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Epigallocatechin-3-gallate (EGCG) is a vital kind of catechin with high bioactive activities, however, limited research has been conducted to elucidate the molecular basis of EGCG biotransformation by Aspergillus niger and the underlying regulatory mechanisms. In this study, A. niger RAF106, isolated from Pu-erh tea, was applied to conduct the EGCG fermentation process, and the samples were collected at different fermentation times to determine the intermediary metabolites of EGCG and the metabolome as well as physiological activity changes of A. niger RAF106. The results demonstrated that EGCG enhances the growth of A. niger RAF106 by promoting conidial germination and hyphae branching. Meanwhile, metabolomic analyses indicated that EGCG significantly regulates the amino acid metabolism of A. niger RAF106. Furthermore, metabolomic analyses also revealed that the levels of original secondary metabolites in the supernatant of the cultures changed significantly from the fermentation stage M2 to M3, in which the main differentially changed metabolites (DCMs) were flavonoids. Most of these flavonoids exhibited antioxidant properties and thus increased the radical scavenging activity of the supernatant of the cultures. In addition, we also found several intermediary metabolites of EGCG, GA, and EGC, including oolonghomobisflavan A, (-)-Epigallocatechin 3, 5-di-gallate, (-)-Epigallocatechin 3-(3-methyl-gallate) (-)-Catechin 3-O-gallate, 4'-Methyl-(-)-epigallocatechin 3-(4-methyl-gallate), myricetin, prodelphinidin B, 7-galloylcatechin, and 3-hydroxyphenylacetic acid. These findings contribute to improving the bioavailability of EGCG and help mine highly active metabolites, which can be used as raw materials for the development of pharmaceutical intermediates or functional foods. In addition, the results also provide a theoretical basis for better control of the risk of A. niger origin and the regulatory mechanisms of the biotransformation process mediated by A. niger.

Citrobacter werkmanii, an aerobe and mesophilic Proteobacterium, is universal in industrial putrefaction, coastal water, and human blood. Our previous studies have discovered that outer membrane protein X (OmpX) of C. werkmanii is involved in calcium response, but the underlying mechanisms and its molecular characteristics remain elusive. To that end, the ompX gene was deleted from the genome of C. werkmanii and its phenotypic variations were thoroughly investigated in conjunction with the wild type (WT) and complementary strains using biochemical and molecular techniques such as RNA-Seq, respectively. The results demonstrated that deleting ompX reduces biofilm formation on polystyrene and glass surfaces. Meanwhile, ΔompX's swimming ability but not for its twitching or swarming abilities, was also reduced on semi-solid plates compared with WT, which was caused by inhibition of flagellar assembly genes, such as flgC, flhB, and fliE, etc. Furthermore, ompX inactivation altered susceptibility to various bactericide classes, as well as responses to Ca

Background Bacterial outer membrane proteins (Omps) play crucial roles in environmental senses, stress responses and substance transportation. Our previous study has found that OmpA contributes to planktonic growth, biocide resistance, biofilm formation and swimming motility in Citrobacter werkmanii, whereas molecular functions of OmpF in this strain remain largely unknown. Hence, in this work, the ompF was firstly knocked out from the genome of C. werkmanii using a homologous recombination method and its phenotypical alternations of ∆ompF were then thoroughly characterized with the parental wild type (WT) and complementary (∆ompF-com) strains using biochemical and molecular approaches. Results The results demonstrated that the swimming ability of ∆ompF was reduced on semi-solid plates compared to WT due to down-regulation of flgC, flgH, fliK and fliF. Meanwhile, the deletion of ompF leads to a decrease of biofilm formation on both glass and polystyrene surfaces because of the reduced cell aggregation. Additionally, the inactivation of ompF also induced different osmotic stress (carbon sources and mental ions) responses of its biofilms comparing with that of WT and ∆ompF-com. Finally, a total of 6 maltose metabolic genes of lamB, malE, malK, malG, malM and malF were all up-regulated in ∆ompF. Gene knockout and HPLC results further revealed that MalEFGK2 cluster mainly contributed to maltose transportation in C. werkmanii. What’s more, for the first time, we have found that the upstream promoter of OmpF and its transcription can be combined with and regulated by MalT. Conclusions Overall, OmpF contributes to a variety of biochemical processes and molecular functions in C. werkmanii and even may be acted as a targeted site to restrain biofilm formation.

Mounting evidence has shown that antimicrobial agents can interfere synergistically with bacterial viability and proliferation when acting together at both the planktonic and biofilm levels without clear underlying molecular mechanisms. Here, multiplexed proteomics by iTRAQ was used to study the interplay between two biocides, the isothiazolone 1,2-benzisothiazolin-3-one (BIT) and the chelating agent disodium ethylenediaminetetraacetic acid (EDTA-2Na), employing the Citrobacter werkmanii as a model system. We first confirmed that these two biocides act synergistically on this bacterial species and then extracted the proteomic profiles of C. werkmanii cells in the presence of BIT, EDTA-2Na, and their combinations. In particular, we identified 43 core proteins that are differentially expressed in all three conditions simultaneously. Meanwhile, we found that these core proteins are consistently up-regulated when these two biocides are present, but not for single biocides, where we found a balanced mix of up- and down-regulation. Meanwhile, most of the deletion mutants of the core DEPs exhibited biofilm growth inhibition under joint biocide action, while their response was very heterogenous, with respect to the wild-type strain. Together, our results show that while BIT and EDTA-2Na act on multiple protein targets, they interact synergistically at the protein level in a very consistent manner. SIGNIFICANCE: Our preliminary experiments have demonstrated that a combination of 1,2-benzisothiazolin-3-one (BIT) and EDTA-2Na shows higher inhibitory effects on planktonic growth and biofilm formation in both C. werkmanii and Staphylococcus aureus than when these two biocides act alone. However, the mechanistic basis of such synergistic interaction is still unknown. Therefore, the key proteins involved in the above-mentioned enhanced antimicrobial synergy were elucidated using multiplexed proteomics analysis by isobaric tags for relative and absolute quantification (iTRAQ). Our results reveal that the joint action of BIT and EDTA-2Na induces consistent protein expression alteration in a set of core proteins of C. werkmanii, which underlies a strong synergistic antimicrobial effect, which increase our understanding of the action modes of BIT and EDTA-2Na as well as their combinations.

Bacterial biofilms, formed on biotic or abiotic surfaces, can lead to serious environmental or medical problems. Therefore, it is necessary to find novel antimicrobial agents to combat biofilms, or more effective combinations of existing biocides. In this study, initial biofilms of Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538 in the presence of xylitol or xylitol and isothiazolones were determined using crystal violet staining in 96-well microplates and confocal laser scanning microscopy. Xylitol and isothiazolones exhibited enhanced synergistic inhibition of initial biofilm formation, and also the structure and production of extracellular polymeric substances by P. aeruginosa ATCC 9027 and S. aureus ATCC 6538 in a dose-dependent manner. In addition, xylitol and isothiazolones inhibited and restored the swimming motility of P. aeruginosa ATCC 9027, respectively. These findings show that a combination of xylitol and isothiazolones exerts pronounced antimicrobial activity against P. aeruginosa and S. aureus biofilms and may be applicable for preventing or reducing bacterial biofilms in vitro.

To screen, identify and study the genes involved in isothiazolone resistance and biofilm formation in Citrobacter werkmanii strain BF-6. A Tn5 transposon library of approximately 900 mutants of C. werkmanii strain BF-6 was generated and screened to isolate 1,2-benzisothiazolin-3-one (BIT) resistant strains. In addition, the tRNA 2-thiocytidine (32) synthetase gene (ttcA) was deleted through homologous recombination and the resulting phenotypic changes of the ΔttcA mutant were studied. A total of 3 genes were successfully identified, among which ΔttcA mutant exhibited a reduction in growth rate and swimming motility. On the other hand, an increase in biofilms formation in ΔttcA were observed but not with a significant resistance enhancement to BIT. This work, for the first time, highlights the role of ttcA gene of C. werkmanii strain BF-6 in BIT resistance and biofilm formation.

Gene ontology (GO) analysis of C. werkmanii BF-6 genome. GO analysis of C. werkmanii BF-6 genome based on GO second level terms, corresponding to 3361 genes for their predicted involvement in biological process (blue), cellular component (brown) and molecular function (yellow). Classified gene objects are depicted as gene numbers. (DOCX 44 kb)

Primers used for amplification of biofilm genes in the qRT-PCR assay. (DOCX 13 kb)

Functional classification of genes encoded by C. werkmanii BF-6 genome based on the COG database. A total of 4234 genes with orthologs in the COG database were classified and the numbers with each classification are indicated. (DOCX 44 kb)

KEGG pathway classifications of genes encoded by the C. werkmanii BF-6 genome based on the KEGG database. Functional classifications were assigned to a total of 3453 genes, and the numbers with each classification are indicated. (DOCX 111 kb)