scholarly journals Identification of a Genetic Locus Essential for Serotype b-Specific Antigen Synthesis in Actinobacillus actinomycetemcomitans

1998 ◽  
Vol 66 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Yasuo Yoshida ◽  
Yoshio Nakano ◽  
Yoshihisa Yamashita ◽  
Toshihiko Koga
Keyword(s):  
High Performance ◽  
Genetic Locus ◽  
Specific Antigen ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Actinobacillus Actinomycetemcomitans ◽  
E Coli ◽  
Chromatography Analysis ◽  
Large Gene ◽  
Serotype B

ABSTRACT A large gene cluster associated with the biosynthesis of the serotype-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans Y4 (serotype b) was cloned and characterized. Western blot analysis showed that Escherichia coli DH5α, containing a plasmid carrying this cluster, produced a polysaccharide which reacted with a monoclonal antibody directed against the SPA of A. actinomycetemcomitans Y4. High-performance liquid chromatography analysis indicated that the polysaccharide produced by an E. coli transformant, as well as A. actinomycetemcomitans Y4 SPA, was composed of rhamnose and fucose. Furthermore, using various derivatives of the plasmid, we demonstrated that the cloned 13-kbBssHII-BspHI fragment was indispensable for SPA synthesis in E. coli DH5α. The 24,909-bp nucleotide sequence, which included this fragment and its flanking regions, was determined. In the sequenced area, 24 open reading frames (ORFs) with the same orientation were found. Most of these were located sequentially within a short distance of each other. Many of the deduced amino acid sequences were similar to the gene products of the polysaccharide synthetic genes of other bacteria. The average G+C content (37.7%) of all 24 ORFs in the sequenced area was lower than that (45.6%) of the whole chromosome of A. actinomycetemcomitans Y4. It is noteworthy the average G+C content of the nine ORFs in the 8.5-kb central region of the 13-kbBssHII-BspHI fragment indispensable for SPA synthesis in E. coli was found to be especially low (27.0%).

scholarly journals EfrAB, an ABC Multidrug Efflux Pump in Enterococcus faecalis

2003 ◽  
Vol 47 (12) ◽  
pp. 3733-3738 ◽  
Author(s):  
Eun-Woo Lee ◽  
M. Nazmul Huda ◽  
Teruo Kuroda ◽  
Tohru Mizushima ◽  
Tomofusa Tsuchiya
Keyword(s):  
Enterococcus Faecalis ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Multidrug Efflux ◽  
Chromosomal Dna ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A DNA fragment responsible for resistance to antimicrobial agents was cloned from the chromosomal DNA of Enterococcus faecalis ATCC 29212 by using drug-hypersensitive mutant Escherichia coli KAM32 as a host cell. Cells of E. coli KAM32 harboring a recombinant plasmid (pAEF82) carrying the DNA fragment became resistant to many structurally unrelated antimicrobial agents, such as norfloxacin, ciprofloxacin, doxycycline, acriflavine, 4′,6-diamidino-2-phenylindole, tetraphenylphosphonium chloride, daunorubicin, and doxorubicin. Since the sequence of the whole genome of E. faecalis is known, we sequenced several portions of the DNA insert in plasmid pAEF82 and identified two open reading frames within the insert. We designated the genes efrA and efrB. A search of the deduced amino acid sequences of EfrA and EfrB revealed that they are similar to each other and that they belong to the ATP-binding cassette (ABC) family of multidrug efflux transporters. Transformed E. coli KAM32 cells harboring efrAB showed energy-dependent efflux of acriflavine. The efflux activity was inhibited by reserpine, verapamil, and sodium-o-vanadate, known inhibitors of ABC efflux pumps.

scholarly journals Two C—P Lyase Operons in Pseudomonas stutzeri and Their Roles in the Oxidation of Phosphonates, Phosphite, and Hypophosphite

2004 ◽  
Vol 186 (14) ◽  
pp. 4730-4739 ◽  
Author(s):  
Andrea K. White ◽  
William W. Metcalf
Keyword(s):  
Insertion Site ◽  
Pseudomonas Stutzeri ◽  
Gene Organization ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Transposon Insertion ◽  
Intergenic Sequences ◽  
Reading Frames

ABSTRACT DNA sequencing and analysis of two distinct C—P lyase operons in Pseudomonas stutzeri WM88 were completed. The htxABCDEFGHIJKLMN operon encodes a hypophosphite-2-oxoglutarate dioxygenase (HtxA), whereas the predicted amino acid sequences of HtxB to HtxN are each homologous to the components of the Escherichia coli phn operon, which encodes C—P lyase, although homologs of E. coli phnF and phnO are absent. The genes in the htx operon are cotranscribed based on gene organization, and the presence of the intergenic sequences is verified by reverse transcription-PCR with total RNA. Deletion of the htx locus does not affect the ability of P. stutzeri to grow on phosphonates, indicating the presence of an additional C—P lyase pathway in this organism. To identify the genes comprising this pathway, a Δhtx strain was mutagenized and one mutant lacking the ability to grow on methylphosphonate as the sole P source was isolated. A ca.-10.6-kbp region surrounding the transposon insertion site of this mutant was sequenced, revealing 13 open reading frames, designated phnCDEFGHIJKLMNP, which were homologous to the E. coli phn genes. Deletion of both the htx and phn operons of P. stutzeri abolishes all growth on methylphosphonate and aminoethylphosphonate. Both operons individually support growth on methylphosphonate; however, the phn operon supports growth on aminoethylphosphonate and phosphite, as well. The substrate ranges of both C—P lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.

scholarly journals TolC-Dependent Exclusion of Porphyrins in Escherichia coli

2008 ◽  
Vol 190 (18) ◽  
pp. 6228-6233 ◽  
Author(s):  
Ryoko Tatsumi ◽  
Masaaki Wachi
Keyword(s):  
Escherichia Coli ◽  
Uv Irradiation ◽  
High Performance ◽  
Aminolevulinic Acid ◽  
Wild Type ◽  
E Coli ◽  
Chromatography Analysis ◽  
Increased Sensitivity ◽  
Mutant Cells ◽  
Reddish Brown Color

ABSTRACT We found that Escherichia coli tolC mutants showed increased sensitivity to 5-aminolevulinic acid (ALA), a precursor of porphyrins. The tolC mutant cells grown in the presence of ALA showed a reddish brown color under visible light and a strong red fluorescence under near-UV irradiation. Fluorescence spectrometry and high-performance liquid chromatography analysis showed that the tolC mutant cells grown in the presence of ALA accumulated a large amount of coproporphyrin(ogen) intracellularly. In contrast, the wild-type cells produced coproporphyrin extracellularly. The tolC mutant cells grown in the presence of ALA, which were capable of surviving in the dark, were killed by near-UV irradiation, suggesting that the intracellular coproporphyrin(ogen) renders these cells photosensitive. These results suggest that the TolC-dependent efflux system is involved in the exclusion of porphyrin(ogen)s in E. coli.

scholarly journals Functional Genomics Enables Identification of Genes of the Arginine Transaminase Pathway in Pseudomonas aeruginosa

2007 ◽  
Vol 189 (11) ◽  
pp. 3945-3953 ◽  
Author(s):  
Zhe Yang ◽  
Chung-Dar Lu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Performance ◽  
Dna Microarrays ◽  
Genetic Locus ◽  
Regulatory Protein ◽  
Chromatography Analysis ◽  
Arginine Catabolism ◽  
Metabolic Versatility ◽  
Phenotype Analysis ◽  
Major Route

ABSTRACT Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of the metabolic versatility of this organism. To identify genes involved in arginine catabolism, we have employed DNA microarrays to analyze the transcriptional profiles of this organism in response to l-arginine. While most of the genes involved in arginine uptake, regulation, and metabolism have been identified as members of the ArgR (arginine-responsive regulatory protein) regulon in our previous study, they did not include any genes of the arginine dehydrogenase (ADH) pathway. In this study, 18 putative transcriptional units of 38 genes, including the two known genes of the ADH pathway, kauB and gbuA, were found to be inducible by exogenous l-arginine in the absence of ArgR. To identify the missing genes that encode enzymes for the initial steps of the ADH pathway, the potential physiological functions of those candidate genes in arginine utilization were studied by growth phenotype analysis of knockout mutants. Expression of these genes was induced by l-arginine in an aruF mutant strain devoid of a functional arginine succinyltransferase pathway, the major route of arginine utilization. Disruption of dadA, a putative catabolic alanine dehydrogenase-encoding gene, in the aruF mutant produced no growth on l-arginine, suggesting the involvement of l-alanine in arginine catabolism. This hypothesis was further supported by the detection of an l-arginine-inducible arginine:pyruvate transaminase activity in the aruF mutant. Knockout of aruH and aruI, which encode an arginine:pyruvate transaminase and a 2-ketoarginine decarboxylase in an operon, also abolished the ability of the aruF mutant to grow on l-arginine. The results of high-performance liquid chromatography analysis demonstrated consumption of 2-ketoarginine and suggested that generation of 4-guanidinobutyraldehyde occurred in the aruF mutant but not in the aruF aruI mutant. These results led us to propose the arginine transaminase pathway that removes the α-amino group of l-arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the arginine transaminase pathway. This work depicted a wider network of arginine metabolism than we previously recognized.

scholarly journals Metagenome-Derived Clones Encoding Two Novel Lactonase Family Proteins Involved in Biofilm Inhibition in Pseudomonas aeruginosa

2008 ◽  
Vol 75 (1) ◽  
pp. 224-233 ◽  
Author(s):  
C. Schipper ◽  
C. Hornung ◽  
P. Bijtenhoorn ◽  
M. Quitschau ◽  
S. Grond ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
High Performance ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Biofilm Inhibition ◽  
Soil Dna ◽  
Isolation And Characterization ◽  
Bacterial Quorum

ABSTRACT Here we report the isolation and characterization of three metagenome-derived clones that interfere with bacterial quorum sensing and degrade N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL). By using a traI-lacZ gene fusion, the metagenome-derived clones were identified from a soil DNA library and analyzed. The open reading frames linked to the 3-oxo-C8-HSL-degrading activities were designated bpiB01, bpiB04, and bpiB07. While the BpiB07 protein was similar to a known lactonase, no significant similarities were observed for the BpiB01 and BpiB04 proteins or the deduced amino acid sequences. High-performance liquid chromatography-mass spectrometry analyses confirmed that the identified genes encode novel lactone-hydrolyzing enzymes. The original metagenome-derived clones were expressed in Pseudomonas aeruginosa and employed in motility and biofilm assays. All clones were able to reproducibly inhibit motility in P. aeruginosa. Furthermore, these genes clearly inhibited biofilm formation in P. aeruginosa when expressed in P. aeruginosa PAO1. Thus, this is the first study in which metagenome-derived proteins have been expressed in P. aeruginosa to successfully inhibit biofilm formation.

scholarly journals Unstable Escherichia coli L Forms Revisited: Growth Requires Peptidoglycan Synthesis

2007 ◽  
Vol 189 (18) ◽  
pp. 6512-6520 ◽  
Author(s):  
Danièle Joseleau-Petit ◽  
Jean-Claude Liébart ◽  
Juan A. Ayala ◽  
Richard D'Ari
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
High Performance ◽  
Specific Inhibitor ◽  
Hypertonic Medium ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Chromatography Analysis ◽  
Peptidoglycan Synthesis ◽  
K 12

ABSTRACT Growing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-type Escherichia coli K-12 strain overnight to a growing L-form-like state by use of the β-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requires d-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the β-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also require d-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells of E. coli—and possibly all L forms—have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.

scholarly journals A Putative Multisubunit Na+/H+ Antiporter fromStaphylococcus aureus

1998 ◽  
Vol 180 (24) ◽  
pp. 6642-6648 ◽  
Author(s):  
Toshiaki Hiramatsu ◽  
Kazuyo Kodama ◽  
Teruo Kuroda ◽  
Tohru Mizushima ◽  
Tomofusa Tsuchiya
Keyword(s):  
Sequence Similarity ◽  
Membrane Vesicles ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Host Cells ◽  
Homology Search ◽  
Upstream Region ◽  
Chromosomal Dna ◽  
E Coli ◽  
Alkaline Conditions

ABSTRACT We cloned several genes encoding an Na+/H+antiporter of Staphylococcus aureus from chromosomal DNA by using an Escherichia coli mutant, lacking all of the major Na+/H+ antiporters, as the host. E. coli cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na+/H+ antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na+/H+antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na+/H+antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na+/H+ antiporter. We observed a large Na+ extrusion activity, driven by respiration in E. coli cells harboring the plasmid carrying the genes. The Na+ extrusion was sensitive to an H+conductor, supporting the idea that the system is not a respiratory Na+ pump but an Na+/H+ antiporter. Introduction of the plasmid into E. coli mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.

scholarly journals Molecular Cloning of the fur Gene from Actinobacillus actinomycetemcomitans

2002 ◽  
Vol 70 (6) ◽  
pp. 3170-3179 ◽  
Author(s):  
V. I. Haraszthy ◽  
E. T. Lally ◽  
G. G. Haraszthy ◽  
J. J. Zambon
Keyword(s):  
Gene Product ◽  
Bacterial Species ◽  
Consensus Sequence ◽  
Actinobacillus Actinomycetemcomitans ◽  
Gene Probe ◽  
Virulence Determinants ◽  
Chromosomal Dna ◽  
E Coli ◽  
Therapeutic Modalities ◽  
Serotype B

ABSTRACT In several bacterial species, iron availability in host tissues is coordinated with the expression of virulence determinants through the fur gene product. Initial experiments showed that a cloned Escherichia coli fur gene probe hybridized to Southern blots of Actinobacillus actinomycetemcomitans strain JP2 (serotype b) chromosomal DNA. The A. actinomycetemcomitans fur gene was then cloned utilizing partial functional complementation of the fur mutant in E. coli strain H1780. Analysis of the cloned DNA sequence revealed a 438-bp open reading frame with a deduced 146-amino-acid sequence exhibiting 80% identity to Haemophilus influenzae Fur and 62% identity to E. coli Fur. The pUC Aafur gene probe (generated from JP2 serotype b) hybridized to representatives from all five A. actinomycetemcomitans serotypes as well as to two strains derived from monkeys, suggesting that fur is widely distributed in A. actinomycetemcomitans. Open reading frames having >70% identity with the E. coli and H. influenzae flavodoxin and gyrase A genes, respectively, were found. Expression of the A. actinomycetemcomitans fur gene product repressed fiu expression and siderophore production in E. coli. A gel shift assay demonstrated that the expressed A. actinomycetemcomitans Fur protein bound the bacterial fur consensus sequence. Further characterization of the fur gene product in A. actinomycetemcomitans may improve our understanding of its role in the pathogenesis of periodontal disease and may lead to specific therapeutic modalities.

scholarly journals CHARACTERIZATION OF THERMOSTABLE BETA-1,4-GALACTANASE AND ITS APPLICATION IN HYDROLYSIS OF PECTIN FROM SWEET POTATO (Ipomoea batatas (L.) Lam) PEELS

2021 ◽  
Vol 83 (5) ◽  
pp. 57-65
Author(s):  
Noor Faizah Ismail ◽  
Dayang Norulfairuz Abang Zaidel ◽  
Mohd Noor Mat Isa
Keyword(s):  
Sweet Potato ◽  
High Performance ◽  
Ipomoea Batatas ◽  
Catalytic Efficiency ◽  
Tween 20 ◽  
E Coli ◽  
Chromatography Analysis ◽  
Potato Peels ◽  
Hydrolysis Of

Galactooligosaccharides (GOS) synthesis has received much attention due to its prebiotic function. Beta-1,4-galactanase responsible for the hydrolysis of galactan plays an important role in producing GOS from biodegradation of this pectin component. In this study, beta-1,4-galactanase (BgcGC) from a thermophilic Geobacillus mahadii Geo-05 was heterologously expressed in Escherichia coli (E. coli) and characterized. The optimum temperature of BgcGC was at 60°C and stable from 20-60°C while optimum pH was at 6 and stable from pH 4-10. BgcGC showed high catalytic efficiency towards potato galactan (873.8 ml mg-1 s-1) and lupin galactan (1694.4 ml mg-1 s-1). The activity of BgcGC was not significantly affected with the presence of 100 mM K+, Tween-20 and 2-mercaptoethanol. Application of BgcGC towards pectin-containing galactan oligomer extracted from sweet potato peels resulted in galactose and GOS synthesis as revealed by high performance liquid chromatography analysis. Thus, this enzyme has a potential to be one of the enzyme candidates involves in pectin complex degradation to produce GOS.

scholarly journals Distinct Actions by Paenibacillus sp. Strain E18 α-l-Arabinofuranosidases and Xylanase in Xylan Degradation

2013 ◽  
Vol 79 (6) ◽  
pp. 1990-1995 ◽  
Author(s):  
Pengjun Shi ◽  
Xiaoyan Chen ◽  
Kun Meng ◽  
Huoqing Huang ◽  
Yingguo Bai ◽  
...  
Keyword(s):  
High Performance ◽  
Synergistic Effects ◽  
Amino Acid Sequences ◽  
Water Soluble ◽  
Open Reading Frames ◽  
Content Type ◽  
Xylan Degradation ◽  
Wheat Arabinoxylan ◽  
Paenibacillus Sp ◽  
Family 10 Xylanase

ABSTRACTWe cloned aPaenibacillussp. strain E18 5.3-kb xylanolytic gene cluster that contains three open reading frames encoding two family 43 α-l-arabinofuranosidases (Abf43A and Abf43B) and one family 10 xylanase (XynBE18). The deduced amino acid sequences of Abf43A and Abf43B were at most 68% and 63% identical to those of two putative family 43 proteins fromClostridiumsp. strain DL-VIII (EHI98634.1 and EHI98635.1), respectively, but were only 11% identical to each other. Recombinant Abf43A and Abf43B had similar activities at 45°C and pH 6.0 but varied in thermostabilities and substrate specificities. Abf43B was active against only 4-nitrophenyl α-l-arabinofuranoside, whereas Abf43A acted on 4-nitrophenyl α-l-arabinofuranoside, wheat arabinoxylan, 4-nitrophenyl α-d-xylopyranoside, and sugar beet arabinan. The sequential and combined effects on xylan degradation by XynBE18, Abf43A, and Abf43B were characterized. For beechwood, birchwood, and oat spelt xylans as the substrates, synergistic effects were found when XynBE18 and Abf43A or Abf43B were incubated together and when the substrates were first incubated with Abf43A or Abf43B and then with XynBE18. Further high-performance liquid chromatography (HPLC) analysis showed that the amounts of xylobiose and xylose increased sharply in the aforementioned reactions. For water-soluble wheat arabinoxylan as the substrate, Abf43A not only released arabinose but also had a synergistic effect with XynBE18. Synergy may arise as the result of removal of arabinose residues from xylans by α-l-arabinofuranosidases, which eliminates steric hindrance caused by the arabinose side chains and which allows xylanases to then degrade the xylan backbone, producing short xylooligosaccharides.

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