Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.     

Effect of temperature on growth and activity of Aeromonas spp. and mixed bacterial populations in the Anacostia River.

During the winter months, total bacterial counts in the water column and in the sediment in the Anacostia River were two- to eightfold higher than at other times of the year, whereas Aeromonas spp. decreased in number of several orders of magnitude. This significant decrease in number in the Anacostia River during the cold months of the year can be explained by the low metabolic activity of Aeromonas at low temperatures.     

Deconvolving sequence variation in mixed DNA populations.

We present an original approach to identifying sequence variants in a mixed DNA population from sequence trace data. The heart of the method is based on parsimony: given a wildtype DNA sequence, a set of observed variations at each position collected from sequencing data, and a complete catalog of all possible mutations, determine the smallest set of mutations from the catalog that could fully explain the observed variations. The algorithmic complexity of the problem is analyzed for several classes of mutations, including block substitutions, single-range deletions, and single-range insertions. The reconstruction problem is shown to be NP-complete for single-range insertions and deletions, while for block substitutions, single character insertion, and single character deletion mutations, polynomial time algorithms are provided. Once a minimum set of mutations compatible with the observed sequence is found, the relative frequency of those mutations is recovered by solving a system of linear equations. Simulation results show the algorithm successfully deconvolving mutations in p53 known to cause cancer. An extension of the algorithm is proposed as a new method of high throughput screening for single nucleotide polymorphisms by multiplexing DNA.     

Rapid Mini-Prep Isolation of High-Quality Plasmid DNA from Lactococcus and Lactobacillus spp.

A simple, rapid plasmid mini-prep procedure for lactococci and lactobacilli which gives high yields and can be performed on overnight broth cultures is presented. Large plasmids were isolated from both lactococci and lactobacilli, including a 70-kb plasmid from Lactobacillus acidophilus C7. The purity of the resulting plasmid DNA makes it suitable for subsequent molecular manipulations. The convenience of the technique makes this rapid mini-prep procedure suitable for routine plasmid isolation from lactic acid bacteria.     

Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.

     

Rapid Mini-Prep Isolation of High-Quality Plasmid DNA from Lactococcus and Lactobacillus spp

A simple, rapid plasmid mini-prep procedure for lactococci and lactobacilli which gives high yields and can be performed on overnight broth cultures is presented. Large plasmids were isolated from both lactococci and lactobacilli, including a 70-kb plasmid from Lactobacillus acidophilus C7. The purity of the resulting plasmid DNA makes it suitable for subsequent molecular manipulations. The convenience of the technique makes this rapid mini-prep procedure suitable for routine plasmid isolation from lactic acid bacteria.     

Use of surface plasmon-coupled emission to measure DNA hybridization

The authors describe a new approach to measuring DNA hybridization based on surface plasmon-coupled emission (SPCE). SPCE is the resonance coupling of excited fluorophores with electron motions in thin metal films, resulting in efficient transfer of energy through the film and radiation into the glass substrate. The authors evaluated the use of SPCE for detection of DNA hybridization. An unlabeled capture biotinylated oligonucleotide was attached near the surface of a thin (50 nm) silver film using streptavidin. The authors then measured the emission intensity of single-stranded Cy5-labeled DNA upon binding to a complementary oligomer attached to a silver film. Hybridization could be detected by an increase in SPCE, which appeared as light radiated into the substrate at a sharply defined angle near 73 degrees from the normal. The largest signals were observed when the excitation angle of incidence equaled the surface plasmon wavelength, but directional emission was also observed without excitation by the surface plasmon evanescent field. The increased intensity is due to proximity to the metal surface, so that hybridization can be detected without a change in the quantum yield of the fluorophore. These results indicate that SPCE can provide highly sensitive real-time measurement of DNA hybridization.     

Detection and characterization of lactose-utilizing Lactococcus spp. in natural ecosystems.

The presence of lactose-utilizing Lactococcus species in nondairy environments was studied by using identification methods based on PCR amplification and (sub)species-specific probes derived from 16S rRNA sequences. Environmental isolates from samples taken on cattle farms and in the waste flow of a cheese production plant were first identified to the genus level, using a Lactococcus genus-specific probe. Isolates which showed a positive signal with this probe were further identified to the (sub)species level. Lactococcus lactis isolates were also characterized at the phenotypic level for the ability to hydrolyze arginine, to ferment citrate, and to produce proteases and bacteriocins. With specific PCR amplifications, the presence of sequences related to citP, coding for citrate permease; prtP, coding for protease; and nisA or nisZ, the structural genes for production of nisin A or nisin Z, respectively, was verified. By these methods, it was possible to isolate lactococci from various environmental sources, such as soil, effluent water, and the skin of cattle. The strains of L. lactis isolated differed in a number of properties, such as the ability to hydrolyze arginine or the absence of citP-related sequences, from those found in industrial starter cultures. The results indicate that the majority of the industrially produced lactococci do not survive outside the dairy environment, although natural niches are available. However, from those niches strains with the potential to be developed into novel starter cultures may be isolated.     

Repair of DNA single-strand breaks in X-irradiated yeast. I. Use of the DNA-unwinding method to measure DNA strand breaks

The DNA-unwinding method developed by Ahnstr?m and his coworkers to measure DNA strand breaks in mammalian cells was used to measure single-strand breaks (SSB) in the DNA of intact yeast cells. DNA unwinding, which took place inside the rigid cell wall of yeast, was investigated as a function of time, radiation dose, and of pH and salt concentration of the alkaline solution. After DNA unwinding had taken place, the cell wall was destroyed by partial enzymatic digestion and sonication in the presence of detergents. Fragments of single- and double-stranded DNA were separated using hydroxylapatite chromatography. In this way the most suitable conditions for DNA unwinding within the cell wall were established. The results show that SSB and double-strand breaks (DSB) give rise to different kinetics of DNA unwinding.     

The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations

Summary Restriction endonucleases and agarose gel electrophoresis have been used to demonstrate extensive nucleotide sequence diversity in mitochondrial DNA (mtDNA) within and between conspecific populations of rodents and other mammals. Cleavage of mtDNA samples with a relatively small number of endonucleases provides information concerning the phylogenetic relatedness of individual organisms which cannot now be readily obtained by any other type of molecular analysis. This information is qualitatively different from that available from the study of nuclear genes or gene products because the mitochondrial genome is inherited intact from the female parent and is not altered by recombination or meiotic segregation.The requirements for large tissue samples and laborious DNA purification procedures have imposed severe limitations on the kinds of population surveys in which this technique could be utilized. Here, we show that these difficulties can be overcome by using DNA-DNA hybridization to detect minute amounts of mtDNA in crude tissue fractions which can be more easily and rapidly prepared from very small amounts of tissue without the use of expensive and immobile laboratory equipment. The techniques are described in detail in an effort to make restriction analysis of mtDNA available to biologists who may be unfamiliar with current DNA technology.     

Polymorphism in Brucella spp. due to highly repeated DNA.

The species of Brucella are very closely related, but Brucella ovis does not express detectable amounts of a protein, designated BCSP31, that is common to the other species. We studied the lack of expression of BCSP31 by Southern analysis. DNAs from the B. ovis culture collection strains and field isolates were probed with a 1.3-kb HindIII fragment encoding BCSP31 of Brucella abortus. The probe hybridized to a 1.6-kb HindIII fragment of all B. ovis strains tested, showing that the gene is present in B. ovis but occurs on a larger restriction fragment. DNA linkage studies and restriction mapping of the cloned polymorphic region of B. ovis showed that the polymorphism was due to a DNA insertion of approximately 0.9 kb at a site downstream of the BCSP31-coding region. When the 1.6-kb polymorphic B. ovis fragment was used to probe a HindIII Southern blot of cellular DNA of strains of B. ovis and of B. abortus, at least 24 fragments of B. ovis and 6 fragments of B. abortus hybridized to the inserted DNA. Specimens of B. ovis collected over a 30-year period on two continents had similar hybridization patterns. The large difference between B. ovis and B. abortus in the number of copies of the repeated DNA is interesting in the context of the closeness of the Brucella species.     

Use of the fluorescent probe LAURDAN to label and measure inner membrane fluidity of endospores of Clostridium spp

A method for measuring the fluidity of inner membranes of populations of endospores of Clostridium spp. with a fluorescent dye was developed. Cells of Clostridium beijerinckii ATCC 8260 and Clostridium sporogenes ATCC 7955 were allowed to sporulate in the presence of 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) on a soil-based media. Labeling of endospores with LAURDAN did not affect endospore viability. Removal of the outer membranes of endospores was done using a chemical treatment and confirmed using transmission electron microscopy (TEM). Two-photon confocal laser scanning microscopy (CLSM), and generalized polarization (GP) measurements were used to assess fluorescence of endospores. Lipid composition analysis of cells and endospores was done to determine whether differences in GP values are attributable to differences in membrane composition. Removal of the outer membranes of endospores did not significantly impact GP values. Decoated, labeled endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 exhibited GP values of 0.77±0.031 and 0.74±0.027 respectively. Differences in ratios of fatty acids between cells and endospores are unlikely to be responsible for high GP values observed in endospores. These GP values indicate high levels of lipid order and the exclusion of water from within inner membranes of endospores.     

Morphological and morphometrical analysis of Heterodera spp. populations in Jordan

Phenotypic diversity of five Jordanian populations of cyst nematodes, Heterodera spp. collected from five regions from Jordan (Ar-Ramtha, Madaba, Dana, Al-Karak, and Jerash) was investigated. Soil samples were collected from one representative field in each region. Morphological and morphometrical characteristics revealed that Heterodera latipons is dominated in cereal fields at Ar-Ramtha, Madaba, Dana and Al-Karak regions and Heterodera schachtii in Jerash. Cysts populations from all cereal fields had bifenestrate vulval cone and a strong underbridge. Wherever, cysts of the cabbage population had ambifenestrate vulval cone with long vulval slit. The bullae were absent in Ar-Ramtha, Madaba and Dana populations, but present in Al-Karak and Jerash. Based on 12 morphometrical characters, the first three functions in canonical discriminant analysis accounted 99.3% of the total variation. Distance from dorsal gland duct opening to stylet base, underbridge length, a = L/W (body length/midbody width) and length of hyaline tail tip had strong and significant contributions in the first function. While the second function was strongly influenced by length of hyaline tail, fenestral length, fenestral width and tail length. However, the third canonical discriminate function was found to be influenced by stylet length, fenestral length, a = L/W (body length/midbody width) and underbridge width. The graphical representation of the distribution of the samples showed that the first canonical discriminant function clearly separated H. schachtii from Jerash from other populations. Whereas, H. latipons collected from Madaba and Dana were clearly separated in the second function. The results indicated that differences at morphological and morphometrical levels revealed diverse populations of Heterodera spp. in Jordan.     

Simultaneous quantification of cyanobacteria and Microcystis spp. using real-time PCR

In order to develop a protocol to quantify cyanobacteria and Microcystis simultaneously, the primers and probe were designed from the conserved regions of 16S rRNA gene sequences of cyanobacteria and Microcystis, respectively. Probe match analysis of the Ribosomal Database Project showed that the primers matched with over 97% of cyanobacterial 16S rRNA genes, indicating these can be used to amplify cyanobacteria specifically. The TaqMan probe, which is located between two primers, matched with 98.2% of sequences in genus GpXI, in which most Microcystis strains are included. The numbers of cyanobacterial genes were estimated with the emission of SYBR Green from the amplicons with two primers, whereas those of Microcystis spp. were measured from the fluorescence of CAL Fluor Gold 540 emitted by exonuclease activity of Taq DNA polymerase in amplification. It is expected that this method enhances the accuracy and reduces the time to count cyanobacteria and potential toxigenic Microcystis spp. in aquatic environmental samples.     

Use of phages to control Campylobacter spp.

The use of phages to control pathogenic bacteria has been investigated since they were first discovered in the beginning of the 1900s. Over the last century we have slowly gained an in-depth understanding of phage biology including which phage properties are desirable when considering phage as biocontrol agents and which phage characteristics to potentially avoid. Campylobacter infections are amongst the most frequently encountered foodborne bacterial infections around the world. Handling and consumption of raw or undercooked poultry products have been determined to be the main route of transmission. The ability to use phages to target these bacteria has been studied for more than a decade and although we have made progress towards deciphering how best to use phages to control Campylobacter associated with poultry production, there is still much work to be done. This review outlines methods to improve the isolation of these elusive phages, as well as methods to identify desirable characteristics needed for a successful outcome. It also highlights the body of research undertaken so far and what criteria to consider when doing in-vivo studies, especially because some in-vitro studies have not been found to translate into to phage efficacy in-vivo.     

Use of pulsed-field gel electrophoresis to measure X-ray-induced double-strand breaks in DNA substituted with BrdU

The substitution of BrdU for TdR in the DNA of Chinese hamster ovary cells caused radiosensitization for both cell killing and an increase in the rate of neutral elution of the DNA. However, no radiosensitization was observed for the amount of DNA that migrated from the plug of agarose gels subjected to pulsed-field gel electrophoresis. An unexpected observation, however, was that the migration rate of BrdU-substituted DNA was relatively independent of radiation dose and was much less than that of unsubstituted DNA which migrated at a faster rate as the radiation dose increased. This difference in migration between TdR- and BrdU-labeled DNA was observed only when electrophoresis conditions were optimized for separating DNA molecules from 1 to 7 Mb. Possibly, the increase in negative charge on BrdU-labeled DNA increases the reorientation time during each pulse, with a resulting decrease in rate of migration, or radiation effects on BrdU-labeled DNA may be responsible for the decrease in migration rate.     

Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium

Plasmid DNA was introduced by electroporation into Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Propionibacterium and Staphylococcus as an alternative to competent-cell or protoplast transformation. Plasmid-containing transformants were recovered in these recipients at frequencies ranging from 10(1) to 10(5) transformants micrograms-1 of pGK12. Several parameters of the protocol, including DNA concentration, voltage, plating regimen and electroporation buffers were evaluated to determine conditions that improved transformation frequencies for Lactobacillus acidophilus. Using optimized conditions, the following plasmids were introduced into L. acidophilus: pAMB1, pC194, pGB354, pGKV1, pSA3, pTRK13, pTV1 and pVA797. The ability to transfer plasmid DNA via eletroporation will greatly facilitate the application of recombinant DNA methodology and transposon technology to Gram-positive bacteria for cloning and analysis of significant genes.     

Between-year variation and spatial dynamics of Cryptosporidium spp. and Giardia spp. infections in naturally infected rodent populations

Prevalence and abundance of Cryptosporidium spp. and Giardia spp. infections were studied over the 8-year period in 3 species of rodents in N.E. Poland (bank vole Myodes glareolus-1523; yellow-necked mouse Apodemus flavicollis- 638; common vole Microtus arvalis- 419). Prevalence was 53.8, 28.1 and 62.3% respectively for Cryptosporidium spp. and 58.3, 24.4 and 74.2% respectively for Giardia spp. Prevalence and abundance of infection varied markedly across 8 years of the study with 1998 and 2002 being years of higher prevalence and abundance, following changes in the densities of host species. The distribution of intestinal protozoa in forest rodents did not vary in the 3 isolated sites during the 4-year study. In the case of Cryptosporidium, fewer older animals carried infection and infections of the oldest bank and common voles were relatively milder. In the case of Giardia in yellow-necked mice, infections were more common in older age classes (2 and 3). The two species showed significant co-occurrence and in animals carrying both species there was a strong significant positive correlation between abundance of infection with each. These data are discussed in relation to the parasite genotypes identified in this region and in respect of the role of various ecological factors in shaping of intestinal protozoa communities.     

Dynamics of mixed cultures of Lactobacillus plantarum and Propionibacterium shermanii

The mixed culture of Lactobacillus plantarum and Propionibacterium shermanii grown anaerobically in glucose minimal medium exhibits features typical of a commensal interaction even though a number of complicating factors, such as a large maintenance requirement of L. plantarum and inhibition of growth of P. shermanii at low pH, are present. A simple mathematical model of the system is presented and is shown to reproduce rather well some of the features of the continuous mixed culture system in both steady-state and transient situations.