Multiple Metal Resistance Research Articles

Multiple metal resistance and antioxidant enzyme production by Klebsiella pneumoniae PbS3A2 isolated from industrial effluent

Multiple metal resistance and antioxidant enzyme production by Klebsiella pneumoniae PbS3A2 isolated from industrial effluent

Complex interactions between diverse mobile genetic elements drive the evolution of metal-resistant bacterial genomes.

In this study, we compared the genomes of three metal-resistant bacteria isolated from mercury-contaminated soil. We identified diverse and novel MGEs with evidence of multiple LGT events shaping their genomic structure and heavy metal resistance. Among the three metal-resistant strains, Sphingobium sp SA2 and Sphingopyxis sp SE2 were resistant to multiple metals including mercury, cadmium, copper, zinc and lead. Pseudoxanthomonas sp SE1 showed resistance to mercury only. Whole genome sequencing by Illumina and Oxford Nanopore technologies was undertaken to obtain comprehensive genomic data. The Sphingobium and Sphingopyxis strains contained multiple chromosomes and plasmids, whereas the Pseudoxanthomonas strain contained one circular chromosome. Consistent with their metal resistance profiles, the strains of Sphingobium and Sphingopyxis contained a higher quantity of diverse metal resistance genes across their chromosomes and plasmids compared to the single-metal resistant Pseudoxanthomonas SE1. In all three strains, metal resistance genes were principally associated with various novel MGEs including genomic islands (GIs), integrative conjugative elements (ICEs), transposons, insertion sequences (IS), recombinase in trio (RIT) elements and group II introns, indicating their importance in facilitating metal resistance adaptation in a contaminated environment. In the Pseudoxanthomonas strain, metal resistance regions were largely situated on a GI. The chromosomes of the strains of Sphingobium and Sphingopyxis contained multiple metal resistance regions, which were likely acquired by several GIs, ICEs, numerous IS elements, several Tn3 family transposons and RIT elements. Two of the plasmids of Sphingobium were impacted by Tn3 family transposons and ISs likely integrating metal resistance genes. The two plasmids of Sphingopyxis harboured transposons, IS elements, an RIT element and a group II intron. This study provides a comprehensive annotation of complex genomic regions of metal resistance associated with novel MGEs. It highlights the critical importance of LGT in the evolution of metal resistance of bacteria in contaminated environments.

Complete Genome Sequence of a Canadian Strain of Escherichia coli with Multiple Metal and Antimicrobial Resistance Genes That Was Isolated from Municipal Biosolids.

This announcement reports the complete genome sequence of a non-Shiga toxin-producing Escherichia coli strain that was isolated from municipal biosolids collected from a Canadian wastewater treatment plant. This strain contains multiple metal, antimicrobial, and heat resistance genes, as determined by genome sequencing, and could be a useful bacterial model for future studies.

A novel strain of Shigella isolated from the gut of Lepidocephalichthys guntea has in its genome a complete gene package for Type ll secretion system, and elaborate repertoire of genes responsible for multiple antibiotic-resistance and metal resistance via specific efflux channels.

The bacterial strain GCP5 was isolated from the gut of a bottom-dwelling fish Lepidocephalichthys guntea, that lives in the Magurmari River near North Bengal University in Siliguri, India. GCP5 was phylogenetically assigned to the Shigella genus using whole genome-based trees, k-mer analysis, the multilocus species tree (MLST), and single nucleotide polymorphism (SNP)-based trees, and the genetic makeup of the isolate was determined following assembly of the genome sequences and genome annotation with several bioinformatics tools. The presence of a complete package of general-secretory-pathway (gsp) genes, grouped in an operon identical to a well-characterized type II secretion system (T2SS), was confirmed by genome mining of Shigella sp. GCP5. The operon's gsp genes shared the most homology with Escherichia coli gsp genes. A few more high-pathogenicity islands (HPIs) in the GCP5 genome were validated using the pan-genomes analysis pipeline (PGAP) and island viewer. Several antibiotic-resistance genes were found in this genome, as well as the existence of key antibiotic efflux pump families, allowing for the creation of a gene network of several antibiotic efflux transporters. In addition, the genome contained genes specific for nickel transport, the nikABCD system, and the RND family transporter cusCFBA, which confers resistance to copper and silver by effluxing out Cu+ and Ag+ ions.

Enzymatic Activity and Horizontal Gene Transfer of Heavy Metals and Antibiotic Resistant Proteus vulgaris from Hospital Wastewater: An Insight.

Globally, the issue of microbial resistance to medicines and heavy metals is getting worse. There are few reports or data available for Proteus vulgaris (P. vulgaris), particularly in India. This investigation intends to reveal the bacteria's ability to transmit genes and their level of resistance as well. The wastewater samples were taken from several hospitals in Lucknow City, India, and examined for the presence of Gram-negative bacteria that were resistant to antibiotics and heavy metals. The microbial population count in different hospital wastewaters decreases with increasing concentrations of metal and antibiotics. Among all the examined metals, Ni and Zn had the highest viable counts, whereas Hg, Cd, and Co had the lowest viable counts. Penicillin, ampicillin, and amoxicillin, among the antibiotics, demonstrated higher viable counts, whereas tetracycline and erythromycin exhibited lower viable counts. The MIC values for the P. vulgaris isolates tested ranged from 50 to 16,00 μg/ml for each metal tested. The multiple metal resistance (MMR) index, which ranged from 0.04 to 0.50, showed diverse heavy metal resistance patterns in all P. vulgaris isolates (in the case of 2-7 metals in various combinations). All of the tested isolates had methicillin resistance, whereas the least number of isolates had ofloxacin, gentamycin, or neomycin resistance. The P. vulgaris isolates displayed multidrug resistance patterns (2-12 drugs) in various antibiotic combinations. The MAR indexes were shown to be between (0.02-0.7). From the total isolates, 98%, 84%, and 80% had urease, gelatinase, and amylase activity, whereas 68% and 56% displayed protease and beta-lactamase activity. Plasmids were present in all the selected resistant isolates and varied in size from 42.5 to 57.0 kb and molecular weight from 27.2 to 37.0 MD. The transmission of the antibiotic/metal resistance genes was evaluated between a total of 7 pairs of isolates. A higher transfer frequency (4.4 × 10-1) was observed among antibiotics, although a lower transfer frequency (1.0 × 10-2) was observed against metals in both the media from the entire site tested. According to exponential decay, the population of hospital wastewater declined in the following order across all sites: Site II > Site IV > Site III > Site I for antibiotics and site IV > site II > site I >site III for metal. Different metal and antibiotic concentrations have varying effects on the population. The metal-tolerant P. vulgaris from hospital wastewater was studied in the current study had multiple distinct patterns of antibiotic resistance. It could provide cutting-edge methods for treating infectious diseases, which are essential for managing and assessing the risks associated with hospital wastewater, especially in the case of P. vulgaris.

Globally Disseminated Multidrug Resistance Plasmids Revealed by Complete Assembly of Multidrug Resistant Escherichia coli and Klebsiella pneumoniae Genomes from Diarrheal Disease in Botswana

Antimicrobial resistance is a disseminated global health challenge because many of the genes that cause resistance can transfer horizontally between bacteria. Despite the central role of extrachromosomal DNA elements called plasmids in driving the spread of resistance, the detection and surveillance of plasmids remains a significant barrier in molecular epidemiology. We assessed two DNA sequencing platforms alone and in combination for laboratory diagnostics in Botswana by annotating antibiotic resistance genes and plasmids in extensively drug resistant bacteria from diarrhea in Botswana. Long-read Nanopore DNA sequencing and high accuracy basecalling effectively estimated the architecture and gene content of three plasmids in Escherichia coli HUM3355 and two plasmids in Klebsiella pneumoniae HUM7199. Polishing the assemblies with Illumina reads increased base calling precision with small improvements to gene prediction. All five plasmids encoded one or more antibiotic resistance genes, usually within gene islands containing multiple antibiotic and metal resistance genes, and four plasmids encoded genes associated with conjugative transfer. Two plasmids were almost identical to antibiotic resistance plasmids sequenced in Europe and North America from human infection and a pig farm. These One Health connections demonstrate how low-, middle-, and high-income countries collectively benefit from increased whole genome sequencing capacity for surveillance and tracking of infectious diseases and antibiotic resistance genes that can transfer between animal hosts and move across continents.

Optimum Biosorption and Resistance of Uranium by Metal-Resistant Bacteria Isolated from Rock Ore

Natural environments contain a variety of microorganisms, of which, a limited number are able to develop bioremediation process. The main goal of the present paper was to perform microbiological investigations to culturable bacteria isolated from uranium ore samples from Aborshid Egypt, and characterize their response to 15 antibiotics and 10 heavy metals beside uranium. Among the potential environmental isolates, there was no detection of any acquired antibiotic resistance , which supports the idea that their resistance mechanisms are mainly intrinsic. On the contrary, the potentially pathogenic isolates presented a broad diversity of acquired antibiotic resistance toward different antibiotic classes. Furthermore, results revealed a varying response of the ore bacteria to the tested heavy metals. All isolates showed multiple metal resistances toward 10 heavy metals, with MIC ranging from 50 to 1000 ppm. We found that optimum temperature for growth of bacteria in general ranges from 30 to 50 °C and bio-sorption of metal increased with increasing pH from 1.0 to 5.0, then decreased upon further increase to 7.0. The decrease in the sorption efficiency at pH higher than 5.0 may be due to the precipitation as hydroxide. We found possible correlation between antibiotic resistance and heavy-metal resistance patterns of Escherichia coli strains isolated from uranium ore; the most potent of the strains in both groups were resistant to Pb, Ni, Cu and Zn. It was concluded that highly metal-resistant bacteria could be used with potential application for treatment of wastewaters.

Complete Genome Sequence of a Mucilaginibacter sp. Strain Isolated from Estuarine Soil Contaminated with Mine Tailings from the Samarco Disaster at Fundão Dam.

ABSTRACTWe report the complete genome sequence of Mucilaginibacter strain 21P, which was isolated from estuarine soil contaminated with mine tailings from the Samarco disaster, which occurred in 2015 in Brazil. The genome sequence comprised 4,739,655 bp, with a G+C content of 43.2%, and harbors multiple antibiotic and metal resistance genes.

Staphylococcus saprophyticus Causing Infections in Humans Is Associated with High Resistance to Heavy Metals.

Staphylococcus saprophyticus is a common pathogen of the urinary tract, a heavy metal-rich environment, but information regarding its heavy metal resistance is unknown. We investigated 422 S. saprophyticus isolates from human infection and colonization/contamination, animals, and environmental sources for resistance to copper, zinc, arsenic, and cadmium using the agar dilution method. To identify the genes associated with metal resistance and assess possible links to pathogenicity, we accessed the whole-genome sequence of all isolates and used in silico and pangenome-wide association approaches. The MIC values for copper and zinc were uniformly high (1,600 mg/liter). Genes encoding copper efflux pumps (copA, copB, copZ, mco, and csoR) and zinc transporters (zinT, czrAB, znuBC, and zur) were abundant in the population (20 to 100%). Arsenic and cadmium showed various susceptibility levels. Genes encoding the ars operon (arsRDABC), an ABC transporter and a two-component permease, were linked to resistance to arsenic (MICs ≥ 1,600 mg/liter; 14% [58/422]; P < 0.05). At least three cad genes (cadA or cadC and cadD-cadX or czrC) and genes encoding multidrug efflux pumps and hyperosmoregulation in acidified conditions were associated with resistance to cadmium (MICs ≥ 200 mg/liter; 20% [85/422]; P < 0.05). These resistance genes were frequently carried by mobile genetic elements. Resistance to arsenic and cadmium were linked to human infection and a clonal lineage originating in animals (P < 0.05). Altogether, S. saprophyticus was highly resistant to heavy metals and accumulated multiple metal resistance determinants. The highest arsenic and cadmium resistance levels were associated with infection, suggesting resistance to these metals is relevant for S. saprophyticus pathogenicity.

How sediment bacterial community shifts along the urban river located in mining city.

Bacterial communities play critical roles in biogeochemical cycles and serve as sensitive indicators of environmental fluctuation. However, the influence of mineral resource exploitation on shaping the bacterial communities in the urban river is still ambiguous. In this study, high-throughput sequencing was used to determine the spatial distribution of the sediment bacterial communities along an urban river in the famous mining city Panzhihua of China. The results showed that mineral resource exploitation had a significant impact on the urban river bacterial community structure but not on the bacterial ecological functions. Distinct families of bacteria often associated with nutrients (i.e., Comamonadaceae and Sphingomonadaceae) and metal contaminants (i.e., Rhodobacteraceae) were more predominant in the residential and mining area, respectively. Relative to dispersal dynamics, environmentally induced species sorting may primarily influence bacterial community structure. Heavy metals and sediment physicochemical properties had both similar and significant influence on shaping bacterial community structure. Among heavy metals, essential metal elements explained more rates of bacterial variation than toxic metals at moderate contaminant levels. Moreover, the bacteria with multiple metal resistances identified in culture-dependent experiments were probably not suitable for indicating heavy metal contamination in field research. Thus, several sensitive bacterial genera such as Rhodobacter, Hylemonella, and Dechloromonas were identified as potential bioindicators to monitor metals (iron and titanium) and nutrients (phosphorus and organic carbon) in the river ecosystem of the Panzhihua region. Together, these results profiled the coupling effect of urbanization and mineral resource utilization on shaping sediment bacterial communities in urban rivers.

Lichenological Practices for Monitoring Atmospheric Pollution and Climate Change in India

Soil microflora is continuously changing with altered soil conditions. These soil alterations are a consequence of heavy metals entering and affecting every sphere of life. Heavy metals are not only hazardous for crops but also affect the soil microbial community. Soil bacteria with the potential of plant growth promotion and multiple metal resistances can be an instrument for crop improvement and heavy metal detoxification. In this study, predominant bacterial community associated with the heavy metal contaminated soil was studied using 16S rRNA gene sequencing in association with culture-based techniques. Elemental metal analysis of collected soil samples showed an elevated level of metal content in the soil. 16S rRNA gene analysis and phylogenetic analysis of 126 bacterial clones revealed the probable predominance of Pseudomonas (40.48%) followed by Flavisolibacter (13.49%). Based on morphological and biochemical characterization, nine Pseudomonas strains were selected from the soil and were further confirmed by 16S rRNA gene sequencing with 92%-100% similarity with Pseudomonas species. The minimum inhibitory concentration (MIC) and maximum tolerance capacity (MTC) of three essential metals Cu, Zn, and Fe were determined individually and in combinations. It was found that Zn is the most toxic metal among the three metals and the metal showed a synergistic effect in inhibiting microbial growth when used in combinations. Presence of three metal resistant/tolerant genes czcA, pcoA and copB were also determined in the isolated Pseudomonas sp. by PCR. The soil in this region has high concentrations of heavy metals. The indigenous Pseudomonas sp. has multiple metal resistances and can be used for bioremediation of heavy metals and microbe assisted phytoremediation.

Complete Sequences of Multiple-Drug Resistant IncHI2 ST3 Plasmids in Escherichia coli of Porcine Origin in Australia

IncHI2 ST3 plasmids are known carriers of multiple antimicrobial resistance genes. Complete plasmid sequences from multiple drug resistant Escherichia coli circulating in Australian swine is however limited. Here we sequenced two related IncHI2 ST3 plasmids, pSDE-SvHI2 and pSDC-F2_12BHI2, from phylogenetically unrelated multiple-drug resistant Escherichia coli strains SvETEC (CC23:O157:H19) and F2_12B (ST93:O7:H4) from geographically disparate pig production operations in New South Wales, Australia. Unicycler was used to co-assemble short read (Illumina) and long read (PacBio SMRT) nucleotide sequence data. The plasmids encoded three drug-resistance loci, two of which carried class 1 integrons. One integron, hosting drfA12-orfF-aadA2, was within a hybrid Tn1721/21, with the second residing within a copper/silver resistance transposon, comprising part of an atypical sul3-associated structure. The third resistance locus was flanked by IS15DI and encoded neomycin resistance (neoR). An oqx-encoding transposon (quinolone resistance), similar in structure to Tn6010, was identified only in pSDC-F2_12BHI2. Both plasmids showed high sequence identity to plasmid pSTM6-275, recently described in Salmonella enterica serotype 1,4,[5],12:i:- that has risen to prominence and become endemic in Australia. IncHI2 ST3 plasmids circulating in commensal and pathogenic E. coli from Australian swine belong to a lineage of plasmids often in association with sul3 and host multiple complex antibiotic and metal resistance structures, formed in part by IS26.

Pseudomonas: A Major Bacteria in Heavy Metal Contaminated Soil of South-West Punjab, India

Soil microflora is continuously changing with altered soil conditions. These soil alterations are a consequence of heavy metals entering and affecting every sphere of life. Heavy metals are not only hazardous for crops but also affect the soil microbial community. Soil bacteria with the potential of plant growth promotion and multiple metal resistances can be an instrument for crop improvement and heavy metal detoxification. In this study, predominant bacterial community associated with the heavy metal contaminated soil was studied using 16S rRNA gene sequencing in association with culture-based techniques. Elemental metal analysis of collected soil samples showed an elevated level of metal content in the soil. 16S rRNA gene analysis and phylogenetic analysis of 126 bacterial clones revealed the probable predominance of Pseudomonas (40.48%) followed by Flavisolibacter (13.49%). Based on morphological and biochemical characterization, nine Pseudomonas strains were selected from the soil and were further confirmed by 16S rRNA gene sequencing with 92%-100% similarity with Pseudomonas species. The minimum inhibitory concentration (MIC) and maximum tolerance capacity (MTC) of three essential metals Cu, Zn, and Fe were determined individually and in combinations. It was found that Zn is the most toxic metal among the three metals and the metal showed a synergistic effect in inhibiting microbial growth when used in combinations. Presence of three metal resistant/tolerant genes czcA, pcoA and copB were also determined in the isolated Pseudomonas sp. by PCR. The soil in this region has high concentrations of heavy metals. The indigenous Pseudomonas sp. has multiple metal resistances and can be used for bioremediation of heavy metals and microbe assisted phytoremediation.

Protocols of Conjugative Plasmid Transfer in Salmonella: Plate, Broth, and Filter Mating Approaches.

Bacterial conjugation is a natural process that allows for horizontal transmission of DNA from one bacterium to another. Several plasmids carry transposons that encode multiple antimicrobial and metal resistance genes. Conjugative plasmid transfer requires intimate cell-to-cell contacts between the donor and the recipient. Self-conjugative plasmids harbor tra genes which facilitate plasmid transfer from donor to recipient bacterial strain. Here we describe different methods of conjugative plasmid transfers via conjugation.

Multiple metal resistance and Cr6+ reduction by bacterium, Staphylococcus sciuri A-HS1, isolated from untreated tannery effluent

A chromium-resistant bacterium was isolated indigenously from tannery effluent and identified as Staphylococcus sciuri. The optimum growth was observed at 37 °C and pH 7. S. sciuri was able to resist Cr6+ (25 mM) and other heavy metals such as As2+ (19 mM), Pb2+ (18.5 mM), Zn2+ (17 mM), Cu2+ (2.5 mM), Cd2+ (3 mM), and Ni2+ (1.5 mM). Biochemical characterization of chromate reductase revealed its optimal pH as 8.0 and temperature as 40 °C. The enzyme activity was stimulated only by Mg2+ among other heavy metals tested. S. sciuri showed chromium biosorption proficiency (q) after 2, 4, 6 and 8 days as 42, 73, 85 and 31 mM/g, respectively. Cr6+ did not stimulate activities of APOX, SOD and CAT significantly, however, only POX showed marked production (86%). Also, enhanced production of glutathione and other non-protein thiols significantly combat metal induced oxidative stress. Pilot study verified that S. sciuri was able to remove 93% Cr6+ from tannery effluent within 6 days of incubation. Thus, S. sciuri could act as a potential candidate for the bioremediation of hexavalent chromium contaminated environmental sites.

Iron (II) and other heavy-metal tolerance in bacteria isolated from rock varnish in the arid region of Al-Jafer Basin, Jordan

Alnaimat S, Shattal SA, AlthunibatO, Alsbou E, Amasha R. 2017. Iron (II) and other heavy-metal tolerance in bacteria isolated from rock varnish in the arid region of Al-Jafer Basin, Jordan. Biodiversitas 18: 1250-1257. This study deals with enumeration, identification and molecular characterization of bacterial species tolerant of iron (II) and other heavy metals that isolated from rock varnish collected from the arid region of Al-Jafer Basin, Jordan. Based on conventional culture-dependent methods, six isolates exhibited a high degree of tolerance to iron (II) and other heavy metals with a minimum inhibitory concentration (550–600 mg/L) to iron, (500-550 mg/L) to lead, (350 mg/L) to copper, (400-450 mg/L) to chromium, (150-200 mg/L) to silver, and (150-200 mg/L) to mercury, in solid media. The results showed that all isolates could tolerate 2000 mg/L Fe2+ expressed as maximum tolerance concentration (MTC). The multiple metal resistances of these isolates were also associated with multiple antibiotic resistances. These iron tolerant isolates were shown to be capable of growth at temperatures ranging from 30 to 40°C and pH values ranging from 5 to 9 with some differences between the six isolates. On the basis of morphology and of 16S rDNA gene sequencing and phylogeny analysis, the iron-tolerant isolates were identified as Bacillus zhangzhouensis (DVCr1), Staphylococcus warneri (DVCr6), Bacillus cereus (DVCr7), Staphylococcus pasteuri (DVCu2), Brevibacterium frigoritolerans (DVPb) and Bacillus altitudinis (DVCr10). The 16S rDNA sequences from five of the six strains were submitted to GenBank and are available under the accession numbers KX781139, KX781140, KX781141, KX781145 and KX781148. The results highlight the high potential of a non-polluted habitat like rock varnish to provide new microbial communities that could be used for enhanced bioremediation of heavy metals from a contaminated site.

Uranium Removal from Wastewater Using Immobilized Multiple Heavy-Metal and Antibiotic Resistance E. coli Isolated from Aborshid Egypt

In this study 6 bacterial isolate, isolated Uranium Ore samples from Aborshid Egypt, were characterized for their response to 15 antibiotics and 10 heavy metals Beside Uranium. The results revealed a varying response of the Ore bacteria to the tested heavy metals. All isolates showed multiple metal resistance towards two to six heavy metals, with MIC ranging from 50 to 1000 ppm. The most potent of the strains in both groups were resistant to Pb, Ni, Cu and Zn. highly metal-resistant bacteria could be used with potential application for treatment of wastewaters, using Immobilized Bacterial cells isolated from Egypt uranium Ore, Uranium removal from uranium refining wastewater, the most potent isolate was identified the Egyptian strains belong to E. coli, based on 16S rRNA gene sequencing the nucleotide sequences reported here were deposited to the NCBI Nucleotide Sequence Database under accession numbers (MF496270) by the name of Mostafa gomaa fadl.

Arbuscular Mycorrhiza-Mediated Alterations in Redox Buffer Synchronize Antioxidant Network to Alleviate Salt Induced Oxidative Burden in Host Legumes and their Nodules : A Review

Soil microflora is continuously changing with altered soil conditions. These soil alterations are a consequence of heavy metals entering and affecting every sphere of life. Heavy metals are not only hazardous for crops but also affect the soil microbial community. Soil bacteria with the potential of plant growth promotion and multiple metal resistances can be an instrument for crop improvement and heavy metal detoxification. In this study, predominant bacterial community associated with the heavy metal contaminated soil was studied using 16S rRNA gene sequencing in association with culture-based techniques. Elemental metal analysis of collected soil samples showed an elevated level of metal content in the soil. 16S rRNA gene analysis and phylogenetic analysis of 126 bacterial clones revealed the probable predominance of Pseudomonas (40.48%) followed by Flavisolibacter (13.49%). Based on morphological and biochemical characterization, nine Pseudomonas strains were selected from the soil and were further confirmed by 16S rRNA gene sequencing with 92%-100% similarity with Pseudomonas species. The minimum inhibitory concentration (MIC) and maximum tolerance capacity (MTC) of three essential metals Cu, Zn, and Fe were determined individually and in combinations. It was found that Zn is the most toxic metal among the three metals and the metal showed a synergistic effect in inhibiting microbial growth when used in combinations. Presence of three metal resistant/tolerant genes czcA, pcoA and copB were also determined in the isolated Pseudomonas sp. by PCR. The soil in this region has high concentrations of heavy metals. The indigenous Pseudomonas sp. has multiple metal resistances and can be used for bioremediation of heavy metals and microbe assisted phytoremediation.

Agromyces aureus sp. nov., isolated from the rhizosphere of Salix caprea L. grown in a heavy-metal-contaminated soil.

A Gram-reaction-positive, motile, yellow-pigmented and rod-shaped bacterial strain, designated AR33T, was isolated from the rhizosphere of Salix caprea L. growing in a former zinc/lead mining and processing site in Austria. A polyphasic approach was applied to determine its taxonomic position. 16S rRNA gene sequence analysis, and morphological and chemotaxonomic properties showed that strain AR33T belongs to the genus Agromyces. Strain AR33T had peptidoglycan type B2γ and the major menaquinones were MK-11, MK-10 and MK-12. The main branched-chain fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Strain AR33T showed catalase and oxidase activity and multiple heavy metal resistances to zinc, lead and cadmium. The DNA G+C content was 70.1 mol%. Levels of 16S rRNA gene sequence similarity with closely related recognized species of the genus Agromyces ranged between 98 and 99 %. However, DNA-DNA hybridization between strain AR33T and the type strains of three Agromyces species showed values lower than 42 % relatedness. Therefore, differential phenotypic characteristics together with DNA-DNA relatedness suggested that strain AR33T can be recognized as representing a distinct Agromyces species, for which the name Agromyces aureus sp. nov. is proposed. The type strain is AR33T (=DSM 101731T=LMG 29235T).

Paralogous Regulators ArsR1 and ArsR2 of Pseudomonas putida KT2440 as a Basis for Arsenic Biosensor Development.

The remarkable metal resistance of many microorganisms is related to the presence of multiple metal resistance operons. Pseudomonas putida KT2440 can be considered a model for these microorganisms since its arsenic resistance is due to the action of proteins encoded by the two paralogous arsenic resistance operons ARS1 and ARS2. Both operons contain the genes encoding the transcriptional regulators ArsR1 and ArsR2 that control operon expression. We show here that purified ArsR1 and ArsR2 bind the trivalent salt of arsenic (arsenite) with similar affinities (~30 μM), whereas no binding is observed for the pentavalent salt (arsenate). Furthermore, trivalent salts of bismuth and antimony showed binding to both paralogues. The positions of cysteines, found to bind arsenic in other homologues, indicate that ArsR1 and ArsR2 employ different modes of arsenite recognition. Both paralogues are dimeric and possess significant thermal stability. Both proteins were used to construct whole-cell, lacZ-based biosensors. Whereas responses to bismuth were negligible, significant responses were observed for arsenite, arsenate, and antimony. Biosensors based on the P. putida arsB1 arsB2 arsenic efflux pump double mutant were significantly more sensitive than biosensors based on the wild-type strain. This sensitivity enhancement by pump mutation may be a convenient strategy for the construction of other biosensors. A frequent limitation found for other arsenic biosensors was their elevated background signal and interference by inorganic phosphate. The constructed biosensors show no interference by inorganic phosphate, are characterized by a very low background signal, and were found to be suitable to analyze environmental samples. Arsenic is at the top of the priority list of hazardous compounds issued by the U.S. Agency for Toxic Substances and Disease. The reason for the stunning arsenic resistance of many microorganisms is the existence of paralogous arsenic resistance operons. Pseudomonas putida KT2440 is a model organism for such bacteria, and their duplicated ars operons and in particular their ArsR transcription regulators have been studied in depth by in vivo approaches. Here we present an analysis of both purified ArsR paralogues by different biophysical techniques, and data obtained provide valuable insight into their structure and function. Particularly insightful was the comparison of ArsR effector profiles determined by in vitro and in vivo experimentation. We also report the use of both paralogues to construct robust and highly sensitive arsenic biosensors. Our finding that the deletion of both arsenic efflux pumps significantly increases biosensor sensitivity is of general relevance in the biosensor field.