Publications by authors named "Ankur B Dalia"

41 Publications

Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor.

Nat Commun 2021 06 18;12(1):3744. Epub 2021 Jun 18.

Department of Biology, Indiana University, Bloomington, IN, USA.

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, and T4P extension is thought to occur through the action of a single, highly conserved motor, PilB. Here, we develop Acinetobacter baylyi as a model to study T4P by employing a recently developed pilus labeling method. By contrast to previous studies of other bacterial species, we find that T4P synthesis in A. baylyi is dependent not only on PilB but also on an additional, phylogenetically distinct motor, TfpB. Furthermore, we identify a protein (CpiA) that inhibits T4P extension by specifically binding and inhibiting PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to disrupt T4P synthesis.
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http://dx.doi.org/10.1038/s41467-021-24124-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8213720PMC
June 2021

Fresh Extension of Vibrio cholerae Competence Type IV Pili Predisposes Them for Motor-Independent Retraction.

Appl Environ Microbiol 2021 06 25;87(14):e0047821. Epub 2021 Jun 25.

Department of Biology, Indiana University, Bloomington, Indiana, USA.

Bacteria utilize dynamic appendages, called type IV pili (T4P), to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can occur with the aid of an ATPase motor or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remain poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization. Extracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions; however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.
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http://dx.doi.org/10.1128/AEM.00478-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231728PMC
June 2021

Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake.

Mol Microbiol 2021 Mar 23. Epub 2021 Mar 23.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA.

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram-positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake.
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http://dx.doi.org/10.1111/mmi.14718DOI Listing
March 2021

The ChiS-Family DNA-Binding Domain Contains a Cryptic Helix-Turn-Helix Variant.

mBio 2021 03 16;12(2). Epub 2021 Mar 16.

Department of Biology, Indiana University, Bloomington, Indiana, USA

Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen , the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a β-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs. Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen , the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.
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http://dx.doi.org/10.1128/mBio.03287-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092284PMC
March 2021

Natural Transformation in a Classical-Biotype Vibrio cholerae Strain.

Appl Environ Microbiol 2021 04 27;87(10). Epub 2021 Apr 27.

South Texas Center for Emerging Infectious Diseases, University of Texas San Antonio, San Antonio, Texas, USA

causes the gastrointestinal illness cholera, which spreads throughout the globe in large pandemics. The current pandemic is caused by O1 El Tor biotype strains, whereas previous pandemics were caused by O1 classical biotype strains. El Tor is noted for its ability to acquire exogenous DNA through chitin-induced natural transformation, which has been exploited for genetic manipulation of El Tor strains in the laboratory. In contrast, the prototypical classical strain O395 lacks this ability, which was suspected to be due to a mutation in the regulatory gene HapR and the regulator TfoX control expression of a third competence regulator, QstR. We found that artificial induction of both TfoX and QstR in the presence of HapR in O395 was required for efficient DNA uptake. However, natural transformation in the classical strain is still orders of magnitude below that of an El Tor strain. O395 expressing HapR could also undergo natural transformation after growth on chitin, which could be increased by artificial induction of TfoX and/or QstR. A plasmid that expresses both TfoX and QstR was created that allowed for consistent DNA uptake in O395 carrying a plasmid. This technique was also used to facilitate cotransformation into O395 of unmarked DNA (Δ, Δ, Δ) for multiplex genome editing by natural transformation (MuGENT). These results demonstrate that the classical biotype O395 strain is functionally capable of DNA uptake, which allows for the rapid genetic manipulation of its genome. Natural transformation (uptake of exogenous DNA) in has contributed to the evolution of these human pathogens. Classical biotype strains were responsible for the first six cholera pandemics but were replaced by El Tor biotype in the current pandemic. This study demonstrates that classical is functionally capable of natural transformation, but inactivation of the transformation regulator HapR and inherent levels of transformation that are lower than those of El Tor suggest that the classical biotype may be less able to utilize natural transformation for horizontal gene transfer.
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http://dx.doi.org/10.1128/AEM.00060-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8117766PMC
April 2021

Prophage-Dependent Neighbor Predation Fosters Horizontal Gene Transfer by Natural Transformation.

mSphere 2020 11 11;5(6). Epub 2020 Nov 11.

Centro de Estudios Científicos, Valdivia, Los Rios, Chile

Natural transformation is a broadly conserved mechanism of horizontal gene transfer (HGT) in bacteria that can shape their evolution through the acquisition of genes that promote virulence, antibiotic resistance, and other traits. Recent work has established that neighbor predation via type VI secretion systems, bacteriocins, and virulent phages plays an important role in promoting HGT. Here, we demonstrate that in chitin estuary microcosms, K139 lysogens exhibit prophage-dependent neighbor predation of nonlysogens to enhance HGT. Through predation of nonlysogens, K139 lysogens also have a fitness advantage under these microcosm conditions. The ecological strategy revealed by our work provides a better understanding of the evolutionary mechanisms used by bacteria to adapt in their natural setting and contributes to our understanding of the selective pressures that may drive prophage maintenance in bacterial genomes. Prophages are nearly ubiquitous in bacterial species. These integrated phage elements have previously been implicated in horizontal gene transfer (HGT) largely through their ability to carry out transduction (generalized or specialized). Here, we show that prophage-encoded viral particles promote neighbor predation leading to enhanced HGT by natural transformation in the waterborne pathogen Our findings contribute to a comprehensive understanding of the dynamic forces involved in prophage maintenance which ultimately drive the evolution of naturally competent bacteria in their natural environment.
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http://dx.doi.org/10.1128/mSphere.00975-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657591PMC
November 2020

Publisher Correction: CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholera.

Nat Commun 2020 Oct 27;11(1):5533. Epub 2020 Oct 27.

Division of Biology and Biological Engineering and Howard Hughes Medical Institute, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA, 91125, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-19389-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591482PMC
October 2020

CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae.

Nat Commun 2020 10 8;11(1):5080. Epub 2020 Oct 8.

Division of Biology and Biological Engineering and Howard Hughes Medical Institute, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA, 91125, USA.

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.
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http://dx.doi.org/10.1038/s41467-020-18866-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545093PMC
October 2020

A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae.

Sci Rep 2020 09 21;10(1):15398. Epub 2020 Sep 21.

Department of Biology, Indiana University, Bloomington, IN, 47405, USA.

The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements P and P to versions that have an additional theophylline-responsive riboswitch (P-riboswitch and P-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with P > P > P-riboswitch > P-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for P > P-riboswitch > P; while the newly developed P-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible P-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of V. cholerae and could be adapted for use in other species.
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http://dx.doi.org/10.1038/s41598-020-72387-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505983PMC
September 2020

ChiS is a noncanonical DNA-binding hybrid sensor kinase that directly regulates the chitin utilization program in .

Proc Natl Acad Sci U S A 2020 08 27;117(33):20180-20189. Epub 2020 Jul 27.

Department of Biology, Indiana University, Bloomington, IN 47405;

Two-component signal transduction systems (TCSs) represent a major mechanism that bacteria use to sense and respond to their environment. Prototypical TCSs are composed of a membrane-embedded histidine kinase, which senses an environmental stimulus and subsequently phosphorylates a cognate partner protein called a response regulator that regulates gene expression in a phosphorylation-dependent manner. uses the hybrid histidine kinase ChiS to activate the expression of the chitin utilization program, which is critical for the survival of this facultative pathogen in its aquatic reservoir. A cognate response regulator for ChiS has not been identified and the mechanism of ChiS-dependent signal transduction remains unclear. Here, we show that ChiS is a noncanonical membrane-embedded one-component system that can both sense chitin and directly regulate gene expression via a cryptic DNA binding domain. Unlike prototypical TCSs, we find that ChiS DNA binding is diminished, rather than stimulated, by phosphorylation. Finally, we provide evidence that ChiS likely activates gene expression by directly recruiting RNA polymerase. This work addresses the mechanism of action for a major transcription factor in and highlights the versatility of signal transduction systems in bacterial species.
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http://dx.doi.org/10.1073/pnas.2001768117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443906PMC
August 2020

Species-Specific Quorum Sensing Represses the Chitobiose Utilization Locus in Vibrio cholerae.

Appl Environ Microbiol 2020 09 1;86(18). Epub 2020 Sep 1.

Department of Biology, Indiana University, Bloomington, Indiana, USA

The marine facultative pathogen forms complex multicellular communities on the chitinous shells of crustacean zooplankton in its aquatic reservoir. -chitin interactions are critical for the growth, evolution, and waterborne transmission of cholera. This is due, in part, to chitin-induced changes in gene expression in this pathogen. Here, we sought to identify factors that influence chitin-induced expression of one locus, the chitobiose utilization operon (), which is required for the uptake and catabolism of the chitin disaccharide. Through a series of genetic screens, we identified that the master regulator of quorum sensing, HapR, is a direct repressor of the operon. We also found that the levels of HapR in are regulated by the ClpAP protease. Furthermore, we show that the canonical quorum sensing cascade in regulates expression in an HapR-dependent manner. Through this analysis, we found that signaling via the species-specific autoinducer CAI-1, but not the interspecies autoinducer AI-2, influences expression. This phenomenon of species-specific regulation may enhance the fitness of this pathogen in its environmental niche. In nature, bacteria live in multicellular and multispecies communities. Microbial species can sense the density and composition of their community through chemical cues using a process called quorum sensing (QS). The marine pathogen is found in communities on the chitinous shells of crustaceans in its aquatic reservoir. interactions with chitin are critical for the survival, evolution, and waterborne transmission of this pathogen. Here, we show that uses QS to regulate the expression of one locus required for -chitin interactions.
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http://dx.doi.org/10.1128/AEM.00915-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480382PMC
September 2020

c-di-GMP modulates type IV MSHA pilus retraction and surface attachment in Vibrio cholerae.

Nat Commun 2020 03 25;11(1):1549. Epub 2020 Mar 25.

Department of Microbiology and Environmental Toxicology, University of California - Santa Cruz, 1156 High St., BioMed 245, Santa Cruz, CA, 95064, USA.

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3',5'-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization.
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http://dx.doi.org/10.1038/s41467-020-15331-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096442PMC
March 2020

A bifunctional ATPase drives tad pilus extension and retraction.

Sci Adv 2019 12 18;5(12):eaay2591. Epub 2019 Dec 18.

Department of Biology, Indiana University, 1001 E. 3rd Street, Bloomington, IN 47405, USA.

A widespread class of prokaryotic motors powered by secretion motor adenosine triphosphatases (ATPases) drives the dynamic extension and retraction of extracellular fibers, such as type IV pili (T4P). Among these, the tight adherence (tad) pili are critical for surface sensing and biofilm formation. As for most other motors belonging to this class, how tad pili retract despite lacking a dedicated retraction motor ATPase has remained a mystery. Here, we find that a bifunctional pilus motor ATPase, CpaF, drives both activities through adenosine 5'-triphosphate (ATP) hydrolysis. We show that mutations within CpaF result in a correlated reduction in the rates of extension and retraction that directly scales with decreased ATP hydrolysis and retraction force. Thus, a single motor ATPase drives the bidirectional processes of pilus fiber extension and retraction.
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http://dx.doi.org/10.1126/sciadv.aay2591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920026PMC
December 2019

Diversity in Natural Transformation Frequencies and Regulation across Species.

mBio 2019 12 17;10(6). Epub 2019 Dec 17.

Department of Biology, Indiana University, Bloomington, Indiana, USA

In species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several species. Here, we show that TfoX expression in strains DS40M4 and NBRC 15631 enables high natural transformation frequencies. Conversely, transformation was not achieved in the model quorum-sensing strain BB120 (previously classified as ). Surprisingly, we find that quorum sensing is not required for transformation in DS40M4 or in contrast to the established regulatory pathway in in which quorum sensing is required to activate the competence regulator QstR. Similar to , expression of both QstR and TfoX is necessary for transformation in DS40M4. There is a wide disparity in transformation frequencies among even closely related strains, with having the lowest functional transformation frequency. Ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in To explore differences in competence regulation, we used previously studied competence genes to inform a comparative genomics analysis coupled with transcriptomics. We find that transformation capability cannot necessarily be predicted by the level of gene conservation but rather correlates with competence gene expression following TfoX induction. Thus, we have uncovered notable species- and strain-level variations in the competence gene regulation pathway across the genus. Naturally transformable, or competent, bacteria are able to take up DNA from their environment, a key method of horizontal gene transfer for acquisition of new DNA sequences. Our research shows that species that inhabit marine environments exhibit a wide diversity in natural transformation capability ranging from nontransformability to high transformation rates in which 10% of cells measurably incorporate new DNA. We show that the role of regulatory systems controlling the expression of competence genes (e.g., quorum sensing) differs throughout both the species and strain levels. We explore natural transformation capabilities of species which have been thus far uncharacterized and find novel regulation of competence. Expression of two key transcription factors, TfoX and QstR, is necessary to stimulate high levels of transformation in and recover low rates of transformation in .
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http://dx.doi.org/10.1128/mBio.02788-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918086PMC
December 2019

Spatiotemporal Analysis of DNA Integration during Natural Transformation Reveals a Mode of Nongenetic Inheritance in Bacteria.

Cell 2019 12;179(7):1499-1511.e10

Department of Biology, Indiana University, Bloomington, IN 47405, USA.

Natural transformation (NT) is a major mechanism of horizontal gene transfer in microbial species that promotes the spread of antibiotic-resistance determinants and virulence factors. Here, we develop a cell biological approach to characterize the spatiotemporal dynamics of homologous recombination during NT in Vibrio cholerae. Our results directly demonstrate (1) that transforming DNA efficiently integrates into the genome as single-stranded DNA, (2) that the resulting heteroduplexes are resolved by chromosome replication and segregation, and (3) that integrated DNA is rapidly expressed prior to cell division. We show that the combination of these properties results in the nongenetic transfer of gene products within transformed populations, which can support phenotypic inheritance of antibiotic resistance in both V. cholerae and Streptococcus pneumoniae. Thus, beyond the genetic acquisition of novel DNA sequences, NT can also promote the nongenetic inheritance of traits during this conserved mechanism of horizontal gene transfer.
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http://dx.doi.org/10.1016/j.cell.2019.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913884PMC
December 2019

The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae.

PLoS Genet 2019 10 28;15(10):e1008362. Epub 2019 Oct 28.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes.
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http://dx.doi.org/10.1371/journal.pgen.1008362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855506PMC
October 2019

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili.

PLoS Genet 2019 10 18;15(10):e1008448. Epub 2019 Oct 18.

Department of Biology, Indiana University, Bloomington, Indiana, United States of America.

Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.
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http://dx.doi.org/10.1371/journal.pgen.1008448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821130PMC
October 2019

Real-time microscopy and physical perturbation of bacterial pili using maleimide-conjugated molecules.

Nat Protoc 2019 06 26;14(6):1803-1819. Epub 2019 Apr 26.

Department of Biology, Indiana University, Bloomington, IN, USA.

Bacteria use surface-exposed, proteinaceous fibers called pili for diverse behaviors, including horizontal gene transfer, surface sensing, motility, and pathogenicity. Visualization of these filamentous nanomachines and their activity in live cells has proven challenging, largely due to their small size. Here, we describe a broadly applicable method for labeling and imaging pili and other surface-exposed nanomachines in live cells. This technique uses a combination of genetics and maleimide-based click chemistry in which a cysteine substitution is made in the major pilin subunit for subsequent labeling with thiol-reactive maleimide dyes. Large maleimide-conjugated molecules can also be used to physically interfere with the dynamic activity of filamentous nanomachines. We describe parameters for selecting cysteine substitution positions, optimized labeling conditions for epifluorescence imaging of pilus fibers, and methods for impeding pilus activity. After cysteine knock-in strains have been generated, this protocol can be completed within 30 min to a few hours, depending on the species and the experiment of choice. Visualization of extracellular nanomachines such as pili using this approach can provide a more comprehensive understanding of the role played by these structures in distinct bacterial behaviors.
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http://dx.doi.org/10.1038/s41596-019-0162-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461830PMC
June 2019

Natural Cotransformation and Multiplex Genome Editing by Natural Transformation (MuGENT) of Vibrio cholerae.

Authors:
Ankur B Dalia

Methods Mol Biol 2018 ;1839:53-64

Department of Biology, Indiana University, Bloomington, IN, USA.

Generating mutant strains is an essential component of microbial genetics. Natural genetic transformation, a process for the uptake and integration of foreign DNA, is shared by diverse microbial species and can be exploited for making mutant strains. Canonically, this process has been used to generate single mutants and sequentially to generate strains with multiple mutations. Recently, we have described a method for multiplex genome editing by natural transformation (MuGENT), which allows the generation of strains with multiple scarless mutations in a single step. Here, we provide a detailed description of the methods used for mutagenesis of the cholera pathogen Vibrio cholerae with a particular emphasis on mutagenesis via MuGENT.
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http://dx.doi.org/10.1007/978-1-4939-8685-9_6DOI Listing
April 2019

Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae.

Nat Microbiol 2018 07 11;3(7):773-780. Epub 2018 Jun 11.

Department of Biology, Indiana University, Bloomington, IN, USA.

Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation ; however, their mechanism of action has remained unclear owing to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus-labelling method, we define the mechanism for type IV competence pilus-mediated DNA uptake during natural transformation. First, we show that type IV competence pili bind to extracellular double-stranded DNA via their tip and demonstrate that this binding is critical for DNA uptake. Next, we show that type IV competence pili are dynamic structures and that pilus retraction brings tip-bound DNA to the cell surface. Finally, we show that pilus retraction is spatiotemporally coupled to DNA internalization and that sterically obstructing pilus retraction prevents DNA uptake. Together, these results indicate that type IV competence pili directly bind to DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfer.
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http://dx.doi.org/10.1038/s41564-018-0174-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582970PMC
July 2018

ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species.

Nucleic Acids Res 2018 07;46(12):6099-6111

Department of Biology, Indiana University, Bloomington, IN 47405, USA.

Acquisition of foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse microbial species. Here, we characterize the mechanism of ComM, a broadly conserved AAA+ protein previously implicated in homologous recombination of transforming DNA (tDNA) in naturally competent Gram-negative bacterial species. In vivo, we found that ComM was required for efficient comigration of linked genetic markers in Vibrio cholerae and Acinetobacter baylyi, which is consistent with a role in branch migration. Also, ComM was particularly important for integration of tDNA with increased sequence heterology, suggesting that its activity promotes the acquisition of novel DNA sequences. In vitro, we showed that purified ComM binds ssDNA, oligomerizes into a hexameric ring, and has bidirectional helicase and branch migration activity. Based on these data, we propose a model for tDNA integration during natural transformation. This study provides mechanistic insight into the enigmatic steps involved in tDNA integration and uncovers the function of a protein required for this conserved mechanism of horizontal gene transfer.
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http://dx.doi.org/10.1093/nar/gky343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158740PMC
July 2018

Natural Transformation in Vibrio parahaemolyticus: a Rapid Method To Create Genetic Deletions.

J Bacteriol 2018 08 10;200(15). Epub 2018 Jul 10.

Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA

The Gram-negative bacterium is an opportunistic human pathogen and the leading cause of seafood-borne acute gastroenteritis worldwide. Recently, this bacterium was implicated as the etiologic agent of a severe shrimp disease with consequent devastating outcomes to shrimp farming. In both cases, acquisition of genetic material via horizontal transfer provided with new virulence tools to cause disease. Dissecting the molecular mechanisms of pathogenesis often requires manipulating its genome. Classically, genetic deletions in are performed using a laborious, lengthy, multistep process. Here, we describe a fast and efficient method to edit this bacterium's genome based on natural competence. Although this method is similar to one previously described, requires counterselection for curing of acquired plasmids due to its recalcitrant nature of retaining extrachromosomal DNA. We believe this approach will be of use to the community. Spreading of vibrios throughout the world correlates with increased global temperatures. As they spread, they find new niches in which to survive, proliferate, and invade. Therefore, genetic manipulation of vibrios is of the utmost importance for studying these species. Here, we have delineated and validated a rapid method to create genetic deletions in This study provides insightful methodology for studies with other species.
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http://dx.doi.org/10.1128/JB.00032-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040184PMC
August 2018

Obstruction of pilus retraction stimulates bacterial surface sensing.

Science 2017 10;358(6362):535-538

Department of Biology, Indiana University, 1001 East 3rd Street, Bloomington, IN 47405, USA.

It is critical for bacteria to recognize surface contact and initiate physiological changes required for surface-associated lifestyles. Ubiquitous microbial appendages called pili are involved in sensing surfaces and facilitating downstream behaviors, but the mechanism by which pili mediate surface sensing has been unclear. We visualized pili undergoing dynamic cycles of extension and retraction. Within seconds of surface contact, these cycles ceased, which coincided with synthesis of the adhesive holdfast required for attachment. Physically blocking pili imposed resistance to pilus retraction, which was sufficient to stimulate holdfast synthesis without surface contact. Thus, to sense surfaces, bacteria use the resistance on retracting, surface-bound pili that occurs upon surface contact.
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http://dx.doi.org/10.1126/science.aan5706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805138PMC
October 2017

Systematic genetic dissection of chitin degradation and uptake in Vibrio cholerae.

Environ Microbiol 2017 10 14;19(10):4154-4163. Epub 2017 Aug 14.

Department of Biology, Indiana University, Bloomington, IN, USA.

Vibrio cholerae is a natural resident of the aquatic environment, where a common nutrient is the chitinous exoskeletons of microscopic crustaceans. Chitin utilization requires chitinases, which degrade this insoluble polymer into soluble chitin oligosaccharides. These oligosaccharides also serve as an inducing cue for natural transformation in Vibrio species. There are 7 predicted endochitinase-like genes in the V. cholerae genome. Here, we systematically dissect the contribution of each gene to growth on chitin as well as induction of natural transformation. Specifically, we created a strain that lacks all 7 putative chitinases and from this strain, generated a panel of strains where each expresses a single chitinase. We also generated expression plasmids to ectopically express all 7 chitinases in our chitinase deficient strain. Through this analysis, we found that low levels of chitinase activity are sufficient for natural transformation, while growth on insoluble chitin as a sole carbon source requires more robust and concerted chitinase activity. We also assessed the role that the three uptake systems for the chitin degradation products GlcNAc, (GlcNAc) and (GlcN) , play in chitin utilization and competence induction. Cumulatively, this study provides mechanistic details for how this pathogen utilizes chitin to thrive and evolve in its environmental reservoir.
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http://dx.doi.org/10.1111/1462-2920.13866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647239PMC
October 2017

The nucleoid occlusion protein SlmA is a direct transcriptional activator of chitobiose utilization in Vibrio cholerae.

PLoS Genet 2017 Jul 6;13(7):e1006877. Epub 2017 Jul 6.

Department of Biology, Indiana University, Bloomington, IN, United States of America.

Chitin utilization by the cholera pathogen Vibrio cholerae is required for its persistence and evolution via horizontal gene transfer in the marine environment. Genes involved in the uptake and catabolism of the chitin disaccharide chitobiose are encoded by the chb operon. The orphan sensor kinase ChiS is critical for regulation of this locus, however, the mechanisms downstream of ChiS activation that result in expression of the chb operon are poorly understood. Using an unbiased transposon mutant screen, we uncover that the nucleoid occlusion protein SlmA is a regulator of the chb operon. SlmA has not previously been implicated in gene regulation. Also, SlmA is a member of the TetR family of proteins, which are generally transcriptional repressors. In vitro, we find that SlmA binds directly to the chb operon promoter, and in vivo, we show that this interaction is required for transcriptional activation of this locus and for chitobiose utilization. Using point mutations that disrupt distinct functions of SlmA, we find that DNA-binding, but not nucleoid occlusion, is critical for transcriptional activation. This study identifies a novel role for SlmA as a transcriptional regulator in V. cholerae in addition to its established role as a cell division licensing factor.
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http://dx.doi.org/10.1371/journal.pgen.1006877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519180PMC
July 2017

Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases.

Nucleic Acids Res 2017 Jul;45(12):7527-7537

Department of Biology, Indiana University, Bloomington, IN 47401, USA.

Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.
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http://dx.doi.org/10.1093/nar/gkx496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499599PMC
July 2017

Multiplex Genome Editing by Natural Transformation (MuGENT) for Synthetic Biology in Vibrio natriegens.

ACS Synth Biol 2017 09 6;6(9):1650-1655. Epub 2017 Jun 6.

Department of Biology, Indiana University , Bloomington, Indiana 47401, United States.

Vibrio natriegens has recently emerged as an alternative to Escherichia coli for molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence in V. natriegens and describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented time scales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts. As a proof-of-concept, we attempted to enhance production of the value added chemical poly-β-hydroxybutyrate (PHB) in V. natriegens by targeting the expression of nine genes involved in PHB biosynthesis via MuGENT. Within 1 week, we isolated edited strains that produced ∼100 times more PHB than the parent isolate and ∼3.3 times more than a rationally designed strain. Thus, the methods described here should extend the utility of this species for diverse academic and industrial applications.
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http://dx.doi.org/10.1021/acssynbio.7b00116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519440PMC
September 2017

Acute Hepatopancreatic Necrosis Disease-Causing Vibrio parahaemolyticus Strains Maintain an Antibacterial Type VI Secretion System with Versatile Effector Repertoires.

Appl Environ Microbiol 2017 07 16;83(13). Epub 2017 Jun 16.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of that have acquired a "selfish plasmid" encoding the deadly binary toxins PirA/PirB To better understand the repertoire of virulence factors in AHPND-causing , we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control. Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Although the plasmid-encoded binary toxins PirA/PirB have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections.
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http://dx.doi.org/10.1128/AEM.00737-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478980PMC
July 2017

Systematic genetic dissection of PTS in Vibrio cholerae uncovers a novel glucose transporter and a limited role for PTS during infection of a mammalian host.

Mol Microbiol 2017 05 28;104(4):568-579. Epub 2017 Feb 28.

Department of Biology, Indiana University, Bloomington, IN, USA.

A common mechanism for high affinity carbohydrate uptake in microbial species is the phosphoenolpyruvate-dependent phosphotransferase system (PTS). This system consists of a shared component, EI, which is required for all PTS transport, and numerous carbohydrate uptake transporters. In Vibrio cholerae, there are 13 distinct PTS transporters. Due to genetic redundancy within this system, the carbohydrate specificity of each of these transporters is not currently defined. Here, using multiplex genome editing by natural transformation (MuGENT), we systematically dissect PTS transport in V. cholerae. Specifically, we generated a mutant strain that lacks all 13 PTS transporters, and from this strain, we created a panel of mutants where each expresses a single transporter. Using this panel, we have largely defined the carbohydrate specificities of each PTS transporter. In addition, this analysis uncovered a novel glucose transporter. We have further defined the mechanism of this transporter and characterized its regulation. Using our 13 PTS transporter mutant, we also provide the first clear evidence that carbohydrate transport by the PTS is not essential during infection in an infant mouse model of cholera. In summary, this study shows how multiplex genome editing can be used to rapidly dissect complex biological systems and genetic redundancy in microbial systems.
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http://dx.doi.org/10.1111/mmi.13646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426971PMC
May 2017

RpoS is required for natural transformation of Vibrio cholerae through regulation of chitinases.

Authors:
Ankur B Dalia

Environ Microbiol 2016 11 30;18(11):3758-3767. Epub 2016 Apr 30.

Department of Biology, Indiana University, Bloomington, Indiana, USA.

Vibrio species naturally reside in the aquatic environment and a major metabolite in this habitat is the chitinous exoskeletons of crustacean zooplankton. In addition to serving as a nutrient, chitin-derived oligosaccharides also induce natural genetic competence in many Vibrio spp., a physiological state in which bacteria take up DNA from the extracellular environment and can integrate it into their chromosome by homologous recombination. Another inducing cue required for competence are quorum-sensing autoinducers. The alternative sigma factor RpoS is critical for natural transformation in Vibrio cholerae, and it was previously presumed to exert this effect through regulation of quorum sensing. Here, we show that RpoS does not affect quorum sensing-dependent regulation of competence. Instead, we show that an rpoS mutant has reduced chitinase activity, which is required to liberate the soluble chitin oligosaccharides that serve as an inducing cue for competence. Consistent with this, we demonstrate that RpoS is required for growth of V. cholerae on insoluble chitin. RpoS also regulates the mucosal escape response in pathogenic strains of V. cholerae. Thus, in addition to promoting egress from its human host, RpoS may also prime this pathogen for successful reentry into the aquatic environment.
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http://dx.doi.org/10.1111/1462-2920.13302DOI Listing
November 2016
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