Characterization of a lytic Lactobacillus plantarum bacteriophage and molecular cloning of a lysin gene in Escherichia coli

Bacteriophage SC921, which can infect Lactobacillus plantarum specifically, was isolated from a fermented vegetable source, Kimchi. This phage is active against six of 11 strains of L. plantarum tested as hosts. Morphologically, it has an isometric head (60 nm in diameter) and a non-contractile tail (260 nm long and 9-11 nm wide), indicating that it belongs to Bradley's group B or the Siphoviridae family according to the International Committee on Taxonomy of Viruses (ICTV). The bouyant density was 1.58 g/cm3. SDS-PAGE experimentation indicated that the phage particle contains two major structural proteins and several minor proteins. The genome was a double stranded linear DNA molecule with cohesive ends and 66.5 kb long by mapping genomic DNA digested with the restriction endonucleases: KpnI, SmaI, and XbaI. The [G + C] content of the phage DNA is 39.4%. For this lysin gene study, 9.4 kb of KpnI-digested DNA fragment was cloned into pUC19 and expressed in Escherichia coli. The KpnI fragment was considered as the genetic element responsible for the lysis gene of L. plantarum bacteriophage. The cloned fragment in pUC19 was hybridized to a 9.4-kb fragment generated by KpnI digestion of SC 921 as a probe. This confirmed that the fragment in pUC19 originated from phage DNA. The lysin gene was near the middle of the phage genome.     

Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella

A phage-based reagent was developed for the detection of Salmonella in food samples. The parental phage was Felix 01, which lyses practically all Salmonella. Using data obtained about the molecular biology of the phage, a recombinant phage that carried the bacterial genes specifying luciferase was produced. The method involved the isolation of amber nonsense mutations and subsequent crosses to render doubly mutant phage with a very low reversion rate on strains lacking an amber suppressor. A plasmid was constructed that contained a segment of Felix 01 DNA with two adjacent genes, one dispensable and the other essential, and their flanking sequences. Recombinant DNA technology was used to remove the two genes and the luxA and luxB genes for luciferase, and a gene specifying a tRNA that recognizes amber codons (supF=tyrT) was put in their stead. This region could be transferred into the genome of the phage by homologous recombination. The recombinant phage cannot grow because it lacks an essential gene. However, it can grow in a host that synthesizes the missing protein. This technique allows the construction of "locked" recombinant phages that carry foreign DNA but which cannot propagate themselves in nature.     

A comparative analysis of distinctive features of yeast protein sequences

The recently published sequence of yeast chromosome III (YCIII) provides the longest continuous stretch of a eukaryotic DNA molecule sequenced to date (315 kb). The sequence contains 116 distinct AUG-initiated open reading frames of at least 200 codons in length, more than 50 of which had not been described previously nor bear significant similarity to known proteins. We have analysed the YCIII known and putative-protein sequences with respect to significant statistical features which might reflect on structural and functional characteristics. The YCIII proteins have striking similarities and differences in their sequence attribute distributions compared to the corresponding distributions for all available yeast sequences and other protein collections. Nine examples of YCIII proteins with distinctive sequence features are discussed in detail.     

The complete sequence of a 15 820 bp segment of Saccharomyces cerevisiae chromosome XI contains the UBI2 and MPL1 genes and three new open reading frames

As part of the EEC yeast genome program, a fragment of 15 820 bp from the right arm of Saccharomyces cerevisiae chromosome XI has been sequenced. This fragment corresponds roughly to the centromere-distal half of cosmid pUKG046 and to a small fragment of cosmid pUKG096, which are located approximately 150 kb from the centromere. It contains four open reading frames (ORFs) which encode potential proteins of more than 100 amino acid residues, as well as the UBI2 gene which carries an intron and does not show up as an ORF in the sequence analysis programs. One of the putative proteins, YKR412, is very rich in serine and has significant homology at the carboxyl end to Nopp140 phosphoprotein. YKR413 has several predicted transmembrane domains. YKR15, which has been recently cloned as the MPL1 gene, encodes a polypeptide that shows homologies to myosin heavy chain and to the cytoskeleton protein Uso1.     

Sequence of a 17·1 kb DNA fragment from chromosome X of Saccharomyces cerevisiae includes the mitochondrial ribosomal protein L8

We have sequenced a continuous segment of 17 137 bp on chromosome X. Sequence analysis of this stretch revealed 14 open reading frames (ORFs) at least 100 amino acids long. One gene, encoding the mitochondrial 60S ribosomal protein L8, had already been sequenced. Four ORF products show weak homologies with known protein sequences. The nine remaining ORF products have no homologies with sequences in data banks. The nucleotide sequence of the 17·1 kb fragment is available through the EMBL data library under Accession Number Z34288.     

Sequence analysis of a 14·2 kb fragment of Saccharomyces cerevisiae chromosome XIV that includes the ypt53, tRNALeu and gsr m2 genes and four new open reading frames

As part of the EU yeast genome program, a fragment of 14 262 bp from the left arm of Saccharomyces cerevisiae chromosome XIV has been sequenced. This fragment corresponds to cosmid 14-14b and is located roughly 130 kb from the centromere. It contains four new open reading frames which encode potential proteins of more than 99 amino acids, as well as the ypt53, tRNA^Leu and gsr m2 genes. The putative protein N2212 is similar to the ribosomal protein S7 from humans. N2215 contains several predicted transmembrane elements. N2231 contains regions which are rich in acidic, as well as basic, residues which could form α-helical structures. Similar regions are found in a variety of proteins including glutamic acid rich protein, trichohyalin, caldesmon, Tb-29 and several cytoskeleton-interacting proteins. The sequence has been entered in the EMBL data library under Accession Number X85811.     

烈性噬菌体vB_VpP_AC2的分离鉴定及生物学特性分析

该研究从水产市场污水中分离纯化副溶血性弧菌噬菌体，并对其中一株噬菌体vB_VpP_AC2（AC2）进行分离鉴定、基因组及部分生物学特性分析，探究该噬菌体作为副溶血性弧菌生防制剂的潜力。噬菌体AC2的噬菌斑透明且边缘清晰，透明部分的直径约为1.12 mm，无可见晕圈。噬菌体AC2的全基因组序列长44 270 bp，鸟嘌呤-胞嘧啶（GC）含量为49.30%，共预测到55个假定的开放阅读框，其中37个与编码已知功能蛋白质的基因相似（占67.27%），包括一个类似holin功能的蛋白（AC2_gp43，UTQ72417.1），该功能蛋白首次在Maculvirus属中被表征。经蛋白网络与末端酶系统发育树分析鉴定，噬菌体AC2属于自转录病毒科（Autograohiviridae），Maculvirus属，与噬菌体vB_VpaP_MGD1的ANIb值最高，为95.62%。在生物学特性方面，噬菌体AC2能够裂解30.77%的受测菌株，其最佳感染复数为0.001~0.1，一步生长曲线显示AC2的潜伏期为20 min，裂解期为35 min，裂解量为143 pfu/cell。总之，噬菌体AC2的分离鉴定不仅可为相关功能蛋白的研究提供参考，还可为水产食品安全提供较高应用价值的噬菌体资源。     

DNA sequencing and analysis of a 24.7 kb segment encompassing centromere CEN11 of Saccharomyces cerevisiae reveals nine previously unknown open reading frames.

A 24.7 kb segment of the cosmid clone pUKG047 containing a Sau3AI-partial fragment from the centromere region of Saccharomyces cerevisiae chromosome XI was sequenced and analysed. A mixed strategy of directed methods including exonuclease III nested deletion, restriction fragment subcloning and oligonucleotide-directed sequences was carried out. Exclusive use was made of the Applied Biosystems Taq DyeDeoxy Terminator Cycle technology and a laser-based AB1373A sequencing system for reactions, gel electrophoresis and automated reading. A total of 12 open reading frames (ORFs) was found. Nine new ORFs (YK102 to YK110) were identified, three of which (YK102, YK107, YK108) showed homologies to proteins of known function from other organisms. In addition, sequence analysis revealed three recently functionally characterized genes (MET14, VPS/SPO15, PAP1), which could be joined to the earlier published CEN11 region.     

VI. Yeast sequencing reports. Sequencing of an 18·8 kb fragment from Saccharomyces cerevisiae chromosome VI

The nucleotide sequence of lambda phage clone 4121, which contains the 18·8 kb fragment of Saccharomyces cerevisiae chromosome VI left arm, was determined. This sequence had seven open reading frames (ORFs), four of which were identical to known genes (ACT1, YPT1, TUB2 and RPO41). Another three ORFs (4121orfR003, 4121orfR004 and 4121orfRN001) were highly homologous to FET3 multi-copper oxidase, glucose transport protein, and hypothetical protein of YIL106w on chromosome IX, respectively. 4121orfRN01 is suggested to contain an intron. The sequence has been submitted to DDBJ/EMBL/GenBank data library under Accession Number D44598.     

A new phage on the ‘Mozzarella’ block: Bacteriophage 5093 shares a low level of homology with other Streptococcus thermophilus phages

Streptococcus thermophilus bacteriophage 5093 is a virulent phage that infects the industrial Mozzarella starter CSK939. The genome of phage 5093 is 37,184 base pairs (bps) containing 50 open reading frames (orfs). Genetic analysis revealed that the genome of phage 5093 is highly mosaic when compared with other sequenced S. thermophilus phages. This is particularly apparent in the late gene cluster with regions displaying high homology to prophage sequences of non-dairy streptococci and limited homology to either pac or cos-type S. thermophilus phages. In addition, a definitive antireceptor gene was not observed – suggesting that phage 5093 may have developed a different system for host recognition. Interestingly, the phage does contain a methylase domain that probably evolved as a phage counter-defence mechanism. These findings suggest that phage 5093 may represent a third group of S. thermophilus phage and provide the link between phages that infect S. thermophilus and its non-dairy ancestors.     

XIV. Yeast sequencing reports. A 21·7 kb DNA segment on the left arm of yeast chromosome XIV carries WHI3, GCR2, SPX18, SPX19, an homologue to the heat shock gene SSB1 and 8 new open reading frames of unknown function

We report the amino acid sequence of 13 open reading frames (ORF > 299 bp) located on a 21·7 kb DNA segment from the left arm of chromosome XIV of Saccharomyces cerevisiae. Five open reading frames had been entirely or partially sequenced previously: WHI3, GCR2, SPX19, SPX18 and a heat shock gene similar to SSB1. The products of 8 other ORFs are new putative proteins among which N1394 is probably a membrane protein. N1346 contains a leucine zipper pattern and the corresponding ORF presents an HAP (global regulator of respiratory genes) upstream activating sequence in the promoting region. N1386 shares homologies with the DNA structure-specific recognition protein family SSRPs and the corresponding ORF is preceded by an MCB (MluI cell cycle box) upstream activating factor. The sequence has been deposited in the EMBL data library under Accession Number X78898.     

Sequence of the CDC10 region at chromosome III of Saccharomyces cerevisiae

A 4.74 kb DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae, adjacent to the centromere region was sequenced. Four open reading frames with an ATG initiation codon and larger than 200 bp were found in this fragment. The largest open reading frame of 966 bp was identified as the CDC10 gene.     

Sequence of a segment of yeast chromosome XI identifies a new mitochondrial carrier, a new member of the G protein family, and a protein with the PAAKK motif of the H1 histones.

We have entirely sequenced an 8.3 kb segment localized on the left arm of chromosome XI of Saccharomyces cerevisiae. Five new open reading frames have been uncovered. One of them encodes a new mitochondrial carrier protein which is dispensable for growth on glycerol medium. Another could be a new member of the G protein family. A third possesses the PAAKK motif common to H1 histones.     

Physical mapping of a centromere-proximal region of chromosome IV-l defines the placement of genes USO1, MBP1, PSA1 and SLC1

A physical map of a 14·5 kb region close to the centromere on the left arm of chromosome IV of Saccharomyces cerevisiae is presented. This map has been constructed by restriction analysis of a clone from a YCp50 genomic library and by use of pre-existing and new sequence data from this region. The map reveals the following gene order (reading from the most centromere-distal to the most centromere-proximal locus): USO1/INT1–MBP1–PSA1–SLC1–YLA1 and defines the size of the open reading frames and intergenic regions.     

Analysis of the complete genome sequence of the lactococcal bacteriophage bIBB29

Bacteriophage bIBB29 was isolated from a whey sample originating from an industrial biotechnological process, disturbed by a bacteriophage attack. Phage bIBB29 was determined to be active against three phage-resistant strains of Lactococcus lactis. It belongs to the 936 species containing virulent phages with isometric head and short non-contractile tail. One-step growth kinetics of bIBB29 phage showed that its latent time was 23 min, and the burst size was about 130 bacteriophages. The complete nucleotide sequence of the virulent L. lactis bacteriophage bIBB29 comprises 29305 nucleotides and is the sixth phage genome of the 936 species published until now. The G+C content of the bIBB29 genome (34.7%) is similar to that of its host and also to that of other phages from the 936 species. The bIBB29 genome counts 54 open reading frames organized in three typical clusters, corresponding to the early, middle and late expressed genes. Only 20 protein products of the predicted genes were found to have their homologs among proteins with known function. The early expressed region in the genomes of 936 group members displays the highest divergence, whereas the late and middle regions share high similarities, with the exception of five genes. The genome of bIBB29 shares the highest overall nucleotide similarity with bIL170 (87%), and the lowest with phage 712 (77%). The host range analysis showed that despite the high level of similarity between the receptor binding protein (RBP) of phage bIBB29 and P475, they have a different host range. This implies that RBP is not a sufficient factor for host range.     

Subtelomeric sequence from the right arm of Schizosaccharomyces pombe chromosome I contains seven permease genes

The sequence has been determined of 80 888 bp of contiguous subtelomeric DNA, including the isp5 gene, from the right arm of chromosome I of Schizosaccharomyces pombe; 27 open reading frames (ORFs) longer than 100 codons are present, giving a density of one gene per 3.0 kb. Seven of the predicted proteins are members of the major facilitator superfamily (MFS) of transport proteins, including four amino acid permease homologues, bringing this family of amino acid permease sequences to 17 in Sz. pombe, and a phylogenetic analysis is presented. Also encoded is an allantoate permease homologue, a sulphate permease homologue and a probable urea active transporter. Predicted non-membrane proteins include a 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), a class III aminotransferase, serine acetyltransferase, protein-L-isoaspartate O-methyltransferase, alpha-glucosidase, alpha-galactosidase, esterase/lipase, oxidoreductase of the short-chain dehydrogenase/reductase (SDR) family, aldehyde dehydrogenase, formamidase, amidase, flavohaemoprotein, a putative translation initiation inhibitor and a protein with similarity to a filamentous fungal conidiation-specific protein. The remaining six ORFs are likely to encode proteins, either because they have sequence similarity with hypothetical proteins or because they are known to be transcribed. Introns are scarce in the sequenced region: only three ORFs contain introns, with only one having multiple introns. The sequenced region also contains a single Tf1 transposon long terminal repeat (LTR). The sequence is derived from cosmid clones c869, c922 and c1039 and has been submitted to the EMBL database under entries SPAC869 (Accession No. AL132779), SPAC922 (AL133522) and SPAC1039 (AL133521).     

The sequence of a 17.5 kb DNA fragment on the left arm of yeast chromosome XI identifies the protein kinase gene ELM1, the DNA primase gene PRI2, a new gene encoding a putative histone and seven new open reading frames

A 17.5 kb DNA fragment of chromosome XI, located between the genetic loci mif2 and mak11 was sequenced and analysed. Ten open reading frames were identified. Two of them are the previously sequenced genes ELM1 and PRI2, two (YKL253 and YKL256) show homologies to proteins from other organisms and one (YKL262) to yeast and mouse histone.     

Characterization of lactococcal bacteriophages isolated from Slovenian dairies

Summary This is the first study on lactic acid bacteria bacteriophages isolated from dairy plants in Slovenia. Over a period of 2 years, thirteen lactococcal phages were isolated from the whey samples taken in three dairy plants. Phages were characterized by morphological properties, host range, restriction patterns, genome size and similarity of phage protein genes. All phages belonged to the Siphoviridae family. Five phages had prolate heads (52–61 × 40–52 nm) and 85–120 nm long non‐contractile tails (morphotype B2). Eight phages had small isometric heads (46–63 nm in diameter) and long non‐contractile tails (morphotype B1). The phages with the prolate heads showed a broader host range than the small isometric‐headed phages. The genome sizes of prolate phages were estimated between 15.9 and 16.3 kb while the genome sizes of small isometric‐headed phages were between 20.0 and 39.9 kb. Based on DNA restriction patterns, genome sizes and multiplex PCR analysis prolate phages were classified into the c2 group and were similar to the phage type c6A while all of the phages with small isometric heads were found to be related to the phage species 936.