Showing papers in "Journal of Bacteriology in 2000"
TL;DR: In most natural environments, association with a surface in a structure known as a biofilm is the prevailing microbial lifestyle and is an efficient means of lingering in a favorable microenvironment rather than being swept away by the current.
Abstract: In most natural environments, association with a surface in a structure known as a biofilm is the prevailing microbial lifestyle. Surface association is an efficient means of lingering in a favorable microenvironment rather than being swept away by the current. Taken to the extreme, we may view the
1,455 citations
TL;DR: Interest in the biodegradation mechanisms and environmental fate of polycyclic aromatic hydrocarbons is prompted by their ubiquitous distribution and their potentially deleterious effects on human health.
Abstract: Interest in the biodegradation mechanisms and environmental fate of polycyclic aromatic hydrocarbons (PAHs) is prompted by their ubiquitous distribution and their potentially deleterious effects on human health. PAHs constitute a large and diverse class of organic compounds and are generally
1,125 citations
TL;DR: Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.
Abstract: We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.
819 citations
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TL;DR: Cell walls are an important structural component of prokaryotic organisms and essential for many aspects of their life, and the diverse structures of the outermost boundary layers strongly reflect adaptations to specific ecological and environmental conditions.
Abstract: Cell walls are an important structural component of prokaryotic organisms and essential for many aspects of their life. Particularly, the diverse structures of the outermost boundary layers strongly reflect adaptations of organisms to specific ecological and environmental conditions ([6][1]).
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757 citations
TL;DR: A novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci is presented and six genetically distinct groups that appear to be derived from clones are identified.
Abstract: Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC1, vrrC2, vrrB1, vrrB2, and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.
720 citations
TL;DR: A simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination is found.
Abstract: Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 20 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments has been visualized by dark-field or differential-interference-contrast microscopy, methods hampered by intense scattering from the cell body or shallow depth of field, respectively. We have found a simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination. We report our initial findings with cells of Escherichia coli. Tumbles (events that enable swimming cells to alter course) are remarkably varied. Not every filament on a cell needs to change its direction of rotation: different filaments can change directions at different times, and a tumble can result from the change in direction of only one. Polymorphic transformations tend to occur in the sequence normal, semicoiled, curly 1, with changes in the direction of movement of the cell body correlated with transformations to the semicoiled form.
698 citations
TL;DR: Surprisingly, it is found that colanic acid production is not required for surface attachment, which is critical for the formation of the complex three-dimensional structure and depth of E. coli biofilms.
Abstract: Although exopolysaccharides (EPSs) are a large component of bacterial biofilms, their contribution to biofilm structure and function has been examined for only a few organisms. In each of these cases EPS has been shown to be required for cellular attachment to abiotic surfaces. Here, we undertook a genetic approach to examine the potential role of colanic acid, an EPS of Escherichia coli K-12, in biofilm formation. Strains either proficient or deficient in colanic acid production were grown and allowed to adhere to abiotic surfaces and were then examined both macroscopically and microscopically. Surprisingly, we found that colanic acid production is not required for surface attachment. Rather, colanic acid is critical for the formation of the complex three-dimensional structure and depth of E. coli biofilms.
663 citations
TL;DR: Galleria mellonella is a good model system for identifying mammalian virulence factors of P. aeruginosa and a novel positive correlation in the virulence patterns was found.
Abstract: Strain PA14, a human clinical isolate of Pseudomonas aeruginosa, is pathogenic in mice and insects (Galleria mellonella). Analysis of 32 different PA14 mutants in these two hosts showed a novel positive correlation in the virulence patterns. Thus, G. mellonella is a good model system for identifying mammalian virulence factors of P. aeruginosa.
525 citations
TL;DR: This study indicates that some strains of V. paradoxus degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources.
Abstract: Acyl-homoserine lactones (acyl-HSLs) serve as dedicated cell-to-cell signaling molecules in many species of the class Proteobacteria. We have addressed the question of whether these compounds can be degraded biologically. A motile, rod-shaped bacterium was isolated from soil based upon its ability to utilize N-(3-oxohexanoyl)-L-homoserine lactone as the sole source of energy and nitrogen. The bacterium was classified as a strain of Variovorax paradoxus. The V. paradoxus isolate was capable of growth on all of the acyl-HSLs tested. The molar growth yields correlated with the length of the acyl group. HSL, a product of acyl-HSL metabolism, was used as a nitrogen source, but not as an energy source. Cleavage and partial mineralization of the HSL ring were demonstrated by using radiolabeled substrate. This study indicates that some strains of V. paradoxus degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources. This study provides clues about the metabolic pathway of acyl-HSL degradation by V. paradoxus.
481 citations
TL;DR: Molecular analyses of biofilm-defective mutants of S. gordonii Challis showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion, which are associated with quorum sensing, osmoadaptation, andAdhesion functions in oral streptococci.
Abstract: Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of S. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of S. gordonii Challis were identified based on Southern hybridization with a Tn916-based probe and DNA sequences of the Tn916-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.
479 citations
TL;DR: The initiation of transcription is a complex process involving many different steps, and many have been exploited by bacteria to give rise to sophisticated regulatory mechanisms that allow the cell to adapt to changing environment.
Abstract: The initiation of transcription is a complex process involving many different steps. These steps are all potential control points for regulating gene expression, and many have been exploited by bacteria to give rise to sophisticated regulatory mechanisms that allow the cell to adapt to changing
TL;DR: Most modern reviews about bacterial cell wall properties use the traditional classification of prokaryotic envelopes as either gram positive or gram negative, but both envelope types show a characteristic difference.
Abstract: Most modern reviews about bacterial cell wall properties use the traditional classification of prokaryotic envelopes as either gram positive or gram negative ([27][1], [30][2]). Mainly based on the variance of their peptidoglycan architecture, both envelope types show a characteristic difference
TL;DR: Cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to visualize CL distribution in Escherichia coli cells of different phospholipid compositions and revealed NAO-binding domains in the plane of the cell membrane.
Abstract: Cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to visualize CL distribution in Escherichia coli cells of different phospholipid compositions. In a filamentous mutant containing only anionic phospholipids, green fluorescent spots were observed along the filaments at approximately regular intervals. Three-dimensional image reconstruction obtained by optical sectioning and a deconvolution algorithm revealed NAO-binding domains in the plane of the cell membrane. Substantial red fluorescence emission of bound NAO supported labeling of CL-containing domains. These structures were not found in mutants deficient in CL biosynthesis. The domains were also observed mostly in the septal region and on the poles in cells of normal size with wild-type phospholipid composition.
TL;DR: It is found that in P. aeruginosa, PQS caused a major induction of rhlI'-lacZ and had lesser effects on the transcription of lasR'-lACZ and rhlR'- lacZ, which implies that PQs acts as a link between the las and rhL quorum-sensing systems and that this signal is not involved in sensing cell density.
Abstract: The opportunistic pathogen Pseudomonas aeruginosa uses intercellular signals to control the density-dependent expression of many virulence factors. The las and rhl quorum-sensing systems function, respectively, through the autoinducers N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone (C(4)-HSL), which are known to positively regulate the transcription of the elastase-encoding gene, lasB. Recently, we reported that a second type of intercellular signal is involved in lasB induction. This signal was identified as 2-heptyl-3-hydroxy-4-quinolone and designated the Pseudomonas quinolone signal (PQS). PQS was determined to be part of the quorum-sensing hierarchy since its production and bioactivity depended on the las and rhl quorum-sensing systems, respectively. In order to define the role of PQS in the P. aeruginosa quorum-sensing cascade, lacZ gene fusions were used to determine the effect of PQS on the transcription of the quorum-sensing system genes lasR, lasI, rhlR, and rhlI. We found that in P. aeruginosa, PQS caused a major induction of rhlI'-lacZ and had lesser effects on the transcription of lasR'-lacZ and rhlR'-lacZ. We also observed that the transcription of both rhlI'-lacZ and lasB'-lacZ was cooperatively effected by C(4)-HSL and PQS. Additionally, we present data indicating that PQS was not produced maximally until cultures reached the late stationary phase of growth. Taken together, our results imply that PQS acts as a link between the las and rhl quorum-sensing systems and that this signal is not involved in sensing cell density.
TL;DR: GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding in Escherichia coli, suggesting that GFP is improperly folded when localized outside of the cytoplasm.
Abstract: The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.
TL;DR: Analysis of the resistance of wild-type, Deltagers3, and Deltager3 spoVF spores to various agents led to the following conclusions: DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; an elevated coreWater content is associated with decreased spore resistant to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine.
Abstract: Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed Deltager3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than Deltager3 spores. The Deltager3 spoVF spores lack DPA and have higher levels of core water than Deltager3 spores, although sporulation with DPA restores close to normal levels of DPA and core water to Deltager3 spoVF spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The Deltager3 spoVF spores also contain minimal levels of the processed active form (termed P(41)) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P(41) formed in Deltager3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-gamma. Analysis of the resistance of wild-type, Deltager3, and Deltager3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than Deltager3 spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.
TL;DR: The genus Agrobacterium has provided excellent models for several aspects of host-pathogen interactions, including intercellular transport of macromolecules, bacterial detection of host organisms, targeting of proteins to plant cell nuclei, and interbacterial chemical signaling via autoinducer-type pheromones.
Abstract: The nine decades since Smith and Townsend demonstrated that Agrobacterium tumefaciens causes plant tumors (95) have been marked by a series of surprises. Among the most important of these was the report in 1958 that these tumors could be excised and propagated in vitro without exogenous plant hormones (7). Equally important were a series of reports beginning about the same time that tumors released compounds that agrobacteria could use as nutrients (24). Perhaps the most exciting discoveries, reported in the 1970s and 1980s, were that tumorigenesis required the transfer of fragments of oncogenic DNA to infected plant cells (10), that this process evolved from a conjugal transfer system (99), and that the genes that direct this process are expressed in response to host-released chemical signals (47). This DNA transfer process has become a cornerstone of plant molecular genetics. The genus Agrobacterium also has provided excellent models for several aspects of host-pathogen interactions, including intercellular transport of macromolecules (11), bacterial detection of host organisms (47), targeting of proteins to plant cell nuclei (3), and interbacterial chemical signaling via autoinducer-type pheromones (120).
TL;DR: The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described, and has been associated with iron transport and metabolism.
Abstract: In Escherichia coli, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the mar regulon, duplicate macroarrays containing 4,290 open reading frames of the E. coli genome were hybridized to radiolabeled cDNA populations derived from mar-deleted and mar-expressing E. coli. Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the soxRS regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described.
TL;DR: This study is the first to show that the capacity for bioluminescence is critical for normal cell-cell interactions between a bacterium and its animal host and presents the first examples of V. fischeri genes that affect normal host tissue development.
Abstract: The bioluminescent bacterium Vibrio fischeri and juveniles of the squid Euprymna scolopes specifically recognize and respond to one another during the formation of a persistent colonization within the host's nascent light-emitting organ. The resulting fully developed light organ contains brightly luminescing bacteria and has undergone a bacterium-induced program of tissue differentiation, one component of which is a swelling of the epithelial cells that line the symbiont-containing crypts. While the luminescence (lux) genes of symbiotic V. fischeri have been shown to be highly induced within the crypts, the role of these genes in the initiation and persistence of the symbiosis has not been rigorously examined. We have constructed and examined three mutants (luxA, luxI, and luxR), defective in either luciferase enzymatic or regulatory proteins. All three are unable to induce normal luminescence levels in the host and, 2 days after initiating the association, had a three- to fourfold defect in the extent of colonization. Surprisingly, these lux mutants also were unable to induce swelling in the crypt epithelial cells. Complementing, in trans, the defect in light emission restored both normal colonization capability and induction of swelling. We hypothesize that a diminished level of oxygen consumption by a luciferase-deficient symbiotic population is responsible for the reduced fitness of lux mutants in the light organ crypts. This study is the first to show that the capacity for bioluminescence is critical for normal cell-cell interactions between a bacterium and its animal host and presents the first examples of V. fischeri genes that affect normal host tissue development.
TL;DR: Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven, suggesting that biofilm bacteria are in a physiological state different from planktonic bacteria.
Abstract: Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.
TL;DR: Long-term survival of the rel(Mtb) mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type, and the mutant was significantly less able to survive extended anaerobic incubation than the wild-type virulent organism.
Abstract: The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (RelMtb) and responds to nutrient starvation by producing (p)ppGpp. A relMtb knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The relMtb mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar RelMtb-independent manner. In vitro growth in liquid media represents a condition that benefits from RelMtb-mediated adaptation. Long-term survival of the relMtb mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anerobic incubation than the wild-type virulent organism. Thus, the RelMtb protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.
TL;DR: The protein-protein interactions involved in chemotactic signaling, including interactions within the supramolecular complexes, interactions between the complexes and the messenger protein CheY, and interactions between CheY and the proteins that regulate its signaling state are reviewed.
Abstract: Chemotaxis is a mechanism by which bacteria efficiently and rapidly respond to changes in the chemical composition of their environment, approaching chemically favorable environments and avoiding unfavorable ones. This behavior is achieved by integrating signals received from receptors that sense the environment and modulating the direction of flagellar rotation accordingly (for reviews, see references 39, 43, and 100). Early studies in the modern era, initiated some 4 decades ago (1), uncovered the behavioral response of cells to changes in the chemical composition of their environment and the correlation between flagellar rotation and the swimming mode of the cells. They also identified most of the gene products involved in chemotaxis (for reviews, see references 50 and 56). The mode of signal transduction began to be understood only in the mid-1980s, when the possibilities of electrical signaling and a direct interaction between the receptors and flagella were eliminated (for a review, see reference 41). The possibility of indirect interaction between the receptors and flagella via a protein that is activated by the receptors and inactivated as it diffuses through the cytoplasm was then raised (96). Subsequently, sequential transient phosphorylation of chemotaxis proteins was found to be a key process in signal transduction (for a review, see reference 25). During the last decade, it was established that the signal in bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium is transduced via protein-protein interactions. These interactions have been extensively studied, contributing greatly to the elucidation of the chemotaxis-signaling cascade. The chemotactic response in bacteria such as E. coli and Salmonella serovar Typhimurium is accomplished by signal transmission between two supramolecular complexes—the receptor complexes, located mainly at the pole(s) of the cell, and the flagellar-motor complexes (usually 5 to 10 complexes per cell), randomly distributed around the cell and embedded within the cell membrane. A messenger protein, CheY, shuttles back and forth between the complexes and transduces the signal from the receptors to the flagella (Fig. 1). The interaction of this messenger protein with the flagellar-motor supramolecular complex increases the probability of shifting the direction of flagellar rotation from the default direction, counterclockwise (CCW), to clockwise (CW) (for a review see reference 38). The consequence of CW rotation is an abrupt turning motion (tumbling), after which (when the default direction resumes) the cell swims in a new direction. Here we review the protein-protein interactions involved in chemotactic signaling, including interactions within the supramolecular complexes, interactions between the complexes and the messenger protein CheY, and interactions between CheY and the proteins that regulate its signaling state. Interactions involved in the signaling pathway leading to adaptation will also be reviewed. We will mainly focus on functional aspects of the interactions. The reader is referred to references 13, 35, 43, 54, and 81 for more-detailed structural aspects. Because this is a minireview, the reference list is incomplete. Whenever possible, reference is made to reviews or papers that provide access to the original literature.
TL;DR: P. putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities and subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds.
Abstract: Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.
TL;DR: It is proposed that nutritional cues are integrated by Crc as part of a signal transduction pathway that regulates biofilm development and is similar to that observed in mutants defective in type IV pilus biogenesis.
Abstract: The transition from a planktonic (free-swimming) existence to growth attached to a surface in a biofilm occurs in response to environmental factors, including the availability of nutrients. We show that the catabolite repression control (Crc) protein, which plays a role in the regulation of carbon metabolism, is necessary for biofilm formation in Pseudomonas aeruginosa. Using phase-contrast microscopy, we found that a crc mutant only makes a dispersed monolayer of cells on a plastic surface but does not develop the dense monolayer punctuated by microcolonies typical of the wild-type strain. This is a phenotype identical to that observed in mutants defective in type IV pilus biogenesis. Consistent with this observation, crc mutants are defective in type IV pilus-mediated twitching motility. We show that this defect in type IV pilus function is due (at least in part) to a decrease in pilA (pilin) transcription. We propose that nutritional cues are integrated by Crc as part of a signal transduction pathway that regulates biofilm development.
TL;DR: Two chromosomal genes are identified from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions that is required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco.
Abstract: Using a genetic screen we have identified two chromosomal genes, cusRS (ylcA ybcZ), from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive gene regulation: PcoR and PcoS from the pco operon of E. coli; CopR and CopS from the cop operon, which provides copper resistance to Pseudomonas syringae; and SilR and SilS from the sil locus, which provides silver ion resistance to Salmonella enterica serovar Typhimurium. The genes cusRS are also required for the copper-dependent expression of at least one chromosomal gene, designated cusC (ylcB), which is allelic to the recently identified virulence gene ibeB in E. coli K1. The cus locus may comprise a copper ion efflux system, because the expression of cusC is induced by high concentrations of copper ions. Furthermore, the translation products of cusC and additional downstream genes are homologous to known metal ion antiporters.
TL;DR: It is concluded that the type III secretion system of SPI-2 is regulated by OmpR, which activates expression of ssrA soon after Salmonella enters the macrophage.
Abstract: Salmonella pathogenicity island 2 (SPI-2) encodes a putative, two-component regulatory system, SsrA-SsrB, which regulates a type III secretion system needed for replication inside macrophages and systemic infection in mice. The sensor and regulator homologs, ssrAB (spiR), and genes within the secretion system, including the structural gene ssaH, are transcribed after Salmonella enters host cells. We have studied the transcriptional regulation of ssrAB and the secretion system by using gfp fusions to the ssrA and ssaH promoters. We found that early transcription of ssrA, after entry into macrophages, is most efficient in the presence of OmpR. An ompR mutant strain does not exhibit replication within cultured macrophages. Furthermore, footprint analysis shows that purified OmpR protein binds directly to the ssrA promoter region. We also show that minimal medium, pH 4.5, induces SPI-2 gene expression in wild-type but not ompR mutant strains. We conclude that the type III secretion system of SPI-2 is regulated by OmpR, which activates expression of ssrA soon after Salmonella enters the macrophage.
TL;DR: Results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites, which depends on phlF function, and may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.
Abstract: The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.
TL;DR: PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes to Salmonella resistance to innate immunity and may be the mechanism of OmpT-mediated resistance to alpha-CAMPs.
Abstract: The outer membrane protein contents of Salmonella enterica serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of the Escherichia coli OmpT and Yersinia pestis Pla family of outer membrane proteases. Salmonella PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (alpha-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue alpha-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to alpha-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes to Salmonella resistance to innate immunity.
TL;DR: A fourth agr specificity group among S. aureus strains is demonstrated and it is shown that most exfoliatin-producing strains belong to this group, which raises the question of the biological significance of the agr autoinduction threshold.
Abstract: The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD. A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non-aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.
TL;DR: It is suggested that the transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.
Abstract: FtsZ assembles in vitro into protofilaments that can adopt two conformations—the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24°. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.