scholarly journals Proteome Analyses of Strains ATCC 51142 and PCC 7822 of the Diazotrophic Cyanobacterium Cyanothece sp. under Culture Conditions Resulting in Enhanced H2Production

2012 ◽  
Vol 79 (4) ◽  
pp. 1070-1077 ◽  
Author(s):  
Uma K. Aryal ◽  
Stephen J. Callister ◽  
Sujata Mishra ◽  
Xiaohui Zhang ◽  
Janani I. Shutthanandan ◽  
...  
Keyword(s):  
Large Scale ◽  
Genetic Manipulation ◽  
Nitrogenase Activity ◽  
Calvin Cycle ◽  
Proteome Analysis ◽  
Culture Conditions ◽  
Pentose Phosphate ◽  
Diurnal Changes ◽  
Content Type ◽  
Diurnal Cycles

ABSTRACTCultures of the cyanobacterial genusCyanothecehave been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N2-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis ofCyanothecesp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N2-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochromecoxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited inCyanotheceATCC 51142 in the presence of glycerol under H2-producing conditions, suggesting a competition between these sources of carbon. However, inCyanothecePCC 7822, the Calvin cycle still played a role in cofactor recycling during H2production. Our data comprise the first comprehensive profiling of proteome changes inCyanothecePCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H2production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H2production.

scholarly journals Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker

2015 ◽  
Vol 197 (6) ◽  
pp. 1135-1144 ◽  
Author(s):  
Jennifer F. Carr ◽  
Michael E. Danziger ◽  
Athena L. Huang ◽  
Albert E. Dahlberg ◽  
Steven T. Gregory
Keyword(s):  
Resistance Gene ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Thermus Thermophilus ◽  
Point Mutations ◽  
Trna Synthetase ◽  
Biological Research ◽  
Lethal Mutant ◽  
Α Subunit ◽  
Content Type

ABSTRACTThermus thermophilusis an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele ofpheSencoding the phenylalanyl-tRNA synthetase α-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analogp-chlorophenylalanine. Insertion of the mutantpheSallele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination usingp-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of therrsBgene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of theT. thermophilusinsertion elements ISTth7and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on β-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome ofT. thermophilusand possibly other thermophiles as well.IMPORTANCEThermus thermophilusis an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the scarcity of genetic tools limits the kinds of manipulations that can currently be performed. We have developed a counterselectable marker that allows the introduction of unmarked deletions and point mutations into theT. thermophilusgenome. We find that this marker can also be used to select large chromosomal deletions apparently resulting from aberrant transposition of endogenous insertion sequences. This system has the potential to advance the genetic manipulation of this important model organism.

scholarly journals Applications of Bacillus subtilis Spores in Biotechnology and Advanced Materials

2020 ◽  
Vol 86 (17) ◽  
Author(s):  
Xiaopei Zhang ◽  
Amal Al-Dossary ◽  
Myer Hussain ◽  
Peter Setlow ◽  
Jiahe Li
Keyword(s):  
Bacillus Subtilis ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Industrial Applications ◽  
Advanced Materials ◽  
Content Type ◽  
Interdisciplinary Approaches ◽  
Food Shortages ◽  
Material Sciences ◽  
Basic Studies

ABSTRACT The bacterium Bacillus subtilis has long been an important subject for basic studies. However, this organism has also had industrial applications due to its easy genetic manipulation, favorable culturing characteristics for large‐scale fermentation, superior capacity for protein secretion, and generally recognized as safe (GRAS) status. In addition, as the metabolically dormant form of B. subtilis, its spores have attracted great interest due to their extreme resistance to many environmental stresses, which makes spores a novel platform for a variety of applications. In this review, we summarize both conventional and emerging applications of B. subtilis spores, with a focus on how their unique characteristics have led to innovative applications in many areas of technology, including generation of stable and recyclable enzymes, synthetic biology, drug delivery, and material sciences. Ultimately, this review hopes to inspire the scientific community to leverage interdisciplinary approaches using spores to address global concerns about food shortages, environmental protection, and health care.

scholarly journals The Path to Triacylglyceride Obesity in the sta6 Strain of Chlamydomonas reinhardtii

2014 ◽  
Vol 13 (5) ◽  
pp. 591-613 ◽  
Author(s):  
Ursula Goodenough ◽  
Ian Blaby ◽  
David Casero ◽  
Sean D. Gallaher ◽  
Carrie Goodson ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Growth Conditions ◽  
Culture Conditions ◽  
Pentose Phosphate ◽  
Small Subset ◽  
Content Type ◽  
Green Microalga ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Tag Accumulation

ABSTRACT When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then “boosted” after 2 days with additional acetate, the cells become “obese” after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of “sensitive” genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes—encoding a diacylglycerol acyltransferase ( DGTT2 ), a glycerol-3-P dehydrogenase ( GPD3 ), and two candidate lipases (Cre03.g155250 and Cre17.g735600)—are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the “sensitive” genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.

scholarly journals Growth Dynamics and Survival of Liberibacter crescens BT-1, an Important Model Organism for the Citrus Huanglongbing Pathogen “Candidatus Liberibacter asiaticus”

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
Marta Sena-Vélez ◽  
Sean D. Holland ◽  
Manu Aggarwal ◽  
Nick G. Cogan ◽  
Mukesh Jain ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Genetic Manipulation ◽  
Model Organism ◽  
Growth Dynamics ◽  
Culture Conditions ◽  
Citrus Greening ◽  
Close Relative ◽  
Logarithmic Phase ◽  
Content Type ◽  
Liberibacter Asiaticus

ABSTRACT Liberibacter crescens is the only cultured member of its genus, which includes the devastating plant pathogen “Candidatus Liberibacter asiaticus,” associated with citrus greening/Huanglongbing (HLB). L. crescens has a larger genome and greater metabolic flexibility than “Ca. Liberibacter asiaticus” and the other uncultured plant-pathogenic Liberibacter species, and it is currently the best model organism available for these pathogens. L. crescens grows slowly and dies rapidly under current culture protocols and this extreme fastidiousness makes it challenging to study. We have determined that a major cause of rapid death of L. crescens in batch culture is its alkalinization of the medium (to pH 8.5 by the end of logarithmic phase). The majority of this alkalinization is due to consumption of alpha-ketoglutaric acid as its primary carbon source, with a smaller proportion of the pH rise due to NH3 production. Controlling the pH rise with higher buffering capacity and lower starting pH improved recoverability of cells from 10-day cultures by >1,000-fold. We have also performed a detailed analysis of L. crescens growth with total cell numbers calibrated to the optical density and the percentage of live and recoverable bacteria determined over 10-day time courses. We modified L. crescens culture conditions to greatly enhance survival and increase maximum culture density. The similarities between L. crescens and the pathogenic liberibacters make this work relevant to efforts to culture the latter organisms. Our results also suggest that growth-dependent pH alteration that overcomes medium buffering should always be considered when growing fastidious bacteria. IMPORTANCE Liberibacter crescens is a bacterium that is closely related to plant pathogens that have caused billions of dollars in crop losses in recent years. Particularly devastating are citrus losses due to citrus greening disease, also known as Huanglongbing, which is caused by “Candidatus Liberibacter asiaticus” and carried by the Asian citrus psyllid. L. crescens is the only close relative of “Ca. Liberibacter asiaticus” that can currently be grown in culture, and it therefore serves as an important model organism for the growth, genetic manipulation, and biological control of the pathogenic species. Here, we show that one of the greatest limitations to L. crescens growth is the sharp increase in alkaline conditions it produces as a consequence of consumption of its preferred nutrient source. In addition to new information about L. crescens growth and metabolism, we provide new guidelines for culture conditions that improve the survival and yield of L. crescens.

scholarly journals Toward Improvement of Erythromycin A Production in an Industrial Saccharopolyspora erythraea Strain via Facilitation of Genetic Manipulation with an ArtificialattBSite for Specific Recombination

2011 ◽  
Vol 77 (21) ◽  
pp. 7508-7516 ◽  
Author(s):  
Jiequn Wu ◽  
Qinglin Zhang ◽  
Wei Deng ◽  
Jiangchao Qian ◽  
Siliang Zhang ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Large Scale ◽  
Genetic Manipulation ◽  
Saccharopolyspora Erythraea ◽  
Scale Production ◽  
Content Type ◽  
Large Scale Production ◽  
Endogenous Genes ◽  
Erythromycin A ◽  
Adenosylmethionine Synthetase

ABSTRACTLarge-scale production of erythromycin A (Er-A) relies on the organismSaccharopolyspora erythraea, in which lack of a typicalattBsite largely impedes the application of phage ΦC31 integrase-mediated recombination into site-specific engineering. We herein report construction of an artificialattBsite in an industrialS. erythraeastrain, HL3168 E3, in an effort to break the bottleneck previously encountered during genetic manipulation mainly from homologous or unpredictable nonspecific integration. Replacement of a cryptic gene,nrps1-1, with a cassette containing eightattBDNA sequences did not affect the high Er-producing ability, setting the stage for precisely engineering the industrial Er-producing strain for foreign DNA introduction with a reliable conjugation frequency. Transfer of either exogenous or endogenous genes of importance to Er-A biosynthesis, including theS-adenosylmethionine synthetase gene for positive regulation,vhbfor increasing the oxygen supply, and two tailoring genes,eryKanderyG, for optimizing the biotransformation at the late stage, was achieved by taking advantage of this facility, allowing systematic improvement of Er-A production as well as elimination of the by-products Er-B and Er-C in fermentation. The strategy developed here can generally be applicable to other strains that lack theattBsite.

scholarly journals Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly

2016 ◽  
Vol 198 (12) ◽  
pp. 1773-1782 ◽  
Author(s):  
Hualan Liu ◽  
W. Keith Ray ◽  
Richard F. Helm ◽  
David L. Popham ◽  
Stephen B. Melville
Keyword(s):  
Heat Resistance ◽  
Clostridium Perfringens ◽  
Protein Separation ◽  
Large Scale ◽  
Food Poisoning ◽  
Proteome Analysis ◽  
Membrane Proteome ◽  
Gene Encoding ◽  
Content Type ◽  
Cyanophycin Synthetase

ABSTRACTHeat-resistant endospore formation plays an important role inClostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores ofC. perfringensstrain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed ofl-arginine-poly(l-aspartic acid), to β-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species ofClostridium, but their role has not been defined. To determine the function of cyanophycin inC. perfringens, a mutation was introduced into thecphAgene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulatingC. perfringenscells, anEscherichia colistrain expressing thecphAgene made copious amounts of cyanophycin, confirming thatcphAencodes a cyanophycin synthetase.IMPORTANCEClostridium perfringensis a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. HowC. perfringenscontrols the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role inC. perfringensthan it does in cyanobacteria.

scholarly journals Influence of Energy and Electron Availability on In Vivo Methane and Hydrogen Production by a Variant Molybdenum Nitrogenase

2019 ◽  
Vol 85 (9) ◽  
Author(s):  
Yanning Zheng ◽  
Caroline S. Harwood
Keyword(s):  
Light Intensity ◽  
Nitrogenase Activity ◽  
Rhodopseudomonas Palustris ◽  
Calvin Cycle ◽  
Major Effect ◽  
Gas Production ◽  
Biofuel Production ◽  
Variant Form ◽  
Content Type

ABSTRACT The anoxygenic phototrophic bacterium Rhodopseudomonas palustris produces methane (CH4) from carbon dioxide (CO2) and hydrogen (H2) from protons (H+) when it expresses a variant form of molybdenum (Mo) nitrogenase that has two amino acid substitutions near its active site. We examined the influence of light energy and electron availability on in vivo production of these biofuels. Nitrogenase activity requires large amounts of ATP, and cells exposed to increasing light intensities produced increasing amounts of CH4 and H2. As expected for a phototroph, intracellular ATP increased with increasing light intensity, but there was only a loose correlation between ATP content and CH4 and H2 production. There was a much stronger correlation between decreased intracellular ADP and increased gas production with increased light intensity, suggesting that the rate-limiting step for CH4 and H2 production by R. palustris is inhibition of nitrogenase by ADP. Increasing the amounts of electrons available to nitrogenase by providing cells with organic alcohols, using nongrowing cells, blocking electrons from entering the Calvin cycle, or blocking H2 uptake resulted in higher yields of H2 and, in some cases, CH4. Our results provide a more complete understanding of the constraints on nitrogenase-based production of biofuels. IMPORTANCE A variant form of Mo nitrogenase catalyzes the conversion of CO2 and protons to the biofuels CH4 and H2. A constant supply of electrons and ATP is needed to drive these reduction reactions. The bacterium R. palustris generates ATP from light and has a versatile metabolism that makes it ideal for manipulating electron availability intracellularly. We therefore explored its potential as a biocatalyst for CH4 and H2 production. We found that intracellular ADP had a major effect on biofuel production, more pronounced than the effect caused by ATP. This is probably due to inhibition of nitrogenase activity by ADP. In general, the amount of CH4 produced by the variant nitrogenase in vivo was affected by electron availability much less than was the amount of H2 produced. This study shows the nature of constraints on in vivo biofuel production by variant Mo nitrogenase.

Financial distress determinants among SMEs: empirical evidence from Sweden

2020 ◽  
Vol 47 (3) ◽  
pp. 547-560 ◽  
Author(s):  
Darush Yazdanfar ◽  
Peter Öhman
Keyword(s):  
Financial Crisis ◽  
Financial Distress ◽  
Large Scale ◽  
Global Financial Crisis ◽  
Binary Logistic Regression ◽  
Data Availability ◽  
Cross Sectional ◽  
Data Set ◽  
Content Type ◽  
The Global Financial Crisis

PurposeThe purpose of this study is to empirically investigate determinants of financial distress among small and medium-sized enterprises (SMEs) during the global financial crisis and post-crisis periods.Design/methodology/approachSeveral statistical methods, including multiple binary logistic regression, were used to analyse a longitudinal cross-sectional panel data set of 3,865 Swedish SMEs operating in five industries over the 2008–2015 period.FindingsThe results suggest that financial distress is influenced by macroeconomic conditions (i.e. the global financial crisis) and, in particular, by various firm-specific characteristics (i.e. performance, financial leverage and financial distress in previous year). However, firm size and industry affiliation have no significant relationship with financial distress.Research limitationsDue to data availability, this study is limited to a sample of Swedish SMEs in five industries covering eight years. Further research could examine the generalizability of these findings by investigating other firms operating in other industries and other countries.Originality/valueThis study is the first to examine determinants of financial distress among SMEs operating in Sweden using data from a large-scale longitudinal cross-sectional database.

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

scholarly journals Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 781-786 ◽  
Author(s):  
Maximo Sánchez ◽  
Martha-Helena Ramírez-Bahena ◽  
Alvaro Peix ◽  
María J. Lorite ◽  
Juan Sanjuán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Lotus Corniculatus ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

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