Publications by authors named "Kevin S Lang"

11 Publications

  • Page 1 of 1

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts.

Cell Rep 2021 Mar;34(9):108797

Department of Biochemistry, Light Hall, Vanderbilt University, Nashville, TN, USA. Electronic address:

Conflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, and evolution across species. Head-on conflicts (lagging-strand genes) are significantly more detrimental than codirectional conflicts (leading-strand genes). The fundamental reason for this difference is unknown. Here, we report that topological stress significantly contributes to this difference. We find that head-on, but not codirectional, conflict resolution requires the relaxation of positive supercoils by the type II topoisomerases DNA gyrase and Topo IV, at least in the Gram-positive model bacterium Bacillus subtilis. Interestingly, our data suggest that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. Altogether, our results reveal a fundamental mechanistic difference between the two types of encounters, addressing a long-standing question in the field of replication-transcription conflicts.
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http://dx.doi.org/10.1016/j.celrep.2021.108797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986047PMC
March 2021

Crystal structure of a membrane-bound O-acyltransferase.

Nature 2018 10 3;562(7726):286-290. Epub 2018 Oct 3.

Department of Biological Structure, University of Washington, Seattle, WA, USA.

Membrane-bound O-acyltransferases (MBOATs) are a superfamily of integral transmembrane enzymes that are found in all kingdoms of life. In bacteria, MBOATs modify protective cell-surface polymers. In vertebrates, some MBOAT enzymes-such as acyl-coenzyme A:cholesterol acyltransferase and diacylglycerol acyltransferase 1-are responsible for lipid biosynthesis or phospholipid remodelling. Other MBOATs, including porcupine, hedgehog acyltransferase and ghrelin acyltransferase, catalyse essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively. Although many MBOAT proteins are important drug targets, little is known about their molecular architecture and functional mechanisms. Here we present crystal structures of DltB, an MBOAT responsible for the D-alanylation of cell-wall teichoic acid in Gram-positive bacteria, both alone and in complex with the D-alanyl donor protein DltC. DltB contains a ring of 11 peripheral transmembrane helices, which shield a highly conserved extracellular structural funnel extending into the middle of the lipid bilayer. The conserved catalytic histidine residue is located at the bottom of this funnel and is connected to the intracellular DltC through a narrow tunnel. Mutation of either the catalytic histidine or the DltC-binding site of DltB abolishes the D-alanylation of lipoteichoic acid and sensitizes the Gram-positive bacterium Bacillus subtilis to cell-wall stress, which suggests cross-membrane catalysis involving the tunnel. Structure-guided sequence comparison among DltB and vertebrate MBOATs reveals a conserved structural core and suggests that MBOATs from different organisms have similar catalytic mechanisms. Our structures provide a template for understanding structure-function relationships in MBOATs and for developing therapeutic MBOAT inhibitors.
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http://dx.doi.org/10.1038/s41586-018-0568-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529733PMC
October 2018

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria.

Annu Rev Microbiol 2018 Sep 1;72:71-88. Epub 2018 Jun 1.

Department of Microbiology, University of Washington, Seattle, Washington 98195, USA; email:

Within the last decade, it has become clear that DNA replication and transcription are routinely in conflict with each other in growing cells. Much of the seminal work on this topic has been carried out in bacteria, specifically, Escherichia coli and Bacillus subtilis; therefore, studies of conflicts in these species deserve special attention. Collectively, the recent findings on conflicts have fundamentally changed the way we think about DNA replication in vivo. Furthermore, new insights on this topic have revealed that the conflicts between replication and transcription significantly influence many key parameters of cellular function, including genome organization, mutagenesis, and evolution of stress response and virulence genes. In this review, we discuss the consequences of replication-transcription conflicts on the life of bacteria and describe some key strategies cells use to resolve them. We put special emphasis on two critical aspects of these encounters: ( a) the consequences of conflicts on replisome stability and dynamics, and ( b) the resulting increase in spontaneous mutagenesis.
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http://dx.doi.org/10.1146/annurev-micro-090817-062514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233710PMC
September 2018

Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis.

Cell 2017 Aug;170(4):787-799.e18

Department of Microbiology, Health Sciences Building - J-wing, University of Washington, Seattle, WA, 98195; Department of Genome Sciences, Foege Building, University of Washington, Seattle, WA 98195. Electronic address:

Replication-transcription collisions shape genomes, influence evolution, and promote genetic diseases. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication and promotes genomic instability. Here, we find that head-on collisions promote R-loop formation in Bacillus subtilis. We show that pervasive R-loop formation at head-on collision regions completely blocks replication, elevates mutagenesis, and inhibits gene expression. Accordingly, the activity of the R-loop processing enzyme RNase HIII at collision regions is crucial for stress survival in B. subtilis, as many stress response genes are head-on to replication. Remarkably, without RNase HIII, the ability of the intracellular pathogen Listeria monocytogenes to infect and replicate in hosts is weakened significantly, most likely because many virulence genes are head-on to replication. We conclude that the detrimental effects of head-on collisions stem primarily from excessive R-loop formation and that the resolution of these structures is critical for bacterial stress survival and pathogenesis.
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http://dx.doi.org/10.1016/j.cell.2017.07.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630229PMC
August 2017

Characterization of Acr2, an H-NS-like protein encoded on A/C2-type plasmids.

Plasmid 2016 Sep - Nov;87-88:17-27. Epub 2016 Aug 1.

University of Minnesota, Department of Veterinary and Biomedical Sciences, St. Paul, MN 55108, United States. Electronic address:

Conjugation plays an important role in the horizontal movement of DNA between bacterial species and even genera. Large conjugative plasmids in Gram-negative bacteria are associated with multi-drug resistance and have been implicated in the spread of these phenotypes to pathogenic organisms. A/C plasmids often carry genes that confer resistance to multiple classes of antibiotics. Recently, transcription factors were characterized that regulate A/C conjugation. In this work, we expanded the regulon of the negative regulator Acr2. We developed an A/C variant, pARK01, by precise removal of resistance genes carried by the plasmid in order to make it more genetically tractable. Using pARK01, we conducted RNA-Seq and ChAP-Seq experiments to characterize the regulon of Acr2, an H-NS-like protein. We found that Acr2 binds several loci on the plasmid. We showed, in vitro, that Acr2 can bind specific promoter regions directly and identify key amino acids which are important for this binding. This study further characterizes Acr2 and suggests its role in modulating gene expression of multiple plasmid and chromosomal loci.
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http://dx.doi.org/10.1016/j.plasmid.2016.07.004DOI Listing
November 2017

Multiple Discharges of Treated Municipal Wastewater Have a Small Effect on the Quantities of Numerous Antibiotic Resistance Determinants in the Upper Mississippi River.

Environ Sci Technol 2015 Oct 10;49(19):11509-15. Epub 2015 Sep 10.

Department of Civil, Environmental, and Geo- Engineering, University of Minnesota , Minneapolis, Minnesota 55455, United States.

This study evaluated multiple discharges of treated wastewater on the quantities of antibiotic resistance genes (ARGs) in the Upper Mississippi River. Surface water and treated wastewater samples were collected along the Mississippi River during three different periods of 4 days during the summer of 2012, and quantitative real-time PCR (qPCR) was used to enumerate several ARGs and related targets. Even though the wastewater effluents contained 75- to 831-fold higher levels of ARGs than the river water, the quantities of ARGs in the Mississippi River did not increase with downstream distance. Plasmids from the incompatibility group A/C were detected at low levels in the wastewater effluents but not in the river water; synthetic DNA containing an ampicillin resistance gene (bla) from cloning vectors was not detected in either the wastewater effluent or river samples. A simple 1D model suggested that the primary reason for the small impact of the wastewater discharges on ARG levels was the large flow rate of the Mississippi River compared to that of the wastewater discharges. Furthermore, this model generally overpredicted the ARG levels in the Mississippi River, suggesting that substantial loss mechanisms (e.g., decay or deposition) were occurring in the river.
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http://dx.doi.org/10.1021/acs.est.5b02803DOI Listing
October 2015

Transcriptome modulations due to A/C2 plasmid acquisition.

Plasmid 2015 Jul 12;80:83-9. Epub 2015 Jun 12.

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA. Electronic address:

Plasmids play an important role in driving the genetic diversity of bacteria. Horizontal gene transfer via plasmids is crucial for the dissemination of antimicrobial resistance genes. Many factors contribute to the persistence of plasmids within bacterial populations, and it has been suggested that epistatic interactions between the host chromosome and plasmid contribute to the fitness of a particular plasmid-host combination. However, such interactions have been shown to differ between bacterial hosts. In this study, RNA-Seq was performed in six different strains, spanning three species, to characterize the influence of host background on the A/C2 plasmid transcriptome. In five of these strains, chromosomal transcriptomes were compared in the presence and absence of A/C2 plasmid pAR060302. Host-specific effects on plasmid gene expression were identified, and acquisition of pAR060302 resulted in changes in the expression of chromosomal genes involved in metabolism and energy production. These results suggest that A/C2 plasmid fitness is, in part, dependent on host chromosome content, as well as environmental factors.
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http://dx.doi.org/10.1016/j.plasmid.2015.05.005DOI Listing
July 2015

IncA/C plasmids: An emerging threat to human and animal health?

Mob Genet Elements 2012 Jan;2(1):55-58

Department of Veterinary and Biomedical Sciences; University of Minnesota; Saint Paul, MN USA.

Incompatibility group IncA/C plasmids are large, low copy, theta-replicating plasmids that have been described in the literature for over 40 years. However, they have only recently been intensively studied on the genomic level because of their associations with the emergence of multidrug resistance in enteric pathogens of humans and animals. These plasmids are unique among other enterobacterial plasmids in many aspects, including their modular structure and gene content. While the IncA/C plasmid genome structure has now been well defined, many questions remain pertaining to their basic biological mechanisms of dissemination and regulation. Here, we discuss the history of IncA/C plasmids in light of our recent understanding of their population distribution, genomics, and effects on host bacteria.
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http://dx.doi.org/10.4161/mge.19626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383451PMC
January 2012

Transcriptome mapping of pAR060302, a blaCMY-2-positive broad-host-range IncA/C plasmid.

Appl Environ Microbiol 2012 May 17;78(9):3379-86. Epub 2012 Feb 17.

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota, USA.

The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, bla(CMY-2), aadA, and aacA. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of floR and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.
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http://dx.doi.org/10.1128/AEM.07199-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346456PMC
May 2012

Novel florfenicol and chloramphenicol resistance gene discovered in Alaskan soil by using functional metagenomics.

Appl Environ Microbiol 2010 Aug 11;76(15):5321-6. Epub 2010 Jun 11.

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA.

Functional metagenomics was used to search for florfenicol resistance genes in libraries of cloned DNA isolated from Alaskan soil. A gene that mediated reduced susceptibility to florfenicol was identified and designated pexA. The predicted PexA protein showed a structure similar to that of efflux pumps of the major facilitator superfamily.
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http://dx.doi.org/10.1128/AEM.00323-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916469PMC
August 2010

Evaluating the effects of chlortetracycline on the proliferation of antibiotic-resistant bacteria in a simulated river water ecosystem.

Appl Environ Microbiol 2007 Sep 6;73(17):5421-5. Epub 2007 Jul 6.

Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA.

Antibiotics and antibiotic metabolites have been found in the environment, but the biological activities of these compounds are uncertain, especially given the low levels that are typically detected in the environment. The objective of this study was to estimate the selection potential of chlortetracycline (CTC) on the antibiotic resistance of aerobic bacterial populations in a simulated river water ecosystem. Six replicates of a 10-day experiment using river water in continuous flow chemostat systems were conducted. Each replicate used three chemostats, one serving as a control to which no antibiotic was added and the other two receiving low and high doses of CTC (8 microg/liter and 800 microg/liter, respectively). The addition of CTC to the chemostats did not impact the overall level of cultivable aerobic bacteria (P = 0.51). The high-CTC chemostat had significantly higher tetracycline-resistant bacterial colony counts than both the low-CTC and the control chemostats (P < 0.035). The differences in resistance between the low-CTC and control chemostats were highly nonsignificant (P = 0.779). In general a greater diversity of tet resistance genes was detected in the high-CTC chemostat and with a greater frequency than in the low-CTC and control chemostats. Low levels of CTC in this in vitro experiment did not select for increased levels of tetracycline resistance among cultivable aerobic bacteria. This finding should not be equated with the absence of environmental risk, however. Low concentrations of antibiotics in the environment may select for resistant bacterial populations once they are concentrated in sediments or other locations.
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http://dx.doi.org/10.1128/AEM.00708-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2042072PMC
September 2007