Characterization of a new operon,as-48EFGH,from the as-48 gene cluster involved in immunity to enterocin AS-48

Enterocin AS-48 is a cyclic peptide produced by Enterococcus faecalis S-48 whose genetic determinants have been identified in the conjugative plasmid pMB2. A region of 7.8 kb, carrying the minimum information required for production of and immunity against AS-48, had been previously cloned and sequenced in pAM401 (pAM401-52). In this region, the as-48A structural gene and as-48B, as-48C, as-48C(1), as-48D, and as-48D(1) genes and open reading frame 6 (ORF6) and ORF7 had been identified. The sequence analysis carried out in this work in the BglII B fragment (6.6-kb) from pMB2 cloned downstream from the last ORF identified (ORF7) revealed the existence of two new ORFs, as-48G and as-48H, necessary for full AS-48 expression. Thus, JH2-2 transformants obtained with the pAM401-81 plasmid became producers and resistant at the wild-type level. Tn5 disruption experiments in the last genes, as-48EFGH, were not able to reproduce these expression levels, confirming that expression of these genes is necessary to get the phenotype conferred by the wild-type pMB2 plasmid. The as-48EFGH operon encodes a new ABC transporter that could be involved in producer self-protection. On the basis of the observed similarities, As-48G would be the ATP-binding domain, the deduced amino acid sequences of As-48E and As48-H could be assigned as transmembrane subunits, and As-48F, with an N-terminal transmembrane segment and a coiled-coil domain, strongly resembles the structure of some known ABC transporter accessory proteins whose localization in the cell is discussed. This cluster of genes is expressed by two polycistronic mRNAs, T(2) and T(3), in JH2-2(pAM401-81) in coordinate expression. Our results also suggest that expression of T(3) could be regulated, because in JH2-2(pAM401(EH)) transformants, T(3) was not detected, suggesting that these genes do not by themselves confer immunity, in accordance with the requirement for the as-48D(1) gene for immunity against AS-48.     

Characterization of a New Operon, as-48EFGH, from the as-48 Gene Cluster Involved in Immunity to Enterocin AS-48

Enterocin AS-48 is a cyclic peptide produced by Enterococcus faecalis S-48 whose genetic determinants have been identified in the conjugative plasmid pMB2. A region of 7.8 kb, carrying the minimum information required for production of and immunity against AS-48, had been previously cloned and sequenced in pAM401 (pAM401-52). In this region, the as-48A structural gene and as-48B, as-48C, as-48C₁, as-48D, and as-48D₁ genes and open reading frame 6 (ORF6) and ORF7 had been identified. The sequence analysis carried out in this work in the BglII B fragment (6.6-kb) from pMB2 cloned downstream from the last ORF identified (ORF7) revealed the existence of two new ORFs, as-48G and as-48H, necessary for full AS-48 expression. Thus, JH2-2 transformants obtained with the pAM401-81 plasmid became producers and resistant at the wild-type level. Tn5 disruption experiments in the last genes, as-48EFGH, were not able to reproduce these expression levels, confirming that expression of these genes is necessary to get the phenotype conferred by the wild-type pMB2 plasmid. The as-48EFGH operon encodes a new ABC transporter that could be involved in producer self-protection. On the basis of the observed similarities, As-48G would be the ATP-binding domain, the deduced amino acid sequences of As-48E and As48-H could be assigned as transmembrane subunits, and As-48F, with an N-terminal transmembrane segment and a coiled-coil domain, strongly resembles the structure of some known ABC transporter accessory proteins whose localization in the cell is discussed. This cluster of genes is expressed by two polycistronic mRNAs, T₂ and T₃, in JH2-2(pAM401-81) in coordinate expression. Our results also suggest that expression of T₃ could be regulated, because in JH2-2(pAM401_EH) transformants, T₃ was not detected, suggesting that these genes do not by themselves confer immunity, in accordance with the requirement for the as-48D₁ gene for immunity against AS-48.     

Enterocin AS-48RJ: a variant of enterocin AS-48 chromosomally encoded by Enterococcus faecium RJ16 isolated from food

The bacteriocinogenic strain RJ16 isolated from goat cheese has been identified as Enterococcusfaecium by species-specific PCR, DNA-rRNA hybridization and rDNA sequencing. Purified bacteriocin from strain RJ16 is a carboxypeptidase A-resistant peptide with a molecular mass (7125 Da) very close to the cyclic peptide enterocin AS-48. Bacteriocin from strain RJ16 and AS-48 show identical antibacterial spectra, although the former is slightly less active on strains of Listeria monocytogenes and Bacillus cereus. Producer strains show cross-immunity. PCR amplification of total DNA from strain RJ16 with primers for the AS-48 structural gene and sequencing of the amplified fragment revealed an almost identical sequence (99.5%), except for a single mutation that predicts the change of Glu residue at position 20 of AS-48 to Val. Therefore, bacteriocin produced by E. faecium RJ16 should be considered a variant of AS-48, which we call AS-48RJ. PCR amplification revealed that strain RJ16 contains the complete as-48. gene cluster. Hybridization with probes for as-48 gene cluster revealed a chromosomal location of as-48 genes in strain RJ16, being the first example of a chromosomal location of this bacteriocin trait. Strain RJ16 produced enzymes of interest in food processing (esterase, esterase lipase and phytase activities), and did not decarboxylate amino acids precursors for biogenic amines. Strain RJ16 did not exhibit haemolytic or gelatinase activities, and PCR amplification revealed the lack of genes encoding for known virulence determinants (aggregation substance, collagen adhesin, enterococcal surface protein, endocarditis antigens, as well as haemolysin and gelatinase production). Strain RJ16 was resistant to ciprofloxacin (MIC > 2 mgl(-1)) and levofloxacin (MIC > 4 mgl(-1)) and showed intermediate resistance to nitrofurantoin and erythromycin, but was sensitive to ampicillin, penicillin, streptomycin, gentamicin, rifampicin, chloramphenicol, tetracycline, quinupristin/dalfopristin, vancomycin and teicoplanin. Altogether, results from this study suggest that this broad-spectrum bacteriocin-producing strain may have a potential use in food preservation.     

Purification of bacteriocin AS-48 from an Enterococcus faecium strain and analysis of the gene cluster involved in its production

The cyclic bacteriocin AS-48 has previously been shown to be produced by Enterococcus faecalis strains. A bacteriocin has been purified from an E. faecium strain (E. faecium 7C5), and it has been found to possess molecular mass, cyclization and amino acid sequence typical of bacteriocin AS-48. In addition to the structural gene as-48A, the sequence analysis of the AS-48 gene cluster present in E. faecium 7C5 has revealed the presence of several putative coding regions presumably involved in bacteriocin production and immunity. The results of DNA hybridization assays have indicated that the AS-48 gene cluster and the gene pd78 are present on the same plasmid, possibly the pPD1 plasmid, in E. faecium 7C5.     

Cloning and Characterization of the Gene Cluster Involved in the Production of the Circular Bacteriocin Carnocyclin A

Carnocyclin A is a circular bacteriocin of 60 amino acids produced by Carnobacterium maltaromaticum UAL307. A region of 12 kb that contained the structural gene for carnocyclin A, cclA, was sequenced using a fosmid library, and 10 genes were identified that could be responsible for carnocyclin A production and immunity. Five of those genes, cclBITCD, were found upstream of cclA: one encodes a protein containing a conserved ATP-binding domain and four encode proteins with putative membrane-spanning domains. CclC shows homology with a family of membrane proteins that contain the domain of unknown function 95 (DUF95). Downstream of cclA four additional genes, cclEFGH, were identified that show similarity to the last four genes, as-48EFGH, of the enterocin AS-48 bacteriocin gene cluster. CclFGH shows sequence homology with As-48FGH. Transformation of C. maltaromaticum UAL26 with cclBITCDA resulted in production of carnocyclin A, indicating that these genes form the minimal requirement for the secretion of fully matured bacteriocin. cclI encodes for a small hydrophobic protein with a high pI, which are characteristic features of known immunity proteins for other circular bacteriocins. Indeed, cloning of cclI behind a constitutive promoter in UAL26 resulted in immunity although the level of resistance was lower than that of UAL26 containing cclBITCDA, indicating that CclI alone is not enough to confer full immunity to carnocyclin A.     

Determination of the gene sequence and the molecular structure of the enterococcal peptide antibiotic AS-48.

The structural gene of the enterococcal peptide antibiotic AS-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the AS-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative AS-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the antibiotic peptide was a 70-residue cyclic molecule. We propose that the AS-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): Polarity and gene organization

We have analyzed the histone genes from the sea urchin Lytechinus pictus. Examination of native DNA from individuals reveals four major Eco RI restriction endonuclease histone gene DNA fragments which have been labeled A (6.0 kb), B (4.1 kb), C (3.1 kb) and D (1.2 kb). The fragments A, B and C have been cloned into E. coli plasmids (pLpA, pLpB and pLpC). These histone gene fragments display length and sequence heterogeneity in different individuals. The plasmid pLpA contains the coding regions for H1, H4, H2B and H3 histones, and we determined that the DNA fragment D is tandem to A in native DNA and that it contains the H2A gene. The plasmids pLpB and pLpC contain the histone genes H2A-H1-H4 and H2B-H3, respectively, and together contain the sequences for the five major histones. Restriction analysis of native L. pictus DNA reveals that B and C are tandem to each other but not intermingled with the A-D-type repeat units, and are thus in separate clusters with a repeat length of 7.2 kb. Since the two cluster types do not segregate, they are not alleles. Hybridization of histone mRNA to exonuclease III-digested linear DNA demonstrated an identical polarity of the histone genes in the A-D- and B-C-type repeat units. This result revealed that the L. pictus histone genes have a polarity which is the same as other sea urchin histone genes examined to date—that is, 3′ H1-H4-H2B-H3-H2A 5′. Restriction endonuclease cleavage patterns of the cloned segments indicate that considerable sequence heterogeneity exists between the two types of histone gene repeat units.     

The ABC Transporter CclEFGH Facilitates the Production of the Circular Bacteriocin Carnocyclin A

Carnocyclin A is a circular bacteriocin produced by Carnobacterium maltaromaticum UAL307. The carnocyclin A gene cluster cclBITCDAEFGH had been previously reported, and it was shown that transformation of C. maltaromaticum UAL26 with cclBITCDA resulted in immunity to, and low production of, carnocyclin A. Here, we demonstrate that full production of carnocyclin A in UAL26 transformants could be achieved when cclBITCDA was complemented with a second plasmid that contains cclEFGH. CclEFGH is a multicomponent ABC transporter that has similarity to As-48EFGH which is involved in the production of enterocin AS-48. Transformation of UAL26 containing cclBITCDA with deletion derivatives of cclEFGH did not increase the production of carnocyclin A, confirming the involvement of CclEFGH in bacteriocin production. Transformants of UAL26 containing cclEFGH showed a slight decrease in sensitivity to carnocyclin A, indicating that CclEFGH might also play a role in immunity.     

Inhibition of Bacillus licheniformis LMG 19409 from ropy cider by enterocin AS-48

AIMS: To determine the activity of enterocin AS-48 against ropy-forming Bacillus licheniformis from cider. METHODS AND RESULTS: Enterocin AS-48 was tested on B. licheniformis LMG 19409 from ropy cider in MRS-G broth, fresh-made apple juice and in two commercial apple ciders (A and B). Bacillus licheniformis was rapidly inactivated in MRS-G by 0.5 microg ml(-1)AS-48 and in fresh-made apple juice by 3 microg ml(-1). Concentration-dependent inactivation of this bacterium in two commercial apple ciders (A and B) stored at 4, 15 and 30 degrees C for 15 days was also demonstrated. Counts from heat-activated endospores in cider A plus AS-48 decreased very slowly. Application of combined treatments of heat (95 degrees C) and enterocin AS-48 reduced the time required to achieved complete inactivation of intact spores in cider A to 4 min for 6 microg ml(-1) and to 1 min for 12 microg ml(-1). D and z values also decreased as the bacteriocin concentration increased. CONCLUSION: Enterocin AS-48 can inhibit ropy-forming B. licheniformis in apple cider and increase the heat sensitivity of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study support the potential use of enterocin AS-48 to control B. licheniformis in apple cider.     

Genomic characterization of the human and mouse protein tyrosine phosphatase-1B genes

PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20 q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3′ end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3′ end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitiative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5′ flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains.     

Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain

AIMS: Control of the enterotoxigenic Staphylococcus aureus CECT 976 strain by enterocin AS-48 in laboratory cultures, and behaviour of the AS-48 activity in the presence of food preservatives. METHODS AND RESULTS: Enterocin AS-48 shows inhibitory activity on the majority of the Staphylococcus species tested. This enterocin has a bactericidal and bacteriolytic mode of action on S. aureus CECT 976, a strain selected for this study by its enterotoxigenic character (SEA production). The inhibitory effect of AS-48 was pH and temperature dependent, and enterocin activity was higher at pH 5. The minimum bactericidal concentration (MBC) of AS-48, decreased from 15 microg ml(-1) at 37 degrees C to 10 microg ml(-1) at 15 degrees C. Sublethally injured cells showed an increased sensitivity with a MBC of 5 microg ml(-1). In this way, the highest effectiveness of Ent AS-48 against S. aureus CECT 976 was obtained at 4 degrees C in combination with high concentrations of NaCl (6 and 7%). Interestingly, enterotoxin SEA production by strain CECT 976 was markedly inhibited by subinhibitory concentrations of Ent AS-48. These low concentrations also provoked a delay of bacterial growth. CONCLUSION: The results presented indicated that Ent AS-48 has a potential for application as a protective agent against S. aureus in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we have established the conditions for an efficient inhibition of growth and enterotoxin production by S. aureus CECT 976 in culture media by a combination of environmental factors and Ent AS-48.     

Analysis of a genomic DNA segment carrying the wheat high-molecular-weight (HMW) glutenin Bx17 subunit and its use as an RFLP marker

Summary A genomic fragment containing the Bx17 high-molecular-weight (HMW) glutenin gene was isolated from a wheat genomic library. The fragment contains a coding region of 2.82kb with 1.98-kb downstream and 12.8-kb upstream flanking regions. The fragment was sequenced and compared with previously published glutenin genes from chromosomes 1A, 1B and 1D using a computer alignment package. The Bx17 gene shows marked similarity to the Bx7 gene sequence. A phenetic tree derived from the alignments is presented. Also shown are restriction fragment length polymorphisms (RFLPs) at the glutenin loci in a set of Australian and international wheat varieties using different regions of the glutenin clone as probes. The RFLPs correlated well with the protein composition in all cultivars analysed.     

Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum

The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44. The gene cluster contains seven genes and spans approx. 56kb. The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules. The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones. EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively. EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro.     

Structure and localization of the human SULT1B1 gene: neighborhood to SULT1E1 and a SULT1D pseudogene.

The soluble sulfotransferases are involved in the elimination of xenobiotics, the activation of procarcinogens, and the regulation of hormones. They comprise a gene superfamily (SULT). The structure and chromosomal location of nine human SULT genes are known. We have characterized a further gene, SULT1B1. Its structure is similar to that of other SULT1 genes. However, the total length of its eight exons and the introns (33.6 kb) is larger than that of other human SULT1 genes (4 to 21 kb). The SULT1B1 gene sequence is part of a sequence entry in the unfinished High-Throughput Genomic Sequences (HTGS) division of GenBank. However, the order and orientation of the SULT1B1 exons are not correct in this entry. SULT1B1 is located on chromosome 4q13.1, nearly 100 kb downstream of SULT1E1 on the same strand. The intervening sequence contains a SULT-like structure showing substantial homology to the mouse SULT1D1 cDNA recently described. However, in humans this structure represents a pseudogene (SULT1D1P) because of mutated splice donors/acceptors and in-frame stop codons in the sequence corresponding to exon II. This SULT gene cluster is located on the minus strand of chromosome 4 with SULT1B1 being closest to the centromer.     

Orientation and molecular map position of the complement genes in the mouse MHC.

Over the past few years six gene clusters have been isolated from the major histocompatibility complex (MHC) of the BALB/c mouse encompassing a total of 1600 kb of DNA and 48 genes. The molecular distances between these gene clusters and the orientation of four of the six clusters on chromosome 17 is not known. Here we use pulse-field gradient gels and Southern blot hybridization to establish large-scale genomic restriction maps covering several hundreds of kb surrounding the three gene clusters located in the K, I, S, and D regions of the MHC. Comparison of the maps orients the complement gene clusters in the S region with the 21-OHB gene pointing towards the K end and the C2 gene pointing towards the D end of the MHC. The distances between the E alpha and 21-OHB genes is 430 kb and between the C2 and TNF-alpha genes at least 420 kb.