Biomedical subjects

Lian-Hui Zhang

Publications and source records attributed to Lian-Hui Zhang.

At least 19 recordsLinked to original sources

PubMed2006

Identification of genetic markers to distinguish the virulent and avirulent subspecies of Pantoea stewartii by comparative proteomics and genetic analysis.

Pantoea stewartii subsp. stewartii (Pnss), the causal agent of Stewart's bacterial wilt and leaf blight of maize and sweet corn, is one of the quarantine pathogens in many countries and regions. In contrast, P. stewartii subsp. indologenes (Pnsi), the closely related subspecies of Pnss, is avirulent on these plants. In this study, the protein expression profiles of these two subspecies were compared using two-dimensional gel electrophoresis analysis. Twenty-one unique protein spots consistently detected in Pnss but not in Pnsi were analyzed by mass spectrometry. Some of these Pnss-specific proteins are known to be essential for virulence and survival in host, such as FoxR and HrcJ, which are the key components of iron uptake and Type III secretion systems, respectively. For further genetic analysis, six Pnss-specific proteins were characterized by peptide sequencing. Southern and Northern blot analyses revealed that the differences in protein expression profiles of the two subspecies were either due to the discrepancy at genome level or because of the variations in transcriptional expression. The results provide novel genetic markers to distinguish the two closely related subspecies and may also serve as useful clues for investigation of the genetic basis accounting for their sharp difference in virulence.

Amino Acid Sequence↗

PubMed2006

Crystal structure and mechanism of TraM2, a second quorum-sensing antiactivator of Agrobacterium tumefaciens strain A6.

Quorum sensing is a community behavior that bacteria utilize to coordinate a variety of population density-dependent biological functions. In Agrobacterium tumefaciens, quorum sensing regulates the replication and conjugative transfer of the tumor-inducing (Ti) plasmid from pathogenic strains to nonpathogenic derivatives. Most of the quorum-sensing regulatory proteins are encoded within the Ti plasmid. Among these, TraR is a LuxR-type transcription factor playing a key role as the quorum-sensing signal receptor, and TraM is an antiactivator that antagonizes TraR through the formation of a stable oligomeric complex. Recently, a second TraM homologue called TraM2, not encoded on the Ti plasmid of A. tumefaciens A6, was identified, in addition to a copy on the Ti plasmid. In this report, we have characterized TraM2 and its interaction with TraR and solved its crystal structure to 2.1 A. Like TraM, TraM2 folds into a helical bundle and exists as homodimer. TraM2 forms a stable complex (K(d) = 8.6 nM) with TraR in a 1:1 binding ratio, a weaker affinity than that of TraM for TraR. Structural analysis and biochemical studies suggest that protein stability may account for the difference between TraM2 and TraM in their binding affinities to TraR and provide a structural basis for L54 in promoting structural stability of TraM.

Agrobacterium tumefaciens↗

PubMed2006

Succinic semialdehyde couples stress response to quorum-sensing signal decay in Agrobacterium tumefaciens.

Quorum sensing (QS) signal decay in Agrobacterium tumefaciens occurs in response to starvation or host signals. We have demonstrated that the gamma-aminobutyric acid (GABA) shunt metabolite links stress response to QS signal decay. Mutation of the aldH gene encoding a succinic semialdehyde dehydrogenase (SSADH) that converts succinic semialdehyde (SSA) to succinic acid results in early expression of the signal degrading enzyme, AttM. Exogenous addition of SSA or its precursor GABA induces AttM expression and abolishes Ti plasmid conjugative transfer. SSA acts by binding to the repressor AttJ that regulates the attKLM operon. attK encodes another SSADH. The stress alarmone ppGpp and SSA modulates separately the expression of the two SSADH enzymes, which might control the intracellular SSA level and hence to switch on/off the QS signal decay system in response to environmental changes. These findings document for the first time a sophisticated signalling mechanism of the widely conserved GABA degradation pathway in prokaryotes.

Agrobacterium tumefaciens↗

PubMed2006

Dual signaling functions of the hybrid sensor kinase RpfC of Xanthomonas campestris involve either phosphorelay or receiver domain-protein interaction.

The hybrid sensor kinase RpfC positively regulates the expression of a range of virulent genes and negatively modulates the synthesis of the quorum sensing signal diffusible signal factor (DSF) in Xanthomonas campestris. Three conserved amino acid residues of RpfC implicated in phosphorelay (His(198) in the histidine kinase domain, Asp(512) in the receiver domain, and His(657) in the histidine phosphotransfer domain) were essential for activation of the production of extracellular enzymes and extracellular polysaccharide (EPS) virulence factors but were not essential for repression of DSF biosynthesis. Domain deletion and subsequent in trans expression analysis revealed that the receiver domain of RpfC alone was sufficient to repress DSF overproduction in an rpfC deletion mutant. Further deletion and alanine scanning mutagenesis analyses identified a peptide of 107 amino acids and three amino acid residues (Gln(496), Glu(504), and Ile(552)) involved in modulating DSF production. Co-immunoprecipitation and far Western blot analyses suggested an interaction between the receiver domain and RpfF, the enzyme involved in DSF biosynthesis. These data support a model in which RpfC modulates two different functions (virulence factor synthesis and DSF synthesis) by utilization of a conserved phosphorelay system and a novel domain-specific protein-protein interaction mechanism, respectively. This latter mechanism represents an added dimension to conventional two-component signaling paradigms.

Amino Acid Sequence↗

PubMed2006

Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover.

HD-GYP is a protein domain of unknown biochemical function implicated in bacterial signaling and regulation. In the plant pathogen Xanthomonas campestris pv. campestris, the synthesis of virulence factors and dispersal of biofilms are positively controlled by a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC and by cell-cell signaling mediated by the diffusible signal molecule DSF (diffusible signal factor). The RpfG/RpfC two-component system has been implicated in DSF perception and signal transduction. Here we show that the role of RpfG is to degrade the unusual nucleotide cyclic di-GMP, an activity associated with the HD-GYP domain. Mutation of the conserved H and D residues of the isolated HD-GYP domain resulted in loss of both the enzymatic activity against cyclic di-GMP and the regulatory activity in virulence factor synthesis. Two other protein domains, GGDEF and EAL, are already implicated in the synthesis and degradation respectively of cyclic di-GMP. As with GGDEF and EAL domains, the HD-GYP domain is widely distributed in free-living bacteria and occurs in plant and animal pathogens, as well as beneficial symbionts and organisms associated with a range of environmental niches. Identification of the role of the HD-GYP domain thus increases our understanding of a signaling network whose importance to the lifestyle of diverse bacteria is now emerging.

Amino Acid Sequence↗

PubMed2006

Regeneration of sugarcane elite breeding lines and engineering of stem borer resistance.

Five elite sugarcane breeding lines were tested for efficiency in embryogenesis and plant regeneration. All of them produced regenerative embryogenic calli but with varied efficiencies. To engineer strongly insect-resistant sugarcanes, the GC content of a truncated cry1Ac gene, which encodes the active region of Cry1Ac insecticidal delta-endotoxin, was increased from the original 37.4 to 47.5% following the sugarcane codon usage pattern. The synthetic cry1Ac gene (s-cry1Ac) was placed under the control of maize ubiquitin promoter and introduced by microprojectile bombardment into the embryogenic calli of sugarcane lines YT79-177 and ROC16. Southern blotting analysis showed that multicopies of s-cry1Ac were integrated into the genomes of transgenic sugarcane lines. Immunoblotting analysis identified 18 transgenic lines expressing detectable levels of s-Cry1Ac, which were estimated in the range of 1.8-10.0 ng mg(-1) total soluble proteins. Four transgenic and two parental lines were assayed for sugarcane stem borer resistance in leaf tissue feeding trials and greenhouse plant assays. The results showed that, while the untransformed control lines were severely damaged in both leaves and stems, the transgenic sugarcane lines expressing high levels of s-Cry1Ac proteins were highly resistant to sugarcane stem borer attack, resulting in complete mortality of the inoculated insects within 1 week after inoculation.

Animals↗

PubMed2006

Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions.

The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis-11-methyl-2-dodecenoic acid, as a cell-cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF-mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild-type strain XC1 and its DSF-deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF-dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell-cell signalling is not only essential for co-ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.

Base Sequence↗

PubMed2006

Dual control of quorum sensing by two TraM-type antiactivators in Agrobacterium tumefaciens octopine strain A6.

Agrobacterium tumefaciens wild-type strains have a unique quorum-sensing (QS)-dependent Ti plasmid conjugative transfer phenotype in which QS signaling is activated by corresponding conjugative opine inducers. Strain K588, with a nopaline-type chromosomal background harboring an octopine-type Ti plasmid, however, is a spontaneous mutant displaying a constitutive phenotype in QS. In this study, we show that a single amino acid mutation (L54P) in the QS antiactivator TraM encoded by the traM gene of Ti plasmid is responsible for the constitutive phenotype of strain K588. Introduction of the L54P point mutation to the TraM of wild-type strain A6 by allelic replacement, however, failed to generate the expected constitutive phenotype in this octopine-type strain. Intriguingly, the QS-constitutive phenotype appeared when the pTiA6 carrying the mutated traM was placed in the chromosomal background of the nopaline-type strain C58C1RS, suggesting an unknown inhibitory factor(s) encoded by the chromosomal background of strain A6 but not by C58C1RS. Low-stringency Southern blotting analysis showed that strain A6, but not strain C58 and its derivatives, contains a second traM homologue. The homologue, designated traM2, has 64% and 65% identities with traM at the DNA and peptide levels, respectively. Similar to TraM, TraM2 is a potent antiactivator that functions by blocking TraR, the QS activator, from specific binding to the tra gene promoters. Deletion of traM2 in strain A6 harboring the mutated traM confers a constitutive QS phenotype. The results demonstrate that the QS system in strain A6 is subjected to the dual control of TraM and TraM2.

Agrobacterium tumefaciens↗

PubMed2005

Transition to quorum sensing in an Agrobacterium population: A stochastic model.

Understanding of the intracellular molecular machinery that is responsible for the complex collective behavior of multicellular populations is an exigent problem of modern biology. Quorum sensing, which allows bacteria to activate genetic programs cooperatively, provides an instructive and tractable example illuminating the causal relationships between the molecular organization of gene networks and the complex phenotypes they control. In this work we--to our knowledge for the first time--present a detailed model of the population-wide transition to quorum sensing using the example of Agrobacterium tumefaciens. We construct a model describing the Ti plasmid quorum-sensing gene network and demonstrate that it behaves as an "on-off" gene expression switch that is robust to molecular noise and that activates the plasmid conjugation program in response to the increase in autoinducer concentration. This intracellular model is then incorporated into an agent-based stochastic population model that also describes bacterial motion, cell division, and chemical communication. Simulating the transition to quorum sensing in a liquid medium and biofilm, we explain the experimentally observed gradual manifestation of the quorum-sensing phenotype by showing that the transition of individual model cells into the "on" state is spread stochastically over a broad range of autoinducer concentrations. At the same time, the population-averaged values of critical autoinducer concentration and the threshold population density are shown to be robust to variability between individual cells, predictable and specific to particular growth conditions. Our modeling approach connects intracellular and population scales of the quorum-sensing phenomenon and provides plausible answers to the long-standing questions regarding the ecological and evolutionary significance of the phenomenon. Thus, we demonstrate that the transition to quorum sensing requires a much higher threshold cell density in liquid medium than in biofilm, and on this basis we hypothesize that in Agrobacterium quorum sensing serves as the detector of biofilm formation.

Agrobacterium tumefaciens↗

PubMed2005

Quorum quenching enzyme activity is widely conserved in the sera of mammalian species.

Acyl-homoserine lactone (AHL) quorum sensing signals play a key role in synchronizing virulence gene expression in Pseudomonas aeruginosa, which could cause fatal bloodstream infections. We showed that AHL inactivation activity, albeit with variable efficiency, was conserved in the serum samples of all the 6 tested mammalian animals. High-performance liquid chromatography and mass spectrometry analyses revealed that mammalian sera had a lactonase-like enzyme(s), which hydrolyzed the lactone ring of AHL to produce acyl homoserine, with enzyme properties reminiscent of paraoxonases (PONs). We further showed that the animal cell lines expressing three mouse PON genes, respectively, displayed strong AHL degradation activities.

4-Butyrolactone↗

PubMed2005

The two-component response regulator PprB modulates quorum-sensing signal production and global gene expression in Pseudomonas aeruginosa.

The response regulator PprB and its cognate sensor PprA were recently reported as a two-component regulatory system that controls membrane permeability and antibiotic sensitivity of Pseudomonas aeruginosa. We found that a Tn5 insertion mutation in pprB caused a drastic reduction in virulence factor production and cell motility. A transcriptome analysis revealed that 175 genes were regulated by PprB. Among the 113 PprB-activated genes, 85.5% are known to be activated by N-3-oxo-dodecanoyl-homoserine lactone (OdDHL) and N-butanoyl-homoserine lactone (BHL). In particular, the expression of lasI, rhlI and rhlR, which encode key components of the las and rhl quorum-sensing (QS) systems, were significantly decreased in the pprB mutant. These data suggest that PprB might regulate QS signal production. Measurement of OdDHL and BHL in cultures of the mutant sustained this hypothesis. By using various OdDHL- or BHL-responsive QS reporter systems, including lasB-lacZ, lasI-lacZ and rsaL-lacZ, we found that the mutation in pprB resulted in a large decrease in the sensitivity of P. aeruginosa to exogenous OdDHL. However, there was no difference in sensitivity to BHL. Further analysis showed that the OdDHL influx was significantly reduced in the pprB mutant. We conclude that PprB is a novel QS modulator that positively regulates N-acylhomoserine lactone production probably by affecting the OdDHL signal influx and thereby influences global expression of the QS-dependent genes.

4-Butyrolactone↗

PubMed2005

VqsM, a novel AraC-type global regulator of quorum-sensing signalling and virulence in Pseudomonas aeruginosa.

Human pathogen Pseudomonas aeruginosa uses quorum-sensing (QS) signalling systems to synchronize the production of virulence factors. There are two interrelated QS systems, las and rhl, in P. aeruginosa. In addition to this complexity, a number of transcriptional regulators were shown to have complicated interplays with las and rhl central QS components. Here, we describe a novel virulence and QS modulator (VqsM) that positively regulates the QS systems in P. aeruginosa. Mutation in vqsM resulted in much reduced production of N-acylhomoserine lactones (AHLs) and extracellular enzymes. Sequence analysis revealed that vqsM encodes a transcriptional regulator with an AraC-type helix-turn-helix DNA binding domain at the C-terminal of the peptide. Global gene expression profile analysis showed at least a total of 302 genes to be influenced, directly or indirectly, by VqsM. Among the 203 VqsM-promoted genes, 52.2% were known to be QS upregulated. Several genes encoding the key regulators implicated in QS, such as rhlR, rsaL, vqsR, mvfR, pprB and rpoS, and two AHL synthesis genes, lasI and rhlI, were suppressed in the vqsM mutant. Similar to the 'AHL-blind' phenotype of vqsR and pprB mutants, vqsM mutant did not respond to external addition of N-3-oxo-dodecanoyl-homoserine lactone signals. Moreover, overexpression of vqsR in vqsM mutant more or less restored the production of both AHL and virulence factors. The results demonstrate that VqsM, largely through modulation of vqsR expression, plays a vital role in regulation of QS signalling in P. aeruginosa.

Animals↗

PubMed2005

Molecular and conformational basis of a specific and high-affinity interaction between AlbA and albicidin phytotoxin.

The albA gene of Klebsiella oxytoca encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 x 10(-8) M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys106, Trp110, Tyr113, Leu114, Tyr126, Pro134, and Trp162 with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His125, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.

Amino Acid Sequence↗

PubMed2005

Quorum sensing and quorum-quenching enzymes.

To gain maximal benefit in a competitive environment, single-celled bacteria have adopted a community genetic regulatory mechanism, known as quorum sensing (QS). Many bacteria use QS signaling systems to synchronize target gene expression and coordinate biological activities among a local population. N-acylhomoserine lactones (AHLs) are one family of the well-characterized QS signals in Gram-negative bacteria, which regulate a range of important biological functions, including virulence and biofilm formation. Several groups of AHL-degradation enzymes have recently been identified in a range of living organisms, including bacteria and eukaryotes. Expression of these enzymes in AHL-dependent pathogens and transgenic plants efficiently quenches the microbial QS signaling and blocks pathogenic infections. Discovery of these novel quorum quenching enzymes has not only provided a promising means to control bacterial infections, but also presents new challenges to investigate their roles in host organisms and their potential impacts on ecosystems.

4-Butyrolactone↗

PubMed2004

Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase).

N-Acyl homoserine lactone (AHL) quorum-sensing signals are the vital elements of bacterial quorum-sensing systems, which regulate diverse biological functions, including virulence. The AHL-lactonase, a quorumquenching enzyme encoded by aiiA from Bacillus sp., inactivates AHLs by hydrolyzing the lactone bond to produce corresponding N-acyl homoserines. To characterize the enzyme, the recombinant AHL-lactonase and its four variants were purified. Kinetic and substrate specificity analysis showed that AHL-lactonase had no or little residue activity to non-acyl lactones and noncyclic esters, but displayed strong enzyme activity toward all tested AHLs, varying in length and nature of the substitution at the C3 position of the acyl chain. The data also indicate that the amide group and the ketone at the C1 position of the acyl chain of AHLs could be important structural features in enzyme-substrate interaction. Surprisingly, although carrying a (104)HX- HXDH(109) short sequence identical to the zinc-binding motif of several groups of metallohydrolytic enzymes, AHL-lactonase does not contain or require zinc or other metal ions for enzyme activity. Except for the amino acid residue His-104, which was shown previously to not be required for catalysis, kinetic study and conformational analysis using circular dichroism spectrometry showed that substitution of the other key residues in the motif (His-106, Asp-108, and His-109), as well as His-169 with serine, respectively, caused conformational changes and significant loss of enzyme activity. We conclude that AHL-lactonase is a highly specific enzyme and that the (106)HXDH(109) approximately H(169) of AHL-lactonase represents a novel catalytic motif, which does not rely on zinc or other metal ions for activity.

Bacillus↗

PubMed2004

A bacterial cell-cell communication signal with cross-kingdom structural analogues.

Extracellular signals are the key components of microbial cell-cell communication systems. This report identified a diffusible signal factor (DSF), which regulates virulence in Xanthomonas campestris pv. campestris, as cis-11-methyl-2-dodecenoic acid, an alpha,beta unsaturated fatty acid. Analysis of DSF derivatives established the double bond at the alpha,beta positions as the most important structural feature for DSF biological activity. A range of bacterial pathogens, including several Mycobacterium species, also displayed DSF-like activity. Furthermore, DSF is structurally and functionally related to farnesoic acid (FA), which regulates morphological transition and virulence by Candida albicans, a fungal pathogen. Similar to FA, which is also an alpha,beta unsaturated fatty acid, DSF inhibits the dimorphic transition of C. albicans at a physiologically relevant concentration. We conclude that alpha,beta unsaturated fatty acids represent a new class of extracellular signals for bacterial and fungal cell-cell communications. As prokaryote-eukaryote interactions are ubiquitous, such cross-kingdom conservation in cell-cell communication systems might have significant ecological and economic importance.

Bacterial Proteins↗

PubMed2004

The quormone degradation system of Agrobacterium tumefaciens is regulated by starvation signal and stress alarmone (p)ppGpp.

A unique signal degradation system has recently been discovered in Agrobacterium tumefaciens. Upon entering stationary phase, A. tumefaciens terminates quorum sensing-dependent Ti-plasmid conjugation by degradation of acyl homoserine lactone (AHL) quormone via the enzyme AttM (AHL-lactonase). attM, together with attK and attL, constitute one transcriptional unit subjected to the control of a common promoter. AttJ, the other member of the signal degradation system, is an IclR-like negative transcriptional factor, which tightly represses the expression of AttM at the early stage of bacterial growth. In this study, we found that this quormone degradation system is activated by either carbon or nitrogen starvation. Quormone degradation was significantly delayed when bacterial culture was supplemented with extra carbon or nitrogen source in the nutrient-limited minimal medium before the onset of stationary phase. To identify the signalling pathway and regulatory mechanisms that mediate quormone degradation, we constructed a reporter strain A6(attKLM::lacZ) in which the promoterless lacZ was transcriptionally fused to the attKLM promoter. Transposon mutagenesis of strain A6(attKLM::lacZ) led to identification of the relA gene, which encodes the stress alarmone (p)ppGpp synthetase. Tn5 knock-out of relA abolished the stationary phase-dependent expression of attM. We concluded that the A. tumefaciens quormone degradation system is coupled to and regulated by the generic (p)ppGpp stress response machinery.

4-Butyrolactone↗

PubMed2004

Quorum sensing and signal interference: diverse implications.

Quorum sensing (QS) is a community genetic regulation mechanism that controls microbiological functions of medical, agricultural and industrial importance. Discovery of microbial QS signals and the signalling mechanisms led to identification of numerous enzymatic and non-enzymatic signal interference mechanisms that quench microbial QS signalling. Evidence is accumulating that such signal interference mechanisms can be developed as promising approaches to control microbial infection and biofilm formation. In addition, these mechanisms exist not only in microorganisms but also in the host organisms of bacterial pathogens, highlighting their potential implications in microbial ecology and in host-pathogen interactions. Investigation of QS and signal interference mechanisms might significantly broaden the scope of research in microbiology.

Animals↗