Fusaric acid is produced by pathogenic fungi of the genus Fusarium, and is toxic to plants and rhizobacteria. Many fluorescent pseudomonads can prevent wilt diseases caused by these fungi. This study was undertaken to evaluate the effect of fusaric acid on P. protegens Pf-5 and elucidate the mechanisms that enable the bacterium to survive in the presence of the mycotoxin. The results confirm that fusaric acid negatively affects growth and motility of P. protegens. Moreover, a notable increase in secretion of the siderophore pyoverdine was observed when P. protegens was grown in the presence of fusaric acid. Concomitantly, levels of enzymes involved in the biosynthesis of pyoverdine and enantio-pyochelin, the second siderophore encoded by P. protegens, increased markedly. Moreover, while similar levels of resistance to fusaric acid were observed for P. protegens mutants unable to synthesize either pyoverdine or enanto-pyochelin and the wild type strain, a double mutant unable to synthesize both kinds of siderophores showed a dramatically reduced resistance to this compound. This reduced resistance was not observed when this mutant was grown under conditions of iron excess. Spectrophotometric titrations revealed that fusaric acid binds not only Fe2+ and Fe3+, but also Zn2+, Mn2+ and Cu2+, with high affinity. Our results demonstrate that iron sequestration accounts at least in part for the deleterious effect of the mycotoxin on P. protegens.
Since its inception, Bacterial Locomotion and Signal Transduction (BLAST) meetings have been the place to exchange and share the latest developments in the field of bacterial signal transduction and motility. At the 12th BLAST meeting, held last January in Tucson AZ, researchers from all over the world met to report and discuss progress in diverse aspects of the field. The majority of these advances, however, came at the level of atomic level structures and their associated mechanisms. This was especially true of the biological machines that sense and respond to environmental changes.
When carbon sources become limiting for growth, bacteria must choose which of the remaining nutrients should be used first. We have identified a nutrient-sensing signaling network in Escherichia coli that is activated at the transition to stationary phase. The network is composed of the two histidine kinase/response regulator systems YehU/YehT and YpdA/YpdB and their target proteins, YjiY and YhjX (both of which are membrane-integrated transporters). The peptide/amino acid-responsive YehU/YehT system was found to have a negative effect on expression of the target gene, yhjX, of the pyruvate-responsive YpdA/YpdB system, while the YpdA/YpdB system stimulated expression of yjiY, the target of the YehU/YehT system. These effects were confirmed in mutants lacking any of the genes for the three primary components of either system. Furthermore, an in vivo interaction assay based on bacterial adenylate cyclase detected heteromeric interactions between the membrane-bound components of the two systems, specifically, between the two histidine kinases and the two transporters, which is compatible with the formation of a larger signaling unit. Finally, the carbon storage regulator A (CsrA) was shown to be involved in posttranscriptional regulation of both yjiY and yhjX.
The polymerization of amino acids into proteins occurs on ribosomes, with the rate influenced by the amino acids being polymerized. The imino acid proline is a poor donor and acceptor for peptide-bond formation, such that translational stalling occurs when three or more consecutive prolines (PPP) are encountered by the ribosome. In bacteria, stalling at PPP motifs is rescued by the elongation factor P (EF-P). Using SILAC mass spectrometry of Escherichia coli strains, we identified a subset of PPP-containing proteins for which the expression patterns remained unchanged or even appeared up-regulated in the absence of EF-P. Subsequent analysis using in vitro and in vivo reporter assays revealed that stalling at PPP motifs is influenced by the sequence context upstream of the stall site. Specifically, the presence of amino acids such as Cys and Thr preceding the stall site suppressed stalling at PPP motifs, whereas amino acids like Arg and His promoted stalling. In addition to providing fundamental insight into the mechanism of peptide-bond formation, our findings suggest how the sequence context of polyproline-containing proteins can be modulated to maximize the efficiency and yield of protein production.
KdpD and KdpE form a histidine kinase/response regulator system that senses K+ limitation and induces the kdpFABC operon, which encodes a high-affinity K+ uptake complex. To define the primary stimulus perceived by KdpD we focused in this study on the dynamics of the Kdp response. Escherichia coli cells were subjected to severe K+ limitation, and all relevant parameters of the Kdp response, i.e., levels of kdpFABC transcripts and KdpFABC proteins, as well as extra- and intracellular K+ concentrations, were quantitatively analysed over time (0 to 180 min). Unexpectedly, induction of kdpFABC was found to follow a non-monotonic time-course. To interpret this unusual behaviour, a mathematical model that adequately captures the dynamics of the Kdp system was established and used for simulations. We found a strong correlation between KdpD/KdpE activation and the intracellular K+ concentration, which is influenced by the uptake of K+ via the KdpFABC complex. Based on these results a model is proposed in which KdpD/KdpE phosphorylation is inversely correlated with the intracellular K+ concentration. To corroborate this hypothesis an isogenic mutant that produces a defective KdpFABC complex, and the trans-complemented mutant that expresses the KtrAB high-affinity K+ uptake system of Vibrio alginolyticus were quantitatively analysed. Experimental data and simulations for the mutants consistently support the tight correlation between KdpD/KdpE activation and the intracellular K+ concentration. This study presents a striking example of the non-intuitive dynamics of a functional unit comprising signalling proteins and a transporter with K+ as mediator.
Inducible switching between phenotypes is a common strategy of bacteria to adapt to fluctuating environments. Here, we analyze the switching kinetics of a paradigmatic inducible system, the arabinose utilization system in E. coli. Using time-lapse fluorescence microscopy of microcolonies in a microfluidic chamber, which permits sudden up- and down-shifts in the inducer arabinose, we characterize the single-cell gene expression dynamics of the araBAD operon responsible for arabinose degradation. While there is significant, inducer-dependent cell-to-cell variation in the timing of the on-switching, the off-switching triggered by sudden removal of arabinose is homogeneous and rapid. We find that rapid off-switching does not depend on internal arabinose degradation. Because the system is regulated via the internal arabinose level sensed by AraC, internal arabinose must be rapidly depleted by leakage or export from the cell, or by degradation via a non-canonical pathway. We explored whether the poorly characterized membrane protein AraJ, which is part of the arabinose regulon and has been annotated as a possible arabinose efflux protein, is responsible for rapid depletion. However, we find that AraJ is not essential for rapid switching to the off-state. We develop a mathematical model for the arabinose system, which quantitatively describes both the heterogeneous on-switching and the homogeneous off-switching. The model also predicts that mutations which disrupt the positive feedback of internal arabinose on the production of arabinose uptake proteins change the heterogeneous on-switching behavior into a homogeneous, graded response. We construct such a mutant and confirm the graded response experimentally. Taken together, our results indicate that the physiological switching behavior of this sugar utilization system is asymmetric, such that off-switching is always rapid and homogeneous, while on-switching is slow and heterogeneously timed at sub-saturating inducer levels.
Phylogeny, ecology, and sensorial constraints are thought to be the most important factors influencing echolocation call design in bats. The Molossidae is a diverse bat family with a majority of species restricted to tropical and subtropical regions. Most molossids are specialized to forage for insects in open space, and thus share similar navigational challenges. We use an unprecedented dataset on the echolocation calls of 8 genera and 18 species of New World molossids to explore how habitat, phylogenetic relatedness, body mass, and prey perception contribute to echolocation call design. Our results confirm that, with the exception of the genus Molossops, echolocation calls of these bats show a typical design for open space foraging. Two lines of evidence point to echolocation call structure of molossids reflecting phylogenetic relatedness. First, such structure is significantly more similar within than among genera. Second, except for allometric scaling, such structure is nearly the same in congeneric species. Despite contrasting body masses, 12 of 18 species call within a relatively narrow frequency range of 20 to 35 kHz, a finding that we explain by using a modeling approach whose results suggest this frequency range to be an adaptation optimizing prey perception in open space. To conclude, we argue that the high variability in echolocation call design of molossids is an advanced evolutionary trait allowing the flexible adjustment of echolocation systems to various sensorial challenges, while conserving sender identity for social communication. Unraveling evolutionary drivers for echolocation call design in bats has so far been hampered by the lack of adequate model organisms sharing a phylogenetic origin and facing similar sensorial challenges. We thus believe that knowledge of the echolocation call diversity of New World molossid bats may prove to be landmark to understand the evolution and functionality of species-specific signal design in bats.
Bat echolocation is primarily used for orientation and foraging but also holds great potential for social communication. The communicative function of echolocation calls is still largely unstudied, especially in the wild. Eavesdropping on vocal signatures encoding social information in echolocation calls has not, to our knowledge, been studied in free-living bats so far. We analysed echolocation calls of the polygynous bat Saccopteryx bilineata and found pronounced vocal signatures encoding sex and individual identity. We showed experimentally that free-living males discriminate approaching male and female conspecifics solely based on their echolocation calls. Males always produced aggressive vocalizations when hearing male echolocation calls and courtship vocalizations when hearing female echolocation calls; hence, they responded with complex social vocalizations in the appropriate social context. Our study demonstrates that social information encoded in bat echolocation calls plays a crucial and hitherto underestimated role for eavesdropping conspecifics and thus facilitates social communication in a highly mobile nocturnal mammal.
eavesdropping; sex-specific signature; individual signature; Saccopteryx bilineata; vocal communication; Chiroptera
We assessed the behavioral flexibility of the trawling long-legged bat, Macrophyllum macrophyllum (Phyllostomidae) in flight cage experiments by exposing it to prey suspended from nylon threads in the air and to food placed onto the water surface at varying distances to clutter-producing background (water plants). The bat revealed flexibility in foraging mode and caught prey in the air (aerial hawking) and from the water surface (trawling). M. macrophyllum was constrained in finding food very near to and within clutter. As echolocation was the prime sensory mode used by M. macrophyllum for detection and localization of food, the bat might have been unable to perceive sufficient information from prey near clutter as background echoes from the water plant increasingly overlapped with echoes from food. The importance of echolocation for foraging is reflected in a stereotypic call pattern of M. macrophyllum that resembles other aerial insectivorous and trawling bats with a pronounced terminal phase (buzz) prior to capture attempts. Our findings contrast studies of other phyllostomid bats that glean prey very near or from vegetation, often using additional sensory cues, such as prey-produced noise, to find food and that lack a terminal phase in echolocation behavior. In M. macrophyllum, acoustic characteristics of its foraging habitat have shaped its sonar system more than phylogeny.
sensory ecology; aerial hawking; gleaning; bat echolocation; clutter; echo overlap
The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we report on one of the rare examples where Gram-positive bacteria, the ‘Staphylococcus intermedius group’ of zoonotic pathogens, excrete two compounds in millimolar concentrations that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini. They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. In vitro studies with the N-acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds caused opposite effects on phosphorylation to those caused by AHL. This explains the quorum quenching activity. Staphylococcal strains producing yayurea A and B clearly benefit from an increased competitiveness in a mixed community.
While studying the potential interaction of staphylococci with Gram-negative bacteria, we came across another communication system in a Staphylococcus species group, which consists of closely related coagulase-positive bacterial species that play a role as zoonotic pathogens. We found that these species excrete two small compounds that inhibit both the expression of QS-controlled toxins and other QS-regulated compounds as well as growth in Gram-negative bacteria. The excreted compounds, which we named yayurea A and B, were isolated from S. delphini and structurally characterized. They represent new bacterial products, which quench the QS regulation in a wide spectrum of Gram-negative bacteria by stimulating the LuxN-mediated phosphorylation of LuxU. Furthermore, growth of yayurea A and B producing S. delphini is not suppressed by respiratory toxins when co-cultured with P. aeruginosa. This suggests that the quorum quenchers have a function in self-protection and competitiveness in natural environments shared with Gram-negatives. Here we show one of the rare cases of inter-phylum interference between firmicutes (Gram-positive) and beta-/gammaproteobacteria (Gram-negative).
Escherichia coli contains 30 two-component systems (TCSs), each consisting of a histidine kinase and a response regulator. Whereas most TCSs are well characterized in this model organism, little is known about the YpdA/YpdB system. To identify YpdB-regulated genes, we compared the transcriptomes of E. coli cells overproducing either YpdB or a control protein. Expression levels of 15 genes differed by more than 1.9-fold between the two strains. A comprehensive evaluation of these genes identified yhjX as the sole target of YpdB. Electrophoretic mobility shift assays with purified YpdB confirmed its interaction with the yhjX promoter. Specifically, YpdB binds to two direct repeats of the motif GGCATTTCAT separated by an 11-bp spacer in the yhjX promoter. yhjX encodes a cytoplasmic membrane protein of unknown function that belongs to the major facilitator superfamily of transporters. Finally, we characterized the pattern of yhjX expression and identified extracellular pyruvate as a stimulus for the YpdA/YpdB system. It is suggested that YpdA/YpdB contributes to nutrient scavenging before entry into stationary phase.
Gleaning insectivorous bats that forage by using echolocation within dense forest vegetation face the sensorial challenge of acoustic masking effects. Active perception of silent and motionless prey in acoustically cluttered environments by echolocation alone has thus been regarded impossible. The gleaning insectivorous bat Micronycteris microtis however, forages in dense understory vegetation and preys on insects, including dragonflies, which rest silent and motionless on vegetation. From behavioural experiments, we show that M. microtis uses echolocation as the sole sensorial modality for successful prey perception within a complex acoustic environment. All individuals performed a stereotypical three-dimensional hovering flight in front of prey items, while continuously emitting short, multi-harmonic, broadband echolocation calls. We observed a high precision in target localization which suggests that M. microtis perceives a detailed acoustic image of the prey based on shape, surface structure and material. Our experiments provide, to our knowledge, the first evidence that a gleaning bat uses echolocation alone for successful detection, classification and precise localization of silent and motionless prey in acoustic clutter. Overall, we conclude that the three-dimensional hovering flight of M. microtis in combination with a frequent emission of short, high-frequency echolocation calls is the key for active prey perception in acoustically highly cluttered environments.
prey perception; echolocation; acoustic clutter; narrow-space active gleaning insectivorous bat; Micronycteris microtis
Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes.
Two-component systems (TCSs) consisting of a membrane-anchored histidine kinase (HK) and a response regulator (RR) with DNA-binding activity. are major players in signal transduction in prokaryotes. Whereas most TCSs in Escherichia coli are well characterized, almost nothing is known about the LytS-like HK YehU and the corresponding LytTR-like RR YehT. To identify YehT-regulated genes, we compared the transcriptomes of E. coli cells overproducing either YehT or the RR KdpE (control). The expression levels of 32 genes differed more than 8-fold between the two strains. A comprehensive evaluation of these genes identified yjiY as a target of YehT. Electrophoretic mobility shift assays with purified YehT confirmed that YehT interacts directly with the yjiY promoter. Specifically, YehT binds to two direct repeats of the motif ACC(G/A)CT(C/T)A separated by a 13-bp spacer in the yjiY promoter. The target gene yjiY encodes an inner membrane protein belonging to the CstA superfamily of transporters. In E. coli cells growing in media containing peptides or amino acids as a carbon source, yjiY is strongly induced at the onset of the stationary-growth phase. Moreover, expression was found to be dependent on cyclic AMP (cAMP)/cAMP receptor protein (CRP). It is suggested that YehU/YehT participates in the stationary-phase control network.
Quorum sensing regulates cell density-dependent phenotypes and involves the synthesis, excretion and detection of so-called autoinducers. Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbellii), one of the best-characterized model organisms for the study of quorum sensing, produces and responds to three autoinducers. HAI-1, AI-2 and CAI-1 are recognized by different receptors, but all information is channeled into the same signaling cascade, which controls a specific set of genes. Here we examine temporal variations of availability and concentration of the three autoinducers in V. harveyi, and monitor the phenotypes they regulate, from the early exponential to the stationary growth phase in liquid culture. Specifically, the exponential growth phase is characterized by an increase in AI-2 and the induction of bioluminescence, while HAI-1 and CAI-1 are undetectable prior to the late exponential growth phase. CAI-1 activity reaches its maximum upon entry into stationary phase, while molar concentrations of AI-2 and HAI-1 become approximately equal. Similarly, autoinducer-dependent exoproteolytic activity increases at the transition into stationary phase. These findings are reflected in temporal alterations in expression of the luxR gene that encodes the master regulator LuxR, and of four autoinducer-regulated genes during growth. Moreover, in vitro phosphorylation assays reveal a tight correlation between the HAI-1/AI-2 ratio as input and levels of receptor-mediated phosphorylation of LuxU as output. Our study supports a model in which the combinations of autoinducers available, rather than cell density per se, determine the timing of various processes in V. harveyi populations.
Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells.
Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon) were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection) and exoproteolytic activity (fluorescence of a promoter::gfp fusion), in single cells provided evidence for functional heterogeneity within a V. harveyi population.
Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population.
Bioluminescence; Exoprotease; Type III secretion; Autoinducer; Division of labor; Subpopulation
Expression of lysP, which encodes the lysine-specific transporter LysP in Escherichia coli, is regulated by the concentration of exogenous available lysine. In this study, the LysR-type transcriptional regulator ArgP was identified as the activator of lysP expression. At lysine concentrations higher than 25 μM, lysP expression was shut off and phenocopied an argP deletion mutant. Purified ArgP-His6 bound to the lysP promoter/control region at a sequence containing a conserved T-N11-A motif. Its affinity increased in the presence of lysine but not in the presence of the other known coeffector, arginine. In vivo data suggest that lysine-loaded ArgP and arginine-loaded ArgP compete at the lysP promoter. We propose that lysine-loaded ArgP prevents lysP transcription at the promoter clearance step, as described for the lysine-dependent regulation of argO (R. S. Laishram and J. Gowrishankar, Genes Dev. 21:1258-1272, 2007). The global regulator Lrp also bound to the lysP promoter/control region. An lrp mutant exhibited reduced lysP expression in the absence of external lysine. These results indicate that ArgP is a major regulator of lysP expression but that Lrp modulates lysP transcription under lysine-limiting conditions.
In an acidic and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, the lysine decarboxylase, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family. Activation of CadC requires two stimuli, lysine and low pH. Whereas lysine is detected by an interplay between CadC and the lysine-specific transporter LysP, pH alterations are sensed by CadC directly. Crystal structural analyses revealed a close proximity between two periplasmic cysteines, Cys208 and Cys272.
Substitution of Cys208 and/or Cys272 by alanine resulted in CadC derivatives that were active in response to only one stimulus, either lysine or pH 5.8. Differential in vivo thiol trapping revealed a disulfide bond between these two residues at pH 7.6, but not at pH 5.8. When Cys208 and Cys272 were replaced by aspartate and lysine, respectively, virtually wild-type behavior was restored indicating that the disulfide bond could be mimicked by a salt bridge.
A disulfide bond was found in the periplasmic domain of CadC that supports an inactive state of CadC at pH 7.6. At pH 5.8 disulfide bond formation is prevented which transforms CadC into a semi-active state. These results provide new insights into the function of a pH sensor.
Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well understood. The T4SS consists of inner- and outer membrane-spanning Cag protein complexes and a surface-located pilus. Previously an arginine-glycine-aspartate (RGD)-dependent typical integrin/ligand type interaction of CagL with α5β1 integrin was reported to be essential for CagA translocation. Here we report a specific binding of the T4SS-pilus-associated components CagY and the effector protein CagA to the host cell β1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to α5β1 integrin is rather strong (dissociation constant, KD of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (KD of 15 nM). For CagA translocation the extracellular part of the β1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of β1 integrin-specific monoclonal antibodies directed against various defined β1 integrin epitopes, such as the PSI, the I-like, the EGF or the β-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of β1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the β1 integrin receptor by an RGD-independent interaction that involves a conformational switch from the open (extended) to the closed (bent) conformation, to initiate effector protein translocation.
Integrins are single transmembrane proteins present on almost all types of cells. They are composed of an α and a β subunit, which together form the ligand binding pocket, able to interact with extracellular matrix proteins. The best known binding domain on integrin ligands is the RGD domain. Many bacterial, but also viral pathogens exploit this ligand-binding domain to interact with integrins on the host cell. Helicobacter pylori, a common bacterial pathogen associated with gastric diseases, was recently added to this list. One of H. pylori's most important factors associated with gastric pathologies is the CagA protein. This protein is directly injected into host cells through the Cag Type IV Secretion System (cag-T4SS). Previous studies demonstrated that the cag-T4SS requires integrins for the injection (translocation) of CagA into cells. We provide evidence that three proteins, CagA, CagI and CagY, interact with integrins in an RGD-independent way. Additionally, our data point out that the Cag apparatus needs the physical capacity of a β1 integrin heterodimer to change from an active/extended conformation to a closed/bent conformation. This novel kind of integrin interaction opens a new way in which pathogens can use receptors on cells.
The KdpD/KdpE two-component system of Escherichia coli regulates expression of the kdpFABC operon encoding the high affinity K+ transport system KdpFABC. The input domain of KdpD comprises a domain that belongs to the family of universal stress proteins (Usp). It has been previously demonstrated that UspC binds to this domain, resulting in KdpD/KdpE scaffolding under salt stress. However the mechanistic significance of this domain for signaling remains unclear. Here, we employed a "domain swapping" approach to replace the KdpD-Usp domain with four homologous domains or with the six soluble Usp proteins of E. coli.
Full response to salt stress was only achieved with a chimera that contains UspC, probably due to unaffected scaffolding of the KdpD/KdpE signaling cascade by soluble UspC. Unexpectedly, chimeras containing either UspF or UspG not only prevented kdpFABC expression under salt stress but also under K+ limiting conditions, although these hybrid proteins exhibited kinase and phosphotransferase activities in vitro. These are the first KdpD derivatives that do not respond to K+ limitation due to alterations in the N-terminal domain. Analysis of the KdpD-Usp tertiary structure revealed that this domain has a net positively charged surface, while UspF and UspG are characterized by net negative surface charges.
The Usp domain within KdpD not only functions as a binding surface for the scaffold UspC, but it is also important for KdpD signaling. We propose that KdpD sensing/signaling involves alterations of electrostatic interactions between the large N- and C-terminal cytoplasmic domains.
Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a pathogenic stage, characterized by release from the nematode into the hemocoel of insect larvae, resulting in rapid insect death caused by bacterial toxins. P. luminescens appears to sense and adapt to the novel host environment upon changing hosts, which facilitates the production of factors involved in survival within the host, host-killing, and -exploitation.
A differential fluorescence induction (DFI) approach was applied to identify genes that are up-regulated in the bacterium after infection of the insect host Galleria mellonella. For this purpose, a P. luminescens promoter-trap library utilizing the mCherry fluorophore as a reporter was constructed, and approximately 13,000 clones were screened for fluorescence induction in the presence of a G. mellonella larvae homogenate. Since P. luminescens has a variety of regulators that potentially sense chemical molecules, like hormones, the screen for up-regulated genes or operons was performed in vitro, excluding physicochemical signals like oxygen, temperature or osmolarity as variables. Clones (18) were obtained exhibiting at least 2.5-fold induced fluorescence and regarded as specific responders to insect homogenate. In combination with a bioinformatics approach, sequence motifs were identified in these DNA-fragments that are similar to 29 different promoters within the P. luminescens genome. By cloning each of the predicted promoters upstream of the reporter gene, induction was verified for 27 promoters in vitro, and for 24 promoters in viable G. mellonella larvae. Among the validated promoters are some known to regulate the expression of toxin genes, including tccC1 (encoding an insecticidal toxin complex), and others encoding putative toxins. A comparably high number of metabolic genes or operons were observed to be induced upon infection; among these were eutABC, hutUH, and agaZSVCD, which encode proteins involved in ethanolamine, histidine and tagatose degradation, respectively. The results reflect rearrangements in metabolism and the use of other metabolites available from the insect. Furthermore, enhanced activity of promoters controlling the expression of genes encoding enzymes linked to antibiotic production and/or resistance was observed. Antibiotic production and resistance may influence competition with other bacteria, and thus might be important for a successful infection. Lastly, several genes of unknown function were identified that may represent novel pathogenicity factors.
We show that a DFI screen is useful for identifying genes or operons induced by chemical stimuli, such as diluted insect homogenate. A bioinformatics comparison of motifs similar to known promoters is a powerful tool for identifying regulated genes or operons. We conclude that signals for the regulation of those genes or operons induced in P. luminescens upon insect infection may represent a wide variety of compounds that make up the insect host. Our results provide insight into the complex response to the host that occurs in a bacterial pathogen, particularly reflecting the potential for metabolic shifts and other specific changes associated with virulence.
Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems.
Here we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red®/ET® Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed.
Chromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before.
The KdpD sensor kinase and the KdpE response regulator control expression of the kdpFABC operon coding for the KdpFABC high-affinity K+ transport system of Escherichia coli. In search of a distinct part of the input domain of KdpD which is solely responsible for K+ sensing, sequences of kdpD encoding the transmembrane region and adjacent N-terminal and C-terminal extensions were subjected to random mutagenesis. Nine KdpD derivatives were identified that had lost tight regulation of kdpFABC expression. They all carried single amino acid replacements located in a region encompassing the fourth transmembrane helix and the adjacent arginine cluster of KdpD. All mutants exhibited high levels of kdpFABC expression regardless of the external K+ concentration. However, 3- to 14-fold induction was observed under extreme K+-limiting conditions and in response to an osmotic upshift when sucrose was used as an osmolyte. These KdpD derivatives were characterized by a reduced phosphatase activity in comparison to the autokinase activity in vitro, which explains constitutive expression. Whereas for wild-type KdpD the autokinase activity and also, in turn, the phosphotransfer activity to KdpE were inhibited by increasing concentrations of K+, both activities were unaffected in the KdpD derivatives. These data clearly show that the extension of the fourth transmembrane helix encompassing the arginine cluster is mainly involved in sensing both K+ limitation and osmotic upshift, which may not be separated mechanistically.
Hydropathy profile analyses of the amino acid sequence of the quorum-sensing hybrid histidine kinase LuxN of Vibrio harveyi predict a periplasmic location of the N terminus. To test this, two-hybrid proteins consisting of LuxN and an N-terminally fused maltose-binding protein with or without a leader sequence were analyzed with regard to the enzymatic activities of LuxN, protease accessibility, and complementation of an Escherichia coli malE mutant. The results strongly support a periplasmic location of the N terminus, implying that LuxN is anchored with nine transmembrane domains in the cytoplasmic membrane.
Escherichia coli lives in the mammalian gastrointestinal tract anaerobically at high osmolarity as well as in the soil aerobically at varying osmolarities. Adaptation to these varying environmental conditions is crucial for growth and survival of E. coli. Two-dimensional protein gels were used to visualize global time-dependent changes (10 to 60 min) in the proteome of cells responding to osmotic stress (0.4 M NaCl or 0.7 M sorbitol) under aerobic or anaerobic conditions. The protein profiles revealed an induction of 12 proteins (Dps, HchA, HdhA, InfB, OsmC, OsmY, ProX, KatE, PspA, TalA, TktB, and TreF) under osmotic stress in an aerobic milieu. Eleven additional proteins (OtsB, YceI, YciE, YciF, YgaU, YjbJ, AcnA, MetL, PoxB, Ssb, and YhbO) were induced by osmotic stress imposed by NaCl. Most of the accumulated proteins were cross-protecting proteins (e.g., OsmY, OsmC, Dps, and KatE) which are regulated at the transcriptional level predominantly by RpoS and other regulators (e.g., integration host factor, OxyR, H-NS, LRP, and FIS). Comparative analysis of the proteome of E. coli grown under aerobic or anaerobic conditions under osmotic stress (NaCl) revealed an overlap of the up-regulated proteins of more than 50%. Ten proteins (PoxB, AcnA, TalA, TktB, KatE, PspA, Ssb, TreF, MetL, and YhbO) were detectable only under aerobic, high-osmolality conditions. Time-dependent alterations of the proteome were monitored, allowing classification of the up-regulated proteins into early, middle, and long-term phases of adaptation. Only a few proteins were found to be down-regulated upon osmotic stress.