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Biomedical subjects

G F Fitzgerald

Publications and source records attributed to G F Fitzgerald.

At least 55 records · Page 3Linked to original sources

Biotechnological approaches to the understanding and improvement of mature cheese flavour.

There have been important milestones in biotechnological practice that have led to the determination and production of superior cheese flavours. Within the past year, the use of gas chromatographic techniques and sensory methodologies has been optimised by several groups in efforts to evaluate the organoleptic properties of a number of mature cheeses. The hydrolysis of milk caseins, small peptides, free amino acids and fatty acids, and the generation of sulfur-containing compounds are uniformly assumed to result in the formation of specific cheese aromas. Giant strides have been taken in molecular technology to aid the dissection and exploitation of the metabolic pathways that lead to the formation of these flavour constituents. Specific advances in molecular technology have included metabolic engineering of lactic acid bacteria for enhanced flavour development.

Biotechnology↗

Novel type I restriction specificities through domain shuffling of HsdS subunits in Lactococcus lactis.

This study identifies a natural system in Lactococcus lactis, in which a restriction modification specificity subunit resident on a 6159 bp plasmid (pAH33) alters the specificity of a functional R/M mechanism encoded by a 20.3 kb plasmid, pAH82. The new specificity was identified after phenotypic and molecular analysis of a 26.5 kb co-integrate plasmid (pAH90), which was detected after bacteriophage challenge of the parent strain. Analysis of the regions involved in the co-integration revealed that two novel hybrid hsdS genes had been formed during the co-integration event. The HsdS chimeras had interchanged the C- and N-terminal variable domains of the parent subunits, generating two new restriction specificities. Comparison of the parent hsdS genes with other type I specificity determinants revealed that the region of the hsdS genes responsible for the co-integration event is highly conserved among lactococcal type I hsdS determinants. Thus, as hsdS determinants are widespread in the genus Lactococcus, new restriction specificities may evolve rapidly after homologous recombination between these genes. This study demonstrates that, similar to previous observations in Gram-negative bacteria, a Gram-positive bacterium can acquire novel restriction specificities naturally through domain shuffling of resident HsdS subunits.

Amino Acid Sequence↗

Investigation of the relationship between lysogeny and lysis of Lactococcus lactis in cheese using prophage-targeted PCR.

The ability of lactococcal strains to lyse (and release intracellular enzymes) during cheese manufacture can be a very desirable trait and has been associated with improvement in flavor and acceleration of cheese ripening. Using a laboratory-scale cheese manufacturing assay, the autolytic behavior of 31 strains of Lactococcus lactis was assessed. In general, marked variation was observed between strains with a 20-fold difference between the best and worst lysing strains based on the release of the intracellular enzyme lactate dehydrogenase. In a parallel experiment, the genomes of these strains were examined for the presence of prophage integrase (int) sequences by using conserved primer sequences from known lysogenic phage. Results demonstrated that the lytic behavior of lactococcal starter strains significantly correlates with the presence of prophage sequences. These results highlight not only the contribution of prophage to starter cell lysis but also the potential of PCR as a useful initial screen to assess strains for this important industrial trait.

Cheese↗

Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray drying.

Spray drying of skim milk was evaluated as a means of preserving Lactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59 degrees C. An air outlet temperature of 80 to 85 degrees C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 x 10(9) CFU/g for NFBC 338 and 5.2 x 10(7) CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at approximately 1 x 10(9) CFU/g during 2 months of powder storage at 4 degrees C, while a decline in the level of survival of approximately 1 log (from 7.2 x 10(7) to 9.5 x 10(6) CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.

Bacteriocins↗

Identification and characterization of an active plasmid partition mechanism for the novel Lactococcus lactis plasmid pCI2000.

The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed. The nucleotide sequence of one 5.6-kb EcoRI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated repA, is indeed required for replication. The results also suggest that the origin of replication is located outside repA. Upstream and divergently transcribed from repA, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified. Gene interruption and transcomplementation experiments showed that this ORF, designated parA, is required for stable inheritance of pCI2000 and is active in trans. This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.

Amino Acid Motifs↗

Food fermentations: role of microorganisms in food production and preservation.

Preservation of foods by fermentation is a widely practiced and ancient technology. Fermentation ensures not only increased shelf life and microbiological safety of a food but also may also make some foods more digestible and in the case of cassava fermentation reduces toxicity of the substrate. Lactic acid bacteria because of their unique metabolic characteristics are involved in many fermentation processes of milk, meats, cereals and vegetables. Although many fermentations are traditionally dependent on inoculation from a previous batch starter cultures are available for many commercial processes such as cheese manufacture thus ensuring consistency of process and product quality. This review outlines the role of lactic acid bacteria in many such fermentations and the mechanisms of antibiosis with particular reference to bacteriocins and gives a brief description of some important fermented foods from various countries. It is anticipated that the contribution of the advances in lactic acid bacteria research towards improvement of strains for use in food fermentation will benefit both the consumer and the producer.

Fermentation↗

Characterisation of Streptococcus thermophilus CNRZ1205 and its cured and re-lysogenised derivatives.

Streptococcus thermophilus CNRZ1205 is the lysogenic host for the temperate phage phi O1205. A derivative of CNRZ1205 was isolated which was cured of phi O1205 and this strain was used to construct a re-lysogenised derivative. Pulse field gel electrophoresis and sequencing of the attachment site regions confirmed that excision and re-integration of the phage was a site-specific event. Interestingly, cells from the cured, as well as its re-lysogenised derivative, were found to have a very long chain length.

DNA Transposable Elements↗

Molecular characterisation of a 5.75-kb cryptic plasmid from Bifidobacterium breve NCFB 2258 and determination of mode of replication.

A small cryptic plasmid originating from Bifidobacterium breve NCFB 2258 was cloned and its complete nucleotide sequence determined. pCIBb1 is a circular DNA molecule, 5750 bp in size with a GC composition of 57%. Computer-assisted analysis identified 10 possible open reading frames (ORFs), seven of which could be assigned no function from homology searches. One ORF, rep (380 amino acids), was postulated to encode a replication protein similar to known replication proteins of rolling circle replicons, particularly those of the pC194 family. Demonstration of single-stranded forms of the plasmid in cell lysates that could be specifically degraded by S1 nuclease provided experimental evidence to substantiate a replication mechanism via single-stranded intermediates. Two other ORFs, par (199 amino acids) and an ftsK-like gene (286 amino acids), were assigned putative functions based on the presence of conserved motifs in their deduced proteins.

Amino Acid Sequence↗

Influence of a probiotic adjunct culture of Enterococcus faecium on the quality of cheddar cheese.

Cheddar cheese has previously been shown to be an effective vehicle for delivery of viable cells of a probiotic Enterococcus faecium strain to the gastrointestinal tract. The particular strain, E. faecium PR88, has proven efficacy in the treatment of irritable bowel syndrome, and in this study it was evaluated for suitability as a starter adjunct for Cheddar cheese manufacture. When added to cheesemilk at an inoculum of 2 x 10(7) cfu/mL, the enterococcal adjunct maintained viability in Cheddar cheese at levels of up to 3 x 10(8) cfu/g during 9 months of ripening. Increased proteolysis and higher levels of some odor-active volatile compounds were observed in Cheddar cheeses containing the PR88 adjunct compared with the control throughout the ripening period. In addition, the enterococcal adjunct strain did not affect cheese composition. Although sensory evaluation showed no significant difference in flavor/aroma and body/texture scores between control and experimental cheeses, repeated comments by the commercial grader consistently described the cheeses containing PR88 as 'more advanced than the control' and as having 'better flavor'. These findings indicate that the presence of the PR88 adjunct strain in Cheddar cheese at levels of >/=10(8) cfu/g may positively influence Cheddar flavor.

Cheese↗

Electrotransformation of industrial strains of Streptococcus thermophilus.

A standard electroporation procedure was utilized to introduce a range of Gram-positive plasmid vectors into nine industrial strains of Streptococcus thermophilus. All the strains were transformable with at least two of the plasmids assessed, but electrotransformation frequencies depended on both the strain and the nature of transforming DNA. In general, small rolling circle (RC) plasmids could be electroporated at high frequency into a wide range of strains with efficiencies of 10(2)-10(5) transformants microgram-1 of transforming DNA. The presence of these plasmids did not influence doubling times during growth in broth, and they were generally extremely stable in slow milk acidifying strains, with 85-100% of transformants retaining the selective markers over 105 generations. Vectors were less stable in fast-growing cultures. Of the three theta-type plasmids assessed, only one, pIL253, could be electroporated at low frequency into some slow growing strains. The presence of this plasmid caused a 40% increase in doubling time and it was lost from cells at a rate of 3% per generation. Attempts to alter the proteolytic status of slow acidifying strains of Strep. thermophilus by the introduction of heterologous proteinase genes are also described.

Electroporation↗

Identification and characterization of a lysis module present in a large proportion of bacteriophages infecting Streptococcus thermophilus.

A lysis module encoded by the temperate bacteriophage phiO1205 was identified. This lysis module contains a lysin gene, designated lyt51, and two putative holin-encoding genes, designated lyt49 and lyt50. lyt51 encodes a lytic enzyme specifically directed against streptococcal cell walls. Similar to other phage-encoded lysins, Lyt51 appears to have a modular design in which the N-terminal portion corresponds to its enzymatic activity while the C-terminal region is responsible for its substrate binding specificity. The two putative holin-encoding genes, lyt49 and lyt50, located immediately upstream of lyt51, were identified on the basis of their homology to other identified holin-encoding genes. Expression of lyt49 or lyt50 in Escherichia coli was shown to cause cell death and leakage of the intracellular enzyme isocitrate dehydrogenase into the growth medium without apparent lysis of the cells. Southern blotting experiments demonstrated that at least one of the three components of the identified lysis module is present in all members of a large collection of bacteriophages, indicating that components of this lysis module are widespread among bacteriophages infecting Streptococcus thermophilus.

Amino Acid Sequence↗

Identification of four phage resistance plasmids from Lactococcus lactis subsp. cremoris HO2.

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage phi712 (936 phage species) and the prolate-headed phage phic2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.

Bacteriophage Typing↗

Molecular characterization of a phage-encoded resistance system in Lactococcus lactis.

A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009 protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.

Bacteriophages↗

Comparison of Streptococcus thermophilus strains by pulse field gel electrophoresis of genomic DNA.

Pulse field gel electrophoresis (PFGE) was utilised to compare the genomes of 16 Streptococcus thermophilus cultures from yoghurt, cheese, laban and dahi after digestion with the restriction endonucleases, SfiI, SmaI and BssHII. PFGE profiles could be used for strain identification and were also useful in predicting relatedness of certain strains. Genetic variations between specific morphotypes of a highly proteolytic culture were not detectable by PFGE in this study. Statistical analysis of SmaI restriction patterns enabled the clustering of strains into two groups which corresponded with biochemical properties of the strains examined and suggested that PFGE profiles could be useful in predicting biochemical characteristics.

DNA Restriction Enzymes↗

Evaluation of bifidobacteria for the production of antimicrobial compounds and assessment of performance in cottage cheese at refrigeration temperature.

Twelve strains of bifidobacteria were identified which exhibited a broad spectrum of antagonistic activity against both Gram-positive and Gram-negative indicators, especially Pseudomonas species, using deferred antagonism spot plate assays. Inhibitory action was shown to be unrelated to hydrogen peroxide production and not solely dependent on acidity. However, attempts to detect inhibitory activity in cell-free supernatant fluids from these strains were unsuccessful. The production of inhibitory compound(s) by Bifidobacterium infantis NCFB 2255 was shown to be an unstable trait resulting in phenotypic alternation between production and non-production. Results from food trials using commercial cottage cheese which was inoculated with the inhibitor-producing strains of Bif. infantis NCFB 2255 and Bif. breve NCFB 2258 indicated that levels of Pseudomonas were reduced, but this observation was species-dependent. The viability of bifidobacteria themselves during storage in cottage cheese at refrigeration temperature was found to be a strain variable trait.

Antibiosis↗

Determination of genetic diversity within the genus Bifidobacterium and estimation of chromosomal size.

Pulsed-field gel electrophoresis was proven to be an efficient means of differentiating 25 strains of Bifidobacterium obtained from culture collections. XbaI, SpeI, DraI restriction enzyme profiles indicated genomic heterogeneity among strains. When seven human isolates of bifidobacteria were compared using the same methods, two individual banding patterns were obtained. However, despite its discriminatory potential, pulsed-field gel electrophoresis was shown to be of no value in taxonomic identification. Genomic sizes estimated for eight Bifidobacterium strains ranged from 1.5 Mb to 2.1 Mb.

Bifidobacterium↗

Identification of a recA homolog (recALP) on the conjugative lactococcal phage resistance plasmid pNP40: evidence of a role for chromosomally encoded recAL in abortive infection.

The determinants for two bacteriophage resistance mechanisms, AbiE and AbiF, are separated by approximately 3,300 nucleotides on the lactococcal plasmid pNP40 (P. Garvey, G.F. Fitzgerald, and C. Hill, Appl. Environ. Microbiol. 61:4321-4328, 1995). DNA sequence analysis of the intervening region led to the identification of two open reading frames (ORFs) which are transcribed in the opposite direction to the Abi determinants. One of these ORFs encodes a recA homolog (designated recALP). This is the first report of a recA-like determinant located to a plasmid. The second ORF (orfU) shares homology with the umuC gene of the SOS response. Analysis of a number of lactococcal strains confirmed the presence of recALP-like sequences in at least two other lactococcal strains. The proximity of the recA and umuC homologs suggested a possible role in the phase resistance encoded by the Abi determinants. However, no evidence was obtained to demonstrate a function for either ORF in the expression of either AbiE or AbiF. Nor could the recALP gene restore resistance to mitomycin in a recA-deficient lactococcal strain, VEL1122. Interestingly, it was shown that the chromosomally encoded recA is necessary for complete expression of the AbiF phenotype, confirming a role for RecA in this abortive infection system.

Amino Acid Sequence↗