Biomedical subjects

Gerald F Fitzgerald

Publications and source records attributed to Gerald F Fitzgerald.

At least 37 records · Page 2Linked to original sources

PubMed2006

Intestinal bifidobacteria that produce trans-9, trans-11 conjugated linoleic acid: a fatty acid with antiproliferative activity against human colon SW480 and HT-29 cancer cells.

Bifidobacterium breve species of human intestinal origin have the ability to synthesize cis-9, trans-11 (c9, t11) conjugated linoleic acid (CLA) from free linoleic acid. In this study, the ability of Bifidobacterium species to isomerize C(18) polyunsaturated fatty acids was investigated, and the antiproliferative activities of the two main microbially produced CLA isomers were assessed. Linoleic acid was converted principally to c9, t11 CLA and lesser amounts of t9, t11 CLA, whereas c9, t11 CLA was converted mainly to t9, t11 CLA. Likewise, t10, c12 CLA was converted principally to t9, t11 CLA, which was incorporated into the bacterial cell membranes. To examine the antiproliferative effect of the two main CLA isomers formed, SW480 and HT-29 human colon cancer cells were cultured in the presence of c9, t11 CLA and t9, t11 CLA. The t9, t11 CLA had a more potent antiproliferative effect than c9, t11 CLA. It is tempting to suggest that the ability of Bifidobacterium to produce such bioactive metabolites may be associated with the beneficial effects of bifidobacteria present in the human gastrointestinal tract.

Antineoplastic Agents↗

PubMed2005

Genome analysis of the obligately lytic bacteriophage 4268 of Lactococcus lactis provides insight into its adaptable nature.

Analysis of the complete nucleotide sequence of the lactococcal phage 4268, which is lytic for the cheese starter Lactococcus lactis DPC4268, is presented. Phage 4268 has a linear genome of 36,596 bp, which is modularly organised and encompasses 49 open reading frames. Putative functions were assigned to approximately 45% of the predicted products of these open reading frames based on sequence similarity with known proteins, N-terminal sequence analysis and identification of conserved domains. Significantly, a segment of the genome has homology to the recently sequenced lysogenic module in lactococcal phage phi31 that contains a lytic switch but no phage integrase or attachment site. This suggests that it is derived from a prophage. A phage 4268-encoded and a host-encoded methylase were found to be highly similar, having only two nucleotide mismatches, suggesting that the phage acquired the methylase gene to protect it from a host endonuclease. Comparative genomic analysis revealed significant homology between phage 4268 and the lactococcal phage BK5-T. The comparative analysis also supported the classification of phage 4268 and other BK5-T-related phage as separate from the proposed P335 species of lactococcal phage.

Bacteriophages↗

PubMed2005

Fermented functional foods based on probiotics and their biogenic metabolites.

The claimed health benefits of fermented functional foods are expressed either directly through the interaction of ingested live microorganisms, bacteria or yeast with the host (probiotic effect) or indirectly as a result of ingestion of microbial metabolites produced during the fermentation process (biogenic effect). Although still far from fully understood, several probiotic mechanisms of action have been proposed, including competitive exclusion, competition for nutrients and/or stimulation of an immune response. The biogenic properties of fermented functional foods result from the microbial production of bioactive metabolites such as certain vitamins, bioactive peptides, organic acids or fatty acids during fermentation.

Animals↗

PubMed2005

The clpB gene of Bifidobacterium breve UCC 2003: transcriptional analysis and first insights into stress induction.

The so-called clp genes, which encode components of the Clp proteolytic complex, are widespread among bacteria. The Bifidobacterium breve UCC 2003 genome contains a clpB gene with significant homology to predicted clpB genes from other members of the Actinobacteridae group. The heat- and osmotic-inducibility of the B. breve UCC 2003 clpB homologue was verified by slot-blot analysis, while Northern blot and primer extension analyses showed that the clpB gene is transcribed as a monocistronic unit with a single promoter. The role of a hspR homologue, known to control the regulation of clpB and dnaK gene expression in other high G+C content bacteria was investigated by gel mobility shift assays. Moreover the predicted 3D structure of HspR provides further insight into the binding mode of this protein to the clpB promoter region, and highlights the key amino acid residues believed to be involved in the protein-DNA interaction.

Bacterial Proteins↗

PubMed2005

A dichotomous epigenetic mechanism governs expression of the LlaJI restriction/modification system.

The LlaJI restriction/modification (R/M) system is comprised of two 5mC MTase-encoding genes, llaJIM1 and llaJIM2, and two genes required for restriction activity, llaJIR1 and llaJIR2. Here, we report the molecular mechanism by which this R/M system is transcriptionally regulated. The recognition sequence for the LlaJI MTases was deduced to be 5'GACGC'3 for M1.LlaJI and 5'GCGTC'3 for M2.LlaJI, thus together constituting an asymmetric complementary recognition site. Two recognition sequences for both LlaJI MTases are present within the LlaJI promoter region, indicative of an epigenetic role. Following in vivo analysis of expression of the LlaJI promoter, we established that both LlaJI MTases were required for complete transcriptional repression. A mutational analysis and DNA binding studies of this promoter revealed that the methylation of two specific cytosines by M2.LlaJI within this region was required to trigger the specific and high affinity binding of M1.LlaJI, which serves to regulate expression of the LlaJI operon. This regulatory system therefore represents the amalgamation of an epigenetic stimulation coupled to the formation of a MTase/repressor:promoter complex.

Base Sequence↗

PubMed2005

Gene structure and transcriptional organization of the dnaK operon of Bifidobacterium breve UCC 2003 and application of the operon in bifidobacterial tracing.

The incorporation and delivery of bifidobacterial strains as probiotic components in many food preparations expose these microorganisms to a multitude of environmental insults, including heat and osmotic stresses. We characterized the dnaK gene region of Bifidobacterium breve UCC 2003. Sequence analysis of the dnaK locus revealed four genes with the organization dnaK-grpE-dnaJ-ORF1, whose deduced protein products display significant similarity to corresponding chaperones found in other bacteria. Northern hybridization and real-time LightCycler PCR analysis revealed that the transcription of the dnaK operon was strongly induced by osmotic shock but was not induced significantly by heat stress. A 4.4-kb polycistronic mRNA, which represented the transcript of the complete dnaK gene region, was detected. Many other small transcripts, which were assumed to have resulted from intensive processing or degradation of this polycistronic mRNA, were identified. The transcription start site of the dnaK operon was determined by primer extension. Phylogenetic analysis of the available bifidobacterial grpE and dnaK genes suggested that the evolutionary development of these genes has been similar. The phylogeny derived from the various bifidobacterial grpE and dnaK sequences is consistent with that derived from 16S rRNA. The use of these genes in bifidobacterial species as an alternative or complement to the 16S rRNA gene marker provides sequence signatures that allow a high level of discrimination between closely related species of this genus.

Adenosine Triphosphatases↗

PubMed2005

Genetic and transcriptional organization of the clpC locus in Bifidobacterium breve UCC 2003.

A homolog of the clpC ATPase gene was identified in the genome of Bifidobacterium breve UCC 2003. Since this gene is very well conserved among eubacteria, we employed a PCR-based approach using primers based on highly conserved regions of ClpC proteins in order to identify homologous genes in other bifidobacterial species. Analysis by slot blot, Northern blot, and primer extension experiments showed that transcription of clpC is induced in response to moderate heat shock regimes. Moreover, we identified in the genome sequence of B. breve UCC 2003 a gene, designated clgR, which is predicted to encode a transcriptional regulator involved in regulation of the bifidobacterial clpC gene. The role of this protein in the regulation of B. breve UCC 2003 clpC gene expression was investigated by performing gel retardation experiments. We show that a biologically active ClgR molecule requires one or more proteinaceous coactivators to assist in the specific binding of ClgR to the clpC promoter region.

Adenosine Triphosphatases↗

PubMed2005

Surface microflora of four smear-ripened cheeses.

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.

Bacteria↗

PubMed2005

Prophage-like elements in bifidobacteria: insights from genomics, transcription, integration, distribution, and phylogenetic analysis.

So far, there is only fragmentary and unconfirmed information on bacteriophages infecting the genus Bifidobacterium. In this report we analyzed three prophage-like elements that are present in the genomes of Bifidobacterium breve UCC 2003, Bifidobacterium longum NCC 2705, and Bifidobacterium longum DJO10A, designated Bbr-1, Bl-1, and Blj-1, respectively. These prophagelike elements exhibit homology with genes of double-stranded DNA bacteriophages spanning a broad phylogenetic range of host bacteria and are surprisingly closely related to bacteriophages infecting low-G+C bacteria. All three prophage-like elements are integrated in a tRNA(Met) gene, which appears to be reconstructed following phage integration. Analysis of the distribution of this integration site in many bifidobacterial species revealed that the attB sites are well conserved. The Blj-1 prophage is 36.9 kb long and was induced when a B. longum DJO10A culture was exposed to mitomycin C or hydrogen peroxide. The Bbr-1 prophage-like element appears to consist of a noninducible 28.5-kb chimeric DNA fragment composed of a composite mobile element inserted into prophage-like sequences, which do not appear to be widely distributed among B. breve strains. Northern blot analysis of the Bbr-1 prophage-like element showed that large parts of its genome are transcriptionally silent. Interestingly, a gene predicted to encode an extracellular beta-glucosidase carried within the Bbr-1 prophage-like element was shown to be transcribed.

Base Sequence↗

PubMed2005

Genetic characterization of the Bifidobacterium breve UCC 2003 hrcA locus.

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and transcriptional regulators, including the DnaJ and the HrcA proteins. Genome analysis of Bifidobacterium breve UCC 2003 revealed a second copy of a dnaJ gene, named dnaJ2, which is flanked by the hrcA gene in a genetic constellation that appears to be unique to the actinobacteria. Phylogenetic analysis using 53 bacterial dnaJ sequences, including both dnaJ1 and dnaJ2 sequences, suggests that these genes have followed a different evolutionary development. Furthermore, the B. breve UCC 2003 dnaJ2 gene seems to be regulated in a manner that is different from that of the previously characterized dnaJ1 gene. The dnaJ2 gene, which was shown to be part of a 2.3-kb bicistronic operon with hrcA, was induced by osmotic shock but not significantly by heat stress. This induction pattern is unlike those of other characterized dnaJ genes and may be indicative of a unique stress adaptation strategy by this commensal microorganism.

Amino Acid Sequence↗

PubMed2005

Transcriptional regulation and characterization of a novel beta-fructofuranosidase-encoding gene from Bifidobacterium breve UCC2003.

An operon involved in fructooligosaccharide breakdown was identified in the genome of Bifidobacterium breve UCC2003. This 2.6-kb transcriptional unit was comprised of three genes that encoded a putative permease, a conserved hypothetical protein, and a beta-fructofuranosidase. Active transcription of the operon was observed when B. breve UCC2003 was grown on sucrose or Actilight, while transcription appeared to be repressed when the organism was grown on glucose, fructose, a combination of glucose and sucrose, or a combination of fructose and sucrose. The beta-fructofuranosidase encoded by this operon was purified and biochemically characterized. The optimum pH and temperature for catalytic activity were determined to be pH 6.0 and 37 degrees C, respectively, and there was a dependence on bivalent cations, particularly manganese. The Km and Vmax values for sucrose hydrolysis were determined to be 25 +/- 2 mM and 24 +/- 3 micromol min(-1) mg(-1), respectively. Interestingly, the enzyme was shown to specifically catalyze cleavage of the beta(2-1) glycosidic bond between glucose and its neighboring fructose moiety in sucrose and other fructooligosaccharides with a relatively low degree of polymerization, and there was no detectable activity towards the beta(2-1) glycosidic bond between two fructose moieties within the same substrate. To our knowledge, such an enzymatic activity has not previously been described in bifidobacteria or other gram-positive bacteria.

Bacterial Proteins↗

PubMed2005

Insertional inactivation of determinants for Mg2+ and Co2+ transport as a tool for screening recombinant Lactococcus species clones.

Insertional inactivation of the plasmid-encoded determinants for Mg(2+) and Co(2+) transport, orf18/corA, provides a tool for screening recombinant clones in Lactococcus, based on the observation that overexpression of orf18/corA results in cell growth inhibition on certain concentrations of CoCl(2). The lacticin 3147 immunity gene, ltnI, was used to insertionally inactivate orf18/corA. The resulting clones were capable of growth on concentrations of CoCl(2) that were inhibitory to the parent strain. Since only 3 of 17 lactococcal starters naturally harbor corA, the system has potential as a screen for selecting recombinant lactococcal clones.

Bacterial Proteins↗

PubMed2005

The ClgR protein regulates transcription of the clpP operon in Bifidobacterium breve UCC 2003.

Five clp genes (clpC, clpB, clpP1, clpP2, and clpX), representing chaperone- and protease-encoding genes, were previously identified in Bifidobacterium breve UCC 2003. In the present study, we characterize the B. breve UCC 2003 clpP locus, which consists of two paralogous genes, designated clpP1 and clpP2, whose deduced protein products display significant similarity to characterized ClpP peptidases. Transcriptional analyses showed that the clpP1 and clpP2 genes are transcribed in response to moderate heat shock as a bicistronic unit with a single promoter. The role of a clgR homologue, known to control the regulation of clpP gene expression in Streptomyces lividans and Corynebacterium glutamicum, was investigated by gel mobility shift assays and DNase I footprint experiments. We show that ClgR, which in its purified form appears to exist as a dimer, requires a proteinaceous cofactor to assist in specific binding to a 30-bp region of the clpP promoter region. In pull-down experiments, a 56-kDa protein copurified with ClgR, providing evidence that the two proteins also interact in vivo and that the copurified protein represents the cofactor required for ClgR activity. The prediction of the ClgR three-dimensional structure provides further insights into the binding mode of this protein to the clpP1 promoter region and highlights the key amino acid residues believed to be involved in the protein-DNA interaction.

Adaptation, Physiological↗

PubMed2004

Molecular and transcriptional analysis of the temperate lactococcal bacteriophage Tuc2009.

The genome of bacteriophage Tuc2009 consists of 38347 base pairs on which 57 open reading frames (ORFs) were identified, divided in two oppositely transcribed regions. The leftward-transcribed region harbors three ORFs, two of which are involved in the establishment of lysogeny. The rightward-transcribed region contains 54 ORFs, which are assumed to be required for the lytic life cycle. An exception to the above organization is ORF 10, of unknown function, located within the rightward-transcribed region that has an orientation opposite to the ORFs surrounding it. Transcriptional analysis of the Tuc2009 genome following infection of a sensitive host revealed that most ORFs are transcribed in a sequential manner. ORFs that are presumed to form (part of) the genetic switch along with the superinfection exclusion-encoding gene are transcribed immediately after infection, followed by transcription of the presumed replication region. Subsequent to this, several small transcripts could be identified followed by a single 24-kb transcript. This latter transcript was shown to specify most of the identified structural proteins as well as two proteins required for host lysis. Interestingly, the 24-kb mRNA was shown to undergo splicing through the activity of a type I intron whose removal from the mRNA resulted in the formation of an ORF specifying a major structural protein. Primer extension analysis was employed to identify the 5' ends of mRNA transcripts and the genome and transcriptional data are discussed in relation to other lactococcal bacteriophages.

Bacteriophages↗

PubMed2004

The impact of bacteriophage genomics.

The discovery of (bacterio)phages revolutionised microbiology and genetics, while phage research has been integral to answering some of the most fundamental biological questions of the twentieth century. The susceptibility of bacteria to bacteriophage attack can be undesirable in some cases, especially in the dairy industry, but can be desirable in others, for example, the use of bacteriophage therapy to eliminate pathogenic bacteria. The relative ease with which entire bacteriophage genome sequences can now be elucidated has had a profound impact on the study of these bacterial parasites.

Animals↗

PubMed2004

Insights into the taxonomy, genetics and physiology of bifidobacteria.

Despite the generally accepted importance of bifidobacteria as probiotic components of the human intestinal microflora and their use in health promoting foods, there is only limited information about their phylogenetic position, physiology and underlying genetics. In the last few years numerous molecular approaches have emerged for the identification and characterization of bifidobacterial strains. Their use, in conjunction with traditional culturing methods, has led to a polyphasic taxonomy which has significantly enhanced our knowledge of the role played by these bacteria in the human intestinal ecosystem. The recent adaptation of culture-independent molecular tools to the fingerprinting of intestinal and food communities offers an exciting opportunity for revealing a more detailed picture of the true complexity of these environments. Furthermore, the availability of bifidobacterial genome sequences has advanced knowledge on the genetics of bifidobacteria and the effects of their metabolic activities on the intestinal ecosystem. The release of a complete Bifidobacterium longum genome sequence and the recent initiative to sequence additional strains are expected to open up a new era of comparative genomics in bifidobacterial biology. Moreover, the use of genomotyping allows a global comparative analysis of gene content between different bifidobacterial isolates of a given species without the necessity of sequencing many strains. Genomotyping provides useful information about the degree of relatedness among various strains of Bifidobacterium species and consequently can be used in a polyphasic identification approach. This review will deal mainly with the molecular tools described for bifidobacterial identification and the first insights into the underlying genetics involved in bifidobacterial physiology as well as genome variability.

Animals↗

PubMed2004

Bifidobacterium psychraerophilum sp. nov. and Aeriscardovia aeriphila gen. nov., sp. nov., isolated from a porcine caecum.

In a previous study that was based primarily on 16S rDNA sequencing, two groups of bifidobacteria that had been recovered from a pig caecum were proposed to belong to two novel species, termed 'Bifidobacterium pyschroaerophilum' and 'Bifidobacterium aerophilum'. In this study, based on DNA G+C content and partial heat-shock protein 60 (HSP60) gene sequences, the assignment of 'B. pyschroaerophilum', corrected to Bifidobacterium pyschraerophilum, to the genus Bifidobacterium was confirmed. The DNA G+C content of 'B. aerophilum' was relatively low, which was consistent with its segregation into subcluster II of the 16S rDNA phylogenetic tree. Based on partial 16S rDNA and HSP60 gene sequences, the species was transferred to a novel genus and reclassified as Aeriscardovia aeriphila gen. nov., sp. nov. Biochemical profiles and growth parameters were established for both novel species. Interestingly, each had a high tolerance to oxygen and grew on agar media under aerobic conditions, a trait that may relate to their caecal habitat. Under aerobic growth conditions, the short-rod morphology of A. aeriphila lengthened considerably. This appeared to arise from incomplete cell division. In addition, B. pyschraerophilum was unusual in that it grew at temperatures as low as 4 degrees C. On the basis of genetic, phylogenetic and phenotypic data, the identities of Bifidobacterium pyschraerophilum sp. nov. (type strain, T16(T)=LMG 21775(T)=NCIMB 13940(T)) and Aeriscardovia aeriphila gen. nov., sp. nov. (type strain, T6(T)=LMG 21773(T)=NCIMB 13939(T)) are confirmed.

Actinobacteria↗

PubMed2004

Variable bacteriocin production in the commercial starter Lactococcus lactis DPC4275 is linked to the formation of the cointegrate plasmid pMRC02.

Lactococcus lactis DPC4275 is a bacteriocin-producing transconjugant of the industrial starter strain DPC4268. Strain DPC4275 was generated through conjugal transfer by mating DPC4268 with L. lactis MG1363 containing the 60-kb plasmid pMRC01, which encodes the genetic determinants for the lantibiotic lacticin 3147 and for a phage resistance mechanism of the abortive infection type. The many significant applications of this strain prompted a genetic analysis of its apparently unstable bacteriocin-producing phenotype. Increased levels of lacticin 3147 produced by DPC4275 were associated with the appearance of an 80-kb plasmid, designated pMRC02, which was derived from DNA originating from pMRC01 (60 kb) and a resident DPC4268 proteinase plasmid, pMT60 (60 kb). Indeed, pMRC02 was shown to be derived from the insertion of a 17-kb fragment of pMRC01, encompassing the lacticin 3147 operon, into pMT60. The presence of pMRC02 at a high copy number was found to correlate with increased levels of lacticin 3147 in DPC4275 compared to the wild-type containing pMRC01. Subsequent transfer of pMRC02 into the plasmid-free strain MG1363 by electroporation allowed a direct phenotypic comparison with pMRC01, also studied in the MG1363 background. Plasmid pMRC02 displayed phage resistance similar to that by pMRC01, although it was less potent, as demonstrated by a larger plaque size for phage c2 infection of MG1363(pMRC02). While this locus is flanked by IS946 elements, the sequencing of pMT60-pMRC01 junction sites established that this event was unlikely to be insertion sequence mediated and most probably occurred by homologous recombination followed by deletion of most of pMRC01. This was not a random occurrence, as nine other transconjugants investigated were found to have the same junction sites. Such derivatives of commercial strains producing increased levels of bacteriocin could be exploited as protection cultures for food applications.

Bacterial Proteins↗