The Genes Coding for Enterocin EJ97 Production by Enterococcus faecalis EJ97 Are Located on a Conjugative Plasmid

ABSTRACT Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.

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Characterization of Novel Carbazole Catabolism Genes from Gram-Positive Carbazole Degrader Nocardioides aromaticivorans IC177

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3321-3329.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3321-3329 ◽

Cited By ~ 40

Author(s):

Kengo Inoue ◽

Hiroshi Habe ◽

Hisakazu Yamane ◽

Hideaki Nojiri

Keyword(s):

Amino Acid ◽

Gene Clusters ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Class Iii ◽

Rt Pcr ◽

Gram Positive ◽

Mononuclear Iron ◽

Reverse Transcription Pcr ◽

Genes Encoding

ABSTRACT Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.

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Characterization of Two Plasmid-borne lpsβ Loci of Rhizobium etli Required for Lipopolysaccharide Synthesis and for Optimal Interaction with Plants

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1997.10.7.891 ◽

1997 ◽

Vol 10 (7) ◽

pp. 891-902 ◽

Cited By ~ 39

Author(s):

Alejandro García-de los Santos ◽

Susana Brom

Keyword(s):

Amino Acid ◽

Dna Sequences ◽

Open Reading Frames ◽

Transcriptional Unit ◽

Rhizobium Etli ◽

Polysaccharide Biosynthesis ◽

Strong Homology ◽

Surface Polysaccharide ◽

Functional Features

In Rhizobium etli CFN42, both the symbiotic plasmid (pd) and plasmid b (pb) are required for effective bean nodulation. This is due to the presence on pb of a region (lpsβ) involved in lipopolysaccharide (LPS) biosynthesis. We report here the genetic array and functional features of this plasmid-borne region. The sequence analysis of a 3,595-bp fragment revealed the presence of a transcriptional unit integrated by two open reading frames (lpsβ1 and lpsβ2) essential for LPS biosynthesis and symbiosis. The lpsβ1 encodes a putative 193 amino acid polypeptide that shows strong homology with glucosyl-1P and galactosyl-1P transferases. The deduced amino acid sequence of the protein encoded by lpsβ2 was very similar to that of proteins involved in surface polysaccharide biosynthesis, such as Pseudomonas aeruginosa WpbM, Bordetella pertussis BplL, and Yersinia enterocolitica TrsG. DNA sequences homologous to lpsβ1 and lpsβ2 of R. etli CFN42 were consistently found in functionally equivalent plasmids of R. etli, R. leguminosarum bv. viciae, and R. leguminosarum bv. trifolii strains, but not in R. meliloti, R. loti, R. tropici, R. fredii, Bradyrhizobium, Azorhizobium, and Agrobacterium tumefaciens. Even though Rhizobium and Agrobacterium do not share lpsβ sequences, their presence is required for crown-gall tumor induction by R. etli transconjugants carrying the Ti plasmid.

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Cloning and Genetic Analyses of the Bacteriocin 41 Determinant Encoded on the Enterococcus faecalis Pheromone-Responsive Conjugative Plasmid pYI14: a Novel Bacteriocin Complemented by Two Extracellular Components (Lysin and Activator)

Journal of Bacteriology ◽

10.1128/jb.01056-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 2075-2085 ◽

Cited By ~ 16

Author(s):

Haruyoshi Tomita ◽

Elizabeth Kamei ◽

Yasuyoshi Ike

Keyword(s):

Enterococcus Faecalis ◽

Sequence Data ◽

Open Reading Frames ◽

Conjugative Plasmid ◽

Cell Surface Receptor ◽

Bacteriocin Activity ◽

Lytic Enzymes ◽

Repeat Structure ◽

Surface Receptor ◽

Reading Frames

ABSTRACT The conjugative plasmid pYI14 (61 kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E. faecalis. Physical mapping of pYI14 showed that it consisted of EcoRI fragments A to P. The clone pHT1100, containing EcoRI fragments A (12.6 kbp) and H (3.5 kbp), conferred the bacteriocin activity on E. faecalis strains. Genetic analysis showed that the determinant was located in a 6.6-kbp region within the EcoRI AH fragments. Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL1 ) ORF8 (bacL2 ), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL1 , bacL2 , bacA, and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL1 and -L2 and bacA mutants. bacL1 and -L2 and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, providing the host with resistance to its own bacteriocin activity. The bacL1 -encoded protein had significant homology with lytic enzymes that attack the gram-positive bacterial cell wall. Sequence data for the deduced bacL1 -encoded protein suggested that it has a domain structure consisting of an N-terminal signal peptide, a second domain with the enzymatic activity, and a third domain with a three-repeat structure directing the proenzyme to its cell surface receptor.

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Identification of Listeria monocytogenes Genes Expressed in Response to Growth at Low Temperature

Applied and Environmental Microbiology ◽

10.1128/aem.68.4.1697-1705.2002 ◽

2002 ◽

Vol 68 (4) ◽

pp. 1697-1705 ◽

Cited By ~ 122

Author(s):

Siqing Liu ◽

James E. Graham ◽

Lance Bigelow ◽

Philip D. Morse ◽

Brian J. Wilkinson

Keyword(s):

Listeria Monocytogenes ◽

Amino Acid ◽

Low Temperature ◽

Cell Surface ◽

Stress Responses ◽

Bacterial Pathogen ◽

Open Reading Frames ◽

Cdna Clones ◽

Adaptive Responses ◽

Significant Similarity

ABSTRACT Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10°C compared to those grown at 37°C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses (flaA and flp), regulatory adaptive responses (rpoN, lhkA, yycJ, bglG, adaB, and psr), general microbial stress responses (groEL, clpP, clpB, flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alterations (fbp, psr, and flaA), and degradative metabolism (eutB, celD, and mleA). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10°C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.

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Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

Journal of Bacteriology ◽

10.1128/jb.181.10.3193-3200.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3193-3200 ◽

Cited By ~ 36

Author(s):

Andrea Wilcks ◽

Lasse Smidt ◽

Ole Andreas Økstad ◽

Anne-Brit Kolstø ◽

Jacques Mahillon ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Dna Sequences ◽

Open Reading Frames ◽

Conjugative Plasmid ◽

Dna Polymerase I ◽

Significant Similarity ◽

Strong Similarity ◽

Stability Functions ◽

Bona Fide ◽

Polymerase I

ABSTRACT A 5.8-kb fragment of the large conjugative plasmid pAW63 fromBacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMβ1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMβ1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMβ1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of theEnterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repCand prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.

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LlaFI, a Type III Restriction and Modification System in Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.65.2.686-693.1999 ◽

1999 ◽

Vol 65 (2) ◽

pp. 686-693 ◽

Cited By ~ 22

Author(s):

Ping Su ◽

Heejeong Im ◽

Hsiaoling Hsieh ◽

Simon Kang’A ◽

Noel W. Dunn

Keyword(s):

Amino Acid ◽

Lactococcus Lactis ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Homology Search ◽

Binding Motif ◽

Significant Similarity ◽

Modification System ◽

Type Iii ◽

Central Regions

ABSTRACT We describe a type III restriction and modification (R/M) system,LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactisLL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence.LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.

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Amino acid sequence of pheromone-inducible surface protein in Enterococcus faecalis , that is encoded on the conjugative plasmid pPD1

FEBS Letters ◽

10.1016/0014-5793(90)80293-r ◽

1990 ◽

Vol 267 (1) ◽

pp. 81-84 ◽

Cited By ~ 7

Author(s):

Jiro Nakayama ◽

Hiromich Nagasawa ◽

Akira Isogai ◽

Don B. Clewell ◽

Akinori Suzuki

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Enterococcus Faecalis ◽

Surface Protein ◽

Conjugative Plasmid

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Enterocin 96, a Novel Class II Bacteriocin Produced by Enterococcus faecalis WHE 96, Isolated from Munster Cheese

Applied and Environmental Microbiology ◽

10.1128/aem.02772-08 ◽

2009 ◽

Vol 75 (13) ◽

pp. 4273-4276 ◽

Cited By ~ 35

Author(s):

Esther Izquierdo ◽

Camille Wagner ◽

Eric Marchioni ◽

Dalal Aoude-Werner ◽

Saïd Ennahar

Keyword(s):

Enterococcus Faecalis ◽

Class Ii ◽

Reversed Phase ◽

Leader Peptide ◽

Edman Degradation ◽

Strong Activity ◽

New Class ◽

Soft Cheese ◽

Wide Range ◽

Activity Spectrum

ABSTRACT Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.

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Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by Enterococcus faecalis BFE 1071

Applied and Environmental Microbiology ◽

10.1128/aem.66.4.1298-1304.2000 ◽

2000 ◽

Vol 66 (4) ◽

pp. 1298-1304 ◽

Cited By ~ 89

Author(s):

E. Balla ◽

L. M. T. Dicks ◽

M. Du Toit ◽

M. J. Van Der Merwe ◽

W. H. Holzapfel

Keyword(s):

Amino Acid ◽

Antimicrobial Peptides ◽

Enterococcus Faecalis ◽

Proteolytic Enzymes ◽

Exchange Chromatography ◽

Open Reading Frames ◽

Mass Spectrometry Analysis ◽

Spectrometry Analysis ◽

Genes Encoding ◽

Cell Supernatant

ABSTRACT The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A andent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type ofE. faecalis bacteriocin.

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Complete Sequence of the Enterocin Q-Encoding Plasmid pCIZ2 from the Multiple Bacteriocin Producer Enterococcus faecium L50 and Genetic Characterization of Enterocin Q Production and Immunity

Applied and Environmental Microbiology ◽

10.1128/aem.00859-06 ◽

2006 ◽

Vol 72 (10) ◽

pp. 6653-6666 ◽

Cited By ~ 41

Author(s):

Raquel Criado ◽

Dzung B. Diep ◽

Ågot Aakra ◽

Jorge Gutiérrez ◽

Ingolf F. Nes ◽

...

Keyword(s):

Enterococcus Faecium ◽

Complete Sequence ◽

Open Reading Frames ◽

Genetic Determinants ◽

Significant Similarity ◽

Minimum Requirement ◽

Initiator Protein ◽

Bacteriocin Producer ◽

Origin Region ◽

Transfer Origin

ABSTRACT The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.

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