Characterization of horA and its flanking regions of Pediococcus damnosus ABBC478 and development of more specific and sensitive horA PCR method

AIMS: To characterize horA and its flanking regions of Pediococcus damnosus ABBC478 and, on the basis of this insight, to develop a more specific and sensitive horA PCR method. METHODS AND RESULTS: A plasmid harbouring the homologue of a hop-resistance gene, horA, was sequenced and designated pRH478. The nucleotide sequence and open reading frame structure of horA and its flanking regions of pRH478 were found to be highly similar to those of pRH45, a horA-harbouring plasmid previously identified in Lactobacillus brevis ABBC45. The nucleotide sequence of the horA homologue of P. damnosus ABBC478 was 99.6% identical with that of horA. Based on this insight, new primers specific to horA were designed and compared with the previously reported specific primer pair. As a consequence, it was demonstrated that the new primer pair is superior in specificity and sensitivity. CONCLUSIONS: The newly developed horA PCR method allows more specific and sensitive determination of the beer-spoilage ability of lactic acid bacteria (LAB). SIGNIFICANCE AND IMPACT OF THE STUDY: The nucleotide sequences of the horA homologues were found to be essentially identical among distinct species of LAB, indicating that horA-specific primers can be designed from almost any region of the horA gene.     

Comparative analysis of conserved genetic markers and adjacent DNA regions identified in beer-spoilage lactic acid bacteria

AIMS: To conduct an inter-species comparative study on the nucleotide sequences of the conserved DNA regions surrounding ORF5, a genetic marker for differentiating beer-spoilage lactic acid bacteria. METHODS AND RESULTS: The conserved DNA regions surrounding ORF5 were examined by PCR analysis, using three beer-spoilage strains, Lactobacillus brevis ABBC45C, L. paracollinoides LA2T and Pediococcus damnosus ABBC478. As a result, the DNA regions containing ORF1-7, originally found in ABBC45C, appeared to be conserved among the three strains, while the downstream region was not found in L. paracollinoides LA2T and P. damnosus ABBC478. The sequencing analysis of the conserved DNA regions of LA2T and ABBC478 revealed ca 99% nucleotide sequence identities with that of ABBC45C. CONCLUSIONS: The nucleotide sequences of the ca 8.2 kb DNA regions containing ORF1-7 were virtually identical among the three strains belonging to different species. The internal organizations of the ORFs were found to be remarkably similar. SIGNIFICANCE AND IMPACT OF THE STUDY: The level of nucleotide sequence identities suggests the DNA regions surrounding ORF5 were horizontally acquired by these beer-spoilage strains belonging to the three different species of lactic acid bacteria.     

Phylogenetic analysis of cyanobacterial strains of genus-Calothrix by single and multiplex randomly amplified polymorphic DNA-PCR

Analyses of molecular polymorphisms in a selected set of Calothrix strains, using primers based on repetitive sequences in the genome, led to the unambiguous differentiation of the strains as well as understanding of their genetic relationships. Seventeen 10 mer random primers were used singly and twelve dual primer combinations were used to examine the phylogenetic relatedness amongst the strains using RAPD- PCR. A total of nine hundred distinct polymorphic DNA fragments (bands), ranging from 0.18 kb to 5.00 kb were produced in PCR reaction with single oligos. A combination of twelve sets of primers generated nine hundred three distinct polymorphic DNA fragments (bands), ranging from 0.13 kb to 6.22 kb, which revealed a wide range of variability amongst the strains. The combined analysis of single and multiplex primer combination showed a maximum correlation coefficient of 0.821 amongst two strains (Ca28 and Ca29) with chlorophyll contents of 4.08 μg/ml and 3.57 μg/ml. These two isolates belonged to same geographical location. The study undertaken has revealed extensive evidence for the applicability of RAPD in cyanobacterial taxonomy, and furthermore, clearly demonstrated the superior discriminative power of RAPD towards the differentiation of geographically unrelated Calothrix strains.     

The effect of chlorogenic, gallic and quinic acids on the growth of spoilage strains of Lactobacillus collinoides and Lactobacillus brevis

The naturally occurring complex organic acids, chlorogenic acid, gallic acid and quinic acid, at concentrations of 100, 500 and 1000 mg l^-1 were evaluated for effects on the growth of three spoilage strains of Lactobacillus collinoides and one of Lact. brevis in acid tomato broth containing 5% (v/v) ethanol at pH 4.8. During early stages of growth, all the complex acids at each concentration stimulated growth of Lact. collinoides but not of Lact. brevis. During stationary phase, chlorogenic and gallic acids produced greater cell densities of all strains, whereas quinic acid generally had less effect. The presence of these complex acids in fruit products may increase the requirement for added preservative in order to prevent spoilage by certain strains of lactic acid bacteria.     

Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis

Lactobacillus brevis ATCC367 was engineered to express pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) genes in order to increase ethanol fermentation from biomass-derived residues. First, a Gram-positive Sarcina ventriculi PDC gene (Svpdc) was introduced into L. brevis ATCC 367 to obtain L. brevis bbc03. The SvPDC was detected by immunoblot using an SvPDC oligo peptide antiserum, but no increased ethanol was detected in L. brevis bbc03. Then, an ADH gene from L. brevis (Bradh) was cloned behind the Svpdc gene that generated a pdc/adh-coupled ethanol cassette pBBC04. The pBBC04 restored anaerobic growth and conferred ethanol production of Escheirichia coli NZN111 (a fermentative defective strain incapable of growing anaerobically). Approximately 58 kDa (SvPDC) and 28 kDa (BrADH) recombinant proteins were observed in L. brevis bbc04. These results indicated that the Gram-positive ethanol production genes can be expressed in L. brevis using a Gram-positive promoter and pTRKH2 shuttle vector. This work provides evidence that expressing Gram-positive ethanol genes in pentose utilizing L. brevis will further aid manipulation of this microbe toward biomass to ethanol production.     

The use of random amplified polymorphic DNA (RAPD) markers to identify strawberry varieties: a forensic application

The random amplified polymorphic DNA (RAPD) technique was applied to settle a lawsuit involving unauthorized commercialization of a patented strawberry variety of high economical relevance ('Marmolada'). Because of economical involvements, the molecular approach was added to the more traditional morphological examination in a double-blind test. All plants belonging to the patented variety were unambiguously identified (13 plants among a total of 31 plants examined). The results were accepted as evidence in the court. This study confirms that the RAPD technique is especially suitable for identification of asexually reproduced plant varieties for forensic or agricultural purposes.     

Screening of Lactobacillus spp. and Pediococcus spp. for glycosidase activities that are important in oenology

AIMS: To assess glycosidase activities from a range of Lactobacillus and Pediococcus species and characterize these activities under conditions pertinent to the wine industry. METHODS AND RESULTS: Lactic acid bacteria were cultured in MRS broth supplemented with apple juice before being harvested, washed and assayed for glycosidase activity using p-nitrophenol-linked substrates. All strains exhibited a detectable capacity for the hydrolysis of the beta- and alpha-d-glucopyranosides. The magnitude of these activities and their response to the physico-chemical parameters investigated varied in a strain-dependent manner. The use of an assay buffer with a pH below 4 generally resulted in a reduced hydrolysis of both substrates while temperature optima ranged between 35 and 45 degrees C. The effect of the inclusion of ethanol in the assay buffer (up to 12%, v/v) ranged from near complete inhibition to increases in activity approaching 80%. With the clear exception of a single strain, glucose and fructose (0.1-20 g l(-1)) acted as inhibitors. An assessment of glycosidase activity during simultaneous exposure to glucose and ethanol at a pH of 3.5 suggested that ethanol decreased loss of activity under these wine-like conditions. CONCLUSIONS: Lactobacillus spp. and Pediococcus spp. possess varying degrees of beta- and alpha-d-glucopyranosidase activities, which in turn are influenced differently by exposure to ethanol and/or sugars, temperature and pH. Several strains appeared suited for further evaluation under winemaking conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the fact that strains of Lactobacillus and Pediococcus have the potential to influence the glycoside composition of wine. Tailoring of wine may therefore be possible through selective application of strains or enzymatic extracts thereof.     

Application of random amplified polymorphic DNA and inter-simple sequence repeat markers in the genus Crataegus

Hawthorn ( Crataegus spp.) has a long history as an ornamental and a source of medicine. We report the use of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers to determine genetic relationships in the genus Crataegus . Twenty-eight accessions, including eight species ( Crataegus pinnatifida , Crataegus bretschneideri , Crataegus maximowiczii , Crataegus kansuensis , Crataegus altaica , Crataegus songarica , Crataegus dahurica and Crataegus sanguinea ) and two botanical varieties ( C. pinnatifida var. major and C. maximowiczii var. ninganensis ) were analysed. Twelve RAPD primers reproducibly and strongly amplified 128 fragments of which 116 were polymorphic; similarly, 13 ISSR primers generated 127 products of which 119 were polymorphic. Dendrograms based on unweighted pair group method with arithmetic average analysis were constructed from both the RAPD and the ISSR data. Similarity coefficient based on RAPD and ISSR markers ranged from 0.22 to 0.98 and 0.23 to 0.98, respectively. The range in similarity coefficient indicated that the genus has a high level of genetic diversity. The Mantel test on the similarity matrices produced by RAPD and ISSR markers gave r  = 0.86, showing high correlation between RAPD and ISSR markers in their ability to detect genetic relationships between Crataegus accessions. RAPD and ISSR appear to be reliable methods for the analysis of genetic relationships among hawthorns.     

Evaluation of purine utilization by Lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine

Abstract

It is well accepted that frequent and heavy intake of purine-rich foods causes elevation of serum uric acid levels, which is a risk factor of hyperuricemia. Reducing intestinal absorption of dietary purines may attenuate the elevation of serum uric acid levels and exacerbation of hyperuricemia. This reduction may be achieved by the ingestion of lactic acid bacteria that take up purines in the intestine. In this study, we investigated the degree of uptake and utilization of purines of three lactobacilli strains. Among them, Lactobacillus gasseri PA-3 (PA-3) showed the greatest incorporation of ¹⁴C-adenine. PA-3 also incorporated ¹⁴C-adenosine and ¹⁴C-AMP. Additionally, using defined growth medium, PA-3 demonstrated greater proliferation in the presence of these purines than in their absence. Although further investigation is required, ingestion of PA-3 may lower serum uric acid levels by reducing intestinal absorption of purines in humans.     

Adsorption of temperate phages of Lactobacillus delbrueckii strains and phage resistance linked to their cell diversity

Aims: The aim of this work was to study the adsorption step of two new temperate bacteriophages (Cb1/204 and Cb1/342) of Lactobacillus delbrueckii and to isolate phage‐resistant derivatives with interesting technological properties. Methods and Results: The effect of divalent cations, pH, temperature and cell viability on adsorption step was analysed. The Ca²⁺ presence was necessary for the phage Cb1/342 but not for the phage Cb1/204. Both phages showed to be stable at pH values between 3 and 8. Their adsorption rates decreased considerably at pH 8 but remained high at acid pH values. The optimum temperatures for the adsorption step were between 30 and 40°C. For the phage Cb1/342, nonviable cells adsorbed a lower quantity of phage particles in comparison with the viable ones, a fact that could be linked to disorganization of phage receptor sites and/or to the physiological cellular state. The isolation of phage‐resistant derivatives with good technological properties from the sensitive strains and their relationship with the cell heterogeneity of the strains were also made. Conclusions: Characterization of the adsorption step for the first temperate Lact. delbrueckii phages isolated in Argentina was made, and phage‐resistant derivatives of their host strains were obtained. Significance and Impact of the Study: Some phage‐resistant derivatives isolated exhibited good technological properties with the prospective to be used at industrial level.     

Expression of Lactobacillus brevis IOEB 9809 tyrosine decarboxylase and agmatine deiminase genes in wine correlates with substrate availability

Aims: Lactobacillus brevis IOEB 9809 is able to produce both tyramine and putrescine via tyrosine decarboxylase and agmatine deiminase enzymes, respectively, when cultured on synthetic media. The aims of this study were to assess the expression of L. brevis IOEB 9809 tdc and aguA1 genes, during wine fermentation and to evaluate the effect of substrate availability and pH on tdc and aguA1 expression, as well as on biogenic amine production and L. brevis viability. Methods and Results: The relative expression of L. brevis IOEB 9809 tdc and aguA1 genes was analysed in wine by quantitative real‐time RT‐PCR (qRT‐PCR) during a period of incubation of 30 days. Cell viability, pH values, putrescine and tyramine concentration were monitored throughout the experiments. Conclusions: The wine trials indicated that L. brevis IOEB 9809 is able to produce both tyramine and putrescine during wine fermentation. Increased cell viability was also observed in wine supplemented with tyrosine or agmatine. qRT‐PCR analysis suggests a strong influence of substrate availability on the expression of genes coding for tyrosine decarboxylase and agmatine deiminase in L. brevis IOEB 9809. Less evident is the relationship between putrescine and tyramine production and tolerance to wine pH. Significance and Impact of Study: To our knowledge, this study represents the first assessment of relative expression of L. brevis IOEB 9809 genes involved in biogenic amine production in wine. Furthermore, an effect of biogenic amine production on viability of L. brevis during wine fermentation was established.     

Isolation of bovine intestinal Lactobacillus plantarum and Pediococcus acidilactici with inhibitory activity against Escherichia coli O157 and F5

Aims:  The growth rate of bovine lactic acid bacteria (LAB) in five different culture conditions, and their inhibitory activity against Escherichia coli O157 and F5 in two assays was assessed to identify LAB for potential prophylactic use in cattle.
Methods and Results:  106 bovine-derived faecal/intestinal LAB were tested in vitro for tolerance to pH 2·0, pH 4·0, 0·15% and 0·3% bile, aerobic incubation, and for inhibitory activity against E. coli O157 ( n  = 3) and F5 ( n  = 1). While no LAB grew at pH 2·0, LAB survivability varied between 35% and 100% on the other tests. Exactly 7·6% (8/106) of LAB supernatants inhibited the growth of E. coli in two assays, whereas 6·6% (7/106) of isolates enhanced the growth of all E. coli strains. Partial 16s rRNA gene sequencing of six best isolates (95th percentile) revealed that five were Lactobacillus plantarum and one Pediococcus acidilactici.
Conclusion:  Lactobacillus plantarum with acid/bile and aerobic resistance and inhibitory activity against E. coli O157 and F5 inhabit the intestinal tract of healthy cattle. Some LAB may enhance E. coli growth.
Significance and Impact of the Study:  Lactobacillus plantarum and P. acidilactici are natural plant micro-organisms and studied silage inoculants. Their identification from gastrointestinal samples of healthy cattle is prophylactically promising.     

Monitoring the bacterial community of maize silage stored in a bunker silo inoculated with Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri

Aims: To monitor variations in the bacterial community and fermentation products of maize silage within and between bunker silos. Methods and Results: Silage samples were collected in 2008 and 2009 from three dairy farms, wherein the farmers arranged for a contractor to produce maize silage using bunker silos. Silage was prepared using a lactic acid bacteria (LAB) inoculant consisting of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri. Eight samples were collected from each bunker silo; 4 ‘outer’ and 4 ‘inner’ samples were collected from near the top and the bottom of the silo. The dry matter, lactic acid, acetic acid, ethanol, 1‐propanol and 1,2‐propanediol contents differed between bunker silos in both sampling years. Higher acetic acid, 1‐propanol and 1,2‐propanediol contents were found in the bottom than the top layers in the 2008 samples, and higher lactic acid content was found in the top than the bottom layers in the 2009 samples. The bacterial community varied more between bunker silos than within a bunker silo in the 2008 samples, whereas differences between the top and the bottom layers were seen across bunker silos in the 2009 samples. The inoculated LAB were uniformly distributed, while several nonconventional silage bacteria were also detected. Lactobacillus acetotolerans, Lactobacillus panis and Acetobacter pasteurianus were detected in both years. Stenotrophomonas maltophilia was detected in the 2008 samples, and Lactobacillus reuteri, Acinetobacter sp. and Rahnella sp. were detected in the 2009 samples. Conclusions: Although differences were seen within and between bunker silos, the bacterial community may indicate a different relationship between bunker silos and sampling locations within a bunker silo from that indicated by the fermentation products. Significance and Impact of the Study: Analysis of bacterial community can help understand how diverse non‐LAB and LAB species are involved in the ensiling process of bunker‐made maize silage.     

Inactivation of oenological lactic acid bacteria (Lactobacillus hilgardii and Pediococcus pentosaceus) by wine phenolic compounds

Aims:  To investigate the inactivation properties of different classes of phenolic compounds present in wine against two wine isolates of Lactobacillus hilgardii and Pediococcus pentosaceus , and to explore their inactivation mechanism.
Methods and Results:  After a first screening of the inactivation potency of 21 phenolic compounds (hydroxybenzoic and hydroxycinnamic acids, phenolic alcohols, stilbenes, flavan-3-ols and flavonols) at specific concentrations, the survival parameters (MIC and MBC) of the most active compounds were determined. For the L. hilgardii strain, the flavonols morin and kaempferol showed the strongest inactivation (MIC values of one and 5 mg l⁻¹, and MBC values of 7·5 and 50 mg l⁻¹, respectively). For the P. pentosaceus strain, flavonols also showed the strongest inactivation effects, with MIC values between one and 10 mg l⁻¹ and MBC values between 7·5 and 300 mg l⁻¹. Observations by epifluorescence and scanning electron microscopy revealed that the phenolics damaged the cell membrane and promoted the subsequent release of the cytoplasm material into the medium.
Conclusions:  The antibacterial activity of wine phenolics against L. hilgardii and P. pentosaceus was dependent on the phenolic compound tested, and led not only to bacteria inactivation, but also to the cell death.
Significance and Impact of the Study:  New information about the inactivation properties of wine lactic acid bacteria by phenolic compounds is presented. It opens up a new area of study for selecting/obtaining wine phenolic preparations with potential applications as a natural alternative to SO₂ in winemaking.     

Use of random amplified polymorphic DNA to identify several novel markers for sex identification in the crested serpent eagle and crested goshawk

The crested serpent eagle (Spilornis cheela hoya) has no distinct sexual dimorphic traits. In the current study, we report the results of an EE0.6 (EcoRI 0.6-kb fragment) sequence applied to S. cheela hoya and a novel random amplified polymorphic DNA (RAPD) marker that can be used to sex individuals within the species S. cheela hoya and Accipiter trivigatus formosae (crested goshawk). We used sex-specific primers for the avian CHD1 (chromo-helicase-DNA-binding 1) gene and the EE0.6 sequence in PCR assays to determine sex. In addition, 120 random primers were used for RAPD fingerprinting to search for novel sex-specific fragments of S. cheela hoya. The OPBB08 random primer generated a 1241-bp sex-specific fragment in all female S. cheela hoya. From the nucleotide sequence, PCR primers were designed to amplify 553-, 895-, and 194-bp sex-specific fragments present in all female S. cheela hoya. One of these primer pairs (ScBB08-7F/R) also amplified a male/female common fragment that can be used as an internal control (543 bp). Moreover, one of the primer pairs (ScBB08-5aF/5bR) could be used to identify genders of A. trivigatus formosae. In conclusion, we identified novel sex-specific DNA markers of S. cheela hoya and A. trivigatus formosae that can be used for rapid and accurate sex identification.     

Draft genome sequences and description of Lactobacillus rhamnosus strains L31, L34, and L35

Lactobacillus rhamnosus is a facultative, lactic acid bacterium in the phylum Firmicutes. Lactobacillus spp. are generally considered beneficial, and specific strains of L. rhamnosus are validated probiotics. We describe the draft genomes of three L. rhamnosus strains (L31, L34, and L35) isolated from the feces of Thai breastfed infants, which exhibit anti-inflammatory properties in vitro. The three genomes range between 2.8 – 2.9 Mb, and contain approximately 2,700 protein coding genes.