Characterization of an Aminopeptidase A from Tetragenococcus halophilus CY54 Isolated from Myeolchi-Jeotgal.

In this study, a pepA gene encoding glutamyl (aspartyl)-specific aminopeptidase (PepA; E.C. 3.4.11.7) was cloned from Tetragenococcus halophilus CY54. The translated PepA from T. halophilus CY54 showed very low similarities with PepAs from Lactobacillus and Lactococcus genera. The pepA from T. halophilus CY54 was overexpressed in E. coli BL21(DE3) using pET26b(+). The recombinant PepA was purified by using an Ni- NTA column. The size of the recombinant PepA was 39.13 kDa as determined by SDS-PAGE, while its optimum pH and temperature were pH 5.0 and 60°C, respectively. In addition, the PepA was completely inactivated by 1 mM EDTA, indicating its metallopeptidase nature. The Km and Vmax of the PepA were 0.98 ± 0.006 mM and 0.1 ± 0.002 mM/min, respectively, when Glu-pNA was used as the substrate. This is the first report on PepA from Tetragenococcus species.

Identification of the putative N-acetylglucosaminidase CseA associated with daughter cell separation in Tetragenococcus halophilus.

The lactic acid bacterium Tetragenococcus halophilus, which is used as a starter to brew soy sauce, comprises both cluster-forming strains and dispersed strains. The cluster-forming strains are industrially useful for obtaining clear soy sauce, because the cell clusters are trapped by filter cloth when the soy sauce mash is pressed. However, the molecular mechanism underlying cell cluster formation is unknown. Whole genome sequence analysis and subsequent target sequence analysis revealed that the cluster-forming strains commonly have functional defects in N-acetylglucosaminidase CseA, a peptidoglycan hydrolase. CseA is a multimodular protein that harbors a GH73 domain and six peptidoglycan-binding LysM domains. Recombinant CseA hydrolyzed peptidoglycan and promoted cell separation. Functional analysis of truncated CseA derivatives revealed that the LysM domains play an important role in efficient peptidoglycan degradation and cell separation. Taken together, the results of this study identify CseA as a factor that greatly affects the cluster formation in T. halophilus.

A frameshift mutation in the rRNA large subunit methyltransferase gene rlmA II determines the susceptibility of a honey bee pathogen Melissococcus plutonius to mirosamicin.

American foulbrood (AFB) and European foulbrood (EFB) caused by Paenibacillus larvae and Melissococcus plutonius, respectively, are major bacterial infections of honey bees. Although macrolides (mirosamicin [MRM] and tylosin) have been used to prevent AFB in Japan, macrolide-resistant P. larvae have yet to be found. In this study, we revealed that both MRM-resistant and -susceptible strains exist in Japanese M. plutonius and that a methyltransferase gene (rlmA II ) was disrupted exclusively in MRM-susceptible strains due to a single-nucleotide insertion. The M. plutonius RlmAII modified G748 of 23S rRNA, and the deletion of rlmA II resulted in increased susceptibility to MRM and the loss of modification at G748, suggesting that methylation at G748 by RlmAII confers MRM resistance in M. plutonius. The single-nucleotide mutation in MRM-susceptible strains was easily repaired by spontaneous deletion of the inserted nucleotide; however, intact rlmA II was only found in Japanese M. plutonius and not in a Paraguayan strain tested or any of the whole-genome-sequenced European strains. MRM has been used in apiculture only in Japan. Although M. plutonius is not the target of this drug, the use of MRM as a prophylactic drug for AFB may have influenced the antibiotic susceptibility of the causative agent of EFB.

Microbial quality of industrial liquid egg white: assumptions on spoiling issues in egg-based chilled desserts.

As a 1st step, this study aimed at investigating the microbial quality of liquid egg white in a French egg processing company. Thirty raw and 33 pasteurized liquid egg white samples were analyzed. Pasteurization was globally found efficient on mesophilic contaminants (1.7 ± 1.6 and 0.8 ± 0.9 log CFU/mL in raw and pasteurized samples, respectively), including for the control of Salmonella. However, Gram-positive enterococci were still detected in the pasteurized samples. As a 2nd step, a representative bacterial collection was built for exploring the spoilage issue in egg-based chilled desserts. Custard cream was chosen as growth medium since this food is widely used for the production of French chilled desserts. All of the 166 isolates of the bacterial collection were shown to be able to grow and to induce spoilage of the custard cream at refrigeration temperature (10 °C). Several spoilage types were highlighted in the custard cream, on the basis of changes regarding pH, consistency, production of holes or gas. As a 3rd step, bacterial enzymatic activities were explored on custard cream-based agar media. The bacterial collection was reduced to 43 isolates, based on further selection regarding the genera and the spoilage types previously highlighted. Albeit to different degrees, all these isolates were able to produce proteases. A large part of these isolates also expressed lipolytic and amylolytic activities. This study emphasizes the need to control egg white contamination and especially with Gram-positive heat-resistant Enterococi, in order to guarantee the shelf life of egg-based chilled desserts.

Diversity of plasmids encoding histidine decarboxylase gene in Tetragenococcus spp. isolated from Japanese fish sauce.

Nineteen isolates of histamine producing halophilic bacteria were isolated from four fish sauce mashes, each mash accumulating over 1000 ppm of histamine. The complete sequences of the plasmids encoding the pyruvoyl dependent histidine decarboxylase gene (hdcA), which is harbored in histamine producing bacteria, were determined. In conjunction, the sequence regions adjacent to hdcA were analyzed to provide information regarding its genetic origin. As reference strains, Tetragenococcus halophilus H and T. muriaticus JCM10006(T) were also studied. Phenotypic and 16S rRNA gene sequence analyses identified all isolates as T. halophilus, a predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR, Southern blot, and complete plasmid sequencing) of the histamine producing isolates confirmed that all the isolates harbored approximately 21-37 kbp plasmids encoding a single copy of the hdc cluster consisting of four genes related to histamine production. Analysis of hdc clusters, including spacer regions, indicated >99% sequence similarity among the isolates. All of the plasmids sequenced encoded traA, however genes related to plasmid conjugation, namely mob genes and oriT, were not identified. Two putative mobile genetic elements, ISLP1-like and IS200-like, respectively, were identified in the up- and downstream region of the hdc cluster of all plasmids. Most of the sequences, except hdc cluster and two adjacent IS elements, were diverse among plasmids, suggesting that each histamine producers harbored a different histamine-related plasmid. These results suggested that the hdc cluster was not spread by clonal dissemination depending on the specific plasmid and that the hdc cluster in tetragenococcal plasmid was likely encoded on transformable elements.

Proteinase-producing halophilic lactic acid bacteria isolated from fish sauce fermentation and their ability to produce volatile compounds.

Halophilic lactic acid bacteria were isolated from fish sauce mashes fermented at 1 to 12 months. Seven out of sixty-four isolates were selected according to their proteolytic activity and growth at 25% NaCl for characterization and investigation of volatile compound production. All selected isolates were Gram-positive cocci with pairs/tetrads and grew at 0-25% NaCl, pH 4.5-9.0. Results of 16S rRNA gene sequence analysis showed 99% homology to Tetragenococcus halophilus ATCC 33315. The restriction fragment length polymorphism (RFLP) patterns of all isolates were also similar to those of T. halophilus ATCC 33315. These isolates were, thus, identified as T. halophilus. All isolates hydrolyzed fish protein in the medium containing 25% NaCl. Intracellular aminopeptidase of 7 isolates exhibited the highest activity of 2.85-3.67 U/ml toward Ala-p-nitroanilide (Ala-pNA). T.halophilus strains MS33 and M11 showed the highest alanyl aminopeptidase activity (P<0.05), and produced histamine in mGYP broth containing 5 and 25% NaCl in the level of 6.62-22.55 and 13.14-20.39 mg/100ml, respectively. Predominant volatile compounds of fish broth containing 25% NaCl inoculated with T. halophilus MS33 and MRC5-5-2 were 1-propanol, 2-methylpropanal, and benzaldehyde, corresponding to major volatile compounds in fish sauce. T.halophilus appeared to play an important role in volatile compound formation during fish sauce fermentation.