Identification of Lactobacillus plantarum genes that are induced in the gastrointestinal tract of mice

Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of environmental niches, including the human gastrointestinal (GI) tract. Moreover, this lactic acid bacterium can survive passage through the human or mouse stomach in an active form. To investigate the genetic background of this persistence, resolvase-based in vivo expression technology (R-IVET) was performed in L. plantarum WCFS1 by using the mouse GI tract as a model system. This approach identified 72 L. plantarum genes whose expression was induced during passage through the GI tract as compared to laboratory media. Nine of these genes encode sugar-related functions, including ribose, cellobiose, sucrose, and sorbitol transporter genes. Another nine genes encode functions involved in acquisition and synthesis of amino acids, nucleotides, cofactors, and vitamins, indicating their limited availability in the GI tract. Four genes involved in stress-related functions were identified, reflecting the harsh conditions that L. plantarum encounters in the GI tract. The four extracellular protein encoding genes identified could potentially be involved in interaction with host specific factors. The rest of the genes are part of several functionally unrelated pathways or encode (conserved) hypothetical proteins. Remarkably, a large number of the functions or pathways identified here have previously been identified in pathogens as being important in vivo during infection, strongly suggesting that survival rather than virulence is the explanation for the importance of these genes during host residence.     

Cloning and expression of cellulase and xylanase genes in Lactobacillus plantarum

Summary Eleven cellulase genes from Gram-positive bacteria were cloned in a Lactobacillus plantarum silage inoculum. Eight of these genes were expressed as active enzymes from their original promotors and translation signals. Where tested, the enzymes produced by transformed L.plantarum had the same temperature and pH optimum as enzymes produced in the original host, or in transformed Escherichia coli. Using chloramphenicol acetyltransferase as a cell-internal marker enzyme, it could be demonstrated that at least endoglucanase D from Clostridium thermocellum was actively secreted by transformed L. plantarum. In growing L. plantarum cultures, most of the enzymes were irreversibly inactivated when the pH decreased below 4.5. If the transformed strains were to be applied as an inoculum in silage, this pH inactivation might be useful in preventing overdigestion of the crop fibre. Offprint requests to: F. Michiels     

Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome

A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria.     

Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome.

A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria.     

Identification and characterization of antibiotic resistance genes in Lactobacillus reuteri and Lactobacillus plantarum

Aims:  The study aimed to identify the resistance genes mediating atypical minimum inhibitory concentrations (MICs) for tetracycline, erythromycin, clindamycin and chloramphenicol within two sets of representative strains of the species Lactobacillus reuteri and Lactobacillus plantarum and to characterize identified genes by means of gene location and sequencing of flanking regions.
Methods and Results:  A tet (W) gene was found in 24 of the 28 Lact. reuteri strains with atypical MIC for tetracycline, whereas four of the six strains with atypical MIC for erythromycin were positive for erm (B) and one strain each was positive for erm (C) and erm (T). The two Lact. plantarum strains with atypical MIC for tetracycline harboured a plasmid-encoded tet (M) gene. The majority of the tet (W)-positive Lact. reuteri strains and all erm -positive Lact. reuteri strains carried the genes on plasmids, as determined by Southern blot and a real-time PCR method developed in this study.
Conclusions:  Most of the antibiotic-resistant strains of Lact. reuteri and Lact. plantarum harboured known plasmid-encoded resistance genes. Examples of putative transfer machineries adjacent to both plasmid- and chromosome-located resistance genes were also demonstrated.
Significance and Impact of the Study:  These data provide some of the knowledge required for assessing the possible risk of using Lact. reuteri and Lact. plantarum strains carrying antibiotic resistance genes as starter cultures and probiotics.     

Bacterial translocation from the gastrointestinal tracts of thymectomized mice

The incidence of translocation of viable indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node, spleen, liver, and kidney was compared in neonatally thymectomized mice and sham-thymectomized specific pathogen-free mice. The immunologic responses of the thymectomized mice to sheep erythrocytes were decreased compared to the responses of sham-thymectomized mice. Strictly anaerobic bacteria were isolated from only 1.8% of the organs from thymectomized mice and from none of the organs of shamthymectomized mice. Aerobic or facultatively anaerobic bacteria were cultured from 27.4% of the organs of thymectomized mice. Of the thymectomized mice, 70.7% contained viable aerobic or facultatively anaerobic bacteria in one or more of their organs tested, compared with only 10% of the sham-thymectomized mice.Escherichia coli was the predominant bacterial species isolated from these organs, althoughStaphylococcus aureus, Streptococcus, andCorynebacterium also were present.Bacteroides were the only strictly anaerobic bacteria cultured. Neonatal thymectomy promotes the translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node, spleen, liver, and kidney.     

Spatial and temporal divergence of expression in duplicated barley germin-like protein-encoding genes

Subfunctionalization is the process by which a pair of duplicated genes, or paralogs, experiences a reduction of individual expression patterns or function while still reproducing the complete expression pattern and function of the ancestral gene. Two germin-like protein (GLP)-encoding genes, GerB and GerF, are paralogs that belong to a small gene family in barley (Hordeum vulgare). Both genes share high nucleotide sequence similarity in coding and noncoding regions and encode identical apoplastic proteins. The use of RNA gel blots, coupled with single-stranded conformation polymorphism (SSCP) analysis of RT-PCR products, elucidated the developmental and tissue-specific expression patterns of each gene. Individual expression patterns provided evidence of both overlapping redundancy and early subfunctionalization. GerB is predominantly expressed in developing shoots, while GerF is predominantly expressed in seedling roots, developing spikes, and pericarp/testa. GerF promoter deletion studies located a region (-356/-97) responsible for high promoter activity and showed the ability of GerB and GerF upstream regions to drive gfp expression in coleoptiles, epicarps, and lemma/palea of developing spikes. The observed expression patterns are consistent with proposed roles in plant development and defense mechanisms for this gene family. These roles may explain why redundancy has been selectively maintained in this duplicate gene pair.     

Spatial and temporal expression patterns of selenoprotein genes during embryogenesis in zebrafish

Selenium is important for embryogenesis in vertebrates but little is known about the expression patterns and biological functions of most selenoprotein genes. Taking advantage of the zebrafish model, systematic analysis of selenoprotein gene expression was performed by in situ hybridization on whole-mount embryos at different developmental stages. Twenty-one selenoprotein mRNAs were analyzed and all of them exhibited expression patterns restricted to specific tissues. Moreover, we demonstrated that highly similar selenoprotein paralogs were expressed within distinct territories. Therefore, tissue- and development-specific expression patterns provided new information for selenoproteins of unknown function.     

Spatial and temporal expression patterns of chitinase genes in developing zebrafish embryos

Chitinases and chitinase like proteins play an important role in mammalian immunity and functions in early zebrafish development have been suggested. Here we report identification of six zebrafish chitinases and chitinase like proteins (called CHIA.1–6) belonging to the glycoside hydrolase family 18, and determine their spatial and temporal expression at 10 stages of zebrafish development.CHIA.4 is highly maternally expressed and it is expressed 100 fold above any other CHIA gene at zygote through to blastula stage. Later, after the maternal to zygotic transition, CHIA.4 expression decreases to the same level as CHIA.5 and CHIA.6. Subsequently, CHIA.1, CHIA.2, CHIA.3 and CHIA.4, CHIA.5, CHIA.6 each follow distinct paths in terms of expression levels.Until 4 days post fertilization the spatial expression patterns of all six CHIA genes overlap extensively, with expression detected predominantly in vascular, ocular and intestinal tissues. At 5 days post fertilization CHIA.1, CHIA.2 and CHIA.3 are expressed almost exclusively in the stomach, whereas CHIA.4, CHIA.5 and CHIA.6 are also prominently expressed in the liver. These different expression patterns may contribute to the establishment of a basis on which functional analysis in older larvae may be founded.     

Immunocytochemical localization of polyamines in the gastrointestinal tracts of rats and mice

An immunocytochemical method using a recently produced monoclonal antibody (ASPM-29) with an antibody specificity to spermine (Spm) and spermidine (Spd) fixed in situ, was used to demonstrate an immunocytochemical localization of polyamine (PA) pools in the gastrointestinal tracts of rats and mice. High PA immunoreactivity was always found in the cytoplasm of cells not only at the cell proliferative zone or the precursor cell zone but also at the neighboring non-proliferative premature cell zone of the epithelium, and a gradient of decreasing PA levels was noticed from these cells to the fully mature differentiated gastric surface mucous cells and absorptive cells of the small and large intestines. Also, strong staining for PAs was seen in the cytoplasm of fully differentiated gastric chief cells and neurons of both the myenteric and submucous plexuses, whereas the nuclei of the cells remained virtually unstained. These results may suggest that PAs are closely associated with the high biosynthetic activity in the cells of the gastrointestinal mucosa of normal rats and mice. This seems to be consistent with the PA imunocytochemical results previously obtained for neoplastic cells and active protein- or peptide-secreting cells, including exocrine or endocrine cell types.     

CcpA affects expression of the groESL and dnaK operons in Lactobacillus plantarum

Background  

Lactic acid bacteria (LAB) are widely used in food industry and their growth performance is important for the quality of the fermented product. During industrial processes changes in temperature may represent an environmental stress to be overcome by starters and non-starters LAB. Studies on adaptation to heat shock have shown the involvement of the chaperon system-proteins in various Gram-positive bacteria. The corresponding operons, namely the dnaK and groESL operons, are controlled by a negative mechanism involving the HrcA repressor protein binding to the cis acting element CIRCE.     

Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

Spatial and temporal expression of the two sucrose synthase genes in maize: immunohistological evidence

Summary The DNAs of two diploid species of Gossypium, G. herbaceum var. africanum (A₁ genome) and G. raimondii (D₅ genome), and the allotetraploid species, G. hirsutum (A_h and D_h genomes), were characterized by kinetic analyses of single copy and repetitive sequences. Estimated haploid genome sizes of A₁ and D₅ were 1.04 pg and 0.68 pg, respectively, in approximate agreement with cytological observations that A genome chromosomes are about twice the size of D genome chromosomes. This differences in genome size was accounted for entirely by differences in the major repetitive fraction (0.56 pg versus 0.20 pg), as single copy fractions of the two genomes were essentially identical (0.41 pg for A₁ and 0.43 pg for D₅). Kinetic analyses and thermal denaturation measurements of single copy duplexes from reciprocal intergenomic hybridizations showed considerable sequence similarity between A₁ and D₅ genomes (77% duplex formation with an average thermal depression of 6 °C). Moreover, little sequence divergence was detectable between diploid single copy sequences and their corresponding genomes in the allotetraploid, consistent with previous chromosome pairing observations in interspecific F₁ hybrids.Journal paper No. 4461 of the Arizona Agricultural Experiment Station