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1. Similar solutions to a common challenge: regulation of genes encoding Ralstonia solanacearum xanthine dehydrogenase

   https://doi.org/10.1093/femsle/fnab022  

   Sivapragasam, Smitha ; Ghosh, Arpita ; Kumar, Sanjay ; Johnson, Danté T ; Grove, Anne ( March 2021 , FEMS Microbiology Letters)

   null (Ed.)

   ABSTRACT The stringent response involves accumulation of (p)ppGpp, and it ensures that survival is prioritized. Production of (p)ppGpp requires purine synthesis, and upregulation of an operon that encodes the purine salvage enzyme xanthine dehydrogenase (Xdh) has been observed during stringent response in some bacterial species, where direct binding of ppGpp to a TetR-family transcription factor is responsible for increased xdh gene expression. We show here that the plant pathogen Ralstonia solanacearum has a regulatory system in which the LysR-family transcription factor XanR controls expression of the xan operon; this operon encodes Xdh as well as other enzymes involved in purine salvage, which favor accumulation of xanthine. XanR bound upstream of the xan operon, a binding that was attenuated on addition of either ppGpp or cyclic di-guanosine monophosphate (c-di-GMP). Using a reporter in which enhanced green fluorescent protein (EGFP) is expressed under control of a modified xan promoter, XanR was shown to repress EGFP production. Our data suggest that R. solanacearum features a regulatory mechanism in which expression of genes encoding purine salvage enzymes is controlled by a transcription factor that belongs to a different protein family, yet performs similar regulatory functions. 

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2. The Global Regulator MftR Controls Virulence and Siderophore Production in Burkholderia thailandensis

   https://doi.org/10.1128/jb.00237-22  

   Thapa, Sudarshan S. ; Al-Tohamy, Ahmed ; Grove, Anne ( November 2022 , Journal of Bacteriology)

   Comstock, Laurie E. (Ed.)

   ABSTRACT Burkholderia thailandensis is a member of the Burkholderia pseudomallei complex. It encodes the transcription factor MftR, which is conserved among the more pathogenic Burkholderia spp. and previously shown to be a global regulator of gene expression. We report here that a B. thailandensis strain in which the mftR gene is disrupted is more virulent in both Caenorhabditis elegans and onion. The Δ mftR strain exhibits a number of phenotypes associated with virulence. It is more proficient at forming biofilm, and the arcDABC gene cluster, which has been linked to anaerobic survival and fitness within a biofilm, is upregulated. Swimming and swarming motility are also elevated in Δ mftR cells. We further show that MftR is one of several transcription factors which control production of the siderophore malleobactin. MftR binds directly to the promoter driving expression of mbaS , which encodes the extracytoplasmic function sigma factor MbaS that is required for malleobactin production. Malleobactin is a primary siderophore in B. thailandensis as evidenced by reduced siderophore production in mbaS ::Tc cells, in which mbaS is disrupted. Expression of mbaS is increased ~5-fold in Δ mftR cells, and siderophore production is elevated. Under iron-limiting conditions, mbaS expression is increased ~150-fold in both wild-type and Δ mftR cells, respectively, reflecting regulation by the ferric uptake regulator (Fur). The mbaS expression profiles also point to repression by a separate, ligand-responsive transcription factor, possibly ScmR. Taken together, these data indicate that MftR controls a number of phenotypes, all of which promote bacterial survival in a host environment. IMPORTANCE Bacterial pathogens face iron limitation in a host environment. To overcome this challenge, they produce siderophores, small iron-chelating molecules. Uptake of iron-siderophore complexes averts bacterial iron limitation. In Burkholderia spp., malleobactin or related compounds are the primary siderophores. We show here that genes encoding proteins required for malleobactin production in B. thailandensis are under the direct control of the global transcription factor MftR. Repression of gene expression by MftR is relieved when MftR binds xanthine, a purine metabolite present in host cells. Our work therefore identifies a mechanism by which siderophore production may be optimized in a host environment, thus contributing to bacterial fitness. 

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3. Transcriptome profiling of type VI secretion system core gene tssM mutant of Xanthomonas perforans highlights regulators controlling diverse functions ranging from virulence to metabolism

   https://doi.org/10.1128/spectrum.02852-23  

   Ramamoorthy, Sivakumar ; Pena, Michelle ; Ghosh, Palash ; Liao, Ying-Yu ; Paret, Mathews ; Jones, Jeffrey B. ; Potnis, Neha ( January 2024 , Microbiology Spectrum)

   Burbank, Lindsey Price (Ed.)

   Type VI secretion system (T6SS) is a versatile, contact-dependent contractile nano-weapon in Gram-negative bacteria that fires proteinaceous effector molecules directly into prokaryotic and eukaryotic cells aiding in manipulation of the host and killing of competitors in complex niches. In plant pathogenic xanthomonads, T6SS has been demonstrated to play these diverse roles in individual pathosystems. However, the molecular network underlying the regulation of T6SS is still elusive inXanthomonasspp. To bridge this knowledge gap, we conducted anin vitrotranscriptome screen using plant apoplast mimicking minimal medium, XVM2 medium, to decipher the effect oftssMdeletion, a core gene belonging to T6SS-cluster i3*, on the regulation of gene expression inXanthomonas perforansstrain AL65. Transcriptomic data revealed that a total of 277 and 525 genes were upregulated, while 307 and 392 genes were downregulated in the mutant strain after 8 and 16 hours of growth in XVM2 medium. The transcript abundance of several genes associated with flagellum and pilus biogenesis as well as type III secretion system was downregulated in the mutant strain. Deletion oftssMof cluster-i3* resulted in upregulation of several T6SS genes belonging to cluster-i3*** and genes involved in biofilm and cell wall biogenesis. Similarly, transcription regulators likerpoN, Pho regulon,rpoE, andcsrAwere identified to be upregulated in the mutant strain. Our results suggest that T6SS modulates the expression of global regulators likecsrA,rpoN, andphoregulons, triggering a signaling cascade, and co-ordinates the expression of suite of virulence factors, stress response genes, and metabolic genes.

   T6SS has received attention due to its significance in mediating interorganismal competition through contact-dependent release of effector molecules into prokaryotic and eukaryotic cells. Reverse-genetic studies have indicated the role of T6SS in virulence in a variety of plant pathogenic bacteria, including the one studied here,Xanthomonas. However, it is not clear whether such effect on virulence is merely due to a shift in the microbiome-mediated protection or if T6SS is involved in a complex virulence regulatory network. In this study, we conducted in vitro transcriptome profiling in minimal medium to decipher the signaling pathways regulated by tssM-i3* inX. perforansAL65. We show that TssM-i3* regulates the expression of a suite of genes associated with virulence and metabolism either directly or indirectly by altering the transcription of several regulators. These findings further expand our knowledge on the intricate molecular circuits regulated by T6SS in phytopathogenic bacteria.

    

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4. Transcription Factor PecS Mediates Agrobacterium fabrum Fitness and Survival

   https://doi.org/10.1128/jb.00478-22  

   Nwokocha, George C. ; Adhikari, Prava ; Iqbal, Asif ; Elkholy, Hannah ; Doerrler, William T. ; Larkin, John C. ; Grove, Anne ( July 2023 , Journal of Bacteriology)

   Becker, Anke (Ed.)

   ABSTRACT The transcriptional regulator PecS is encoded by select bacterial pathogens. For instance, in the plant pathogen Dickeya dadantii , PecS controls a range of virulence genes, including pectinase genes and the divergently oriented gene pecM , which encodes an efflux pump through which the antioxidant indigoidine is exported. In the plant pathogen Agrobacterium fabrum (formerly named Agrobacterium tumefaciens ), the pecS-pecM locus is conserved. Using a strain of A. fabrum in which pecS has been disrupted, we show here that PecS controls a range of phenotypes that are associated with bacterial fitness. PecS represses flagellar motility and chemotaxis, which are processes that are important for A. fabrum to reach plant wound sites. Biofilm formation and microaerobic survival are reduced in the pecS disruption strain, whereas the production of acyl homoserine lactone (AHL) and resistance to reactive oxygen species (ROS) are increased when pecS is disrupted. AHL production and resistance to ROS are expected to be particularly relevant in the host environment. We also show that PecS does not participate in the induction of vir genes. The inducing ligands for PecS, urate, and xanthine, may be found in the rhizosphere, and they accumulate within the plant host upon infection. Therefore, our data suggest that PecS mediates A. fabrum fitness during its transition from the rhizosphere to the host plant. IMPORTANCE PecS is a transcription factor that is conserved in several pathogenic bacteria, where it regulates virulence genes. The plant pathogen Agrobacterium fabrum is important not only for its induction of crown galls in susceptible plants but also for its role as a tool in the genetic manipulation of host plants. We show here that A. fabrum PecS controls a range of phenotypes, which would confer the bacteria an advantage while transitioning from the rhizosphere to the host plant. This includes the production of signaling molecules, which are critical for the propagation of the tumor-inducing plasmid. A more complete understanding of the infection process may inform approaches by which to treat infections as well as to facilitate the transformation of recalcitrant plant species. 

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5. A role for Vibrio vulnificus PecS during hypoxia

   https://doi.org/10.1038/s41598-019-39095-4  

   Bhattacharyya, Nabanita ; Lemon, Tiffany L. ; Grove, Anne ( February 2019 , Scientific Reports)

   The genusVibrioincludes serious human pathogens, and mollusks are a significant reservoir for species such asV.vulnificus.Vibriospecies encode PecS, a member of the multiple antibiotic resistance regulator (MarR) family of transcription factors;pecSis divergently oriented topecM, which encodes an efflux pump. We report here thatVibriospecies feature frequent duplications ofpecS-pecMgenes, suggesting evolutionary pressures to respond to distinct environmental situations. The singleV.vulnificusPecS binds two sites within thepecS-pecMintergenic region with K_d = 0.3 ± 0.1 nM, a binding that is attenuated by the ligands xanthine and urate, except when promoter DNA is saturated with PecS. A unique target is found in the intergenic region between genes encoding the nitric oxide sensing transcription factor, NsrR, andnod; thenod-encoded nitric oxide dioxygenase is important for preventing nitric oxide stress. Reporter gene assays show that PecS-mediated repression of gene expression can be relieved in presence of ligand. Since xanthine and urate are produced as part of the oxidative burst during host defenses and under molluscan hypoxia, we propose that these intermediates in the host purine degradation pathway function to promote bacterial survival during hypoxia and oxidative stress.

    

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