scholarly journals Diazotrophic Anaeromyxobacter Isolates from Soils

2020 ◽  
Vol 86 (16) ◽  
Author(s):  
Yoko Masuda ◽  
Haruka Yamanaka ◽  
Zhen-Xing Xu ◽  
Yutaka Shiratori ◽  
Toshihiro Aono ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Paddy Soils ◽  
Soil Environment ◽  
Soil Dna ◽  
Content Type ◽  
Environmental Function ◽  
Culture Independent ◽  
Nitrogenase Genes ◽  
Dependent Growth

ABSTRACT Biological nitrogen fixation is an essential reaction in a major pathway for supplying nitrogen to terrestrial environments. Previous culture-independent analyses based on soil DNA/RNA/protein sequencing could globally detect the nitrogenase genes/proteins of Anaeromyxobacter (in the class Deltaproteobacteria), commonly distributed in soil environments and predominant in paddy soils; this suggests the importance of Anaeromyxobacter in nitrogen fixation in soil environments. However, direct experimental evidence is lacking; there has been no research on the genetic background and ability of Anaeromyxobacter to fix nitrogen. Therefore, we verified the diazotrophy of Anaeromyxobacter based on both genomic and culture-dependent analyses using Anaeromyxobacter sp. strains PSR-1 and Red267 isolated from soils. Based on the comparison of nif gene clusters, strains PSR-1 and Red267 as well as strains Fw109-5, K, and diazotrophic Geobacter and Pelobacter in the class Deltaproteobacteria contain the minimum set of genes for nitrogenase (nifBHDKEN). These results imply that Anaeromyxobacter species have the ability to fix nitrogen. In fact, Anaeromyxobacter PSR-1 and Red267 exhibited N2-dependent growth and acetylene reduction activity (ARA) in vitro. Transcriptional activity of the nif gene was also detected when both strains were cultured with N2 gas as a sole nitrogen source, indicating that Anaeromyxobacter can fix and assimilate N2 gas by nitrogenase. In addition, PSR-1- or Red267-inoculated soil showed ARA activity and the growth of the inoculated strains on the basis of RNA-based analysis, demonstrating that Anaeromyxobacter can fix nitrogen in the paddy soil environment. Our study provides novel insights into the pivotal environmental function, i.e., nitrogen fixation, of Anaeromyxobacter, which is a common soil bacterium. IMPORTANCE Anaeromyxobacter is globally distributed in soil environments, especially predominant in paddy soils. Current studies based on environmental DNA/RNA analyses frequently detect gene fragments encoding nitrogenase of Anaeromyxobacter from various soil environments. Although the importance of Anaeromyxobacter as a diazotroph in nature has been suggested by culture-independent studies, there has been no solid evidence and validation from genomic and culture-based analyses that Anaeromyxobacter fixes nitrogen. This study demonstrates that Anaeromyxobacter harboring nitrogenase genes exhibits diazotrophic ability; moreover, N2-dependent growth was demonstrated in vitro and in the soil environment. Our findings indicate that nitrogen fixation is important for Anaeromyxobacter to survive under nitrogen-deficient environments and provide a novel insight into the environmental function of Anaeromyxobacter, which is a common bacterium in soils.

scholarly journals Small-Molecule Inhibition of Choline Catabolism in Pseudomonas aeruginosa and Other Aerobic Choline-Catabolizing Bacteria

2011 ◽  
Vol 77 (13) ◽  
pp. 4383-4389 ◽  
Author(s):  
Liam F. Fitzsimmons ◽  
Stevenson Flemer ◽  
A. Sandy Wurthmann ◽  
P. Bruce Deker ◽  
Indra Neil Sarkar ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Burkholderia Cepacia ◽  
Sinorhizobium Meliloti ◽  
Growth Inhibitor ◽  
Bioinformatic Analysis ◽  
Content Type ◽  
Genes Encoding ◽  
Dependent Growth

ABSTRACTCholine is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolismin vitroandin situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation inPseudomonas aeruginosa. We used genetic analyses and13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor inP. aeruginosabut did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, includingPseudomonas mendocina,Pseudomonas fluorescens,Pseudomonas putida,Burkholderia cepacia,Burkholderia ambifaria, andSinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolismin situ.

scholarly journals Vaginal Gel Component Hydroxyethyl Cellulose Significantly Enhances the Infectivity ofChlamydia trachomatisSerovars D and E

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Tímea Raffai ◽  
Katalin Burián ◽  
László Janovák ◽  
Anita Bogdanov ◽  
Johannes H. Hegemann ◽  
...  
Keyword(s):  
Fold Increase ◽  
Hydroxyethyl Cellulose ◽  
Gelling Agent ◽  
Maximal Increase ◽  
Content Type ◽  
Enhancing Effect ◽  
Vaginal Gels ◽  
Dependent Growth

ABSTRACTThe transmission of the urogenital serovars ofChlamydia trachomatiscan be significantly influenced by vaginal gels. Hydroxyethyl cellulose is a commonly used gelling agent that can be found in vaginal gels. Hydroxyethyl cellulose showed a concentration-dependent growth-enhancing effect onC. trachomatisserovars D and E, with a 26.1-fold maximal increasein vitroand a 2.57-fold increasein vivo.

scholarly journals Wide Variation in Antibiotic Resistance Proteins Identified by Functional Metagenomic Screening of a Soil DNA Library

2012 ◽  
Vol 78 (6) ◽  
pp. 1708-1714 ◽  
Author(s):  
Kelly M. McGarvey ◽  
Konstantin Queitsch ◽  
Stanley Fields
Keyword(s):  
Antibiotic Resistance ◽  
Protein Function ◽  
Short Form ◽  
Soil Dna ◽  
Large Variability ◽  
Content Type ◽  
Resistance Proteins ◽  
Dna Library ◽  
Culture Independent ◽  
Dihydrofolate Reductases

ABSTRACTMost genes for antibiotic resistance present in soil microbes remain unexplored because most environmental microbes cannot be cultured. Only recently has the identification of these genes become feasible through the use of culture-independent methods. We screened a soil metagenomic DNA library in anEscherichia colihost for genes that can confer resistance to kanamycin, gentamicin, rifampin, trimethoprim, chloramphenicol, or tetracycline. The screen revealed 41 genes that encode novel protein variants of eight protein families, including aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases, dihydrofolate reductases, and transporters. Several proteins of the same protein family deviate considerably from each other yet confer comparable resistance. For example, five dihydrofolate reductases sharing at most 44% amino acid sequence identity in pairwise comparisons were equivalent in conferring trimethoprim resistance. We identified variants of aminoglycoside acetyltransferases and transporters that differ in the specificity of the drugs for which they confer resistance. We also found wide variation in protein structure. Two forms of rifampin ADP-ribosyltransferases, one twice the size of the other, were similarly effective at conferring rifampin resistance, although the short form was expressed at a much lower level. Functional metagenomic screening provides insight into the large variability in antibiotic resistance protein sequences, revealing divergent variants that preserve protein function.

scholarly journals Microbial gas vesicles as nanotechnology tools: exploiting intracellular organelles for translational utility in biotechnology, medicine and the environment

Microbiology ◽  
2020 ◽  
Vol 166 (6) ◽  
pp. 501-509 ◽  
Author(s):  
Amy M. Hill ◽  
George P. C. Salmond
Keyword(s):  
Cyanobacterial Blooms ◽  
Gas Vesicles ◽  
Reporter System ◽  
Viral Pathogens ◽  
Content Type ◽  
Environmental Function ◽  
Link Type ◽  
Intracellular Organelles

A range of bacteria and archaea produce gas vesicles as a means to facilitate flotation. These gas vesicles have been purified from a number of species and their applications in biotechnology and medicine are reviewed here. Halobacterium sp. NRC-1 gas vesicles have been engineered to display antigens from eukaryotic, bacterial and viral pathogens. The ability of these recombinant nanoparticles to generate an immune response has been quantified both in vitro and in vivo. These gas vesicles, along with those purified from Anabaena flos-aquae and Bacillus megaterium , have been developed as an acoustic reporter system. This system utilizes the ability of gas vesicles to retain gas within a stable, rigid structure to produce contrast upon exposure to ultrasound. The susceptibility of gas vesicles to collapse when exposed to excess pressure has also been proposed as a biocontrol mechanism to disperse cyanobacterial blooms, providing an environmental function for these structures.

scholarly journals NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Yuhua Zhan ◽  
Zhiping Deng ◽  
Yongliang Yan ◽  
Hongyang Zhang ◽  
Chao Lu ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Stress Responses ◽  
Posttranscriptional Regulation ◽  
Noncoding Rna ◽  
Nitrogenase Activity ◽  
Pseudomonas Stutzeri ◽  
Fine Tuning ◽  
Coding Region ◽  
Content Type ◽  
Nitrogenase Genes

ABSTRACT Expression of nitrogenase genes (nifHDK) is strictly regulated at both transcriptional and posttranscriptional levels. Efficient nitrogenase activity requires maintaining sufficient levels of nif mRNAs, yet the underlying mechanism is not fully understood due to its complexity. We have previously shown that a novel regulatory noncoding RNA (ncRNA), NfiS, optimizes nitrogen fixation through targeting nifK mRNA in Pseudomonas stutzeri A1501. Here, we report the identification and characterization of a second ncRNA inducible under nitrogen fixation conditions (nitrogen-free and microaerobic conditions), termed NfiR (for nitrogen fixation condition-inducible ncRNA), the expression of which is dependent on two global regulators, NtrC and Hfq. Comparative phenotypic and proteomic analyses of an nfiR mutant identify a role of NfiR in regulating the expression of nitrogenase genes. Further microscale thermophoresis and genetic complementation showed that an 11-nucleotide (nt) sequence in the stem-loop structure of NfiR (nucleotides 12 to 22) pairs with its counterpart in the coding region of nifD mRNA (nucleotides 1194 to 1207) by eight nucleotides. Significantly, deletion of nfiR caused a 60% reduction of nitrogenase activity, and the half-life of nifD mRNA was reduced from 20 min for the wild type to 15 min for the ΔnfiR mutant. With regard to nitrogenase activity and stability of the nifD and nifK transcripts, phenotypes were more severe for the double deletion mutant lacking nfiR and nfiS, suggesting that NfiR, in concert with NfiS, optimizes nitrogenase production at the posttranscriptional level. IMPORTANCE Biological nitrogen fixation is an energy-expensive process requiring the hydrolysis of 16 ATPs. Consequently, the expression of nif genes is highly regulated at both transcriptional and posttranscriptional levels through complex regulatory networks. Global regulation involves a number of regulatory proteins, such as the nif-specific activator NifA and the global nitrogen regulator NtrC, as well as various regulatory ncRNAs. We show that the two P. stutzeri ncRNAs, namely NfiS and NfiR (for nitrogen fixation condition-inducible ncRNA), optimize nitrogen fixation and environmental stress responses. NfiS and NfiR respond differently to various environmental signals and differ in their secondary structures. In addition, the two ncRNAs target the mRNAs of nifK and nifD, respectively. Such ncRNA-based posttranscriptional regulation of nitrogenase expression might be an evolved survival strategy, particularly in nitrogen-limiting environments. This study not only highlights the significant roles of regulatory ncRNAs in the coordination and fine tuning of various physiological processes but also provides a new paradigm for posttranscriptional regulation in nitrogen-fixing bacteria.

scholarly journals The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Christopher A. Lopez ◽  
William N. Beavers ◽  
Andy Weiss ◽  
Reece J. Knippel ◽  
Joseph P. Zackular ◽  
...  
Keyword(s):  
Content Type ◽  
Novel Treatments ◽  
Clostridioides Difficile ◽  
Limiting Nutrients ◽  
Pathogen Fitness ◽  
Gene Transcripts ◽  
Dependent Growth ◽  
Pathogen Colonization ◽  
The Impact

ABSTRACT The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In medium containing CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select C. difficile transcripts in a mouse CDI model relative to expression in vitro. Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain C. difficile proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts C. difficile proline fermentation. Overall, these results highlight how C. difficile colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response. IMPORTANCE Clostridioides difficile infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling C. difficile colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control C. difficile metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of C. difficile genes necessary for proline fermentation. Paradoxically, this leads to reduced C. difficile proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce C. difficile fitness in the intestines. These results emphasize the complexities of how nutrient availability influences C. difficile colonization and provide insight into critical metabolic processes that drive the pathogen’s growth.

scholarly journals Human Milk Oligosaccharides Promote the Growth of Staphylococci

2012 ◽  
Vol 78 (14) ◽  
pp. 4763-4770 ◽  
Author(s):  
K. M. Hunt ◽  
J. Preuss ◽  
C. Nissan ◽  
C. A. Davlin ◽  
J. E. Williams ◽  
...  
Keyword(s):  
Human Milk ◽  
Bacterial Communities ◽  
Bacterial Species ◽  
Human Milk Oligosaccharides ◽  
Milk Oligosaccharides ◽  
Content Type ◽  
Nutritional Conditions ◽  
Culture Independent ◽  
Lactating Mammary Gland

ABSTRACTHuman milk oligosaccharides (HMO), which constitute a major component of human milk, promote the growth of particular bacterial species in the infant's gastrointestinal tract. We hypothesized that HMO also interact with the bacterial communities present in human milk. To test this hypothesis, two experiments were conducted. First, milk samples were collected from healthy women (n= 16); culture-independent analysis of the bacterial communities was performed, HMO content was analyzed, and the relation between these factors was investigated. A positive correlation was observed between the relative abundance ofStaphylococcusand total HMO content (r= 0.66). In a follow-up study, we conducted a series ofin vitrogrowth curve experiments utilizingStaphylococcus aureusorStaphylococcus epidermidisand HMO isolated from human milk. HMO exhibited stimulatory effects on bacterial growth under various nutritional conditions. Analysis of culture supernatants from these experiments revealed that HMO did not measurably disappear from the culture medium, indicating that the growth-enhancing effects were not a result of bacterial metabolism of the HMO. Instead, stimulation of growth caused greater utilization of amino acids in minimal medium. Collectively, the data provide evidence that HMO may promote the growth ofStaphylococcusspecies in the lactating mammary gland.

scholarly journals Culture-Independent Characterization of a Gene Responsible for Nitrogen Fixation in the Symbiotic Microbial Community in the Gut of the Termite Neotermes koshunensis

1999 ◽  
Vol 65 (11) ◽  
pp. 4935-4942 ◽  
Author(s):  
Satoko Noda ◽  
Moriya Ohkuma ◽  
Ron Usami ◽  
Koki Horikoshi ◽  
Toshiaki Kudo
Keyword(s):  
Nitrogen Fixation ◽  
Gene Organization ◽  
Rt Pcr ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Culture Independent ◽  
Pcr Products ◽  
Nitrogenase Genes ◽  
Alternative Nitrogenase ◽  
Reading Frames

ABSTRACT Expression of the nitrogen fixation gene, nifH, in the gut of the termite Neotermes koshunensis was characterized without cultivation. nifH cDNA was directly amplified from mRNA of the mixed microbial population in the gut by reverse transcription (RT)-PCR. Analyses of the RT-PCR products revealed that, among the diverse nifH sequences, only a few corresponding to an alternative nitrogenase (encoded by the anf gene) were preferentially transcribed in the termite gut. Expression of theanf gene was further investigated quantitatively under several termite feeding conditions by competitive PCR. The levels of expression of the anf gene were largely congruent with the nitrogen fixation activity displayed by the termite. The amounts of the genomic anf gene in the population showed no significant change, indicating that the level of expression was critical for nitrogen fixation activity. Interestingly, no significant decrease in the expression level was observed when the diet contained molybdenum (Mo), which represses ordinary anf genes. A 3.6-kb DNA region downstream of the anf gene was isolated and found to contain reading frames homologous to anfH,anfD, and anfG of the Bacteriadomain which encode subunits of an alternative nitrogenase having no Mo as a cofactor. This DNA region also contained reading frames encodingglnB-like proteins, which is a common feature of the nitrogenase genes of the Archaea domain. These results indicate that the anf group of nitrogenase genes is the most important group of genes responsible for nitrogen fixation inN. koshunensis and that the anf gene possesses novel features with respect to the regulation of its expression and its gene organization.

scholarly journals Culture-Based and Culture-Independent Bacteriologic Analysis of Cystic Fibrosis Respiratory Specimens

2015 ◽  
Vol 54 (3) ◽  
pp. 613-619 ◽  
Author(s):  
Mona A. Mahboubi ◽  
Lisa A. Carmody ◽  
Bridget K. Foster ◽  
Linda M. Kalikin ◽  
Donald R. VanDevanter ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Sequence Analysis ◽  
Dna Sequence ◽  
Bacterial Species ◽  
Bacterial Load ◽  
Considerable Proportion ◽  
Content Type ◽  
Culture Independent ◽  
Respiratory Specimens

Cystic fibrosis (CF) is characterized by chronic infection and inflammation of the airways.In vitroculture of select bacterial species from respiratory specimens has been used to guide antimicrobial therapy in CF for the past few decades. More recently, DNA sequence-based, culture-independent approaches have been used to assess CF airway microbiology, although the role that these methods will (or should) have in routine microbiologic analysis of CF respiratory specimens is unclear. We performed DNA sequence analyses to detect bacterial species in 945 CF sputum samples that had been previously analyzed by selective CF culture. We determined the concordance of results based on culture and sequence analysis, highlighting the comparison of the results for the most prevalent genera. Although overall prevalence rates were comparable between the two methods, results varied by genus. While sequence analysis was more likely to detectAchromobacter,Stenotrophomonas, andBurkholderia, it was less likely to detectStaphylococcus.Streptococcusspp. were rarely reported in culture results but were the most frequently detected species by sequence analysis. A variety of obligate and facultative anaerobic species, not reported by culture, was also detected with high prevalence by sequence analysis. Sequence analysis indicated that in a considerable proportion of samples, taxa not reported by selective culture constituted a relatively high proportion of the total bacterial load, suggesting that routine CF culture may underrepresent significant segments of the bacterial communities inhabiting CF airways.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

Sign in / Sign up

Export Citation Format

Share Document