Microcin E492 is a pore-forming bacteriocin with toxic activity against Enterobacteriaceae, which undergoes amyloid aggregation as a mechanism to regulate its toxicity. To be active, it requires the posttranslational attachment to the C-terminus of a glycosylated enterochelin derivative (salmochelin), a process carried out by the proteins MceC, MceI and MceJ encoded in the MccE492 gene cluster. Both microcin E492 and salmochelin have a proposed role in the virulence of the bacterial pathogen Klebsiella pneumoniae. Besides, enterochelin is produced as a response to low iron availability and its synthesis is controlled by the global iron regulator Fur. Since the production of active microcin E492 depends on enterochelin biosynthesis, both processes could be coordinately regulated. In this work, we investigated the role of Fur in the expression of the microcin E492 maturation genes mceCJI. mceC was not regulated by Fur as it occurs with its homolog iroB in Salmonella enterica. We demonstrated that mceJI along with the previously uncharacterized gene mceX are transcribed as a single mRNA, and that Fur binds in vivo to a Fur box located upstream of the mceX-mceJI unit. Also, we established that the expression of these genes decreased in a condition of high iron availability, while this effect is abrogated in a Δfur background. Furthermore, our results indicated that MceX acts as a negative regulator of microcin E492 structural gene expression, coupling its synthesis to the iron-dependent regulatory circuit. Consequently, fur or mceX overexpression led to a significant decrease in the antibacterial activity of cells producing microcin E492. Altogether these results show that both the expression of microcin E492 maturation genes mceJI, and MceX the negative regulator of microcin E492 synthesis, are coordinated with the enterochelin production by Fur, depending on the iron levels in the medium.

An increased abundance of antibiotic resistance genes (ARGs) in aquatic environments has been linked to environmental pollution. Mining polluted sites with high concentration of metals could favor the in situ coselection of ARGs, whereas wastewater discharges release fecal antibiotic resistant bacteria in the environment. To study the effect of human fecal contamination and mining pollution, water and sediment samples affected by mining activities and sewage discharges were collected from three lakes in Bolivia, the pristine Andean lake Pata Khota, the Milluni Chico lake directly impacted by acid mine drainage, and the Uru-Uru lake located close to Oruro city and highly polluted by mining activities and human wastewater discharges. Physicochemical parameters, including metal composition, were analyzed in water and sediment samples. ARGs were screened for and verified by quantitative polymerase chain reaction (PCR) together with the mobile element class 1 integron (intl1), as well as crAssphage, a marker of human fecal pollution. The gene intl1 was positively correlated with sul1, sul2, tetA, and blaOXA-2. CrAssphage was only detected in the Uru-Uru lake, and its tributaries and significantly higher abundance of ARGs were found in these sites. Multivariate analysis showed that crAssphage abundance, electrical conductivity, and pH were positively correlated with higher levels of intl1 and ARGs. Taken together, our results suggest that fecal pollution is the major driver of higher levels of ARGs and intl1 in environments contaminated by wastewater and mining activities.

Here, we report the draft genome sequence of the Gram-negative strain Klebsiella pneumoniae RYC492, which produces the amyloid-forming and antibacterial peptide microcin E492. The sequenced genome consists of a 5,095,761-bp assembled open chromosome where the gene cluster for microcin production is located in a putative 31-kb genomic island flanked by sequence repeats and containing a putative integrase-coding gene.

26Water and sediment samples affected by mining activities were collected from 27 three lakes in Bolivia, the pristine Andean lake Pata Khota, the Milluni Chico lake 28 directly impacted by acid mine drainage, and the Uru-Uru lake located close to 29 Oruro city and highly polluted by mining activities and human wastewater 30 discharges. Physicochemical parameters, including metal compositions, were 31 analyzed in water and sediment samples. Antibiotic resistance genes (ARGs), 32 were screened for, and verified by quantitative PCR together with the mobile 33 element class 1 integron (intl1) as well as crAssphage, a marker of human fecal 34 pollution. The gene intl1 showed a positive correlation with sul1, sul2, tetA and 35 blaOXA-2. CrAssphage was only detected in Uru-Uru lake and its tributaries and 36 significantly higher abundance of ARGs were found in these sites. Multivariate 37 analysis showed that crAssphage abundance, electrical conductivity and pH were 38 positively correlated with higher levels of intl1 and ARGs. Taken together our 39 results suggest that fecal pollution is the major driver of higher ARGs and intl1 in 40 wastewater and mining contaminated environments. 41 3 48 can be incorporated and replicated in environmental microorganisms, thereby 49 increasing their concentration (3). 50 It has been reported that anthropogenic activities cause pollution of aquatic 51 environments with ARGs and MGEs (4). Wastewater discharges cause co-52 occurrence of MGEs and different ARGs in water and sediments (5). At a 53 continental scale, ARGs in sediments are strongly correlated with MGEs and 54antibiotic residues (6). Recently, it has been observed that microorganisms living in 55 aquatic microbial communities that came from wastewater were able to transfer 56 ARGs via horizontal gene transfer (HGT) after exposure to low levels of antibiotics 57 and biocides (7). Many of the ARGs that can be found in clinical settings have also 58 been found in the environment, suggesting the possibility of movement and 59 dissemination between these two scenarios (8). 60Mining activities cause contamination of downstream water with dissolved 61 metals (9) where heavy metals tend to accumulate in sediments (10). It has been 62 suggested that heavy metals can favor selection of ARGs via co-selection, i.e. the 63 simultaneous acquisition of both, ARG and metal resistance genes, where the 64 presence of metals constitutes the selective pressure (11). Several studies support 65 this relation. Urban soil samples of Belfast in Northern Ireland, exhibited a pattern 66 of co-occurrence between metals (Zn, Cu, Cd, Co, Ni, Hg, Cr and As) and many 67 ARGs. Moreover, the degree of metal toxicity was positively correlated with the 68 abundance of MGEs, and ARGs (12). Metals, as Cu and Zncan in some cases 69 exert stronger selection pressure over soil microbial communities for the selection 70 of resistant bacteria, even more than specific antibiotics (13). In the Dongying river 71 in China, the levels of Cu and Cr were positively c...

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