Biomedical subjects

Lian-Hui Zhang

Publications and source records attributed to Lian-Hui Zhang.

31 records · Page 2Linked to original sources

PubMed2004

Insecticidal Bacillus thuringiensis silences Erwinia carotovora virulence by a new form of microbial antagonism, signal interference.

It is commonly known that bacteria may produce antibiotics to interfere with the normal biological functions of their competitors in order to gain competitive advantages. Here we report that Bacillus thuringiensis suppressed the quorum-sensing-dependent virulence of plant pathogen Erwinia carotovora through a new form of microbial antagonism, signal interference. E. carotovora produces and responds to acyl-homoserine lactone (AHL) quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B. thuringiensis strains possess AHL-lactonase, which is a potent AHL-degrading enzyme. B. thuringiensis did not seem to interfere with the normal growth of E. carotovora; rather, it abolished the accumulation of AHL signal when they were cocultured. In planta, B. thuringiensis significantly decreased the incidence of E. carotovora infection and symptom development of potato soft rot caused by the pathogen. The biocontrol efficiency is correlated with the ability of bacterial strains to produce AHL-lactonase. While all the seven AHL-lactonase-producing B. thuringiensis strains provided significant protection against E. carotovora infection, Bacillus fusiformis and Escherichia coli strains that do not process AHL-degradation enzyme showed little effect in biocontrol. Mutation of aiiA, the gene encoding AHL-lactonase in B. thuringiensis, resulted in a substantial decrease in biocontrol efficacy. These results suggest that signal interference mechanisms existing in natural ecosystems could be explored as a new version of antagonism for prevention of bacterial infections.

4-Butyrolactone↗

PubMed2004

MorA defines a new class of regulators affecting flagellar development and biofilm formation in diverse Pseudomonas species.

Assembly of bacterial flagella is developmentally important during both planktonic cell growth and biofilm formation. Flagellar biogenesis is complex, requiring coordinated expression of over 40 genes, and normally commences during the log-to-stationary transition phase. We describe here a novel membrane-localized regulator, MorA, that controls the timing of flagellar development and affects motility, chemotaxis, and biofilm formation in Pseudomonas putida. MorA is conserved among diverse Pseudomonas species, and homologues are present in all Pseudomonas genomes sequenced thus far. In P. putida, the absence of MorA derepresses flagellar development, which leads to constitutive formation of flagella in the mutant cells in all growth phases. In Pseudomonas aeruginosa, the absence of MorA led to a reduction in biofilm formation. However, unlike the motility of P. putida, the motility of the P. aeruginosa mutants was unaffected. Our data illustrate a novel developmentally regulated sensory and signaling pathway for several properties required for virulence and ecological fitness of Pseudomonas species.

Amino Acid Sequence↗

PubMed2003

Cloning and characterization of a novel lipase from Vibrio harveyi strain AP6.

A lipolytic enzyme designated Vst, was cloned from Vibrio harveyi strain AP6. The vst gene was 1650 bp and was predicted to encode a 549 amino acid precursor with a molecular mass of about 61 kDa. The predicted polypeptide shared about 30% sequence identity with Bacillus esterases. Sequence alignment of Vst and related esterases revealed the presence of an active site serine located in the middle of the GESAG motif, at positions 212-216. This motif resembled the GXSXG consensus motif characteristic of lipolytic enzymes. Vst was expressed as a carboxy-terminal 6 x His tagged recombinant enzyme from E. coli TOP10 cells. SDS-PAGE and Western blot analysis using anti-His antibodies confirmed that the size of the mature protein was about 61 kDa. Substrate specificity of Vst was investigated using p -nitrophenyl (p NP) esters with varying carbon chain lengths, from C2 to C18. Vst showed the highest activity with the long chain p -nitrophenyl ester p NPC12 but was able to hydrolyze longer chain esters (p NPC14-p NPC16) as well as short and medium chain esters (p NPC4 and p NPC8).

Amino Acid Motifs↗

PubMed2003

Isomaltulose synthase (PalI) of Klebsiella sp. LX3. Crystal structure and implication of mechanism.

Isomaltulose synthase from Klebsiella sp. LX3 (PalI, EC 5.4.99.11) catalyzes the isomerization of sucrose to produce isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) and trehalulose (alpha-D-glucosylpyranosyl-1,1-d-fructofuranose). The PalI structure, solved at 2.2-A resolution with an R-factor of 19.4% and Rfree of 24.2%, consists of three domains: an N-terminal catalytic (beta/alpha)8 domain, a subdomain between N beta 3 and N alpha 3, and a C-terminal domain having seven beta-strands. The active site architecture of PalI is identical to that of other glycoside hydrolase family 13 members, suggesting a similar mechanism in substrate binding and hydrolysis. However, a unique RLDRD motif in the proximity of the active site has been identified and shown biochemically to be responsible for sucrose isomerization. A two-step reaction mechanism for hydrolysis and isomerization, which occurs in the same pocket is proposed based on both the structural and biochemical data. Selected C-terminal truncations have been shown to reduce and even abolish the enzyme activity, consistent with the predicted role of the C-terminal residues in the maintenance of enzyme conformation and active site topology.

Amino Acid Sequence↗

PubMed2003

Microbial diversity and prevalence of virulent pathogens in biofilms developed in a water reclamation system.

Bacterial biofilm is a common phenomenon in both natural and engineered systems which often becomes a source of contamination and microbially influenced corrosion. It is thought that formation of biofilm in the monoculture of several bacterial species is regulated by acylhomoserine lactone (AHL) quorum-sensing signals. In this study, we investigated the microbial diversity and existence of AHL-producing and AHL-degrading bacterial species in the biofilm samples from a water reclamation system located in a tropical environment. 16S ribosomal DNA sequencing analysis indicated the presence of at least 11 bacterial species, including the frequently encountered bacterial pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae, and several rare pathogens. We showed that only two groups of isolates, belonging to P. aeruginosa and Enterobacter agglomerans, produced AHL signals. We also found that three bacterial isolates, i.e., Agrobacterium tumefaciens XJ01, Bacillus cereus XJ08, and Ralstonia sp. XJ12, expressed AHL degradation enzymes. Furthermore, we showed that P. aeruginosa isolate HL43 was virulent against animal model Caenorhabditis elegans and released 2-6-fold more pyocyanin cytotoxin than P. aeruginosa strains PA01 and PA14, the two commonly used laboratory strains. These data indicate the complexity and importance of biofilm research in water reclamation.

Animals↗

PubMed2003

Quorum quenching and proactive host defense.

Both plants and humans have inducible defense mechanisms. This passive defense strategy leaves the host unprotected for a period of time until resistance is activated. Moreover, many bacterial pathogens have evolved cell-cell communication (quorum-sensing) mechanisms to mount population-density-dependent attacks to overwhelm the host's defense responses. Several chemicals and enzymes have been investigated for years for their potential to target the key components of bacterial quorum-sensing systems. These quorum-quenching reagents, which block bacterial cell-cell communications, can disintegrate a bacterial population-density-dependent attack. It has now been shown that a quorum-quenching mechanism can be engineered in plants and might be used as a strategy in controlling bacterial pathogens and to build up a proactive defense barrier.

Bacteria↗

PubMed2003

Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes.

N-acylhomoserine lactones (AHLs) are used as signal molecules by many quorum-sensing Proteobacteria. Diverse plant and animal pathogens use AHLs to regulate infection and virulence functions. These signals are subject to biological inactivation by AHL-lactonases and AHL-acylases. Previously, little was known about the molecular details underlying the latter mechanism. An AHL signal-inactivating bacterium, identified as a Ralstonia sp., was isolated from a mixed-species biofilm. The signal inactivation encoding gene from this organism, which we call aiiD, was cloned and successfully expressed in Escherichia coli and inactivated three AHLs tested. The predicted 794-amino-acid polypeptide was most similar to the aculeacin A acylase (AAC) from Actinoplanes utahensis and also shared significant similarities with cephalosporin acylases and other N-terminal (Ntn) hydrolases. However, the most similar homologues of AiiD are deduced proteins of undemonstrated function from available Ralstonia, Deinococcus and Pseudomonas genomes. LC-MS analyses demonstrated that AiiD hydrolyses the AHL amide, releasing homoserine lactone and the corresponding fatty acid. Expression of AiiD in Pseudomonas aeruginosa PAO1 quenched quorum sensing by this bacterium, decreasing its ability to swarm, produce elastase and pyocyanin and to paralyze nematodes. Thus, AHL-acylases have fundamental implications and hold biotechnological promise in quenching quorum sensing.

4-Butyrolactone↗

PubMed2003

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics.

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K. oxytoca ATCC 13182(T) to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His(78), His(125), His(141) and His(189)) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95 %. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that His(125) might be the residue required for albicidin binding. Mutation of His(125) to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His(125) totally abolished binding activity. Mutation of His(125) to arginine and tyrosine had no effect. These results indicate that His(125) plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

Anti-Bacterial Agents↗

PubMed2003

Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1.

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.

4-Butyrolactone↗

PubMed2002

Expression, crystallization and preliminary X-ray analysis of isomaltulose synthase (PalI) from Klebsiella sp. LX3.

Isomaltulose synthase (PalI) catalyzes the hydrolysis of the alpha-1,2 bond between the glucose and fructose moieties of sucrose and the formation of alpha-1,6 and alpha-1,1 bonds between the two components to produce isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofranose) and trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofranose), respectively. The PalI protein has been overexpressed, purified and crystallized at 295 K using the hanging-drop vapour-diffusion method. The crystals diffract to 2.2 A resolution using synchrotron radiation and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 59.239, b = 94.153, c = 111.294 A.

Amino Acid Sequence↗

PubMed2002

Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens.

A signal turnover system is an essential component of many genetic regulatory mechanisms. The best-known example is the ubiquitin-dependent protein degradation system that exists in many organisms. We found that Agrobacterium tumefaciens adopts a unique signal turnover system to control exiting from a quorum-sensing mode. A. tumefaciens regulates Ti plasmid conjugal transfer by a quorum-sensing signal, N-3-oxo-octanoyl homoserine lactone (3OC8HSL), also known as Agrobacterium autoinducer. By using Tn5 mutagenesis and a functional cloning approach, we identified two genes that are involved in switching from a conjugal quorum-sensing mode to a nonconjugal mode at the onset of stationary phase. First, we located attJ, which codes for an IclR-type suppressor that regulates the second gene attM. The latter encodes a homologue of N-acylhomoserine lactone (AHL)-lactonase. Mass spectrometry analysis shows that the enzyme encoded by attM is an AHL-lactonase that hydrolyzes the lactone ring of 3OC8HSL. In wild-type A. tumefaciens, attM expression is initially suppressed by AttJ but significantly elevated at the stationary phase accompanied a sharp decline in 3OC8HSL. DNA gel retardation analysis shows that AttJ specifically binds to the promoter that controls AHL-lactonase expression. Mutation of attJ resulted in constitutive production of AHL-lactonase that abolishes 3OC8HSL accumulation and Ti plasmid transfer. These data suggest that A. tumefaciens has a sophisticated multicomponent quorum-sensing signal turnover system, allowing the cell to sense a change in growth and adjust cellular activities accordingly.

4-Butyrolactone↗

PubMed2002

Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species.

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.

4-Butyrolactone↗

PubMed2002

Isomaltulose synthase from Klebsiella sp. strain LX3: gene cloning and characterization and engineering of thermostability.

The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an alpha-amylase domain and (beta/alpha)(8)-barrel structures, suggesting that it belongs to the alpha-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in alpha-amylases and glucosyltransferases (Asp(241), Glu(295), Asp(369), His(145), and His(368)) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a Km of 54.6 +/- 1.7 mM for sucrose, and maximum activity (approximately 328.0 +/- 2.5 U/mg) at pH 6.0 and 35 degrees C. PalI activity was strongly inhibited by Fe3+ and Hg2+ and was enhanced by Mn2+ and Mg2+. The half-life of PalI was 1.8 min at 50 degrees C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu(498) and Arg(310) with proline resulted in an 11-fold increase in the half-life of PalI at 50 degrees C.

Amino Acid Sequence↗