Taxonomic studies on some leuconostoc-like organisms from fermented sausages: description of a new genus Weissella for the Leuconostoc paramesenteroides group of species

Taxonomic studies were performed on some unknown Leuconostoc -like organisms from fermented Greek sausage. Comparative 16S rRNA sequence analysis showed the unidentified organisms represent a new line within the Leuconostoc paramesenteroides group of species. On the basis of the results of this and earlier phylogenetic investigations, it is proposed that Leuconostoc paramesenteroides and related species be reclassified in a new genus Weissella. In addition a new species, Weissella hellenica , is proposed for the isolates from fermented sausage.     

Identification of Carnobacterium spp. and Leuconostoc spp. in meat by genus-specific 16S rRNA probes

Oligonucleotide probes specific for Carnobacterium and Leuconostoc species were constructed from the variable regions of 16S rRNA obtained from the literature and sequence data bases. The probes were hybridized with crude nucleic acid extract from 32 type strains of lactic acid bacteria (LAB) commonly found on meat. Two of the probes hybridized only to the four Carnobacterium species whereas the other two hybridized only to five of the six Leuconostoc species tested. The probes were also hybridized with nucleic acids from unknown strains of LAB. The identification was consistent with the results of biochemical tests used to characterize the two genera.     

Ruminococcus luti sp. nov., isolated from a human faecal sample

A strain of an unidentified strictly anoxic, gram-postive, non-motile Ruminococcus-like bacterium was isolated from a human faecal sample. The organism used carbohydrates as fermentable substrates, produced acetate, succinate, and hydrogen as the major products of glucose metabolism, and possessed a G + C content of 43.3 mol%. The morphological and biochemical characteristics of the organism were consistent with its assignment to the genus Ruminococcus but it did not correspond to any recognized species of this genus. Comparative 16S rRNA gene sequencing showed the unidentified bacterium represents a previously unrecognised sub-line within the Clostridium coccoides rRNA group of organisms. The nearest relative of the unknown bacterium corresponded to Ruminococcus obeum but a 16S rRNA sequence divergence value of >3% demonstrated it represents a different species. Based on the presented findings a new species, Ruminococcus luti, is described. The type strain of Ruminococcus luti is BInIX(T) (DSM 14534T, CCUG 45635T).     

The phosphoenolpyruvate carboxylase from Methanothermobacter thermautotrophicus has a novel structure

In Methanothermobacter thermautotrophicus, oxaloacetate synthesis is a major and essential CO(2)-fixation reaction. This methanogenic archaeon possesses two oxaloacetate-synthesizing enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxylase. The phosphoenolpyruvate carboxylase from this organism was purified to homogeneity. The subunit size of this homotetrameric protein was 55 kDa, which is about half that of all known bacterial and eukaryotic phosphoenolpyruvate carboxylases (PPCs). The NH(2)-terminal sequence identified this enzyme as the product of MTH943, an open reading frame with no assigned function in the genome sequence. A BLAST search did not show an obvious sequence similarity between MTH943 and known PPCs, which are generally well conserved. This is the first report of a new type of phosphoenolpyruvate carboxylase that we call PpcA ("A" for "archaeal"). Homologs to PpcA were present in most archaeal genomic sequences, but only in three bacterial (Clostridium perfringens, Oenococcus oeni, and Leuconostoc mesenteroides) and no eukaryotic genomes. PpcA was the only recognizable oxaloacetate-producing enzyme in Methanopyrus kandleri, a hydrothermal vent organism. Each PpcA-containing organism lacked a PPC homolog. The activity of M. thermautotrophicus PpcA was not influenced by acetyl coenzyme A and was about 50 times less sensitive to aspartate than the Escherichia coli PPC. The catalytic core (including His(138), Arg(587), and Gly(883)) of the E. coli PPC was partly conserved in PpcA, but three of four aspartate-binding residues (Lys(773), Arg(832), and Asn(881)) were not. PPCs probably evolved from PpcA through a process that added allosteric sites to the enzyme. The reverse is also equally possible.     

A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice

E thiraj , S. & S uresh , E.R. 1985. A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice. Journal of Applied -Bacteriology 59 , 239–242.
A strain of Leuconostoc oenos was isolated from a blown can of mango juice. Various tests to identify and characterize the bacterium suggested that it could be a strain of L. oenos . This is the first report of L. oenos as a spoilage organism in fruit products other than wine.     

Cetobacterium somerae sp. nov. from human feces and emended description of the genus Cetobacterium

Phenorypic and phylogenetic studies were performed on four isolates of an unidentified gram-negative, microaerotolerant, non-spore-forming, rod-shaped bacterium isolated from the feces of children. The unknown organism was bile resistant and produced acetic acid as the major end product of metabolism of peptides and carbohydrates. It possessed a low DNA G + C content of 31 mol %. Comparative 16S rRNA gene sequencing demonstrated that the four isolates were phylogenetically identical (100% 16S rRNA sequence similarity) and represent a hitherto unknown sub-line within the genus Cetobacterium. The novel bacterium displayed approximately 5% sequence divergence with Cetobacterium ceti, and can be readily distinguished from the latter by physiological and biochemical criteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown fecal bacterium be classified in the genus Cetobacterium, as Cetobacterium somerae sp. nov. The proposed type strain of Cetobacterium somerae is WAL 14325(T) (ATCC BAA-474(T) = CCUG 46254T).     

Characterization of Leucocin B-KM432Bz from Leuconostoc pseudomesenteroides Isolated from Boza,and Comparison of its Efficiency to Pediocin PA-1

A bacteriocin-producing bacterium was isolated from boza and identified as Leuconostoc pseudomesenteroides KM432Bz. The antimicrobial peptide was purified and shown to be identical to other class IIa bacteriocins: leucocin A from Leuconostoc gelidum UAL-187 and Leuconostoc pseudomesenteroides QU15 and leucocin B from Leuconostoc carnosum Ta11a. The bacteriocin was named leucocin B-KM432Bz. Leucocin B-KM432Bz gene cluster encodes the bacteriocin precursor (lcnB), the immunity protein (lcnI) and the dedicated export machinery (lcnD and lcnE). A gene of unknown and non-essential function (lcnC), which is interrupted by an insertion sequence of the IS30 family, is localized between lcnB and lcnD. The activity of leucocin B-KM432Bz requires subunit C of the EII^t _Man mannose permease, which is the receptor for entry into target cells. The determination of the minimum inhibitory concentrations revealed the lowest values for leucocin B-KM432Bz over Listeria strains, with 4 to 32 fold better efficiency than pediocin PA-1.     

Characterization of a psychrotrophic Clostridium causing spoilage in vacuum-packed cooked pork: description of Clostridium algidicarnis sp. nov.

A Clostridium species causing spoilage of vacuum-packed refrigerated pork was isolated and characterized. The unknown organism differed phenotypically from other clostridial species usually associated with spoilage. Phylogenetic analyses based on 16S rRNA gene sequencing demonstrated that the psychrotroph represents a distinct line of descent within the genus Clostridium. It is proposed that the organism be classified as a new species of the genus Clostridium, Clostridium algidicarnis .     

Methanosarcina lacustris sp. nov., a new psychrotolerant methanogenic archaeon from anoxic lake sediments

A new psychrotolerant methanogenic archaeon strain ZS was isolated from anoxic lake sediments (Switzerland). The cells of the organism were non-motile cocci, 1.5-3.5 microm in diameter. The cells aggregated and formed pseudoparenchyma. The cell wall was Gram-positive. The organism utilized methanol, mono-, di-, trimethylamine and H2/CO2 with methane production. The temperature range for growth was 1-35 degrees C with an optimum at 25 degrees C. The DNA G+C content of the organism was 43.4. mol%. Analysis of the 16S rRNA gene sequence showed that strain ZS was phylogenetically closely related to members of the genus Methanosarcina, but clearly differed from all described species of this genus (95.6-97.6% of sequence similarity). The level of DNA-DNA hybridization of strain ZS with Methanosarcina barkeri and Methanosarcina mazei was 15 and 31%, respectively. Based on the results of physiological and phylogenetic studies strain ZS can be assigned to a new species of the genus Methanasarcina. The name Methanosarcina lacustris sp. nov. is proposed. The type strain is ZS (= DSM 13486T, VKM B-2268).     

Characterization of Leuconostoc gasicomitatum sp. nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions

Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product. Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called "protein swell." This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L. gelidum was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L. gelidum type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the L. gelidum type strain. These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp. nov. for this spoilage-associated Leuconostoc species.     

Mycobacterium confluentis sp. nov.

A new rapidly growing mycobacterium was isolated from human sputum. This organism grew at 22, 31, 37, and 41 degrees C and possessed catalase, acid phosphatase, acetamidase, urease, nicotinamidase, pyrazinamidase, and nitrate reductase activities. It did not produce nicotinic acid, hydrolyze Tween, or have benzamidase, isonicotinamidase, succinidamidase, and arylsulfatase activities. A mycolic acid analysis revealed a simple, unique pattern. The organism is susceptible to antituberculotic drugs. A comparative 16S rRNA sequence analysis placed this organism within the confines of the genus Mycobacterium, most closely related to the thermotolerant rapidly growing species. On the basis of the pattern of enzymatic activities and metabolic properties, as well as the unique 16S rRNA sequence, we propose that our single strain represents a new species, for which we propose the name Mycobacterium confluentis. The type strain is strain 1389/90; a culture of this strain has been deposited in the German Collection of Microorganisms and Cell Cultures as strain DSM 44017.     

Leuconostoc mesenteroides subsp. mesenteroides FR52 synthesizes two distinct bacteriocins

A.M. REVOL-JUNELLES, R. MATHIS, F. KRIER, Y. FLEURY, A. DELFOUR AND G. LEFEBVRE. 1996. Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, was purified from producing cells by the adsorption-desorption method, combined with reverse-phase high-performance liquid chromatography. The elution profile revealed the presence o two inhibitory peaks of activity, each displaying different inhibitory spectra. Mesenterocin 52A possessed a broad inhibitory spectrum, including anti- Listeria activity, while Mesenterocin 52B was only active against Leuconostoc spp. The amino acid sequence and M_r of Mesenterocin 52A appeared identical to the previously described Mesentericing Y105. In contrast, Mesenerocin 52B possessed a M_r of 3446 Da, corresponding to 32 amino acids and a sequence that shared no homology with known bacteriocins:     

A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice

A strain of Leuconostoc oenos was isolated from a blown can of mango juice. Various tests to identify and characterize the bacterium suggested that it could be a strain of L. oenos. This is the first report of L. oenos as a spoilage organism in fruit products other than wine.     

Identification and characterization of Lactic Acid Bacteria isolated from Tibetan Qula cheese

Fourteen strains of Lactic Acid Bacteria (LAB) isolated from Qula, a Tibetan traditional yak cheese, were divided into four groups (A-D) according to morphological and biochemical characteristics. On the basis of the 16S rRNA gene sequence analysis, group A and group B strains were placed in the cluster making up the genus Leuconostoc, which together with Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, formed a distinct cluster. The group C strain was clearly identified as Enterococcus faecium by forming a very well defined cluster with this species. The group D strains were placed in the lactobacilli cluster with Lactobacillus plantarum and Lactobacillus pentosus being the closely related species. On the basis of DNA-DNA hybridization, strains in the groups A, B, C and D were identified as Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc pseudomesenteroides, Enterococcus faecium and Lactobacillus plantarum, respectively. Leuconostoc mesenteroides subsp. dextranicum was the dominate member of the population.     

Identification of a replicon from pTXL1, a small cryptic plasmid from Leuconostoc mesenteroides subsp. mesenteroides Y110, and development of a food-grade vector

A 2,665-bp cryptic plasmid, pTXL1, isolated from Leuconostoc mesenteroides subsp. mesenteroides Y110 was identified. This plasmid harbors a replicon localized on a 1,300-bp fragment. Two observations suggested that pTXL1 does not belong to rolling-circle replication (RCR)-type plasmids and most likely replicates via a theta mechanism. These hypotheses are supported by the observation that no detectable single-stranded intermediate was found for the replicon and that, unlike in RCR-type plasmids, the pTXL1 replicon sequence lacks an open reading frame encoding a replicase. The small-sized pTXL1 plasmid is stable and, according to its origin, can be considered in the "generally recognized as safe" category. Its ability to replicate in several lactic acid bacteria was exploited to develop a vector producing mesentericin Y105, a class II anti-Listeria bacteriocin. With this new vector, a recombinant industrial Leuconostoc cremoris strain able to produce mesentericin Y105 was constructed.     

A cryptic miniplasmid from the hyperthermophilic bacterium Thermotoga sp. strain RQ7.

An 846-bp cryptic plasmid has been discovered in the hyperthermophilic bacterium Thermotoga sp. strain RQ7. This is the first plasmid described for an organism from this ancient bacterial lineage and the smallest plasmid described to date for any organism. Nucleotide sequencing revealed a single open reading frame possibly encoding a 25,460-Da basic protein (212 amino acids). Upstream of the putative promoter lie five 11-bp direct repeats, each separated by 1 to 4 bp, while between the promoter and the open reading frame lies an 11-bp palindromic sequence. Its mode of replication is unknown, but its sequence bears similarities to those of plasmids which replicate by a rolling-circle mechanism.     

Subdoligranulum variabile gen. nov., sp. nov. from human feces

During studies on the microflora of human feces we have isolated a strictly anaerobic, non-spore-forming, Gram-negative staining organism which exhibits a somewhat variable coccus-shaped morphology. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism is phylogenetically a member of the Clostridium leptum supra-generic rRNA cluster and displays a close affinity to some rDNA clones derived from human and pig feces. The nearest named relatives of the unidentified isolate corresponded to Faecalibacterium prausnitzii (formerly Fusobacterium prausnitzii) displaying a 16S rRNA sequence divergence of approximately 9%, with Anaerofilum agile and A. pentosovorans the next closest relatives of the unidentified bacterium (sequence divergence approximately 10%). Based on phenotypic and phylogenetic considerations, it is proposed that the unusual coccoid-shaped organism be classified as a new genus and species, Subdoligranulum variabile. The type strain of S. variabile is BI 114(T) (=CCUG 47106(T)=DSM 15176(T)).     

Ponticoccus gilvus gen. nov., sp. nov., a novel member of the family Propionibacteriaceae from seawater

A novel actinobacterium, designated strain MSW-19^T, was isolated from a seawater sample in Republic of Korea. Cells were aerobic, Gram-positive, non-endospore-forming, and non-motile cocci. Colonies were circular, convex, opaque, and vivid yellow in colour. A phylogenetic tree based on 16S rRNA gene sequences exhibited that the organism formed a distinct clade within the radius encompassing representatives of the family Propionibacteriaceae. The phylogenetic neighbors were the type strains of the genera Friedmanniella, Microlunatus, Micropruina, Propionicicella, and Propionicimonas. Levels of 16S rRNA gene sequence similarity between the isolate and members of the family were less than 95.3%. The cell wall peptidoglycan of the organism contained LL-diaminopimelic acid as the diagnostic diamino acid. The isolate contained MK-9(H₄) as the predominant menaquinone, ai-C_15:0 as the major fatty acid and polar lipids including phosphatidylglycerol, phosphatidylethanolamine, and an unknown phospholipid. The G+C content of the DNA was 69.6 mol%. On the basis of the phenotypic and phylogenetic data presented here, the isolate is considered to represent a novel genus and species in the family Propionibacteriaceae, for which the name Ponticoccus gilvus gen. nov., sp. nov. is proposed. The type strain is strain MSW-19^T (= KCTC 19476^T= DSM 21351^T).     

Characterization of plasmid-encoded citrate permease (citP) genes from Leuconostoc species reveals high sequence conservation with the Lactococcus lactis citP gene.

The citrate permease determinant (citP) in several Leuconostoc strains was demonstrated to be plasmid encoded by curing experiments and hybridization studies with a DNA fragment containing the citP gene from Lactococcus lactis subsp. lactis biovar diacetylactis NCDO176. Cloning and nucleotide sequence analysis of Leuconostoc lactis NZ6070 citP revealed almost complete identity to lactococcal citP.