scholarly journals Sustainable Growth of Dehalococcoides mccartyi 195 by Corrinoid Salvaging and Remodeling in Defined Lactate-Fermenting Consortia

2014 ◽  
Vol 80 (7) ◽  
pp. 2133-2141 ◽  
Author(s):  
Yujie Men ◽  
Erica C. Seth ◽  
Shan Yi ◽  
Robert H. Allen ◽  
Michiko E. Taga ◽  
...  
Keyword(s):  
Cell Growth ◽  
Abc Transporter ◽  
De Novo ◽  
Sustainable Growth ◽  
Desulfovibrio Vulgaris ◽  
Physiological Level ◽  
Content Type ◽  
Dehalococcoides Mccartyi ◽  
Microbe Interactions ◽  
Abc Transporter Genes

ABSTRACTCorrinoids are essential cofactors of reductive dehalogenases inDehalococcoides mccartyi, an important bacterium in bioremediation, yet sequencedD. mccartyistrains do not possess the complete pathway forde novocorrinoid biosynthesis.Pelosinussp. andDesulfovibriosp. have been detected in dechlorinating communities enriched from contaminated groundwater without exogenous cobalamin corrinoid. To investigate the corrinoid-related interactions among key members of these communities, we constructed consortia by growingD. mccartyistrain 195 (Dhc195) in cobalamin-free, trichloroethene (TCE)- and lactate-amended medium in cocultures withDesulfovibrio vulgarisHildenborough (DvH) orPelosinus fermentansR7 (PfR7) and with both in tricultures. Only the triculture exhibited sustainable dechlorination and cell growth when a physiological level of 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, was provided. In the triculture, DvH provided hydrogen while PfR7 provided corrinoids to Dhc195, and the initiation of dechlorination and Dhc195 cell growth was highly dependent on the growth of PfR7. Corrinoid analysis indicated that Dhc195 imported and remodeled the phenolic corrinoids produced by PfR7 into cobalamin in the presence of DMB. Transcriptomic analyses of Dhc195 showed the induction of the CbiZ-dependent corrinoid-remodeling pathway and BtuFCD corrinoid ABC transporter genes during corrinoid salvaging and remodeling. In contrast, another operon annotated to encode a putative iron/cobalamin ABC transporter (DET1174-DET1176) was induced when cobalamin was exogenously provided. Interestingly, a global upregulation of phage-related genes was observed when PfR7 was present. These findings provide insights into both the gene regulation of corrinoid salvaging and remodeling in Dhc195 when it is grown without exogenous cobalamin and microbe-to-microbe interactions in dechlorinating microbial communities.

scholarly journals Metagenomic and Metatranscriptomic Analyses Reveal the Structure and Dynamics of a Dechlorinating Community Containing Dehalococcoides mccartyi and Corrinoid-Providing Microorganisms under Cobalamin-Limited Conditions

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Yujie Men ◽  
Ke Yu ◽  
Jacob Bælum ◽  
Ying Gao ◽  
Julien Tremblay ◽  
...  
Keyword(s):  
De Novo ◽  
Early Stage ◽  
Microbial Interactions ◽  
Community Members ◽  
Content Type ◽  
Dehalococcoides Mccartyi ◽  
Level Information ◽  
Microbe Interactions ◽  
The One ◽  
Shed Light

ABSTRACT The aim of this study is to obtain a systems-level understanding of the interactions between Dehalococcoides and corrinoid-supplying microorganisms by analyzing community structures and functional compositions, activities, and dynamics in trichloroethene (TCE)-dechlorinating enrichments. Metagenomes and metatranscriptomes of the dechlorinating enrichments with and without exogenous cobalamin were compared. Seven putative draft genomes were binned from the metagenomes. At an early stage (2 days), more transcripts of genes in the Veillonellaceae bin-genome were detected in the metatranscriptome of the enrichment without exogenous cobalamin than in the one with the addition of cobalamin. Among these genes, sporulation-related genes exhibited the highest differential expression when cobalamin was not added, suggesting a possible release route of corrinoids from corrinoid producers. Other differentially expressed genes include those involved in energy conservation and nutrient transport (including cobalt transport). The most highly expressed corrinoid de novo biosynthesis pathway was also assigned to the Veillonellaceae bin-genome. Targeted quantitative PCR (qPCR) analyses confirmed higher transcript abundances of those corrinoid biosynthesis genes in the enrichment without exogenous cobalamin than in the enrichment with cobalamin. Furthermore, the corrinoid salvaging and modification pathway of Dehalococcoides was upregulated in response to the cobalamin stress. This study provides important insights into the microbial interactions and roles played by members of dechlorinating communities under cobalamin-limited conditions. IMPORTANCE The key chloroethene-dechlorinating bacterium Dehalococcoides mccartyi is a cobalamin auxotroph, thus acquiring corrinoids from other community members. Therefore, it is important to investigate the microbe-microbe interactions between Dehalococcoides and the corrinoid-providing microorganisms in a community. This study provides systems-level information, i.e., taxonomic and functional compositions and dynamics of the supportive microorganisms in dechlorinating communities under different cobalamin conditions. The findings shed light on the important roles of Veillonellaceae species in the communities compared to other coexisting community members in producing and providing corrinoids for Dehalococcoides species under cobalamin-limited conditions.

scholarly journals A Low-Affinity Penicillin-Binding Protein 2x Variant Is Required for Heteroresistance in Streptococcus pneumoniae

2014 ◽  
Vol 58 (7) ◽  
pp. 3934-3941 ◽  
Author(s):  
Hansjürg Engel ◽  
Moana Mika ◽  
Dalia Denapaite ◽  
Regine Hakenbeck ◽  
Kathrin Mühlemann ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Abc Transporter ◽  
Binding Protein ◽  
Population Analysis ◽  
Resistance Testing ◽  
Penicillin Binding Protein ◽  
Secondary Resistance ◽  
Content Type ◽  
Abc Transporter Genes

ABSTRACTHeteroresistance to penicillin inStreptococcus pneumoniaeis the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain23F2349in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found thatpbp2x, but notpbp2borpbp1aalone, conferred heteroresistance to R6. However, a change ofpbp2xexpression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP onciaRdisruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinitypbp2xundergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded bypstS,phoU,pstB, andpstCin these highly resistant subpopulations. In conclusion, the presence of low-affinitypbp2xenables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.

scholarly journals Ligands of Thermophilic ABC Transporters Encoded in a Newly Sequenced Genomic Region of Thermotoga maritima MSB8 Screened by Differential Scanning Fluorimetry

2011 ◽  
Vol 77 (18) ◽  
pp. 6395-6399 ◽  
Author(s):  
Nathalie Boucher ◽  
Kenneth M. Noll
Keyword(s):  
Abc Transporter ◽  
Thermotoga Maritima ◽  
Binding Constants ◽  
Genomic Region ◽  
Genomic Research ◽  
Gene Encoding ◽  
Content Type ◽  
Differential Scanning Fluorimetry ◽  
Beta Glucosidase ◽  
Abc Transporter Genes

ABSTRACTThe chromosome ofThermotoga maritimastrain MSB8 was found to have an 8,870-bp region that is not present in its published sequence. The isolate that was sequenced by The Institute for Genomic Research (TIGR) in 1999 is apparently a laboratory variant of the isolate deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 3109) in 1986. This newly sequenced region from the DSMZ culture was located between TM1848 (cbp, cellobiose phosphorylase) and TM1847 (the 3′ end of a truncated ROK regulator). The new region contained seven genes: a beta glucosidase gene (bglA), three trehalose ABC transporter genes (treEFG), three xylose ABC transporter genes (xylE2F2K2), and the 5′ end of a gene encoding the ROK regulator TM1847. We present a new differential scanning fluorimetry method using a low pH that was necessary to screen potential ligands of these exceptionally thermostable periplasmic substrate-binding proteins. This method showed that trehalose, sucrose, and glucose stabilized TreE, and their binding was confirmed by measuring changes in intrinsic fluorescence upon ligand binding. Binding constants of 0.024 μM, 0.300 μM, and 56.78 μM at 60°C, respectively, were measured. XylE2 ligands were similarly determined and xylose, glucose, and fucose bound withKd(dissociation constant) values of 0.042 μM, 0.059 μM, and 1.436 μM, respectively. Since there is no discernible phenotypic difference between the TIGR isolate and the DSMZ isolate despite the variance in their genomes, we propose that they be called genomovars:T. maritimaMSB8 genomovar TIGR andT. maritimaMSB8 genomovar DSM 3109, respectively.

scholarly journals Unexpected Specificity of Interspecies Cobamide Transfer from Geobacter spp. to Organohalide-Respiring Dehalococcoides mccartyi Strains

2012 ◽  
Vol 78 (18) ◽  
pp. 6630-6636 ◽  
Author(s):  
Jun Yan ◽  
Kirsti M. Ritalahti ◽  
Darlene D. Wagner ◽  
Frank E. Löffler
Keyword(s):  
Reductive Dechlorination ◽  
De Novo ◽  
Synthetic Medium ◽  
Geobacter Sulfurreducens ◽  
Rrna Gene ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Mccartyi ◽  
Gene Copies ◽  
Pure Cultures

ABSTRACTDehalococcoides mccartyistrains conserve energy from reductive dechlorination reactions catalyzed by corrinoid-dependent reductive dehalogenase enzyme systems.Dehalococcoideslacks the ability forde novocorrinoid synthesis, and pure cultures require the addition of cyanocobalamin (vitamin B12) for growth. In contrast,Geobacter lovleyi, which dechlorinates tetrachloroethene tocis-1,2-dichloroethene (cis-DCE), and the nondechlorinating speciesGeobacter sulfurreducenshave complete sets of cobamide biosynthesis genes and produced 12.9 ± 2.4 and 24.2 ± 5.8 ng of extracellular cobamide per liter of culture suspension, respectively, during growth with acetate and fumarate in a completely synthetic medium.G. lovleyi-D. mccartyistrain BAV1 or strain FL2 cocultures provided evidence for interspecies corrinoid transfer, andcis-DCE was dechlorinated to vinyl chloride and ethene concomitant withDehalococcoidesgrowth. In contrast, negligible increase inDehalococcoides16S rRNA gene copies and insignificant dechlorination occurred inG. sulfurreducens-D. mccartyistrain BAV1 or strain FL2 cocultures. Apparently,G. lovleyiproduces a cobamide that complementsDehalococcoides' nutritional requirements, whereasG. sulfurreducensdoes not. Interestingly,Dehalococcoidesdechlorination activity and growth could be restored inG. sulfurreducens-Dehalococcoidescocultures by adding 10 μM 5′,6′-dimethylbenzimidazole. Observations made with theG. sulfurreducens-Dehalococcoidescocultures suggest that the exchange of the lower ligand generated a cobalamin, which supportedDehalococcoidesactivity. These findings have implications forin situbioremediation and suggest that the corrinoid metabolism ofDehalococcoidesmust be understood to faithfully predict, and possibly enhance, reductive dechlorination activities.

scholarly journals Versatility in Corrinoid Salvaging and Remodeling Pathways Supports Corrinoid-Dependent Metabolism in Dehalococcoides mccartyi

2012 ◽  
Vol 78 (21) ◽  
pp. 7745-7752 ◽  
Author(s):  
Shan Yi ◽  
Erica C. Seth ◽  
Yu-Jie Men ◽  
Sally P. Stabler ◽  
Robert H. Allen ◽  
...  
Keyword(s):  
De Novo ◽  
Chlorinated Solvents ◽  
Axial Ligand ◽  
Content Type ◽  
Dehalococcoides Mccartyi ◽  
Axial Ligands ◽  
The Common ◽  
Enzyme Cofactors ◽  
Lower Ligand ◽  
Insight Into

ABSTRACTCorrinoids are cobalt-containing molecules that function as enzyme cofactors in a wide variety of organisms but are produced solely by a subset of prokaryotes. Specific corrinoids are identified by the structure of their axial ligands. The lower axial ligand of a corrinoid can be a benzimidazole, purine, or phenolic compound. Though it is known that many organisms obtain corrinoids from the environment, the variety of corrinoids that can serve as cofactors for any one organism is largely unstudied. Here, we examine the range of corrinoids that function as cofactors for corrinoid-dependent metabolism inDehalococcoides mccartyistrain 195.Dehalococcoidesbacteria play an important role in the bioremediation of chlorinated solvents in the environment because of their unique ability to convert the common groundwater contaminants perchloroethene and trichloroethene to the innocuous end product ethene. All isolatedD. mccartyistrains require exogenous corrinoids such as vitamin B12for growth. However, like many other corrinoid-dependent bacteria, none of the well-characterizedD. mccartyistrains has been shown to be capable of synthesizing corrinoidsde novo. In this study, we investigate the ability ofD. mccartyistrain 195 to use specific corrinoids, as well as its ability to modify imported corrinoids to a functional form. We show that strain 195 can use only specific corrinoids containing benzimidazole lower ligands but is capable of remodeling other corrinoids by lower ligand replacement when provided a functional benzimidazole base. This study of corrinoid utilization and modification byD. mccartyiprovides insight into the array of strategies that microorganisms employ in acquiring essential nutrients from the environment.

scholarly journals Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Mohd Farid Abdul-Halim ◽  
Stefan Schulze ◽  
Anthony DiLucido ◽  
Friedhelm Pfeiffer ◽  
Alexandre Wilson Bisson Filho ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Surface ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
De Novo ◽  
Hot Spot ◽  
Control Cell ◽  
Haloferax Volcanii ◽  
Surface Proteins ◽  
Content Type

ABSTRACT The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii. ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an ΔartA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the ΔhvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ΔhvpssA and ΔhvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination. IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.

scholarly journals Loss of Mitochondrial Functions Associated with Azole Resistance in Candida glabrata Results in Enhanced Virulence in Mice

2011 ◽  
Vol 55 (5) ◽  
pp. 1852-1860 ◽  
Author(s):  
Sélène Ferrari ◽  
Maurizio Sanguinetti ◽  
Flavia De Bernardis ◽  
Riccardo Torelli ◽  
Brunella Posteraro ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Abc Transporter ◽  
Candida Glabrata ◽  
Selective Advantage ◽  
Azole Resistance ◽  
Content Type ◽  
Mitochondrial Functions ◽  
Abc Transporter Genes

ABSTRACTMitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur inCandida glabrata. Cells with mitochondrial DNA deficiency (so-called “petite mutants”) upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of suchC. glabratamutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and relatedC. glabrataisolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genesC. glabrata CDR1(CgCDR1),CgCDR2, andCgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even within vitrogrowth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays duringin vitrogrowth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selectedin vivounder azole therapy, even if strongly affectingin vitrogrowth characteristics, can confer a selective advantage under host conditions, allowing theC. glabratamutant to be more virulent than wild-type isolates.

scholarly journals Effect of Cell Growth on Hepatitis C Virus (HCV) Replication and a Mechanism of Cell Confluence-Based Inhibition of HCV RNA and Protein Expression

2006 ◽  
Vol 80 (3) ◽  
pp. 1181-1190 ◽  
Author(s):  
Heather B. Nelson ◽  
Hengli Tang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Growth ◽  
De Novo ◽  
Physiological State ◽  
Inhibitory Effect ◽  
Cell Number ◽  
Serum Starvation ◽  
Hcv Rna ◽  
Cell Growth Arrest

ABSTRACT An intimate relationship between hepatitis C virus (HCV) replication and the physiological state of the host liver cells has been reported. In particular, a highly reproducible and reversible inhibitory effect of high cell density on HCV replication was observed: high levels of HCV RNA and protein can be detected in actively growing cells but decline sharply when the replicon cells reach confluence. Arrested cell growth of confluent cells has been proposed to be responsible for the inhibitory effect. Indeed, other means of arresting cell growth have also been shown to inhibit HCV replication. Here, we report a detailed study of the effect of cell growth and confluence on HCV replication using a flow cytometry-based assay that is not biased against cytostasis and reduced cell number. Although we readily reproduced the inhibitory effect of cell confluence on HCV replication, we found no evidence of inhibition by serum starvation, which arrested cell growth as expected. In addition, we observed no inhibitory effect by agents that perturb the cell cycle. Instead, our results suggest that the reduced intracellular pools of nucleosides account for the suppression of HCV expression in confluent cells, possibly through the shutoff of the de novo nucleoside biosynthetic pathway when cells become confluent. Adding exogenous uridine and cytidine to the culture medium restored HCV replication and expression in confluent cells. These results suggest that cell growth arrest is not sufficient for HCV replicon inhibition and reveal a mechanism for HCV RNA inhibition by cell confluence.

scholarly journals Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

2013 ◽  
Vol 79 (23) ◽  
pp. 7360-7370 ◽  
Author(s):  
John Seip ◽  
Raymond Jackson ◽  
Hongxian He ◽  
Quinn Zhu ◽  
Seung-Pyo Hong
Keyword(s):  
Yarrowia Lipolytica ◽  
Lipid Accumulation ◽  
Oleaginous Yeast ◽  
De Novo ◽  
Lipid Synthesis ◽  
Omega 3 ◽  
Wild Type ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

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