Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus helveticus

Abstract Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus . Only Lb. delbrückii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis . No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains.     

Genetic Markers Unique to Listeria monocytogenes Serotype 4b Differentiate Epidemic Clone II (Hot Dog Outbreak Strains) from Other Lineages

A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.     

Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus

Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus. Only Lb. delbrückii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis. No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains.     

Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus,Lactobacillus crispatus,Lactobacillus amylovorus,Lactobacillus gallinarum,Lactobacillus gasseri,and Lactobacillus johnsonii

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.     

Screening of Lactobacilli derived from chicken feces and partial characterization of Lactobacillus acidophilus A12 as an animal probiotics

This study was performed to screen and select Lactobacillus strains from chicken feces for probiotic use in animals. Of these strains, strain A12 had the highest immunostimulatory effect. Therefore, strain A12 was characterized as a potential probiotic. Strain A12 was tentatively identified as Lactobacillus acidophilus A12, using the API 50 CHL kit based on a 99.9% homology. L. acidophilus A12 was highly resistant to artificial gastric juice (pH 2.5) and bile acid (oxgall). Based on results from the API ZYM kit, leucine arylamidase, crystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, alpha-galactosidase, beta- galactosidase, alpha-glucosidase, beta-glucosidase, and N-acetyl-beta- glucosamidase were produced by strain A12. L. acidophilus A12 showed resistance to several antibiotics (nisin, gentamycin, and erythromycin). The amount of interleukin (IL)-1alpha in 20x concentrated supernatant from L. acidophilus A12 was approximately 156 pg/ml. With regard to antioxidant activity, L. acidophilus A12 supernatant showed 60.6% DPPH radical scavenging activity. These results demonstrate the potential use of L. acidophilus A12 as a health-promoting probiotics.     

Evaluation of Numerical Analysis of Random Amplified Polymorphic DNA (RAPD)-PCR as a Method to Differentiate Lactobacillus plantarum and Lactobacillus pentosus

Lactobacillus plantarum and Lactobacillus pentosus grouped into one protein profile cluster at r ≥ 0.70, separate from Lactobacillus casei, Lactobacillus sake, and Lactobacillus curvatus. Similar sugar fermentation reactions were recorded for representative strains of L. plantarum and L. pentosus. Representative strains, including the type of each species, were selected from the different protein profile clusters and their genetic relatedness determined by using numerical analysis of random amplified polymorphic DNA (RAPD)-PCR. The type strains of L. plantarum (ATCC 14917^T) and L. pentosus (NCFB 363^T) displayed different RAPD profiles and grouped into two independent clusters, well separated from L. casei, L. curvatus, and L. sake. Numerical analysis of RAPD-PCR proved a reliable and accurate method to distinguish between strains of L. plantarum and L. pentosus.     

Development and Use of a Polyvalent Conjugate to Differentiate Histoplasma capsulatum and Histoplasma duboisii from Other Pathogens

Previous investigations have demonstrated the existence of five Histoplasma capsulatum serotypes. Available specific fluorescent-antibody reagents stain only four of the five serotypes. Antibodies produced against the most complete H. capsulatum serotype were labeled with fluorescein isothiocyanate to develop a reagent specific for H. capsulatum that was reactive with all the known serotypes. The unadsorbed reagent not only stained all the H. capsulatum serotypes, but it also stained cultures of Blastomyces dermatitidis, H. duboisii, several Candida species, and a variety of other fungi. Adsorption of the conjugate with antigens of C. albicans produced a reagent that intensely stained only H. capsulatum, H. duboisii, and B. dermatitidis. Differentiation of B. dermatitidis from the Histoplasma species was accomplished by application of a B. dermatitidis specific fluorescent antibody to antigens positive with the H. capsulatum reagent. At present, differentiation of H. capsulatum from H. duboisii may be accomplished only by animal inoculation. Our data substantiate the antigenic relationships hypothesized earlier, and they indicate that H. capsulatum shares at least two antigens with the other fungi that were studied.     

Vibrio vulnificus Phage PV94 Is Closely Related to Temperate Phages of V. cholerae and Other Vibrio Species

Background

Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1) infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species.

Results

In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5′-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218.

Conclusion

We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species.     

Genetic diversity in the lactose operons of Lactobacillus helveticus strains and its relationship to the role of these strains as commercial starter cultures

Two novel insertion sequence elements, ISLhe1 and ISLhe15, were located upstream of the genes encoding the beta-galactosidase enzyme in Lactobacillus helveticus commercial starter strains. Strains with the IS982 family element, ISLhe1, demonstrated reduced beta-galactosidase activity compared to the L. helveticus type strain, whereas strains with the ISLhe15 element expressed beta-galactosidase in the absence of lactose.     

Genetic Diversity in the Lactose Operons of Lactobacillus helveticus Strains and Its Relationship to the Role of These Strains as Commercial Starter Cultures

Two novel insertion sequence elements, ISLhe1 and ISLhe15, were located upstream of the genes encoding the β-galactosidase enzyme in Lactobacillus helveticus commercial starter strains. Strains with the IS982 family element, ISLhe1, demonstrated reduced β-galactosidase activity compared to the L. helveticus type strain, whereas strains with the ISLhe15 element expressed β-galactosidase in the absence of lactose.     

Two Closely Related Novel Picornaviruses in Cattle and Sheep in Hungary from 2008 to 2009, Proposed as Members of a New Genus in the Family Picornaviridae

Two novel picornaviruses were serendipitously identified in apparently healthy young domestic animals—cattle (Bos taurus) and, subsequently, sheep (Ovis aries)—in Hungary during 2008 and 2009. Complete genome sequencing and comparative analysis showed that the two viruses are related to each other and have identical genome organizations, VPg + 5′ UTR^IRES-II[L/1A-1B-1C-1D-2A^NPG↓P/2B-2C/3A-3B^VPg-3C^pro-3D^pol] 3′ UTR-poly(A). We suggest that they form two novel viral genotypes/serotypes, bovine hungarovirus 1 (BHuV-1; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"JQ941880","term_id":"406066046","term_text":"JQ941880"}}JQ941880) and ovine hungarovirus 1 (OHuV-1; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"HM153767","term_id":"404897591","term_text":"HM153767"}}HM153767), which may belong to a potential novel picornavirus genus in the family Picornaviridae. The genome lengths of BHuV-1 and OHuV-1 are 7,583 and 7,588 nucleotides, each comprising a single open reading frame encoding 2,243 and 2,252 amino acids, respectively. In the 5′ untranslated regions (5′ UTRs), both hungaroviruses are predicted to have a type II internal ribosome entry site (IRES). The nucleotide sequence and the secondary RNA structure of the hungarovirus IRES core domains H-I-J-K-L are highly similar to that of human parechovirus (HPeV) (genus Parechovirus), especially HPeV-3. However, in the polyprotein coding region, the amino acid sequences are more closely related to those of porcine teschoviruses (genus Teschovirus). Hungaroviruses were detected in 15% (4/26) and 25% (4/16) of the fecal samples from cattle and sheep, respectively. This report describes the discovery of two novel picornaviruses in farm animals, cattle and sheep. The mosaic genetic pattern raises the possibility that hungaroviruses, human parechoviruses, and porcine teschoviruses may be linked to each other by modular recombination of functional noncoding RNA elements.     

A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by halA from the Cyanobacterium Arthrospira platensis Is Closely Related to Its Counterparts from Yeasts and Plants

3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg²⁺ for its activity and could use PAP or 3′-phosphoadenosine-5′-phosphosulfate as a substrate. HalA is sensitive to Li⁺ (50% inhibitory concentration [IC₅₀] = 3.6 mM) but only slightly sensitive to Na⁺ (IC₅₀ = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li⁺ and Na⁺, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li⁺ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.     

Protein hydrolysate from organic fraction of municipal solid waste compost as nitrogen source to produce lactic acid by Lactobacillus fermentum ATCC 9338 and Lactobacillus plantarum NCIMB 8826

In this work a strategy for obtaining free amino-acids concentrate from an organic fraction of municipal solid waste compost and its use as a nitrogen source for lactic acid production, a compound widely used in different industries, using L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 strains is described. Enzymatic digestion is based on the combined action of endoprotease Alcalase 1.5 MG and exoprotease Flavourzyme 500 MG. The highest degree of hydrolysis obtained under the optimal conditions was 41%. The use of glucanase Viscozyme L prior to protein hydrolysis helped to reduce the viscosity of the solution and promote the action of proteases, increasing its hydrolysis degree by 76%. The hydrolysate contained all 21 amino-acids, making it ideal for lactic acid bacteria growth. During shake flask cultivations the culture media was complemented with glucose as carbon source. Finally, with the hydrolysate, a maximum lactic acid concentration of 9.0 ± 0.2 g·L⁻¹ and 11.1 ± 0.1 g·L⁻¹ for L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 respectively was obtained after 27 h. The innovation of the approach lies in exploiting the overproduction of compost for the production of lactic acid.     

Lactobacillus pentosus CECT 4023 T co-utilizes glucose and xylose to produce lactic acid from wheat straw hydrolysate: Anaerobiosis as a key factor

Lactic acid is a versatile chemical that can be produced via fermentation of lignocellulosic materials. The heterolactic strain Lactobacillus pentosus CECT 4023 T, that can consume glucose and xylose, was studied to produce lactic acid from steam exploded wheat straw prehydrolysate. The effect of temperature and pH on bacterial growth was analyzed. Besides, the effect of oxygen on lactic acid production was tested and fermentation yields were compared in different scenarios. This strain showed very high tolerance to the inhibitors contained in the wheat straw prehydrolysate. The highest lactic acid yields based on present sugar, around 0.80 g g⁻¹, were obtained from glucose in presence of 25%, 50%, and 75% v v⁻¹ of prehydrolysate in strict anaerobiosis. Lactic fermentation of wheat straw hydrolysate obtained after enzymatic hydrolysis of the prehydrolysate yielded 0.39 g of lactic acid per gram of released sugars, which demonstrated the high potential of L. pentosus to produce lactic acid from hemicellulosic hydrolysates. Results presented herein not only corroborated the ability of L. pentosus to grow using mixtures of sugars, but also demonstrated the suitability of this strain to be applied as an efficient lactic acid producer in a lignocellulosic biorefinery approach. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2739, 2019     

Evolutionary Insights from Bat Trypanosomes: Morphological, Developmental and Phylogenetic Evidence of a New Species, Trypanosoma (Schizotrypanum) erneyi sp. nov., in African Bats Closely Related to Trypanosoma (Schizotrypanum) cruzi and Allied Species

Parasites of the genus Trypanosoma are common in bats and those of the subgenus Schizotrypanum are restricted to bats throughout the world, with the exception of Trypanosoma (Schizotrypanum) cruzi that also infects other mammals and is restricted to the American Continent. We have characterized trypanosome isolates from Molossidae bats captured in Mozambique, Africa. Morphology and behaviour in culture, supported by phylogenetic inferences using SSU (small subunit) rRNA, gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cyt b (cytochrome b) genes, allowed to classify the isolates as a new Schizotrypanum species named Trypanosoma (Schizotrypanum) erneyi sp. nov. This is the first report of a Schizotrypanum species from African bats cultured, characterized morphologically and biologically, and positioned in phylogenetic trees. The unprecedented finding of a new species of the subgenus Schizotrypanum from Africa that is closest related to the America-restricted Trypanosoma (Schizotrypanum) cruzi marinkellei and T. cruzi provides new insights into the origin and evolutionary history of T. cruzi and closely related bat trypanosomes. Altogether, data from our study support the hypothesis of an ancestor trypanosome parasite of bats evolving to infect other mammals, even humans, and adapted to transmission by triatomine bugs in the evolutionary history of T. cruzi in the New World.     

Evaluation of numerical analyses of RAPD and API 50 CH patterns to differentiate Lactobacillus plantarum, Lact. fermentum, Lact. rhamnosus, Lact. sake, Lact. parabuchneri, Lact. gallinarum, Lact. casei, Weissella minor and related taxa isolated from kocho and tef

AIM: The study was carried out to assess the agreement of API 50 CH fermentation data of food lactobacilli with their RAPD profiles to determine whether the system could be used alone as a reliable taxonomic tool for this genus. METHODS AND RESULTS: API 50 CH, RAPD and DNA:DNA reassociation data for 42 lactobacilli from tef and kocho were compared with 30 type strains. Discrepancies were observed between the three methods in assigning strains of Lactobacillus plantarum, Lact. fermentum, Weissella minor and Lact. gallinarum, and Lact. fermentum, Lact. amylophilus, Lact. casei subsp. pseudoplantarum and Lact. rhamnosus. DNA reassociation data agreed well with RAPD results. CONCLUSIONS: API 50 CH profiles should be complemented with molecular genetic results for effective identification in Lactobacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: The study suggested less dependability of metabolic data alone as an identification tool.     

Stratigraphy of Cladocera (Crustacea) and Chironomidae (Insecta: Diptera) in Three Sediment Cores from the Central Baltic Sea as Related to Paleo-salinity

In three sediment cores from the Central Baltic Sea (Bornholm Basin, Gdansk Bay) corresponding horizons with maximum abundance of remains of a limnetic fauna (Cladocera, Chironomidae) were found. These associations indicate the existence of an oligotrophic freshwater lake. However, this horizon does not coincide with the Ancylus stage but is located in sediment layers from the very end of that period, or from the Mastogloia stage, which was a transitional brackish water phase.     

Lipid Composition of Whole Roots and of Ca2+, Mg2+-activated Adenosine Triphosphatases from Wheat and Oat as Related to Mineral Nutrition

Lipid composition of whole roots of wheat (Triticum vulgare Vill. cv. Svenno Spring Wheat) and oat (Avena sativa L. cv. Brighton) and of cell wall fractions, mitochondrial fractions and microsomal fractions of these roots were studied. Lipid composition depended upon the level of mineral nutrition. In wheat total phospholipids, phosphatidyl choline and sulfolipid content was highest in the roots grown at the higher salt concentration, while the reverse was true for oat roots. In both species glycolipid and sterol content was lower in the high salt roots, at the same time as higher proportions of them were built into the microsomal fraction. Phosphatidyl choline content of the wheat root membrane fractions increased with the salt level, while the opposite occurred in the oat roots. The phosphatidyl choline content may be correlated with the (Ca²⁺, Mg²⁺)-stimulated ATPase activity.