Alan J. Hillier

Analysis of the DNA sequence, gene expression, origin of replication and modular structure of the Lactococcus lactis lytic bacteriophage sk1

Bacteriophage sk1 is a small isometric‐headed lytic phage belonging to the 936 species. It infects Lactococcus lactis, a commonly used dairy starter organism. Nucleotide sequence data analysis indicated that the sk1 genome is 28 451 nucleotides long and contains 54 open reading frames (ORFs) of 30 or more codons, interspersed with three large intergenic regions. The nucleotide sequence of several of the sk1 ORFs demonstrated significant levels of identity to genes (many encoding proteins of unknown function) in other lactococcal phages of both small isometric‐headed and prolate‐headed morphotype. Based on this identity and predicted peptide structures, sk1 genes for the terminase, major structural protein and DNA polymerase have been putatively identified. Genes encoding holin and lysin were also identified, subcloned into an Escherichia coli expression vector, and their function demonstrated in vivo. The sk1 origin of replication was located by identifying sk1 DNA fragments able to support the maintenance in L. lactis of a plasmid lacking a functional Gram‐positive ori. The minimal fragment conferring replication origin function contained a number of direct repeats and 179 codons of ORF47. Although no similarity between phage sk1 and coliphage λ at the nucleotide or amino acid sequence level was observed, an alignment of the sk1 late region ORFs with the λ structural and packaging genes revealed a striking correspondence in both ORF length and isoelectric point of the ORF product. It is proposed that this correspondence is indicative of a strong conservation in gene order within these otherwise unrelated isometric‐headed phages that can be used to predict the functions of the sk1 gene products.

Genomic organization of lactic acid bacteria.

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.

The nature of the stimulation of the growth of Streptococcus lactis by yeast extract

Yeast extract was fractionated on Sephadex G-25 into 7 fractions. The fraction most stimulatory to the growth of Streptococcus lactis C10 contained over 70% of the amino N present in yeast extract and consisted of a wide variety of free amino acids and a small amount of peptide material. Examination of possible replacement factors for this fraction revealed that the amino -acid material present was largely responsible for the stimulation of Str. lactis C10. Purine and pyrimidine bases and inorganic constituents also contributed to the stimulation. In addition, yeast extract contained a component which decomposed H2O2, a metabolite which accumulates in the growth medium under aerobic conditions and inhibits growth. The nature of the stimulation was studied by isolating slow and fast acid-producing colonies of Str. lactis C10. It appeared that yeast extract and other amino-acid supplements prevented an observed inhibition of the growth of the slow variants below pH 6.0, apparently by satisfying a nutritional deficiency caused by a drop in pH.

Construction of plasmid vectors for the detection of streptococcal promoters

Plasmid vectors have been constructed for detecting DNA fragments that exhibit promoter activity in Streptococcus sanguis. The plasmids are able to replicate in both S. sanguis and Escherichia coli, and contain an erythromycin resistance marker which is expressed in both hosts. Selection for promoter activity is dependent upon the insertion of appropriate DNA fragments upstream from a promoterless chloramphenicol acetyl transferase gene (cat) from Staphylococcus aureus. To facilitate this insertion, a pair of vectors, pMU1327 and pMU1328, were constructed with the polylinker from M13mp 18 in either orientation. The to transcriptional terminator of phage lambda is present downstream from cat. Translation stop codons in all reading frames are located between the polylinker and the initiation codon of cat. These plasmids have been used to isolate DNA fragments from S. sanguis, S. lactis and S. cremoris that exhibit promoter activity in S. sanguis.

[63] l-Lactate dehydrogenase,1 FDP-activated, from Streptococcus cremoris

Publisher Summary This chapter discusses the assay method and properties of L-lactate dehydrogenase, fructose- 1,6-diphosphate (FDP)-activated, from Streptococcus cremoris . L-lactate dehydrogenase activity is measured spectrophotometrically at 340 nm by following either the oxidation of nicotinamide adenine dinucleotide dehydrogenase (NADH) in the presence of pyruvate and FDP, or the reduction of NAD in the presence of L-lactate and FDP. The steps involved in the purification procedureof L-lactate dehydrogenase are (1) the growth of organism, (2) the preparation of cell-free extracts, (3) fractionation with streptomycin sulfate, (4) first and second fractionation with ammonium sulfate, (5) chromatography on diethylaminoethyl (DEAE)-cellulose, and (6) chromatography on DEAE-Sephadex. The stability of the enzyme is dependent on both the type and pH of the buffer in which it is stored. Cell-free extracts prepared in acidic to neutral buffers and stored at 5°C and retain more lactate dehydrogenase activity than those prepared in alkaline buffers. The activation of lactate dehydrogenase activity by FDP is inhibited by phosphate and by the treatment of the enzyme with pyridoxal phosphate followed by reduction with sodium borohydride.

Sequence Analysis and Molecular Characterization of the Lactococcus lactis Temperate Bacteriophage BK5-T

ABSTRACT The Lactococcus lactis temperate bacteriophage BK5-T is one of twelve type phages that define L. lactis phage species. This paper describes the nucleotide sequence and analysis of a 21-kbp region of the BK5-T genome and completes the nucleotide sequence of the genome of this phage. The 40,003-nucleotide linear genome encodes 63 open reading frames. Sequence runoff experiments showed that the cohesive ends of the BK5-T genome contained a 12-bp 3′ single-stranded overhang with the sequence 5′-CACACACATAGG-3′. Two major BK5-T structural proteins, of approximately 30 and 20 kDa, were identified, and N-terminal sequence analysis determined that they were encoded by orf7 and orf12, respectively. A 169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in trans to the host cell and is likely a part of the BK5-T origin of replication (ori).

Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.

Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

ABSTRACT Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.

Characterization and Genomic Analysis of Phage asccφ28, a Phage of the Family Podoviridae Infecting Lactococcus lactis

ABSTRACT Bacteriophage asccφ28 infects dairy fermentation strains of Lactococcus lactis. This report describes characterization of asccφ28 and its full genome sequence. Phage asccφ28 has a prolate head, whiskers, and a short tail (C2 morphotype). This morphology and DNA hybridization to L. lactis phage P369 DNA showed that asccφ28 belongs to the P034 phage species, a group rarely encountered in the dairy industry. The burst size of asccφ28 was found to be 121 ± 18 PFU per infected bacterial cell after a latent period of 44 min. The linear genome (18,762 bp) contains 28 possible open reading frames (ORFs) comprising 90% of the total genome. The ORFs are arranged bidirectionally in recognizable functional modules. The genome contains 577 bp inverted terminal repeats (ITRs) and putatively eight promoters and four terminators. The presence of ITRs, a phage-encoded DNA polymerase, and a terminal protein that binds to the DNA, along with BLAST and morphology data, show that asccφ28 more closely resembles streptococcal phage Cp-1 and the φ29-like phages that infect Bacillus subtilis than it resembles common lactococcal phages. The sequence of this phage is the first published sequence of a P034 species phage genome.

Metabolism of [ 14 C]bicarbonate by Streptococcus lactis : identification and distribution of labelled compounds

Streptococcus lactis C10, grown in tryptone–yeast extract–lactose broth containing [ 14 C] bicarbonate, incorporated radioactivity into the protein and nucleic acid fractions of the cell as well as into compounds which were excreted by the organism into the growth medium. Aspartic acid was the first compound to be labelled and was the only amino acid labelled in the cell protein. All 4 bases were labelled in the cell RNA. Aspartic, succinic and lactic acids were the radioactive compounds excreted into the growth medium.