Cloning and sequence analysis of the hemB gene of Staphylococcus aureus

1994 ◽  
Vol 40 (8) ◽  
pp. 651-657 ◽  
Author(s):  
Badria Kafala ◽  
A. Sasarman
Keyword(s):  
Staphylococcus Aureus ◽  
Biosynthetic Pathway ◽  
Aminolevulinic Acid ◽  
Colony Growth ◽  
Reading Frame ◽  
E Coli ◽  
Preliminary Identification ◽  
The Family ◽  
Genes Encoding ◽  
Porphobilinogen Synthase

The hemB gene is a member of the family of genes encoding enzymes of the porphyrin biosynthetic pathway and codes for the enzyme porphobilinogen synthase, which is responsible for the conversion of Δ-aminolevulinic acid to porphobilinogen. To clone the hemB gene of Staphylococcus aureus we used Tn917-mediated transposon mutagenesis. Tn917 confers resistance to erythromycin and is carried by plasmid pTV1ts, which has thermosensitive replication. Hem mutants were selected by growth in the presence of kanamycin and erythromycin at 43 °C. Preliminary identification of the hem mutants was based on their dwarf colony growth, which could be restored to normal by hemin. DNA extracted from one of the hem mutants was digested with several restriction endonucleases and hybridized to a probe representing the XbaI–AvaI end of Tn917. A BglII–EcoRI fragment of 4.5 kb gave a positive signal and was cloned into pUC18. Transformants were identified by colony hybridization with the Tn917 probe. The positive clones were sequenced, starting from the transposon end. The results allowed us to identify an open reading frame whose nucleotide sequence presented a homology of 63% to the sequence of the hemB gene of Bacillus subtilis and of 55% to the sequence of the hemB gene of Escherichia coli K12. No other nucleotide sequences, except those belonging to known hemB genes, presented significant homologies to our sequence. The cloning of the hemB gene of S. aureus was confirmed by the ability of the gene to complement a hemB mutant of E. coli K12. To our knowledge, this is the first report of the cloning of a hem gene in S. aureus.Key words: Δ-aminolevulinic acid dehydratase, hemB gene, S. aureus, heme, porphyrins.

scholarly journals Dissemination of CTX-M-Type β-Lactamases among Clinical Isolates of Enterobacteriaceae in Paris, France

2004 ◽  
Vol 48 (4) ◽  
pp. 1249-1255 ◽  
Author(s):  
C. Eckert ◽  
V. Gautier ◽  
M. Saladin-Allard ◽  
N. Hidri ◽  
C. Verdet ◽  
...  
Keyword(s):  
Insertion Sequence ◽  
Dna Fingerprint ◽  
Clinical Isolates ◽  
Pcr Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Paris Area ◽  
The Family ◽  
Genes Encoding ◽  
Cephalosporin Resistance

ABSTRACT We analyzed 19 clinical isolates of the family Enterobacteriaceae (16 Escherichia coli isolates and 3 Klebsiella pneumoniae isolates) collected from four different hospitals in Paris, France, from 2000 to 2002. These strains had a particular extended-spectrum cephalosporin resistance profile characterized by a higher level of resistance to cefotaxime and aztreonam than to ceftazidime. The bla CTX-M genes encoding these β-lactamases were involved in this resistance, with a predominance of bla CTX-M-15. Ten of the 19 isolates produced both TEM-1- and CTX-M-type enzymes. One strain (E. coli TN13) expressed CMY-2, TEM-1, and CTX-M-14. bla CTX-M genes were found on large plasmids. In 15 cases the same insertion sequence, ISEcp1, was located upstream of the 5′ end of the bla CTX-M gene. In one case we identified an insertion sequence designated IS26. Examination of the other three bla CTX-M genes by cloning, sequencing, and PCR analysis revealed the presence of a complex sul1-type integron that includes open reading frame ORF513, which carries the bla gene and the surrounding DNA. Five isolates had the same plasmid DNA fingerprint, suggesting clonal dissemination of CTX-M-15-producing strains in the Paris area.

scholarly journals Identification and Transcriptional Control of the Genes Encoding the Caulobacter crescentus ClpXP Protease

1999 ◽  
Vol 181 (10) ◽  
pp. 3039-3050 ◽  
Author(s):  
Magne Østerås ◽  
Agathe Stotz ◽  
Stefanie Schmid Nuoffer ◽  
Urs Jenal
Keyword(s):  
Heat Shock ◽  
Transcriptional Control ◽  
Caulobacter Crescentus ◽  
Activity Patterns ◽  
Amino Acid Sequences ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Reading Frame ◽  
E Coli ◽  
Genes Encoding

ABSTRACT The region of the Caulobacter crescentus chromosome harboring the genes for the ClpXP protease was isolated and characterized. Comparison of the deduced amino acid sequences of theC. crescentus ClpP and ClpX proteins with those of their homologues from several gram-positive and gram-negative bacteria revealed stronger conservation for the ATPase regulatory subunit (ClpX) than for the peptidase subunit (ClpP). The C. crescentus clpX gene was shown by complementation analysis to be functional in Escherichia coli. However, clpX fromE. coli was not able to substitute for the essential nature of the clpX gene in C. crescentus. TheclpP and clpX genes are separated on theC. crescentus chromosome by an open reading frame pointing in the opposite direction from the clp genes, and transcription of clpP and clpX was found to be uncoupled. clpP is transcribed as a monocistronic unit with a promoter (PP1) located immediately upstream of the 5′ end of the gene and a terminator structure following its 3′ end. PP1 is under heat shock control and is induced upon entry of the cells into the stationary phase. At least three promoters forclpX (PX1, PX2, and PX3) were mapped in the clpP-clpX intergenic region. In contrast to PP1, the clpX promoters were found to be downregulated after heat shock but were also subject to growth phase control. In addition, the clpP andclpX promoters showed different activity patterns during the cell cycle. Together, these results demonstrate that the genes coding for the peptidase and the regulatory subunits of the ClpXP protease are under independent transcriptional control in C. crescentus. Determination of the numbers of ClpP and ClpX molecules per cell suggested that ClpX is the limiting component compared with ClpP.

scholarly journals Plasmodium falciparum hydroxymethylbilane synthase does not house any cosynthase activity within the haem biosynthetic pathway

2021 ◽  
Author(s):  
Alan F. Scott ◽  
Evelyne Deery ◽  
Andrew D. Lawrence ◽  
Martin J. Warren
Keyword(s):  
Plasmodium Falciparum ◽  
Hplc Analysis ◽  
Biosynthetic Pathway ◽  
Gene Encoding ◽  
E Coli ◽  
Aminolaevulinic Acid ◽  
Porphobilinogen Synthase

AbstractThe production of uroporphyrinogen III, the universal progenitor of macrocyclic, modified tetrapyrroles, is produced from aminolaevulinic acid (ALA) by a conserved pathway involving three enzymes: porphobilinogen synthase (PBGS), hydroxymethylbilane synthase (HmbS) and uroporphyrinogen III synthase (UroS). The gene encoding uroporphyrinogen III synthase has not yet been identified in Plasmodium falciparum but it has been suggested that this activity is housed inside a bifunctional hybroxymethylbilane synthase (HmbS). In this present study it is demonstrated that P. falciparum HmbS does not have uroporphyrinogen III synthase activity. This was demonstrated by the failure of a codon optimised P. falciparum hemC gene, encoding HmbS, to compliment a defined E. coli hemD- mutant (SASZ31) deficient in uroporphyrinogen III synthase activity. Furthermore, HPLC analysis of the oxidsed reaction product from recombinant, purified HmbS showed that only uroporphyrin I could be detected (corresponding to hydroxymethylbilane production). No uroporphyrin III was detected, thus showing that P. falciparum HmbS does not have UroS activity and can only catalyse the formation of hydroxymethylbilane from porphobilinogen.

scholarly journals Bioaktivitas Turunan Metil Sinamat Terhadap Pertumbuhan Bakteri Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aureugenosa dan Jamur Candida albicans

2016 ◽  
pp. 60-64
Author(s):  
Teni Ernawati ◽  
Andri Budiana ◽  
Teni Ernawati
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacillus Subtilis ◽  
Candida Albicans ◽  
Cinnamic Acid ◽  
Microbial Growth ◽  
Test Results ◽  
Methyl Cinnamate ◽  
E Coli ◽  
The Family

Methyl cinnamic is a compound isolated from Alpinia malaccensis included in the family Zingiberaceae. A. malaccensis in Indonesia known as galangal forest. Some studies inform that ginger has anti-bacterial and pharmacologically galangal act as an antifungal. In this study the bioactivity of the compound methyl cinnamate and methyl cinnamic derivative which results cinnamic methyl esterification compound on the growth of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aureugenosa and fungus Candida albicans. Methyl cinnamic derivative compounds tested are; cinnamic acid, cinnamic ethyl, butyl and 2-butyl cinnamic cinnamic. Anti-microbial test results showed that of the samples tested, cinnamic acid is able to inhibit microbial growth of S. aureus, B. subtilis, E. coli and P. aureugenosa and fungi C.albicans.DOI :http://dx.doi.org/10.15408/jkv.v0i0.3176 

Revealing the promoting effect of betaine on vitamin B12 biosynthetic pathway of Pseudomonas denitrificans by using a proteomics analysis

2021 ◽  
Vol 22 ◽  
Author(s):  
Kun-tai Li ◽  
Yong Yang ◽  
Xin Cheng
Keyword(s):  
Vitamin B12 ◽  
Biosynthetic Pathway ◽  
Aminolevulinic Acid ◽  
Fermentation Medium ◽  
Promoting Effect ◽  
Pseudomonas Denitrificans ◽  
Methyl Group ◽  
Integral Effect ◽  
Comparative Metabolomics ◽  
Porphobilinogen Synthase

Background: Our previous comparative metabolomics research revealed that betaine (N,N,N-trimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had a powerful promoting effect on the generation of vitamin B12 precursors and intermediates in vitamin B12-producing Pseudomonas denitrificans. However, the integral effect of betaine on the vitamin B12 biosynthetic pathway is still unclear. Objective: Considering the vitamin B12 biosynthetic pathway of P. denitrificans as a whole, this work aimed to reveal the biological function of betaine on the vitamin B12 biosynthetic pathway in P. denitrificans, which would sharpen and expand the understanding of betaine as the methyl-group donor for vitamin B12 biosynthesis. Materials and Methods: By using a proteomics method based on the iTRAQ technique, the present study compared and analyzed the differential expression of proteins involved in vitamin B12 biosynthetic pathway under 10 g/L betaine addition to P. denitrificans fermentation medium. Results: The results showed that betaine could significantly up-regulate the expression of proteins related to the vitamin B12 biosynthetic pathway, which was mainly reflected in the following three aspects: 1) the δ-aminolevulinic acid (ALA) synthase and porphobilinogen synthase that were responsible for the formation of the committed precursors for tetrapyrrole-derived macrocycle in vitamin B12 molecule; 2) the C-methylation-related enzymes (such as precorrin-4 C(11)-methyltransferase, Precorrin-2 C(20)-methyltransferase, Precorrin-8X methylmutase, and Precorrin-6Y C5,15-methyltransferase) and methionine synthase that were crucial to the C-methylation reactions for vitamin B12 biosynthesis; 3) the late-stage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. Conclusions: The present study clearly demonstrated that betaine could significantly promote the expression of the integral enzymes involved in the vitamin B12 biosynthetic pathway of P. denitrificans, thus promoting vitamin B12 biosynthesis.

scholarly journals Clustering of the YNA1 gene encoding a Zn(II)2Cys6 transcriptional factor in the yeast Hansenula polymorpha with the nitrate assimilation genes YNT1, YNI1 and YNR1, and its involvement in their transcriptional activation

1998 ◽  
Vol 335 (3) ◽  
pp. 647-652 ◽  
Author(s):  
Julio ÁVILA ◽  
Celedonio GONZÁLEZ ◽  
Nélida BRITO ◽  
José M. SIVERIO
Keyword(s):  
Transcriptional Activation ◽  
Hansenula Polymorpha ◽  
Nitrate Assimilation ◽  
Transcriptional Factors ◽  
Gene Encoding ◽  
Reading Frame ◽  
Transcription Start Sites ◽  
The Family ◽  
Genes Encoding ◽  
Free Media

The genes encoding the nitrate transporter (YNT1), nitrite reductase (YNI1) and nitrate reductase (YNR1) are clustered in the yeast Hansenula polymorpha. In addition, DNA sequencing of the region containing these genes demonstrated that a new open reading frame called YNA1 (yeast nitrate assimilation) was located between YNR1 and YNI1. The YNA1 gene encodes a protein of 529 residues belonging to the family of Zn(II)2Cys6 fungal transcriptional factors, and has the highest similarity to the transcriptional factors encoded by nirA, and to a smaller extent to nit-4, involved in the nitrate induction of the gene involved in the assimilation of this compound in filamentous fungi. Northern blot analysis showed the presence of the YNA1 transcript in cells incubated in nitrate, nitrate plus ammonium, ammonium, and nitrogen-free media, with a decrease in its levels in those cells incubated in ammonium. In nitrate the strain Δyna1::URA3, with a disrupted YNA1 gene, neither grew nor expressed the genes YNT1, YNI1 and YNR1. In the gene cluster YNT1-YNI1-YNA1-YNR1, the four genes were transcribed independently in the YNT1 → YNR1 direction and the transcription start sites were determined by primer extension.

scholarly journals Pas, a Novel Protein Required for Protein Secretion and Attaching and Effacing Activities of Enterohemorrhagic Escherichia coli

1998 ◽  
Vol 180 (17) ◽  
pp. 4370-4379 ◽  
Author(s):  
Andreas U. Kresse ◽  
Kai Schulze ◽  
Christina Deibel ◽  
Frank Ebel ◽  
Manfred Rohde ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Enterohemorrhagic Escherichia Coli ◽  
Host Cells ◽  
Reading Frame ◽  
E Coli ◽  
Genes Encoding ◽  
Enterocyte Effacement ◽  
Putative Member

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic E. coli. We have identified an open reading frame (ORF) which is located upstream of the espA, espB, andespD genes on the complementary strand and which exhibits high homology to the genes spiB fromSalmonella, yscD from Yersinia, andpscD from Pseudomonas. Localization studies showed that the encoded product is present in the cytoplasmic and inner membrane fractions of EHEC. The construction and characterization of a recombinant clone containing an in-frame deletion of this ORF demonstrated that the encoded product is a putative member of a type III system required for protein secretion. Disruption of this ORF, designated pas (protein associated with secretion), abolished the secretion of Esp proteins. The mutant adhered only poorly and lost its capacities to trigger attaching and effacing activity and to invade HeLa cells. These results demonstrate that Pas is a virulence-associated factor that plays an essential role in EHEC pathogenesis.

Crystallization and preliminary X-ray diffraction studies of E. coli porphobilinogen synthase and its heavy-atom derivatives

1998 ◽  
Vol 54 (3) ◽  
pp. 438-440 ◽  
Author(s):  
L. Shimoni-Livny ◽  
H. L. Carrell ◽  
T. Wagner ◽  
A. Kaufman Katz ◽  
C. Afshar ◽  
...  
Keyword(s):  
Active Sites ◽  
Aminolevulinic Acid ◽  
Heavy Atom ◽  
Unit Cell ◽  
X Ray Diffraction ◽  
X Ray ◽  
E Coli ◽  
Cell Dimensions ◽  
Porphobilinogen Synthase ◽  
Unit Cell Dimensions

Porphobilinogen synthase (PBGS) catalyzes the condensation of two identical substrate molecules, 5-aminolevulinic acid (ALA), in an asymmetric manner to form porphobilinogen. E. coli PBGS is an homooctameric enzyme. The number of active sites is not clear, but each subunit binds one ZnII ion and one MgII ion. Diffraction-quality crystals of native E. coli PBGS have been obtained, and unit-cell dimensions (a = 130.8, c = 144.0 Å) are reported. These crystals diffract to about 3.0 Å resolution.

scholarly journals TmSpz6 Is Essential for Regulating the Immune Response to Escherichia coli and Staphylococcus aureus Infection in Tenebrio molitor

Insects ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 105 ◽  
Author(s):  
Tariku Tesfaye Edosa ◽  
Yong Hun Jo ◽  
Maryam Keshavarz ◽  
Young Min Bae ◽  
Dong Hyun Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Immune Response ◽  
Expression Analysis ◽  
Fungal Infections ◽  
Specific Expression ◽  
Reading Frame ◽  
E Coli ◽  
Aureus Infection ◽  
Toll Receptor

Spätzle is an extracellular protein that activates the Toll receptor during embryogenesis and immune responses in Drosophila. However, the functions of the spätzle proteins in the innate immune response against bacteria or fungi in T. molitor are not well understood. Therefore, in this study, the open reading frame (ORF) of TmSpz6 was identified and its function in the response to bacterial and fungal infections in T. molitor was investigated using RNAi. The highest expression of TmSpz6 was in prepupae, and 3- and 6-day-old pupae, while remarkable expression was also observed in other stages. The tissue-specific expression analysis showed that TmSpz6 expression was highest in the hemocytes of larvae. TmSpz6 expression was highly induced when challenged with Escherichia coli, Staphylococcus aureus, or Candida albicans at 6 h post-injection; however, TmSpz6-silenced larvae were significantly more susceptible to only E. coli and S. aureus infection. The antimicrobial peptides (AMPs) gene expression analysis results show that TmSpz6 mainly positively regulated the expression of TmTencin-2 and -3 in response to E. coli and S. aureus infection. Collectively, these results suggest that TmSpz6 plays an important role in regulating AMP expression and increases the survival of T. molitor against E. coli and S. aureus.

scholarly journals The Biosynthetic Pathway for Myxol-2′ Fucoside (Myxoxanthophyll) in the Cyanobacterium Synechococcus sp. Strain PCC 7002

2009 ◽  
Vol 191 (10) ◽  
pp. 3292-3300 ◽  
Author(s):  
Joel E. Graham ◽  
Donald A. Bryant
Keyword(s):  
Biosynthetic Pathway ◽  
Chemical Characterization ◽  
Open Reading Frame ◽  
Biotechnological Applications ◽  
Reading Frame ◽  
Genes Encoding ◽  
Synechococcus Sp ◽  
Related Compounds ◽  
Β Carotene

ABSTRACT Synechococcus sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic β-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2′ fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2′-OH rather than on the 1′-OH position of the ψ end of the molecule. In this study, the genes encoding two enzymes that modify the ψ end of myxoxanthophyll in Synechococcus sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed cruF, encodes a 1′-hydroxylase and that open reading frame SynPCC7002_A2031, renamed cruG, encodes a 2′-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when cruF was expressed in a lycopene-producing strain of Escherichia coli. Database searches showed that homologs of cruF and cruG occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain crtC homologs also contain cruF orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.

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