Conserved ESX-1 Substrates EspE and EspF Are Virulence Factors That Regulate Gene Expression

ABSTRACT Mycobacterium tuberculosis, the cause of human tuberculosis, and Mycobacterium marinum, a nontubercular pathogen with a broad host range, require the ESX-1 secretion system for virulence. The ESX-1 system secretes proteins which cause phagosomal lysis within the macrophage via an unknown mechanism. As reported elsewhere (R. E. Bosserman et al., Proc Natl Acad Sci U S A 114:E10772–E10781, 2017, https://doi.org/10.1073/pnas.1710167114), we recently discovered that the ESX-1 system regulates gene expression in M. marinum This finding was confirmed in M. tuberculosis in reports by C. Sala et al. (PLoS Pathog 14:e1007491, 2018, https://doi.org/10.1371/journal.ppat.1007491) and A. M. Abdallah et al. (PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). We further demonstrated that a feedback control mechanism connects protein secretion to WhiB6-dependent expression of the esx-1 genes via an unknown mechanism. Here, we connect protein secretion and gene expression by showing for the first time that specific ESX-1 substrates have dual functions inside and outside the mycobacterial cell. We demonstrate that the EspE and EspF substrates negatively control esx-1 gene expression in the M. marinum cytoplasm through the conserved WhiB6 transcription factor. We found that EspE and EspF are required for virulence and promote lytic activity independently of the major EsxA and EsxB substrates. We show that the dual functions of EspE and EspF are conserved in the orthologous proteins from M. tuberculosis. Our findings support a role for EspE and EspF in virulence that is independent of the EsxA and EsxB substrates and demonstrate that ESX-1 substrates have a conserved role in regulating gene expression.

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EspM Is a Conserved Transcription Factor That Regulates Gene Expression in Response to the ESX-1 System

mBio ◽

10.1128/mbio.02807-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 3

Author(s):

Kevin G. Sanchez ◽

Micah J. Ferrell ◽

Alexandra E. Chirakos ◽

Kathleen R. Nicholson ◽

Robert B. Abramovitch ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Protein Transport ◽

Transcriptional Response ◽

Secretion System ◽

Pathogenic Species ◽

Macrophage Infection ◽

Content Type ◽

Link Type ◽

Phagosomal Membrane

ABSTRACT Pathogenic mycobacteria encounter multiple environments during macrophage infection. Temporally, the bacteria are engulfed into the phagosome, lyse the phagosomal membrane, and interact with the cytosol before spreading to another cell. Virulence factors secreted by the mycobacterial ESX-1 (ESAT-6-system-1) secretion system mediate the essential transition from the phagosome to the cytosol. It was recently discovered that the ESX-1 system also regulates mycobacterial gene expression in Mycobacterium marinum (R. E. Bosserman, T. T. Nguyen, K. G. Sanchez, A. E. Chirakos, et al., Proc Natl Acad Sci U S A 114:E10772–E10781, 2017, https://doi.org/10.1073/pnas.1710167114), a nontuberculous mycobacterial pathogen, and in the human-pathogenic species M. tuberculosis (A. M. Abdallah, E. M. Weerdenburg, Q. Guan, R. Ummels, et al., PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). It is not known how the ESX-1 system regulates gene expression. Here, we identify the first transcription factor required for the ESX-1-dependent transcriptional response in pathogenic mycobacteria. We demonstrate that the gene divergently transcribed from the whiB6 gene and adjacent to the ESX-1 locus in mycobacterial pathogens encodes a conserved transcription factor (MMAR_5438, Rv3863, now espM). We prove that EspM from both M. marinum and M. tuberculosis directly and specifically binds the whiB6-espM intergenic region. We show that EspM is required for ESX-1-dependent repression of whiB6 expression and for the regulation of ESX-1-associated gene expression. Finally, we demonstrate that EspM functions to fine-tune ESX-1 activity in M. marinum. Taking the data together, this report extends the esx-1 locus, defines a conserved regulator of the ESX-1 virulence pathway, and begins to elucidate how the ESX-1 system regulates gene expression. IMPORTANCE Mycobacterial pathogens use the ESX-1 system to transport protein substrates that mediate essential interactions with the host during infection. We previously demonstrated that in addition to transporting proteins, the ESX-1 secretion system regulates gene expression. Here, we identify a conserved transcription factor that regulates gene expression in response to the ESX-1 system. We demonstrate that this transcription factor is functionally conserved in M. marinum, a pathogen of ectothermic animals; M. tuberculosis, the human-pathogenic species that causes tuberculosis; and M. smegmatis, a nonpathogenic mycobacterial species. These findings provide the first mechanistic insight into how the ESX-1 system elicits a transcriptional response, a function of this protein transport system that was previously unknown.

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Pathovar Transcriptomes

mSystems ◽

10.1128/msystems.00049-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 1

Author(s):

Amy Platenkamp ◽

Jay L. Mellies

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Regulatory Networks ◽

Virulence Gene ◽

Model System ◽

Genomic Sequences ◽

Bacterial Strains ◽

Content Type ◽

Virulence Gene Expression ◽

Link Type

ABSTRACT Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression, and they found variation among individual isolates. Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression. They also found variation among individual isolates. Their work illustrates the importance of moving beyond observing regulatory phenomena of a limited number of regulons in a few archetypal strains, with the possibility of correlating clinical symptoms to key transcriptional pathways across lineages and phylogroups.

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Construction of a Broad-Host-Range Tn7-Based Vector for Single-Copy PBAD-Controlled Gene Expression in Gram-Negative Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02926-12 ◽

2012 ◽

Vol 79 (2) ◽

pp. 718-721 ◽

Cited By ~ 20

Author(s):

F. Heath Damron ◽

Elizabeth S. McKenney ◽

Herbert P. Schweizer ◽

Joanna B. Goldberg

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Host Range ◽

Single Copy ◽

Broad Host Range ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Delivery Vector ◽

Inducible Gene

ABSTRACTWe describe a mini-Tn7-based broad-host-range expression cassette for arabinose-inducible gene expression from the PBADpromoter. This delivery vector, pTJ1, can integrate a single copy of a gene into the chromosome of Gram-negative bacteria for diverse genetic applications, of which several are discussed, usingPseudomonas aeruginosaas the model host.

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Molecular characterization of a novel bla CTX-M-3-carrying Tn6741 transposon in Morganella morganii isolated from swine

Journal of Medical Microbiology ◽

10.1099/jmm.0.001235 ◽

2020 ◽

Vol 69 (8) ◽

pp. 1089-1094

Author(s):

Xingwei Luo ◽

Yajun Zhai ◽

Dandan He ◽

Xiaodie Cui ◽

Yingying Yang ◽

...

Keyword(s):

Type Species ◽

Susceptibility Testing ◽

Morganella Morganii ◽

Ionization Time ◽

Content Type ◽

Genetic Environment ◽

Link Type ◽

Flight Mass Spectrometry ◽

First Time

Introduction. The bla CTX-M-3 gene has rarely been reported in Morganella morganii strains and its genetic environment has not yet been investigated. Aim. To identify the bla CTX-M-3 gene in M. morganii isolated from swine and characterize its genetic environment. Methodology. A M. morganii isolate (named MM1L5) from a deceased swine was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing. The bla genes were detected and then the genetic location and environment of bla CTX-M-3 were investigated by Southern blot and PCR mapping, respectively. The M. morganii bla CTX-M-3 gene was cloned and expressed in Escherichia coli . Results. Isolate MM1L5 harboured the bla CTX-M-3 and bla TEM-1 genes. The bla CTX-M-3 gene, located on the chromosome, was co-carried with an IS26 and bla TEM-1 gene by a novel 6361 bp IS26-flanked composite transposon, designated Tn6741. This transposon consisted of a novel bla CTX-M-3-containing module, IS26-ΔISEcp1-bla CTX-M-3-Δorf477-IS26 (named Tn6710), and a bla TEM-1-containing module, IS26-Δorf477-bla TEM-1-tnpR-IS26, differing from previous reports. Phylogenetic analysis showed a significant variation based on the sequence of Tn6741, as compared to those of other related transposons. Interestingly, although the cloned bla CTX-M-3 gene could confer resistance to ceftiofur, cefquinome, ceftriaxone and cefotaxime, one amino acid substitution (Ile-142-Thr) resulted in a significant reduction of resistance to these antimicrobials. Conclusion. This is the first time that bla CTX-M-3 has been identified on a chromosome from a M. morganii isolate. Furthermore, the bla CTX-M-3 gene was located with an IS26 element and bla TEM-1 gene on a novel IS26-flanked composite transposon, Tn6741, suggesting that Tn6741 might act as a reservoir for the bla CTX-M-3 and bla TEM-1 genes and may become an important vehicle for their dissemination among M. morganii .

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An Aminotransferase Is Responsible for the Deamination of the N-Terminal Leucine and Required for Formation of Oxazolone Ring A in Methanobactin of Methylosinus trichosporium OB3b

Applied and Environmental Microbiology ◽

10.1128/aem.02619-16 ◽

2016 ◽

Vol 83 (1) ◽

Cited By ~ 10

Author(s):

Wenyu Gu ◽

Bipin S. Baral ◽

Alan A. DiSpirito ◽

Jeremy D. Semrau

Keyword(s):

Gene Expression ◽

Gene Cluster ◽

Wilson Disease ◽

Critical Role ◽

Broad Host Range ◽

Copper Binding ◽

Copper Uptake ◽

Methylosinus Trichosporium ◽

Content Type ◽

Oxazolone Ring

ABSTRACT Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper-regulating expression of alternative forms of methane monooxygenase. A copper-binding compound, or chalkophore, called methanobactin plays a key role in copper uptake in methanotrophs. Methanobactin is a ribosomally synthesized and posttranslationally modified peptide (RiPP) with two heterocyclic rings with an associated thioamide for each ring, formed from X-Cys dipeptide sequences that bind copper. The gene coding for the precursor polypeptide of methanobactin, mbnA, is part of a gene cluster, but the role of other genes in methanobactin biosynthesis is unclear. To begin to elucidate the function of these genes, we constructed an unmarked deletion of mbnABCMN in Methylosinus trichosporium OB3b and then homologously expressed mbnABCM using a broad-host-range cloning vector to determine the function of mbnN, annotated as coding for an aminotransferase. Methanobactin produced by this strain was found to be substantially different from wild-type methanobactin in that the C-terminal methionine was missing and only one of the two oxazolone rings was formed. Rather, in place of the N-terminal 3-methylbutanoyl-oxazolone-thioamide group, a leucine and a thioamide-containing glycine (Gly-Ψ) were found, indicating that MbnN is used for deamination of the N-terminal leucine of methanobactin and that this posttranslational modification is critical for closure of the N-terminal oxazolone ring in M. trichosporium OB3b. These studies provide new insights into methanobactin biosynthesis and also provide a platform for understanding the function of other genes in the methanobactin gene cluster. IMPORTANCE Methanotrophs, microbes that play a critical role in the carbon cycle, are influenced by copper, with gene expression and enzyme activity changing as copper levels change. Methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin for copper uptake, and methanobactin plays a key role in controlling methanotrophic activity. Methanobactin has also been shown to be effective in the treatment of Wilson disease, an autosomal recessive disorder where the human body cannot correctly assimilate copper. It is important to characterize the methanobactin biosynthesis pathway to understand how methanotrophs respond to their environment as well as to optimize the use of methanobactin for the treatment of copper-related diseases such as Wilson disease. Here we show that mbnN, encoding an aminotransferase, is involved in the deamination of the N-terminal leucine and necessary for the formation of one but not both of the heterocyclic rings in methanobactin that are responsible for copper binding.

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TetR-Based Gene Regulation Systems for Francisella tularensis

Applied and Environmental Microbiology ◽

10.1128/aem.01679-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 6883-6889 ◽

Cited By ~ 17

Author(s):

Eric D. LoVullo ◽

Cheryl N. Miller ◽

Martin S. Pavelka ◽

Thomas H. Kawula

Keyword(s):

Gene Expression ◽

Francisella Tularensis ◽

Protein Function ◽

Expression System ◽

Promoter Sequence ◽

Growth Defect ◽

Regulate Gene Expression ◽

Content Type ◽

System A ◽

Tet Repressor

ABSTRACTThere are a number of genetic tools available for studyingFrancisella tularensis, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems forF. tularensisbased on the Tet repressor, TetR. For the inducible system, atetoperator sequence was cloned into a modifiedF. tularensis groESLpromoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon,luxCDABE, was cloned under the hybridFrancisellatetracycline-regulated promoter (FTRp), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association withtetO.Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml−1. In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists ofFTRpregulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with theluxreporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploringF. tularensisprotein function.

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Regulation by the Modulation of Gene Expression Variability

Journal of Bacteriology ◽

10.1128/jb.00235-15 ◽

2015 ◽

Vol 197 (12) ◽

pp. 1974-1975 ◽

Cited By ~ 2

Author(s):

David Dubnau

Keyword(s):

Gene Expression ◽

Regulation Of Gene Expression ◽

Content Type ◽

Expression Variability ◽

Link Type ◽

Article Type ◽

Modulation Of Gene Expression ◽

The Mean ◽

Cell Changes ◽

Expression Variance

Classically, transcription is regulated so that the average expression per cell changes, often with a distribution that extends across the population. Roggiani and Goulian (M. Roggiani and M. Goulian, J. Bacteriol. 197:1976–1987, 2015, doi:http://dx.doi.org/10.1128/JB.00074-15) have shown that this is what happens when thetorCADoperon ofEscherichia coliis induced anaerobically by the addition of trimethylamine-N-oxide (TMAO). However, when the same inducer is added to aerobically growing cells, only a subset of the cells respond, although the mean expression per cell is similar to that obtained anaerobically. Thus, in the presence of oxygen, the variance but not the expression mean is altered. The regulation of gene expression variance appears to be due to noise in the phosphorelay that governstorCADtranscription.

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Transcriptome profiling of Variovorax paradoxus EPS under different growth conditions reveals regulatory and structural novelty in biofilm formation

Access Microbiology ◽

10.1099/acmi.0.000121 ◽

2020 ◽

Vol 2 (6) ◽

Author(s):

Richard J. Fredendall ◽

Jenny L. Stone ◽

Michael J. Pehl ◽

Paul M. Orwin

Keyword(s):

Gene Expression ◽

Stationary Phase ◽

Exponential Growth ◽

Biofilm Formation ◽

Type Species ◽

Extracellular Dna ◽

Differentially Expressed ◽

Content Type ◽

Variovorax Paradoxus ◽

Link Type

We used transcriptome analysis by paired-end strand-specific RNA-seq to evaluate the specific changes in gene expression associated with the transition to static biofilm growth in the rhizosphere plant growth-promoting bacterium Variovorax paradoxus EPS. Triplicate biological samples of exponential growth, stationary phase and static biofilm samples were examined. DESeq2 and Rockhopper were used to identify robust and widespread shifts in gene expression specific to each growth phase. We identified 1711 protein-coding genes (28%) using DESeq2 that had altered expression greater than twofold specifically in biofilms compared to exponential growth. Fewer genes were specifically differentially expressed in stationary-phase culture (757, 12%). A small set of genes (103/6020) were differentially expressed in opposing fashions in biofilm and stationary phase, indicating potentially substantial shifts in phenotype. Gene-ontology analysis showed that the only class of genes specifically upregulated in biofilms was associated with nutrient transport, highlighting the importance of nutrient uptake in the biofilm. The biofilm-specific genes did not overlap substantially with the loci identified by mutagenesis studies, although some were present in both sets. The most highly upregulated biofilm-specific gene is predicted to be a part of the RNA degradosome, which indicates that RNA stability is used to regulate the biofilm phenotype. Two small putative proteins, Varpa_0407 and Varpa_3832, are highly expressed specifically in biofilms and are predicted to be secreted DNA-binding proteins, which may stabilize extracellular DNA as a component of the biofilm matrix. An flp/tad type-IV pilus locus (Varpa_5148–60) is strongly downregulated specifically in biofilms, in contrast with results from other systems for these pili. Mutagenesis confirms that this locus is important in surface motility rather than biofilm formation. These experimental results suggest that V. paradoxus EPS biofilms have substantial regulatory and structural novelty.

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Detection of cytosine methylation in Burkholderia cenocepacia by single-molecule real-time sequencing and whole-genome bisulfite sequencing

Microbiology ◽

10.1099/mic.0.001027 ◽

2021 ◽

Author(s):

Ian Vandenbussche ◽

Andrea Sass ◽

Filip Van Nieuwerburgh ◽

Marta Pinto-Carbó ◽

Olga Mannweiler ◽

...

Keyword(s):

Gene Expression ◽

Single Molecule ◽

Type Species ◽

Cytosine Methylation ◽

Regulation Of Gene Expression ◽

Burkholderia Cenocepacia ◽

Smrt Sequencing ◽

Content Type ◽

Link Type ◽

Genome Bisulfite Sequencing

Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in Burkholderia cenocepacia , an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the B. cenocepacia genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGCC sequences. Methylation motif GCGGCCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in CpG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when B. cenocepacia strains J2315 and K56-2 were compared, which suggests that GGCC methylation patterns in B. cenocepacia are strain-specific. It seems likely that 4mC methylation of GGCC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of B. cenocepacia .

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Reclassification of Rhizobium tropici type A strains as Rhizobium leucaenae sp. nov.

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.032912-0 ◽

2012 ◽

Vol 62 (Pt_5) ◽

pp. 1179-1184 ◽

Cited By ~ 75

Author(s):

Renan Augusto Ribeiro ◽

Marco A. Rogel ◽

Aline López-López ◽

Ernesto Ormeño-Orrillo ◽

Fernando Gomes Barcellos ◽

...

Keyword(s):

Type Species ◽

Novel Species ◽

Type A ◽

Gliricidia Sepium ◽

Phenotypic Traits ◽

Broad Host Range ◽

Rhizobium Tropici ◽

Content Type ◽

Link Type ◽

Well Differentiated

Rhizobium tropici is a well-studied legume symbiont characterized by high genetic stability of the symbiotic plasmid and tolerance to tropical environmental stresses such as high temperature and low soil pH. However, high phenetic and genetic variabilities among R. tropici strains have been largely reported, with two subgroups, designated type A and B, already defined within the species. A polyphasic study comprising multilocus sequence analysis, phenotypic and genotypic characterizations, including DNA–DNA hybridization, strongly supported the reclassification of R. tropici type A strains as a novel species. Type A strains formed a well-differentiated clade that grouped with R. tropici , Rhizobium multihospitium , Rhizobium miluonense , Rhizobium lusitanum and Rhizobium rhizogenes in the phylogenies of the 16S rRNA, recA, gltA, rpoA, glnII and rpoB genes. Several phenotypic traits differentiated type A strains from all related taxa. The novel species, for which the name Rhizobium leucaenae sp. nov. is proposed, is a broad host range rhizobium being able to establish effective root-nodule symbioses with Leucaena leucocephala, Leucaena esculenta, common beans (Phaseolus vulgaris) and Gliricidia sepium. Strain CFN 299T ( = USDA 9039T = LMG 9517T = CECT 4844T = JCM 21088T = IAM 14230T = SEMIA 4083T = CENA 183T = UMR1026T = CNPSo 141T) is designated the type strain of Rhizobium leucaenae sp. nov.

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