Integrated co‐regulation of bacterial arsenic and phosphorus metabolisms

Kang, Yoon‐Suk; Heinemann, Joshua; Bothner, Brian; Rensing, Christopher; McDermott, Timothy R.

doi:10.1111/j.1462-2920.2012.02881.x

Cited by 42 publications

(108 citation statements)

References 50 publications

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“…Plasmid isolation, gel electrophoresis, transformation, and PCR amplification of DNA were performed as previously described (46). The broad-host-range plasmid pAT28 (47) was used as a vector backbone for plasmid-borne, bio-reporter expression in Gram-negative and Gram-positive bacterial cells (Fig.…”

Section: Methodsmentioning

confidence: 99%

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

Kang

2017

Infect Immun

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We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila. Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.KEYWORDS Brucella, Legionella pneumophila, coinfection, fluorescent image analysis, intracellular pathogens, pathogenesis, phagosomes, reporter genes, type IV secretion system B rucella species are Gram-negative Alphaproteobacteria that cause chronic, systemic infections in mammals and zoonotic infections in humans (1). These pathogens are known to infect the reticuloendothelial system and proliferate significantly in macrophage-rich organs such as liver, spleen, and bone marrow. Chronic, often debilitating bloodstream infection is typical. In humans, a chronic course punctuated by spikes in body temperature is underscored by the descriptive name, undulant fever, given to disease caused by these organisms. Endovascular infection and osteomyelitis are concerning sequelae. Humans may acquire Brucella from airborne exposure related to large quantities of organisms shed from birthing livestock or from ingesting unpasteurized dairy products.B. abortus, B. melitensis, and B. suis, the species responsible for human zoonotic infection, are facultative intracellular pathogens (1). Intracellular growth is thought critical to successful infection of the host. More particularly, each of these pathogens deploys a type IV secretion system (T4SS), a molecular syringe, to inject virulence

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Section: Methodsmentioning

confidence: 99%

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

Kang

2017

Infect Immun

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“…E. coli was grown with 50 g/ml Km, 20 g/ml Gen, or 20 g/ml Tc, as required. Constructs were mobilized into A. tumefaciens strains by conjugation with E. coli S17-1 (20,30).…”

Section: Methodsmentioning

confidence: 99%

“…Bacterial growth was monitored via measurements of the culture optical density by use of a SpectraMax microtiter plate reader (Molecular Devices, CA). Plasmid isolation, gel electrophoresis, transformation, PCR amplification of DNA, and reporter gene assays were conducted as previously described (20,30). When required, the MMNH 4 agar medium was supplemented with 500 g/ml kanamycin (Km) for selection and maintenance of reporter constructs derived from pLSP-KT2lacZ or with 80 g/ml gentamicin (Gen), 20 g/ml tetracycline (Tc), or 15% sucrose to select for/against pJQ200SK for the generation of deletion mutations (see below).…”

Section: Methodsmentioning

confidence: 99%

“…As more genomes are sequenced, it has become increasingly apparent that many ars genes are reiterated, with some microorganisms containing two or more ars operons (18)(19)(20)(21)(22)(23)(24)(25)(26). The relative importance of such reiteration has been examined only sparingly.…”

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confidence: 99%

“…ArsR1 also represses the expression of the nearby phoB1 and pstS1 genes ( Fig. 1), which are both required for optimal expression of aioBA [structural genes encoding As(III) oxidase] (20) and are also controlled by the phosphorus stress response (20). In the current study, we examined the contributions of the four ArsR proteins to regulating each arsR gene, phoB1, and pstS1, and we briefly assessed their structural similarities.…”

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Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A

Kang

Brame

Jetter

et al. 2016

Appl Environ Microbiol

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ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCEGiven the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR. This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate ars operons, encode proteins exhibiting various degrees of functional overlap with respect to autoregulation and cross-regulation, as well as control of other functional genes. In some cases, differences in regulatory activity are associated with only limited differences in protein primary structure. The experiments summarized herein also present evidence that ArsR proteins appear to have activator functions, representing novel regulatory activities for ArsR, previously known only to be a repressor. In reaction to arsenic in their environment, microorganisms orchestrate an organized response that may involve arsenite [As(III)] oxidation, arsenate [As(V)] reduction, or both. These redox reactions serve to detoxify or protect the organism or to generate energy, depending on the organism and the genes involved. Current models depict As(III) being taken up into the cell via aquaglyceroporins (e.g., reviewed in references 1, 2, and 3), where it then interacts with a DNA-binding repressor protein, ArsR, resulting in a conformational change in ArsR and causing it...

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A Random Biogeochemical Walk into Three Soda Lakes of the Western USA: With an Introduction to a Few of Their Microbial Denizens

Oremland

2013

Cellular Origin, Life in Extreme Habitats and Astrobiology

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Integrated co‐regulation of bacterial arsenic and phosphorus metabolisms

Cited by 42 publications

References 50 publications

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila

Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A

A Random Biogeochemical Walk into Three Soda Lakes of the Western USA: With an Introduction to a Few of Their Microbial Denizens

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