Genome Sequence of Lactococcus garvieae IPLA 31405, a Bacteriocin-Producing, Tetracycline-Resistant Strain Isolated from a Raw-Milk Cheese

This work describes the draft genome sequence of Lactococcus garvieae IPLA 31405, isolated from a traditional Spanish cheese. The genome contains a lactose-galactose operon, a bacteriocin locus, two integrated phages, a transposon harboring an active tet(M) gene, and two theta-type plasmid replicons. Genes encoding virulence factors were not recorded.     

Controlled gene expression in bifidobacteria by use of a bile-responsive element

The promoter activity of the upstream region of the bile-inducible gene betA from Bifidobacterium longum subsp. longum NCC2705 was characterized. DNA fragments were cloned into the reporter vector pMDYAbfB, and the arabinofuranosidase activity was determined under different in vitro conditions. A segment of 469 bp was found to be the smallest operational unit that retains bile inducibility. The reporter activity was strongly affected by the presence of ox gall, cholate, and conjugated cholate, but not by other bile salts and cell-surface-acting compounds. Remarkably, this bile-inducible system was also active in other bifidobacteria containing betA homologs.     

Sequencing and analysis of three plasmids from <Emphasis Type="Italic">Lactobacillus casei</Emphasis> TISTR1341 and development of plasmid-derived <Emphasis Type="Italic">Escherichia coli–L. casei</Emphasis> shuttle vectors

Pyrosequencing followed by conventional PCR and sequencing was used to determine the complete nucleotide sequence of three plasmids (pRCEID2.9, pRCEID3.2, and pRCEID13.9) from the Lactobacillus casei strain TISTR1341. The plasmid sequences were found to be almost identical, respectively, to those of pLA106, pLA105, and pLA103 from Lactobacillus acidophilus strain TK8912, suggesting that these strains may be related. Sequence analysis and comparison indicated that pRCEID2.9 replicates by a rolling circle (RC) mechanism, while pRCEID3.2 and pRCEID13.9 probably follow a theta-type mode of replication. Replicons of pRCEID2.9 and pRCEID13.9 were used to develop Escherichia coli/L. casei compatible shuttle vectors, which were stably maintained in different genetic backgrounds. Real-time quantitative PCR analysis showed copy numbers of around 4 and 15, respectively, for the pRCEID13.9- and pRCEID2.9-derived shuttle vectors per chromosome equivalent. The functionality of vector pRCEID-LC13.9 was proved by cloning and expressing in L. casei of a green fluorescent protein gene variant from Aequorea victoria under the control of the promoter from a homologous lactate dehydrogenase gene. The new vectors might complement those currently in use for the exploitation of L. casei as a cellular factory and in other biotechnological applications.     

Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua

Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae.     

Potential of the Bacillus thuringiensis Toxin Reservoir for the Control of Lobesia botrana (Lepidoptera: Tortricidae), a Major Pest of Grape Plants

The potential of Bacillus thuringiensis Cry proteins to control the grape pest Lobesia botrana was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from B. thuringiensis, was the protein most toxic to L. botrana larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 μg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 μg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of B. thuringiensis sprays or expressed together in transgenic plants.     

Development of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> LL441 and its plasmid-cured derivatives in cheese

A wild Lactobacillus plantarum strain and two of its plasmid-cured derivatives were separately used as adjunct cultures in the manufacture of a Gouda-like traditional Spanish cheese. The wild strain, LL441, harbours seven plasmids and produces a lantibiotic-like bacteriocin. The LL441-B2 derivative has lost plasmids of 40 and 80 kb and the bacteriocin-producing capability. The LL441-B11 derivative has lost in addition a 70 kb plasmid encoding active α- and β-galactosidases. All three strains could be used as adjunct cultures as none of the technological and biochemical parameters of the cheeses was affected. Both the wild-type and the two derivatives were recovered from experimental cheeses up to 30 days after manufacture at similar rates of nearly 20%. Thus, the phenotypic traits under examination were not essential for L. plantarum to grow into the cheese matrix. Electronic Publication     

Changes in Permeability of Brush Border Membrane Vesicles from Spodoptera littoralis Midgut Induced by Insecticidal Crystal Proteins from Bacillus thuringiensis

Bacillus thuringiensis insecticidal crystal proteins (ICPs) are thought to induce pore formation in midgut cell membranes of susceptible insects. Cry1Ca, which is significantly active in Spodoptera littoralis, made brush border membrane vesicles permeable to KCl (osmotic swelling was monitored by the light scattering technique); the marginally active ICPs Cry1Aa, Cry1Ab, and Cry1Ac did not.     

Molecular Analysis of tet(W) Gene-Mediated Tetracycline Resistance in Dominant Intestinal Bifidobacterium Species from Healthy Humans

tet(W) was found responsible for tetracycline resistance (MICs, 4 to ≥32 μg ml⁻¹) in dominant bifidobacterial species from the gastrointestinal tracts of healthy humans. The gene from Bifidobacterium longum H66 proved to be identical over a 2.6-kbp region to the recently described tet(W) determinant of Butyrivibrio fibrisolvens.