Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.623
Filtrar
1.
Nat Microbiol ; 7(5): 695-706, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35505245

RESUMO

Gut bacteria face a key problem in how they capture enough energy to sustain their growth and physiology. The gut bacterium Clostridium sporogenes obtains its energy by utilizing amino acids in pairs, coupling the oxidation of one to the reduction of another-the Stickland reaction. Oxidative pathways produce ATP via substrate-level phosphorylation, whereas reductive pathways are thought to balance redox. In the present study, we investigated whether these reductive pathways are also linked to energy generation and the production of microbial metabolites that may circulate and impact host physiology. Using metabolomics, we find that, during growth in vitro, C. sporogenes produces 15 metabolites, 13 of which are present in the gut of C. sporogenes-colonized mice. Four of these compounds are reductive Stickland metabolites that circulate in the blood of gnotobiotic mice and are also detected in plasma from healthy humans. Gene clusters for reductive Stickland pathways suggest involvement of electron transfer proteins, and experiments in vitro demonstrate that reductive metabolism is coupled to ATP formation and not just redox balance. Genetic analysis points to the broadly conserved Rnf complex as a key coupling site for energy transduction. Rnf complex mutants show aberrant amino acid metabolism in a defined medium and are attenuated for growth in the mouse gut, demonstrating a role of the Rnf complex in Stickland metabolism and gut colonization. Our findings reveal that the production of circulating metabolites by a commensal bacterium within the host gut is linked to an ATP-yielding redox process.


Assuntos
Clostridium , Metabolômica , Trifosfato de Adenosina/metabolismo , Animais , Bactérias/metabolismo , Clostridium/genética , Clostridium/metabolismo , Fermentação , Camundongos
2.
Bioresour Technol ; 353: 127138, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35405210

RESUMO

In this study, continuous cultivations of C.carboxidivorans to study heterotrophic and mixotrophic conversion of glucose and H2, CO2, and CO were established. Glucose fermentations at pH 6 showed a high ratio of alcohol-to-acid production of 2.79 mol mol-1. While H2 or CO2 were not utilized together with glucose, CO feeding drastically increased the combined alcohol titer to 9.1 g l-1. Specifically, CO enhanced acetate (1.9-fold) and ethanol (1.7-fold) production and triggered chain elongation to butanol (1.5-fold) production but did not change the alcohol:acid ratio. Flux balance analysis showed that CO served both as a carbon and energy source, and CO mixotrophy displayed a carbon and energy efficiency of 45 and 77%, respectively. This study expands the knowledge on physiology and metabolism of C.carboxidivorans and can serve as the starting point for rational engineering and process intensification to establish efficient production of alcohols and acids from carbon waste.


Assuntos
Butanóis , Monóxido de Carbono , 1-Butanol/metabolismo , Butanóis/metabolismo , Dióxido de Carbono/metabolismo , Monóxido de Carbono/metabolismo , Clostridium/metabolismo , Etanol/metabolismo , Fermentação , Glucose/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-35451952

RESUMO

A Gram-stain-positive, anaerobic, spore-forming, rod-shaped (0.4-0.6 µm×2.5-3.2 µm), flagellated bacterium, designated strain YB-6T, was isolated from activated sludge of an anaerobic tank at Weizhou marine oil mining wastewater treatment plant in Beihai, Guangxi, PR China. The culture conditions were 25-45 °C (optimum, 37 °C), pH 4-12 (pH 7.0) and NaCl concentration of 0-7 % w/v (0%). Strain YB-6T grew slowly in petroleum wastewater and removed 68.2 % of the total organic carbon in petroleum wastewater within 10 days. Concentrations of naphthalene, anthracene and phenanthrene at an initial concentration of 50 mg l-1 were reduced by 32.8, 40.4 and 14.6 %, respectively, after 7 days. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain YB-6T belongs to Clostridium cluster I and is most closely related to Clostridium uliginosum CK55T (98.5 % similarity). The genome size of strain YB-6T was 3.96 Mb, and the G+C content was 26.5 mol%. The average nucleotide identity value between strain YB-6T and C. uliginosum CK55T was 81.9 %. The major fatty acids in strain YB-6T were C14 : 0 FAME, C16 : 0 FAME and summed feature 4 (unknown 14.762 and/or C15 : 2 FAME). The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, five unidentified aminophospholipids, one unidentified glycolipid and one unidentified aminolipid. Diaminopimelic acid was not detected in the strain YB-6T cell walls. Whole-cell sugars mainly consisted of ribose and galactose. Based on the results of phenotypic and genotypic analyses, strain YB-6T represents a novel species of the genus Clostridium, for which the name Clostridium weizhouense sp. nov. is proposed. The type strain is YB-6T (=GDMCC 1.2529T=JCM 34754T).


Assuntos
Petróleo , Esgotos , Anaerobiose , Bactérias Anaeróbias/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , China , Clostridium , DNA Bacteriano/genética , Ácidos Graxos/química , Fosfolipídeos/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Esgotos/microbiologia , Águas Residuárias/microbiologia
4.
Microbiol Spectr ; 10(2): e0228821, 2022 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-35412381

RESUMO

Transcription initiation is a tightly regulated process that is crucial for many aspects of prokaryotic physiology. High-throughput transcription start site (TSS) mapping can shed light on global and local regulation of transcription initiation, which in turn may help us understand and predict microbial behavior. In this study, we used Capp-Switch sequencing to determine the TSS positions in the genomes of three model solventogenic clostridia: Clostridium acetobutylicum ATCC 824, C. beijerinckii DSM 6423, and C. beijerinckii NCIMB 8052. We first refined the approach by implementing a normalization pipeline accounting for gene expression, yielding a total of 12,114 mapped TSSs across the species. We further compared the distributions of these sites in the three strains. Results indicated similar distribution patterns at the genome scale, but also some sharp differences, such as for the butyryl-CoA synthesis operon, particularly when comparing C. acetobutylicum to the C. beijerinckii strains. Lastly, we found that promoter structure is generally poorly conserved between C. acetobutylicum and C. beijerinckii. A few conserved promoters across species are discussed, showing interesting examples of how TSS determination and comparison can improve our understanding of gene expression regulation at the transcript level. IMPORTANCE Solventogenic clostridia have been employed in industry for more than a century, initially being used in the acetone-butanol-ethanol (ABE) fermentation process for acetone and butanol production. Interest in these bacteria has recently increased in the context of green chemistry and sustainable development. However, our current understanding of their genomes and physiology limits their optimal use as industrial solvent production platforms. The gene regulatory mechanisms of solventogenesis are still only partly understood, impeding efforts to increase rates and yields. Genome-wide mapping of transcription start sites (TSSs) for three model solventogenic Clostridium strains is an important step toward understanding mechanisms of gene regulation in these industrially important bacteria.


Assuntos
Acetona , Clostridium acetobutylicum , Acetona/metabolismo , Bactérias Anaeróbias , Butanóis/metabolismo , Clostridium/genética , Clostridium/metabolismo , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Fermentação
5.
Chemosphere ; 299: 134425, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35351479

RESUMO

Biofuels, such as ethanol and butanol, obtained from carbon monoxide-rich gas or syngas bioconversion (solventogenesis) are an attractive alternative to traditional fermentation processes with merits of no competition with food production and sustainability. However, there is a lack of comprehensive understanding of some key process parameters and mechanisms enhancing solventogenesis during the fermentation process. This review provides an overview of the current state of the art of the main influencing factors during the syngas fermentation process catalyzed by acetogenic species as well as undefined mixed cultures. The role of syngas pressure, syngas components, fermentation pH, temperature, trace metals, organic compounds and additional materials is overviewed. As a so far hardly considered approach, thermodynamic calculations of the Gibbs free energy of CO conversion to acetic acid, ethanol, butyric acid and butanol under different CO pressures and pH at 25, 33 and 55 °C are also addressed and reviewed. Strategies for enhancing mass transfer and longer carbon chain solvent production are considered as well.


Assuntos
Reatores Biológicos , Clostridium , 1-Butanol , Ácido Acético , Butanóis , Monóxido de Carbono , Etanol , Fermentação , Termodinâmica
6.
Nucleic Acids Res ; 50(6): 3523-3534, 2022 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-35258601

RESUMO

RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a 'Quick' method to generate a single PAM prediction or in a 'Comprehensive' method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.


Assuntos
Sistemas CRISPR-Cas , Biologia Computacional/métodos , Sistemas CRISPR-Cas/genética , Clostridium/genética , Biblioteca Gênica , Motivos de Nucleotídeos
7.
Appl Environ Microbiol ; 88(7): e0247921, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35285680

RESUMO

The majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source. IMPORTANCE Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO2 into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.


Assuntos
Monóxido de Carbono , Clostridium , Processos Autotróficos , Monóxido de Carbono/metabolismo , Clostridium/metabolismo , Elementos de DNA Transponíveis , Genoma Bacteriano , Mutagênese Insercional
8.
Appl Environ Microbiol ; 88(7): e0241921, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35311509

RESUMO

Butyrate is produced by chemical synthesis based on crude oil, produced by microbial fermentation, or extracted from animal fats (M. Dwidar, J.-Y. Park, R. J. Mitchell, and B.-I. Sang, The Scientific World Journal, 2012:471417, 2012, https://doi.org/10.1100/2012/471417). Butyrate production by anaerobic bacteria is highly favorable since waste or sustainable resources can be used as the substrates. For this purpose, the native hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was used as a chassis strain due to its broad substrate spectrum. BLASTp analysis of the predicted proteome of C. saccharoperbutylacetonicum N1-4(HMT) resulted in the identification of gene products potentially involved in acetone-butanol-ethanol (ABE) fermentation. Their participation in ABE fermentation was either confirmed or disproven by the parallel production of acids or solvents and the respective transcript levels obtained by transcriptome analysis of this strain. The genes encoding phosphotransacetylase (pta) and butyraldehyde dehydrogenase (bld) were deleted to reduce acetate and alcohol formation. The genes located in the butyryl-CoA synthesis (bcs) operon encoding crotonase, butyryl-CoA dehydrogenase with electron-transferring protein subunits α and ß, and 3-hydroxybutyryl-CoA dehydrogenase were overexpressed to channel the flux further towards butyrate formation. Thereby, the native hyper-butanol producer C. saccharoperbutylacetonicum N1-4(HMT) was converted into the hyper-butyrate producer C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_PbgaL]. The transcription pattern following deletion and overexpression was characterized by a second transcriptomic study, revealing partial compensation for the deletion. Furthermore, this strain was characterized in pH-controlled fermentations with either glucose or Excello, a substrate yielded from spruce biomass. Butyrate was the main product, with maximum butyrate concentrations of 11.7 g·L-1 and 14.3 g·L-1, respectively. Minimal amounts of by-products were detected. IMPORTANCE Platform chemicals such as butyrate are usually produced chemically from crude oil, resulting in the carry-over of harmful compounds. The selective production of butyrate using sustainable resources or waste without harmful by-products can be achieved by bacteria such as clostridia. The hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was converted into a hyper-butyrate producer. Butyrate production with very small amounts of by-products was established with glucose and the sustainable lignocellulosic sugar substrate Excello extracted from spruce biomass by the biorefinery Borregaard (Sarpsborg, Norway).


Assuntos
Butiratos , Petróleo , 1-Butanol/metabolismo , Acetona/metabolismo , Butanóis/metabolismo , Butiratos/metabolismo , Clostridium/genética , Clostridium/metabolismo , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Lignina , Petróleo/metabolismo , Açúcares/metabolismo
9.
Mol Immunol ; 145: 124-138, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35349868

RESUMO

BACKGROUND AND OBJECTIVE: Asthma is one of the most common chronic inflammatory diseases of the respiratory tract. Previous studies have shown that the reduction of regulatory B cells (Bregs) can increase inflammation of the body and promote the formation of chronic airway inflammation in asthma, but the detailed mechanisms have not been fully elucidated. The intestinal flora Clostridium leptum (CL) has been reported to modulate immune regulatory cells in the body, but the specific mechanisms are not clear. This study aimed to investigate the effects of CL on the differentiation of interleukin (IL)- 10+ Bregs and the regulation of the asthmatic inflammation-associated immune network. METHODS: The abundances of CL and the frequencies of blood Bregs from asthmatic patients and healthy controls were compared. The house dust mite (HDM)-induced asthma model was established in mice. The effects of CL exposure and B cell infusion on Breg differentiation, T cell cytokine production, and inflammatory cell infiltration in mouse lungs were examined. Bregs were cocultured with regulatory T cells (Tregs) and CD4+ non-Tregs to evaluate their roles on Foxp3 expression and T cell differentiation, respectively. RESULTS: Compared with healthy controls, asthmatic patients had significantly reduced frequencies of blood Bregs and abundances of fecal CL, and these two parameters were positively correlated. In the asthma model, the frequencies of Bregs in lungs were significantly reduced; while the infusion of Bregs isolated from CL- supplemented mice significantly reduced airway inflammation and hyperresponsiveness. In addition, Bregs inhibited the differentiation of cocultured non-Tregs into multiple effector cells and enhanced Foxp3 expression in cocultured Tregs. CONCLUSION: Bregs contribute to the alleviation of airway inflammation, which provides insight on implementing CL-based microbial induction of Bregs in asthma therapy.


Assuntos
Asma , Linfócitos B Reguladores , Animais , Clostridium/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Inflamação , Interleucina-10/metabolismo , Pulmão , Camundongos , Linfócitos T Reguladores
10.
Anaerobe ; 74: 102550, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35331913

RESUMO

BACKGROUND: Clostridioides (Clostridium) difficile commonly causes hospital-acquired infection which can range from mild diarrhoea to life-threatening toxic megacolon and even death. Reports on C. difficile infection (CDI) in Vietnam are limited, so this study was designed to evaluate the prevalence, molecular epidemiology and antimicrobial susceptibility of C. difficile isolated from children with diarrhoea in Vietnam. Infants are often colonised with C. difficile and it was hypothesised that those colonising strains would represent strains of C. difficile circulating in the hospital/region at the time, however, this was not an attempt to determine if C. difficile was the cause of the diarrhoea. METHODS: Diarrhoeal stool samples collected at two children's hospitals in northern Vietnam from October 1, 2020 to February 28, 2021 were transported to Perth, Western Australia, for culture of C. difficile and further investigations on isolates; PCR ribotyping, toxin gene profiling and antimicrobial susceptibility testing. RESULTS: From these hospitals, 370 diarrhoeal stool samples were collected, most from children aged 1-15 months (71.9%; 266/370). The overall prevalence of C. difficile in stool samples from children aged ≤16 years was 37.8% (140/370) and the highest prevalence was in the 2-12 months age group (52.9%; 74/140). In total, 151 isolates of C. difficile were recovered; the proportion of toxigenic isolates was 16.6% (25/151). Of the 25 toxigenic C. difficile isolates, the toxin gene profiles A+B+CDT- and A-B+CDT- comprised 72% and 28%, respectively. The four most prevalent C. difficile ribotypes (RTs) were QX 011 (25/151), RT 010 (25/151), QX 107 (12/151) and RT 012 (11/151). All isolates were susceptible to vancomycin, metronidazole and fidaxomicin, while there was significant resistance to clindamycin (90.1%), and some to moxifloxacin (6.6%) and rifaximin (3.3%). CONCLUSION: The prevalence of C. difficile in children with diarrhoea was high (37.8%) although the proportion of toxigenic strains was comparatively low. The clinical significance of any isolate needs to be determined.


Assuntos
Clostridioides difficile , Infecções por Clostridium , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Criança , Clostridioides , Clostridioides difficile/genética , Clostridium/genética , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/epidemiologia , Diarreia/tratamento farmacológico , Diarreia/epidemiologia , Humanos , Lactente , Testes de Sensibilidade Microbiana , Ribotipagem , Vietnã/epidemiologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-35353674

RESUMO

An acid/alcohol-producing, Gram-stain-positive, obligately anaerobic, rod-shaped, non-motile, non-spore forming acetogen, designated as strain P21T, was isolated from old hay after enrichment with CO as the substrate. Spores not observed even after prolonged incubation (30 days). Phylogenetic analysis of the 16S rRNA gene sequence of strain P21T showed it was closely related to Clostridium carboxidivorans DSM 15243T (97.9%), Clostridium scatologenes DSM 757T (97.7 %) and Clostridium drakei DSM 12750T (97.7 %). The genome is 5.6 Mb and the G+C content is 29.4 mol%. Average nucleotide identity between strain P21T, C. carboxidivorans, C. scatologenes and C. drakei was 87.1, 86.4, 86.4 %, respectively. Strain P21T grew on CO:CO2, H2:CO2, l-arabinose, ribose, xylose, fructose, galactose, glucose, lactose, mannose, cellobiose, sucrose, cellulose, starch, pyruvate, choline, glutamate, histidine, serine, threonine and casamino acids. End products of metabolism were acetate, butyrate, caproate, ethanol and hexanol. Dominant cellular fatty acids (>10 %) were C16 : 0 (41.5 %), C16 : 1 ω7c/C16 : 1 ω6c (10.0 %), and a summed feature containing cyclo C17 : 1/C18 : 0 (17.3 %). Based on the phenotypic, chemotaxonomic, phylogenetic and phylogenomic analyses, strain P21T represents a new species in the genus Clostridium, for which the name Clostridium muellerianum sp. nov. is proposed. The type strain is P21T (=DSM 111390T=NCIMB 15261T).


Assuntos
Monóxido de Carbono , Ácidos Graxos , Técnicas de Tipagem Bacteriana , Composição de Bases , Clostridium , DNA Bacteriano/genética , Ácidos Graxos/química , Oxirredução , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
12.
Enzyme Microb Technol ; 157: 110023, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35247829

RESUMO

Thermoanaerobacter thermocopriae-derived thermostable cycloisomaltooligosaccharide (CI)-forming enzymes catalyze the production of CIs from dextran. The primary structure of the enzyme is comprised of CI glucanotransferase (TtCITase) at the N-terminal region and long isomaltooligosaccharide-forming enzyme (TtTGase) at the C-terminal region connected by carbohydrate-binding module family 35 (CBM, TtCBM). Three truncated mutants of CI-forming enzymes were successfully produced in Corynebacterium glutamicum, a food-grade host system, and their biochemical properties were characterized. The enzymes had optimum at pH 6.0 and pH-stability (5.0-12.0). Three enzymes had optimum temperature over 55 °C and they maintained 80% activity at 55 °C for 2 h, 12 h, and 18 h, respectively. Enzymes without CBM showed weaker allosteric behavior than those of other enzymes, which suggests the important role of CBM in allosteric behavior. However, CBM bearing enzymes showed high production of CIs with various degree of polymerization. These enzymes have potential application as the encapsulating material for insoluble pharmaceutical biomaterials.


Assuntos
Glucosiltransferases , Thermoanaerobacter , Carboidratos , Clostridium , Glucosiltransferases/química , Glucosiltransferases/genética , Thermoanaerobacter/genética
13.
Biochem Soc Trans ; 50(2): 867-876, 2022 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-35356968

RESUMO

Biobutanol is gaining much attention as a potential biofuel due to its superior properties over ethanol. Butanol has been naturally produced via acetone-butanol-ethanol (ABE) fermentation by many Clostridium species, which are not very user-friendly bacteria. Therefore, to improve butanol titers and yield, various butanol synthesis pathways have been engineered in Escherichia coli, a much more robust and convenient host than Clostridium species. This review mainly focuses on the biosynthesis of n-butanol in engineered E. coli with an emphasis on efficient enzymes for butanol production in E. coli, butanol competing pathways, and genome engineering of E. coli for butanol production. In addition, the use of alternate strategies for butanol biosynthesis/enhancement, alternate substrates for the low cost of butanol production, and genetic improvement for butanol tolerance in E. coli have also been discussed.


Assuntos
1-Butanol , Butanóis , 1-Butanol/metabolismo , Butanóis/metabolismo , Clostridium/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/metabolismo , Fermentação , Engenharia Metabólica
14.
mSphere ; 7(2): e0092621, 2022 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-35350846

RESUMO

An intact gut microbiota confers colonization resistance against Clostridioides difficile through a variety of mechanisms, likely including competition for nutrients. Recently, proline was identified as an important environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, trans-4-hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate trans-4-hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. Therefore, we constructed a C. difficile ΔhypD mutant and found that it was modestly impaired in fitness in a mouse model of infection, and was associated with an altered microbiota when compared to mice challenged with the wild-type strain. Changes in the microbiota between the two groups were largely driven by members of the Lachnospiraceae family and the Clostridium genus. We found that C. difficile and type strains of three commensal Clostridia had significant alterations to their metabolic gene expression in the presence of trans-4-hydroxyproline in vitro. The proline reductase (prd) genes were elevated in C. difficile, consistent with the hypothesis that trans-4-hydroxyproline is used by C. difficile to supply proline for energy metabolism. Similar transcripts were also elevated in some commensal Clostridia tested, although each strain responded differently. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by trans-4-hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important in determining their role in nutrient competition against C. difficile. IMPORTANCE Proline is an essential environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. We found that C. difficile and three commensal Clostridia strains had significant, but different, alterations to their metabolic gene expression in the presence of hydroxyproline in vitro. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important to determining their role in nutrient competition against C. difficile.


Assuntos
Clostridioides difficile , Infecções por Clostridium , Animais , Clostridioides , Clostridioides difficile/genética , Clostridium , Infecções por Clostridium/metabolismo , Hidroxiprolina/química , Hidroxiprolina/metabolismo , Camundongos , Peptídeo Hidrolases , Prolina/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-35258450

RESUMO

Non-human primates harbour diverse microbiomes in their guts. As a part of the China Microbiome Initiatives, we cultivated and characterized the gut microbiome of cynomolgus monkeys (Macaca fascicularis). In this report, we communicate the characterization and taxonomy of eight bacterial strains that were obtained from faecal samples of captive cynomolgus monkeys. The results revealed that they represented eight novel bacterial species. The proposed names of the eight novel species are Alkaliphilus flagellatus (type strain MSJ-5T=CGMCC 1.45007T=KCTC 15974T), Butyricicoccus intestinisimiae MSJd-7T (MSJd-7T=CGMCC 1.45013T=KCTC 25112T), Clostridium mobile (MSJ-11T=CGMCC 1.45009T=KCTC 25065T), Clostridium simiarum (MSJ-4T=CGMCC 1.45006T=KCTC 15975T), Dysosmobacter acutus (MSJ-2T=CGMCC 1.32896T=KCTC 15976T), Paenibacillus brevis MSJ-6T (MSJ-6T=CGMCC 1.45008T=KCTC 15973T), Peptoniphilus ovalis (MSJ-1T=CGMCC 1.31770T=KCTC 15977T) and Tissierella simiarum (MSJ-40T=CGMCC 1.45012T=KCTC 25071T).


Assuntos
Paenibacillus , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Clostridium , DNA Bacteriano/genética , Ácidos Graxos/química , Fezes , Haplorrinos , Fosfolipídeos , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
16.
Anaerobe ; 74: 102539, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35217150

RESUMO

OBJECTIVES: Carriage of Clostridioides difficile by different species of animals has led to speculation that animals could represent a reservoir of this pathogen for human infections. The objective of this study was to compare C. difficile isolates from humans, dogs, and cattle from a restricted geographic area. METHODS: C. difficile isolates from 36 dogs and 15 dairy calves underwent whole genome sequencing, and phenotypic assays assessing growth and virulence were performed. Genomes of animal-derived isolates were compared to 29 genomes of isolates from a pediatric population as well as 44 reference genomes. RESULTS: Growth rates and relative cytotoxicity of isolates were significantly higher and lower, respectively, in bovine-derived isolates compared to pediatric- and canine-derived isolates. Analysis of core genes showed clustering by host species, though in a few cases, human strains co-clustered with canine or bovine strains, suggesting possible interspecies transmission. Geographic differences (e.g., farm, litter) were small compared to differences between species. In an analysis of accessory genes, the total number of genes in each genome varied between host species, with 6.7% of functional orthologs differentially present/absent between host species and bovine-derived strains having the lowest number of genes. Canine-derived isolates were most likely to be non-toxigenic and more likely to carry phages. A targeted study of episomes identified in local pediatric strains showed sharing of a methicillin-resistance plasmid with dogs, and historic sharing of a wide range of episomes across hosts. Bovine-derived isolates harbored the widest variety of antibiotic-resistance genes, followed by canine CONCLUSIONS: While C. difficile isolates mostly clustered by host species, occasional co-clustering of canine and pediatric-derived isolates suggests the possibility of interspecies transmission. The presence of a pool of resistance genes in animal-derived isolates with the potential to appear in humans given sufficient pressure from antibiotic use warrants concern.


Assuntos
Clostridioides difficile , Infecções por Clostridium , Animais , Antibacterianos/farmacologia , Bovinos , Criança , Clostridioides , Clostridioides difficile/genética , Clostridium , Infecções por Clostridium/epidemiologia , Cães , Humanos
17.
Artigo em Inglês | MEDLINE | ID: mdl-35188883

RESUMO

Some species of the genus Clostridium are efficient acetate producers and have been deemed useful for upgrading industrial biogas. An acetogenic, strictly anaerobic, Gram-stain-positive, subterminal endospore-forming bacterium designated strain PL3T was isolated from peatland soil enrichments with H2 and CO2. Cells of strain PL3T were 0.8-1.0×4.0-10.0 µm in size and rod-shaped. Growth of strain PL3T occurred at pH 6.0-7.5 (optimum, pH 7.0), at 20-40 °C (optimum, 30 °C) and with 0-1.5 % (w/v) NaCl (optimum, 0.5%). Biochemical analyses revealed that strain PL3T metabolized lactose, maltose, raffinose, rhamnose, lactic acid, sorbitol, arabinose and glycerol. Acetic acid was the predominant metabolite under anaerobic respiration with H2/CO2. The major cellular fatty acids were C16 : 0, C16 : 1 cis 9 and C17 : 0 cyc. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, aminolipid and aminophospholipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PL3T belongs to the genus Clostridium with the highest sequence similarity to Clostridium aciditolerans DSM 17425T (98.6 %) followed by Clostridium nitrophenolicum (97.8 %). The genomic DNA G+C content of strain PL3T was 31.1 mol%.The genomic in silico DNA-DNA hybridization value between strain PL3T and C. aciditolerans DSM 17425T was 25.1 %, with an average nucleotide identity of 80.2 %. Based on phenotypic, chemotaxonomic and phylogenetic differences, strain PL3T was suggested to represent a novel species of the genus Clostridium, for which the name Clostridium thailandense sp. nov. is proposed. The type strain is PL3T (=DSM 111812T=TISTR 2984T).


Assuntos
Dióxido de Carbono , Clostridium/classificação , Filogenia , Microbiologia do Solo , Sphagnopsida/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , Dióxido de Carbono/metabolismo , Clostridium/isolamento & purificação , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
18.
Appl Environ Microbiol ; 88(6): e0239321, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35138930

RESUMO

Using the Wood-Ljungdahl pathway, acetogens can nonphotosynthetically fix gaseous C1 molecules, preventing them from entering the atmosphere. Many acetogens can also grow on liquid C1 compounds such as formate and methanol, which avoid the storage and mass transfer issues associated with gaseous C1 compounds. Substrate redox state also plays an important role in acetogen metabolism and can modulate products formed by these organisms. Butyribacterium methylotrophicum is an acetogen known for its ability to synthesize longer-chained molecules such as butyrate and butanol, which have significantly higher values than acetate or ethanol, from one-carbon (C1) compounds. We explored B. methylotrophicum's C1 metabolism by varying substrates, substrate concentrations, and substrate feeding strategies to improve four-carbon product titers. Our results showed that formate utilization by B. methylotrophicum favored acetate production and methanol utilization favored butyrate production. Cofeeding of both substrates produced a high butyrate titer of 4 g/liter when methanol was supplied in excess to formate. Testing of formate feeding strategies, in the presence of methanol, led to further increases in the butyrate to acetate ratio. Mixotrophic growth of liquid and gaseous C1 substrates expanded the B. methylotrophicum product profile, as ethanol, butanol, and lactate were produced under these conditions. We also showed that B. methylotrophicum is capable of producing caproate, a six-carbon product, presumably through chain elongation cycles of the reverse ß-oxidation pathway. Furthermore, we demonstrated butanol production via heterologous gene expression. Our results indicate that both selection of appropriate substrates and genetic engineering play important roles in determining titers of desired products. IMPORTANCE Acetogenic bacteria can fix single-carbon (C1) molecules. However, improvements are needed to overcome poor product titers. Butyribacterium methylotrophicum can naturally ferment C1 compounds into longer-chained molecules such as butyrate alongside traditional acetate. Here, we show that B. methylotrophicum can effectively grow on formate and methanol to produce high titers of butyrate. We improved ratios of butyrate to acetate through adjusted formate feeding strategies and produced higher-value six-carbon molecules. We also expanded the B. methylotrophicum product profile with the addition of C1 gases, as the organism produced ethanol, butanol, and lactate. Furthermore, we developed a transformation protocol for B. methylotrophicum to facilitate genetic engineering of this organism for the circular bioeconomy.


Assuntos
Monóxido de Carbono , Clostridium , Acetatos/metabolismo , Monóxido de Carbono/metabolismo , Clostridium/metabolismo , Metanol/metabolismo
19.
Bioresour Technol ; 348: 126832, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35149183

RESUMO

With concerns over global warming and climate change, many efforts have been devoted to mitigate atmospheric CO2 level. As a CO2 utilization strategy, formate dehydrogenase (FDH) from Clostridium species were explored to discover O2-tolerant and efficient FDHs that can catalyze CO2 to formate (i.e. CO2 reductase). With FDH from Clostridium ljungdahlii (ClFDH) that plays as a CO2 reductase previously reported as the reference, FDH from C.autoethanogenum (CaFDH), C. coskatii (CcFDH), and C. ragsdalei (CrFDH) were newly discovered via genome-mining. The FDHs were expressed in Escherichia coli and the recombinant FDHs successfully catalyzed CO2 reduction with a specific activity of 15 U g-1-CaFDH, 17 U g-1-CcFDH, and 8.7 U g-1-CrFDH. Interestingly, all FDHs newly discovered retain their catalytic activity under aerobic condition, although Clostridium species are strict anaerobe. The results discussed herein can contribute to biocatalytic CO2 utilization.


Assuntos
Dióxido de Carbono , Clostridium/enzimologia , Formiato Desidrogenases , Proteínas de Bactérias/genética , Dióxido de Carbono/metabolismo , Catálise , Clostridium/genética , Formiato Desidrogenases/genética , Formiatos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...