Arnold Geis

Taxonomic Differentiation of Bacteriophages of Lactococcus lactis by Electron Microscopy, DNA-DNA Hybridization, and Protein Profiles

SUMMARY: Thirty-seven virulent and 19 temperate bacteriophages of Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were classified in a taxonomic system on the basis of morphology, DNA-DNA hybridization, and protein composition. As judged from electron microscopy and susceptibility to cleavage by restriction endonucleases, the genome of all the bacteriophages investigated is composed of double stranded DNA. Seven virulent phage groups were recognized: types P034 (genome size 18·1 kilobase pairs, kb), P001 (20·2 kb), P008 (29·7 kb), P335 (36·4 kb), P026 (51·5 kb), P107 (51·5 kb), and P087 (54·5 kb). In addition, two temperate phage groups were established: types TP-40-3 (genome size 42·1 kb) and TP-936-1 (37·8 kb). Phages within each group revealed strong DNA homology and similar protein compositions, whereas no significant DNA homology and different proteins were found in phages of different groups. Virulent phages of group P335 exhibited strong DNA homology with the temperate phages of group TP-936-1.

Purification of an X-prolyl-dipeptidyl aminopeptidase from the cell wall proteolytic system of Lactococcus lactis subsp. cremoris

SummaryThe β-casein specific cell wall proteolytic system of Lactococcus lactis subsp. cremoris P8-2-47 contains a metal-independent X-prolyl-dipeptidyl-aminopeptidase. Suitable substrates for its assay are Gly-Pro-nitroanilide and Ala-Pronitroanilide. It is suggested that the function of the enzyme is to cleave the proline-rich sequences of β-casein, as shown by the degradation of β-casomorphin. It is a serine proteinase with a monomer molecular mass of about 90 000 daltons, a temperature optimum of 45°–50°C, and a pH optimum of about 7.

Sequence analysis and characterization of plasmids from Streptococcus thermophilus

The nucleotide sequences of eight plasmids isolated from seven Streptococcus thermophilus strains have been determined. Plasmids pSt04, pER1-1, and pJ34 are related and replicate via a rolling circle mechanism. Plasmid pJ34 encodes for a replication initiation protein (RepA) and a small polypeptide with unknown function. Plasmids pSt04 and pER1-1 carry in addition to repA genes coding for small heat shock proteins (sHsp). Expression of these proteins is induced at elevated temperatures or low pH and increases the thermo- and acid resistance. Plasmids pER1-2 and pSt22-2 show identical sequences with five putative open reading frames (ORFs). The gene products of ORF1 and ORF4 reveal some similarities to transposon encoded proteins of Bacillus subtilis and Tn916. ORF1 of plasmid pSt106 encodes a protein similar to resolvases of different Gram-positive bacteria. Integrity of ORF2 and 3, encoding a putative DNA primase and a replication protein, is essential for replication. ORF1 to 3 of plasmid pSt08, which are organized in a tricistronic operon, encode a RepA protein, an adenosine-specific methyltransferase, and a type II restriction endonuclease. Another type II restriction-modification (R/M) system is encoded on plasmid pSt0 which is highly similar to those encoded on lactococcal plasmid pHW393 and B. subtilis plasmid pXH13. Plasmid-free derivatives of strains St0 and St08 show increased phage sensitivity, indicating that in the wild-type strains the R/M systems are functionally expressed. Recombinant plasmids based on the replicons of plasmids pSt04, pJ34, pSt106, pSt08, and pSt0, are able to replicate in Lactococcus lactis and B. subtilis, respectively, whereas constructs carrying pER1-2 only replicate in S. thermophilus.

Sequencing and characterization of pST1, a cryptic plasmid from Streptococcus thermophilus

Eighty-six strains of S. thermophilus were examined for their plasmid content. Thirteen strains were found to contain one or two plasmids ranging in size from 2.1 to 7.4 kb. DNA-DNA hybridization analysis revealed the presence of five distinct groups of DNA homology. The complete nucleotide sequence of plasmid pST1 (Accession number X65856), which belongs to the major homology group, was determined. It has a molecular size of 2093 bp, a GC content of 35% and contains one major open reading frame of 945 bp (ORF A). The predicted protein, designated Rep A, showed sequence homology with replication proteins from a group of plasmids which are known to replicate via single-stranded DNA intermediates (ssDNA plasmids).

Application of the shsp Gene, Encoding a Small Heat Shock Protein, as a Food-Grade Selection Marker for Lactic Acid Bacteria

ABSTRACT Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60°C or pH 3.5 and in the ability to grow at 52°C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Emr) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 × 104 and 1.0 × 104 CFU/0.5 μg of DNA, with standard deviations of 0.54 × 104 and 0.32 × 104, for shsp and Emr selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 × 104 and 3.8 × 103 CFU/0.5 μg of DNA, with standard deviations of 0.63 × 104 and 3.48 × 103, for shsp and Emr selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.

Cloning of Escherichia coli lacZ and lacY Genes and Their Expression in Gluconobacter oxydans and Acetobacter liquefaciens

ABSTRACT An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G. oxydans and A. liquefaciens. Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both lac genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.

Conjugation, a common plasmid transfer mechanism in lactic acid streptococci of dairy starter cultures

Summary 82 strains of mesophilic lactic acid streptococci (including 30 Streptococcus lactis , 13 S. lactis subsp. ‘diacety lactis’ and 39 5. cremoris) isolated from starter and Kefir cultures were examined as donor strains in agar-surface-matings for their potential to transfer lactose fermentation ability (Lac + phenotype) to the plasmid-free recipient strain S. lactis subsp. ‘diacetylactis’ Bu2–60. 10 S. lactis, 5 S. lactis subsp. ‘ diacetylactis ’ and 5 S. cremoris strains did transfer the Lac + phenotype to the plasmid-free recipient with frequencies between 2 × 10 −4 and 5 × 10 −9 per donor, and between 10 −3 and 4 × 10 −9 per recipient, respectively. Mating and curing experiments provided evidence that the transfer of the Lac + phenotype was due to conjugative lactose plasmids ranging in size from 20 to 67 megadaltons (Mdal). In many matings, additional plasmids were cotransferred with the lactose plasmids, e.g. a 5.3 megadalton citrate plasmid from 3 S. lactis subsp. ‘ diacetylactis ’ strains, several cryptic plasmids from all three species and a 41 megadalton bacteriophage resistance plasmid from one S. lactis subsp. ‘ diacetylactis ’ strain. Conjugal transfer of lactose plasmids was also observed in milk under suitable conditions.

Bacillus subtilis develops competence for uptake of plasmid DNA when growing in milk products

Transformation with plasmid DNA of naturally competent cells of Bacillus subtilis 168 in milk products was studied. Plasmid pMG36enpr, a broad host-range lactococcal vector carrying an erythromycin resistance and the B. subtilis npr gene encoding neutral protease, was taken up by B. subtilis cells grown in UHT chocolate milk. Under these conditions competence was optimal during transition from exponential to stationary growth phase, resulting in 9 x 10(1) transformants per 0.01 microgram DNA. No manipulation of the cells was necessary for competence to develop. When cells were pregrown in synthetic medium, higher transformation rates were obtained in assays, where the subsequent transformation experiments were either done in chocolate milk diluted 1:1 (v/v) with synthetic growth medium (up to 8 x 10(2) transformants) or in undiluted chocolate milk (1 x 10(2) transformants). The number of transformants was reduced to 4 x 10 (1), when diluted milk or flavored milks were used. No transformants were obtained in diluted yoghurt. Controls, in which both the preculturing and the transformation assays were done in synthetic medium, gave the maximum number of transformants (4 x 10(3) transformants per 0.01 microgram DNA).

Plasmid Transfer via Transduction from Streptococcus thermophilus to Lactococcus lactis

Using Streptococcus thermophilus phages, plasmid transduction in Lactococcus lactis was demonstrated. The transduction frequencies were 4 orders of magnitude lower in L. lactis than in S. thermophilus. These results are the first evidence that there is phage-mediated direct transfer of DNA from S. thermophilus to L. lactis. The implications of these results for phage evolution are discussed.

Behaviour of Genetically Modified Microorganisms in Yoghurt

Summary The microflora of traditional yoghurt consists of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus . Using two promiscuitive plasmds, conjugational pAMβ1 and non-conjugational pNZ18, both bacterial species were analyzed for their abilities to participate in gene transfer by conjugation, transduction or transformation. While no transfer at all into L. delbrueckii subsp. bulgaricus was observed under any of the conditions tested, S. thermophilus was able to act as recipient or donor in conjugation and transduction. Transduction even occurred in yoghurt. Transformation rates observed for S. thermophilus were low and may be at least partly attributed to the presence of surface associated nuclease activities. Segregational stability of pAMβ1 was shown to depend on environmental factors and on the host bacteria harbouring the plasmid. The data are discussed with respect to safety evaluation and risk analyses of genetically modified starter bacteria, and demands for future research on safety evaluation of novel foods are addressed.