Mark W. Lubbers

Cloning and Expression of an Oligopeptidase, PepO, with Novel Specificity from Lactobacillus rhamnosus HN001 (DR20)

ABSTRACT Oligopeptidases of starter and nonstarter lactic acid bacteria contribute to the proteolytic events important in maturation and flavor development processes in cheese. This paper describes the molecular cloning, expression, and specificity of the oligopeptidase PepO from the probiotic nonstarter strain Lactobacillus rhamnosus HN001 (DR20). The pepO gene encodes a protein of 70.9 kDa, whose primary sequence includes the HEXXH motif present in certain classes of metallo-oligopeptidases. The pepO gene was cloned in L. rhamnosus HN001 and overexpressed in pTRKH2 from its own promoter, which was mapped by primer extension. It was further cloned in both pNZ8020 and pNZ8037 and overexpressed in Lactococcus lactis subsp. cremoris NZ9000 from the nisA promoter. The purified PepO enzyme demonstrated unique cleavage specificity for αs1-casein fragment 1–23, hydrolyzing the bonds Pro-5-Ile-6, Lys-7-His-8, His-8-Gln-9, and Gln-9-Gly-10. The impact of this enzyme in cheese can now be assessed.

Direct selection and phage display of a Gram-positive secretome

Surface, secreted and transmembrane protein-encoding open reading frames, collectively the secretome, can be identified in bacterial genome sequences using bioinformatics. However, functional analysis of translated secretomes is possible only if many secretome proteins are expressed and purified individually. We have now developed and applied a phage display system for direct selection, identification, expression and purification of bacterial secretome proteins.

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2.

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3′ extended DNAs, with the following sequence: 5′-GTTAGGCTT-3′ 3′-CAATCCGAA-5′. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3′ extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5′ extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.

Isolation of Lactococcal Prolate Phage-Phage Recombinants by an Enrichment Strategy Reveals Two Novel Host Range Determinants

Virulent lactococcal prolate (or c2-like) phages are the second most common phage group that causes fermentation failure in the dairy industry. We have mapped two host range determinants in two lactococcal prolate phages, c2 and 923, for the host strains MG1363 and 112. Each phage replicates on only one of the two host strains: c2 on MG1363 and 923 on 112. Phage-phage recombinants that replicated on both strains were isolated by a new method that does not require direct selection but rather employs an enrichment protocol. After initial mixed infection of strain 112, two rotations, the first of which was carried out on strain MG1363 and the second on 112, permitted continuous amplification of double-plating recombinants while rendering one of the parent phages unamplified in each of the two rotations. Mapping of the recombination endpoints showed that the presence of the N-terminal two-thirds of the tail protein L10 of phage c2 and a 1,562-bp cosR-terminal fragment of phage 923 genome overcame blocks of infection in strains MG1363 and 112, respectively. Both infection inhibition mechanisms act at the stage of DNA entry; in strain MG1363, the infection block acts early, before phage DNA enters the cytoplasm, and in strain 112, it acts late, after most of the DNA has entered the cell but before it undergoes cos-end ligation. These are the first reported host range determinants in bacteriophage of lactic acid bacteria required for overcoming inhibition of infection at the stage of DNA entry and cos-end ligation.

Examination of lactococcal bacteriophage c2 DNA replication using two-dimensional agarose gel electrophoresis

The ori locus of the prolate-headed lactococcal bacteriophage c2 supports plasmid replication in Lactococcus lactis in the absence of phage infection. To determine whether phage c2 DNA replication is initiated at the ori locus in vivo and to investigate the mechanism of phage DNA replication, replicating intermediates of phage c2 were analyzed using neutral/neutral two-dimensional agarose gel electrophoresis (2D). The 2D data revealed that c2 replicates via a theta mechanism and localized the initiation of theta replication to the ori region of the c2 genome.

Sequence Diversity and Functional Conservation of the Origin of Replication in Lactococcal Prolate Phages

ABSTRACT Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium Lactococcus lactis. In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found. The nonconserved sequence was confined to the template for an untranslated transcript, PE1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved. All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant PE1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins. Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the PE1-T transcript is required for replication. This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.

Cloning and characterisation of the main intracellular esterase from Lactobacillus rhamnosus HN001

In cheese, the breakdown of milk fat into free fatty acids (FFAs) and esters by lipases and esterases contributes to flavour development. Short-chain FFAs and short-chain ethyl esters are important for the flavour of Italian-style cheeses such as Parmesan, Grana Padano, Romano and Provolone. Esterase activity has been identified in the lactic acid bacteria (LAB) used as starter bacteria and in the adjunct microflora during cheese manufacture. A novel esterase gene, designated AA7, was identified in the genome sequence of Lactobacillus rhamnosus HN001. AA7 was shown to be the main intracellular esterase of L. rhamnosus HN001. The enzyme was characterised for both hydrolytic release of FFAs and alcoholytic synthesis of ethyl esters from synthetic substrates. AA7 was shown to be particularly active on short-chain acyl substrates and had a preference for monoacylglycerol substrates. AA7 was used in cheese manufacture and catalysed an accumulation of butyric acid and ethyl esters in cheese, two of the most important flavour compounds in Italian-style cheeses.