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 Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

scholarly journals Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants

2015 ◽  
Vol 81 (11) ◽  
pp. 3728-3736 ◽  
Author(s):  
Rie Ukuda-Hosokawa ◽  
Yasutsune Sadoyama ◽  
Misaki Kishaba ◽  
Takashi Kuriwada ◽  
Hisashi Anbutsu ◽  
...  
Keyword(s):  
Bacterial Pathogen ◽  
Threshold Level ◽  
Pathogen Transmission ◽  
Citrus Greening ◽  
Diaphorina Citri ◽  
Infection Level ◽  
Content Type ◽  
Liberibacter Asiaticus ◽  
Density Dynamics ◽  
Infection Density

ABSTRACTHuanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “CandidatusLiberibacter asiaticus,” and vectored by a phloem-sucking insect,Diaphorina citri. We investigated the infection density dynamics of “Ca. Liberibacter asiaticus” in field populations ofD. citriwith experiments using field-collected insects to address how “Ca. Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “Ca. Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “Ca. Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 103to 107bacteria per insect. In spring, the infection densities were low in March, at ∼103bacteria per insect, increasing up to 106to 107bacteria per insect in April and May, and decreasing to 105to 106bacteria per insect in late May, whereas the infection densities were constantly ∼106to 107bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “Ca. Liberibacter asiaticus” infection densities in fieldD. citripopulations. Inoculation experiments with citrus seedlings using field-collected “Ca. Liberibacter asiaticus”-infected insects suggested that (i) “Ca. Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼106bacteria per insect) of “Ca. Liberibacter asiaticus” density inD. citriis required for successful transmission to citrus plants, and (iii)D. citriattaining the threshold infection level transmits “Ca. Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.

scholarly journals SufT is required for growth of Mycobacterium smegmatis under iron limiting conditions

Microbiology ◽  
2020 ◽  
Vol 166 (3) ◽  
pp. 296-305 ◽  
Author(s):  
Tsaone Tamuhla ◽  
Lydia Joubert ◽  
Danicke Willemse ◽  
Monique J. Williams
Keyword(s):  
Mycobacterium Smegmatis ◽  
Type Species ◽  
Model Organism ◽  
Culture Conditions ◽  
Growth Defect ◽  
Content Type ◽  
Link Type ◽  
Targeted Gene Deletion ◽  
Accessory Factor

Iron-sulphur (FeS) clusters are versatile cofactors required for a range of biological processes within cells. Due to the reactive nature of the constituent molecules, assembly and delivery of these cofactors requires a multi-protein machinery in vivo. In prokaryotes, SufT homologues are proposed to function in the maturation and transfer of FeS clusters to apo-proteins. This study used targeted gene deletion to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. Deletion of the sufT gene in M. smegmatis had no impact on growth under standard culture conditions and did not significantly alter activity of the FeS cluster dependent enzymes succinate dehydrogenase (SDH) and aconitase (ACN). Furthermore, the ΔsufT mutant was no more sensitive than the wild-type strain to the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), or the anti-tuberculosis drugs isoniazid, clofazimine or rifampicin. In contrast, the ΔsufT mutant displayed a growth defect under iron limiting conditions, and an increased requirement for iron during biofilm formation. This data suggests that SufT is an accessory factor in FeS cluster biogenesis in mycobacteria which is required under conditions of iron limitation.

scholarly journals Hybrid genome assembly and annotation of Danionella translucida, a transparent fish with the smallest known vertebrate brain

2019 ◽  
Author(s):  
Mykola Kadobianskyi ◽  
Lisanne Schulze ◽  
Markus Schuelke ◽  
Benjamin Judkewitz
Keyword(s):  
Large Scale ◽  
Genetic Manipulation ◽  
Large Fraction ◽  
Model Organism ◽  
Close Relative ◽  
Vertebrate Brain ◽  
Hybrid Genome ◽  
Long Read ◽  
Optical Turbidity ◽  
Low Coverage

Studying the activity of distributed neuronal circuits at a cellular resolution in vertebrates is very challenging due to the size and optical turbidity of their brains. We recently presented Danionella translucida, a close relative of zebrafish, as a model organism suited for studying large-scale neural network interactions in adult individuals. Danionella remains transparent throughout its life, has the smallest known vertebrate brain and possesses a rich repertoire of complex behaviours. Here we sequenced, assembled and annotated the Danionella translucida genome employing a hybrid Illumina/Nanopore read library as well as RNA-seq of embryonic, larval and adult mRNA. We achieved high assembly continuity using low-coverage long-read data and annotated a large fraction of the transcriptome. This dataset will pave the way for molecular research and targeted genetic manipulation of the smallest known vertebrate brain.

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 Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

2015 ◽  
Vol 81 (12) ◽  
pp. 4120-4129 ◽  
Author(s):  
Raúl Castanera ◽  
Leticia López-Varas ◽  
Antonio G. Pisabarro ◽  
Lucía Ramírez
Keyword(s):  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Pleurotus Ostreatus ◽  
Reference Genes ◽  
Time Course ◽  
Extracellular Enzymes ◽  
Expression Patterns ◽  
Model Organism ◽  
Culture Conditions ◽  
Content Type

ABSTRACTRecently, the lignin-degrading basidiomycetePleurotus ostreatushas become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we tested the suitability of 13 candidate genes for their use as reference genes inP. ostreatustime course cultures for enzyme production. We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase (lacc10andmnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal controls leads to biased results and misinterpretations of the biological responses underlying expression changes.

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).

scholarly journals Parasexuality and Ploidy Change in Candida tropicalis

2013 ◽  
Vol 12 (12) ◽  
pp. 1629-1640 ◽  
Author(s):  
Riyad N. H. Seervai ◽  
Stephen K. Jones ◽  
Matthew P. Hirakawa ◽  
Allison M. Porman ◽  
Richard J. Bennett
Keyword(s):  
Sexual Reproduction ◽  
Candida Tropicalis ◽  
Culture Conditions ◽  
Sexual Cycle ◽  
Chromosome Loss ◽  
Parasexual Cycle ◽  
Content Type ◽  
Ploidy Changes ◽  
Diploid Cells

ABSTRACTCandidaspecies exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation.Candida albicans, the most studiedCandidaspecies and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes inCandida tropicalis, a closely related species toC. albicansthat was recently revealed to undergo sexual mating.C. tropicalisdiploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) ofC. tropicalistetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels ofC. tropicalisdiploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cellsin vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur inC. tropicalisand indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.

scholarly journals Involvement of RpoN in Regulating Bacterial Arsenite Oxidation

2012 ◽  
Vol 78 (16) ◽  
pp. 5638-5645 ◽  
Author(s):  
Yoon-Suk Kang ◽  
Brian Bothner ◽  
Christopher Rensing ◽  
Timothy R. McDermott
Keyword(s):  
Binding Site ◽  
Sigma Factor ◽  
Model Organism ◽  
Coding Region ◽  
Rt Pcr ◽  
Obvious Effect ◽  
Content Type ◽  
Definitive Evidence ◽  
Relative Contribution ◽  
Insertional Inactivation

ABSTRACTIn this study with the model organismAgrobacterium tumefaciens, we used a combination oflacZgene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of AsIIIoxidation. A deletion mutation that removed the RpoN binding site from theaioBApromoter and anaacC3(gentamicin resistance) cassette insertional inactivation of therpoNcoding region eliminatedaioBAexpression and AsIIIoxidation, althoughrpoNexpression was not related to cell exposure to AsIII. Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters foraioB,phoB1,pstS1,dctA,glnA,glnB, andflgBthat were examined by using qualitative RT-PCR andlacZreporter fusions to assess the relative contribution of RpoN to their transcription. The expressions ofaioBanddctAin the wild-type strain were considerably enhanced in cells exposed to AsIII, and both genes were silent in therpoN::aacC3mutant regardless of AsIII. The expression level ofglnAwas not influenced by AsIIIbut was reduced (but not silent) in therpoN::aacC3mutant and further reduced in the mutant under N starvation conditions. TherpoN::aacC3mutation had no obvious effect on the expression ofglnB,pstS1,phoB1, orflgB. These experiments provide definitive evidence to document the requirement of RpoN for AsIIIoxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by AsIIIor by this sigma factor.

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