scholarly journals Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

2013 ◽  
Vol 79 (17) ◽  
pp. 5321-5328 ◽  
Author(s):  
Anne Krog ◽  
Tonje Marita Bjerkan Heggeset ◽  
Trond Erling Ellingsen ◽  
Trygve Brautaset
Keyword(s):  
Functional Characterization ◽  
Methylotrophic Bacterium ◽  
Content Type ◽  
Key Enzymes ◽  
Bacillus Methanolicus ◽  
Bacterium Bacillus ◽  
Glutamate Synthesis ◽  
Synthesis And Degradation

ABSTRACTBacillus methanolicuswild-type strain MGA3 secretes 59 g/liter−1ofl-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved inl-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded byyweBand two glutamate synthases (GOGATs) that are encoded by thegltABoperon and bygltA2were found, in contrast toBacillus subtilis, which has two different GDHs and only one GOGAT.B. methanolicushas a glutamine synthetase (GS) that is encoded byglnAand a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by theodhABoperon. TheyweB,gltA,gltB, andgltA2gene products were purified and characterized biochemicallyin vitro. YweB has a lowKmvalue for ammonium (10 mM) and a highKmvalue forl-glutamate (250 mM), and theVmaxvalue is 7-fold higher forl-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similarKmvalues (1 to 1.4 mM) andVmaxvalues (4 U/mg) for bothl-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymesin vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activityin vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium.In vivoexperiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulatingl-glutamate production in this organism.

scholarly journals Expression and Functional Characterization of the First Bacteriophage-Encoded Chaperonin

2012 ◽  
Vol 86 (18) ◽  
pp. 10103-10111 ◽  
Author(s):  
Lidia P. Kurochkina ◽  
Pavel I. Semenyuk ◽  
Victor N. Orlov ◽  
Johan Robben ◽  
Nina N. Sykilinda ◽  
...  
Keyword(s):  
Thermal Inactivation ◽  
Functional Characterization ◽  
Content Type ◽  
Chaperonin Groel ◽  
Phage Propagation ◽  
Physicochemical Methods ◽  
First Time

Chaperonins promote protein foldingin vivoand are ubiquitously found in bacteria, archaea, and eukaryotes. The first viral chaperonin GroEL ortholog, gene product 146 (gp146), whose gene was earlier identified in the genome of bacteriophage EL, has been shown to be synthesized during phage propagation inPseudomonas aeruginosacells. The recombinant gp146 has been expressed inEscherichia coliand characterized by different physicochemical methods for the first time. Using serum against the recombinant protein, gp146's native substrate, the phage endolysin gp188, has been immunoprecipitated from the lysate of EL-infected bacteria and identified by mass spectrometry.In vitroexperiments have shown that gp146 has a protective effect against endolysin thermal inactivation and aggregation, providing evidence of its chaperonin function. The phage chaperonin has been found to have the architecture and some properties similar to those of GroEL but not to require cochaperonin for its functional activity.

scholarly journals Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus

2006 ◽  
Vol 188 (8) ◽  
pp. 3063-3072 ◽  
Author(s):  
Øyvind M. Jakobsen ◽  
Aline Benichou ◽  
Michael C. Flickinger ◽  
Svein Valla ◽  
Trond E. Ellingsen ◽  
...  
Keyword(s):  
Methylotrophic Bacterium ◽  
Primary Metabolism ◽  
Wild Type ◽  
Fructose Bisphosphate ◽  
Methanol Tolerance ◽  
Oxidation Of Methanol ◽  
Bacillus Methanolicus ◽  
Multiple Copies ◽  
Bacterium Bacillus

ABSTRACT The natural plasmid pBM19 carries the key mdh gene needed for the oxidation of methanol into formaldehyde by Bacillus methanolicus. Five more genes, glpX, fba, tkt, pfk, and rpe, with deduced roles in the cell primary metabolism, are also located on this plasmid. By using real-time PCR, we show that they are transcriptionally upregulated (6- to 40-fold) in cells utilizing methanol; a similar induction was shown for two chromosomal genes, hps and phi. These seven genes are involved in the fructose bisphosphate aldolase/sedoheptulose bisphosphatase variant of the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation. Curing of pBM19 causes higher methanol tolerance and reduced formaldehyde tolerance, and the methanol tolerance is reversed to wild-type levels by reintroducing mdh. Thus, the RuMP pathway is needed to detoxify the formaldehyde produced by the methanol dehydrogenase-mediated conversion of methanol, and the in vivo transcription levels of mdh and the RuMP pathway genes reflect the methanol tolerance level of the cells. The transcriptional inducer of hps and phi genes is formaldehyde, and not methanol, and introduction of multiple copies of these two genes into B. methanolicus made the cells more tolerant of growth on high methanol concentrations. The recombinant strain also had a significantly higher specific growth rate on methanol than the wild type. While pBM19 is critical for growth on methanol and important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for B. methanolicus when growing on mannitol. Our data contribute to a new and fundamental understanding of the regulation of B. methanolicus methylotrophy.

scholarly journals An Inducible and Secreted Eukaryote-Like Serine/Threonine Kinase of Salmonella enterica Serovar Typhi Promotes Intracellular Survival and Pathogenesis

2014 ◽  
Vol 83 (2) ◽  
pp. 522-533 ◽  
Author(s):  
Nagaraja Theeya ◽  
Atri Ta ◽  
Sayan Das ◽  
Rahul S. Mandal ◽  
Oishee Chakrabarti ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Functional Characterization ◽  
Systemic Infection ◽  
Serine Threonine Kinase ◽  
Content Type ◽  
Threonine Kinase ◽  
Universal Substrate

Eukaryote-like serine/threonine kinases (eSTKs) constitute an important family of bacterial virulence factors. Genome analysis had predicted putative eSTKs inSalmonella entericaserovar Typhi, although their functional characterization and the elucidation of their role in pathogenesis are still awaited. We show here that the primary sequence and secondary structure of thet4519locus ofSalmonellaTyphi Ty2 have all the signatures of eukaryotic superfamily kinases.t4519encodes a ∼39-kDa protein (T4519), which shows serine/threonine kinase activitiesin vitro. Recombinant T4519 (rT4519) is autophosphorylated and phosphorylates the universal substrate myelin basic protein. Infection of macrophages results in decreased viability of the mutant (Ty2Δt4519) strain, which is reversed by gene complementation. Moreover, reactive oxygen species produced by the macrophages signal to the bacteria to induce T4519, which is translocated to the host cell cytoplasm. That T4519 may target a host substrate(s) is further supported by the activation of host cellular signaling pathways and the induction of cytokines/chemokines. Finally, the role of T4519 in the pathogenesis ofSalmonellaTyphi is underscored by the significantly decreased mortality of mice infected with the Ty2Δt4519strain and the fact that the competitive index of this strain for causing systemic infection is 0.25% that of the wild-type strain. This study characterizes the first eSTK ofSalmonellaTyphi and demonstrates its role in promoting phagosomal survival of the bacteria within macrophages, which is a key determinant of pathogenesis. This, to the best of our knowledge, is the first study to describe the essential role of eSTKs in thein vivopathogenesis ofSalmonellaspp.

scholarly journals RNA Sequencing Reveals Differences between the Global Transcriptomes of Salmonella enterica Serovar Enteritidis Strains with High and Low Pathogenicities

2013 ◽  
Vol 80 (3) ◽  
pp. 896-906 ◽  
Author(s):  
Devendra H. Shah
Keyword(s):  
Rna Sequencing ◽  
Salmonella Enterica ◽  
Negative Binomial ◽  
Phenotypic Diversity ◽  
Functional Characterization ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Content Type ◽  
Expression Of Genes

ABSTRACTSalmonella entericaserovar Enteritidis is one of the important causes of bacterial food-borne gastroenteritis worldwide. Field strains ofS. Enteritidis are relatively genetically homogeneous; however, they show extensive phenotypic diversity and differences in virulence potential. RNA sequencing (RNA-Seq) was used to characterize differences in the global transcriptome between several genetically similar but phenotypically diverse poultry-associated field strains ofS. Enteritidis grown in laboratory medium at avian body temperature (42°C). TheseS. Enteritidis strains were previously characterized as high-pathogenicity (HP;n= 3) and low-pathogenicity (LP;n= 3) strains based on bothin vitroandin vivovirulence assays. Using the negative binomial distribution-based statistical tools edgeR and DESeq, 252 genes were identified as differentially expressed in LP strains compared with their expression in the HP strains (P< 0.05). A majority of genes (235, or 93.2%) showed significantly reduced expression, whereas a few genes (17, or 6.8%) showed increased expression in all LP strains compared with HP strains. LP strains showed a unique transcriptional profile that is characterized by significantly reduced expression of several transcriptional regulators and reduced expression of genes involved in virulence (e.g.,Salmonellapathogenicity island 1 [SPI-1], SPI-5, and fimbrial and motility genes) and protection against osmotic, oxidative, and other stresses, such as iron-limiting conditions commonly encountered within the host. Several functionally uncharacterized genes also showed reduced expression. This study provides a first concise view of the global transcriptional differences between field strains ofS. Enteritidis with various levels of pathogenicity, providing the basis for future functional characterization of several genes with potential roles in virulence or stress regulation ofS. Enteritidis.

scholarly journals Overexpression of Wild-Type Aspartokinase Increases l-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus

2008 ◽  
Vol 75 (3) ◽  
pp. 652-661 ◽  
Author(s):  
�yvind M. Jakobsen ◽  
Trygve Brautaset ◽  
Kristin F. Degnes ◽  
Tonje M. B. Heggeset ◽  
Simone Balzer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Methylotrophic Bacterium ◽  
Wild Type ◽  
Lysine Production ◽  
Bacillus Methanolicus ◽  
Semialdehyde Dehydrogenase ◽  
Aspartate Pathway ◽  
The Impact

ABSTRACT Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V max values (between 47 and 58 μmol/min/mg protein) and Km values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC50], 0.1 mM) and by l-lysine (IC50, 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC50, 4 mM) and by l-lysine (IC50, 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

scholarly journals Functional characterization of multiple PAS domain-containing diguanylate cyclases in Synechocystis sp. PCC 6803

Microbiology ◽  
2020 ◽  
Vol 166 (7) ◽  
pp. 659-668
Author(s):  
Ko Ishikawa ◽  
Chihiro Chubachi ◽  
Saeko Tochigi ◽  
Naomi Hoshi ◽  
Seiji Kojima ◽  
...  
Keyword(s):  
Salt Stress ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
Active Form ◽  
Guanosine Monophosphate ◽  
Content Type ◽  
Link Type ◽  
Pcc 6803

Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger known to control a variety of bacterial processes. The model cyanobacterium, Synechocystis sp. PCC 6803, has a score of genes encoding putative enzymes for c-di-GMP synthesis and degradation. However, most of them have not been functionally characterized. Here, we chose four genes in Synechocystis (dgcA–dgcD), which encode proteins with a GGDEF, diguanylate cyclase (DGC) catalytic domain and multiple Per-ARNT-Sim (PAS) conserved regulatory motifs, for detailed analysis. Purified DgcA, DgcB and DgcC were able to catalyze synthesis of c-di-GMP from two GTPs in vitro. DgcA had the highest activity, compared with DgcB and DgcC. DgcD did not show detectable activity. DgcA activity was specific for GTP and stimulated by the divalent cations, magnesium or manganese. Full activity of DgcA required the presence of the multiple PAS domains, probably because of their role in protein dimerization or stability. Synechocystis mutants carrying single deletions of dgcA–dgcD were not affected in their growth rate or biofilm production during salt stress, suggesting that there was functional redundancy in vivo. In contrast, overexpression of dgcA resulted in increased biofilm formation in the absence of salt stress. In this study, we characterize the enzymatic and physiological function of DgcA–DgcD, and propose that the PAS domains in DgcA function in maintaining the enzyme in its active form.

scholarly journals Env7p Associates with the Golgin Protein Imh1 at the trans-Golgi Network in Candida albicans

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Kongara Hanumantha Rao ◽  
Swagata Ghosh ◽  
Asis Datta
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Functional Characterization ◽  
Evolutionary Divergence ◽  
Content Type ◽  
Membrane Asymmetry ◽  
Specific Localization ◽  
Golgi Network

ABSTRACT A multitier regulation exists at the trans-Golgi network in all higher organisms. We report a palmitoylated protein kinase, Env7, that functions at the TGN interface by interacting with two more TGN-resident proteins, namely, Imh1 and Arl1. Palmitoylation seems to be important for the specific localization. This study focuses on the involvement of a ubiquitous protein kinase, whose substrates had not yet been reported from any organism, as an upstream signaling component that modulates the activity of the Imh1-Arl1 complex crucial for maintaining membrane asymmetry. Virulence is significantly diminished in an Env7 mutant. The functioning of this protein in C. albicans seems to be quite different from its nearest homologue in S. cerervisiae, which reflects the evolutionary divergence between these two organisms. Vesicular dynamics is one of the very important aspects of cellular physiology, an imbalance of which leads to the disorders or diseases in higher eukaryotes. We report the functional characterization of a palmitoylated protein kinase from Candida albicans whose homologue in Saccharomyces cerevisiae has been reported to be involved in negative regulation of membrane fusion and was named Env7. However, the downstream target of this protein remains to be identified. Env7 in C. albicans (CaEnv7) could be isolated from the membrane fraction and localized to vesicular structures associated with the Golgi apparatus. Our work reports Env7 in C. albicans as a new player involved in maintaining the functional dynamics at the trans-Golgi network (TGN) by interacting with two other TGN-resident proteins, namely, Imh1p and Arl1p. Direct interaction could be detected between Env7p and the golgin protein Imh1p. Env7 is itself phosphorylated (Env7p) and phosphorylates Imh1 in vivo. An interaction between Env7 and Imh1 is required for the targeted localization of Imh1. CaEnv7 has a putative palmitoylation site toward both N and C termini. An N-terminal palmitoylation-defective strain retains its ability to phosphorylate Imh1 in vitro. An ENV7 homozygous mutant showed compromised filamentation in solid media and attenuated virulence, whereas an overexpressed strain affected cell wall integrity. Thus, Env7 plays a subtle but important role at the level of multitier regulation that exists at the TGN. IMPORTANCE A multitier regulation exists at the trans-Golgi network in all higher organisms. We report a palmitoylated protein kinase, Env7, that functions at the TGN interface by interacting with two more TGN-resident proteins, namely, Imh1 and Arl1. Palmitoylation seems to be important for the specific localization. This study focuses on the involvement of a ubiquitous protein kinase, whose substrates had not yet been reported from any organism, as an upstream signaling component that modulates the activity of the Imh1-Arl1 complex crucial for maintaining membrane asymmetry. Virulence is significantly diminished in an Env7 mutant. The functioning of this protein in C. albicans seems to be quite different from its nearest homologue in S. cerervisiae, which reflects the evolutionary divergence between these two organisms.

scholarly journals Atypical HemolyticListeria innocuaIsolates Are Virulent, albeit Less thanListeria monocytogenes

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Alexandra Moura ◽  
Olivier Disson ◽  
Morgane Lavina ◽  
Pierre Thouvenot ◽  
Lei Huang ◽  
...  
Keyword(s):  
Human Exposure ◽  
Functional Characterization ◽  
Listeria Innocua ◽  
Whole Genome ◽  
Gene Products ◽  
Content Type ◽  
Functional Assays

ABSTRACTListeria innocuais considered a nonpathogenicListeriaspecies. Natural atypical hemolyticL. innocuaisolates have been reported but have not been characterized in detail. Here, we report the genomic and functional characterization of representative isolates from the two known natural hemolyticL. innocuaclades. Whole-genome sequencing confirmed the presence ofListeriapathogenicity islands (LIPI) characteristic ofListeria monocytogenesspecies. Functional assays showed that LIPI-1 andinlAgenes are transcribed, and the corresponding gene products are expressed and functional. Usingin vitroandin vivoassays, we show that atypical hemolyticL. innocuais virulent, can actively cross the intestinal epithelium, and spreads systemically to the liver and spleen, albeit to a lesser degree than the referenceL. monocytogenesEGDe strain. Although human exposure to hemolyticL. innocuais likely rare, these findings are important for food safety and public health. The presence of virulence traits in someL. innocuaclades supports the existence of a common virulent ancestor ofL. monocytogenesandL. innocua.

scholarly journals Heterologous Production and Functional Characterization of Ageritin, a Novel Type of Ribotoxin Highly Expressed during Fruiting of the Edible Mushroom Agrocybe aegerita

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
Annageldi Tayyrov ◽  
Sophie Azevedo ◽  
Robert Herzog ◽  
Eva Vogt ◽  
Simon Arzt ◽  
...  
Keyword(s):  
Protein Biosynthesis ◽  
Functional Characterization ◽  
Edible Mushroom ◽  
Defense Proteins ◽  
Content Type ◽  
Phosphodiester Bond ◽  
Agrocybe Aegerita ◽  
Protein Toxin

ABSTRACT Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita. The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro. Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development. IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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