zntA Gene Research Articles

Profiling of intracellular and extracellular antibiotic resistance genes in municipal wastewater treatment plant and their effluent-receiving river.

The presence of antibiotic resistance genes (ARGs) and heavy metal resistance genes (MRGs) in extracellular and intracellular DNA (eDNA and iDNA) has received considerable attention in recent years owing to the potential threat to human health and the ecosystem. As a result, we investigated six ARGs, three MRGs, and two mobile genetic elements (MGEs) in the municipal wastewater treatment plant (MWWTP) and its adjacent environments. Results revealed that the absolute abundances of eARGs and eMRGs were lower than iARGs and iMRGs in MWWTP. By contrast, eARGs and eMRGs were higher in river sediments. Among ARGs, aminoglycoside resistance genes (aadA) was the most abundant gene (3.13 × 102 to 2.31 × 106 copies/mL in iDNA; 1.27 × 103 to 7.23 × 105 copies/mL in eDNA) in MWWTP, while zntA gene (9.4 × 102 to 3.97 × 106 copies/mL in iDNA; 3.2 × 103 to 6 × 105 copies/mL in eDNA) was amongst the MRGs. Notably, intI1 was enriched and positively correlated with iDNA (tetA, sul1, blaCTX-M, ermB, and merA) and eDNA (blaCTX-M, ermB, and merA), demonstrating its function in the proliferation of resistance genes. This widespread distribution of ARGs, MRGs, and MGEs in MWWTP and its adjacent river sediments will help clarify the transmission routes within these environments and provide a theoretical basis for better monitoring and mitigation of such dissemination.

Characterization and whole-genome sequencing of an extreme arsenic tolerant Citrobacter freundii SRS1 strain isolated from Savar area in Bangladesh.

Citrobacter freundii SRS1, Gram-negative bacteria, were isolated from Savar, Bangladesh. The strain could tolerate up to 80 mM sodium arsenite, 400 mM sodium arsenate, 5 mM manganese sulfate, 3 mM lead nitrate, 2.5 mM cobalt chloride, 2.5 mM cadmium acetate, and 2.5 mM chromium chloride. The whole-genome sequencing revealed that the genome size of C. freundii SRS1 is estimated to be 5.4 Mbp long, and the G+C content is 51.7%. The genome of C. freundii SRS1 contains arsA. arsB, arsC, arsD, arsH, arsR and acr3 genes for arsenic resistance; czcA, czcD, cbiN, and cbiM genes for cobalt; chrA, and chrB genes for chromium; mntH, sitA, sitB, sitC, and sitD genes for manganese; and zntA genes for lead and cadmium resistance. This novel acr3 gene has never previously been reported in any C. freundii strain except SRS1. A set of 130 completely sequenced strains of C. freundii were selected for phylogenomic analysis. The phylogenetic tree showed that the SRS1 strain is closely related to the C. freundii 62 strain. Further analyses of the genes involved in metal and metalloid resistance might facilitate identifying the mechanisms and pathways involved in high metal resistance in the C. freundii SRS1 strain.

Molecular characterization, antibiogram and distribution of zntA gene in zinc-resistant Escherichia coli population recovered from anthropogenically-influenced surface water sources in Nigeria

BackgroundAnthropogenic activities have been largely responsible for the elevated concentration of metals in the environment. The presence of these metals has necessitated the acquisition of resistance mechanisms by bacteria to metals and other environmental toxicants. This study aimed at investigating the occurrence and distribution of zntA gene, and antibiogram of zinc-resistant Escherichia coli (E. coli) from selected anthropogenically-influenced surface waters in Ibadan, Nigeria. MethodsWater samples were collected and analyzed for zinc concentration using atomic absorption spectrophotometer. Zinc-resistant E. coli were isolated on CHROMagar E. coli incorporated with 1 mM of zinc salt and antibiotic susceptibility testing on the isolates was carried out using the disc diffusion method. The identity of the isolates was confirmed by detecting the presence of uidA gene using specific primers, while the detection of zinc resistance gene (zntA) was done using polymerase chain reaction (PCR). ResultsThe zinc concentrations in the surface water samples were: AG (6.12 mg/L), ZAR (5.54 mg/L), UB (0.01 mg/L) and OG (6.61 mg/L). A total of fifty-four zinc-resistant E. coli were obtained. The percentage resistance to the antibiotics was: ampicillin (92.6%), imipenem (77.8%), amoxicillin-clavulanate (68.5%), sulfamethoxazole-trimethoprim (53.7%), doxycycline (68.5%), cefotaxime (27.8%), ciprofloxacin (7.41%), colistin sulphate (13%), chloramphenicol (1.9%) and gentamicin (0%). zntA gene was detected in fifteen (27.8%) of the isolates, while fifty-one (94.4%) of the isolates were multidrug resistant (MDR). ConclusionThis study has confirmed that surface waters in South-west Nigeria could be potential reservoirs of metal and antibiotic resistant bacteria and their genes.

Inorganic Cadmium Detection Using a Fluorescent Whole-Cell Bacterial Bioreporter

Cadmium pollution has become a serious environmental issue due to its toxicity and frequent entrance into environment components such as soil, water, and air via many anthropogenic sources. Over the last two decades, whole-cell bacterial bioreporters have been acknowledged as bio-sentinels for the determination of toxic heavy metals. Herein a sensitive and quite specific bacterial bioreporter was developed to cope with the need for the rapid and simple determination of cadmium. The construction and characterization of a fluorescence-based whole-cell cadmium bioreporter strain, Escherichia coli MG1655 (pBR-PzntA), was described which is based on the expression of green fluorescent protein under the control of the zntA gene promoter of heavy metal resistance determinant. The developed bioreporter was able determine cadmium at 5 µg/L after 3.5 hours of induction in a defined medium while the cadmium detection limit was improved to 2 µg/L after 1.5 hours by the use of an inorganic phosphate-limiting defined medium. Drastic changes in cadmium sensitivity were obtained between bioreporter cells induced at different growth phases. The maximum fluorescence performance was obtained for early exponential growth phase cells. This cadmium bioreporter was found to be more sensitive and specific to cadmium ions than to a wide range of heavy metals and was sensitive to only cadmium at drinking water quality standard concentrations. These findings will lead to future studies including integration of the bioreporter cells into a portable device to assess bioavailable cadmium levels in environmental samples which will provide a rapid and practical field detection technique.

Occurrence of metal and antibiotic resistant Escherichia coli harbouring zntA and copA genes in selected surface water in Ibadan, South-west Nigeria

ABSTRACT Escherichia coli strains were isolated from surface water collected from three sampling points and cultured on CHROMAgar E. coli. The zinc and copper concentrations of the water samples were determined using Atomic Absorption Spectrophotometer. Colonies of Escherichia coli were subjected to increasing concentration (0.1–1.0 mM) of copper and zinc in the isolation medium. Isolates showing resistance to both metals at 1 mM were identified using conventional methods and by targeting the uidA gene, while PCR amplification of the copA and zntA genes was carried out using specific primers. The selected bacteria were subjected to 10 classes of antibiotics using the Kirby–Bauer disc diffusion technique. Five E. coli showing resistance to copper and zinc and possessing uidA gene were obtained. Of the five isolates, three possessed both zntA and copA genes, while zntA was absent in the remaining two. All the isolates peculiarly showed resistance to ampicillin and doxycycline, while three were multi-drug resistant (MDR).

Phenotypic and genotypic investigation of the heavy metal resistance in Escherichia coliisolates recovered from cattle stool samples

The purpose of this study was to examine resistance against cadmium (Cd$^{+2}$), copper (Cu$^{+2}$), lead (Pb$^{+2}$), mercury (Hg$^{+2}$), and manganese (Mn$^{+2}$) in Escherichia coli isolates recovered from cattle stool using phenotypic (agar dilution) and genotypic [polymerase chain reaction (PCR)] methods. In addition, the isolates were genotyped via enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Escherichia coli was isolated and identified from the 100 stool samples that were examined in the study. It was determined phenotypically that all isolates were sensitive to mercury; 97% of them showed resistance to cadmium; 69% showed resistance to copper; 24% showed resistance to lead; and 20% showed resistance to manganese. In the isolates found resistant by phenotypic method, the presence of the zntA gene provided common resistance for Cd$^{+2}$ and Pb$^{+2}$; pcoR gene provided resistance for Cu$^{+2}$; and mntR gene for Mn$^{+2}$ in both genomic DNA and plasmid DNA. Since all isolates were sensitive to Hg$^{+2}$, the presence of merA gene was not examined. While the target genes that were examined for lead, cadmium, and manganese were detected in all isolates, the pcoR gene for copper was detected in 53.6% of the phenotypic-resistant isolates. As a result of the statistical analysis, it was determined that the phenotypic resistance rates of the isolates did not vary according to age group, county, or city at a significant level (P > 0.05). The high metal resistance detected in the present study led us to conclude that heavy metal contamination around cattle farms may be common. Metals are used as an additive substance in animal husbandry. Using the correct fertilizer to minimize contamination sources or limiting the use of materials that contain metals may be useful, and new legal rules may be required.

Analysis of zntA gene in environmental Escherichia coli and additional implications on its role in zinc translocation

Escherichia coli strains from sewage sample were screened for the presence and expression of heavy metal-translocating zntA gene by PCR and RT-PCR analysis with type culture of K-12 as standard strain. The strain which showed high level of gene expression (SBVP1) was chosen to further study the growth and heavy metal translocation. This superior strain was grown in the presence of ZnSO4, Pb (CH3COO)2 and mixture of ZnSO4, Pb(CH3COO)2 metal salts and the growth was observed at different time points. The cell pellet fraction was found to have more of zinc than lead as determined by atomic absorption spectroscopy indicating the translocation of these metals from media to the cells. However, the intracellular translocation of zinc is affected by the presence of lead in the media. Expression of the zntA gene in bacteria grown in the presence of ZnSO4 was also studied and the molecular analysis results correlate with spectroscopic observations.

Roles of Agrobacterium tumefaciens C58 ZntA and ZntB and the transcriptional regulator ZntR in controlling Cd2+/Zn2+/Co2+ resistance and the peroxide stress response.

The putative zinc exporters ZntA (a P1B-type ATPase) and ZntB (2-TM-GxN family) in Agrobacterium tumefaciens were characterized. The expression of the zntA gene is inducible by CdCl2, ZnCl2 and CoCl2, of which CdCl2 is the most potent inducer, whereas zntB is constitutively expressed. The metal-induced expression of zntA is controlled by the MerR-like regulator ZntR. The zntA and zntR mutants were highly sensitive to CdCl2 and ZnCl2, and CoCl2 sensitivity was demonstrated to a lesser extent. By contrast, the zntB mutant showed similar levels of metal resistance to the WT strain. Even in the zntA mutant background, zntB did not play an apparent role in metal resistance under the conditions tested. The inactivation of zntA increased the accumulation of intracellular cadmium and zinc, and conferred hyper-resistance to H2O2. Thus, the metal transporter ZntA and its regulator ZntR are important for controlling zinc homeostasis and cadmium and cobalt detoxification. The loss of either the zntA or zntR gene did not affect the virulence of A. tumefaciens in Nicotiana benthamiana.

Metal transport ATPase genes from Cupriavidus metallidurans CH34: a transcriptomic approach

Cupriavidus metallidurans strain CH34 is a multiple metal resistant β-proteobacterium isolated from the sediments of a zinc metallurgical plant. This strain possesses a large diversity of heavy-metal-resistance genes that are located mostly on two large plasmids, pMOL28 and pMOL30. The metal efflux ATPases constitute the major mechanism by which metal ions are removed and pumped out from the cytoplasmic pool of thiol groups. The genome of C. metallidurans CH34 contains eight such P1-ATPases. This high number of ATPases, compared with the P1-ATPase content in other micro-organisms, is a typical feature of the adaptation of C. metallidurans CH34 to metal-rich biotopes. We performed a phylogenetic analysis, and used quantitative PCR to study the metal-specific induction of the ATPases genes and, for some of them, their neighbouring genes. One of the ATPases, a homologue of the E. coli zntA gene, was characterized in detail by analysing the phenotypes of zntA mutants in different genetic backgrounds. In addition to the already-reported sensitivity to Zn(II), Cd(II), and Pb(II), zntA mutants also displayed a higher sensitivity to Tl(I) and Bi(III), indicating that the ZntA protein might play a role in the tolerance of very large cations.

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host .zntA gene product.

ZntR is a Zn(II)-responsive MerR-like transcriptional regulator of zntA in Escherichia coli.

We have identified the promoter/operator region of the zntA gene of Escherichia coli and shown that Zn(II) is the primary inducer of expression of this Zn(II)/Cd(II) export gene. The promoter PzntA shows sequence similarities to the promoters of mercury resistance (mer) operons, including a long spacer region containing an inverted repeat sequence. The gene encoding the transcriptional regulator of PzntA, designated zntR, has been identified from genome sequence data, by expression of the gene product and by insertional inactivation/complementation. The ZntR product is a member of the MerR family of transcriptional regulators and appears to act as a hypersensitive transcriptional switch. A hybrid MerR/ZntR protein has been constructed and indicates that the C-terminal region of ZntR recognizes Zn(II).

A Zn(II)-translocating P-type ATPase from Proteus mirabilis

A mutant of Proteus mirabilis had been previously isolated as defective in swarming. The mutation had been found to be in a gene related to the Escherichia coli zntA gene, which encodes the ZntA Zn(II)-translocating P-type ATPase. In this study the P. mirabilis gene was expressed in an E. coli strain in which the zntA gene had been disrupted. The P. mirabilis gene complemented the sensitivity to salts of zinc and cadmium. Everted membrane vesicles from the zntA-disrupted strain lost ATP-driven 65Zn(II) uptake. Membranes from the complemented strain had restored 65Zn(II) transport. These results demonstrate that the P. mirabilis homologue of ZntA is a Zn(II)-translocating P-type ATPase.

Molecular characterization of a chromosomal determinant conferring resistance to zinc and cobalt ions in Staphylococcus aureus.

A DNA fragment conferring resistance to zinc and cobalt ions was isolated from a genomic DNA library of Staphylococcus aureus RN450. The DNA sequence analysis revealed two consecutive open reading frames, designated zntR and zntA. The predicted ZntR and ZntA showed significant homology to members of ArsR and cation diffusion families, respectively. A mutant strain containing the null allele of zntA was more sensitive to zinc and cobalt ions than was the parent strain. The metal-sensitive phenotype of the mutant was complemented by a 2.9-kb DNA fragment containing zntR and zntA. An S. aureus strain harboring multiple copies of zntR and zntA showed an increased resistance to zinc. The resistance to zinc in the wild-type strain was inducible. Transcriptional analysis indicated that zntR and zntA genes were cotranscribed. The zinc uptake studies suggested that the zntA product was involved in the export of zinc ions out of cells.