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Microbes and Environments Dec 2019Horizontal gene transfer (HGT) between bacteria with different habitats and nutritional requirements is important for the spread of antibiotic resistance genes (ARG)....
Horizontal gene transfer (HGT) between bacteria with different habitats and nutritional requirements is important for the spread of antibiotic resistance genes (ARG). The objective of the present study was to clarify the effects of organic matter on HGT between nourished and starved bacteria. We demonstrated that conjugation ability is affected by the nutritional conditions of the cell and environment. A filter mating HGT experiment was performed using Photobacterium damselae ssp. damselae, strain 04Ya311, a marine-origin bacterium possessing the multidrug-resistance plasmid pAQU1, as the donor, and Escherichia coli as the recipient. The donor and recipient were both prepared as nutrient-rich cultured and starved cells. Filter mating was performed on agar plates with and without organic nutrients. The transcription of the plasmid-borne genes tet(M) and traI was quantitated under eutrophic and oligotrophic conditions. The donor P. damselae transferred the plasmid to E. coli at a transfer rate of 10 under oligotrophic and eutrophic conditions. However, when the donor was starved, HGT was not detected under oligotrophic conditions. The addition of organic matter to starved cells restored conjugative HGT even after 6 d of starvation. The transcription of traI was not detected in starved cells, but was restored upon the addition of organic matter. The HGT rate appears to be affected by the transcription of plasmid-associated genes. The present results suggest that the HGT rate is low in starved donors under oligotrophic conditions, but is restored by the addition of organic matter.
Topics: Conjugation, Genetic; Culture Media; Drug Resistance, Multiple, Bacterial; Escherichia coli; Gene Transfer, Horizontal; Genes, Bacterial; Nutrients; Photobacterium; Plasmids; Transcription, Genetic
PubMed: 31631079
DOI: 10.1264/jsme2.ME19099 -
MicrobiologyOpen Mar 2021Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that can be manufactured sustainably and represent a promising green alternative to petrochemical-based...
Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that can be manufactured sustainably and represent a promising green alternative to petrochemical-based plastics. Here, we describe the complete genome of a new marine PHA-producing bacterium-Photobacterium ganghwense (strain C2.2), which we have isolated from the Black Sea seashore. This new isolate is psychrotolerant and accumulates PHA when glycerol is provided as the main carbon source. Transmission electron microscopy, specific staining with Nile Red visualized via epifluorescence microscopy and gas chromatography analysis confirmed the accumulation of PHA. This is the only PHA-producing Photobacterium for which we now have a complete genome sequence, allowing us to investigate the pathways for PHA production and other secondary metabolite synthesis pathways. The de novo assembly genome, obtained using open-source tools, comprises two chromosomes (3.5, 2 Mbp) and a megaplasmid (202 kbp). We identify the entire PHA synthesis gene cluster that encodes a class I PHA synthase, a phasin, a 3-ketothiolase, and an acetoacetyl-CoA reductase. No conventional PHA depolymerase was identified in strain C2.2, but a putative lipase with extracellular amorphous PHA depolymerase activity was annotated, suggesting that C2.2 is unable to degrade intracellular PHA. A complete pathway for the conversion of glycerol to acetyl-CoA was annotated, in accordance with its ability to convert glycerol to PHA. Several secondary metabolite biosynthetic gene clusters and a low number of genes involved in antibiotic resistance and virulence were also identified, indicating the strain's suitability for biotechnological applications.
Topics: Acetyl Coenzyme A; Acetyl-CoA C-Acyltransferase; Acyltransferases; Alcohol Oxidoreductases; Aquatic Organisms; Biosynthetic Pathways; Drug Resistance, Bacterial; Genome, Bacterial; Glycerol; Photobacterium; Plant Lectins; Plasmids; Polyhydroxyalkanoates; Soil Microbiology; Virulence; Whole Genome Sequencing
PubMed: 33970538
DOI: 10.1002/mbo3.1182 -
Angewandte Chemie (International Ed. in... Dec 2021Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in-vivo activity of EPO is...
Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life. EPO carries three N-glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline-assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor.
Topics: Erythropoietin; Glycosylation; Humans; Molecular Structure; N-Acetylneuraminic Acid; Photobacterium; Sialyltransferases; beta-D-Galactoside alpha 2-6-Sialyltransferase
PubMed: 34523784
DOI: 10.1002/anie.202110013 -
International Journal of Food... Nov 2016The processing environment of salmon processing plants represents a potential major source of bacteria causing spoilage of fresh salmon. In this study, we have...
The processing environment of salmon processing plants represents a potential major source of bacteria causing spoilage of fresh salmon. In this study, we have identified major contamination routes of important spoilage associated species within the genera Pseudomonas, Shewanella and Photobacterium in pre-rigor processing of salmon. Bacterial counts and culture-independent 16S rRNA gene analysis on salmon fillet from seven processing plants showed higher levels of Pseudomonas spp. and Shewanella spp. in industrially processed fillets compared to salmon processed under strict hygienic conditions. Higher levels of Pseudomonas spp. and Shewanella spp. were found on fillets produced early on the production day compared to later processed fillets. The levels of Photobacterium spp. were not dependent on the processing method or time of processing. In follow-up studies of two plants, bacterial isolates (n=2101) from the in-plant processing environments (sanitized equipment/machines and seawater) and from salmon collected at different sites in the production were identified by partial 16S rRNA gene sequencing. Pseudomonas spp. dominated in equipment/machines after sanitation with 72 and 91% of samples from the two plants being Pseudomonas-positive. The phylogenetic analyses, based on partial 16S rRNA gene sequencing, showed 48 unique sequence profiles of Pseudomonas of which two were dominant. Only six profiles were found on both machines and in fillets in both plants. Shewanella spp. were found on machines after sanitation in the slaughter department while Photobacterium spp. were not detected after sanitation in any parts of the plants. Shewanella spp. and Photobacterium spp. were found on salmon in the slaughter departments. Shewanella was frequently present in seawater tanks used for bleeding/short term storage. In conclusion, this study provides new knowledge on the processing environment as a source of contamination of salmon fillets with Pseudomonas spp. and Shewanella spp., while Photobacterium spp. most likely originate from the live fish and seawater. The study show that strict hygiene during processing is a prerequisite for optimal shelf life of salmon fillets and that about 90% reductions in the initial levels of bacteria on salmon fillets can be obtained using optimal hygienic conditions.
Topics: Animals; Bacterial Load; Fish Products; Food Contamination; Food Handling; Food Industry; Food Microbiology; Food Preservation; Norway; Photobacterium; Phylogeny; Pseudomonas; RNA, Ribosomal, 16S; Salmon; Seawater; Shewanella
PubMed: 27552347
DOI: 10.1016/j.ijfoodmicro.2016.08.016 -
MSystems 2017Members of the family are often associated with chitin-containing organisms, and they are thought to play a major role in chitin degradation. The purpose of the present...
Members of the family are often associated with chitin-containing organisms, and they are thought to play a major role in chitin degradation. The purpose of the present study was to determine how chitin affects the transcriptome and metabolome of two bioactive strains, and . We focused on chitin degradation genes and secondary metabolites based on the assumption that these molecules in nature confer an advantage to the producer. Growth on chitin caused upregulation of genes related to chitin metabolism and of genes potentially involved in host colonization and/or infection. The expression of genes involved in secondary metabolism was also significantly affected by growth on chitin, in one case being 34-fold upregulated. This was reflected in the metabolome, where the antibiotics andrimid and holomycin were produced in larger amounts on chitin. Other polyketide synthase/ nonribosomal peptide synthetase (PKS-NRPS) clusters in were also strongly upregulated on chitin. Collectively, this suggests that both and have a specific lifestyle for growth on chitin and that their secondary metabolites likely play a crucial role during chitin colonization. The bacterial family (vibrios) is considered a major player in the degradation of chitin, the most abundant polymer in the marine environment; however, the majority of studies on the topic have focused on a small number of species. In this study, we analyzed the genomes of two vibrios to assess their genetic potential for the degradation of chitin. We then used transcriptomics and metabolomics to demonstrate that chitin strongly affects these vibrios at both the transcriptional and metabolic levels. We observed a strong increase in production of secondary metabolites, suggesting an ecological role for these molecules during chitin colonization in the marine environment.
PubMed: 28066819
DOI: 10.1128/mSystems.00141-16 -
Applied and Environmental Microbiology Jul 2022Photobacterium damselae comprises two subspecies, P. damselae subsp. damselae and P. damselae subsp. , that contrast remarkably despite their taxonomic relationship. The...
Photobacterium damselae comprises two subspecies, P. damselae subsp. damselae and P. damselae subsp. , that contrast remarkably despite their taxonomic relationship. The former is opportunistic and free-living but can cause disease in compromised individuals from a broad diversity of taxa, while the latter is a highly specialized, primary fish pathogen. Here, we employ new closed curated genome assemblies from Australia to estimate the global phylogenetic structure of the species P. damselae. We identify genes responsible for the shift from an opportunist to a host-adapted fish pathogen, potentially via an arthropod vector as fish-to-fish transmission was not achieved in repeated cohabitation challenges despite high virulence for . Acquisition of ShdA adhesin and of thiol peroxidase may have allowed the environmental, generalist ancestor to colonize zooplankton and to occasionally enter in fish host sentinel cells. As dependence on the host has increased, P. damselae has lost nonessential genes, such as those related to nitrite and sulfite reduction, urea degradation, a type 6 secretion system (T6SS) and several toxin-antitoxin (TA) systems. Similar to the evolution of Yersinia pestis, the loss of urease may be the crucial event that allowed the pathogen to stably colonize zooplankton vectors. Acquisition of host-specific genes, such as those required to form a sialic acid capsule, was likely necessary for the emergent P. damselae subsp. to become a highly specialized, facultative intracellular fish pathogen. Processes that have shaped P. damselae subsp. from subsp. damselae are similar to those underlying evolution of Yersinia pestis from Y. pseudotuberculosis. Photobacterium damselae subsp. damselae is a ubiquitous marine bacterium and opportunistic pathogen of compromised hosts of diverse taxa. In contrast, its sister subspecies P. damselae subsp. () is highly virulent in fish. has evolved from a single subclade of through gene loss and acquisition. We show that fish-to-fish transmission does not occur in repeated infection models in the primary host and present genomic evidence for vector-borne transmission, potentially via zooplankton. The broad genomic changes from generalist to specialist parallel those of the environmental opportunist Yersinia pseudotuberculosis to vector-borne plague bacterium Y. pestis and demonstrate that evolutionary processes in bacterial pathogens are universal between the terrestrial and marine biosphere.
Topics: Animals; Fish Diseases; Fishes; Gram-Negative Bacterial Infections; Photobacterium; Phylogeny
PubMed: 35862683
DOI: 10.1128/aem.00222-22 -
Foods (Basel, Switzerland) Jul 2021Improved quality control and prolonged shelf life are important actions in preventing food waste. To get an overview of the bacterial diversity of fillets from live...
Improved quality control and prolonged shelf life are important actions in preventing food waste. To get an overview of the bacterial diversity of fillets from live stored mature Atlantic cod, bacterial isolates were identified before and after storage (air and vacuum) and freezing/thawing. Based on the load of dominating bacteria, the effect of different packaging methods and a short freezing/thawing process on prolonged shelf-life was evaluated (total viable counts, bacteriota, sensory attributes, and volatile components). Hand filleted (strict hygiene) cod fillets had a low initial bacterial load dominated by the spoilage organism , whereas industrially produced fillets had higher bacterial loads and diversity (, , , ). The identified bacteria after storage in vacuum or air were similar to the initially identified bacteria. Bacteriota analysis showed that a short time freezing/thawing process reduced while modified atmosphere packaging (MAP; 60% CO/40% O or 60% CO/40% N) inhibited the growth of important spoilage bacteria () and allowed the growth of / and Despite being dominated by fresh fillets stored in MAP 60% CO/40% N demonstrated better sensory quality after 13 days of storage than fillets stored in MAP 60% CO/40% O (dominated by ). spp. or other members of may therefore be potential spoilage organisms in cod when other spoilage bacteria are reduced or inhibited.
PubMed: 34441531
DOI: 10.3390/foods10081754 -
International Journal of Molecular... Mar 2023Lumazine protein from marine luminescent bacteria of species bind with very high affinity to the fluorescent chromophore 6,7-dimethyl-8-ribitylumazine. The light...
Lumazine protein from marine luminescent bacteria of species bind with very high affinity to the fluorescent chromophore 6,7-dimethyl-8-ribitylumazine. The light emission of bacterial luminescent systems is used as a sensitive, rapid, and safe assay for an ever-increasing number of biological systems. Plasmid pRFN4, containing the genes encoding riboflavin from the operon of , was designed for the overproduction of lumazine. To construct fluorescent bacteria for use as microbial sensors, novel recombinant plasmids (pRFN4-Pp N-P and pRFN4-Pp LP N-P) were constructed by amplifying the DNA encoding the N-P gene (L) from and the promoter region (LP) present upstream of the operon of the gene by PCR and ligating into the pRFN4-Pp N-P plasmid. A new recombinant plasmid, pRFN4-Pp LP-N-P, was constructed with the expectation that the fluorescence intensity would be further increased when transformed into . When this plasmid was transformed into 43R, the fluorescence intensity of transformants was 500 times greater than that of alone. As a result, the recombinant plasmid in which the gene encoding N-LumP and DNA containing the promoter exhibited expression that was so high as to show fluorescence in single cells. The fluorescent bacterial systems developed in the present study using and riboflavin genes can be utilized in the future as biosensors with high sensitivity and rapid analysis times.
Topics: Escherichia coli; Riboflavin; Plasmids; Promoter Regions, Genetic; Operon; Luminescent Measurements; Bacterial Proteins
PubMed: 36982169
DOI: 10.3390/ijms24065096 -
Foods (Basel, Switzerland) Jul 2023Broomcorn millet Huangjiu brewing is usually divided into primary fermentation and post-fermentation. Microbial succession is the major factor influencing the...
Broomcorn millet Huangjiu brewing is usually divided into primary fermentation and post-fermentation. Microbial succession is the major factor influencing the development of the typical Huangjiu flavor. Here, we report the changes in flavor substances and microbial community during the primary fermentation of broomcorn millet Huangjiu. Results indicated that a total of 161 volatile flavor compounds were measured during primary fermentation, and estragole was detected for the first time in broomcorn millet Huangjiu. A total of 82 bacteria genera were identified. , , and were the dominant genera. and were dominant among the 30 fungal genera. Correlation analysis showed that 102 microorganisms were involved in major flavor substance production during primary fermentation, , , , , , , , and were most associated with flavoring substances. Four bacteria, (R1), (R2), (R3), and (R4), were isolated and identified from wheat Qu, which were added to wine Qu to prepare four kinds of fortified Qu (QR1, QR2, QR3, QR4). QR1 and QR2 fermentation can enhance the quality of Huangjiu. This work reveals the correlation between microorganisms and volatile flavor compounds and is beneficial for regulating the micro-ecosystem and flavor of the broomcorn millet Huangjiu.
PubMed: 37509772
DOI: 10.3390/foods12142680 -
Frontiers in Microbiology 2019Histamine poisoning is the most common cause of human foodborne illness due to the consumption of fish products. An enzyme-based amperometric biosensor was developed to...
Histamine poisoning is the most common cause of human foodborne illness due to the consumption of fish products. An enzyme-based amperometric biosensor was developed to be used as a screening tool to detect histamine and histamine-producing bacteria (HPB) in tuna. It was developed by immobilizing histidine decarboxylase and horseradish peroxidase on the surface of screen-printed electrodes through a cross-linking procedure employing glutaraldehyde and bovine serum albumin. The signal generated in presence of histamine at the surface of the electrode was measured by chronoamperometry at in presence of a soluble redox mediator. The sensitivity of the electrode was 1.31-1.59 μA/mM, with a linear range from 2 to 20 μg/ml and detection limit of 0.11 μg/ml. In this study fresh tuna filets purchased in supermarkets in different days ( = 8) were analyzed to detect HPB. Samples with different concentration of histamine were analyzed with culture-based counting methods, biosensor and HPLC and also a challenge test was made. Recovery of histamine from cultures and tuna samples was also assessed. The presence of , , and was detected using culture- and PCR-based methods. At the time of purchase these tuna samples had histamine concentrations from below the limit of detection (LOD) to 60 μg/g. HPLC and biosensor methods provided similar results in the range from zero to 432 μg/g (correlation coefficient, = 0.990) and the recovery of histamine from cultures and tuna samples was very high (mean bias -12.69 to 1.63%, with root-mean-square error <12%). These results clearly show that fresh tuna is commonly contaminated with strong HPB. The histamine biosensor can be used by the Food Business Operators as a screening tool to detect their presence and to determine whether their process controls are adequate or not.
PubMed: 31507542
DOI: 10.3389/fmicb.2019.01844