FARS2 Deficiency Causes Cardiomyopathy by Disrupting Mitochondrial Homeostasis and the Mitochondrial Quality Control System.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a common heritable heart disease. Although HCM has been reported to be associated with many variants of genes involved in sarcomeric protein biomechanics, pathogenic genes have not been identified in patients with partial HCM. FARS2 (the mitochondrial phenylalanyl-tRNA synthetase), a type of mitochondrial aminoacyl-tRNA synthetase, plays a role in the mitochondrial translation machinery. Several variants of FARS2 have been suggested to cause neurological disorders; however, FARS2-associated diseases involving other organs have not been reported. We identified FARS2 as a potential novel pathogenic gene in cardiomyopathy and investigated its effects on mitochondrial homeostasis and the cardiomyopathy phenotype. METHODS: FARS2 variants in patients with HCM were identified using whole-exome sequencing, Sanger sequencing, molecular docking analyses, and cell model investigation. Fars2 conditional mutant (p.R415L) or knockout mice, fars2-knockdown zebrafish, and Fars2-knockdown neonatal rat ventricular myocytes were engineered to construct FARS2 deficiency models both in vivo and in vitro. The effects of FARS2 and its role in mitochondrial homeostasis were subsequently evaluated using RNA sequencing and mitochondrial functional analyses. Myocardial tissues from patients were used for further verification. RESULTS: We identified 7 unreported FARS2 variants in patients with HCM. Heart-specific Fars2-deficient mice presented cardiac hypertrophy, left ventricular dilation, progressive heart failure accompanied by myocardial and mitochondrial dysfunction, and a short life span. Heterozygous cardiac-specific Fars2R415L mice displayed a tendency to cardiac hypertrophy at age 4 weeks, accompanied by myocardial dysfunction. In addition, fars2-knockdown zebrafish presented pericardial edema and heart failure. FARS2 deficiency impaired mitochondrial homeostasis by directly blocking the aminoacylation of mt-tRNAPhe and inhibiting the synthesis of mitochondrial proteins, ultimately contributing to an imbalanced mitochondrial quality control system by accelerating mitochondrial hyperfragmentation and disrupting mitochondrion-related autophagy. Interfering with the mitochondrial quality control system using adeno-associated virus 9 or specific inhibitors mitigated the cardiac and mitochondrial dysfunction triggered by FARS2 deficiency by restoring mitochondrial homeostasis. CONCLUSIONS: Our findings unveil the previously unrecognized role of FARS2 in heart and mitochondrial homeostasis. This study may provide new insights into the molecular diagnosis and prevention of heritable cardiomyopathy as well as therapeutic options for FARS2-associated cardiomyopathy.

α-Phenylalanyl tRNA synthetase competes with Notch signaling through its N-terminal domain.

The alpha subunit of the cytoplasmic Phenylalanyl tRNA synthetase (α-PheRS, FARSA in humans) displays cell growth and proliferation activities and its elevated levels can induce cell fate changes and tumor-like phenotypes that are neither dependent on the canonical function of charging tRNAPhe with phenylalanine nor on stimulating general translation. In intestinal stem cells of Drosophila midguts, α-PheRS levels are naturally slightly elevated and human FARSA mRNA levels are elevated in multiple cancers. In the Drosophila midgut model, elevated α-PheRS levels caused the accumulation of many additional proliferating cells resembling intestinal stem cells (ISCs) and enteroblasts (EBs). This phenotype partially resembles the tumor-like phenotype described as Notch RNAi phenotype for the same cells. Genetic interactions between α-PheRS and Notch suggest that their activities neutralize each other and that elevated α-PheRS levels attenuate Notch signaling when Notch induces differentiation into enterocytes, type II neuroblast stem cell proliferation, or transcription of a Notch reporter. These non-canonical functions all map to the N-terminal part of α-PheRS which accumulates naturally in the intestine. This truncated version of α-PheRS (α-S) also localizes to nuclei and displays weak sequence similarity to the Notch intracellular domain (NICD), suggesting that α-S might compete with the NICD for binding to a common target. Supporting this hypothesis, the tryptophan (W) residue reported to be key for the interaction between the NICD and the Su(H) BTD domain is not only conserved in α-PheRS and α-S, but also essential for attenuating Notch signaling.

Contribution of upregulated aminoacyl-tRNA biosynthesis to metabolic dysregulation in gastric cancer.

BACKGROUND AND AIM: Metabolic reprogramming is characterized by dysregulated levels of metabolites and metabolic enzymes. Integrated metabolomic and transcriptomic data analysis can help to elucidate changes in the levels of metabolites and metabolic enzymes, screen the core metabolic pathways, and develop novel therapeutic strategies for cancer. METHODS: Here, the metabolome of gastric cancer tissues was determined using liquid chromatography-mass spectrometry. The transcriptome data from The Cancer Genome Atlas dataset were integrated with the liquid chromatography-mass spectrometry data to identify the common dysregulated gastric cancer-specific metabolic pathways. Additionally, the protein expression and clinical significance of key metabolic enzymes were examined using a gastric cancer tissue array. RESULTS: Metabolomic analysis of 16 gastric cancer tissues revealed that among the 15 dysregulated metabolomic pathways, the aminoacyl-tRNA biosynthesis pathway in the gastric tissues was markedly upregulated relative to that in the adjacent noncancerous tissues, which was consistent with the results of transcriptome analysis. Bioinformatic analysis revealed that among the key regulators in the aminoacyl-tRNA biosynthesis pathway, the expression levels of threonyl-tRNA synthetase (TARS) and phenylalanyl-tRNA synthetase (FARSB) were correlated with tumor grade and poor survival, respectively. Additionally, gastric tissue array data analysis indicated that TARS and FARSB were upregulated in gastric cancer tissues and were correlated with poor prognosis and tumor metastasis. CONCLUSIONS: This study demonstrated that the aminoacyl-tRNA biosynthesis pathway is upregulated in gastric cancer and both TARS and FARSB play key roles in the progression of gastric cancer. Additionally, a novel therapeutic strategy for gastric cancer was proposed that involves targeting the aminoacyl-tRNA biosynthesis pathway.

Early-onset epileptic encephalopathy with migrating focal seizures associated with a FARS2 homozygous nonsense variant.

Epilepsy of infancy with migrating focal seizures (EIMFS) is now a well-recognized early-onset syndrome included in the ILAE classification of the epilepsies. KCNT1 gain-of-function variants are identified in about half of patients. In the remaining cases, the underlying genetic component is far more heterogeneous with sporadic mutations occasionally reported in SCN1A, SCN2A, SLC12A5, TBC1D24, PLCB1, SLC25A22, and KCNQ2. Here, we report, for the first time, a homozygous deleterious variant in the FARS2 gene, identified using a 115-gene panel for monogenic epilepsies, in a patient with EIMFS. This boy was the second child born to healthy consanguineous parents. The first seizures occurred at six weeks of age. The patient rapidly developed severe epilepsy with focal discharges on EEG, migrating from one brain region to another, highly suggestive of EIMFS. At five months of age, he had daily multifocal clonic seizures and erratic myoclonic fits, which were not consistently related to spikes or spike-and-wave discharges. Neurological status was severely abnormal from onset and the patient died at 10 months of age from respiratory distress. Using the gene panel, a homozygous missense variant of FARS2 was identified, at Chr6 (GRCh37):g.5404829C>T, c.667C>T (NM_001318872.1), inherited from both parents, leading to an arginine-to-cysteine substitution, p.(Arg223Cys). FARS2 is a member of the mitochondrial aminoacyl tRNA transferase (ARS) enzymes. ARS variants are increasingly recognized causes of early-onset epileptic and neurodevelopmental encephalopathies, however, the associated epileptic phenotype is not completely described. This case shows that FARS2-related seizures can mimic EIMFS in the early stage of the disease. Furthermore, in the setting of migrating focal seizures of infancy, FARS2 should be considered as a further candidate gene, and increased lactate level and occurrence of refractory myoclonic seizures are possible key features to suspect FARS deficiency.

A patient with juvenile-onset refractory status epilepticus caused by two novel compound heterozygous mutations in FARS2 gene.

FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase and is implicated in autosomal recessive combined oxidative phosphorylation deficiency 14. The clinical manifestation can be divided into early onset epileptic phenotype and spastic paraplegia phenotype. The purpose of this study was to report a case of juvenile manifesting refractory epilepsy caused by two novel compound heterozygous mutations in the FARS2 gene. Microscopic and histochemical examination as well as next-generation sequencing and reconstruction of the three-dimensional structure of FARS2 protein were performed. A 17-year-old man with no developmental delays suffered from generalized tonic-clonic convulsion since 12 years of age and developed refractory status epilepticus 5 years later. No specific etiology was found following brain imaging, muscle biopsy and metabolic studies. DNA sequencing identified two novel compound heterozygous mutations in FARS2, (p.V197M and p.F402S), derived from each parents, respectively. These mutations affected the structure or thermodynamic stability of the protein. This is a case report of juvenile-onset refractory epilepsy caused by two novel compound heterozygous mutations in the FARS2 gene. This case confirms and expands the clinicalphenotype and the genotypic spectrum of the FARS2 gene.

FARSA mutations mimic phenylalanyl-tRNA synthetase deficiency caused by FARSB defects.

Pathogenic variants in genes encoding aminoacyl-tRNA synthetases cause numerous disorders characterized by involvement of neurons, muscles, lungs and liver. Recently, biallelic FARSB defects have been shown to cause severe growth restriction with combined brain, liver and lung involvement (Rajab interstitial lung disease [ILD] with brain calcifications). Herein, for the first time, we present a patient with similar condition associated with biallelic mutations in FARSA (NM_004461.3: c.766T>C:p.Phe256Leu and c.1230C>A:p.Asn410Lys). Both detected FARSA variants are ultrarare and predicted to be damaging by in silico programs. Furthermore, they are both located in the active site of phenylalanyl-tRNA synthetase (PheRS) with Asn410Lys directly affecting a residue forming the wall of the phenylalanine-binding pocket. Clinical features shared between our patient and the FARSB syndrome include ILD with cholesterol pneumonitis, growth delay, hypotonia, brain calcifications with cysts and liver dysfunction. Our findings indicate that a disease similar to a syndrome associated with FARSB defects can also be caused by biallelic FARSA mutations. These findings are consistent with molecular structure of PheRS which is a tetramer including both FARSA and FARSB proteins.

Biophysical characterization Of Alpers encephalopathy associated mutants of human mitochondrial phenylalanyl-tRNA synthetase.

Mutations in nucleus-encoded mitochondrial aminoacyl-tRNA synthetases (mitaaRSs) lead to defects in mitochondrial translation affecting the expression and function of 13 subunits of the respiratory chain complex leading to diverse pathological conditions. Mutations in the FARS2 gene encoding human mitochondrial phenylalanyl-tRNA synthetase (HsmitPheRS) have been found to be associated with two different clinical representations, infantile Alpers encephalopathy and spastic paraplegia. Here we have studied three pathogenic mutants (Tyr144Cys, Ile329Thr, and Asp391Val) associated with Alpers encephalopathy to understand how these variants affect the biophysical properties of the enzyme. These mutants have already been reported to have reduced aminoacylation activity. Our study established that the mutants are significantly more thermolabile compared to the wild-type enzyme with reduced solubility in vitro. The presence of aggregation-prone insoluble HsmitPheRS variants could have a detrimental impact on organellar translation, and potentially impact normal mitochondrial function. © 2019 IUBMB Life, 71(8): 1141-1149, 2019 © 2019 IUBMB Life, 71(8):1141-1149, 2019.

Quantitative Lipidomics in Pulmonary Alveolar Proteinosis.

Rationale: Pulmonary alveolar proteinosis (PAP) is characterized by filling of the alveolar spaces by lipoprotein-rich material of ill-defined composition, and is caused by molecularly different and often rare diseases that occur from birth to old age.Objectives: To perform a quantitative lipidomic analysis of lipids and the surfactant proteins A, B, and C in lavage fluids from patients with proteinosis of different causes in comparison with healthy control subjects.Methods: During the last two decades, we have collected BAL samples from patients with PAP due to autoantibodies against granulocyte-macrophage colony-stimulating factor; genetic mutations in CSF2RA (colony-stimulating factor 2 receptor α-subunit), MARS (methionyl aminoacyl-tRNA synthetase), FARSB (phenylalanine-tRNA synthetase, ß-subunit), and NPC2 (Niemann-Pick disease type C2); and secondary to myeloid leukemia. Their lipid composition was quantified.Measurements and Main Results: Free cholesterol was largely increased by 60-fold and cholesteryl esters were increased by 24-fold. There was an excessive, more than 130-fold increase in ceramide and other sphingolipids. In particular, the long-chain ceramides d18:1/20:0 and d18:1/24:0 were elevated and likely contributed to the proapoptotic environment observed in PAP. Cellular debris lipids such as phosphatidylethanolamine and phosphatidylserine were only moderately increased, by four- to sevenfold. The surfactant lipid class phosphatidylcholine expanded 17-fold, lysophosphatidylcholine expanded 54-fold, and the surfactant proteins A, B, and C expanded 144-, 4-, and 17-fold, respectively. These changes did not differ among the various diseases that cause PAP.Conclusions: This insight into the alveolar lipidome may provide monitoring tools and lead to new therapeutic strategies for PAP.

Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase.

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA(Phe) We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA(Phe) with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNA(Phe) species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress.

Novel Compound Heterozygous Mutations Expand the Recognized Phenotypes of FARS2-Linked Disease.

Mutations in mitochondrial aminoacyl-tRNA synthetases are an increasingly recognized cause of human diseases, often arising in individuals with compound heterozygous mutations and presenting with system-specific phenotypes, frequently neurologic. FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase (mtPheRS), perturbations of which have been reported in 6 cases of an infantile, lethal disease with refractory epilepsy and progressive myoclonus. Here the authors report the case of juvenile onset refractory epilepsy and progressive myoclonus with compound heterozygous FARS2 mutations. The authors describe the clinical course over 6 years of care at their institution and diagnostic studies including electroencephalogram (EEG), brain magnetic resonance imaging (MRI), serum and cerebrospinal fluid analyses, skeletal muscle biopsy histology, and autopsy gross and histologic findings, which include features shared with Alpers-Huttenlocher syndrome, Leigh syndrome, and a previously published case of FARS2 mutation associated infantile onset disease. The authors also present structure-guided analysis of the relevant mutations based on published mitochondrial phenylalanyl transfer RNA synthetase and related protein crystal structures as well as biochemical analysis of the corresponding recombinant mutant proteins.

Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code.

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.

Aminoacylation of tRNA 2'- or 3'-hydroxyl by phosphoseryl- and pyrrolysyl-tRNA synthetases.

Class I and II aminoacyl-tRNA synthetases (AARSs) attach amino acids to the 2'- and 3'-OH of the tRNA terminal adenosine, respectively. One exception is phenylalanyl-tRNA synthetase (PheRS), which belongs to Class II but attaches phenylalanine to the 2'-OH. Here we show that two Class II AARSs, O-phosphoseryl- (SepRS) and pyrrolysyl-tRNA (PylRS) synthetases, aminoacylate the 2'- and 3'-OH, respectively. Structure-based-phylogenetic analysis reveals that SepRS is more closely related to PheRS than PylRS, suggesting that the idiosyncratic feature of 2'-OH acylation evolved after the split between PheRS and PylRS. Our work completes the understanding of tRNA aminoacylation positions for the 22 natural AARSs.

Enterococcus alcedinis sp. nov., isolated from common kingfisher (Alcedo atthis).

Two Gram-positive, catalase-negative bacterial strains were isolated from the cloaca of common kingfishers (Alcedo atthis). Repetitive sequence-based PCR fingerprinting using the (GTG)5 primer grouped these isolates into a single cluster separated from all known enterococcal species. The two strains revealed identical 16S rRNA gene sequences placing them within the genus Enterococcus with Enterococcus aquimarinus LMG 16607(T) as the closest relative (97.14 % similarity). Further taxonomic investigation using sequencing of the genes for the superoxide dismutase (sodA), phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) as well as application of whole-cell protein fingerprinting, automated ribotyping and extensive phenotyping confirmed that both strains belong to the same species. Based on data from this polyphasic study, these strains represent a novel species of the genus Enterococcus, for which the name Enterococcus alcedinis sp. nov. is proposed. The type strain is L34(T) (= CCM 8433(T) = LMG 27164(T)).

Leuconostoc mesenteroides subsp. suionicum subsp. nov.

Strains LMG 8159 and LMG 11499 were reclassified by a polyphasic approach, including 16S rRNA gene sequence analysis, 16S-23S rRNA intergenic spacer (IGS) sequence analysis, (GTG)(5)-PCR fingerprinting, RAPD fingerprinting, fatty acid methyl ester analysis and an analysis of phenotypic features using API 50 CH. The two strains were closely related to the type strains of the three defined subspecies of Leuconostoc mesenteroides, showing 99.7-99.9% 16S rRNA gene sequence similarity, 99.2% 16S-23S rRNA gene intergenic spacer sequence similarity, 97.1-97.4% pheS gene sequence similarity and 98.0-98.2% rpoA gene sequence similarity. Low atpA gene sequence similarity (91.4-91.7%), (GTG)(5)-PCR fingerprinting, RAPD fingerprinting, fatty acid compositions and phenotypic features allowed us to differentiate strains LMG 8159 and LMG 11499 from all established subspecies within L. mesenteroides. Based upon the data obtained in the present and previous studies, a novel subspecies is proposed within the species L. mesenteroides, Leuconostoc mesenteroides subsp. suionicum subsp. nov., with the type strain LMG 8159(T) (=ATCC 9135(T) =DSM 20241(T) =NCIMB 6992(T)).

Identification of enterococci from broiler products and a broiler processing plant and description of Enterococcus viikkiensis sp. nov.

In two previous studies dealing with lactic acid bacteria (LAB) from modified-atmosphere-packaged (MAP) broiler products and a broiler processing plant, several isolates remained unidentified. According to 16S rRNA gene sequence analysis, 36 isolates were assigned to the genus Enterococcus. Numerical analysis of combined HindIII and EcoRI ribopatterns of these isolates resulted in species-specific clusters that were congruent with the clusters obtained by both DNA-directed RNA polymerase subunit A (rpoA) and phenylalanyl-tRNA synthetase α chain (pheS) housekeeping gene analyses. In the analyses, a group of five isolates distinct from any known enterococcal species clustered together. The five isolates were positioned in the Enterococcus avium group, with E. devriesei being the closest phylogenetic neighbor. The DNA-DNA hybridization levels with E. devriesei ranged from 28.8 to 54.3% and indicated that these strains represented a novel species. The name Enterococcus viikkiensis sp. nov. is proposed, with strain DSM 24043(T) (LMG 26075(T)) being the type strain. Our study demonstrated that the identification of enterococci within the E. avium phylogenetic group demands polyphasic taxonomic approaches. The rpoA and pheS gene similarities (99.0 to 99.2% and 94.3 to 95.4%, respectively) between E. viikkiensis and its closest phylogenetic neighbor, E. devriesei, were higher than those previously reported within the enterococci. In addition, the phenotypic profiles of the species in the E. avium group were also highly similar, and some traits were found to be misleading for enterococci, such as E. viikkiensis does not grow at 45°C. The numerical analysis of combined HindIII and EcoRI ribopatterns was of considerable assistance in distinguishing enterococcal species within the E. avium group.

Polyphasic taxonomic characterization of Lactobacillus rossiae isolates from Belgian and Italian sourdoughs reveals intraspecific heterogeneity.

(GTG)5-PCR fingerprinting and pheS sequence analysis of 18 Lactobacillus rossiae isolates, mainly originating from Belgian and Italian artisan sourdoughs, revealed intraspecies grouping as evidenced by the delineation of three and two subgroups, respectively. On the other hand, 16S rRNA and rpoA gene sequence analysis and DNA-DNA hybridizations supported the accommodation of all isolates in a single species. No correlation between genetic and phenotypic heterogeneity was observed. Collectively, these data do not warrant taxonomic division of L. rossiae. On the other hand, the considerable differences in intraspecies sequence variation of L. rossiae isolates displayed by the pheS (9.8%) and rpoA (1.1%) genes highlight that the discriminatory power of housekeeping genes as alternative genomic markers for the 16S rRNA gene in the identification of Lactobacillus species may significantly differ from gene to gene. In conclusion, this study has demonstrated that a polyphasic approach remains highly useful for identification of isolates belonging to genotypically heterogeneous species such as L. rossiae.

Lactobacillus sobrius Konstantinov et al. 2006 is a later synonym of Lactobacillus amylovorus Nakamura 1981.

While studying the taxonomy of six lactic acid bacterium isolates from Finnish porcine intestine and faeces, the taxonomic positions of Lactobacillus sobrius type strain DSM 16698T and strain AD5 based on comparative 16S rRNA sequence analysis were found to be controversial, as they showed high similarity to Lactobacillus amylovorus strains. Therefore, the taxonomy of these species was addressed in a polyphasic taxonomy study that included, in addition to re-evaluating the 16S rRNA gene sequence and DNA-DNA reassociation results, multilocus sequence analysis (MLSA) of the housekeeping genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) as well as numerical analysis of HindIII and EcoRI ribotypes. 16S rRNA gene sequence analysis demonstrated a very high similarity between the L. sobrius and L. amylovorus type and reference strains and representative Finnish porcine isolates (99.6-99.9 %). The MLSA data showed the close phylogenetic relationship of these strains; pheS and rpoA gene sequence similarities were 98.5-100 % and 99.6-99.8 %, respectively. Numerical analyses of HindIII/EcoRI ribotypes placed these strains in a single cluster by both enzymes. Finally, the DNA-DNA reassociation experiments revealed high reassociation levels (higher than 79 %) between the strains. These results indicate that DSM 16698T, AD5 and the related porcine lactobacilli strains from Finland constitute a single species, Lactobacillus amylovorus, and that the name Lactobacillus sobrius should be considered as a later synonym of Lactobacillus amylovorus.

Influence of geographical origin and flour type on diversity of lactic acid bacteria in traditional Belgian sourdoughs.

A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.

Lactobacillus crustorum sp. nov., isolated from two traditional Belgian wheat sourdoughs.

A polyphasic taxonomic study of the lactic acid bacteria (LAB) population in three traditional Belgian sourdoughs, sampled between 2002 and 2004, revealed a group of isolates that could not be assigned to any recognized LAB species. Initially, sourdough isolates were screened by means of (GTG)(5)-PCR fingerprinting. Four isolates displaying unique (GTG)(5)-PCR patterns were further investigated by means of phenylalanyl-tRNA synthase (pheS) gene sequence analysis and represented a bifurcated branch that could not be allocated to any LAB species present in the in-house pheS database. Their phylogenetic affiliation was determined using 16S rRNA gene sequence analysis and showed that the four sourdough isolates belong to the Lactobacillus plantarum group with Lactobacillus mindensis, Lactobacillus farciminis and Lactobacillus nantensis as closest relatives. Further genotypic and phenotypic studies, including whole-cell protein analysis (SDS-PAGE), amplified fragment length polymorphism (AFLP) fingerprinting, DNA-DNA hybridization, DNA G+C content analysis, growth characteristics and biochemical features, demonstrated that the new sourdough isolates represent a novel Lactobacillus species for which the name Lactobacillus crustorum sp. nov. is proposed. The type strain of the new species is LMG 23699(T) (=CCUG 53174(T)).

Reclassification of Lactobacillus amylophilus LMG 11400 and NRRL B-4435 as Lactobacillus amylotrophicus sp. nov.

The taxonomic position of six Lactobacillus amylophilus strains isolated from swine waste-corn fermentations was reinvestigated. All strains were included in a multilocus sequence analysis (MLSA) study for species identification of Lactobacillus using the genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA). Partial pheS and rpoA gene sequences showed that strains LMG 11400 and NRRL B-4435 represent a separate lineage that is distantly related to the type strain of L. amylophilus, LMG 6900T, and to three other strains of the species. The MLSA data showed that the two strains LMG 11400 and NRRL B-4435 constituted a distinct cluster, sharing 100% pheS and rpoA gene sequence similarity. The other reference strains clustered together with the type strain of L. amylophilus, LMG 6900T, and were clearly differentiated from strains LMG 11400 and NRRL B-4435 (80 and 89% pheS and rpoA gene sequence similarity, respectively). The 16S rRNA gene sequences of the latter two strains are 100% identical, with the nearest phylogenetic neighbour L. amylophilus LMG 6900T showing only 97.2% 16S rRNA gene sequence similarity. Further polyphasic taxonomic study based on whole-cell protein fingerprinting, DNA-DNA hybridization and biochemical features demonstrated that the two strains represent a single, novel Lactobacillus species, for which the name Lactobacillus amylotrophicus sp. nov. is proposed. The type strain is LMG 11400T (=NRRL B-4436T=DSM 20534T).