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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 17 July 2018, Vol.115(29), pp.7587-7592
    Description: Many species of produce acyl-homoserine lactone (AHL) compounds as quorum-sensing (QS) signals for cell density-dependent gene regulation. Most known AHL synthases, LuxI-type enzymes, produce fatty AHLs, and the fatty acid moiety is derived from an acyl-acyl carrier protein (ACP) intermediate in fatty acid biosynthesis. Recently, a class of LuxI homologs has been shown to use CoA-linked aromatic or amino acid substrates for AHL synthesis. By using an informatics approach, we found the CoA class of LuxI homologs exists primarily in α-Proteobacteria. The genome of , a dimorphic prosthecate bacterium, possesses a like AHL synthase gene that we predicted to encode a CoA-utilizing enzyme. We show the LuxI homolog catalyzes synthesis of phenylacetyl-homoserine lactone (PA-HSL). Our experiments show obtains phenylacetate from its environment and uses a CoA ligase to produce the phenylacetyl-CoA substrate for the LuxI homolog. By using an AHL degrading enzyme, we showed that PA-HSL controls aggregation, biofilm formation, and pigment production in These findings advance a limited understanding of the CoA-dependent AHL synthases. We describe how to identify putative members of the class, we describe a signal synthesized by using an environmental aromatic acid, and we identify phenotypes controlled by the aryl-HSL.
    Keywords: Prosthecomicrobium ; Bacterial Communication ; Phenylacetate ; Sociomicrobiology ; Α-Proteobacteria ; Bacterial Proteins ; Carrier Proteins ; 4-Butyrolactone -- Analogs & Derivatives ; Alphaproteobacteria -- Physiology ; Biofilms -- Growth & Development ; Quorum Sensing -- Physiology ; Signal Transduction -- Physiology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    In: Nature, 2014, Vol.506(7489), p.489
    Description: What mechanisms underlie the transitions responsible for the diverse shapes observed in the living world? Although bacteria exhibit a myriad of morphologies (1), the mechanisms responsible for the evolution of bacterial cell shape are not understood. We investigated morphological diversity in a group of bacteria that synthesize an appendage-like extension of the cell envelope called the stalk (2,3). The location and number of stalks varies among species, as exemplified by three distinct subcellular positions of stalks within a rod-shaped cell body: polar in the genus Caulobacter and subpolar or bilateral in the genus Asticcacaulis (4). Here we show that a developmental regulator of Caulobacter crescentus, SpmX (5), is co-opted in the genus Asticcacaulis to specify stalk synthesis either at the subpolar or bilateral positions. We also show that stepwise evolution of a specific region of SpmX led to the gain of a new function and localization of this protein, which drove the sequential transition in stalk positioning. Our results indicate that changes in protein function, co-option and modularity are key elements in the evolution of bacterial morphology. Therefore, similar evolutionary principles of morphological transitions apply to both single-celled prokaryotes and multicellular eukaryotes.
    Keywords: Evolution (Biology) – Research ; Bacteria – Physiological Aspects ; Morphology (Biology) – Research;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 3
    In: Molecular Microbiology, May 2013, Vol.88(3), pp.486-500
    Description: Bacterial exopolysaccharide synthesis is a prevalent and indispensible activity in many biological processes, including surface adhesion and biofilm formation. In , surface attachment and subsequent biofilm growth depend on the ability to synthesize an adhesive polar polysaccharide known as the holdfast. In this work, we show that polar polysaccharide synthesis is a conserved phenomenon among lphaproteobacterial species closely related to . Among them, mutagenesis of showed that disruption of the gene, which encodes a putative polysaccharide deacetylase, leads to accumulation of holdfast in the culture supernatant. Examination of the deletion mutant in revealed that this strain synthesizes holdfast; however, like the   mutant, the holdfasts are shed into the medium and have decreased adhesiveness and cohesiveness. Site‐directed mutagenesis at the predicted catalytic site of   phenocopied the mutant and abolished the esterase activity of . In contrast, overexpression of increased cell adherence without increasing holdfast synthesis. We conclude that the polysaccharide deacetylase activity of is required for the adhesive and cohesive properties of the holdfast, as well as for the anchoring of the holdfast to the cell envelope.
    Keywords: Microbial Polysaccharides -- Analysis;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 4
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 31 January 2012, Vol.109(5), pp.1697-701
    Description: Elongation of many rod-shaped bacteria occurs by peptidoglycan synthesis at discrete foci along the sidewall of the cells. However, within the Rhizobiales, there are many budding bacteria, in which new cell growth is constrained to a specific region. The phylogeny of the Rhizobiales indicates that this mode of zonal growth may be ancestral. We demonstrate that the rod-shaped bacterium Agrobacterium tumefaciens grows unidirectionally from the new pole generated after cell division and has an atypical peptidoglycan composition. Polar growth occurs under all conditions tested, including when cells are attached to a plant root and under conditions that induce virulence. Finally, we show that polar growth also occurs in the closely related bacteria Sinorhizobium meliloti, Brucella abortus, and Ochrobactrum anthropi. We find that unipolar growth is an ancestral and conserved trait among the Rhizobiales, which includes important mutualists and pathogens of plants and animals.
    Keywords: Alphaproteobacteria -- Growth & Development ; Rhizobiaceae -- Growth & Development
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 5
    Language: English
    In: Applied and environmental microbiology, 15 August 2016, Vol.82(16), pp.5015-25
    Description: Mechanistic studies of many processes in Agrobacterium tumefaciens have been hampered by a lack of genetic tools for characterization of essential genes. In this study, we used a Tn7-based method for inducible control of transcription from an engineered site on the chromosome. We demonstrate that this method enables tighter control of inducible promoters than plasmid-based systems and can be used for depletion studies. The method enables the construction of depletion strains to characterize the roles of essential genes in A. tumefaciens Here, we used the strategy to deplete the alphaproteobacterial master regulator CtrA and found that depletion of this essential gene results in dramatic rounding of cells, which become nonviable. Agrobacterium tumefaciens is a bacterial plant pathogen and natural genetic engineer. Thus, studies of essential processes, including cell cycle progression, DNA replication and segregation, cell growth, and division, may provide insights for limiting disease or improving biotechnology applications.
    Keywords: Gene Expression Regulation, Bacterial ; Genes, Essential ; Agrobacterium Tumefaciens -- Genetics ; Bacterial Proteins -- Genetics
    ISSN: 00992240
    E-ISSN: 1098-5336
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  • 6
    Language: English
    In: Applied and environmental microbiology, 01 December 2017, Vol.83(23)
    Description: To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogen Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of The coinoculation of with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages. attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative hage eptidoglycan ydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of in the absence of additional phage genes causes a block in cell division and subsequent lysis of cells. When the presumed active site of the -acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic. The characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as , may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The specificity of bacteriophage treatment for the host is an asset in complex communities, such as in orchards where it would be detrimental to harm the symbiotic bacteria in the environment. Second, bacteriophages are potential sources of enzymes that efficiently lyse bacterial cells. These phage proteins may have a broad specificity, but since proteins do not replicate as phages do, their effect is highly localized, providing an alternative to traditional antibiotic treatments. Thus, studies of lytic bacteriophages that infect may provide insights for designing preventative strategies against bacterial pathogens.
    Keywords: Agrobacterium ; Bacteriophage ; Endolysin ; Lysis ; Phage Genomics ; Bacteriolysis ; Gene Expression ; Agrobacterium Tumefaciens -- Physiology ; Bacteriophages -- Enzymology ; N-Acetylmuramoyl-L-Alanine Amidase -- Metabolism ; Viral Proteins -- Metabolism
    ISSN: 00992240
    E-ISSN: 1098-5336
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  • 7
    Language: English
    In: Applied microbiology and biotechnology, July 2018, Vol.102(14), pp.6023-6038
    Description: A novel whole-cell biosensor was developed to noninvasively and simultaneously monitor the in situ genetic activities of the four quorum sensing (QS) networks in Pseudomonas aeruginosa PAO1, including the las, rhl, pqs, and iqs systems. P. aeruginosa PAO1 is a model bacterium for studies of biofilm and pathogenesis while both processes are closely controlled by the QS systems. This biosensor worked well by selectively monitoring the expression of one representative gene from each network. In the biosensor, the promoter regions of lasI, rhlI, pqsA, and ambB (QS genes) controlled the fluorescent reporter genes of Turbo YFP, mTag BFP2, mNEON Green, and E2-Orange, respectively. The biosensor was successful in monitoring the impact of an important environmental factor, salt stress, on the genetic regulation of QS networks. High salt concentrations (≥ 20 g·L) significantly downregulated rhlI, pqsA, and ambB after the biosensor was incubated for 17 h to 18 h at 37 °C, resulting in slow bacterial growth.
    Keywords: Fluorescence ; Gene Expression ; Pseudomonas Aeruginosa Pao1 ; Quorum Sensing ; Salt Stress ; Whole-Cell Biosensor ; Bacterial Proteins -- Genetics ; Biosensing Techniques -- Methods ; Pseudomonas Aeruginosa -- Physiology ; Quorum Sensing -- Genetics
    ISSN: 01757598
    E-ISSN: 1432-0614
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  • 8
    Language: English
    In: Journal of bacteriology, 01 September 2017, Vol.199(17)
    Description: is a rod-shaped bacterium that grows by polar insertion of new peptidoglycan during cell elongation. As the cell cycle progresses, peptidoglycan synthesis at the pole ceases prior to insertion of new peptidoglycan at midcell to enable cell division. The homolog of the polar organelle development protein PopZ has been identified as a growth pole marker and a candidate polar growth-promoting factor. Here, we characterize the function of PopZ in cell growth and division of Consistent with previous observations, we observe that PopZ localizes specifically to the growth pole in wild-type cells. Despite the striking localization pattern of PopZ, we find the absence of the protein does not impair polar elongation or cause major changes in the peptidoglycan composition. Instead, we observe an atypical cell length distribution, including minicells, elongated cells, and cells with ectopic poles. Most minicells lack DNA, suggesting a defect in chromosome segregation. Furthermore, the canonical cell division proteins FtsZ and FtsA are misplaced, leading to asymmetric sites of cell constriction. Together, these data suggest that PopZ plays an important role in the regulation of chromosome segregation and cell division. is a bacterial plant pathogen and a natural genetic engineer. However, very little is known about the spatial and temporal regulation of cell wall biogenesis that leads to polar growth in this bacterium. Understanding the molecular basis of growth may allow for the development of innovations to prevent disease or to promote growth during biotechnology applications. Finally, since many closely related plant and animal pathogens exhibit polar growth, discoveries in may be broadly applicable for devising antimicrobial strategies.
    Keywords: Agrobacterium ; Popz ; Cell Division ; Cell Polarity ; Chromosome Segregation ; Growth Polarity ; Asymmetric Cell Division ; Cell Division ; Chromosome Segregation ; Agrobacterium Tumefaciens -- Cytology ; Bacterial Proteins -- Metabolism ; Cell Cycle Proteins -- Metabolism
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 9
    Language: English
    In: Analytical chemistry, 16 October 2012, Vol.84(20), pp.8571-8
    Description: We report the development of an automated microfluidic "baby machine" to synchronize the bacterium Caulobacter crescentus on-chip and to move the synchronized populations downstream for analysis. The microfluidic device is fabricated from three layers of poly(dimethylsiloxane) and has integrated pumps and valves to control the movement of cells and media. This synchronization method decreases incubation time and media consumption and improves synchrony quality compared to the conventional plate-release technique. Synchronized populations are collected from the device at intervals as short as 10 min and at any time over four days. Flow cytometry and fluorescence cell tracking are used to determine synchrony quality, and cell populations synchronized in minimal growth medium with 0.2% glucose (M2G) and peptone yeast extract (PYE) medium contain 〉70% and 〉80% swarmer cells, respectively. Our on-chip method overcomes limitations with conventional physical separation methods that consume large volumes of media, require manual manipulations, have lengthy incubation times, are limited to one collection, and lack precise temporal control of collection times.
    Keywords: Lab-On-A-Chip Devices ; Caulobacter Crescentus -- Growth & Development ; Microfluidic Analytical Techniques -- Instrumentation
    ISSN: 00032700
    E-ISSN: 1520-6882
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  • 10
    Language: English
    In: Analytical chemistry, 15 December 2015, Vol.87(24), pp.12032-9
    Description: In the environment, most bacteria form surface-attached cell communities called biofilms. The attachment of single cells to surfaces involves an initial reversible stage typically mediated by surface structures such as flagella and pili, followed by a permanent adhesion stage usually mediated by polysaccharide adhesives. Here, we determine the absolute and relative timescales and frequencies of reversible and irreversible adhesion of single cells of the bacterium Caulobacter crescentus to a glass surface in a microfluidic device. We used fluorescence microscopy of C. crescentus expressing green fluorescent protein to track the swimming behavior of individual cells prior to adhesion, monitor the cell at the surface, and determine whether the cell reversibly or irreversibly adhered to the surface. A fluorescently labeled lectin that binds specifically to polar polysaccharides, termed holdfast, discriminated irreversible adhesion events from reversible adhesion events where no holdfast formed. In wild-type cells, the holdfast production time for irreversible adhesion events initiated by surface contact (23 s) was 30-times faster than the holdfast production time that occurs through developmental regulation (13 min). Irreversible adhesion events in wild-type cells (3.3 events/min) are 15-times more frequent than in pilus-minus mutant cells (0.2 events/min), indicating the pili are critical structures in the transition from reversible to irreversible surface-stimulated adhesion. In reversible adhesion events, the dwell time of cells at the surface before departing was the same for wild-type cells (12 s) and pilus-minus mutant cells (13 s), suggesting the pili do not play a significant role in reversible adhesion. Moreover, reversible adhesion events in wild-type cells (6.8 events/min) occur twice as frequently as irreversible adhesion events (3.3 events/min), demonstrating that most cells contact the surface multiple times before transitioning from reversible to irreversible adhesion.
    Keywords: Bacterial Adhesion ; Microfluidic Analytical Techniques ; Caulobacter Crescentus -- Metabolism
    ISSN: 00032700
    E-ISSN: 1520-6882
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