Microbial ß-Galactosidase of Pediococcus pentosaceus ID-7: Isolation, Cloning, and Molecular Characterization.

Pediococcus pentosaceus ID-7 was isolated from kimchi, a Korean fermented food, and it showed high activity for lactose hydrolysis. The ß-galactosidase of P. pentosaceus ID-7 belongs to the GH2 group, which is composed of two distinct proteins. The heterodimeric LacLM type of ß-galactosidase found in P. pentosaceus ID-7 consists of two genes partially overlapped, lacL and lacM encoding LacL (72.2 kDa) and LacM (35.4 kDa). In this study, Escherichia coli MM294 was used for the production of LacL, LacM, and LacLM. These three types of recombinant proteins were expressed, purified, and characterized. The specific activities of LacLM and LacL were 339 and 31 U/mg, respectively. However, activity was not detected with LacM alone. The optimal pH of LacLM and LacL was pH 7.5 and pH 7.0, and the optimal temperature of LacLM and LacL was 40°C and 50°C, respectively. The optimal temperature changes indicate that LacLM is able to achieve higher activity at a relatively lower temperature. LacLM was strongly activated by Mg2+, Mn2+, and Zn2+, which was not true for LacL. Consistent with this, EDTA strongly inactivated LacLM and LacL, but the presence of reducing agents did not dramatically alter the activity. Taken together, multiple alignment of amino acid sequences and phylogenetic analysis results of LacL and LacM of P. pentosaceus ID-7 suggest the evolution of LacL into LacLM and that the use of divalent metal ions results in higher activity.

Mucosally administered Lactobacillus surface-displayed influenza antigens (sM2 and HA2) with cholera toxin subunit A1 (CTA1) Induce broadly protective immune responses against divergent influenza subtypes.

The development of a universal influenza vaccine that provides broad cross protection against existing and unforeseen influenza viruses is a critical challenge. In this study, we constructed and expressed conserved sM2 and HA2 influenza antigens with cholera toxin subunit A1 (CTA1) on the surface of Lactobacillus casei (pgsA-CTA1sM2HA2/L. casei). Oral and nasal administrations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and their isotypes (IgG1 & IgG2a) as well as mucosal IgA. The mucosal administration of pgsA-CTA1sM2HA2/L. casei may also significantly increase the levels of sM2- or HA2-specific cell-mediated immunity because increased release of both IFN-γ and IL-4 was observed. The recombinant pgsA-CTA1sM2HA2/L. casei provided better protection of BALB/c mice against 10 times the 50% mouse lethal doses (MLD50) of homologous A/EM/Korea/W149/06(H5N1) or A/Aquatic bird/Korea/W81/2005 (H5N2) and heterologous A/Puerto Rico/8/34(H1N1), or A/Chicken/Korea/116/2004(H9N2) or A/Philippines/2/08(H3N2) viruses, compared with L. casei harboring sM2HA2 and also the protection was maintained up to seven months after administration. These results indicate that recombinant L. casei expressing the highly conserved sM2, HA2 of influenza and CTA1 as a mucosal adjuvant could be a potential mucosal vaccine candidate or tool to protect against divergent influenza viruses for human and animal.

Transcriptional regulatory cascade for elastase production in Vibrio vulnificus: LuxO activates luxT expression and LuxT represses smcR expression.

Vibrio vulnificus causes diseases through actions of various virulence factors, including the elastase encoded by the vvpE gene. Through transposon mutagenesis of V. vulnificus, vvpE expression was shown to be increased by luxO mutation. Since the vvpE gene is known to be positively regulated by SmcR via direct binding to the vvpE promoter, the role of LuxO in smcR expression was investigated. The luxAB-transcriptional fusions containing different lengths of the smcR promoter region indicated that the smcR transcription was negatively regulated by LuxO and that a specific upstream region of the smcR gene was required for this repression. Since LuxO is a known member of positive regulators, the negative regulation of smcR transcription by LuxO prompted us to identify the factor(s) linking LuxO and smcR transcription. LuxT was isolated in a ligand fishing experiment using the smcR upstream region as bait, and smcR expression was increased by luxT mutation. Recombinant LuxT bound to a specific upstream region of the smcR gene, -154 to -129 relative to the smcR transcription start site. The expression of luxT was positively regulated by LuxO, and the luxT promoter region contained a putative LuxO-binding site. Mutagenesis of the LuxO-binding site in the luxT promoter region resulted in a loss of transcriptional control by LuxO. Therefore, this study demonstrates a transcriptional regulatory cascade for elastase production, where LuxO activates luxT transcription and LuxT represses smcR transcription.