scholarly journals Mapping the genetic landscape of biomineralization in Magnetospirillum magneticum AMB-1 with RB-Tnseq

2021 ◽  
Author(s):  
Hayley C McCausland ◽  
Kelly M Wetmore ◽  
Adam P. Arkin ◽  
Arash Komeili
Keyword(s):  
Essential Gene ◽  
Growth Conditions ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
High Iron ◽  
Genetic Landscape ◽  
Transposon Insertions ◽  
Magnetospirillum Magneticum ◽  
Markerless Deletion

Magnetotactic bacteria (MTB) are a phylogenetically diverse group of bacteria remarkable for their ability to biomineralize magnetite (Fe3O4) or greigite (Fe3S4) in organelles called magnetosomes. The majority of genes required for magnetosome formation are encoded by a magnetosome gene island (MAI). Here, we conducted random barcoded transposon mutagenesis (RB-TnSeq) in Magnetospirillum magneticum AMB-1 to identify the global genetic requirements for magnetosome formation under different growth conditions. We generated a library of 184,710 unique strains in a wild-type background, generating ~34 mutant strains for each gene. RB-TnSeq also allowed us to determine the essential gene set of AMB-1 under standard laboratory growth conditions. To pinpoint novel genes that are important for magnetosome formation, we subjected the library to magnetic selection screens in varied growth conditions. We compared biomineralization in standard growth conditions to biomineralization in high iron and anaerobic conditions, respectively. Strains with transposon insertions in the MAI gene mamT had an exacerbated biomineralization defect under both high iron and anerobic conditions compared to standard conditions, adding to our knowledge of the role of MamT in magnetosome formation. Mutants in amb4151, a gene outside of the MAI, are more magnetic than wild-type cells under anaerobic conditions. All three of these phenotypes were validated by creating a markerless deletion strain of the gene and evaluating with TEM imaging. Overall, our results indicate that growth conditions affect which genes are required for biomineralization and that some MAI genes may have more nuanced functions than was previously understood.

scholarly journals Genomic Analyses of Anaerobically Induced Genes in Saccharomyces cerevisiae: Functional Roles of Rox1 and Other Factors in Mediating the Anoxic Response

2002 ◽  
Vol 184 (1) ◽  
pp. 250-265 ◽  
Author(s):  
Kurt E. Kwast ◽  
Liang-Chuan Lai ◽  
Nina Menda ◽  
David T. James ◽  
Susanne Aref ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Large Fraction ◽  
Growth Conditions ◽  
Oxygen Availability ◽  
Dna Arrays ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Induced Genes

ABSTRACT DNA arrays were used to investigate the functional role of Rox1 in mediating acclimatization to anaerobic conditions in Saccharomyces cerevisiae. Multiple growth conditions for wild-type and rox1 null strains were used to identify open reading frames with a statistically robust response to this repressor. These results were compared to those obtained for a wild-type strain in response to oxygen availability. Transcripts of nearly one-sixth of the genome were differentially expressed (P < 0.05) with respect to oxygen availability, the majority (>65%) being down-regulated under anoxia. Of the anaerobically induced genes, about one-third (106) contain putative Rox1-binding sites in their promoters and were significantly (P < 0.05) up-regulated in the rox1 null strains under aerobiosis. Additional promoter searches revealed that nearly one-third of the anaerobically induced genes contain an AR1 site(s) for the Upc2 transcription factor, suggesting that Upc2 and Rox1 regulate the majority of anaerobically induced genes in S. cerevisiae. Functional analyses indicate that a large fraction of the anaerobically induced genes are involved in cell stress (∼1/3), cell wall maintenance (∼1/8), carbohydrate metabolism (∼1/10), and lipid metabolism (∼1/12), with both Rox1 and Upc2 predominating in the regulation of this latter group and Upc2 predominating in cell wall maintenance. Mapping the changes in expression of functional regulons onto metabolic pathways has provided novel insight into the role of Rox1 and other trans-acting factors in mediating the physiological response of S. cerevisiae to anaerobic conditions.

scholarly journals MtBZR1 Plays an Important Role in Nodule Development in Medicago truncatula

2019 ◽  
Vol 20 (12) ◽  
pp. 2941
Author(s):  
Can Cui ◽  
Hongfeng Wang ◽  
Limei Hong ◽  
Yiteng Xu ◽  
Yang Zhao ◽  
...  
Keyword(s):  
Medicago Truncatula ◽  
Sinorhizobium Meliloti ◽  
Normal Growth ◽  
Dry Mass ◽  
Growth Conditions ◽  
Nodule Development ◽  
Functional Study ◽  
Loss Of Function ◽  
Wild Type

Brassinosteroid (BR) is an essential hormone in plant growth and development. The BR signaling pathway was extensively studied, in which BRASSINAZOLE RESISTANT 1 (BZR1) functions as a key regulator. Here, we carried out a functional study of the homolog of BZR1 in Medicago truncatula R108, whose expression was induced in nodules upon Sinorhizobium meliloti 1021 inoculation. We identified a loss-of-function mutant mtbzr1-1 and generated 35S:MtBZR1 transgenic lines for further analysis at the genetic level. Both the mutant and the overexpression lines of MtBZR1 showed no obvious phenotypic changes under normal growth conditions. After S. meliloti 1021 inoculation, however, the shoot and root dry mass was reduced in mtbzr1-1 compared with the wild type, caused by partially impaired nodule development. The transcriptomic analysis identified 1319 differentially expressed genes in mtbzr1-1 compared with wild type, many of which are involved in nodule development and secondary metabolite biosynthesis. Our results demonstrate the role of MtBZR1 in nodule development in M. truncatula, shedding light on the potential role of BR in legume–rhizobium symbiosis.

scholarly journals Comparison of genes required for H2O2 resistance in Streptococcus gordonii and Streptococcus sanguinis

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2627-2638 ◽  
Author(s):  
Yifan Xu ◽  
Andreas Itzek ◽  
Jens Kreth
Keyword(s):  
Growth Conditions ◽  
Streptococcus Gordonii ◽  
Aerobic Growth ◽  
Anaerobic Conditions ◽  
Oral Biofilm ◽  
Streptococcus Sanguinis ◽  
Functional Studies ◽  
Stress Protection ◽  
H2o2 Resistance

Hydrogen peroxide (H2O2) is produced by several members of the genus Streptococcus mainly through the pyruvate oxidase SpxB under aerobic growth conditions. The acute toxic nature of H2O2 raises the interesting question of how streptococci cope with intrinsically produced H2O2, which subsequently accumulates in the microenvironment and threatens the closely surrounding population. Here, we investigate the H2O2 susceptibility of oral Streptococcus gordonii and Streptococcus sanguinis and elucidate potential mechanisms of how they protect themselves from the deleterious effect of H2O2. Both organisms are considered primary colonizers and occupy the same intraoral niche making them potential targets for H2O2 produced by other species. We demonstrate that S. gordonii produces relatively more H2O2 and has a greater ability for resistance to H2O2 stress. Functional studies show that, unlike in Streptococcus pneumoniae, H2O2 resistance is not dependent on a functional SpxB and confirms the important role of the ferritin-like DNA-binding protein Dps. However, the observed increased H2O2 resistance of S. gordonii over S. sanguinis is likely to be caused by an oxidative stress protection machinery present even under anaerobic conditions, while S. sanguinis requires a longer period of time for adaptation. The ability to produce more H2O2 and be more resistant to H2O2 might aid S. gordonii in the competitive oral biofilm environment, since it is lower in abundance yet manages to survive quite efficiently in the oral biofilm.

scholarly journals Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

2009 ◽  
Vol 76 (3) ◽  
pp. 670-679 ◽  
Author(s):  
Eva González ◽  
M. Rosario Fernández ◽  
Didac Marco ◽  
Eduard Calam ◽  
Lauro Sumoy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Growth Conditions ◽  
Wild Type ◽  
Genome Expression ◽  
Whole Genome Expression ◽  
Histidine Tag ◽  
Butanediol Dehydrogenase

ABSTRACT NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.

Relationship of the Escherichia coli TrkA System of Potassium Ion Uptake with the F0F1-ATPase Under Growth Conditions Without Anaerobic or Aerobic Respiration

1998 ◽  
Vol 18 (3) ◽  
pp. 143-154 ◽  
Author(s):  
Armen Trchounian ◽  
Yelena Ohanjanyan ◽  
Karine Bagramyan ◽  
Vitya Vardanian ◽  
Eleonora Zakharyan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Transport Systems ◽  
Aerobic Respiration ◽  
Anaerobic Conditions ◽  
Intact Cells ◽  
Potassium Ion Uptake ◽  
Relationship Of ◽  
The Relationship

K+ uptake by the Escherichia coli TrkA system is unusual in that it requires both ATP and ΔμH; a relation withH+ circulation through the membrane is thereforesuggested. The relationship of this system with the F0F1-ATPase was studied in intact cells grownunder different conditions. A significant increase of the N,N'-dicyclohexylcarbodiimide(DCCD)-inhibitedH+ efflux through the F0F1 by 5 mMK+, but not by Na+ added into thepotassium-free medium was revealed only in fermenting wild-type orparent cells, that were grown under anaerobic conditions withoutanaerobic or aerobic respiration and with the production of H2. Such an increase disappeared in the Δunc or the trkA mutants that have alteredF0F1 or defective TrkA, respectively. This finding indicates a closed relationship between TrkA andF0F1, with these transport systems beingassociated in a single mechanism that functions as an ATP-driven H+–K+-exchanging pump. ADCCD-inhibited H+–K+-exchangethrough these systems with the fixed stoichiometry of H+and K+ fluxes(2H+/K+) and a higherK+ gradient between the cytoplasm and the externalmedium were also found in these bacteria. They were not observed incells cultured under anaerobic conditions in the presence of nitrate orunder aerobic conditions with respiration and without production of H2. The role of anaerobic or aerobic respiration as adeterminant of the relationship of the TrkA with the F0F1 is postulated. Moreover, an increase of DCCD-inhibited H+ efflux by added K+, aswell as the characteristics of DCCD-sensitiveH+–K+-exchange found in a parentstrain, were lost in the arcA mutant with a defective Arc system, suggesting a repression of enzymes in respiratorypathways. In addition, K+ influx in the latest mutantwas not markedly changed by valinomycin or with temperature. The arcA gene product or the Arc system is proposed to beimplicated in the regulation of the relationship between TrkA and F0F1.

scholarly journals A Crucial Role of Mitochondrial Dynamics in Dehydration Resistance in Saccharomyces cerevisiae

2021 ◽  
Vol 22 (9) ◽  
pp. 4607
Author(s):  
Chang-Lin Chen ◽  
Ying-Chieh Chen ◽  
Wei-Ling Huang ◽  
Steven Lin ◽  
Rimantas Daugelavičius ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Survival Rate ◽  
Mitochondrial Dynamics ◽  
Growth Conditions ◽  
Stationary Growth Phase ◽  
Yeast Cells ◽  
Mitochondrial Activity ◽  
Wild Type ◽  
Stationary Growth

Mitochondria are dynamic organelles as they continuously undergo fission and fusion. These dynamic processes conduct not only mitochondrial network morphology but also activity regulation and quality control. Saccharomyces cerevisiae has a remarkable capacity to resist stress from dehydration/rehydration. Although mitochondria are noted for their role in desiccation tolerance, the mechanisms underlying these processes remains obscure. Here, we report that yeast cells that went through stationary growth phase have a better survival rate after dehydration/rehydration. Dynamic defective yeast cells with reduced mitochondrial genome cannot maintain the mitochondrial activity and survival rate of wild type cells. Our results demonstrate that yeast cells balance mitochondrial fusion and fission according to growth conditions, and the ability to adjust dynamic behavior aids the dehydration resistance by preserving mitochondria.

scholarly journals Chemotaxis of Escherichia coli to Pyrimidines: a New Role for the Signal Transducer Tap

2007 ◽  
Vol 190 (3) ◽  
pp. 972-979 ◽  
Author(s):  
Xianxian Liu ◽  
Rebecca E. Parales
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Growth Conditions ◽  
Wild Type ◽  
E Coli ◽  
C Terminus ◽  
Signaling Domain ◽  
Chemotaxis Proteins ◽  
Periplasmic Domain

ABSTRACT Escherichia coli exhibits chemotactic responses to sugars, amino acids, and dipeptides, and the responses are mediated by methyl-accepting chemotaxis proteins (MCPs). Using capillary assays, we demonstrated that Escherichia coli RP437 is attracted to the pyrimidines thymine and uracil and the response was constitutively expressed under all tested growth conditions. All MCP mutants lacking the MCP Tap protein showed no response to pyrimidines, suggesting that Tap, which is known to mediate dipeptide chemotaxis, is required for pyrimidine chemotaxis. In order to confirm the role of Tap in pyrimidine chemotaxis, we constructed chimeric chemoreceptors (Tapsr and Tsrap), in which the periplasmic and cytoplasmic domains of Tap and Tsr were switched. When Tapsr and Tsrap were individually expressed in an E. coli strain lacking all four native MCPs, Tapsr mediated chemotaxis toward pyrimidines and dipeptides, but Tsrap did not complement the chemotaxis defect. The addition of the C-terminal 19 amino acids from Tsr to the C terminus of Tsrap resulted in a functional chemoreceptor that mediated chemotaxis to serine but not pyrimidines or dipeptides. These results indicate that the periplasmic domain of Tap is responsible for detecting pyrimidines and the Tsr signaling domain confers on Tapsr the ability to mediate efficient chemotaxis. A mutant lacking dipeptide binding protein (DBP) was wild type for pyrimidine taxis, indicating that DBP, which is the primary chemoreceptor for dipeptides, is not responsible for detecting pyrimidines. It is not yet known whether Tap detects pyrimidines directly or via an additional chemoreceptor protein.

scholarly journals Anoxia-induced mitophagy in the yeast Kluyveromyces marxianus

2020 ◽  
Vol 20 (7) ◽  
Author(s):  
Hisashi Hoshida ◽  
Shota Kagawa ◽  
Kentaro Ogami ◽  
Rinji Akada
Keyword(s):  
Kluyveromyces Marxianus ◽  
Nitrogen Starvation ◽  
Glucose Consumption ◽  
Growth Conditions ◽  
Mitochondrial Morphology ◽  
Wild Type ◽  
Aerobic Growth ◽  
Anaerobic Conditions ◽  
Quantitative Analyses ◽  
Membrane Components

ABSTRACT Kluyveromyces marxianus is a thermotolerant, ethanol-producing yeast that requires oxygen for efficient ethanol fermentation. Under anaerobic conditions, glucose consumption and ethanol production are retarded, suggesting that oxygen affects the metabolic state of K. marxianus. Mitochondria require oxygen to function, and their forms and number vary according to environmental conditions. In this study, the effect of anoxia on mitochondrial behavior in K. marxianus was examined. Under aerobic growth conditions, mitochondria-targeted GFP exhibited a tubular and dotted localization, representing a typical mitochondrial morphology, but under anaerobic conditions, GFP localized in vacuoles, suggesting that mitophagy occurs under anaerobic conditions. To confirm mitophagy induction, the ATG32, ATG8, ATG11 and ATG19 genes were disrupted. Vacuolar localization of mitochondria-targeted GFP under anaerobic conditions was interrupted in the Δatg32 and Δatg8 strains but not the Δatg11 and Δatg19 strains. Electron microscopy revealed mitochondria-like membrane components in the vacuoles of wild-type cells grown under anaerobic conditions. Quantitative analyses using mitochondria-targeted Pho8 demonstrated that mitophagy was induced in K. marxianus by anoxia but not nitrogen starvation. To the best of our knowledge, this is the first demonstration of anoxia-induced mitophagy in yeasts.

scholarly journals The essential function of Swc4p - a protein shared by two chromatin-modifying complexes of the yeast Saccharomyces cerevisiae - resides within its N-terminal part.

2008 ◽  
Vol 55 (3) ◽  
pp. 603-612 ◽  
Author(s):  
Arkadiusz Miciałkiewicz ◽  
Anna Chełstowska
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Random Mutagenesis ◽  
Growth Conditions ◽  
Histone H2a ◽  
Terminal Part ◽  
Yeast Saccharomyces Cerevisiae ◽  
Chromatin Remodeling Complexes ◽  
Essential Function

The Swc4p protein, encoded by an essential gene, is shared by two chromatin-remodeling complexes in Saccharomyces cerevisiae cells: NuA4 (nucleosome acetyltransferase of H4) and SWR1. The SWR1 complex catalyzes ATP-dependent exchange of the nucleosomal histone H2A for H2AZ (Htz1p). The activity of NuA4 is responsible mainly for the acetylation of the H4 histone but also for the acetylation of H2A and H2AZ. In this work we investigated the role of the Swc4p protein. Using random mutagenesis we isolated a collection of swc4 mutants and showed that the essential function of Swc4p resides in its N-terminal part, within the first 269 amino acids of the 476-amino acid-long protein. We also demonstrated that Swc4p is able to accommodate numerous mutations without losing its functionality under standard growth conditions. However, when swc4 mutants were exposed to methyl methanesulfonate (MMS), hydroxyurea or benomyl, severe growth deficiencies appeared, pointing to an involvement of Swc4p in many chromatin-based processes. The mutants' phenotypes did not result from an impairment of histone acetylation, as in the mutant which bears the shortest isolated variant of truncated Swc4p, the level of overall H4 acetylation was unchanged.

scholarly journals The Critical Role of embC in Mycobacterium tuberculosis

2008 ◽  
Vol 190 (12) ◽  
pp. 4335-4341 ◽  
Author(s):  
Renan Goude ◽  
Anita G. Amin ◽  
Delphi Chatterjee ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Essential Gene ◽  
Critical Role ◽  
Normal Growth ◽  
Growth Conditions ◽  
Northern Analysis ◽  
Immunomodulatory Effects ◽  
Key Genes

ABSTRACT Arabinan polymers are major components of the cell wall in Mycobacterium tuberculosis and are involved in maintaining its structure, as well as playing a role in host-pathogen interactions. In particular, lipoarabinomannan (LAM) has multiple immunomodulatory effects. In the nonpathogenic species Mycobacterium smegmatis, EmbC has been identified as a key arabinosyltransferase involved in the incorporation of arabinose into LAM, and an embC mutant is viable but lacks LAM. In contrast, we demonstrate here that in M. tuberculosis, embC is an essential gene under normal growth conditions, suggesting a more crucial role for LAM in the pathogenic mycobacteria. M. tuberculosis EmbC has an activity similar to that of M. smegmatis EmbC, since we were able to complement an embC mutant of M. smegmatis with embCMtb , confirming that it encodes a functional arabinosyltransferase. In addition, we observed that the size of LAM produced in M. smegmatis was dependent on the level of expression of embCMtb . Northern analysis revealed that embC is expressed as part of a polycistronic message encompassing embC and three upstream genes. The promoter region for this transcript was identified and found to be up-regulated in stationary phase but down-regulated during hypoxia-induced nonreplicating persistence. In conclusion, we have identified one of the key genes involved in LAM biosynthesis in M. tuberculosis and confirmed its essential role in this species.

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