Cloning, nucleotide sequence, and expression of the gene encoding the bacteriocin boticin B from Clostridium botulinum strain 213B

Boticin B is a heat-stable bacteriocin produced by Clostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18. 8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene, btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing the HindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.     

Atypical Genetic Locus Associated with Constitutive Production of Enterocin B by Enterococcus faecium BFE 900

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287–2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.     

Characterization of ard B and ard C actin gene loci of Physarum polycephalum

Summary Physarum polycephalum (strain M₃CVIII) contains four unlinked actin gene loci, each with two alleles (ard A1, ard A2, ard B1, ard B2, ard C1, ard C2, ard D1 and ard D2). The 4.8 kbp HindIII component of the ard C2 locus was isolated as a recombinant phage-, after HindIII fragments of Physarum DNA ranging from 4.3 kbp to 5.5 kbp were cloned into phage- NM1149. The fraction of Physarum DNA cloned contained the ard C locus, and no other actin locus. Small inserts were favoured to reduce the probability of cloning a complete repetitive element, because such elements have been found to adversely affect the stability of recombinants.The coding sequences of the actin gene (approximately 1.1 kbp) spanned more than 3 kbp indicating the presence of introns. A 1.6 kbp HindIII/EcoRI fragment of the ard C locus, which contained some coding sequences, hybridized extensively with HindIII fragments of genomic DNA indicating the presence of repetitive sequences. A 2.3 kbp HindIII/EcoRI fragment containing most of the coding sequences of the C2 allele of the ard C locus hybridized with the C1, allele and both alleles of the ard B locus, but not with the ard A locus or ard D locus. This distinction was used to establish for the ard B and ard C loci the relationship between the EcoRI and HindIII fragments that define an ard locus. The ability to distinguish between ard loci may facilitate studies of the expression of particular actin loci.     

PCR-Restriction Fragment Length Polymorphism Analysis of the Phospholipase B (PLB1) Gene for Subtyping of Cryptococcus neoformans Isolates

Cryptococcus neoformans is a pathogenic yeast that is currently divided into three varieties, five serotypes, and eight molecular types. The following report describes the use of PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) as a simple tool to differentiate between C. neoformans subgroups. A PLB1 fragment, 1,970 bp, was amplified and digested with either AvaI or HindIII. Both sets of profiles grouped the isolates into their respective varieties, but only the AvaI profiles allowed for the identification of the eight molecular types via the corresponding RFLP profiles A1 to A8. Digestion of the same fragments with HindIII resulted in RFLP profiles H1 to H5, which distinguished only between serotype A, AD, D, and B/C. Neither enzyme distinguished serotype B from serotype C. The serotype AD profile was a composite of the serotype A and D profiles. Further investigation showed that the serotype AD isolates used in this study are heterozygous, with one allele of PLB1 originating from a serotype A parent and the other from a serotype D parent.     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.     

A plasmid cloning vector for Kpnl-cleaved DNA

A plasmid cloning vector containing a single site for KpnI has been generated by insertion of a 3.5-kb EcoRI/HindIII fragment of pCR1 into the EcoRI/HindIII sites of pBR322. KpnI cleavage yields 3′ rather than 5′ “sticky ends” which allows reconstitution of the recognition site after cloning by a homopolymer joining procedure. This is an advantage shared with only one or two other commercially available restriction enzymes.     

Similarity in Nucleotide Sequence of the Gene Encoding Nontoxic Component of Botulinum Toxin Produced by Toxigenic Clostridium butyricum Strain BL6340 and Clostridium botulinum Type E Strain Mashike

The complete nucleotide and deduced amino acid sequence of the nontoxic component of botulinum type E progenitor toxin is determined in recombinant plasmid pU9BUH containing about 6.0 kb HindIII fragment obtained from chromosomal DNA of Clostridium butyricum strain BL6340. The open reading frame (ORF) of this nontoxic component gene is composed of 3,486 nucleotide bases (1,162 amino acid residues). The molecular weight calculated from deduced amino acid residues is estimated 13,6810.1. The present study revealed that 33 nucleotide bases of 3,486 are different in the nontoxic component gene between C.butyricum strain BL6340 and C. botulinum type E strain Mashike. This corresponds to the difference of 17 amino acid residues in these nontoxic component.     

A Novel Lantibiotic,Nukacin ISK-1, of Staphylococcus warneri ISK-1: Cloning of the Structural Gene and Identification of the Structure

Staphylococcus warneri ISK-1, which we had previously reported as Pediococcus sp. ISK-1, produces a novel bacteriocin, nukacin ISK-1. Edman degradation of the chemically reduced nukacin ISK-1 produced a sequence of 27 amino acids, 7 of which were unidentified. Using single-specific-primer-PCR product as a probe, a 3.6-kb HindIII fragment containing the nukacin ISK-1 structural gene (nukA) was cloned and sequenced. The deduced amino acid sequence of nukacin ISK-1 had 57 amino acids, including a 30-amino acid leader region. The propeptide sequence showed significant similarity to those of lacticin-481 type lantibiotics. In the region upstream of nukA, a part of a long open reading frame (ORF), designated as nukM, encoding a putative modification enzyme was oriented in the opposite direction. In the region downstream of nukA, ORF1 was found in which the sequence of the putative translational product was similar to various response regulatory proteins.     

Differentiation of the Gene Clusters Encoding Botulinum Neurotoxin Type A Complexes in Clostridium botulinum Type A, Ab, and A(B) Strains

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.     

Development of a Genetic Transformation System for an Alga-Lysing Bacterium

Four marine bacteria, Alteromonas sp. strains A27, A28, A29, and A30, that lyse the diatom Skeletonema costatum NIES-324 were isolated from coastal seawater samples. They were also able to lyse the diatoms Thalassiosira sp. and Eucampia zodiacs and the raphidophycean flagellate Chattonella antiqua. Cryptic indigenous plasmids, designated pAS28 and pAS29, were detected in Alteromonas sp. strains A28 and A29, respectively. These plasmids appeared to be similar based on size and restriction site analysis. A shuttle vector that replicates in Escherichia coli and Alteromonas sp. strain A28 was constructed by fusing pAS28 and E. coli vector pCRIIc. The 16-kbp chimeric plasmid, designated pASS1, had the ability to transform strain A28 at a frequency of 10⁶ transformants per μg of DNA. Deletion analysis of pASS1 showed that the 4.7-kb EcoRI-HindIII region of pAS28 was essential for plasmid maintenance in strain A28. This EcoRI-HindIII fragment contained an open reading frame which appeared to encode a 708-amino-acid protein.     

Duplicate Copies of Genes Encoding Methanesulfonate Monooxygenase in Marinosulfonomonas methylotropha Strain TR3 and Detection of Methanesulfonate Utilizers in the Environment

Marinosulfonomonas methylotropha strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from M. methylotropha strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp HindIII fragment contained msmA, msmB, and msmC, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp HindIII fragment contained duplicate copies of msmA and msmB, as well as msmD, encoding the reductase component of methanesulfonate. Both sets of msmA and msmB genes were virtually identical, and the derived msmA and msmB sequences of M. methylotropha strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer Methylosulfonomonas methylovora strain M2 were found to be 82 and 69% identical. The msmA gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of msmA which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding msmA genes from newly isolated Hyphomicrobium, Methylobacterium, and Pedomicrobium species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these msmA gene fragments, compared to msmA sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology.     

MspI andHindIII restriction fragment length polymorphisms at the human Na,K-ATPase β-subunit (ATP1B) gene locus

The authors report on four restriction fragment length polymorphisms detected at the ATP1B gene locus with the restriction endonucleasesHindIII andMspI.     

Characterization of the Genes Encoding the Botulinum Neurotoxin Complex in a Strain of Clostridium botulinum Producing Type B and F Neurotoxins

The organization of the clusters of genes encoding proteins of the botulinum neurotoxin (BoNT) progenitor complex was elucidated in a strain of Clostridium botulinum producing type B and F neurotoxins. With PCR and sequencing strategies, the type B BoNT-gene cluster was found to be composed of genes encoding BoNT/B, nontoxic nonhemagglutinin component (NTNH), P-21, and the hemagglutinins HA-33, HA-17, and HA-70, whereas the type F BoNT-gene cluster has genes encoding BoNT/F, NTNH, P-47, and P-21. Comparative sequence analysis showed that BoNT/F in type BF strain 3281 shares highest homology with BoNT/F of non-proteolytic (group II) C. botulinum whereas NTNH and P-21 in the type F cluster of strain 3281 are more similar to the corresponding proteins in proteolytic (group I) type F C. botulinum. These findings indicate diverse evolutionary origins for genes encoding BoNT/F and its associated non-toxic proteins, although the genes are contiguous. By contrast, sequence comparisons indicate that genes encoding BoNT/B and associated non-toxic proteins in strain 3281 possess a similar evolutionary origin. It was demonstrated that the genes present in the BoNT/B gene cluster of this type BF strain show exceptionally high homology with the equivalent genes in the silent BoNT/B gene cluster of C. botulinum type A(B), possibly indicating their common ancestry. Received: 30 March 1998 / Accepted: 21 May 1998     

Gene Organization and Sequence Determination of the Two Botulinum Neurotoxin Gene Clusters in Clostridium botulinum Type A(B) Strain NCTC 2916

The gene organization and nucleotide sequence of the type A and B BoNT-gene clusters in Clostridium botulinum strain NCTC 2916 were studied. The aim was to clarify the organization of genes within C. botulinum type A strains possessing an unexpressed BoNT/B gene. The BoNT/A-gene cluster includes genes encoding BoNT, NTNH and a part of P-47 (the gene for this protein was reported in strains of C. botulinum types E and F). Clustered with the silent BoNT/B gene were genes encoding NTNH, P-21 and HA-33. Sequencing analysis of the NTNHs revealed the presence of 471 amino acids identical in the type B and A gene clusters. This gene organization contrasts markedly with the purported organization in strain NCTC 2916 described by Henderson et al. (FEMS Microbiol. Lett. 140, 151–158). In type A(B) strain NCTC 2916, the neurotoxin gene is of type BoNT/A1 within a gene cluster that has identical organization to that found in BoNT/A2 type strains; these observations may be significant in establishing the origin of the BoNT-gene cluster. Received: 28 July 1997 / Accepted: 15 October 1997     

Mapping of Escherichia coli RNA polymerase binding sites on 2-micrometers DNA from Saccharomyces cerevisiae. Heterogeneity within the inverted duplication and evidence for an eukaryotic invertible DNA sequence.

The 2-μm DNA plasmids from Saccharomyces cerevisiae strain H1 and strain HQ/5C were analyzed by electron microscopy for the presence of Escherichia coli RNA polymerase binding sites. On native 2-μm DNA isolated from strain HQ/5C five RNA polymerase binding sites were detected. One further site was mapped on cloned 2-μm DNA type 23 from S. cerevisiae strain H1. This additional site is located at a distance of 2.15 kilobases from EcoRI site B inside one of the inverted duplication (id) sequences. No such binding site could be detected in the other id sequence of the type 23 molecule, thus indicating that the two id sequences of strain H1 differ in at least one short region. The location of the id sequence carrying the RNA polymerase binding site was analyzed in native 2-μm DNA isolated from strain H1 and found to be present on HindIII fragment 2 and absent from HindIII fragment 5. This indicates that at least a part of the id sequences has a fixed position with respect to the unique S segment and further suggests a site specific recombination mechanism for the inversion of one of the unique segments. As a control for the specificity of RNA polymerase binding, we have mapped binding sites on vectors pBR313 and pBR322. The location of the E. coli RNA polymerase binding sites on 2-μm DNA is discussed in relation to the DNA regions expressed in E. coli minicells.     

Analysis of a polymorphic region of the Euglena gracilis chloroplast genome

The circular chloroplast genome of Euglena gracilis is known to carry a single region of variable length (polymorphic region) located on BglII·Z fragments (6.1–6.9 kbp). We now have cloned a BglII·Z fragment (6.35 kbp) using as vector a modified pBR322. The cloned BglIl·Z fragment is split by HindIII into fragments of 1.0, 2.6 and 2.75 kbp and by HaeIII into fragments of 0.05, 1.35 and 4.95 kbp, respectively. The zone of variable length on this BglIl·Z fragment can be confined to a BglII-HindIII fragment of 2.6 kbp which is next to the previously mapped BglIl·G₁ (4.7 kbp) and about 4.5 kbp away from the 5′ end of the ‘extra’ 16 S rRNA gene. Two HindIII fragments from the polymorphic region of 5.9 and 6.1 kbp, respectively, were cloned and used in electron microscopic studies. Heteroduplexes formed between the two cloned HindIII fragments show the expected size difference of about 200 bp. Single strand reannealing experiments allow us to place the polymorphic region between two previously mapped short inverted repeats and partial DNA melting experiments indicate that the polymorphic region is very rich in dA-dT.     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.