scholarly journals Catabolism of Serine by Pediococcus acidilactici and Pediococcus pentosaceus

2012 ◽  
Vol 79 (4) ◽  
pp. 1309-1315 ◽  
Author(s):  
Stefan Irmler ◽  
Tharmatha Bavan ◽  
Andrea Oberli ◽  
Alexandra Roetschi ◽  
René Badertscher ◽  
...  
Keyword(s):  
Pediococcus Acidilactici ◽  
Gene Encoding ◽  
Pediococcus Pentosaceus ◽  
Content Type ◽  
Cell Extracts ◽  
Serine Dehydratase ◽  
Mutant Strains ◽  
Amino Acid Profiles ◽  
Culture Supernatants ◽  
Dehydratase Activity

ABSTRACTThe ability to produce diacetyl from pyruvate andl-serine was studied in various strains ofPediococcus pentosaceusandPediococcus acidilacticiisolated from cheese. After being incubated on both substrates, onlyP. pentosaceusproduced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified inP. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functionaldsdAwas cloned fromP. pentosaceusFAM19132 and expressed inEscherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate froml- andd-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants fromdsdAwild-type anddsdAmutant strains ofP. pentosaceusdid not show differences in serine levels. In contrast,P. acidilacticidegraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate.

scholarly journals Characterization of a Feruloyl Esterase from Lactobacillus plantarum

2013 ◽  
Vol 79 (17) ◽  
pp. 5130-5136 ◽  
Author(s):  
María Esteban-Torres ◽  
Inés Reverón ◽  
José Miguel Mancheño ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Bacterial Species ◽  
Hydrolytic Activity ◽  
Feruloyl Esterase ◽  
Gene Encoding ◽  
Content Type ◽  
Cell Extracts ◽  
Methyl Ferulate ◽  
Methyl Caffeate

ABSTRACTLactobacillus plantarumis frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant.L. plantarumWCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methylp-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methylp-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present amongL. plantarumstrains. The present study constitutes the description of feruloyl esterase activity inL. plantarumand provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species.

scholarly journals Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972

2012 ◽  
Vol 78 (9) ◽  
pp. 3469-3472 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Dolores Gutiérrez ◽  
Beatriz Martínez ◽  
Ana Rodríguez ◽  
Pilar García
Keyword(s):  
Staphylococcus Aureus ◽  
Lactococcus Lactis ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Content Type ◽  
Inducible Promoters ◽  
Secretion Signal ◽  
Cell Extracts ◽  
Culture Supernatants ◽  
Secretion Signal Sequence

ABSTRACTBacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded byStaphylococcus aureusphage vB_SauS-phi-IPLA88, was expressed and secreted inLactococcus lactisusing the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts.

scholarly journals Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation

2015 ◽  
Vol 81 (18) ◽  
pp. 6380-6392 ◽  
Author(s):  
Manuel Nieto-Domínguez ◽  
Laura I. de Eugenio ◽  
Jorge Barriuso ◽  
Alicia Prieto ◽  
Beatriz Fernández de Toro ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Intronless Gene ◽  
Content Type ◽  
Alkyl Glycosides ◽  
Beechwood Xylan ◽  
Dimeric Form ◽  
Culture Supernatants ◽  
Hydrolysis Of

ABSTRACTThis paper reports on a novel β-xylosidase from the hemicellulolytic fungusTalaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu2+tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, andKmandVmaxvalues of 0.17 mM and 52.0 U/mg, respectively, using commercialp-nitrophenyl-β-d-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.

scholarly journals CsrA Supports both Environmental Persistence and Host-Associated Growth of Acinetobacter baumannii

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
John M. Farrow ◽  
Greg Wells ◽  
Samantha Palethorpe ◽  
Mark D. Adams ◽  
Everett C. Pesci
Keyword(s):  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Bacterial Pathogen ◽  
Multidrug Resistant ◽  
Growth Defect ◽  
Gene Encoding ◽  
Content Type ◽  
Indirect Transmission ◽  
Mutant Strains ◽  
Carbon Storage Regulator

ABSTRACT Acinetobacter baumannii is an opportunistic and frequently multidrug-resistant Gram-negative bacterial pathogen that primarily infects critically ill individuals. Indirect transmission from patient to patient in hospitals can drive infections, supported by this organism’s abilities to persist on dry surfaces and rapidly colonize susceptible individuals. To investigate how A. baumannii survives on surfaces, we cultured A. baumannii in liquid media for several days and then analyzed isolates that lost the ability to survive drying. One of these isolates carried a mutation that affected the gene encoding the carbon storage regulator CsrA. As we began to examine the role of CsrA in A. baumannii, we observed that the growth of ΔcsrA mutant strains was inhibited in the presence of amino acids. The ΔcsrA mutant strains had a reduced ability to survive drying and to form biofilms but an improved ability to tolerate increased osmolarity compared with the wild type. We also examined the importance of CsrA for A. baumannii virulence. The ΔcsrA mutant strains had a greatly reduced ability to kill Galleria mellonella larvae, could not replicate in G. mellonella hemolymph, and also had a growth defect in human serum. Together, these results show that CsrA is essential for the growth of A. baumannii on host-derived substrates and is involved in desiccation tolerance, implying that CsrA controls key functions involved in the transmission of A. baumannii in hospitals.

scholarly journals Discovery of a Gene Involved in a Third Bacterial Protoporphyrinogen Oxidase Activity through Comparative Genomic Analysis and Functional Complementation

2011 ◽  
Vol 77 (14) ◽  
pp. 4795-4801 ◽  
Author(s):  
Tye O. Boynton ◽  
Svetlana Gerdes ◽  
Sarah H. Craven ◽  
Ellen L. Neidle ◽  
John D. Phillips ◽  
...  
Keyword(s):  
Oxidase Activity ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Predicted Amino Acid Sequence ◽  
Acinetobacter Baylyi ◽  
Gene Encoding ◽  
Oxidase Gene ◽  
Protoporphyrinogen Oxidase ◽  
Content Type ◽  
Cell Extracts

ABSTRACTTetrapyrroles are ubiquitous molecules in nearly all living organisms. Heme, an iron-containing tetrapyrrole, is widely distributed in nature, including most characterized aerobic and facultative bacteria. A large majority of bacteria that contain heme possess the ability to synthesize it. Despite this capability and the fact that the biosynthetic pathway has been well studied, enzymes catalyzing at least three steps have remained “missing” in many bacteria. In the current work, we have employed comparative genomics via the SEED genomic platform, coupled with experimental verification utilizingAcinetobacter baylyiADP1, to identify one of the missing enzymes, a new protoporphyrinogen oxidase, the penultimate enzyme in heme biosynthesis. COG1981 was identified by genomic analysis as a candidate protein family for the missing enzyme in bacteria that lacked HemG or HemY, two known protoporphyrinogen oxidases. The predicted amino acid sequence of COG1981 is unlike those of the known enzymes HemG and HemY, but in some genomes, the gene encoding it is found neighboring other heme biosynthetic genes. When the COG1981 gene was deleted from the genome ofA. baylyi, a bacterium that lacks bothhemGandhemY, the organism became auxotrophic for heme. Cultures accumulated porphyrin intermediates, and crude cell extracts lacked protoporphyrinogen oxidase activity. The heme auxotrophy was rescued by the presence of a plasmid-borne protoporphyrinogen oxidase gene from a number of different organisms, such ashemGfromEscherichia coli,hemYfromMyxococcus xanthus, or the human gene for protoporphyrinogen oxidase.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals PacBio Amplicon Sequencing Method To Measure Pilin Antigenic Variation Frequencies of Neisseria gonorrhoeae

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Egon A. Ozer ◽  
Lauren L. Prister ◽  
Shaohui Yin ◽  
Billy H. Ward ◽  
Stanimir Ivanov ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Amplicon Sequencing ◽  
Common Mechanism ◽  
Macrophage Infection ◽  
Gene Encoding ◽  
Transmitted Infection ◽  
Variable Regions ◽  
Content Type ◽  
Strain Fa1090

ABSTRACT Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE. Antigenic variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE. We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection. IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin antigenic variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.

scholarly journals Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

2012 ◽  
Vol 11 (8) ◽  
pp. 1055-1066 ◽  
Author(s):  
Matthias Kretschmer ◽  
Jana Klose ◽  
James W. Kronstad
Keyword(s):  
Ustilago Maydis ◽  
Short Chain Fatty Acids ◽  
Phytopathogenic Fungi ◽  
Metabolic Adaptation ◽  
In Planta ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Content Type ◽  
C Terminus ◽  
Fungal Phytopathogens

ABSTRACTAn understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogenUstilago maydis. However,mfe2mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in thehad1gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog,had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of anmfe2Δ mutant. We also show that short-chain fatty acids induce cell death inU. maydisand that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms byU. maydisthat includes potential metabolic contributions to proliferationin plantaand an effect on virulence-related morphogenesis.

scholarly journals Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

2014 ◽  
Vol 80 (20) ◽  
pp. 6465-6472 ◽  
Author(s):  
Sarah L. Robinson ◽  
Daniel G. Panaccione
Keyword(s):  
Aspergillus Fumigatus ◽  
Candidate Genes ◽  
Mass Spectra ◽  
Ergot Alkaloids ◽  
Lysergic Acid ◽  
Precursor Feeding ◽  
Gene Encoding ◽  
Content Type ◽  
Multiple Reactions ◽  
Important Pathway

ABSTRACTDifferent lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungusEpichloë festucaevar.lolii×Epichloë typhinaisolate Lp1 (henceforth referred to asEpichloësp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate geneseasHandcloAwere expressed in a mutant strain of the moldAspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with theEpichloësp. Lp1 allele ofeasA, which encodes an enzyme that catalyzed the synthesis of agroclavine from anA. fumigatusintermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressingeasAandcloAfromEpichloësp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result ofEpichloësp. Lp1easAexpression in the absence ofcloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.

scholarly journals Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

2016 ◽  
Vol 55 (3) ◽  
pp. 844-858 ◽  
Author(s):  
Per Sikora ◽  
Sofia Andersson ◽  
Jadwiga Winiecka-Krusnell ◽  
Björn Hallström ◽  
Cecilia Alsmark ◽  
...  
Keyword(s):  
Parasite Burden ◽  
Genomic Variation ◽  
Loss Of Function ◽  
Oocyst Wall ◽  
Gene Encoding ◽  
Cryptosporidium Hominis ◽  
Content Type ◽  
Successful Isolation ◽  
Target Dna ◽  
Stool Samples

ABSTRACTIn order to improve genotyping and epidemiological analysis ofCryptosporidiumspp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 differentCryptosporidium hominispatient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17× and 490× coverage. Sequence homology-based functional annotation identified several genes of interest, including the gene encodingCryptosporidiumoocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to theCryptosporidium parvumreference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in theC. hoministree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencingCryptosporidiumdirectly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes ofCryptosporidiumfrom human fecal samples, while alluding to the potential for a higher degree of genotyping withinCryptosporidiumepidemiology.

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