterized. The recombinant enzyme was determined as a monomer with a molecular mass of 75 kDa. SaAS protein exhibited the maximum total and polymerization activities at pH 9.0 and maximum hydrolysis activity at pH 8.0. The optimum temperature for total, polymerization, and hydrolysis activities were 40, 40, and 45 °C, respectively. Under the optimal pH and temperature, SaAS had a specific activity of 108.2 U/mg. SaAS also showed excellent salt tolerance and could retain 77.4% of its original total activity at 4.0 M NaCl. The addition of Mg2+, Ba2+, and Ca2+ enhanced the total activity of SaAS. When the conversion of 0.1 M and 1.0 M sucrose was catalyzed at pH 9.0 and 40 °C for 24 h, the ratios of hydrolysis, polymerization, and isomerization reactions were 11.9:77.4:10.7 and 15.3:53.5:31.2, respectively. The α-arbutin yield of 60.3% was achieved from 20 mM sucrose and 5 mM hydroquinone catalyzed by SaAS. KEY POINTS: • A novel amylosucrase from Salinispirillum sp. LH10-3-1 (SaAS) was characterized. • SaAS has the highest specific enzyme activity among all known amylosucrase. • SaAS has hydrolysis, polymerization, isomerization, and glucosyltransferase activities.

Modeling the growth of diverse microorganisms during feast-famine enrichment.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can decrease the severe environmental pollution of petroleum plastics. PHA production by mixed microbial communities has been extensively studied to lower the high PHA prices. However, the competition between distinct microbial communities during the enrichment of PHA accumulators in mixed cultures has not been widely investigated. Thus, in this work, we developed a mathematical model for the competition between PHA accumulators and non-PHA accumulators in the feast-famine enrichment strategy. The developed model successfully simulated published lab-scale experimental data for Plasticicumulans acidivorans, a well-studied PHA accumulator that can store PHA up to 90% of the cell weight. The growth kinetics for both PHA and non-PHA accumulators were estimated and compared to the values in the literature. The uncertainties in the model kinetics were studied by expanding the model to include additional sub-biomass components for each heterotrophic group. As a result, the microbial diversity of microbial communities was observed to influence the enrichment of PHA accumulators in mixed cultures. Additionally, the calibrated model was applied to investigate the cultivation conditions, such as cycle lengths, carbon-to-nitrogen ratio, and solids retention time for successful P. acidivorans enrichment in mixed cultures. The developed model can be applied to control the cultivation and enrichment of PHA accumulators in large-scale PHA production systems. PRACTITIONER POINTS: A new model for the enrichment of PHA accumulators was developed. The model can simulate PHA accumulation by enriched cultures. The model was calibrated and validated for Plasticicumulans acidivorans. The impact of microbial diversity on enriching PHA accumulators was investigated. Short cycles (<12 h) and SRT (<10 d) are suggested for successful enrichment.

Genome-centric insight into metabolically active microbial population in shallow-sea hydrothermal vents.

BACKGROUND: Geothermal systems have contributed greatly to both our understanding of the functions of extreme life and the evolutionary history of life itself. Shallow-sea hydrothermal systems are ecological intermediates of deep-sea systems and terrestrial springs, harboring unique and complexed ecosystems, which are well-lit and present physicochemical gradients. The microbial communities of deep-sea and terrestrial geothermal systems have been well-studied at the population genome level, yet little is known about the communities inhabiting the shallow-sea hydrothermal systems and how they compare to those inhabiting other geothermal systems. RESULTS: Here, we used genome-resolved metagenomic and metaproteomic approaches to probe into the genetic potential and protein expression of microorganisms from the shallow-sea vent fluids off Kueishantao Island. The families Nautiliaceae and Campylobacteraceae within the Epsilonbacteraeota and the Thiomicrospiraceae within the Gammaproteobacteria were prevalent in vent fluids over a 3-year sampling period. We successfully reconstructed the in situ metabolic modules of the predominant populations within the Epsilonbacteraeota and Gammaproteobacteria by mapping the metaproteomic data back to metagenome-assembled genomes. Those active bacteria could use the reductive tricarboxylic acid cycle or Calvin-Benson-Bassham cycle for autotrophic carbon fixation, with the ability to use reduced sulfur species, hydrogen or formate as electron donors, and oxygen as a terminal electron acceptor via cytochrome bd oxidase or cytochrome bb3 oxidase. Comparative metagenomic and genomic analyses revealed dramatic differences between submarine and terrestrial geothermal systems, including microbial functional potentials for carbon fixation and energy conversion. Furthermore, shallow-sea hydrothermal systems shared many of the major microbial genera that were first isolated from deep-sea and terrestrial geothermal systems, while deep-sea and terrestrial geothermal systems shared few genera. CONCLUSIONS: The metabolic machinery of the active populations within Epsilonbacteraeota and Gammaproteobacteria at shallow-sea vents can mirror those living at deep-sea vents. With respect to specific taxa and metabolic potentials, the microbial realm in the shallow-sea hydrothermal system presented ecological linkage to both deep-sea and terrestrial geothermal systems. Video Abstract.

Urease-negative uropathogen Kalamiella piersonii YU22 metabolizes urea by urea carboxylase and allophanate hydrolase enzyme system.

Urea is one of the major components of the human urine and its breakdown by the uropathogens occurs mainly through the activity of the enzyme urease. However, a few reports suggest the presence of an alternate enzyme system for urea breakdown namely urea carboxylase (UC) and allophanate hydrolase (AH). We have previously reported the UC and AH system in the genome of a urease-negative uropathogen Kalamiella piersonii YU22 of the novel genus Kalamiella (reclassified recently as Pantoea).To validate the UC and AH activity in the presence of urea, we investigated the growth and urea utilization patterns of this bacterium. Growth kinetics, variations in media pH, NH4-N generation and UC and AH gene expressions were probed using urea-containing media. YU22 was able to grow in M9 media containing urea and increase the pH of the media due to the urea breakdown. Further, significantly higher concentrations of extracellular NH4-N (p < 0.001) was also detected in the cultures along with over-expression of UC and AH genes. The bacterium formed biofilm, and displayed swimming and swarming motilities in presence of urea. Additional glucose supply to urea boosted the colonization but ameliorated the media alkalization and ammonification through suppression of gene expressions encoding UC and AH. These results show that the urease-negative strain YU22 can utilize the UC and AH system for urea metabolism. We propose to further investigate the UC and AH system in other urease-negative uropathogens and its implications for pathogenicity and urinary tract colonization.

Diaphorin, a Polyketide Produced by a Bacterial Symbiont of the Asian Citrus Psyllid, Inhibits the Growth and Cell Division of Bacillus subtilis but Promotes the Growth and Metabolic Activity of Escherichia coli.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a notorious agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Diaphorin belongs to the pederin family of bioactive agents found in various host-symbiont systems, including beetles, lichens, and sponges, harboring phylogenetically diverse bacterial producers. Previous studies showed that diaphorin, which is present in D. citri at concentrations of 2 to 20 mM, has inhibitory effects on various eukaryotes, including the natural enemies of D. citri. However, little is known about its effects on prokaryotic organisms. To address this issue, the present study assessed the biological activities of diaphorin on two model prokaryotes, Escherichia coli (Gammaproteobacteria: Enterobacterales) and Bacillus subtilis (Firmicutes: Bacilli). Their growth and morphological features were analyzed using spectrophotometry, optical microscopy followed by image analysis, and transmission electron microscopy. The metabolic activity of E. coli was further assessed using the ß-galactosidase assay. The results revealed that physiological concentrations of diaphorin inhibit the growth and cell division of B. subtilis but promote the growth and metabolic activity of E. coli. This finding implies that diaphorin functions as a defensive agent of the holobiont (host plus symbionts) against some bacterial lineages but is metabolically beneficial for others, which potentially include obligate symbionts of D. citri. IMPORTANCE Certain secondary metabolites, including antibiotics, evolve to mediate interactions among organisms. These molecules have distinct spectra for microorganisms and are often more effective against Gram-positive bacteria than Gram-negative ones. However, it is rare that a single molecule has completely opposite activities on distinct bacterial lineages. The present study revealed that a secondary metabolite synthesized by an organelle-like bacterial symbiont of psyllids inhibits the growth of Gram-positive Bacillus subtilis but promotes the growth of Gram-negative Escherichia coli. This finding not only provides insights into the evolution of microbiomes in animal hosts but also may potentially be exploited to promote the effectiveness of industrial material production by microorganisms.

Oceanospirillales containing the DMSP lyase DddD are key utilisers of carbon from DMSP in coastal seawater.

BACKGROUND: Ubiquitous and diverse marine microorganisms utilise the abundant organosulfur molecule dimethylsulfoniopropionate (DMSP), the main precursor of the climate-active gas dimethylsulfide (DMS), as a source of carbon, sulfur and/or signalling molecules. However, it is currently difficult to discern which microbes actively catabolise DMSP in the environment, why they do so and the pathways used. RESULTS: Here, a novel DNA-stable isotope probing (SIP) approach, where only the propionate and not the DMS moiety of DMSP was 13C-labelled, was strategically applied to identify key microorganisms actively using DMSP and also likely DMS as a carbon source, and their catabolic enzymes, in North Sea water. Metagenomic analysis of natural seawater suggested that Rhodobacterales (Roseobacter group) and SAR11 bacteria were the major microorganisms degrading DMSP via demethylation and, to a lesser extent, DddP-driven DMSP lysis pathways. However, neither Rhodobacterales and SAR11 bacteria nor their DMSP catabolic genes were prominently labelled in DNA-SIP experiments, suggesting they use DMSP as a sulfur source and/or in signalling pathways, and not primarily for carbon requirements. Instead, DNA-SIP identified gammaproteobacterial Oceanospirillales, e.g. Amphritea, and their DMSP lyase DddD as the dominant microorganisms/enzymes using DMSP as a carbon source. Supporting this, most gammaproteobacterial (with DddD) but few alphaproteobacterial seawater isolates grew on DMSP as sole carbon source and produced DMS. Furthermore, our DNA-SIP strategy also identified Methylophaga and other Piscirickettsiaceae as key bacteria likely using the DMS, generated from DMSP lysis, as a carbon source. CONCLUSIONS: This is the first study to use DNA-SIP with 13C-labelled DMSP and, in a novel way, it identifies the dominant microbes utilising DMSP and DMS as carbon sources. It highlights that whilst metagenomic analyses of marine environments can predict microorganisms/genes that degrade DMSP and DMS based on their abundance, it cannot disentangle those using these important organosulfur compounds for their carbon requirements. Note, the most abundant DMSP degraders, e.g. Rhodobacterales with DmdA, are not always the key microorganisms using DMSP for carbon and releasing DMS, which in this coastal system were Oceanospirillales containing DddD. Video abstract.

A previously uncharacterized gene, PA2146, contributes to biofilm formation and drug tolerance across the ɣ-Proteobacteria.

Transcriptomic studies have revealed a large number of uncharacterized genes that are differentially expressed in biofilms, which may be important in regulating biofilm phenotypes such as resistance to antimicrobial agents. To identify biofilm genes of unknown function in P. aeruginosa, we made use of RNA-seq and selected 27 uncharacterized genes that were induced upon biofilm growth. Biofilms by respective mutants were subsequently analyzed for two biofilm characteristics, the biofilm architecture and drug susceptibility. The screen revealed 12 out of 27 genes to contribute to biofilm formation and 13 drug susceptibility, with 8 genes affecting both biofilm phenotypes. Amongst the genes affecting both biofilm phenotypes was PA2146, encoding a small hypothetical protein that exhibited some of the most substantial increases in transcript abundance during biofilm growth by P. aeruginosa PAO1 and clinical isolates. PA2146 is highly conserved in É£-proteobacteria. Inactivation of PA2146 affected both biofilm phenotypes in P. aeruginosa PAO1, with inactivation of homologs in Klebsiella pneumoniae and Escherichia coli having similar effects. Heterologous expression of PA2146 homologs complemented the P. aeruginosa ∆PA2146, suggesting that PA2146 homologs substitute for and play a similar role as PA2146 in P. aeruginosa.

Discovery of lignin-transforming bacteria and enzymes in thermophilic environments using stable isotope probing.

Characterizing microorganisms and enzymes involved in lignin biodegradation in thermal ecosystems can identify thermostable biocatalysts. We integrated stable isotope probing (SIP), genome-resolved metagenomics, and enzyme characterization to investigate the degradation of high-molecular weight, 13C-ring-labeled synthetic lignin by microbial communities from moderately thermophilic hot spring sediment (52 °C) and a woody "hog fuel" pile (53 and 62 °C zones). 13C-Lignin degradation was monitored using IR-GCMS of 13CO2, and isotopic enrichment of DNA was measured with UHLPC-MS/MS. Assembly of 42 metagenomic libraries (72 Gb) yielded 344 contig bins, from which 125 draft genomes were produced. Fourteen genomes were significantly enriched with 13C from lignin, including genomes of Actinomycetes (Thermoleophilaceae, Solirubrobacteraceae, Rubrobacter sp.), Firmicutes (Kyrpidia sp., Alicyclobacillus sp.) and Gammaproteobacteria (Steroidobacteraceae). We employed multiple approaches to screen genomes for genes encoding putative ligninases and pathways for aromatic compound degradation. Our analysis identified several novel laccase-like multi-copper oxidase (LMCO) genes in 13C-enriched genomes. One of these LMCOs was heterologously expressed and shown to oxidize lignin model compounds and minimally transformed lignin. This study elucidated bacterial lignin depolymerization and mineralization in thermal ecosystems, establishing new possibilities for the efficient valorization of lignin at elevated temperature.

Dynamic changes in the microbial community in the surface seawater of Jiaozhou Bay after crude oil spills: An in situ microcosm study.

The changes in the composition and structure of microbial communities in Jiaozhou Bay are strongly affected by marine oil pollution, but the outcomes of the microbial responses and effects of dispersant application remain unclear. Herein, we performed an in situ microcosm study to investigate the response of the indigenous microbial community under crude oil alone and combined oil and dispersant treatment in the surface seawater of a semi-enclosed marine area of Jiaozhou Bay. The dynamics of the bacterial classification based on 16s rDNA sequencing were used to assess the changes with the crude oil concentration, dispersant use, and time. The crude oil resulted in a high abundance of the genera Pseudohongiella, Cycloclasticus, Marivita, and C1-B045 from the Gammaproteobacteria and Alphaproteobacteria classes, suggesting for hydrocarbon degradation. However, the dispersant treatment was more advantageous for Pacificibacter, Marivita, and Loktanella. Besides accelerating the rate of bacterial community succession, the dispersants had significantly stronger effects on the structure of the bacterial community and the degradation functions than the oil. A higher dose of oil exposure corresponded to fewer dominant species with a high relative abundance. Our study provides information for screening potential degradation bacteria and assessing the risks that oil spills pose to marine ecosystems.

Identification of new Dickeya dadantii virulence factors secreted by the type 2 secretion system.

Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for Dickeya virulence. Previous studies showed that the D. dadantii T2SS secretes a wide range of plant cell wall degrading enzymes, including pectinases and a cellulase. However, the full repertoire of exoproteins it can secrete has probably not yet been identified. Secreted proteins possess a signal peptide and are first addressed to the periplasm before their recruitment by Out. T2SS-specific secretion signals remain unknown which prevents in silico identification of T2SS substrates. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced and encoding proteins with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, the putative virulence factor VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and that svf genes are present in a variable number of copies in other Pectobacteriaceae, up to three in D. fanghzongdai. This work opens the way to the study of the role of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.

GATD3A, a mitochondrial deglycase with evolutionary origins from gammaproteobacteria, restricts the formation of advanced glycation end products.

BACKGROUND: Functional complexity of the eukaryotic mitochondrial proteome is augmented by independent gene acquisition from bacteria since its endosymbiotic origins. Mammalian homologs of many ancestral mitochondrial proteins have uncharacterized catalytic activities. Recent forward genetic approaches attributed functions to proteins in established metabolic pathways, thereby limiting the possibility of identifying novel biology relevant to human disease. We undertook a bottom-up biochemistry approach to discern evolutionarily conserved mitochondrial proteins with catalytic potential. RESULTS: Here, we identify a Parkinson-associated DJ-1/PARK7-like protein-glutamine amidotransferase-like class 1 domain-containing 3A (GATD3A), with bacterial evolutionary affinities although not from alphaproteobacteria. We demonstrate that GATD3A localizes to the mitochondrial matrix and functions as a deglycase. Through its amidolysis domain, GATD3A removes non-enzymatic chemical modifications produced during the Maillard reaction between dicarbonyls and amines of nucleotides and amino acids. GATD3A interacts with factors involved in mitochondrial mRNA processing and translation, suggestive of a role in maintaining integrity of important biomolecules through its deglycase activity. The loss of GATD3A in mice is associated with accumulation of advanced glycation end products (AGEs) and altered mitochondrial dynamics. CONCLUSIONS: An evolutionary perspective helped us prioritize a previously uncharacterized but predicted mitochondrial protein GATD3A, which mediates the removal of early glycation intermediates. GATD3A restricts the formation of AGEs in mitochondria and is a relevant target for diseases where AGE deposition is a pathological hallmark.

Genomic Features and Pervasive Negative Selection in Rhodanobacter Strains Isolated from Nitrate and Heavy Metal Contaminated Aquifer.

Rhodanobacter species dominate in the Oak Ridge Reservation (ORR) subsurface environments contaminated with acids, nitrate, metal radionuclides, and other heavy metals. To uncover the genomic features underlying adaptations to these mixed-waste environments and to guide genetic tool development, we sequenced the whole genomes of eight Rhodanobacter strains isolated from the ORR site. The genome sizes ranged from 3.9 to 4.2 Mb harboring 3,695 to 4,035 protein-coding genes and GC contents approximately 67%. Seven strains were classified as R. denitrificans and one strain, FW510-R12, as R. thiooxydans based on full length 16S rRNA sequences. According to gene annotation, the top two Cluster of Orthologous Groups (COGs) with high pan-genome expansion rates (Pan/Core gene ratio) were "replication, recombination and repair" and "defense mechanisms." The denitrifying genes had high DNA homologies except the predicted protein structure variances in NosZ. In contrast, heavy metal resistance genes were diverse with between 7 to 34% of them were located in genomic islands, and these results suggested origins from horizontal gene transfer. Analysis of the methylation patterns in four strains revealed the unique 5mC methylation motifs. Most orthologs (78%) had ratios of nonsynonymous to synonymous substitutions (dN/dS) less than one when compared to the type strain 2APBS1, suggesting the prevalence of negative selection. Overall, the results provide evidence for the important roles of horizontal gene transfer and negative selection in genomic adaptation at the contaminated field site. The complex restriction-modification system genes and the unique methylation motifs in Rhodanobacter strains suggest the potential recalcitrance to genetic manipulation. IMPORTANCE Despite the dominance of Rhodanobacter species in the subsurface of the contaminated Oak Ridge Reservation (ORR) site, very little is known about the mechanisms underlying their adaptions to the various stressors present at ORR. Recently, multiple Rhodanobacter strains have been isolated from the ORR groundwater samples from several wells with varying geochemical properties. Using Illumina, PacBio, and Oxford Nanopore sequencing platforms, we obtained the whole genome sequences of eight Rhodanobacter strains. Comparison of the whole genomes demonstrated the genetic diversity, and analysis of the long nanopore reads revealed the heterogeneity of methylation patterns in strains isolated from the same well. Although all strains contained a complete set of denitrifying genes, the predicted tertiary structures of NosZ differed. The sequence comparison results demonstrate the important roles of horizontal gene transfer and negative selection in adaptation. In addition, these strains may be recalcitrant to genetic manipulation due to the complex restriction-modification systems and methylations.