Growth of facultatively heterofermentative lactobacilli on starter cell suspensions

The growth of facultatively heterofermentative lactobacilli (FHL) on cell suspensions of the homofermentative Lactobacillus helveticus was investigated. Osmotic lysis of L. helveticus led to a significant increase of ribose. It decreased steadily in parallel with the growth of FHL, strongly suggesting that the bacteria used ribose as a growth substrate.     

Phenotypic and Genotypic Analysis of Amino Acid Auxotrophy in Lactobacillus helveticus CNRZ 32

The conversion of amino acids into volatile and nonvolatile compounds by lactic acid bacteria in cheese is thought to represent the rate-limiting step in the development of mature flavor and aroma. Because amino acid breakdown by microbes often entails the reversible action of enzymes involved in biosynthetic pathways, our group investigated the genetics of amino acid biosynthesis in Lactobacillus helveticus CNRZ 32, a commercial cheese flavor adjunct that reduces bitterness and intensifies flavor notes. Most lactic acid bacteria are auxotrophic for several amino acids, and L. helveticus CNRZ 32 requires 14 amino acids. The reconstruction of amino acid biosynthetic pathways from a draft-quality genome sequence for L. helveticus CNRZ 32 revealed that amino acid auxotrophy in this species was due primarily to gene absence rather than point mutations, insertions, or small deletions, with good agreement between gene content and phenotypic amino acid requirements. One exception involved the phenotypic requirement for Asp (or Asn), which genome predictions suggested could be alleviated by citrate catabolism. This prediction was confirmed by the growth of L. helveticus CNRZ 32 after the addition of citrate to a chemically defined medium that lacked Asp and Asn. Genome analysis also predicted that L. helveticus CNRZ 32 possessed ornithine decarboxylase activity and would therefore catalyze the conversion of ornithine to putrescine, a volatile biogenic amine. However, experiments to confirm ornithine decarboxylase activity in L. helveticus CNRZ 32 by the use of several methods were unsuccessful, which indicated that this bacterium likely does not contribute to putrescine production in cheese.     

Deficiency of the LIM-Only Protein FHL2 Reduces Intestinal Tumorigenesis in Apc Mutant Mice

Background

The four and a half LIM-only protein 2 (FHL2) is capable of shuttling between focal adhesion and nucleus where it signals through direct interaction with a number of proteins including β-catenin. Although FHL2 activation has been found in various human cancers, evidence of its functional contribution to carcinogenesis has been lacking.

Methodology/Principal Findings

Here we have investigated the role of FHL2 in intestinal tumorigenesis in which activation of the Wnt pathway by mutations in the adenomatous polyposis coli gene (Apc) or in β-catenin constitutes the primary transforming event. In this murine model, introduction of a biallelic deletion of FHL2 into mutant Apc^Δ14/+ mice substantially reduces the number of intestinal adenomas but not tumor growth, suggesting a role of FHL2 in the initial steps of tumorigenesis. In the lesions, Wnt signalling is not affected by FHL2 deficiency, remaining constitutively active. Nevertheless, loss of FHL2 activity is associated with increased epithelial cell migration in intestinal epithelium, which might allow to eliminate more efficiently deleterious cells and reduce the risk of tumorigenesis. This finding may provide a mechanistic basis for tumor suppression by FHL2 deficiency. In human colorectal carcinoma but not in low-grade dysplasia, we detected up-regulation and enhanced nuclear localization of FHL2, indicating the activation of FHL2 during the development of malignancy.

Conclusions/Significance

Our data demonstrate that FHL2 represents a critical factor in intestinal tumorigenesis.     

Transport and Metabolism of Lactose, Glucose, and Galactose in Homofermentative Lactobacilli

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties, whereas L. bulgaricus, L. lactis, and L. acidophilus strains metabolized only the glucose moiety and released galactose into the growth medium. All four species tested contained β-galactosidase activity, and no significant phospho-β-galactosidase activity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate (PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose phosphotransferase or PEP:galactose phosphotransferase system was obtained.     

LIM domain protein FHL1B interacts with PP2A catalytic β subunit--a novel cell cycle regulatory pathway

Four-and-a-half LIM domain protein 1B (FHL1B) is an alternatively-spliced isoform of FHL1. In this study, FHL1B was demonstrated to interact with the β catalytic subunit (Cβ) of a type 2A protein phosphatase (PP2A) by yeast two-hybrid screening. Domain studies using a small-scale yeast two-hybrid interaction assay revealed the mediation of protein-protein interaction by FHL1B’s C-terminus. Interaction between FHL1B and PP2A was further verified by co-immunoprecipitation. FHL1B was also shown to shuttle between nucleus and cytoplasm at different phases of the cell cycle. These data suggest that the FHL1B/PP2A_Cβ interaction may illustrate a novel cell-cycle regulatory pathway.

Structured summary

MINT-8044739: FHL1B (uniprotkb:Q13642-2) physically interacts (MI:0915) with PP2Acbeta (uniprotkb:P62714) by two hybrid (MI:0018)MINT-8044769, MINT-8044778: FHL1B (uniprotkb:Q13642-2) physically interacts (MI:0915) with PP2Acbeta (uniprotkb:P62714) by anti bait coimmunoprecipitation (MI:0006)     

Deletion of the FHL2 gene attenuates the formation of atherosclerotic lesions after a cholesterol-enriched diet

AimsFHL2, a member of the four and a half LIM domain (FHL) family of proteins, may play an important role in the circulatory system and in particular atherosclerosis.Main methodsTo investigate the role of FHL2 in atherogenesis, FHL2-null and wild-type control male mice were fed either a normal chow (NC) or a cholesterol-enriched diet (CED).Key findingsAt 3 months post CED, aortic atherosclerotic plaques were observed in both control and FHL2-null mice. Lesions in control mice increased dramatically by 6 months of CED. In contrast, lesion size did not increase during this time in CED-fed FHL2-null mice. Relative to control mice on a normal chow of diet (NCD), control mice on a CED exhibited lower circulating nitric oxide (NO) levels, and decreased expression of connexin37 (Cx37) and Cx40 in aortic endothelium. In contrast, FHL2-null mice on a CED maintained similar levels of circulating NO as FHL2-null mice fed a NCD. Cxs levels in aortic endothelium of FHL2-null mutants on a NCD were lower relative to control mice on a NCD, and did not decrease with CED.SignificanceOur data demonstrate a role for FHL2 in atherogenesis, the regulation of circular NO release, and expression of gap junctions within aortic endothelium.     

Subtypes of familial hemophagocytic lymphohistiocytosis in Japan based on genetic and functional analyses of cytotoxic T lymphocytes

Background

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disease of infancy or early childhood. To clarify the incidence and subtypes of FHL in Japan, we performed genetic and functional analyses of cytotoxic T lymphocytes (CTLs) in Japanese patients with FHL.

Design and Methods

Among the Japanese children with hemophagocytic lymphohistiocytosis (HLH) registered at our laboratory, those with more than one of the following findings were eligible for study entry under a diagnosis of FHL: positive for known genetic mutations, a family history of HLH, and impaired CTL-mediated cytotoxicity. Mutations of the newly identified causative gene for FHL5, STXBP2, and the cytotoxicity and degranulation activity of CTLs in FHL patients, were analyzed.

Results

Among 31 FHL patients who satisfied the above criteria, PRF1 mutation was detected in 17 (FHL2) and UNC13D mutation was in 10 (FHL3). In 2 other patients, 3 novel mutations of STXBP2 gene were confirmed (FHL5). Finally, the remaining 2 were classified as having FHL with unknown genetic mutations. In all FHL patients, CTL-mediated cytotoxicity was low or deficient, and degranulation activity was also low or absent except FHL2 patients. In 2 patients with unknown genetic mutations, the cytotoxicity and degranulation activity of CTLs appeared to be deficient in one patient and moderately impaired in the other.

Conclusions

FHL can be diagnosed and classified on the basis of CTL-mediated cytotoxicity, degranulation activity, and genetic analysis. Based on the data obtained from functional analysis of CTLs, other unknown gene(s) responsible for FHL remain to be identified.     

Impact of Proteolytic <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> MTCC5463 on Production of Bioactive Peptides Derived from Honey Based Fermented Milk

In this study, Lactobacillus helveticus MTCC5463 was evaluated for its proteolytic activity and production of bioactive peptides during fermentation of honey supplemented milk under specified growth conditions. Generally, lactic acid bacteria have a strong proteolytic system. However, L. helveticus MTCC5463 showed maximum proteolytic activity at 4 % level of honey supplementation compared to 6 % and control. Similarly, water soluble extract derived from fermented honey based milks exhibited different level of bioactive peptides productions during fermentation. L. helveticus MTCC5463 showed maximum peptides production at 4 % level of honey supplementation compared to control during HPLC analysis and LC–MS analysis.     

Genetic Characterization and Physiological Role of Endopeptidase O from Lactobacillus helveticus CNRZ32

A previously identified insert expressing an endopeptidase from a Lactobacillus helveticus CNRZ32 genomic library was characterized. Nucleotide sequence analysis revealed an open reading frame of 1,941 bp encoding a putative protein of 71.2 kDa which contained a zinc-protease motif. Protein homology searches revealed that this enzyme has 40% similarity with endopeptidase O (PepO) from Lactococcus lactis P8-2-47. Northern hybridization revealed that pepO is monocistronic and is expressed throughout the growth phase. CNRZ32 derivatives lacking PepO activity were constructed via gene replacement. Enzyme assays revealed that the PepO mutant had significantly reduced endopeptidase activity when compared to CNRZ32 with two of the three substrates examined. Growth studies indicated that PepO has no detectable effect on growth rate or acid production by Lactobacillus helveticus CNRZ32 in amino acid defined or skim milk medium.     

Influence of an initial addition of lactic acid on growth, acid production and their coupling for batch cultures of Lactobacillus helveticus

Growth and production coupling for L. helveticus growing at constant pH?on a medium largely supplemented with yeast extract and peptons has been examined as a function of initial lactic acid addition: these conditions allowed to avoid side effects of cultivation pH?and nitrogen source deficiency on coupling. Growth of L. helveticus strain milano was reduced by half for a free lactic acid concentration of 0.34?g.l^?1; both growth and production were fully inhibited for a critical end-product concentration of 83?g.l^?1, despite the fact that lactose was still available. The extent of autolysis 8?h after growth ceased was practically independent of initial lactic acid concentration: the same conclusion held for the part played by a growth-associated mechanism in total acid production. During the slowing down phase of growth, production was non-growth-associated: production rate remained close to its maximal value, while growth rate declined.     

Heterogeneity of Putative Surface Layer Proteins in Lactobacillus helveticus

The S-layer-encoding genes of 21 Lactobacillus helveticus strains were characterized. Phylogenetic analysis based on the identified S-layer genes revealed two main clusters, one which includes a sequence similar to that of the slpH1 gene of L. helveticus CNRZ 892 and a second cluster which includes genes similar to that of prtY. These results were further confirmed by Southern blot hybridization. This study demonstrates S-layer gene variability in the species L. helveticus.     

Isolation and Characterization of a Slowly Milk-Coagulating Variant of Lactobacillus helveticus Deficient in Purine Biosynthesis

A slowly milk-coagulating variant (Fmc⁻) of Lactobacillus helveticus CRL 1062, designated S1, was isolated and characterized. Strain S1 possessed all the known essential components required to utilize casein as a nitrogen source, which include functional proteinase and peptidase activities as well as functional amino acid, di- and tripeptide, and oligopeptide transport systems. The amino acid requirements of strain S1 were similar to those of the parental strain. However, on a purine-free, chemically defined medium, the growth rate of the Fmc⁻ strain was threefold lower than that of the wild-type strain. L. helveticus S1 was found to be defective in IMP dehydrogenase activity and therefore was deficient in the ability to synthesize XMP and GMP. This conclusion was further supported by the observation that the addition of guanine or xanthine to milk, a substrate poor in purine compounds, restored the Fmc⁺ phenotype of L. helveticus S1.     

A Lactobacillus helveticus-Specific DNA Probe Detects Restriction Fragment Length Polymorphisms in This Species

A cloned 2-kb EcoRI fragment (fragment f) from a 34-kb plasmid of Lactobacillus helveticus CNRZ 1094 was shown by dot blot to specifically hybridize to total DNAs of 75 L. helveticus strains. No hybridization was found with L. acidophilus, L. crispatus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. gasseri, or L. intestinalis strains. When Southern blots of EcoRI digests of L. helveticus strains were probed with fragment f, these strains displayed restriction fragment length polymorphisms on the basis of which they could be grouped into several clusters.     

Mutations of the FHL1 Gene Cause Emery-Dreifuss Muscular Dystrophy

Emery-Dreifuss muscular dystrophy (EDMD) is a rare disorder characterized by early joint contractures, muscular dystrophy, and cardiac involvement with conduction defects and arrhythmias. So far, only 35% of EDMD cases are genetically elucidated and associated with EMD or LMNA gene mutations, suggesting the existence of additional major genes. By whole-genome scan, we identified linkage to the Xq26.3 locus containing the FHL1 gene in three informative families belonging to our EMD- and LMNA-negative cohort. Analysis of the FHL1 gene identified seven mutations, in the distal exons of FHL1 in these families, three additional families, and one isolated case, which differently affect the three FHL1 protein isoforms: two missense mutations affecting highly conserved cysteines, one abolishing the termination codon, and four out-of-frame insertions or deletions. The predominant phenotype was characterized by myopathy with scapulo-peroneal and/or axial distribution, as well as joint contractures, and associated with a peculiar cardiac disease characterized by conduction defects, arrhythmias, and hypertrophic cardiomyopathy in all index cases of the seven families. Heterozygous female carriers were either asymptomatic or had cardiac disease and/or mild myopathy. Interestingly, four of the FHL1-mutated male relatives had isolated cardiac disease, and an overt hypertrophic cardiomyopathy was present in two. Expression and functional studies demonstrated that the FHL1 proteins were severely reduced in all tested patients and that this was associated with a severe delay in myotube formation in the two patients for whom myoblasts were available. In conclusion, FHL1 should be considered as a gene associated with the X-linked EDMD phenotype, as well as with hypertrophic cardiomyopathy.     

Complete Genome Sequence of Lactobacillus helveticus R0052, a Commercial Probiotic Strain

Lactobacillus helveticus R0052 is a commercially available strain that is widely used in probiotic preparations. The genome sequence consisted of 2,129,425 bases. Comparative analysis showed that it was unique among L. helveticus strains in that it contained genes encoding mucus-binding proteins similar to those found in Lactobacillus acidophilus.     

FHL1 Reduces Dystrophy in Transgenic Mice Overexpressing FSHD Muscular Dystrophy Region Gene 1 (FRG1)

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.