Biomedical subjects

Gerald F Fitzgerald

Publications and source records attributed to Gerald F Fitzgerald.

At least 19 recordsLinked to original sources

PubMed2006

Identification and characterization of a fructose phosphotransferase system in Bifidobacterium breve UCC2003.

In silico analysis of the Bifidobacterium breve UCC2003 genome allowed identification of four genetic loci, each of which specifies a putative enzyme II (EII) protein of a phosphoenolpyruvate:sugar phosphotransferase system. The EII encoded by fruA, a clear homologue of the unique EIIBCA enzyme encoded by the Bifidobacterium longum NCC2705 genome, was studied in more detail. The fruA gene is part of an operon which contains fruT, which is predicted to encode a homologue of the Bacillus subtilis antiterminator LicT. Transcriptional analysis showed that the fru operon is induced by fructose. The genetic structure, complementation studies, and the observed transcription pattern of the fru operon suggest that the EII encoded in B. breve is involved in fructose transport and that its expression is controlled by an antiterminator mechanism. Biochemical studies unequivocally demonstrated that FruA phosphorylates fructose at the C-6 position.

Amino Acid Sequence↗

PubMed2006

Genomics as a means to understand bacterial phylogeny and ecological adaptation: the case of bifidobacteria.

The field of microbiology has in recent years been transformed by the ever increasing number of publicly available whole-genome sequences. This sequence information has significantly enhanced our understanding of the physiology, genetics and evolutionary development of bacteria. Among the latter group of microorganisms, bifidobacteria represent important human commensals because of their perceived contribution to maintaining a balanced gastrointestinal tract microbiota. In recent years bifidobacteria have drawn much scientific attention because of their use as live bacteria in numerous food preparations with various health-related claims. For this reason, these bacteria constitute a growing area of interest with respect to genomics, molecular biology and genetics. Recent genome sequencing of a number of bifidobacterial species has allowed access to the complete genetic make-up of these bacteria. In this review we will discuss how genomic data has allowed us to understand bifidobacterial evolution, while also revealing genetic functions that explains their presence in the particular ecological environment of the gastrointestinal tract.

Acclimatization↗

PubMed2006

Growth and colour development of some surface ripening bacteria with Debaryomyces hansenii on aseptic cheese curd.

The growth of five bacteria isolated from red-smear cheeses, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variabile, Microbacterium gubbeenense and Staphylococcus saprophyticus in mixed cultures with Debaryomyces hansenii on aseptic model cheese curd at 10 and 14 degrees C was investigated. At both temperatures, C. casei and Micro. gubbeenense had a longer lag phase than C. variabile, Brevi. aurantiacum and Staph. saprophyticus. In all cultures, lactose was utilised first and was consumed more rapidly at 14 degrees C than at 10 degrees C, i.e., 6 d at 14 degrees C and 10 d at 10 degrees C. This utilisation coincided with the exponential growth of Deb. hansenii on the cheese surface. Lactate was also used as a carbon source and was totally consumed after 21 d at 14 degrees C and approximately 90% was consumed after 21 d at 10 degrees C regardless of the ripening culture. Small differences (<0.5 pH unit) in the surface-pH during ripening were noticeable between ripening cultures. Differences in the colour development of the mixed cultures with the yeast control were only noticeable after 15 d for Brevi. aurantiacum and after 21 d for the other bacteria. Regardless of the organisms tested, colour development and colour intensity were also greater at 14 degrees C than at 10 degrees C. This study has provided useful information on the growth and contribution to colour development of these bacteria on cheese.

Brevibacterium↗

PubMed2006

A genetic dissection of the LlaJI restriction cassette reveals insights on a novel bacteriophage resistance system.

BACKGROUND: Restriction/modification systems provide the dual function of protecting host DNA against restriction by methylation of appropriate bases within their recognition sequences, and restriction of foreign invading un-methylated DNA, such as promiscuous plasmids or infecting bacteriphage. The plasmid-encoded LlaJI restriction/modification system from Lactococcus lactis recognizes an asymmetric, complementary DNA sequence, consisting of 5'GACGC'3 in one strand and 5'GCGTC'3 in the other and provides a prodigious barrier to bacteriophage infection. LlaJI is comprised of four similarly oriented genes, encoding two 5mC-MTases (M1.LlaJI and M2.LlaJI) and two subunits responsible for restriction activity (R1.LlaJI and R2.LlaJI). Here we employ a detailed genetic analysis of the LlaJI restriction determinants in an attempt to characterize mechanistic features of this unusual hetero-oligomeric endonuclease. RESULTS: Detailed bioinformatics analysis confirmed the presence of a conserved GTP binding and hydrolysis domain within the C-terminal half of the R1.LlaJI amino acid sequence whilst the N-terminal half appeared to be entirely unique. This domain architecture was homologous with that of the "B" subunit of the GTP-dependent, methyl-specific McrBC endonuclease from E.coli K-12. R1.LlaJI did not appear to contain a catalytic centre, whereas this conserved motif; PD....D/EXK, was clearly identified within the amino acid sequence for R2.LlaJI. Both R1.LlaJI and R2.LlaJI were found to be absolutely required for detectable LlaJI activity in vivo. The LlaJI restriction subunits were purified and examined in vitro, which allowed the assignment of R1.LlaJI as the sole specificity determining subunit, whilst R2.LlaJI is believed to mediate DNA cleavage. CONCLUSION: The hetero-subunit structure of LlaJI, wherein one subunit mediates DNA binding whilst the other subunit is predicted to catalyze strand hydrolysis distinguishes LlaJI from previously characterized restriction-modification systems. Furthermore, this distinction is accentuated by the fact that whilst LlaJI behaves as a conventional Type IIA system in vivo, in that it restricts un-methylated DNA, it resembles the Type IV McrBC endonuclease, an enzyme specific for methylated DNA. A number of similar restriction determinants were identified in the database and it is likely LlaJI together with these homologous systems, comprise a new subtype of the Type II class incorporating features of Type II and Type IV systems.

Amino Acid Sequence↗

PubMed2006

Multireplicon genome architecture of Lactobacillus salivarius.

Lactobacillus salivarius subsp. salivarius strain UCC118 is a bacteriocin-producing strain with probiotic characteristics. The 2.13-Mb genome was shown by sequencing to comprise a 1.83 Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids. Megaplasmids previously have not been characterized in lactic acid bacteria or intestinal lactobacilli. Annotation of the genome sequence indicated an intermediate level of auxotrophy compared with other sequenced lactobacilli. No single-copy essential genes were located on the megaplasmid. However, contingency amino acid metabolism genes and carbohydrate utilization genes, including two genes for completion of the pentose phosphate pathway, were megaplasmid encoded. The megaplasmid also harbored genes for the Abp118 bacteriocin, a bile salt hydrolase, a presumptive conjugation locus, and other genes potentially relevant for probiotic properties. Two subspecies of L. salivarius are recognized, salivarius and salicinius, and we detected megaplasmids in both subspecies by pulsed-field gel electrophoresis of sizes ranging from 100 kb to 380 kb. The discovery of megaplasmids of widely varying size in L. salivarius suggests a possible mechanism for genome expansion or contraction to adapt to different environments.

Amino Acids↗

PubMed2006

Characterization of the lytic-lysogenic switch of the lactococcal bacteriophage Tuc2009.

Tuc2009 is a temperate bacteriophage of Lactococcus lactis subsp. cremoris UC509 which encodes a CI- and Cro-type lysogenic-lytic switch region. A helix-swap of the alpha3 helices of the closely related CI-type proteins from the lactococcal phages r1t and Tuc2009 revealed the crucial elements involved in DNA recognition while also pointing to conserved functional properties of phage CI proteins infecting different hosts. CI-type proteins have been shown to bind to specific sequences located in the intergenic switch region, but to date, no detailed binding studies have been performed on lactococcal Cro analogues. Experiments shown here demonstrate alternative binding sites for these two proteins of Tuc2009. CI2009 binds to three inverted repeats, two within the intergenic region and one within the cro2009 gene. This DNA-binding pattern appears to be conserved among repressors of lactococcal and streptococcal phages. The Cro2009 protein appears to bind to three direct repeats within the intergenic region causing distortion of the bound DNA.

DNA, Viral↗

PubMed2006

Analysis of bifidobacterial evolution using a multilocus approach.

Bifidobacteria represent one of the most numerous groups of bacteria found in the gastrointestinal tract of humans and animals. In man, gastrointestinal bifidobacteria are associated with health effects and for this reason they are often used as functional ingredients in food and pharmaceutical products. Such applications may benefit from or require a clear and reliable bifidobacterial species identification. The increasing number of available bacterial genome sequences has provided a large amount of housekeeping gene sequences that can be used both for identification of bifidobacterial species as well as for understanding bifidobacterial evolution. In order to assess their relative positions in the evolutionary process, fragments from seven conserved genes, clpC, dnaB, dnaG, dnaJ1, purF, rpoC and xfp, were sequenced from each of the currently described type strains of the genus Bifidobacterium. The results demonstrate that the concatenation of these seven gene sequences for phylogenetic purposes allows a significant increase in the discriminatory power between taxa.

Animals↗

PubMed2006

Polyphasic analysis indicates that Lactobacillus salivarius subsp. salivarius and Lactobacillus salivarius subsp. salicinius do not merit separate subspecies status.

Lactobacillus salivarius Rogosa et al. 1953 was described as a homofermentative lactobacillus with two varieties: salivarius, typified inter alia by the ability to ferment rhamnose, and salicinius, characterized by the ability to ferment the glucoside salicin. These varieties have become accepted as subspecies divisions. We have examined the relatedness of 32 L. salivarius strains by a polyphasic approach. Carbohydrate fermentation profile analysis did not support clear distinction of the two subspecies. L. salivarius UCC118 was shown to be facultatively heterofermentative, confirming in silico genome analysis. 16S rRNA gene sequences and 16S-23S rRNA intergenic spacer region sequences provided no discrimination between any of the strains or subspecies. Broad subdivisions were distinguishable by pulsed-field gel genomic digest patterns, but they did not allow subspecific or phenotypic distinctions. A phylogeny based upon groEL gene sequences was discordant with rhamnose or salicin fermentation data for many taxa, and no reliable phenotypic correlations could be established. In the absence of meaningful taxonomic criteria, we therefore propose that Lactobacillus salivarius comprises a single species with no infraspecific taxa. Based on the present study and literature data, an emended description of the species Lactobacillus salivarius is provided.

Aged↗

PubMed2006

Diversity of the genus Lactobacillus revealed by comparative genomics of five species.

The genus Lactobacillus contains over 80 recognized species, and is characterized by a high level of diversity, reflected in its complex phylogeny. The authors' recent determination of the genome sequence of Lactobacillus salivarius means that five complete genomes of Lactobacillus species are available for comparative genomics: L. salivarius, L. plantarum, L. acidophilus, L. johnsonii and L. sakei. This paper now shows that there is no extensive synteny of the genome sequences of these five lactobacilli. Phylogeny based on whole-genome alignments suggested that L. salivarius was closer to L. plantarum than to L. sakei, which was closest to Enterococcus faecalis, in contrast to 16S rRNA gene relatedness. A total of 593 orthologues common to all five species were identified. Species relatedness based on this protein set was largely concordant with genome synteny-based relatedness. A Lactobacillus supertree, combining individual phylogenetic trees from each of 354 core proteins, had four main branches, comprising L. salivarius-L. plantarum; L. sakei; E. faecalis; and L. acidophilus-L. johnsonii. The extreme divergence of the Lactobacillus genomes analysed supports the recognition of new subgeneric divisions.

Genes, Bacterial↗

PubMed2006

Sequence and comparative genomic analysis of lactococcal bacteriophages jj50, 712 and P008: evolutionary insights into the 936 phage species.

The complete genome sequences of three lactococcal 936-type bacteriophages, 712, jj50 and P008, were determined. Comparative genomic analysis of these phages with the previously sequenced 936-type phages, sk1 and bIL170, reveals a strict conservation of the overall genetic organization of this geographically diverse phage group. Genetic divergence was mainly observed in the early expressed region of the phage genomes, where a number of deletions, exchanges and insertions appear to have occurred. These genetic differences may be responsible for the observed differential sensitivity to the lactococcal DNA injection blocking protein, Sie(2009), and the abortive infection system, AbiA.

Bacterial Proteins↗

PubMed2006

Plasmids of lactococci - genetic accessories or genetic necessities?

Lactococci are one of the most exploited microorganisms used in the manufacture of food. These intensively used cultures are generally characterized by having a rich plasmid complement. It could be argued that it is the plasmid complement of commercially utilized cultures that gives them their technical superiority and individuality. Consequently, it is timely to reflect on the desirable characteristics encoded on lactococcal plasmids. It is argued that plasmids play a key role in the evolution of modern starter strains and are a lot more than just selfish replicosomes but more essential necessities of intensively used commercial starters. Moreover, the study of plasmid biology provides a genetic blueprint that has proved essential for the generation of molecular tools for the genetic improvement of Lactococcus lactis.

Genetic Markers↗

PubMed2006

How high G+C Gram-positive bacteria and in particular bifidobacteria cope with heat stress: protein players and regulators.

The Actinobacteridae group of bacteria includes pathogens, plant commensals, endosymbionts as well as inhabitants of the gastrointestinal tract. For various reasons, these microorganisms represent a growing area of interest with respect to genomics, molecular biology and genetics. This review will discuss the current knowledge on the molecular players that allow actinobacteria to contend with heat stress, with an emphasis on bifidobacteria. We describe the principal molecular chaperones involved in heat stress. Temporal expression of heat-shock genes based on functional genomics in members of the Actinobacteridae group is also discussed, as well as the emerging molecular mechanisms controlling the heat-stress response.

Actinobacteria↗

PubMed2006

Screening for and identification of starch-, amylopectin-, and pullulan-degrading activities in bifidobacterial strains.

Forty-two bifidobacterial strains were screened for alpha-amylase and/or pullulanase activity by investigating their capacities to utilize starch, amylopectin, or pullulan. Of the 42 bifidobacterial strains tested, 19 were capable of degrading potato starch. Of these 19 strains, 11 were able to degrade starch and amylopectin, as well as pullulan. These 11 strains, which were shown to produce extracellular starch-degrading activities, included 5 strains of Bifidobacterium breve, 1 B. dentium strain, 1 B. infantis strain, 3 strains of B. pseudolongum, and 1 strain of B. thermophilum. Quantitative and qualitative enzyme activities were determined by measuring the concentrations of released reducing sugars and by high-performance thin-layer chromatography, respectively. These analyses confirmed both the inducible nature and the extracellular nature of the starch- and pullulan-degrading enzyme activities and showed that the five B. breve strains produced an activity that is consistent with type II pullulanase (amylopullulanase) activity, while the remaining six strains produced an activity with properties that resemble those of type III pullulan hydrolase.

Amino Acid Sequence↗

PubMed2006

Comparative and functional analysis of sortase-dependent proteins in the predicted secretome of Lactobacillus salivarius UCC118.

Surface proteins are important factors in the interaction of probiotic and pathogenic bacteria with their environment or host. We performed a comparative bioinformatic analysis of four publicly available Lactobacillus genomes and the genome of Lactobacillus salivarius subsp. salivarius strain UCC118 to identify secreted proteins and those linked to the cell wall. Proteins were identified which were predicted to be anchored by WXL-binding domains, N- or C-terminal anchors, GW repeats, lipoprotein anchors, or LysM-binding domains. We identified 10 sortase-dependent surface proteins in L. salivarius UCC118, including three which are homologous to mucus-binding proteins (LSL_0152, LSL_0311, and LSL_1335), a collagen-binding protein homologue (LSL_2020b), two hypothetical proteins (LSL_1838 and LSL_1902b), an enterococcal surface protein homologue (LSL_1085), a salivary agglutinin-binding homologue (LSL_1832b), an epithelial binding protein homologue (LSL_1319), and a proteinase homologue (LSL_1774b). However, two of the genes are gene fragments and four are pseudogenes, suggesting a lack of selection for their function. Two of the 10 genes were not transcribed in vitro, and 1 gene showed a 10-fold increase in transcript level in stationary phase compared to logarithmic phase. The sortase gene was deleted, and three genes encoding sortase-dependent proteins were disrupted. The sortase mutant and one sortase-dependent protein (mucus-binding homologue) mutant showed a significant reduction in adherence to human epithelial cell lines. The genome-wide investigation of surface proteins can thus help our understanding of their roles in host interaction.

Aminoacyltransferases↗

PubMed2006

Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118.

Given the increasing commercial and clinical relevance of probiotic cultures, improving the technological robustness of what are often process-sensitive cultures is an important biological goal. The nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strain Lactobacillus salivarius UCC118. Following nisin induction, strains expressing betL exhibited a significant increase in resistance to several stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray- and freeze-drying. The ability to confer additional stress tolerance on a probiotic culture may be an important step in delivering viable cultures for maximal efficacy.

Anti-Bacterial Agents↗

PubMed2006

Comparative genomics and transcriptional analysis of prophages identified in the genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei.

Lactobacillus gasseri ATCC 33323, Lactobacillus salivarius subsp. salivarius UCC 118, and Lactobacillus casei ATCC 334 contain one (LgaI), four (Sal1, Sal2, Sal3, Sal4), and one (Lca1) distinguishable prophage sequences, respectively. Sequence analysis revealed that LgaI, Lca1, Sal1, and Sal2 prophages belong to the group of Sfi11-like pac site and cos site Siphoviridae, respectively. Phylogenetic investigation of these newly described prophage sequences revealed that they have not followed an evolutionary development similar to that of their bacterial hosts and that they show a high degree of diversity, even within a species. The attachment sites were determined for all these prophage elements; LgaI as well as Sal1 integrates in tRNA genes, while prophage Sal2 integrates in a predicted arginino-succinate lyase-encoding gene. In contrast, Lca1 and the Sal3 and Sal4 prophage remnants are integrated in noncoding regions in the L. casei ATCC 334 and L. salivarius UCC 118 genomes. Northern analysis showed that large parts of the prophage genomes are transcriptionally silent and that transcription is limited to genome segments located near the attachment site. Finally, pulsed-field gel electrophoresis followed by Southern blot hybridization with specific prophage probes indicates that these prophage sequences are narrowly distributed within lactobacilli.

Animals↗

PubMed2006

Anatomy of a lactococcal phage tail.

Bacteriophages of the Siphoviridae family utilize a long noncontractile tail to recognize, adsorb to, and inject DNA into their bacterial host. The tail anatomy of the archetypal Siphoviridae lambda has been well studied, in contrast to phages infecting gram-positive bacteria. This report outlines a detailed anatomical description of a typical member of the Siphoviridae infecting a gram-positive bacterium. The tail superstructure of the lactococcal phage Tuc2009 was investigated using N-terminal protein sequencing, Western blotting, and immunogold transmission electron microscopy, allowing a tangible path to be followed from gene sequence through encoded protein to specific architectural structures on the Tuc2009 virion. This phage displays a striking parity with lambda with respect to tail structure, which reenforced a model proposed for Tuc2009 tail architecture. Furthermore, comparisons with lambda and other lactococcal phages allowed the specification of a number of genetic submodules likely to encode specific tail structures.

Electrophoresis, Polyacrylamide Gel↗

PubMed2006

Sequence analysis of the lactococcal plasmid pNP40: a mobile replicon for coping with environmental hazards.

The conjugative lactococcal plasmid pNP40, identified in Lactococcus lactis subsp. diacetylactis DRC3, possesses a potent complement of bacteriophage resistance systems, which has stimulated its application as a fitness-improving, food-grade genetic element for industrial starter cultures. The complete sequence of this plasmid allowed the mapping of previously known functions including replication, conjugation, bacteriocin resistance, heavy metal tolerance, and bacteriophage resistance. In addition, functions for cold shock adaptation and DNA damage repair were identified, further confirming pNP40's contribution to environmental stress protection. A plasmid cointegration event appears to have been part of the evolution of pNP40, resulting in a "stockpiling" of bacteriophage resistance systems.

Adaptation, Physiological↗