Egon Bech Hansen

Commercial bacterial starter cultures for fermented foods of the future

Starter cultures for fermented foods are today developed mainly by design rather than by screening. The design principles are based on knowledge of bacterial metabolism and physiology as well as on the interaction with the food product. In the genomics era, we will obtain a wealth of data making design on a rational basis even simpler. The design tools available are food grade tools for genetic, metabolic and protein engineering and an increased use of laboratory automation and high throughput screening methods. The large body of new data will influence the future patterns of regulation. It is currently difficult to predict in what direction the future regulatory requirements will influence innovation in the food industry. It can either become a promoting force for the practical use of biotechnology to make better and safer products, or it can be limiting the use of starter cultures to a few strains with official approval. Successful cultures based on modern technology is expected to be launched in the areas of: probiotics, bioprotection, general improvement of yield and performance for the existing culture market and probably the introduction of cultures for fermenting other food products. A scientific basis for dramatic innovations that could transform the culture industry is currently being established.

Isolation of Lactococcus lactis nonsense suppressors and construction of a food‐grade cloning vector

Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined. One suppressor is an ochre suppressor with an altered tRNAgin and the other an amber suppressor with an altered tRNAser. The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food‐grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following etectroporation of a suppressible purine auxotroph, can be done on purine‐free medium. The pepN gene from L. lactis Wg2 was subcloned resulting in a food‐grade plasmid giving a four‐ to fivefold increase in lysine aminopeptidase activity.

Physical mapping and nucleotide sequence of the rnpA gene that encodes the protein component of ribonuclease P in Escherichia coli

The rnpA gene, coding for the protein component of ribonuclease P (RNase P), was allocated to the dnaA region at 83 min of the E. coli K-12 map. This was accomplished through analysis of recombinant pBR322 plasmids, some of which complemented the temperature sensitivity of a strain carrying the rnpA 49 allele and restored the RNA processing activity. Although the temperature sensitivity of a strain carrying the rnp-241 allele could not be complemented by the rnpA+ plasmid, the RNA-processing activity was restored, suggesting that the rnp-241 mutation is allelic with rnpA 49. In this analysis we also found two genes coding for proteins (60 and 50 kDal) of unknown function. The order of the genes located in this region is in the clockwise orientation: rpmH (5.4 kDal; ribosomal protein L34), rnpA (14 kDal; protein component of RNase P), a gene for a 60-kDal protein (inner membrane protein), a gene for a 50-kDal protein, and tnaA. All these genes are expressed in the clockwise orientation. From the DNA sequence of the rnpA gene region a very basic polypeptide with an Mr of 13773 could be deduced. We conclude that this polypeptide is the rnpA gene product, and is the protein component of RNase P. Comparison with previously published data on the transcription of rpmH suggests that the rnpA gene is the second gene in the rpmH operon.

Effect of four probiotic strains and Escherichia coli O157:H7 on tight junction integrity and cyclo-oxygenase expression.

Controversy exists as to whether contact between a probiotic bacterial cell and an epithelial cell in the gut is needed to confer beneficial effects of probiotics, or whether metabolites from probiotics are sufficient to cause this effect. To address this question, Caco-2 cells were treated with cell-free supernatants of four probiotics, Bifidobacterium lactis 420, Bifidobacterium lactis HN019, Lactobacillus acidophilus NCFM, Lactobacillus salivarius Ls-33, and by a cell-free supernatant of a pathogenic bacteria, Escherichia coli O157:H7 (EHEC). Tight junction integrity as well as expression of cyclo-oxygenases, which are prostaglandin-producing enzymes, were measured. Probiotic-specific as well as EHEC-specific effects on tight junction integrity and cyclo-oxygenase expression were evident, indicating that live bacterial cells were not necessary for the manifestation of the effects. B. lactis 420 cell-free supernatant increased tight junction integrity, while EHEC cell-free supernatant induced damage on tight junctions. In general, EHEC and probiotics had opposite effects upon cyclo-oxygenase expression. Furthermore, B. lactis 420 cell-free supernatant protected the tight junctions from EHEC-induced damage when administered prior to the cell-free supernatant of EHEC. These results indicate that probiotics produce bioactive metabolites, suggesting that consumption of specific probiotic bacteria might be beneficial in protecting intestinal epithelial cells from the deleterious effects of pathogenic bacteria.

The nucleotide sequence of the dnaA gene promoter and of the adjacent rpmH gene, coding for the ribosomal protein L34, of Escherichia coli.

The nucleotide sequence was determined of a 945‐bp EcoRI fragment from the Escherichia coli K‐12 chromosome at 82 min containing the promoter region of the dnaA gene. This nucleotide sequence contained a coding sequence identical to the amino acid sequence of the ribosomal protein L34, designated rpmH . The rimA mutation, which affects the maturation of 50S ribosomal particles, may be an allele of the rpmH gene since it maps close to, or within, the L34 coding sequence. The rpmH gene and the dnaA gene are transcribed in the clockwise and counter‐clockwise direction, respectively. Nuclease S1 mapping of transcripts indicated the existence of two major promoters for the L34 gene and two promoters for the dnaA gene within the 945‐bp EcoRI fragment.

Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli

SummaryA fine structure genetic map of several mutations in the dnaA gene of Escherichia coli was constructed by the use of recombinant λ and M13 phages. The dnaA508 mutation was found to be the mutation most proximal to the promoter, while the dnaA203 mutation was found to be the most distal one. The order of mutations established in this analysis was: dnaA508, dnaA167, (dnaA5, dnaA46, dnaA211), dnaA205, dnaA204, dnaA203. The mutations dnaA601, dnaA602, dnaA603, dnaA604 and dnaA606 were found to map very close to each other and close to dnaA205 in the middle third of the dnaA gene. In analysing the dominance relationship all 13 dnaA mutations were found to be recessive to the wild type. Characteristic phenotypes of the dnaA(Ts) mutants, like reversibility of the temperature inactivation of the dnaA protein, cold sensivity of haploid or of merodiploid strains and suppressibility by rpoB mutations, are found to correlate with clusters of mutations within the gene.

Cloning of the lysA gene from Mycobacterium tuberculosis

The lysA and proC genes of Mycobacterium tuberculosis were cloned by screening of a recombinant lambda gt11 M. tuberculosis DNA library for phages able to complement lysA and proC Escherichia coli mutants. The lysA gene encodes diaminopimelic acid decarboxylase which catalyzes the conversion of diaminopimelic acid (DAP) to lysine. The lysA gene from M. tuberculosis encodes a 44-kDa protein, as determined by maxicell experiments. The nucleotide sequence of the structural gene was established. The deduced amino acid sequence was found to exhibit significant homology (from 55% to 73% similarity, and from 27% to 53% identity) to DAP decarboxylase sequences from other bacterial species.

Comparative Evaluation of the Antimicrobial Activity of Different Antimicrobial Peptides against a Range of Pathogenic Bacteria

Analysis of a Selected Set of Antimicrobial Peptides The rapid emergence of resistance to classical antibiotics has increased the interest in novel antimicrobial compounds. Antimicrobial peptides (AMPs) represent an attractive alternative to classical antibiotics and a number of different studies have reported antimicrobial activity data of various AMPs, but there is only limited comparative data available. The mode of action for many AMPs is largely unknown even though several models have suggested that the lipopolysaccharides (LPS) play a crucial role in the attraction and attachment of the AMP to the bacterial membrane in Gram-negative bacteria. We compared the potency of Cap18, Cap11, Cap11-1-18m2, Cecropin P1, Cecropin B, Bac2A, Bac2A-NH2, Sub5-NH2, Indolicidin, Melittin, Myxinidin, Myxinidin-NH2, Pyrrhocoricin, Apidaecin and Metalnikowin I towards Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Aeromonas salmonicida, Listeria monocytogenes, Campylobacter jejuni, Flavobacterium psychrophilum, Salmonella typhimurium and Yersinia ruckeri by minimal inhibitory concentration (MIC) determinations. Additional characteristics such as cytotoxicity, thermo and protease stability were measured and compared among the different peptides. Further, the antimicrobial activity of a selection of cationic AMPs was investigated in various E. coli LPS mutants. Cap18 Shows a High Broad Spectrum Antimicrobial Activity Of all the tested AMPs, Cap18 showed the most efficient antimicrobial activity, in particular against Gram-negative bacteria. In addition, Cap18 is highly thermostable and showed no cytotoxic effect in a hemolytic assay, measured at the concentration used. However, Cap18 is, as most of the tested AMPs, sensitive to proteolytic digestion in vitro. Thus, Cap18 is an excellent candidate for further development into practical use; however, modifications that should reduce the protease sensitivity would be needed. In addition, our findings from analyzing LPS mutant strains suggest that the core oligosaccharide of the LPS molecule is not essential for the antimicrobial activity of cationic AMPs, but in fact has a protective role against AMPs.

Exponential sinusoidal modeling of transitional speech segments

A generalized sinusoidal model for speech signal processing is studied. The main feature of the model is that the amplitude of each sinusoidal component is allowed to vary exponentially with time. We propose to use the model in transitional speech segments such as speech onsets and voiced/unvoiced transitions. Computer simulations with natural speech signals indicate substantial better modeling performance in both transitional and voiced regions compared with the traditional constant-amplitude sinusoidal model.

Structure and regulation of the lytic replicon of phage P1

Three replicons, R, L and P1dR, have been previously identified in bacteriophage P1, but only the R (or plasmid) replicon has been functionally and structurally characterized. Evidence is provided here that the L-replicon is the principal replicon used for DNA replication during the lytic cycle. The L-replicon (exclusive of its promoter) is shown to be contained within a 1093-base-pair DNA segment that includes a 281-codon open reading frame, designated repL. L-replicon function requires transcription in the direction that should generate translatable repL message. This transcription is normally under the control of the phage c1 repressor, but a deletion that places the functional L-replicon under alternative control can be constructed. The DNA sequence of the replicon and surrounding regions was established. The sequenced region contains the c4 and ant genes and a hitherto unidentified gene, kilA, which is immediately upstream of repL and is controlled by the c1-regulated promoter. The kilA gene was shown to be non-essential for both replication and lytic development whereas the repL gene probably encodes an essential replication protein.