Iron is an essential element because it functions as a cofactor of many enzymes, but excess iron causes cell damage. Iron hemostasis in Escherichia coli was transcriptionally maintained by the ferric uptake regulator (Fur). Despite having been studied extensively, the comprehensive physiological roles and mechanisms of Fur-coordinated iron metabolism still remain obscure. In this work, by integrating a high-resolution transcriptomic study of the Fur wild-type and knockout Escherichia coli K-12 strains in the presence or absence of iron with high-throughput ChIP-seq assay and physiological studies, we revisited the regulatory roles of iron and Fur systematically and discovered several intriguing features of Fur regulation. The size of the Fur regulon was expanded greatly, and significant discrepancies were observed to exist between the regulations of Fur on the genes under its direct repression and activation. Fur showed stronger binding strength to the genes under its repression, and genes that were repressed by Fur were more sensitive to Fur and iron regulation as compared to the genes that were activated by Fur. Finally, we found that Fur linked iron metabolism to many essential processes, and the systemic regulations of Fur on carbon metabolism, respiration, and motility were further validated or discussed. These results highlight how Fur and Fur-controlled iron metabolism affect many cellular processes in a systematic way.

Bacillus strains are widely distributed in terrestrial and marine environments, and some of them are used as biocontrol organisms for their biofilm-formation ability. In Bacillus subtilis, biofilm formation is fine-tuned by a complex network, a clear understanding of which still requires study. In bacteria, tmRNA, encoded by the ssrA gene, catalyzes trans-translation that can rescue ribosomes stalled on mRNA transcripts lacking a functional stop codon. tmRNA also affects physiological bioprocesses in some bacteria. In this study, we constructed a ssrA mutant in B. subtilis and found that the biofilm formation in the ssrA mutant was largely impaired. Moreover, we isolated a biofilm-formation suppressor of ssrA, in which the biofilm formation was restored to a level even stronger than that in the wild type. We further performed RNAseq assays with the wild type, ssrA mutant, and suppressor of ssrA for comparisons of their transcriptomes. By analyzing the transcriptomic data, we predicted the possible functions of some differentially expressed genes (DEGs) in the tmRNA regulation of biofilm formation in B. subtilis. Finally, we found that the overexpression of two DEGs, acoA and yhjR, could restore the biofilm formation in the ssrA mutant, indicating that AcoA and YhjR were immediate regulators involved in the tmRNA regulatory web controlling biofilm formation in B. subtilis. Our data can improve the knowledge about the molecular network involved in Bacillus biofilm formation and provide new targets for manipulation of Bacillus biofilms for future investigation.

Background: Enterobacter bugandensis is an emerging human pathogen in which multidrug resistant strains have been continuously isolated from various environments. Thus, this organism possesses the potential to pose challenges in human healthcare. However, the mechanisms, especially the efflux pumps, responsible for the multidrug resistance in E. bugandensis remain to be well elucidated.Methods: The Enterobacter strain CMCC(B) 45301 was specifically identified using whole genome sequencing. The specific CMCC(B) 45301 homologues of the TolC dependent efflux-pump genes characterized in Escherichia coli were identified. The tolC deletion mutant in CMCC(B) 45301 was constructed and subjected to susceptibility tests using 26 different antimicrobial agents, along with the wild type strain. The synergistic effects combining the Bacillus crude extract (BCE) and several other TolC-affected compounds against CMCC(B) 45301 were assayed.Results: We reclassified the Enterobacter CMCC(B) 45301 strain from species cloacae to bugandensis, on the basis of its whole genome sequence. We found that the CMCC(B) 45301 TolC, AcrAB, AcrD, AcrEF, MdtABC, EmrAB, and MacAB exhibit high similarity with their respective homologues in E. coli and Enterobacter cloacae. Our results for the susceptibility tests revealed that lacking tolC causes 4- to 256-fold decrease in the minimal inhibitory concentrations of piperacillin, gentamicin, kanamycin, tetracycline, norfloxacin, ciprofloxacin, chloramphenicol, and erythromycin against CMCC(B) 45301. In addition, the inhibition zones formed by cefuroxime, cefoperazone, amikacin, streptomycin, minocycline, doxycycline, levofloxacin, florfenicol, trimethoprim-sulfamethoxazole, azithromycin, lincomycin, and clindamycin for the tolC mutant were larger or more obvious than that for the parent. Our data suggested the important role played by TolC in CMCC(B) 45301 susceptibility to common antibiotic families covering ß-lactam, aminoglycoside, tetracycline, fluoroquinolone, phenicol, folate pathway antagonist, macrolide, and lincosamide. Deletion for tolC also increased the susceptibility of CMCC(B) 45301 to berberine hydrochloride and BCE, two natural product-based agents. Finally, we found that erythromycin, norfloxacin, and ciprofloxacin can potentiate the antibacterial activity of BCE against CMCC(B) 45301.Discussion: The present study elaborated the comprehensive TolC effect on the antimicrobial susceptibility profile in E. bugandensis, which might contribute to the development of more therapeutic options against this nosocomial pathogen.

Metschnikowia bicuspidata is the specific pathogen for “milky disease” in the Chinese mitten crab (Eriocheir sinensis), accounting for huge losses to the industry. And yet, there is no precise study describing the pathogenesis of M. bicuspidata, largely hindering the development of novel control methods against its causing diseases. Here, we compared the transcriptomes of M. bicuspidata cells collected from a control group (cultured without E. sinensis hemocytes) and a treatment group (cultured with E. sinensis hemocytes), using RNA sequencing. Through comprehensively analyzing the differentially expressed genes (DEGs), both the most regulated ones and the ones involved in crucial enriched KEGG pathways, we found that certain processes might be required for M. bicuspidata’s survival under hemocyte stress. Key genes involved in oxidative phosphorylation, fatty acid metabolism, upper glycolysis, and gluconeogenesis were upregulated, and those for β-glucan unmasking, autophagy, and cell polarity were downregulated, in the treatment group. Our results suggest that M. bicuspidata colonizes and therefore establishes an infection in E. sinensis via enhancing aerobic respiration, glucose-6-phosphate accumulation, and cell-wall masking. In addition, we applied multiple means to evaluate a series of candidate reference genes and found that PMA1 in combination with ACT1 is the most suitable choice for accurate normalization in quantitative real-time PCR (qRT-PCR) assays. Thus, we used this combination as the reference and performed qRT-PCR verification of several DEGs. It is shown that the expression trends of these tested DEGs in qRT-PCR assays are the same as those in RNA-Seq assays. This study not only provides insights into strategies facilitating M. bicuspidata’s survival within E. sinensis, initially elucidating the pathogenesis of this yeast, but also recommends a useful molecular tool regarding qRT-PCR assays in this pathogen.