Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3′ degenerate core based on four highly conserved amino acids and a longer 5′ consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS⁻) and EPS-producing (EPS⁺) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS⁺ bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS⁺ strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for Lactococcus lactis

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.     

Downregulation of Salmonella Virulence Gene Expression During Invasion of Epithelial Cells Treated with Lactococcus lactis subsp. cremoris JFR1 Requires OppA

Invasion of Salmonella into host intestinal epithelial cells requires the expression of virulence genes. In this study, cell culture models of human intestinal cells (mucus-producing HT29-MTX cells, absorptive Caco-2 cells, and combined cocultures of the two) were used to determine the effects of Lactococcus lactis subsp. cremoris treatments (exopolysaccharide producing and nonproducing strains) on the virulence gene expression of Salmonella Typhimurium and its mutant lacking the oligopeptide permease subunit A (ΔoppA). During the course of epithelial cell (HT29-MTX, Caco-2, and combined) infection by Salmonella Typhimurium DT104, improved barrier function was reflected by increased transepithelial electrical resistance in cells treated with both strains of L. lactis subsp. cremoris. In addition, virulence gene expression was downregulated, accompanied with lower numbers of invasive bacteria into epithelial cells in the presence of L. lactis subsp. cremoris treatments. Similarly, virulence gene expression of Salmonella was also suppressed when coincubated with overnight cultures of both L. lactis subsp. cremoris strains in the absence of epithelial cells. However, in medium or in the presence of cell cultures, Salmonella lacking the OppA permease function remained virulent. HT29-MTX cells and combined cultures stimulated by Salmonella Typhimurium DT104 showed significantly lower secretion levels of pro-inflammatory cytokine IL-8 after treatment with L. lactis subsp. cremoris cell suspensions. Contrarily, these responses were not observed during infection with S. Typhimurium ΔoppA. Both the exopolysaccharide producing and nonproducing strains of L. lactis subsp. cremoris JFR1 exhibited an antivirulence effect against S. Typhimurium DT104 although no significant difference between the two strains was observed. Our results show that an intact peptide transporter is essential for the suppression of Salmonella virulence genes which leads to the protection of the barrier function in the cell culture models studied.

     

Modeling the impacts of global warming on predation and biotic resistance: mosquitoes, damselflies and avian malaria in Hawaii

Biotic resistance from native predators can play an important role in regulating or limiting exotic prey. We investigate how global warming potentially alters the strength and spatial extent of these predator–prey interactions in aquatic insect ecosystems. As a simple model system, we use rock pools in streams of rainforests of Hawaii, which contain the beautiful Hawaiian damselfly Megalagrion calliphya as predator and the invasive southern house mosquito Culex quinquefasciatus as prey. This abundant mosquito is the major vector of avian malaria transmission to native forest birds. We use mathematical modeling to evaluate the potential impacts of damselfly predation and temperature on mosquito population dynamics. We model this predator–prey system along an elevational gradient (749-1952 m elevation) and assess the effect of 1°C and 2°C climate warming scenarios as well as the effects of El Niño and La Niña oscillations, on predator–prey dynamics. Our results indicate that the strength of biotic resistance of native predators on invasive prey may decrease with increasing temperature because demographic rates of predator and prey are differentially affected by temperature. Future warming could therefore increase the abundance of invasive species by releasing them from predation pressure. If the invasive species is a disease vector, these shifts could increase the impact of disease on both humans and wildlife.     

Production,purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1

Background  

The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances.     

Cloning, sequencing, and expression in Escherichia coli of the D-hydantoinase gene from Pseudomonas putida and distribution of homologous genes in other microorganisms.

Pseudomonas putida DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubstituted hydantoins. The gene encoding this D-hydantoinase enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.8-kb insert of subclone pGES19 was determined. One open reading frame of 1,104 bp was found and was predicted to encode a polypeptide with a molecular size of 40.5 kDa. Local regions of identity between the predicted amino acid sequence and that of other known amidohydrolases (two other D-hydantoinases, allantionase and dihydroorotase) were found. The D-hydantoinase gene was used as a probe to screen DNA isolated from diverse organisms. Within Pseudomonas strains of rRNA group I, the probe was specific. The probe did not detect D-hydantoinase genes in pseudomonads not in rRNA group I, other bacteria, or plants known to express D-hydantoinase activity.     

Seasonal surveillance confirms the range expansion of Aedes japonicus japonicus (Theobald) (Diptera: Culicidae) to the Hawaiian Islands of Oahu and Kauai

The Asian bush mosquito, Aedes japonicus japonicus (Theobald) was not known to occur in the Hawaii archipelago until it was identified on the island of Hawaii in 2003. This mosquito species remained undetected on the neighboring islands for 8?years before it was discovered at the Honolulu International Airport on Oahu in 2012. By 2015, four Ae. j. japonicus mosquitoes were collected in the western mountains of Oahu and one was collected in the central mountains of Kauai. The collection of this invasive mosquito species across the neighboring Hawaiian Islands of Oahu and Kauai indicated the need for increased seasonal surveillance on these islands. Following nearly four years of surveillance, Ae. j. japonicus was also confirmed to occur in the eastern mountains of Oahu and in the central mountainous region of Kauai. To expand the knowledge of the spread of invasive mosquitoes species further surveillance is necessary to identify all possible areas where populations of Ae. j. japonicus and other invasive mosquito species occur in Hawaiian archipelago.     

Use of a polymerase-chain-reaction-amplified DNA probe from Pseudomonas putida to detect d-hydantoinase-producing microorganisms by direct colony hybridization

Pseudomonas putida strain DSM 84 produces N-carbamyl-d-amino acids from the corresponding d-5-monosubstituted hydantoins. The sequence of the d-hydantoinase gene from this strain (GenBank accession number L24157) was used to develop a DNA probe of 122 base pairs (bp) that could detect d-hydantoinase genes in other bacterial genera by DNA and by colony hybridization. Under conditions tolerating 32% mismatch, the probe was specific for all strains that expressed d-hydantoinase activity. These include Pseudomonadaceae of all rRNA groups, and bacteria belonging to the genera Agrobacterium, Serratia, Corynebacterium, and Arthrobacter. Environmental sampling was simulated by screening a mixture of unknown microorganims from commercial inocula for the biodegradation of industrial, municipal and domestic wastes. The 122-bp probe was specific for microorganisms that subsequently demonstrated d-hydantoinase activity. Bacterial species from four different genera were detected, which were Pseudomonas, Klebsiella, Enterobacter, and Enterococcus.     

PARASITIC INTERACTIONS AND VEGETATIVE ULTRASTRUCTURE OF CHOREOAEMA THURETII (CORALLINALES,RHODOPHYTA)1

The monotypic coralline red alga, Choreonema thuretii (Bornet) Schmitz (Choreonematoideae), grows endophytically within three geniculate genera of the Corallinoideae. Although the thallus of Choreonema is reduced, lacks differentiated plastids, and is endophytic except for its conceptacles, its status as a parasite has been questioned because cellular connections to the host had not been ob served. Transmission electron microscopy, however, disclosed a previously undescribed type of parasitic interaction in which Choreonema interacts with its host through specialized cells known as lenticular cells. These small, lens-shaped cells are produced from the single file of host-penetrating vegetative cells. Pit plug morphology between vegetative and lenticular cells is polarized. Plug caps facing the vegetative cell have normal coralline morphology, while those facing the lenticular cell are composed of three layers. Regions of lenticular cells near host cells protrude toward the host cell; upon encountering the host cell wall, the prolrusion produces numerous finger-like fimbriate processes that make cellular connections with the host cell. Lenticular cells may extend several protrusions toward a host cell or penetrate more than one host cell; two or more lenticular cells may also penetrate the same host cell. The lack of secondary pit connections, cell fusions, and passage of parasitic nuclei suggest that this parasitic relationship may be evolutionarily older than previously reported cases of parasitism in red algae.