RESUMEN
Staphylococcus aureus is a well-documented bacterial contaminant in platelet concentrates (PCs), a blood component used to treat patients with platelet deficiencies. This bacterium can evade routine PC culture screening and cause septic transfusion reactions. Here, we investigated the gene expression modulation within the PC niche versus trypticase soy media (TSB) of S. aureus CBS2016-05, a strain isolated from a septic reaction, in comparison to PS/BAC/317/16/W, a strain identified during PC screening. RNA-seq analysis revealed upregulation of the capsule biosynthesis operon (capA-H), surface adhesion factors (sasADF), clumping factor A (clfA), protein A (spa), and anaerobic metabolism genes (pflAB, nrdDG) in CBS2016-05 when grown in PCs versus TSB, implying its enhanced pathogenicity in this milieu, in contrast to the PS/BAC/317/16/W strain. Furthermore, we investigated the impact of S. aureus CBS2016-05 on platelet functionality in spiked PCs versus non-spiked PC units. Flow cytometry analyses revealed a significant decrease in glycoprotein (GP) IIb (CD41) and GPIbα (CD42b) expression, alongside increased P-selectin (CD62P) and phosphatidylserine (annexin V) expression in spiked PCs compared to non-spiked PCs (p = 0.01). Moreover, spiked PCs exhibited a drastic reduction in MitoTrack Red FM and Calcein AM positive platelets (87.3% vs. 29.4%, p = 0.0001 and 95.4% vs. 24.7%, p = 0.0001) in a bacterial cell density manner. These results indicated that S. aureus CBS2016-05 triggers platelet activation and apoptosis, and compromises mitochondrial functionality and platelet viability, in contaminated PCs. Furthermore, this study enhanced our understanding of the effects of platelet-bacteria interactions in the unique PC niche, highlighting S. aureus increased pathogenicity and deleterious effect on platelet functionality in a strain specific manner. Our novel insights serve as a platform to improve PC transfusion safety.
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Plaquetas , Regulación Bacteriana de la Expresión Génica , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Plaquetas/microbiología , Plaquetas/metabolismo , Humanos , Virulencia/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Infecciones Estafilocócicas/microbiologíaRESUMEN
Campylobacter jejuni (C. jejuni) is one of the most common causes of foodborne infections worldwide and a major contributor to diarrheal diseases. This study aimed to explore the ability of commensal gut bacteria to control C. jejuni infection. Bacterial strains from the intestinal mucosa of broilers were screened in vitro against C. jejuni ATCC BAA1153. The cell-free supernatant (CFS) of Ligilactobacillus salivarius UO.C249 showed potent dose-dependent antimicrobial activity against the pathogen, likely due to the presence of bacteriocin-like moieties, as confirmed by protease treatment. Genome and exoproteome analyses revealed the presence of known bacteriocins, including Abp118. The genome of Lg. salivarius UO.C249 harbors a 1.8-Mb chromosome and a 203-kb megaplasmid. The strain was susceptible to several antibiotics and had a high survival rate in the simulated chicken gastrointestinal tract (GIT). Post-protease treatment revealed residual inhibitory activity, suggesting alternative antimicrobial mechanisms. Short-chain fatty acid (SCFA) quantification confirmed non-inhibitory levels of acetic (24.4 ± 1.2 mM), isovaleric (34 ± 1.0 µM), and butyric (32 ± 2.5 µM) acids. Interestingly, extracellular vesicles (EVs) isolated from the CFS of Lg. salivarius UO.C249 were found to inhibit C. jejuni ATCC BAA-1153. Proteome profiling of these EVs revealed the presence of unique proteins distinct from bacteriocins identified in CFS. The majority of the identified proteins in EVs are located in the membrane and play roles in transmembrane transport and peptidoglycan degradation, peptidase, proteolysis, and hydrolysis. These findings suggest that although bacteriocins are a primary antimicrobial mechanism, EV production also contributes to the inhibitory activity of Lg. salivarius UO.C249 against C. jejuni. IMPORTANCE: Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and a global public health concern. The increasing antibiotic resistance and lack of effective alternatives in livestock production pose serious challenges for controlling C. jejuni infections. Therefore, alternative strategies are needed to control this pathogen, especially in the poultry industry where it is prevalent and can be transmitted to humans through contaminated food products. In this study, Ligilactobacillus salivarius UO.C249 isolated from broiler intestinal mucosa inhibited C. jejuni and exhibited important probiotic features. Beyond bacteriocins, Lg. salivarius UO.C249 secretes antimicrobial extracellular vesicles (EVs) with a unique protein set distinct from bacteriocins that are involved in transmembrane transport and peptidoglycan degradation. Our findings suggest that beyond bacteriocins, EV production is also a distinct inhibitory signaling mechanism used by Lg. salivarius UO.C249 to control C. jejuni. These findings hold promise for the application of probiotic EVs for pathogen control.
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Bacteriocinas , Campylobacter jejuni , Pollos , Vesículas Extracelulares , Ligilactobacillus salivarius , Probióticos , Bacteriocinas/farmacología , Bacteriocinas/metabolismo , Bacteriocinas/genética , Probióticos/farmacología , Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Animales , Pollos/microbiología , Ligilactobacillus salivarius/fisiología , Antibacterianos/farmacología , Infecciones por Campylobacter/microbiología , Infecciones por Campylobacter/veterinaria , Infecciones por Campylobacter/prevención & controlRESUMEN
Staphylococcus epidermidis is one of the predominant bacterial contaminants in platelet concentrates (PCs), a blood component used to treat bleeding disorders. PCs are a unique niche that triggers biofilm formation, the main pathomechanism of S. epidermidis infections. We performed whole genome sequencing of four S. epidermidis strains isolated from skin of healthy human volunteers (AZ22 and AZ39) and contaminated PCs (ST10002 and ST11003) to unravel phylogenetic relationships and decipher virulence mechanisms compared to 24 complete S. epidermidis genomes in GenBank. AZ39 and ST11003 formed a separate unique lineage with strains 14.1 .R1 and SE95, while AZ22 formed a cluster with 1457 and ST10002 closely grouped with FDAAGOS_161. The four isolates were assigned to sequence types ST1175, ST1174, ST73 and ST16, respectively. All four genomes exhibited biofilm-associated genes ebh, ebp, sdrG, sdrH and atl. Additionally, AZ22 had sdrF and aap, whereas ST10002 had aap and icaABCDR. Notably, AZ39 possesses truncated ebh and sdrG and harbours a toxin-encoding gene. All isolates carry multiple antibiotic resistance genes conferring resistance to fosfomycin (fosB), ß-lactams (blaZ) and fluoroquinolones (norA). This study reveales a unique lineage for S. epidermidis and provides insight into the genetic basis of virulence and antibiotic resistance in transfusion-associated S. epidermidis strains.
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In this study, we aimed to develop a protective probiotic coculture to inhibit the growth of Salmonella enterica serovar Typhimurium in the simulated chicken gut environment. Bacterial strains were isolated from the digestive mucosa of broilers and screened in vitro against Salmonella Typhimurium ATCC 14028. A biocompatibility coculture test was performed, which identified two biocompatible strains, Ligilactobacillus salivarius UO.C109 and Ligilactobacillus saerimneri UO.C121 with high inhibitory activity against Salmonella. The cell-free supernatant (CFS) of the selected isolates exhibited dose-dependent effects, and the inhibitory agents were confirmed to be proteinaceous by enzymatic and thermal treatments. Proteome and genome analyses revealed the presence of known bacteriocins in the CFS of L. salivarius UO.C109, but unknown for L. saerimneri UO.C121. The addition of these selected probiotic candidates altered the bacterial community structure, increased the diversity of the chicken gut microbiota challenged with Salmonella, and significantly reduced the abundances of Enterobacteriaceae, Parasutterlla, Phascolarctobacterium, Enterococcus, and Megamonas. It also modulated microbiome production of short-chain fatty acids (SCFAs) with increased levels of acetic and propionic acids after 12 and 24 h of incubation compared to the microbiome challenged with S. Typhimurium. Furthermore, the selected probiotic candidates reduced the adhesion and invasion of Salmonella to Caco-2 cells by 37-39% and 51%, respectively, after 3 h of incubation, compared to the control. These results suggest that the developed coculture probiotic strains has protective activity and could be an effective strategy to control Salmonella infections in poultry.
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BACKGROUND AND OBJECTIVES: Staphylococcus aureus is a predominant contaminant of platelet concentrates (PCs) that can evade detection during screening with culture methods. Importantly, S. aureus produces staphylococcal enterotoxins (SEs) during PC storage, which are linked to slow growth and enhanced biofilm formation. This study investigated timing of SE production during PC storage and feasibility of SE detection as a PC safety strategy. MATERIALS AND METHODS: Genomic and transcriptomic data of transfusion-relevant S. aureus PS/BAC/169/17/W, PS/BAC/317/16/W, CI/BAC/25/13/W and CBS2016-05 were used to determine the presence and differential expression of exotoxin genes in PCs. Trypticase soy broth (TSB) and PCs were inoculated with 1.0E+06 cfu/mL of S. aureus PS/BAC/169/17/W and CBS2016-05. Expression of SEs at different growth phases was confirmed with Western blotting. PCs were inoculated with 30 cfu/unit of the same strains, and SE detection during PC storage was optimized with a sandwich dot-ELISA assay. RESULTS: S. aureus genomes contain multiple exotoxin genes including those encoding for SEs. Transcriptome data revealed significant upregulation (0.5-6.7-fold, p < 0.05) of SE genes in PCs versus TSB. Western blots demonstrated SE production at all growth phases. Notably, dot-ELISA detected clinically relevant concentrations of SEs (~0.2 µg/mL) at 32 h of PC storage when S. aureus PS/BAC/169/17/W and CBS2016-05 counts were 1.8E+04 and 1.4E+04 cfu/mL, respectively. CONCLUSION: Genomic analyses revealed that staphylococcal exotoxins are widely distributed and highly conserved among transfusion-relevant S. aureus isolates. Furthermore, SEs are significantly upregulated in PCs and detected at 30 h of PC storage. Therefore, bacterial toxin detection could supplement mitigation strategies to enhance PC safety.
Asunto(s)
Enterotoxinas , Infecciones Estafilocócicas , Humanos , Enterotoxinas/genética , Enterotoxinas/metabolismo , Staphylococcus aureus/genética , Infecciones Estafilocócicas/prevención & control , Infecciones Estafilocócicas/microbiologíaRESUMEN
We announce the draft genome sequences of 12 Bacteroides, 4 Phocaeicola, and 2 Parabacteroides strains, among which was a newly isolated species, Bacteroidaceae bacterium UO.H1004. These isolates produce health-benefiting short-chain fatty acids (SCFAs) and the neurotransmitter γ-aminobutyric acid (GABA) in various concentrations.
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BACKGROUND AND OBJECTIVES: Platelet concentrates (PCs) contaminated with Staphylococcus aureus can escape detection during PC screening, causing septic transfusion reactions. This study aimed to determine the impact of S. aureus contamination on platelet metabolism and functionality during PC storage. MATERIALS AND METHODS: Targeted metabolomics (N = 3) was performed on non-spiked PCs and PCs inoculated with 10-20 colony-forming units (CFU)/bag of S. aureus. Metabolites were quantified at 0, 48 and 144 h using high-performance mass spectrometry (MS). Additionally, PCs spiked with approximately 20 CFU/bag of S. aureus were sampled every 24 h for up to 144 h to evaluate platelet functionality using flow cytometry (N = 2). RESULTS: Eight metabolites had significantly different levels in spiked PCs (log2 fold-change ≤ or ≥±1) versus non-spiked units at 48 and 144 h. Xanthine, uridine, serine, glutamine and threonine were increased, whereas orotic acid, dihydroorotic acid and aspartic acid were decreased. Flow cytometry showed a significant decrease in expression of GPIIb while P-selectin expression was significantly increased in spiked PCs after 72 h of storage when S. aureus concentration was ≥10E+08 CFU/ml. Additionally, phosphatidylserine exposure was significantly increased after 48 h of PC storage, when S. aureus had reached a concentration of 2E+06. CONCLUSION: Contamination with S. aureus exacerbates platelet storage lesions in contaminated PCs but only when the bacterium has reached clinically significant levels.
Asunto(s)
Plaquetas , Staphylococcus aureus , Humanos , Plaquetas/microbiología , Pruebas de Función Plaquetaria , Contaminación de Medicamentos , Bacterias , Transfusión de PlaquetasRESUMEN
Anti-virulence agents are non-bacteriostatic and non-bactericidal emerging therapeutic options which hamper the production of virulence factors in pathogenic flora. In Staphylococcus aureus and Enterococcus faecalis, regulation of virulence genes' expression occurs through the cyclic peptide-mediated accessory gene regulator (agr) and its ortholog fsr quorum sensing systems, respectively. In the present study, we screened a set of 54 actinomycetales secondary metabolites as novel anti-virulence compounds targeting quorum sensing system of the Gram-positive bacteria. The results indicated that four compounds, Phenalinolactones A-D, BU-4664LMe, 4,5-dehydrogeldamycin, and Questinomycin A, potentially inhibit the agr quorum sensing system and hemolytic activity of S. aureus. On the other hand, Decatromicin A and B, Okilactomycin, Rishirilide A, Abyssomicin I, and Rebeccamycin selectively blocked the fsr quorum sensing system and the gelatinase production in E. faecalis at sub-lethal concentrations. Interestingly, Synerazol uniquely showed the capability to inhibit both fsr and agr quorum sensing systems. Further, in silico molecular docking studies were performed which provided closer insights into the mode of action of these compounds and proposed that the inhibitory activity of these compounds could be attributed to their potential ability to bind to the ATP-active site of S. aureus AgrA. Taken together, our study highlights the potential of actinomycetales secondary metabolites with diverse structures as anti-virulence quorum sensing inhibitors.
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We present the genome sequence of Staphylococcus aureus strain PS/BAC/317/16/W, which was isolated from contaminated platelet concentrates by the National Health Service Blood and Transplant in England (2017). Genome sequence analysis revealed the presence of one chromosome (2,665,983 bp) and two plasmids (4,265 bp and 2,921 bp) in this strain.
RESUMEN
We present the genome sequence of Staphylococcus aureus strain CBS2016-05, which was isolated from contaminated platelet concentrates by Canadian Blood Services in 2016. This strain caused a septic reaction in an acute leukemia patient. Genome sequence analysis revealed the presence of one chromosome (2,766,936 bp) and one plasmid (36,441 bp).
RESUMEN
BACKGROUND: Staphylococcus epidermidis forms surface-attached aggregates (biofilms) when grown in platelet concentrates (PCs). Comparative transcriptome analyses were undertaken to investigate differential gene expression of S. epidermidis biofilms grown in PCs. STUDY DESIGN AND METHODS: Two S. epidermidis strains isolated from human skin (AZ22 and AZ39) and one strain isolated from contaminated PCs (ST02) were grown in glucose-supplemented Trypticase Soy Broth (TSBg) and PCs. RNA was extracted and sequenced using Illumina HiSeq. Differential expression analysis was done using DESeq, and significantly differentially expressed genes (DEGs) were selected. DEGs were subjected to Kyoto encyclopedia of genes and genomes and Gene Ontology analyses. Differential gene expression was validated with quantitative reverse transcription-PCR. RESULTS: A total of 436, 442, and 384 genes were expressed in AZ22, AZ39, and ST02, respectively. DEG analysis showed that 170, 172, and 117 genes were upregulated in PCs in comparison to TSBg, whereas 120, 135, and 89 genes were downregulated (p < .05) in mature biofilms of AZ22, AZ39, and ST02, respectively. Twenty-seven DEGs were shared by all three strains. While 76 DEGs were shared by AZ22 and AZ39, only 34 and 21 DEGs were common between ST02, and AZ22 and AZ39, respectively. Significant transcriptional expression changes were observed in genes involved in platelet-bacteria interaction, biofilm formation, production of virulence factors, and resistance to antimicrobial peptides and antibiotics. CONCLUSION: Differential gene expression in S. epidermidis is triggered by the stressful PC storage environment. Upregulation of virulence and antimicrobial resistance genes could have clinical implications for transfusion patients.
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Bacteriemia/microbiología , Biopelículas/crecimiento & desarrollo , Plaquetas/microbiología , Regulación Bacteriana de la Expresión Génica , Staphylococcus epidermidis/genética , Secuencia de Bases , Conservación de la Sangre , Farmacorresistencia Microbiana/genética , Ontología de Genes , Humanos , ARN Bacteriano/biosíntesis , ARN Bacteriano/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/microbiología , Staphylococcus epidermidis/efectos de los fármacos , Staphylococcus epidermidis/crecimiento & desarrollo , Staphylococcus epidermidis/aislamiento & purificación , TranscriptomaRESUMEN
Clostridium perfringens produces various exotoxins and enzymes that cause food poisoning and gas gangrene. The genes involved in virulence are regulated by the agr-like quorum sensing (QS) system, which consists of a QS signal synthesis system and a VirSR two-component regulatory system (VirSR TCS) which is a global regulatory system composed of signal sensor kinase (VirS) and response regulator (VirR). We found that the perfringolysin O gene (pfoA) was transiently expressed during mid-log phase of bacterial growth; its expression was rapidly shut down thereafter, suggesting the existence of a self-quorum quenching (sQQ) system. The sQQ system was induced by the addition of stationary phase culture supernatant (SPCS). Activity of the sQQ system was heat stable, and was present following filtration through the ultrafiltration membrane, suggesting that small molecules acted as sQQ agents. In addition, sQQ was also induced by pure acetic and butyric acids at concentrations equivalent to those in the stationary phase culture, suggesting that organic acids produced by C. perfringens were involved in sQQ. In pH-controlled batch culture, sQQ was greatly diminished; expression level of pfoA extended to late-log growth phase, and was eventually increased by one order of magnitude. Furthermore, hydrochloric acid induced sQQ at the same pH as was used in organic acids. SPCS also suppressed the expression of genes regulated by VirSR TCS. Overall, the expression of virulence factors of C. perfringens was downregulated by the sQQ system, which was mediated by primary acidic metabolites and acidic environments. This suggested the possibility of pH-controlled anti-virulence strategies.
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Ácidos/farmacología , Clostridium perfringens/efectos de los fármacos , Clostridium perfringens/metabolismo , Redes y Vías Metabólicas/fisiología , Percepción de Quorum/efectos de los fármacos , Percepción de Quorum/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Clostridium perfringens/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Reguladores/efectos de los fármacos , Genes Reguladores/fisiología , Proteínas Hemolisinas/genética , Concentración de Iones de Hidrógeno , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Percepción de Quorum/genética , Factores de Virulencia/genéticaRESUMEN
The aim of this study was to compare the antibacterial properties of the surfaces of copper plates that were rolled to a thickness of 25 and 100 µm. Differences in topology of 25- and 100-µm-thick copper plates were studied using scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray diffraction (XRD). Antibacterial activity of the copper surfaces was tested against strains of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Streptococcus sp. BY1, Enterococcus sp. BY2, and Bacillus cereus BY3. Changes in viable cell numbers were determined by plating onto optimal growth media and staining with LIVE/DEAD BacLight™. Changes in metabolic activity were recorded by expression of the luciferase (lux) gene. Cell morphology was studied using SEM. Accumulation and diffusion of copper from cells were recorded using inductively coupled plasma mass spectroscopy (ICP-MS). Lipid and protein oxidation were recorded spectrophotometrically. Surfaces of 25-µm-thick copper plates were rough compared to that of 100-µm-thick copper plates. For most species, a five-log reduction in cell numbers, cell membrane instability, and a decline in metabolic activity were recorded after 15 min of exposure to 25-µm-thick copper plates. Copper accumulated in the cells, and lipids and proteins were oxidized. The rough surface of thinner copper plates (25 µm thick) released more copper and was more antimicrobial compared to thicker (100 µm) copper plates. Cell death was attributed to destabilization of the cell membrane, lipid peroxidation, and protein oxidation.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Cobre/farmacología , Antibacterianos/química , Bacterias/metabolismo , Bacterias/ultraestructura , Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Peroxidación de Lípido/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/ultraestructura , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Propiedades de Superficie , Difracción de Rayos XRESUMEN
The Gulf of Cambay is a trumpet-shaped inlet of the Arabian Sea, located along the west coast of India and confronts a high tidal range with strong water currents. The region belongs to a semi-arid zone and saline alkaline intertidal soils are considered biologically extreme. The selected four soil types (S1-S4) were affected by salinity, alkalinity and sodicity. Soil salinity ranged from 20 to 126 dS/m, soil pH 8.6-10.0 with high sodium adsorption ratio (SAR) and exchangeable sodium percentage (ESP). Abundance of the key functional genes like cbbL, nifH, amoA and apsA involved in biogeochemical cycling were targeted using qPCR, which varied from (2.36 ± 0.03) × 10(4) to (2.87 ± 0.26) × 10(8), (1.18 ± 0.28) × 10(6) to (1.01 ± 0.26) × 10(9), (1.41 ± 0.21) × 10(6) to (1.29 ± 0.05) × 10(8) and (8.47 ± 0.23) × 10(4) to (1.73 ± 0.01) × 10(6) per gram dry weight, respectively. The microbial community structure revealed that soils S1 and S3 were dominated by phylum Firmicutes whereas S4 and S2 showed an abundance of Proteobacterial clones. These soils also represented Bacteroidetes, Chloroflexi, Actinobacteria, Planctomycetes and Acidobacteria clones. Molecular phylogeny showed a significant variation in the bacterial community distribution among the intertidal soil types. A high number of novel taxonomic units were observed which makes the intertidal zone a unique reservoir of unidentified bacterial taxa that may be explored further.
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Bacterias/clasificación , Bacterias/genética , Biota , Genes Bacterianos , Sedimentos Geológicos/microbiología , Microbiología del Suelo , Bacterias/aislamiento & purificación , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Concentración de Iones de Hidrógeno , India , Datos de Secuencia Molecular , ARN Ribosómico 16S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Salinidad , Análisis de Secuencia de ADN , Suelo/químicaRESUMEN
Bacterial autotrophy contributes significantly to the overall carbon balance, which stabilises atmospheric CO2 concentration and decelerates global warming. Little attention has been paid to different modes of carbon/sulphur metabolism mediated by autotrophic bacterial communities in terrestrial soil ecosystems. We studied these pathways by analysing the distribution and abundance of the diagnostic metabolic marker genes cbbM, apsA and soxB, which encode for ribulose-1,5-bisphosphate carboxylase/oxygenase, adenosine phosphosulphate reductase and sulphate thiohydrolase, respectively, among different contrasting soil types. Additionally, the abundance of community members was assessed by quantifying the gene copy numbers for 16S rRNA, cbbL, cbbM, apsA and soxB. Distinct compositional differences were observed among the clone libraries, which revealed a dominance of phylotypes associated with carbon and sulphur cycling, such as Gammaproteobacteria (Thiohalomonas, Allochromatium, Chromatium, Thiomicrospira) and Alphaproteobacteria (Rhodopseudomonas, Rhodovulum, Paracoccus). The rhizosphere soil was devoid of sulphur metabolism, as the soxB and apsA genes were not observed in the rhizosphere metagenome, which suggests the absence or inadequate representation of sulphur-oxidising bacteria. We hypothesise that the novel Gammaproteobacteria sulphur oxidisers might be actively involved in sulphur oxidation and inorganic carbon fixation, particularly in barren saline soil ecosystems, suggesting their significant putative ecological role and contribution to the soil carbon pool.
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Carbono/metabolismo , Ecosistema , Metagenoma , Microbiota , Microbiología del Suelo , Suelo/química , Azufre/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Biodiversidad , Genes Bacterianos , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16SRESUMEN
Diazotrophs are key players of the globally important biogeochemical nitrogen cycle, having a significant role in maintaining ecosystem sustainability. Saline soils are pristine and unexplored habitats representing intriguing ecosystems expected to harbour potential diazotrophs capable of adapting in extreme conditions, and these implicated organisms are largely obscure. Differential occurrence of diazotrophs was studied by the nifH gene-targeted clone library approach. Four nifH gene clone libraries were constructed from different soil niches, that is saline soils (low and high salinity; EC 3.8 and 7.1 ds m(-1) ), and agricultural and rhizosphere soil. Additionally, the abundance of diazotrophic community members was assessed using quantitative PCR. Results showed environment-dependent metabolic versatility and the presence of nitrogen-fixing bacteria affiliated with a range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Cyanobacteria and Firmicutes. The analyses unveiled the dominance of Alphaproteobacteria and Gammaproteobacteria (Pseudomonas, Halorhodospira, Ectothiorhodospira, Bradyrhizobium, Agrobacterium, Amorphomonas) as nitrogen fixers in coastal-saline soil ecosystems, and Alphaproteobacteria and Betaproteobacteria (Bradyrhizobium, Azohydromonas, Azospirillum, Ideonella) in agricultural/rhizosphere ecosystems. The results revealed a repertoire of novel nitrogen-fixing bacterial guilds particularly in saline soil ecosystems.
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Biota , Fijación del Nitrógeno , Oxidorreductasas/genética , Microbiología del Suelo , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Sequestration of CO(2) by autotrophic bacteria is a key process of biogeochemical carbon cycling in soil ecosystem. Rhizosphere is a rich niche of microbial activity and diversity, influenced by change in atmospheric CO(2). Structural changes in rhizosphere composition influence microbial communities and the nutrient cycling. In the present study, the bacterial diversity and population dynamics were established using cbbL and 16S rRNA gene targeted metagenomics approach from the rhizosphere of Arachis hypogaea. A total of 108 cbbL clones were obtained from the rhizospheric soil which revealed predominance of cbbL sequences affiliated to Rhizobium leguminosarum, Bradyrhizobium sp., Sinorhizobium meliloti, Ochrobactrum anthropi and a variety of uncultured cbbL harboring bacteria. The 16S rRNA gene clone library exhibited the dominance of Firmicutes (34.4%), Proteobacteria (18.3%), Actinobacteria (17.2%) and Bacteroidetes (16.1%). About 43% nucleotide sequences of 16S rRNA gene clone library were novel genera which showed <95% homology with published sequences. Gene copy number of cbbL and 16S rRNA genes, determined by quantitative real-time PCR (qRT PCR), was 9.38 ± 0.75 × 10(7) and 5.43 ± 0.79 × 10(8) (per g dry soil), respectively. The results exhibited bacterial community structure with high bacterial diversity and abundance of CO(2)-fixing bacteria, which can be explored further for their role in carbon cycling, sustainable agriculture and environment management.
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Arachis/genética , Arachis/microbiología , Genes Bacterianos , Rizosfera , Arachis/metabolismo , Secuencia de Bases , Ciclo del Carbono/genética , Dióxido de Carbono/metabolismo , Clonación Molecular , Dosificación de Gen , Biblioteca de Genes , Metagenómica , Datos de Secuencia Molecular , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genéticaRESUMEN
BACKGROUND: Soils harbour high diversity of obligate as well as facultative chemolithoautotrophic bacteria that contribute significantly to CO2 dynamics in soil. In this study, we used culture dependent and independent methods to assess the community structure and diversity of chemolithoautotrophs in agricultural and coastal barren saline soils (low and high salinity). We studied the composition and distribution of chemolithoautotrophs by means of functional marker gene cbbL encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a phylogenetic marker 16S rRNA gene. The cbbL form IA and IC genes associated with carbon fixation were analyzed to gain insight into metabolic potential of chemolithoautotrophs in three soil types of coastal ecosystems which had a very different salt load and sulphur content. RESULTS: In cbbL libraries, the cbbL form IA was retrieved only from high saline soil whereas form IC was found in all three soil types. The form IC cbbL was also amplified from bacterial isolates obtained from all soil types. A number of novel monophyletic lineages affiliated with form IA and IC phylogenetic trees were found. These were distantly related to the known cbbL sequences from agroecosystem, volcanic ashes and marine environments. In 16S rRNA clone libraries, the agricultural soil was dominated by chemolithoautotrophs (Betaproteobacteria) whereas photoautotrophic Chloroflexi and sulphide oxidizers dominated saline ecosystems. Environmental specificity was apparently visible at both higher taxonomic levels (phylum) and lower taxonomic levels (genus and species). The differentiation in community structure and diversity in three soil ecosystems was supported by LIBSHUFF (P = 0.001) and UniFrac. CONCLUSION: This study may provide fundamentally new insights into the role of chemolithoautotrophic and photoautotrophic bacterial diversity in biochemical carbon cycling in barren saline soils. The bacterial communities varied greatly among the three sites, probably because of differences in salinity, carbon and sulphur contents. The cbbL form IA-containing sulphide-oxidizing chemolithotrophs were found only in high saline soil clone library, thus giving the indication of sulphide availability in this soil ecosystem. This is the first comparative study of the community structure and diversity of chemolithoautotrophic bacteria in coastal agricultural and saline barren soils using functional (cbbL) and phylogenetic (16S rDNA) marker genes.