The pickle, a traditional fermented vegetable product, is popular in Sichuan Province of China. The objective of this study was to investigate the diversity of dominant lactic acid bacteria (LAB) in pickles by analyzing 36 samples collected from 6 different regions in Sichuan Province. The LAB counts in these samples varied from 3.90 to 8.40 log cfu ml−1. In total, 185 presumptive LAB with Gram-positive and catalase-negative properties were obtained from these samples using MRS agar, and those strains were identified at the species level by physiological tests, 16S rRNA gene sequencing and multiplex PCR assay. The results revealed that all isolates were accurately identified as Enterococcus thailandicus (2 strains), Lactobacillus alimentarius (16 strains), L. brevis (24 strains), L. paracasei (9 strains), L. plantarum (81 strains), L. pentosus (38 strains), L. sakei (8 strains), L. spicheri (1 strain), Leuconostoc lactis (1 strain) and Pediococcus ethanolidurans (5 strains). The predominant LAB in Sichuan pickle was L. plantarum, which were isolated from most samples. The results also demonstrated that different regions in Sichuan Province have complex compositions of LAB species, and such a rich resource of LAB strains provides raw data for further studies involving probiotic strain selection.
Polyhydroxyalkanoates (PHAs) accumulating bacteria were isolated under various selective conditions such as pH, salt concentrations and types of heavy metal. Fifty strains of bacterial isolates were found to belong to Bacillus, Proteus, Pseudomonas, Aeromonas, Alcaligenes and Chromobacterium, based on phenotypical features and genotypic investigation. Only twenty five bacterial isolates were selected and observed for the production of PHAs. Interestingly, bacteria belonging to Firmucutes Bacillus sp. produced a high amount of PHAs. The maximum PHAs were accumulated by B. licheniformis PHA 007 at 68.80% of dry cell weight (DCW). Pseudomonas sp., Aeromonas sp., Alcaligenes sp. and Chromobacterium sp. were recorded to produce a moderate amount of PHAs, varying from 10.00−44.32% of DCW. The enzymatic activity was preliminarily analyzed by the ratio of the clear zone diameter to colony diameter. Bacillus gave the highest ratio of hydrolysis zone which corresponds to the highest hydrolytic enzyme activities. Bacillus licheniformis PHA 007 had the highest lipase and protease activity at 2.1 and 5.1, respectively. However, the highest amylase activity was observed in Bacillus sp. PHA 023 at 1.4. Determination of metabolic characteristics was also investigated to check for their ability to consume a wide range of substrates. Bacillus, Aeromonas sp. and Alcaligenes sp. had great ability to utilize a variety of substrates. To decrease high PHA cost, different sources of cheap substrates were tested for the production of PHAs. Bacillus cereus PHA 008 gave the maximal yield of PHA production (64.09% of DCW) when cultivated in anaerobically treated POME. In addition, the accumulation of PHA copolymers such as 3-hydroxyvalerate and 3-hydroxyhexanoate was also observed in Bacillus and Pseudomomas sp. strain 012 and 045, respectively. Eight of the nine isolates accumulated a significant amount of PHAs when inexpensive carbon sources were used as substrates. Here it varied from 1.69% of DCW by B. licheniformis PHA 007 to 64.09% of DCW by B. cereus PHA 008.
A cyaA-deficient Escherichia coli strain was transformed by a plasmid carrying the gene for BsPAC, a photoactivated adenylyl cyclase identified from a Beggiatoa sp., and was subjected to an antibiotic susceptibility assay and biofilm formation assay under a light or dark condition. Cells expressing BsPAC that were incubated under blue light (470 nm) were more susceptible to fosfomycin, nalidixic acid and streptomycin than were cells incubated in the dark. Cells expressing BsPAC formed more biofilms when incubated under the light than did cells cultured in the dark. We concluded from these observations that it is possible to determine the importance of cAMP in antibiotic susceptibility and biofilm formation of E. coli by photomanipulating the cellular cAMP level by the use of BsPAC. A site-directed mutant of BsPAC in which Tyr7 was replaced by Phe functioned even in the dark, indicating that Tyr7 plays an important role in photoactivation of BsPAC. Results of mutational analysis of BsPAC should contribute to an understanding of the molecular basis for photoactivation of the protein.
A strictly aerobic, Gram-negative, orange-pigmented, rod-shaped, non-motile and chemoheterotrophic bacteria representing a new genus and species, designated YM8-076T, was isolated from lake water collected at a harbor on Lake Notoro, Hokkaido, Japan. Preliminary analysis based on the 16S rRNA gene sequence revealed that the novel isolate could be affiliated with the family Saprospiraceae of the phylum Bacteroidetes and that it showed highest sequence similarity (88.5%) to Haliscomenobacterhydrossis ATCC 27775T. The strain could be differentiated phenotypically from recognized members of the family Saprospiraceae. The G+C content of DNA was 53.7 mol%, MK-7 was the major menaquinone and iso-C15：0, iso-C15：1 and iso-C17：0 3-OH were the major cellular fatty acids. On the basis of polyphasic taxonomic studies, it was concluded that strain YM8-076T represents a new genus and species of the family Saprospiraceae. We propose the name Portibacterlacus gen. nov., sp. nov. for this strain; its type strain is YM8-076T (=KCTC 23747T=NBRC 108769T).
Proteolytic degradation is one of the serious bottlenecks limiting the yields of heterologous protein production by Aspergillus oryzae. In this study, we selected a tripeptidyl peptidase gene AosedD (AO090166000084) as a candidate potentially degrading the heterologous protein, and performed localization analysis of the fusion protein AoSedD-EGFP in A. oryzae. As a result, the AoSedD-EGFP was observed in the septa and cell walls as well as in the culture medium, suggesting that AoSedD is a secretory enzyme. An AosedD disruptant was constructed to investigate an effect of AoSedD on the production level of heterologous proteins and protease activity. Both of the total protease and tripeptidyl peptidase activities in the culture medium of the AosedD disruptant were decreased as compared to those of the control strain. The maximum yields of recombinant bovine chymosin (CHY) and human lysozyme (HLY) produced by the AosedD disruptants showed approximately 2.9- and 1.7-fold increases, respectively, as compared to their control strains. These results suggest that AoSedD is one of the major proteases involved in the proteolytic degradation of recombinant proteins in A. oryzae.
Three strains of aerobic chemoorganotrophic naphthalene-degrading bacteria (designated TSY03bT, TSY04, and TSW01) isolated from sediment of a polychlorinated-dioxin-transforming microcosm were characterized. These strains had Gram-negative-stained, rod-shaped cells measuring 0.6‒0.9 μm in width and 1.2‒3.0 μm in length and were motile by means of peritrichous flagella. Naphthalene was utilized as the sole carbon and energy source, and the transcription of a putative aromatic-ring hydroxylating gene was inducible by naphthalene. The major component of cellular fatty acids was summed feature 8 (C18:1ω7c and/or C18:1ω6c), and significant proportions of C18:0 and C19:0 cyclo ω8cis were also found. The major respiratory quinone was ubiquinone-10. The G+C content of the DNA was 60.3‒60.9 mol%. Phylogenetic analyses by studying sequence information on the housekeeping atpD, dnaK, glnII, gyrB, and recA genes as well as on 16S rRNA genes and the 16S-23S rDNA internal transcribed spacer region revealed that the strains grouped with members of the genus Rhizobium, with Rhizobiumselenitireducens as their closest relative but formed a distinct lineage at the species level. This was confirmed by genomic DNA-DNA hybridization studies. These phenotypic, genotypic, and phylogenetic data strongly suggest that our isolates should be classified under a novel species of the genus Rhizobium. Thus, we propose the name Rhizobium naphthalenivorans sp. nov. to accommodate the novel isolates. The type strain is TSY03bT (= NBRC 107585T = KCTC 23252T).
This study aimed to evaluate the inheritance of the trait ochratoxin A adsorption in two wine strains of Saccharomyces cerevisiae and their 46 descendants. Each strain was inoculated in triplicate in test tubes containing 10 ml of must obtained from the Calabrian Zibibbo white grape variety, artificially contaminated with ochratoxin A to reach a total content of 4.10 ng/ml. The microvinification trials were performed at 25°C. After 30 days, ochratoxin A values ranged from 0.74 to 3.18 ng/ml, from 0.01 to 2.69 ng/ml, and from 0.60 to 2.95 ng/ml respectively in wines, in lees after washing, and in the saline solution used to wash the lees. The analysis of OTA in wines was performed to find the residual toxin content after yeast activity, thus obtaining technological evidence of yeast influence on wine detoxification. The analysis of OTA in lees after washing was performed to distinguish the OTA linked to cells. The analysis of OTA in the saline solution used to wash the lees was performed to distinguish the OTA adsorbed on yeast cell walls and removed by washing, thus focusing on the adsorption activity of wine yeast through electrostatic and ionic interactions between parietal mannoproteins and OTA. Ploidy of the two parental strains was controlled by flow cytometry. Results demonstrated that the ochratoxin A adsorption is genetically controlled and is a polygenic inheritable trait of wine yeasts. The majority of the descendants are characterized by a great and significant diversity compared to their parents. Both the parental strains had genome sizes consistent with their being diploid, so validating the observed results. These findings constitute an initial step to demonstrate the mechanisms of inheritance and establish breeding strategies to improve the ochratoxin A adsorption trait in wine yeasts. This will allow a decrease in the ochratoxin A content of contaminated musts during winemaking, by using genetically improved wine yeasts.
Eleven strains of acetic acid bacteria were isolated from stems of sugarcane, fruits, and a flower in Japan. The isolates were separated into three groups, Groups I, II, and III, in the genus Acetobacter according to phylogenetic analysis based on 16S rRNA sequences. The isolates had sequence similarities of 99.8-100% within the Group, 99.3-99.6% to those of the type strains of each related Acetobacter species, and less than 98.4% to those of the type strains of other Acetobacter species. Genomic DNA G+C contents of Groups I, II, and III were 59.2-59.4, 60.5-60.7, and 58.7-58.9 mol%, respectively. The isolates in the Group showed high values of DNA-DNA relatedness to each other, but low values less than 46% to the type strains of related Acetobacter species. A good correlation was found between the three Groups and groups based on DNA G+C contents and DNA-DNA relatedness. All the strains had Q-9 as the main component, and Q-8 and Q-10 as minor components. The isolates in the three Groups did not completely match with any Acetobacter species on catalase reaction, the production of ketogluconic acids from D-glucose, growth on ammoniac nitrogen with ethanol (Hoyer-Frateur medium and Frateur modified Hoyer medium), growth on 30% (w/v) D-glucose, growth in 10% (v/v) ethanol, or DNA G+C contents. On the basis of phylogenetic relationships in the genus Acetobacter and chemosystematic and phenotypic characteristics, the three Groups were regarded as novel species in the genus Acetobacter. Acetobacter okinawensis sp. nov. is proposed for Group I, Acetobacter papayae sp. nov. for Group II, and Acetobacter persicus sp. nov. for Group III.