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A microarray method was developed for the detection of 40 bacterial species reported in the literature to be predominant in the human gastrointestinal tract. The 40 species include seven species each of Bacteroides and Clostridium, six species of Ruminococcus, five species of Bifidobacterium, four species of Eubacterium, two species each of Fusobacterium, Lactobacillus and Enterococcus, and single species each of Collinsella, Eggerthella, Escherichia, Faecalibacterium and Finegoldia. Three 40-mer oligos specific for each bacterial species were designed based on comparison of the 16S rDNA sequences available in the GenBank database, and were used to make the DNA-array on epoxy slides. Using two universal primers, the 16S rRNA gene from bacteria present in fecal samples were amplified and labeled with Cyanine5-dCTP by PCR, and then hybridized to the DNA-array. After resolving some difficulties caused by sequence conflicts in GenBank and inaccurate reference strains, all 40 bacterial reference species gave positive results. The microarray method was used to screen fecal samples obtained from 11 healthy human volunteers for the presence of these intestinal bacteria. The results indicated that 25-37 of the 40 species could be detected in each fecal sample and that 33 of the species were found in a majority of the samples.
Mol Cell Probes 2004 Aug
PMID:DNA microarray analysis of predominant human intestinal bacteria in fecal samples. 1527 82

Two families of genes related to, and including, rolling circle replication initiator protein (Rep) genes were defined by sequence similarity and by evidence of intergene family recombination. The Rep genes of circoviruses were the best characterized members of the "RecRep1 family." Other members of the RecRep1 family were Rep-like genes found in the genomes of the Canarypox virus, Entamoeba histolytica, and Giardia duodenalis and in a plasmid, p4M, from the Gram-positive bacterium, Bifidobacterium pseudocatenulatum. The "RecRep2 family" comprised some previously identified Rep-like genes from plasmids of phytoplasmas and similar Rep-like genes from the genomes of Lactobacillus acidophilus, Lactococcus lactis, and Phytoplasma asteris. Both RecRep1 and RecRep2 proteins have a nucleotide-binding domain significantly similar to the helicases (2C proteins) of picorna-like viruses. On the N-terminal side of the nucleotide binding domain, RecRep1 proteins have a domain significantly similar to one found in nanovirus Reps, whereas RecRep2 proteins have a domain significantly similar to one in the Reps of pLS1 plasmids. We speculate that RecRep genes have been transferred from viruses or plasmids to parasitic protozoan and bacterial genomes and that Rep proteins were themselves involved in the original recombination events that generated the ancestral RecRep genes.
Mol Biol Evol 2006 Jun
PMID:Two families of rep-like genes that probably originated by interspecies recombination are represented in viral, plasmid, bacterial, and parasitic protozoan genomes. 1653 8

A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.
Mol Cell Proteomics 2006 Jun
PMID:A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705. 1654 25

Competitive exclusion treatment is able to increase the pathogen colonization resistance of day-old chicks by applying probiotic bacteria stabilizing the indigenous microflora. In order to develop a safe microbial feed additive, various bacterial strains were isolated out of the gastrointestinal tract of healthy chickens. One hundred twenty-one representatives were selected based on differences in whole-cell protein patterns and screened for antagonistic properties. Five effective strains (Pediococcus acidilactici, Enterococcus faecium, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, and Lactobacillus salivarius ssp. salivarius) exhibited in vitro the ability to inhibit a range of common pathogens and were evaluated with regard to the risks associated with genetic transfer of antibiotic resistances from animals to humans via the food chain. The probiotic strains were sensitive to several clinically effective antibiotics, though some of them showed single resistances. None of the vancomycin-resistant (R) strains carried the enterococcal vanA gene. Two tetracycline R strains were shown to harbor a tet(M)-associated resistance. The strains contained no extrachromosomal DNA and were not able to transfer the resistance by means of conjugation. On basis of the collected data the presence of easy transferable resistances was excluded and the chicken strains were considered to be suitable for the use as feed additive.
Mol Nutr Food Res 2006 May
PMID:Development of a competitive exclusion product for poultry meeting the regulatory requirements for registration in the European Union. 1667 74

The chlamydiae are important human and animal pathogens which form a phylogentically distinct lineage within the Bacteria. There is evidence that some genes in these obligate intracellular parasites have undergone lateral exchange with other free-living organisms. In the present work, we describe two interesting cases of lateral gene transfer between chlamydiae and actinobacteria, which have been identified based on the shared presence of conserved inserts in two important proteins. In the enzyme serine hydroxymethyltransferase (SHMT or GlyA protein), which links amino acid and nucleotide metabolisms by generating the key intermediate for one-carbon transfer reactions, two conserved inserts of 3 and 31 amino acids (aa) are uniquely present in various chlamydiae species as well as in a subset of Actinobacteria and in the Treponema species. Similarly, in the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which is involved in the synthesis of cell wall peptidoglycan, a 16-aa conserved insert is specifically present in various sequenced chlamydiae and a subset of actinobacteria (i.e., Streptomyces, Actinomyces, Tropheryma, Bifidobacterium, Leifsonia, Arthrobacter, and Brevibacterium). To determine the phylogenetic depths of the GlyA and MurA inserts, the fragments of these genes from two chlamydiae-like species, Simkania negevensis and Waddlia chondrophila, were PCR amplified and sequenced. The presence of the corresponding inserts in both these species strongly indicates that these inserts are distinctive characteristics of the Chlamydiales order. In phylogenetic trees based on GlyA and MurA protein sequences, the chlamydiae species (and also the Treponema species in the case of GlyA) branched with a high affinity with various insert-containing actinobacteria within a clade of other actinobacteria. These results provide strong evidence that the shared presence of these indels in these bacteria is very likely a consequence of ancient lateral gene transfers from actinobacteria to chlamydiae. Pairwise sequence identity and the branching pattern of the GlyA homologues in the phylogenetic tree indicates that the glyA gene was initially transferred from an actinobacteria to an ancestor of the Treponema genus and from there it was acquired by the common ancestor of the Chlamydiales.
J Mol Evol 2006 Aug
PMID:Lateral transfers of serine hydroxymethyltransferase (glyA) and UDP-N-acetylglucosamine enolpyruvyl transferase (murA) genes from free-living Actinobacteria to the parasitic chlamydiae. 1683 93

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.
J Mol Microbiol Biotechnol 2007
PMID:Sugar transport systems of Bifidobacterium longum NCC2705. 1718 7

Actinobacteria constitute one of the largest phyla among bacteria and represent gram-positive bacteria with a high G+C content in their DNA. This bacterial group includes microorganisms exhibiting a wide spectrum of morphologies, from coccoid to fragmenting hyphal forms, as well as possessing highly variable physiological and metabolic properties. Furthermore, Actinobacteria members have adopted different lifestyles, and can be pathogens (e.g., Corynebacterium, Mycobacterium, Nocardia, Tropheryma, and Propionibacterium), soil inhabitants (Streptomyces), plant commensals (Leifsonia), or gastrointestinal commensals (Bifidobacterium). The divergence of Actinobacteria from other bacteria is ancient, making it impossible to identify the phylogenetically closest bacterial group to Actinobacteria. Genome sequence analysis has revolutionized every aspect of bacterial biology by enhancing the understanding of the genetics, physiology, and evolutionary development of bacteria. Various actinobacterial genomes have been sequenced, revealing a wide genomic heterogeneity probably as a reflection of their biodiversity. This review provides an account of the recent explosion of actinobacterial genomics data and an attempt to place this in a biological and evolutionary context.
Microbiol Mol Biol Rev 2007 Sep
PMID:Genomics of Actinobacteria: tracing the evolutionary history of an ancient phylum. 1780 69

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.
J Mol Microbiol Biotechnol 2008
PMID:Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria. 1795 5

Extracellular proteins of Bifidobacterium longum may mediate important interactions with the host. Here, we report on a comprehensive analysis of such proteins by using protein-free culture conditions and two-dimensional gel electrophoresis followed by mass spectrometry for protein identification. Seventeen proteins were detected in the culture supernatant, and 14 of them could be identified. Among these were 3 hypothetical solute-binding proteins of ABC transporters, an invasion-associated protein homolog, putative enzymes catalyzing cell wall turnover, several polypeptides with similarity to bacterial conjugation proteins, and 3 proteins of unknown function. Surprisingly, aldolase, usually considered as a cytoplasmic protein, was found in the culture supernatant. All proteins, excluding aldolase, were predicted to contain a signal peptide and a signal peptide cleavage site in their immature form. Some of the excreted proteins are interesting targets for further genetic and physiological studies.
J Mol Microbiol Biotechnol 2008
PMID:A preliminary analysis of Bifidobacterium longum exported proteins by two-dimensional electrophoresis. 1795 13

The aim of this study was to evaluate the survival of Lactobacillus rhamnosus R11 and Lactobacillus acidophilus R52 in the human digestive tract and their effects on the microbiota homeostasis. We designed an open human trial including 14 healthy volunteers. A 3-week exclusion period of fermented products was followed by a 12-day consumption period of 4 capsules daily containing 2 x 10(9)L. rhamnosus R11 and 1 x 10(8)L. acidophilus R52, and a 12-day wash-out period. The 2 strains and dominant bacterial groups of the microbiota were quantified by real-time polymerase chain reaction. At the end of the capsule consumption period, high levels of L. rhamnosus R11 were detected in faecal samples from all volunteers, reaching a mean value of 7.1 log(10) colony-forming unit (CFU) equivalents/g of stool. L. acidophilus R52 was detected in the stools of only 1 volunteer, reaching a maximum level of 6.1 log(10) CFU equivalents/g of stool. Dilution plating enumerations performed in parallel provided less consistent and generally lower levels. No significant effect of capsule consumption was observed on microbiota homeostasis for the dominant faecal populations. Mean values of 8.8, 9.2, 9.9 and 10.6 log(10) CFU equivalents/g of stool were obtained for the Clostridium coccoides, Bifidobacterium sp., Bacteroides sp. and Clostridium leptum groups, respectively.
J Mol Microbiol Biotechnol 2008
PMID:Lactobacillus rhamnosus R11 consumed in a food supplement survived human digestive transit without modifying microbiota equilibrium as assessed by real-time polymerase chain reaction. 1795 15


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