Vladimir V. Smeianov

Comparative High-Density Microarray Analysis of Gene Expression during Growth of Lactobacillus helveticus in Milk versus Rich Culture Medium

ABSTRACT Lactobacillus helveticus CNRZ32 is used by the dairy industry to modulate cheese flavor. The compilation of a draft genome sequence for this strain allowed us to identify and completely sequence 168 genes potentially important for the growth of this organism in milk or for cheese flavor development. The primary aim of this study was to investigate the expression of these genes during growth in milk and MRS medium by using microarrays. Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays. Additionally, the entire draft genome sequence was used to produce tiled microarrays in which noninterrupted sequence contigs were covered by consecutive 24-mer probes and associated mismatch probe sets. Total RNA isolated from cells grown in skim milk or in MRS to mid-log phase was used as a template to synthesize cDNA, followed by Cy3 labeling and hybridization. An analysis of data from annotated gene probes identified 42 genes that were upregulated during the growth of CNRZ32 in milk (P < 0.05), and 25 of these genes showed upregulation after applying Bonferronis adjustment. The tiled microarrays identified numerous additional genes that were upregulated in milk versus MRS. Collectively, array data showed the growth of CNRZ32 in milk-induced genes encoding cell-envelope proteinases, oligopeptide transporters, and endopeptidases as well as enzymes for lactose and cysteine pathways, de novo synthesis, and/or salvage pathways for purines and pyrimidines and other functions. Genes for a hypothetical phosphoserine utilization pathway were also differentially expressed. Preliminary experiments indicate that cheese-derived, phosphoserine-containing peptides increase growth rates of CNRZ32 in a chemically defined medium. These results suggest that phosphoserine is used as an energy source during the growth of L. helveticus CNRZ32.

Anti-inflammatory properties of intestinal Bifidobacterium strains isolated from healthy infants

Certain Bifidobacterium strains have been shown to inhibit inflammatory responses in intestinal epithelial cells. However, the precise mechanisms of these effects, including the chemical nature of the active compounds, remain to be elucidated. Here partial characterization of the anti‐inflammatory properties of Bifidobacterium strains isolated from feces of healthy infants is reported. It was found that conditioned media (CM) of all strains studied are capable of attenuating tumor necrosis factor‐α (TNF‐α) and lipopolysaccharide‐ (LPS) induced inflammatory responses in the HT‐29 cell line. In contrast, neither killed bifidobacterial cells, nor cell‐free extracts showed such activities. Further investigations resulted in attribution of this activity to heat‐stable, non‐lipophilic compound(s) resistant to protease and nuclease treatments and of molecular weight less than 3  kDa. The anti‐inflammatory effects were dose‐ and time‐dependent and associated with inhibition of IκB phosphorylation and nuclear factor‐κ light chain enhancer of activated B cells (NF‐κB)‐dependent promoter activation. The combined treatments of cells with CMs and either LPS or TNF‐α, but not with CMs alone, resulted in upregulation of transforming growth factor‐β1, IκBζ, and p21CIP mRNAs. Our data suggest certain species‐specificities of the anti‐inflammatory properties of bifidobacteria. This observation should prompt additional validation studies using larger set of strains and employing the tools of comparative genomics.

Characterization of plasmids from human infant Bifidobacterium strains : Sequence analysis and construction of E. coli-Bifidobacterium shuttle vectors

A survey of infant fecal Bifidobacterium isolates for plasmid DNA revealed that a significant portion of the strains, 17.6%, carry small plasmids. The majority of plasmid-harboring strains belonged to the Bifidobacterium longum/infantis group. Most of the plasmids could be assigned into two groups based on their sizes and the restriction profiles. Three plasmids, pB44 (3.6 kb) from B. longum, pB80 (4.9 kb) from Bifidobacterium bifidum, and pB21a (5.2kb) from Bifidobacterium breve were sequenced. While the former two plasmids were found to be highly similar to previously characterized rolling-circle replicating pKJ36 and pKJ56, respectively, the third plasmid, pB21a, does not share significant nucleotide homology with known plasmids. However, it might be placed into the pCIBb1-like group of bifidobacterial rolling-plasmids based on the homology of its Rep protein and the overall molecular organization. Two sets of Escherichia coli-Bifidobacterium shuttle vectors constructed based on pB44 and pB80 replicons were capable of transforming B. bifidum and B. breve strains with efficiency up to 3x10(4)cfu/microg DNA. Additionally, an attempt was made to employ a broad host range conjugation element, RP4, in developing of E. coli-Bifidobacterium gene transfer system.

Production of human basic fibroblast growth factor (FGF-2) in Bifidobacterium breve using a series of novel expression/secretion vectors.

Four E. coli-Bifidobacterium shuttle vectors were constructed using Bifidobacterium plasmids, pB44 and pB80. The vectors carry two bifidobacterial promoters, a signal peptide-encoding sequence, sec2, of Bifidobacterium breve, and a transcriptional terminator from hup gene of Bifidobacterium longum. Functionality of the constructs were tested using human FGF-2 gene. The expression of FGF-2 was detected by Western blotting in B. breve transformed with three of the vectors. The highest amount of FGF-2 was produced upon transformation with pESH86, which is a pB80-based plasmid carrying FGF-2 under control of a hup promoter (Phup). Similarly, the level of FGF-2 mRNA transcribed from pESH86 was approximately threefold higher, 882 ± 70 AU (arbitrary units), when compared to those transcribed from pB44-based pESH46 (Phup) (289 ± 65 AU) and pESH47 (Pgap) (282 ± 37 AU). These results suggest the vectors have the potential for production of exported fusion proteins in bifidobacteria and the expression levels can be regulated through the employment of different bifidobacterial promoters and/or replicons.

Application of Several Molecular Techniques to Study Numerically Predominant Bifidobacterium spp. and Bacteroidales Order Strains in the Feces of Healthy Children

Bifidobacteria and Bacteroides-like bacteria are strictly anaerobic nonpathogenic members of human intestinal microflora. Here we describe an analysis of the species and subspecies composition of these bacterial populations in healthy children using a combination of culture and molecular methods at two different time points. It was found that B. bifidum and B. longum are the most common dominant taxons in infants aged between 8 and 16 months. The majority of the infants carried several dominant Bifidobacterium strains belonging to different species. Examination of the dominant bifidoflora in some of these children after a 5-year period showed major shifts in both species and strain composition, but the dominant strains remained unchanged in two children. The majority of dominant Bacteroides-like isolates belonged to species B. vulgatus and B. uniformis, but members of genera Alistipes and Barnesiella were common too. In addition, a novel approach to species identification of Bacteroidales order bacteria using amplified ribosomal DNA restriction analysis (ARDRA) is described.

Intraspecies Genomic Diversity and Long-Term Persistence of Bifidobacterium longum

Members of genus Bifidobacterium are Gram-positive bacteria, representing a large part of the human infant microbiota and moderately common in adults. However, our knowledge about their diversity, intraspecific phylogeny and long-term persistence in humans is still limited. Bifidobacterium longum is generally considered to be the most common and prevalent species in the intestinal microbiota. In this work we studied whole genome sequences of 28 strains of B. longum, including 8 sequences described in this paper. Part of these strains were isolated from healthy children during a long observation period (up to 10 years between isolation from the same patient). The three known subspecies (longum, infantis and suis) could be clearly divided using sequence-based phylogenetic methods, gene content and the average nucleotide identity. The profiles of glycoside hydrolase genes reflected the different ecological specializations of these three subspecies. The high impact of horizontal gene transfer on genomic diversity was observed, which is possibly due to a large number of prophages and rapidly spreading plasmids. The pan-genome characteristics of the subspecies longum corresponded to the open pan-genome model. While the major part of the strain-specific genetic loci represented transposons and phage-derived regions, a large number of cell envelope synthesis genes were also observed within this category, representing high variability of cell surface molecules. We observed the cases of isolation of high genetically similar strains of B. longum from the same patients after long periods of time, however, we didn’t succeed in the isolation of genetically identical bacteria: a fact, reflecting the high plasticity of microbiota in children.

Search for Protein Adhesin Gene in Bifidobacterium Longum Genome Using Surface Phage Display Technology

A fragment of the nucleotide sequence encoding polypeptide binding to HT-29 epithelial cells was cloned from VMKB44 Bifidobacterium longum genome library using surface phage display technology. Sequencing of this polypeptide consisting of 26 amino acid residues showed that it is an extracellular fragment of a large BL0155 transmembrane protein belonging to the ABC transport protein superfamily. The genes encoding homologues of this protein were detected in genomes of not only bifidobacteria of different species, but also in many other enteric commencals and pathogens.

Correction to: Intrinsic and inducible resistance to hydrogen peroxide in Bifidobacterium species

In the published article, the co-author Abdelmoneim Abdalla’s affiliation has been published incompletely. The additional affiliation is given below: