scholarly journals Characterization of key enzymes involved in triacylglycerol biosynthesis in mycobacteria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Agostina Crotta Asis ◽  
Franco Savoretti ◽  
Matías Cabruja ◽  
Hugo Gramajo ◽  
Gabriela Gago
Keyword(s):  
Phosphatidic Acid ◽  
Growth Phase ◽  
De Novo ◽  
Cell Envelope ◽  
Exponential Growth Phase ◽  
Genes Encoding ◽  
Concomitant Reduction ◽  
Tag Biosynthesis ◽  
Odd Chain Fatty Acids

AbstractPhosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. PAP activity has a key role in the regulation of PA flux towards TAG or glycerophospholipid synthesis. In this work we have characterized two Mycobacterium smegmatis genes encoding for functional PAP proteins. Disruption of both genes provoked a sharp reduction in de novo TAG biosynthesis in early growth phase cultures under stress conditions. In vivo labeling experiments demonstrated that TAG biosynthesis was restored in the ∆PAP mutant when bacteria reached exponential growth phase, with a concomitant reduction of phospholipid synthesis. In addition, comparative lipidomic analysis showed that the ∆PAP strain had increased levels of odd chain fatty acids esterified into TAGs, suggesting that the absence of PAP activity triggered other rearrangements of lipid metabolism, like phospholipid recycling, in order to maintain the wild type levels of TAG. Finally, the lipid changes observed in the ∆PAP mutant led to defective biofilm formation. Understanding the interaction between TAG synthesis and the lipid composition of mycobacterial cell envelope is a key step to better understand how lipid homeostasis is regulated during Mycobacterium tuberculosis infection.

scholarly journals The Putrescine Importer PuuP of Escherichia coli K-12

2009 ◽  
Vol 191 (8) ◽  
pp. 2776-2782 ◽  
Author(s):  
Shin Kurihara ◽  
Yuichi Tsuboi ◽  
Shinpei Oda ◽  
Hyeon Guk Kim ◽  
Hidehiko Kumagai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Growth Phase ◽  
Exponential Growth ◽  
Minimal Medium ◽  
Exponential Growth Phase ◽  
E Coli ◽  
Genes Encoding ◽  
K 12 ◽  
Utilization Pathway

ABSTRACT The Puu pathway is a putrescine utilization pathway involving gamma-glutamyl intermediates. The genes encoding the enzymes of the Puu pathway form a gene cluster, the puu gene cluster, and puuP is one of the genes in this cluster. In Escherichia coli, three putrescine importers, PotFGHI, PotABCD, and PotE, were discovered in the 1990s and have been studied; however, PuuP had not been discovered previously. This paper shows that PuuP is a novel putrescine importer whose kinetic parameters are equivalent to those of the polyamine importers discovered previously. A puuP + strain absorbed up to 5 mM putrescine from the medium, but a ΔpuuP strain did not. E. coli strain MA261 has been used in previous studies of polyamine transporters, but PuuP had not been identified previously. It was revealed that the puuP gene of MA261 was inactivated by a point mutation. When E. coli was grown on minimal medium supplemented with putrescine as the sole carbon or nitrogen source, only PuuP among the polyamine importers was required. puuP was expressed strongly when putrescine was added to the medium or when the puuR gene, which encodes a putative repressor, was deleted. When E. coli was grown in M9-tryptone medium, PuuP was expressed mainly in the exponential growth phase, and PotFGHI was expressed independently of the growth phase.

scholarly journals Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Wenchao Feng ◽  
Dylan Minor ◽  
Mengyao Liu ◽  
Jinquan Li ◽  
Suzanne L. Ishaq ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Phase ◽  
Virulence Genes ◽  
Expression Patterns ◽  
Mouse Skin ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Subcutaneous Infection ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [SseA+] and normal SpeB activity [SpeBA+]). SseA+ SpeBA+ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an SseA+ SpeBA+ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other SseA+ SpeBA+ isolates also had nonsense mutations or small indels in rocA. RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.

Defects in pyruvate kinase cause a conditional increase of thiamine synthesis inSalmonella typhimurium

1999 ◽  
Vol 45 (7) ◽  
pp. 565-572 ◽  
Author(s):  
Todd Christian ◽  
Diana M Downs
Keyword(s):  
Pyruvate Kinase ◽  
De Novo ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Purine Biosynthesis ◽  
Phenotypic Analysis ◽  
Metabolic Defects ◽  
Genes Encoding ◽  
Metabolic Interactions

As genomic sequence data become more prevalent, the challenges in microbial physiology shift from identifying biochemical pathways to understanding the interactions that occur between them to create a robust but responsive metabolism. One of the most powerful methods to identify such interactions is in vivo phenotypic analysis. We have utilized thiamine synthesis as a model to detect subtle metabolic interactions due to the sensitivity allowed by the small cellular requirement for this vitamin. Although purine biosynthesis produces an intermediate in thiamine synthesis, mutants blocked in the first step of de novo purine biosynthesis (PurF) are able to grow in the absence of thiamine owing to an alternative synthesis. A number of general metabolic defects have been found to prevent PurF-independent thiamine synthesis. Here we report stimulation of thiamine-independent growth caused by a mutation in one or both genes encoding the pyruvate kinase isozymes. The results presented herein represent the first phenotype described for mutants defective in pykA or pykF, and thus identify metabolic interactions that exist in vivo.Key words: thiamine synthesis, metabolic integration.

In vivo analyses of constitutive and regulated promoters in halophilic archaea

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Dagmar Gregor ◽  
Felicitas Pfeifer
Keyword(s):  
Growth Phase ◽  
Promoter Activity ◽  
Halophilic Archaea ◽  
Stationary Growth Phase ◽  
Halobacterium Salinarum ◽  
Exponential Growth Phase ◽  
Haloferax Mediterranei ◽  
Basal Promoter Activity ◽  
Transcriptional Activator Protein

The two gvpA promoters PcA and PpA of Halobacterium salinarum, and the PmcA promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with β-galactosidase activity, as reporter. For comparison, the Pfdx promoter of the ferredoxin gene of Hbt. salinarum and the PbgaH promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed. Pfdx , driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas PbgaH and the three gvpA promoters yielded the largest activities during the stationary growth phase. Compared to Pfdx , the basal promoter activities of PpA and PmcA were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The PcA promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the PcA -TATA box and the BRE element were the reason for the lack of the basal PcA activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger PpA promoter and investigated in Hfx. volcanii transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the PpA -BRE element was substituted for the PcA -BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring PpA -BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of PmcA -BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of PmcA .

scholarly journals Characterization and regulation of inositol monophosphatase activity in Mycobacterium smegmatis

2002 ◽  
Vol 361 (2) ◽  
pp. 385-390 ◽  
Author(s):  
Jérôme NIGOU ◽  
Gurdyal S. BESRA
Keyword(s):  
Mycobacterium Smegmatis ◽  
De Novo ◽  
Ph Dependence ◽  
Exponential Growth Phase ◽  
Inositol Monophosphatase ◽  
Bivalent Cation ◽  
Inositol 1 ◽  
Genus Mycobacterium ◽  
Phosphatidylinositol Mannosides

Mycobacterium tuberculosis and related members of the genus Mycobacterium contain a number of inositol-based lipids, such as phosphatidylinositol mannosides, lipomannan and lipoarabinomannan. The synthesis of phosphatidylinositol in M. smegmatis is essential for growth and myo-inositol is a key metabolite for mycobacteria. Little is known about the biosynthesis of inositol in mycobacteria and the only known de novo pathway for myo-inositol biosynthesis involves a two-step process. First, cyclization of glucose 6-phosphate to afford myo-inositol 1-phosphate via inositol-1-phosphate synthase and, secondly, dephosphorylation of myo-inositol 1-phosphate by inositol monophosphatase (IMP) to afford myo-inositol. The following report examines IMP activity in M. smegmatis extracts, with regard to pH dependence, bivalent cation re quirement, univalent cation inhibition, regulation by growth and carbon source. We show that IMP activity, which is optimal at the end of the exponential growth phase in Sauton's medium, is Mg2+-dependent. Moreover, IMP activity is inhibited by Li+ and Na+, with Li+ also being able to inhibit growth of M. smegmatis in vivo. This study represents a first step in the delineation of myo-inositol biosynthesis in mycobacteria.

scholarly journals Adaptive evolution within the gut microbiome of individual people

2017 ◽  
Author(s):  
Shijie Zhao ◽  
Tami D. Lieberman ◽  
Mathilde Poyet ◽  
Sean M. Gibbons ◽  
Mathieu Groussin ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Evolutionary History ◽  
De Novo ◽  
Cell Envelope ◽  
Adaptive Dynamics ◽  
Mobile Element ◽  
Healthy People ◽  
Selective Forces ◽  
Key Genes

AbstractIndividual bacterial lineages stably persist for years in the human gut microbiome1–3. However, the potential of these lineages to adapt during colonization of healthy people is not well understood2,4. Here, we assess evolution within individual microbiomes by sequencing the genomes of 602Bacteroides fragilisisolates cultured from 12 healthy subjects. We find thatB. fragiliswithin-subject populations contain substantialde novonucleotide and mobile element diversity, which preserve years of within-person evolutionary history. This evolutionary history contains signatures of within-person adaptation to both subject-specific and common selective forces, including parallel mutations in sixteen genes. These sixteen genes are involved in cell-envelope biosynthesis and polysaccharide utilization, as well as yet under-characterized pathways. Notably, one of these genes has been shown to be critical forB. fragiliscolonization in mice5, indicating that key genes have not already been optimized for survivalin vivo. This lack of optimization, given historical signatures of purifying selection in these genes, suggests that varying selective forces with discordant solutions act uponB. fragilis in vivo. Remarkably, in one subject, twoB. fragilissublineages coexisted at a stable relative frequency over a 1.5-year period despite rapid adaptive dynamics within one of the sublineages. This stable coexistence suggests that competing selective forces can lead toB. fragilisniche-differentiation even within a single person. We conclude thatB. fragilisadapts rapidly within the microbiomes of individual healthy people, providing a new route for the discovery of key genes in the microbiome and implications for microbiome stability and manipulation.

scholarly journals Transcription and Analysis of Polymorphism in a Cluster of Genes Encoding Surface-Associated Proteins of Clostridium difficile

2003 ◽  
Vol 185 (15) ◽  
pp. 4461-4470 ◽  
Author(s):  
Marie-Pierre Savariau-Lacomme ◽  
Carole Lebarbier ◽  
Tuomo Karjalainen ◽  
Anne Collignon ◽  
Claire Janoir
Keyword(s):  
Clostridium Difficile ◽  
Growth Phase ◽  
Cysteine Proteases ◽  
Exponential Growth Phase ◽  
Polycistronic Transcript ◽  
Conserved Sequence ◽  
Domain Structures ◽  
Genes Encoding ◽  
Associated Proteins ◽  
Insight Into

ABSTRACT Recent investigations of the Clostridium difficile genome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins. Two of these genes have been proven to encode proteins involved in cell adherence: slpA encodes the precursor of the two proteins of the S-layer, P36 and P47, whereas cwp66 encodes the Cwp66 adhesin. To gain further insight into the function of this cluster, we further focused on slpA, cwp66, and cwp84, the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases. It displayed nonspecific proteolytic activity when expressed as a recombinant protein in Escherichia coli. Polymorphism of cwp66 and cwp84 genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots. The slpA gene is strongly transcribed during the entire growth phase as a bicistronic transcript; cwp66 is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream. The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence. No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains. The cwp84 gene is strongly transcribed as a monocistronic message. This feature, together with the highly conserved sequence pattern of cwp84, suggests a significant role in the physiopathology of C. difficile for the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster.

scholarly journals Dynamism of Metabolic Carbon Flow of Starch and Lipids in Chlamydomonas debaryana

2021 ◽  
Vol 12 ◽  
Author(s):  
Naoki Sato ◽  
Masakazu Toyoshima
Keyword(s):  
Fatty Acids ◽  
Growth Phase ◽  
De Novo ◽  
Membrane Lipids ◽  
Carbon Flow ◽  
Added Value ◽  
Gas Chromatography Mass Spectrometry ◽  
Exponential Growth Phase ◽  
Alternative Source ◽  
Metabolic Carbon

Microalgae have the potential to recycle CO2 as starch and triacylglycerol (TAG), which provide alternative source of biofuel and high added-value chemicals. Starch accumulates in the chloroplast, whereas TAG accumulates in the cytoplasmic lipid droplets (LD). Preferential accumulation of starch or TAG may be achieved by switching intracellular metabolic carbon flow, but our knowledge on this control remains limited. Are these two products mutually exclusive? Or, does starch act as a precursor to TAG synthesis, or vice versa? To answer these questions, we analyzed carbon flow in starch and lipids using a stable isotope 13C in Chlamydomonas debaryana NIES-2212, which accumulates, without nutrient limitation, starch in the exponential growth phase and TAG in the stationary phase. Pulse labeling experiments as well as pulse labeling and chase experiments were conducted, and then, gas chromatography-mass spectrometry (GC-MS) analysis was performed on starch-derived glucose and lipid-bound fatty acids. We exploited the previously developed method of isotopomer analysis to estimate the proportion of various pools with different isotopic abundance. Starch turned over rapidly to provide carbon for the synthesis of fatty acids in the exponential phase cells. Most fatty acids showed rapid and slow components of metabolism, whereas oleic acid decayed according to a single exponential curve. Highly labeled population of fatty acids that accumulated during the initial labeling decreased rapidly, and replaced by low abundance population during the chase time, indicating that highly labeled fatty acids were degraded and the resulting carbons were re-used in the re-synthesis with about 9-fold unlabeled, newly fixed carbons. Elongation of C16–C18 acids in vivo was indicated by partially labeled C18 acids. The accumulation of TAG in the stationary growth phase was accounted for by both de novo synthesis and remodeling of membrane lipids. These results suggest that de novo synthesis of starch and TAG was rapid and transient, and also almost independent to each other, but there is a pool of starch quickly turning over for the synthesis of fatty acids. Fatty acids were also subject to re-synthesis. Evidence was also provided for remodeling of lipids, namely, re-use of acyl groups in polar lipids for TAG synthesis.

scholarly journals Temporal Expression of the Bacillus subtilis secAGene, Encoding a Central Component of the Preprotein Translocase

1999 ◽  
Vol 181 (2) ◽  
pp. 493-500 ◽  
Author(s):  
Markus Herbort ◽  
Michael Klein ◽  
Erik H. Manting ◽  
Arnold J. M. Driessen ◽  
Roland Freudl
Keyword(s):  
Bacillus Subtilis ◽  
Growth Phase ◽  
Exponential Growth ◽  
De Novo ◽  
Stationary Growth Phase ◽  
Extracellular Proteins ◽  
Exponential Growth Phase ◽  
Central Component ◽  
Temporal Expression ◽  
Stationary Growth

ABSTRACT In Bacillus subtilis, the secretion of extracellular proteins strongly increases upon transition from exponential growth to the stationary growth phase. It is not known whether the amounts of some or all components of the protein translocation apparatus are concomitantly increased in relation to the increased export activity. In this study, we analyzed the transcriptional organization and temporal expression of the secA gene, encoding a central component of the B. subtilis preprotein translocase. We found that secA and the downstream gene (prfB) constitute an operon that is transcribed from a vegetative (ςA-dependent) promoter located upstream ofsecA. Furthermore, using different independent methods, we found that secA expression occurred mainly in the exponential growth phase, reaching a maximal value almost precisely at the transition from exponential growth to the stationary growth phase. Following to this maximum, the de novo transcription ofsecA sharply decreased to a low basal level. Since at the time of maximal secA transcription the secretion activity of B. subtilis strongly increases, our results clearly demonstrate that the expression of at least one of the central components of the B. subtilis protein export apparatus is adapted to the increased demand for protein secretion. Possible mechanistic consequences are discussed.

A KCNC1 mutation in Epilepsy of Infancy with Focal Migrating Seizures produces functional channels that fail to be regulated by phosphorylation

2021 ◽  
Author(s):  
Yalan Zhang ◽  
Syed R Ali ◽  
Rima Nabbout ◽  
Giulia Barcia ◽  
Leonard K. Kaczmarek
Keyword(s):  
De Novo ◽  
Channel Modulation ◽  
Progressive Myoclonus Epilepsy ◽  
C Terminus ◽  
Myoclonus Epilepsy ◽  
Voltage Dependent ◽  
Genes Encoding ◽  
Clear Change

Channelopathies caused by mutations in genes encoding ion channels generally produce a clear change in channel function. Accordingly, mutations in KCNC1, which encodes the voltage-dependent Kv3.1 potassium channel, result in Progressive Myoclonus Epilepsy as well as other Developmental and Epileptic Encephalopathies, and these have been shown to reduce or fully abolish current amplitude. One exception to this is the mutation A513V Kv3.1b, located in the cytoplasmic C-terminal domain of the channel protein. This de novo variant was detected in a patient with Epilepsy of Infancy with Focal Migrating Seizures (EIFMS) but no difference could be detected between A513V Kv3.1 current and that of wild type Kv3.1. Using both biochemical and electrophysiological approaches, we have now confirmed that this variant produces functional channels but find that the A513V mutation renders the channel completely insensitive to regulation by phosphorylation at S503, a nearby regulatory site in the C-terminus. In this respect, the mutation resembles those in another channel, KCNT1, which are the major cause of EIFMS. Because the amplitude of Kv3.1 current is constantly adjusted by phosphorylation in vivo, our findings suggest that loss of such regulation contributes to EIFMS phenotype and emphasize the role of channel modulation for normal neuronal function.

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