Publications by authors named "Michael J Benedik"

46 Publications

Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation.

Environ Microbiol 2021 Nov 19;23(11):7245-7254. Epub 2021 Oct 19.

Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania, 16802-4400, USA.

Cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as an active stress response. How these phage fossils affect persister cell resuscitation has, however, not been explored. Persister cells form as a result of stresses such as starvation, antibiotics and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis and Lyme disease. Deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system resulting from inactivating the transcriptional regulator AlpA of the CP4-57 cryptic prophage. Deletion of alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Both phosphate regulators PhoP and PhoB stimulate resuscitation. This suggests a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.
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http://dx.doi.org/10.1111/1462-2920.15816DOI Listing
November 2021

Xenogeneic silencing relies on temperature-dependent phosphorylation of the host H-NS protein in Shewanella.

Nucleic Acids Res 2021 04;49(6):3427-3440

Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, Innovation Academy of South China Sea Ecology and Environmental Engineering, South China Sea Institute of Oceanology, Chinese Academy of Sciences, No. 1119, Haibin Road, Nansha District, Guangzhou 511458, China.

Lateral gene transfer (LGT) plays a key role in shaping the genome evolution and environmental adaptation of bacteria. Xenogeneic silencing is crucial to ensure the safe acquisition of LGT genes into host pre-existing regulatory networks. We previously found that the host nucleoid structuring protein (H-NS) silences prophage CP4So at warm temperatures yet enables this prophage to excise at cold temperatures in Shewanella oneidensis. However, whether H-NS silences other genes and how bacteria modulate H-NS to regulate the expression of genes have not been fully elucidated. In this study, we discovered that the H-NS silences many LGT genes and the xenogeneic silencing of H-NS relies on a temperature-dependent phosphorylation at warm temperatures in S. oneidensis. Specifically, phosphorylation of H-NS at Ser42 is critical for silencing the cold-inducible genes including the excisionase of CP4So prophage, a cold shock protein, and a stress-related chemosensory system. By contrast, nonphosphorylated H-NS derepresses the promoter activity of these genes/operons to enable their expression at cold temperatures. Taken together, our results reveal that the posttranslational modification of H-NS can function as a regulatory switch to control LGT gene expression in host genomes to enable the host bacterium to react and thrive when environmental temperature changes.
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http://dx.doi.org/10.1093/nar/gkab137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034616PMC
April 2021

Persister Cells Resuscitate Using Membrane Sensors that Activate Chemotaxis, Lower cAMP Levels, and Revive Ribosomes.

iScience 2020 Jan 21;23(1):100792. Epub 2019 Dec 21.

Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802-4400, USA; The Huck Institute of the Life Sciences, Pennsylvania State University, University Park, PA 16802-4400, USA. Electronic address:

Persistence, the stress-tolerant state, is arguably the most vital phenotype since nearly all cells experience nutrient stress, which causes a sub-population to become dormant. However, how persister cells wake to reconstitute infections is not understood well. Here, using single-cell observations, we determined that Escherichia coli persister cells resuscitate primarily when presented with specific carbon sources, rather than spontaneously. In addition, we found that the mechanism of persister cell waking is through sensing nutrients by chemotaxis and phosphotransferase membrane proteins. Furthermore, nutrient transport reduces the level of secondary messenger cAMP through enzyme IIA; this reduction in cAMP levels leads to ribosome resuscitation and rescue. Resuscitating cells also immediately commence chemotaxis toward nutrients, although flagellar motion is not required for waking. Hence, persister cells wake by perceiving nutrients via membrane receptors that relay the signal to ribosomes via the secondary messenger cAMP, and persisters wake and utilize chemotaxis to acquire nutrients.
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http://dx.doi.org/10.1016/j.isci.2019.100792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957856PMC
January 2020

Cyanide-degrading nitrilases in nature.

J Gen Appl Microbiol 2018 05 30;64(2):90-93. Epub 2017 Dec 30.

Structural Biology Research Unit, Institute for Infectious Diseases and Molecular Medicine, University of Cape Town.

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http://dx.doi.org/10.2323/jgam.2017.06.002DOI Listing
May 2018

Cyanide bioremediation: the potential of engineered nitrilases.

Appl Microbiol Biotechnol 2017 Apr 6;101(8):3029-3042. Epub 2017 Mar 6.

Department of Biology, Texas A&M University, College Station, TX, 77843-3258, USA.

The cyanide-degrading nitrilases are of notable interest for their potential to remediate cyanide contaminated waste streams, especially as generated in the gold mining, pharmaceutical, and electroplating industries. This review provides a brief overview of cyanide remediation in general but with a particular focus on the cyanide-degrading nitrilases. These are of special interest as the hydrolysis reaction does not require secondary substrates or cofactors, making these enzymes particularly good candidates for industrial remediation processes. The genetic approaches that have been used to date for engineering improved enzymes are described; however, recent structural insights provide a promising new approach.
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http://dx.doi.org/10.1007/s00253-017-8204-xDOI Listing
April 2017

Residue Y70 of the Nitrilase Cyanide Dihydratase from Is Critical for Formation and Activity of the Spiral Oligomer.

J Microbiol Biotechnol 2016 Dec;26(12):2179-2183

Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.

Nitrilases pose attractive alternatives to the chemical hydrolysis of nitrile compounds. The activity of bacterial nitrilases towards substrate is intimately tied to the formation of large spiral-shaped oligomers. In the nitrilase CynD (cyanide dihydratase) from , mutations in a predicted oligomeric surface region altered its oligomerization and reduced its activity. One mutant, CynD Y70C, retained uniform oligomer formation however it was inactive, unlike all other inactive mutants throughout that region all of which significantly perturbed oligomer formation. It was hypothesized that Y70 is playing an additional role necessary for CynD activity beyond influencing oligomerization. Here, we performed saturation mutagenesis at residue 70 and demonstrated that only tyrosine or phenylalanine is permissible for CynD activity. Furthermore, we show that other residues at this position are not only inactive, but have altered or disrupted oligomer conformations. These results suggest that Y70's essential role in activity is independent of its role in the formation of the spiral oligomer.
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http://dx.doi.org/10.4014/jmb.1606.06035DOI Listing
December 2016

Bacillus pumilus Cyanide Dihydratase Mutants with Higher Catalytic Activity.

Front Microbiol 2016 12;7:1264. Epub 2016 Aug 12.

Department of Biology, Texas A&M University, College Station TX, USA.

Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied. K93R and D172N increased the enzyme's thermostability whereas E327K mutation had a less pronounced effect on stability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 but lowered its k cat. However, the A202T mutation, located in the dimerization or the A surface, destabilized the protein and abolished its activity. No significant effect on activity at alkaline pH was observed for any of the purified mutants. These mutations help confirm the model of CynD and are discussed in the context of the protein-protein interfaces leading to the protein quaternary structure.
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http://dx.doi.org/10.3389/fmicb.2016.01264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981594PMC
August 2016

Probing an Interfacial Surface in the Cyanide Dihydratase from Bacillus pumilus, A Spiral Forming Nitrilase.

Front Microbiol 2015 5;6:1479. Epub 2016 Jan 5.

Department of Biology, Texas A&M University, College Station TX, USA.

Nitrilases are of significant interest both due to their potential for industrial production of valuable products as well as degradation of hazardous nitrile-containing wastes. All known functional members of the nitrilase superfamily have an underlying dimer structure. The true nitrilases expand upon this basic dimer and form large spiral or helical homo-oligomers. The formation of this larger structure is linked to both the activity and substrate specificity of these nitrilases. The sequences of the spiral nitrilases differ from the non-spiral forming homologs by the presence of two insertion regions. Homology modeling suggests that these regions are responsible for associating the nitrilase dimers into the oligomer. Here we used cysteine scanning across these two regions, in the spiral forming nitrilase cyanide dihydratase from Bacillus pumilus (CynD), to identify residues altering the oligomeric state or activity of the nitrilase. Several mutations were found to cause changes to the size of the oligomer as well as reduction in activity. Additionally one mutation, R67C, caused a partial defect in oligomerization with the accumulation of smaller oligomer variants. These results support the hypothesis that these insertion regions contribute to the unique quaternary structure of the spiral microbial nitrilases.
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http://dx.doi.org/10.3389/fmicb.2015.01479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700190PMC
January 2016

Orphan toxin OrtT (YdcX) of Escherichia coli reduces growth during the stringent response.

Toxins (Basel) 2015 Jan 29;7(2):299-321. Epub 2015 Jan 29.

Department of Chemical Engineering, the Pennsylvania State University, University Park, PA 16802-4400, USA.

Toxin/antitoxin (TA) systems are nearly universal in prokaryotes; toxins are paired with antitoxins which inactivate them until the toxins are utilized. Here we explore whether toxins may function alone; i.e., whether a toxin which lacks a corresponding antitoxin (orphan toxin) is physiologically relevant. By focusing on a homologous protein of the membrane-damaging toxin GhoT of the Escherichia coli GhoT/GhoS type V TA system, we found that YdcX (renamed OrtT for orphan toxin related to tetrahydrofolate) is toxic but is not part of TA pair. OrtT is not inactivated by neighboring YdcY (which is demonstrated to be a protein), nor is it inactivated by antitoxin GhoS. Also, OrtT is not inactivated by small RNA upstream or downstream of ortT. Moreover, screening a genomic library did not identify an antitoxin partner for OrtT. OrtT is a protein and its toxicity stems from membrane damage as evidenced by transmission electron microscopy and cell lysis. Furthermore, OrtT reduces cell growth and metabolism in the presence of both antimicrobials trimethoprim and sulfamethoxazole; these antimicrobials induce the stringent response by inhibiting tetrahydrofolate synthesis. Therefore, we demonstrate that OrtT acts as an independent toxin to reduce growth during stress related to amino acid and DNA synthesis.
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http://dx.doi.org/10.3390/toxins7020299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344625PMC
January 2015

Probing C-terminal interactions of the Pseudomonas stutzeri cyanide-degrading CynD protein.

Appl Microbiol Biotechnol 2015 Apr 31;99(7):3093-102. Epub 2014 Dec 31.

Department of Biology, Texas A&M University, College Station, TX, 77843-3258, USA.

The cyanide dihydratases from Bacillus pumilus and Pseudomonas stutzeri share high amino acid sequence similarity throughout except for their highly divergent C-termini. However, deletion or exchange of the C-termini had different effects upon each enzyme. Here we extended previous studies and investigated how the C-terminus affects the activity and stability of three nitrilases, the cyanide dihydratases from B. pumilus (CynDpum) and P. stutzeri (CynDstut) and the cyanide hydratase from Neurospora crassa. Enzymes in which the C-terminal residues were deleted decreased in both activity and thermostability with increasing deletion lengths. However, CynDstut was more sensitive to such truncation than the other two enzymes. A domain of the P. stutzeri CynDstut C-terminus not found in the other enzymes, 306GERDST311, was shown to be necessary for functionality and explains the inactivity of the previously described CynDstut-pum hybrid. This suggests that the B. pumilus C-terminus, which lacks this motif, may have specific interactions elsewhere in the protein, preventing it from acting in trans on a heterologous CynD protein. We identify the dimerization interface A-surface region 195-206 (A2) from CynDpum as this interaction site. However, this A2 region did not rescue activity in C-terminally truncated CynDstutΔ302 or enhance the activity of full-length CynDstut and therefore does not act as a general stability motif.
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http://dx.doi.org/10.1007/s00253-014-6335-xDOI Listing
April 2015

The MqsR/MqsA toxin/antitoxin system protects Escherichia coli during bile acid stress.

Environ Microbiol 2015 Sep 14;17(9):3168-81. Epub 2015 Feb 14.

Department of Chemical Engineering, Pennsylvania State University, University Park, PA, 16802-4400, USA.

Toxin/antitoxin (TA) systems are ubiquitous within bacterial genomes, and the mechanisms of many TA systems are well characterized. As such, several roles for TA systems have been proposed, such as phage inhibition, gene regulation and persister cell formation. However, the significance of these roles is nebulous due to the subtle influence from individual TA systems. For example, a single TA system has only a minor contribution to persister cell formation. Hence, there is a lack of defining physiological roles for individual TA systems. In this study, phenotype assays were used to determine that the MqsR/MqsA type II TA system of Escherichia coli is important for cell growth and tolerance during stress from the bile salt deoxycholate. Using transcriptomics and purified MqsR, we determined that endoribonuclease toxin MqsR degrades YgiS mRNA, which encodes a periplasmic protein that promotes deoxycholate uptake and reduces tolerance to deoxycholate exposure. The importance of reducing YgiS mRNA by MqsR is evidenced by improved growth, reduced cell death and reduced membrane damage when cells without ygiS are stressed with deoxycholate. Therefore, we propose that MqsR/MqsA is physiologically important for E. coli to thrive in the gallbladder and upper intestinal tract, where high bile concentrations are prominent.
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http://dx.doi.org/10.1111/1462-2920.12749DOI Listing
September 2015

Phosphodiesterase DosP increases persistence by reducing cAMP which reduces the signal indole.

Biotechnol Bioeng 2015 Mar 21;112(3):588-600. Epub 2014 Oct 21.

Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania.

Persisters are bacteria that are highly tolerant to antibiotics due to their dormant state and are of clinical significance owing to their role in infections. Given that the population of persisters increases in biofilms and that cyclic diguanylate (c-di-GMP) is an intracellular signal that increases biofilm formation, we sought to determine whether c-di-GMP has a role in bacterial persistence. By examining the effect of 30 genes from Escherichia coli, including diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that breakdown c-di-GMP, we determined that DosP (direct oxygen sensing phosphodiesterase) increases persistence by over a thousand fold. Using both transcriptomic and proteomic approaches, we determined that DosP increases persistence by decreasing tryptophanase activity and thus indole. Corroborating this effect, addition of indole reduced persistence. Despite the role of DosP as a c-di-GMP phosphodiesterase, the decrease in tryptophanase activity was found to be a result of cyclic adenosine monophosphate (cAMP) phosphodiesterase activity. Corroborating this result, the reduction of cAMP via CpdA, a cAMP-specific phosphodiesterase, increased persistence and reduced indole levels similarly to DosP. Therefore, phosphodiesterase DosP increases persistence by reducing the interkingdom signal indole via reduction of the global regulator cAMP.
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http://dx.doi.org/10.1002/bit.25456DOI Listing
March 2015

Toxin YafQ increases persister cell formation by reducing indole signalling.

Environ Microbiol 2015 Apr 8;17(4):1275-85. Epub 2014 Aug 8.

Department of Chemical Engineering and, Pennsylvania State University, University Park, PA, 16802-4400, USA.

Persister cells survive antibiotic and other environmental stresses by slowing metabolism. Since toxins of toxin/antitoxin (TA) systems have been postulated to be responsible for persister cell formation, we investigated the influence of toxin YafQ of the YafQ/DinJ Escherichia coli TA system on persister cell formation. Under stress, YafQ alters metabolism by cleaving transcripts with in-frame 5'-AAA-G/A-3' sites. Production of YafQ increased persister cell formation with multiple antibiotics, and by investigating changes in protein expression, we found that YafQ reduced tryptophanase levels (TnaA mRNA has 16 putative YafQ cleavage sites). Consistently, TnaA mRNA levels were also reduced by YafQ. Tryptophanase is activated in the stationary phase by the stationary-phase sigma factor RpoS, which was also reduced dramatically upon production of YafQ. Tryptophanase converts tryptophan into indole, and as expected, indole levels were reduced by the production of YafQ. Corroborating the effect of YafQ on persistence, addition of indole reduced persistence. Furthermore, persistence increased upon deleting tnaA, and persistence decreased upon adding tryptophan to the medium to increase indole levels. Also, YafQ production had a much smaller effect on persistence in a strain unable to produce indole. Therefore, YafQ increases persistence by reducing indole, and TA systems are related to cell signalling.
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http://dx.doi.org/10.1111/1462-2920.12567DOI Listing
April 2015

Draft Genome Sequence of Cupriavidus sp. Strain SK-3, a 4-Chlorobiphenyl- and 4-Clorobenzoic Acid-Degrading Bacterium.

Genome Announc 2014 Jul 3;2(4). Epub 2014 Jul 3.

We report the draft genome sequence of Cupriavidus sp. strain SK-3, which can use 4-chlorobiphenyl and 4-clorobenzoic acid as the sole carbon source for growth. The draft genome sequence allowed the study of the polychlorinated biphenyl degradation mechanism and the recharacterization of the strain SK-3 as a Cupriavidus species.
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http://dx.doi.org/10.1128/genomeA.00664-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082005PMC
July 2014

Draft Genome Sequence of Cupriavidus sp. Strain SK-4, a di-ortho-Substituted Biphenyl-Utilizing Bacterium Isolated from Polychlorinated Biphenyl-Contaminated Sludge.

Genome Announc 2014 May 22;2(3). Epub 2014 May 22.

Cupriavidus sp. strain SK-4 is a bacterium capable of growing aerobically on monochlorobiphenyls and dichlorobiphenyls as the sole carbon sources for growth. Here, we report its draft genome sequence with the aim of facilitating an understanding of polychlorinated biphenyl biodegradation mechanisms.
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http://dx.doi.org/10.1128/genomeA.00474-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031344PMC
May 2014

RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli.

Nucleic Acids Res 2014 Jun 19;42(10):6448-62. Epub 2014 Apr 19.

Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China

For toxin/antitoxin (TA) systems, no toxin has been identified that functions by cleaving DNA. Here, we demonstrate that RalR and RalA of the cryptic prophage rac form a type I TA pair in which the antitoxin RNA is a trans-encoded small RNA with 16 nucleotides of complementarity to the toxin mRNA. We suggest the newly discovered antitoxin gene be named ralA for RalR antitoxin. Toxin RalR functions as a non-specific endonuclease that cleaves methylated and unmethylated DNA. The RNA chaperone Hfq is required for RalA antitoxin activity and appears to stabilize RalA. Also, RalR/RalA is beneficial to the Escherichia coli host for responding to the antibiotic fosfomycin. Hence, our results indicate that cryptic prophage genes can be functionally divergent from their active phage counterparts after integration into the host genome.
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http://dx.doi.org/10.1093/nar/gku279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041452PMC
June 2014

Toxin GhoT of the GhoT/GhoS toxin/antitoxin system damages the cell membrane to reduce adenosine triphosphate and to reduce growth under stress.

Environ Microbiol 2014 Jun 21;16(6):1741-54. Epub 2014 Jan 21.

Department of Chemical Engineering, Pennsylvania State University, State College, PA, 16802, USA.

Toxin/antitoxin (TA) systems perhaps enable cells to reduce their metabolism to weather environmental challenges although there is little evidence to support this hypothesis. Escherichia coli GhoT/GhoS is a TA system in which toxin GhoT expression is reduced by cleavage of its messenger RNA (mRNA) by antitoxin GhoS, and TA system MqsR/MqsA controls GhoT/GhoS through differential mRNA decay. However, the physiological role of GhoT has not been determined. We show here through transmission electron microscopy, confocal microscopy and fluorescent stains that GhoT reduces metabolism by damaging the membrane and that toxin MqsR (a 5'-GCU-specific endoribonuclease) causes membrane damage in a GhoT-dependent manner. This membrane damage results in reduced cellular levels of ATP and the disruption of proton motive force (PMF). Normally, GhoT is localized to the pole and does not cause cell lysis under physiological conditions. Introduction of an F38R substitution results in loss of GhoT toxicity, ghost cell production and membrane damage while retaining the pole localization. Also, deletion of ghoST or ghoT results in significantly greater initial growth in the presence of antimicrobials. Collectively, these results demonstrate that GhoT reduces metabolism by reducing ATP and PMF and that this reduction in metabolism is important for growth with various antimicrobials.
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http://dx.doi.org/10.1111/1462-2920.12373DOI Listing
June 2014

Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan.

Nucleic Acids Res 2014 Jan 16;42(2):1245-56. Epub 2013 Oct 16.

Department of Biology, Texas A&M University, College Station, TX 77843, USA, Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, AL 35899, USA, Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA, Quantitative Proteomics Center, Department of Biological Sciences, Columbia University, New York, NY 10027, USA and Department of Biology, Stanford University, Stanford, CA 94305, USA.

A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome's ability to sense free L-Trp. We show that the mutational change A2058U in 23S rRNA reduces the concentration dependence of L-Trp-mediated tna operon induction, whereas the TnaC I19L change suppresses this phenotype, restoring the sensitivity of the translating A2058U mutant ribosome to free L-Trp. These findings suggest that interactions between TnaC residue I19 and 23S rRNA nucleotide A2058 contribute to the creation of a regulatory L-Trp binding site within the translating ribosome.
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http://dx.doi.org/10.1093/nar/gkt923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3902921PMC
January 2014

Catabolic plasmid specifying polychlorinated biphenyl degradation in Cupriavidus sp. strain SK-4: mobilization and expression in a pseudomonad.

J Basic Microbiol 2015 Mar 21;55(3):338-45. Epub 2013 Jun 21.

Department of Microbiology, University of Lagos, Akoka, Lagos, Nigeria.

Strain SK-4, a polychlorinated biphenyl (PCB) degrader previously reported to utilize di-ortho-substituted biphenyl, was genotypically re-characterized as a species of Cupriavidus. The bacterium harbored a single plasmid (pSK4), which resisted curing and which, after genetic marking by a transposon (SK4Tn5), could be mobilized into a pseudomonad. Analysis of pSK4 in both the transconjugant and the wild type revealed that it specifies the genes coding for 2-hydroxy-2,4-pentadienoate degradation in addition to those of the upper biphenyl pathway. Expression of the benzoate metabolic pathway in the transconjugant is evidence suggesting that the benzoate catabolic genes are also localized on the plasmid. This implies that pSK4 codes for all the genes involved in biphenyl mineralization. It is therefore reasonable to propose that the plasmid is the determinant for the unique metabolic capabilities known to exist in Cupriavidus sp. strain SK-4.
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http://dx.doi.org/10.1002/jobm.201200807DOI Listing
March 2015

Arrested protein synthesis increases persister-like cell formation.

Antimicrob Agents Chemother 2013 Mar 7;57(3):1468-73. Epub 2013 Jan 7.

Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA.

Biofilms are associated with a wide variety of bacterial infections and pose a serious problem in clinical medicine due to their inherent resilience to antibiotic treatment. Within biofilms, persister cells comprise a small bacterial subpopulation that exhibits multidrug tolerance to antibiotics without undergoing genetic change. The low frequency of persister cell formation makes it difficult to isolate and study persisters, and bacterial persistence is often attributed to a quiescent metabolic state induced by toxins that are regulated through toxin-antitoxin systems. Here we mimic toxins via chemical pretreatments to induce high levels of persistence (10 to 100%) from an initial population of 0.01%. Pretreatment of Escherichia coli with (i) rifampin, which halts transcription, (ii) tetracycline, which halts translation, and (iii) carbonyl cyanide m-chlorophenylhydrazone, which halts ATP synthesis, all increased persistence dramatically. Using these compounds, we demonstrate that bacterial persistence results from halted protein synthesis and from environmental cues.
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http://dx.doi.org/10.1128/AAC.02135-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591907PMC
March 2013

Type II toxin/antitoxin MqsR/MqsA controls type V toxin/antitoxin GhoT/GhoS.

Environ Microbiol 2013 Jun 4;15(6):1734-44. Epub 2013 Jan 4.

Key Laboratory of Marine Bio-Resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.

Toxin endoribonucleases of toxin/antitoxin (TA) systems regulate protein production by selectively degrading mRNAs but have never been shown to control other TA systems. Here we demonstrate that toxin MqsR of the MqsR/MqsA system enriches toxin ghoT mRNA in vivo and in vitro, since this transcript lacks the primary MqsR cleavage site 5'-GCU. GhoT is a membrane toxin that causes the ghost cell phenotype, and is part of a type V TA system with antitoxin GhoS that cleaves specifically ghoT mRNA. Introduction of MqsR primary 5'-GCU cleavage sites into ghoT mRNA reduces ghost cell production and cell death likely due to increased degradation of the altered ghoT mRNA by MqsR. GhoT also prevents cell elongation upon the addition of low levels of ampicillin. Therefore, during stress, antitoxin GhoS mRNA is degraded by toxin MqsR allowing ghoT mRNA translation to yield another free toxin that forms ghost cells and increases persistence. Hence, we show that GhoT/GhoS is the first TA system regulated by another TA system.
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http://dx.doi.org/10.1111/1462-2920.12063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3620836PMC
June 2013

Draft genome sequence of the cyanide-utilizing bacterium Pseudomonas fluorescens strain NCIMB 11764.

J Bacteriol 2012 Dec;194(23):6618-9

Department of Biological Sciences, University of North Texas, Denton, Texas, USA.

We report here the 6.97-Mb draft genome sequence of Pseudomonas fluorescens strain NCIMB 11764, which is capable of growth on cyanide as the sole nitrogen source. The draft genome sequence allowed the discovery of several genes implicated in enzymatic cyanide turnover and provided additional information contributing to a better understanding of this organism's unique cyanotrophic ability. This is the first sequenced genome of a cyanide-assimilating bacterium.
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http://dx.doi.org/10.1128/JB.01670-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497540PMC
December 2012

A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS.

Nat Chem Biol 2012 Oct;8(10):855-61

Key Laboratory of Marine Bio-Resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.

Among bacterial toxin-antitoxin systems, to date no antitoxin has been identified that functions by cleaving toxin mRNA. Here we show that YjdO (renamed GhoT) is a membrane lytic peptide that causes ghost cell formation (lysed cells with damaged membranes) and increases persistence (persister cells are tolerant to antibiotics without undergoing genetic change). GhoT is part of a new toxin-antitoxin system with YjdK (renamed GhoS) because in vitro RNA degradation studies, quantitative real-time reverse-transcription PCR and whole-transcriptome studies revealed that GhoS masks GhoT toxicity by cleaving specifically yjdO (ghoT) mRNA. Alanine substitutions showed that Arg28 is important for GhoS activity, and RNA sequencing indicated that the GhoS cleavage site is rich in U and A. The NMR structure of GhoS indicates it is related to the CRISPR-associated-2 RNase, and GhoS is a monomer. Hence, GhoT-GhoS is to our knowledge the first type V toxin-antitoxin system where a protein antitoxin inhibits the toxin by cleaving specifically its mRNA.
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http://dx.doi.org/10.1038/nchembio.1062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514572PMC
October 2012

Bacterial persistence increases as environmental fitness decreases.

Microb Biotechnol 2012 Jul 6;5(4):509-22. Epub 2012 Jan 6.

Departments of Chemical Engineering Biology, Texas A&M University, College Station, TX 77843-3122, USA.

Since persister cells cause chronic infections and since Escherichia coli toxin MqsR increases persisters, we used protein engineering to increase the toxicity of MqsR to gain insights into persister cell formation. Through two amino acid replacements that increased the stability of MqsR, toxicity and persistence were increased. A whole-transcriptome study revealed that the MqsR variant increased persistence by repressing genes for acid resistance, multidrug resistance and osmotic resistance. Corroborating these microarray results, deletion of rpoS, as well as the genes that the master stress response regulator RpoS controls, increased persister formation dramatically to the extent that nearly the whole population became persistent. Furthermore, wild-type cells stressed by prior treatment to acid or hydrogen peroxide increased persistence 12 000-fold. Whole-transcriptome analyses of persister cells generated by two different methods (wild-type cells pretreated with hydrogen peroxide and the rpoS deletion) corroborated the importance of suppressing RpoS in persister cell formation. Therefore, the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance and osmotic resistance systems.
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http://dx.doi.org/10.1111/j.1751-7915.2011.00327.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323757PMC
July 2012

Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function.

Nucleic Acids Res 2012 Mar 21;40(5):2247-57. Epub 2011 Nov 21.

Department of Biology, Texas A&M University, College Station, TX 77843, USA.

Translation of the TnaC nascent peptide inhibits ribosomal activity in the presence of l-tryptophan, inducing expression of the tnaCAB operon in Escherichia coli. Using chemical methylation, this work reveals how interactions between TnaC and the ribosome are affected by mutations in both molecules. The presence of the TnaC-tRNA(Pro) peptidyl-tRNA within the ribosome protects the 23S rRNA nucleotide U2609 against chemical methylation. Such protection was not observed in mutant ribosomes containing changes in 23S rRNA nucleotides of the A748-A752 region. Nucleotides A752 and U2609 establish a base-pair interaction. Most replacements of either A752 or U2609 affected Trp induction of a TnaC-regulated LacZ reporter. However, the single change A752G, or the dual replacements A752G and U2609C, maintained Trp induction. Replacements at the conserved TnaC residues W12 and D16 also abolished the protection of U2609 by TnaC-tRNA(Pro) against chemical methylation. These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive. This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.
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http://dx.doi.org/10.1093/nar/gkr1052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3299997PMC
March 2012

Antitoxin DinJ influences the general stress response through transcript stabilizer CspE.

Environ Microbiol 2012 Mar 26;14(3):669-79. Epub 2011 Oct 26.

Department of Chemical Engineering, Texas A & M University, College Station, TX 77843-3122, USA.

Antitoxins are becoming recognized as proteins that regulate more than their own synthesis; for example, we found previously that antitoxin MqsA of the Escherichia coli toxin/antitoxin (TA) pair MqsR/MqsA directly represses the gene encoding the stationary-phase sigma factor RpoS. Here, we investigated the physiological role of antitoxin DinJ of the YafQ/DinJ TA pair and found DinJ also affects the general stress response by decreasing RpoS levels. Corroborating the reduced RpoS levels upon producing DinJ, the RpoS-regulated phenotypes of catalase activity, cell adhesins and cyclic diguanylate decreased while swimming increased. Using a transcriptome search and DNA-binding assays, we determined that the mechanism by which DinJ reduces RpoS is by repressing cspE at the LexA palindrome; cold-shock protein CspE enhances translation of rpoS mRNA. Inactivation of CspE abolishes the ability of DinJ to influence RpoS. Hence, DinJ influences the general stress response indirectly by regulating cspE.
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http://dx.doi.org/10.1111/j.1462-2920.2011.02618.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261204PMC
March 2012

Engineering pH-tolerant mutants of a cyanide dihydratase.

Appl Microbiol Biotechnol 2012 Apr 13;94(1):131-40. Epub 2011 Oct 13.

Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.

Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynD(pum)) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynD(pum) mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.
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http://dx.doi.org/10.1007/s00253-011-3620-9DOI Listing
April 2012

Antitoxin MqsA helps mediate the bacterial general stress response.

Nat Chem Biol 2011 Jun 24;7(6):359-66. Epub 2011 Apr 24.

Department of Chemical Engineering, Texas A&M University, College Station, Texas, USA.

Although it is well recognized that bacteria respond to environmental stress through global networks, the mechanism by which stress is relayed to the interior of the cell is poorly understood. Here we show that enigmatic toxin-antitoxin systems are vital in mediating the environmental stress response. Specifically, the antitoxin MqsA represses rpoS, which encodes the master regulator of stress. Repression of rpoS by MqsA reduces the concentration of the internal messenger 3,5-cyclic diguanylic acid, leading to increased motility and decreased biofilm formation. Furthermore, the repression of rpoS by MqsA decreases oxidative stress resistance via catalase activity. Upon oxidative stress, MqsA is rapidly degraded by Lon protease, resulting in induction of rpoS. Hence, we show that external stress alters gene regulation controlled by toxin-antitoxin systems, such that the degradation of antitoxins during stress leads to a switch from the planktonic state (high motility) to the biofilm state (low motility).
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http://dx.doi.org/10.1038/nchembio.560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097263PMC
June 2011

Rapid generation of random mutant libraries.

Bioeng Bugs 2010 Sep-Oct;1(5):337-40

Department of Biology, Texas A&M University, College Station, TX, USA.

A simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification is described. This will be especially useful for generating large pools of randomly mutagenized clones after error-prone PCR mutagenesis. Here we investigate various parameters to optimize this approach and we demonstrate that as little as 1 pmole of PCR fragment can generate a library with greater than 104 clones in a single transformation without ligation.
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http://dx.doi.org/10.4161/bbug.1.5.12942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037583PMC
January 2012
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