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Dickeya zeae is the causal agent of rice foot rot and maize stalk rot diseases, which could cause severe economic losses. The pathogen is known to produce two phytotoxins known as zeamine and zeamine II which are also potent antibiotics against both gram-positive and gram-negative bacteria pathogens. Zeamine II is a long-chain aminated polyketide and zeamine shares the same polyketide structure as zeamine II, with an extra valine derivative moiety conjugated to the primary amino group of zeamine II. In this study, we have identified a gene designated as zmsK encoding a putative nonribosomal peptide synthase (NRPS) by screening of the transposon mutants defective in zeamine production. Different from most known NRPS enzymes, which are commonly multidomain proteins, ZmsK contains only a condensation domain. High-performance liquid chromatography and mass spectrometry analyses showed that the ZmsK deletion mutant produced only zeamine II but not zeamine, suggesting that ZmsK catalyzes the amide bond formation by using zeamine II as a substrate to generate zeamine. We also present evidence that a partially conserved catalytic motif within the condensation domain is critical for zeamine production. Furthermore, we show that deletion of zmsK substantially decreased the total antimicrobial activity and virulence of D. zeae. Our findings provide a new insight into the biosynthesis pathway of zeamines and the virulence mechanisms of the bacterial pathogen D. zeae.

Background P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues. Methodology/Findings Intriguingly, however, in this study we noticed that while a low cell density inoculum of P. aeruginosa caused severe cytotoxicity against human lung tissue cell line A549, increasing the cell density of bacterial inoculum led to decreased cytotoxicity. Addition of the supernatants from high density bacterial culture to low cell density inoculum protected the human cells from bacterial cytotoxic damage, suggesting that P. aeruginosa may produce and accumulate an inhibitory molecule(s) counteracting its pathogenic infection. The inhibitor was purified from the stationary-phase culture supernatants of P. aeruginosa strain PAO1 using bioassay-guided high performance liquid chromatography (HPLC), and characterized to be phenylacetic acid (PAA) by mass spectrometry and nuclear magnetic resonance spectroscopy. Microarray analysis revealed that treatment of P. aeruginosa with PAA down-regulated the transcriptional expression of Type III secretion system (T3SS) genes and related regulatory genes including rsmA and vfr, which were confirmed by transcriptional and translational analysis. Conclusions Identification of bacterial metabolite PAA as a T3SS-specific inhibitor explains this intriguing inverse cell-density-dependent-cytotoxicity phenomenon as T3SS is known to be a key virulence factor associated with cytotoxicity and acute infection. The findings may provide useful clues for design and development of new strategies to combat this formidable bacterial pathogen.

Emerging resistance to current antibiotics raises the need for new microbial drug targets. We show that targeting branched-chain amino acid (BCAA) biosynthesis using sulfonylurea herbicides, which inhibit the BCAA biosynthetic enzyme acetohydroxyacid synthase (AHAS), can exert bacteriostatic effects on several pathogenic bacteria, including Burkholderia pseudomallei , Pseudomonas aeruginosa , and Acinetobacter baumannii . Our results suggest that targeting biosynthetic enzymes like AHAS, which are lacking in humans, could represent a promising antimicrobial drug strategy.

Quorum sensing in Burkholderia cenocepacia H111 involves two signalling systems that depend on different signal molecules, namely N-acyl homoserine lactones (AHLs) and the diffusible signal factor cis-2-dodecenoic acid (BDSF). Previous studies have shown that AHLs and BDSF control similar phenotypic traits, including biofilm formation, proteolytic activity and pathogenicity. In this study we mapped the BDSF stimulon by RNA-Seq and shotgun proteomics analysis. We demonstrate that a set of the identified BDSF-regulated genes or proteins are also controlled by AHLs, suggesting that the two regulons partially overlap. The detailed analysis of two mutually regulated operons, one encoding three lectins and the other one encoding the large surface protein BapA and its type I secretion machinery, revealed that both AHLs and BDSF are required for full expression, suggesting that the two signalling systems operate in parallel. In accordance with this, we show that both AHLs and BDSF are required for biofilm formation and protease production.

The diffusible factor synthase XanB2, originally identified in Xanthomonas campestris pv. campestris (Xcc), is highly conserved across a wide range of bacterial species, but its substrate and catalytic mechanism have not yet been investigated. Here, we show that XanB2 is a unique bifunctional chorismatase that hydrolyses chorismate, the end-product of the shikimate pathway, to produce 3-hydroxybenzoic acid (3-HBA) and 4-HBA. 3-HBA and 4-HBA are respectively associated with the yellow pigment xanthomonadin biosynthesis and antioxidant activity in Xcc. We further demonstrate that XanB2 is a structurally novel enzyme with three putative domains. It catalyses 3-HBA and 4-HBA biosynthesis via a unique mechanism with the C-terminal YjgF-like domain conferring activity for 3-HBA biosynthesis and the N-terminal FGFG motif-containing domain responsible for 4-HBA biosynthesis. Furthermore, we show that Xcc produces coenzyme Q8 (CoQ8) via a new biosynthetic pathway independent of the key chorismate-pyruvate lyase UbiC. XanB2 is the alternative source of 4-HBA for CoQ8 biosynthesis. The similar CoQ8 biosynthetic pathway, xanthomonadin biosynthetic gene cluster and XanB2 homologues are well conserved in the bacterial species within Xanthomonas, Xylella, Xylophilus, Pseudoxanthomonas, Rhodanobacter, Frateuria, Herminiimonas and Variovorax, suggesting that XanB2 may be a conserved metabolic link between the shikimate pathway, ubiquinone and xanthomonadin biosynthetic pathways in diverse bacteria.

Many bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals in modulation of virulence and biofilm formation. Previous work on Xanthomonas campestris showed that the RpfC/RpfG two-component system is involved in sensing and responding to DSF signals, but little is known in other microorganisms. Here we show that in Burkholderia cenocepacia the DSF-family signal cis -2-dodecenoic acid (BDSF) negatively controls the intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) level through a receptor protein RpfR, which contains Per/Arnt/Sim (PAS)-GGDEF-EAL domains. RpfR regulates the same phenotypes as BDSF including swarming motility, biofilm formation, and virulence. In addition, the BDSF − mutant phenotypes could be rescued by in trans expression of RpfR, or its EAL domain that functions as a c-di-GMP phosphodiesterase. BDSF is shown to bind to the PAS domain of RpfR with high affinity and stimulates its phosphodiesterase activity through induction of allosteric conformational changes. Our work presents a unique and widely conserved DSF-family signal receptor that directly links the signal perception to c-di-GMP turnover in regulation of bacterial physiology.

The polymorphism and similarity relationships among 35 mating-type isolates of Ustilago scitaminea collected from Southern China were determined with random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses. These fungal isolates were collected from 16 sugarcane cultivars including F134 that is resistant to the physiological race 1 but susceptible to the race 2 of U. scitaminea, and N: Co376 that is immune to both races 1 and 2. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis revealed that the U. scitaminea isolates could be divided into 2 groups with a coefficient of 0.74. The first group comprises two isolates collected from the sugarcane cultivar F134, while the remaining 33 isolates were clustered into the second group. The second group was further divided into two subgroups with most of the isolates from Guangdong Province which clustered in the same subgroup, and all the isolates from Guangxi and Yunnan Provinces were clustered in another subgroup. Given that the member of the second group could infect the cultivar N:Co376, which is immune to the races 1 and 2, our results suggest that majority of U. scitaminea in sugarcane-producing regions of Southern China may belong to or genetically similar to race 3. Key words: Ustilago scitaminea , sugarcane, inter-simple sequence repeat (ISSR), random amplified polymorphic deoxyribonucleic acid (DNA) (RAPD), genetic diversity.

Secretory effector proteins expressed within the esophageal glands of root-knot nematodes (Meloidogyne spp.) are thought to play key roles in nematode invasion of host roots and in formation of feeding sites necessary for nematodes to complete their life cycle. In this study, a novel effector protein gene designated as Mj-nulg1a, which is expressed specifically within the dorsal gland of Meloidogyne javanica, was isolated through suppression subtractive hybridization. Southern blotting and BLAST search analyses showed that Mj-nulg1a is unique for Meloidogyne spp. A real-time reverse-transcriptase polymerase chain reaction assay showed that expression of Mj-nulg1a was upregulated in parasitic second-stage juveniles and declined in later parasitic stages. MJ-NULG1a contains two putative nuclear localization signals and, consistently, in planta immunolocalization analysis showed that MJ-NULG1a was localized in the nuclei of giant cells during nematode parasitism. In planta RNA interference targeting Mj-nulg1a suppressed the expression of Mj-nulg1a in nematodes and attenuated parasitism ability of M. javanica. In contrast, transgenic Arabidopsis expressing Mj-nulg1a became more susceptible to M. javanica infection than wild-type control plants. These results depict a novel nematode effector that is targeted to giant cell nuclei and plays a critical role in M. javanica parasitism.

Current methods for identifying transcription start sites (TSSs) of specific genes in bacteria usually require adaptors or radioactive labeling. These approaches can be technically demanding and environmentally unfriendly. Here we present a method for identifying TSS called ARF-TSS, which is based on cDNA generation, circularization, PCR amplification, and DNA sequencing to determine the 5′-end of transcripts, thus circumventing the need for adaptors and radioactive labeling. We validated the method using the gene lasI from the bacterial pathogen Pseudomonas aeruginosa. Our results show that ARF-TSS could be a good alternative to traditional methods for bacterial TSS analysis.

The type III secretion system (T3SS) of Pseudomonas aeruginosa is a key virulence determinant whose expression is induced by polyamine signals from mammalian host. SpuD and SpuE were postulated to be spermidine-preferential binding proteins, which regulate the polyamine content in this bacterial pathogen. In this study, we found that SpuD is a putrescine-preferential binding protein, while SpuE binds to spermidine exclusively. We have determined the crystal structures of SpuD in free form and in complex with putrescine and SpuE in free form and in complex with spermidine. Upon ligand binding, SpuD and SpuE undergo an "open-to-closed" conformational switch with the resultant closed ligand-bound forms, SpuD-putrescine and SpuE-spermidine, similar to their Escherichia coli counterparts PotF-putrescine and PotD-spermidine, respectively. Structural comparison suggested that two aromatic residues, Trp271 of SpuE and Phe273 of SpuD in segment II region, are the key structural determinants for putrescine/spermidine recognition specificity. Mutagenesis combined with isothermal titration calorimetry showed that substitution of Trp271 by Phe enabled SpuE to gain substantial binding affinity for putrescine, while replacement of Phe273 by Trp reduced the binding affinity of SpuD toward putrescine by 250-fold. Altogether, these results revealed the molecular mechanism governing polyamine recognition specificity by SpuD and SpuE and provide the basis for further structural and functional studies of polyamine signal importation system in P. aeruginosa.

Conjugal transfer of the nopaline-type Agrobacterium Ti plasmid pTiC58 is regulated by a transcriptional activator, TraR, and a diffusible signal molecule, conjugation factor (CF). CF is a member of a family of substituted homoserine lactones (HSLs) that act as coinducers for regulating gene expression in diverse Gram-negative bacteria by a mechanism called autoinduction. In Vibrio fischeri HSL production is conferred by the luxI gene. Homologues of this gene are responsible for HSL production by other Gram-negative bacteria. A gene that we call traI, conferring production of material with CF activity, was localized to a 1-kb region at the upstream end of tra3 of pTiC58. Spectroscopy showed that the activity was authentic CF. Sequence analysis showed that traI could encode a protein of 211 amino acids, TraI, that is related to the proteins responsible for HSL production by other bacteria. A second, partial open reading frame immediately downstream of traI could encode a protein related to TrbB of plasmid RP4, which is required for conjugal transfer. Transcription of traI and of the downstream tra3 genes requires TraR and CF and initiates from the traI promoter. The results show that traI is responsible for CF production, that it is the first gene of the tra3 operon, and that expression of this operon is regulated by autoinduction.

1. Introduction 1602. Chemistry of DSF-Family Signals 1612.1. Detection of DSF-Family Signals 1612.2. Purification and Structural Characterization ofDSF-Family Signals1622.3. Synthesis of DSF-Family Signals 1632.4. Nomenclature 1632.5. Structural Features and Biological Activity 1633. DSF Signaling Mechanisms in

Findings from previous studies suggest that the quorum sensing signal DSF (diffusible signal factor) negatively regulates biofilm formation in Xanthomonas campestris pv. campestris (Xcc) by affecting the expression of manA encoding biofilm dispersion and an unknown factor(s). In this study, by analysing the double deletion mutant ΔrpfFΔmanA, in which DSF biosynthesis gene rpfF and biofilm dispersal gene manA were deleted, we found that DSF modulated biofilm development by suppression of a mechanism essential for biofilm formation. Transposon mutagenesis of ΔrpfFΔmanA and subsequent analyses led to the identification of a novel gene locus xagABC encoding a putative glycosyl transferase system. Genetic analysis revealed that the transcriptional expression of xagABC was negatively regulated by DSF through the RpfC/RpfG two-component regulatory system. Deletion of the xag genes resulted in decreased extracellular polysaccharide production, abolished Xcc biofilm formation and attenuated the bacterial resistance to oxidative stress. Furthermore, we provide evidence that xagABC and manA were differentially expressed in Xcc and the biofilm formed by overexpression of xagABC in wild-type Xcc could be dispersed by ManA. These results provide new insight into the molecular mechanisms by which Xcc switches between planktonic growth and biofilm lifestyle.

Previous work has shown that Burkholderia cenocepacia produces the diffusible signal factor (DSF) family signal cis -2-dodecenoic acid (C 12 :Δ 2 , also known as BDSF), which is involved in the regulation of virulence. In this study, we determined whether C 12 :Δ 2 production is conserved in other members of the Burkholderia cepacia complex (Bcc) by using a combination of high-performance liquid chromatography, mass spectrometry, and bioassays. Our results show that five Bcc species are capable of producing C 12 :Δ 2 as a sole DSF family signal, while four species produce not only C 12 :Δ 2 but also a new DSF family signal, which was identified as cis , cis -11-methyldodeca-2,5-dienoic acid (11-Me-C 12 :Δ 2,5 ). In addition, we demonstrate that the quorum-sensing signal cis -11-methyl-2-dodecenoic acid (11-Me-C 12 :Δ 2 ), which was originally identified in Xanthomonas campestris supernatants, is produced by Burkholderia multivorans . It is shown that, similar to 11-Me-C 12 :Δ 2 and C 12 :Δ 2 , the newly identified molecule 11-Me-C 12 :Δ 2,5 is a potent signal in the regulation of biofilm formation, the production of virulence factors, and the morphological transition of Candida albicans . These data provide evidence that DSF family molecules are highly conserved bacterial cell-cell communication signals that play key roles in the ecology of the organisms that produce them.

Ustilago maydis is an important model fungal organism for diverse studies. Little improvement has been made in the method for its transformation since the PEG-mediated transfection of spheroplasts that was reported more than 20years ago. We have constructed binary T-DNA vectors carrying Hygromycin and Nourseothricin resistance gene cassettes and have developed a highly efficient method for transformation of this fungus based on Agrobacterium tumefaciens-mediated transformation (ATMT). Through a series of optimization, at least 1x10(4) Hygromycin B resistant colony forming units (CFU) have been achieved on each 90mm agar plate using 10(6) sporidia. Optimal pH value for ATMT is approximately 5.6. Approximately 96% Hygromycin B-resistant transformants contain a single-copy T-DNA inserted into the nuclear genome. Analysis of 204 T-DNA flanking sequences showed that 15.2% of them were found in the coding sequences and a further 37.25% within 0.5kb from the coding sequences at the 5' UTR or promoter regions. In addition, a method for preparation and preservation of transformation-ready T-DNA donor and receptor cells has been developed allowing gene tagging experiments to be performed on-demand. An initial screening of 5000 mutants resulted in the identification of a putative farnesyl transferase beta subunit and a PRE6 homologue as new players of sexual mating in U. maydis.

Background Many gram-negative bacterial pathogens employ Type III secretion systems (T3SS) to inject effector proteins into host cells in infectious processes. Methodology/Principal Findings By screening a transposon mutant library of P. aeruginosa, we found that mutation of spuDEFGH, which encode a major spermidine uptake system, abolished the expression of the exsCEBA operon that codes for key T3SS regulators under inducing conditions (low calcium). Whole genome microarray analysis revealed that inactivation of the spermidine uptake system significantly decreased the transcriptional expression of most, if not all, T3SS genes. Consistently, the spermidine uptake mutants showed decreased expression of the T3SS genes in responding to host cell extract and attenuated cytotoxicity. Furthermore, exogenous addition of spermidine to the wild type strain PAO1 enhanced the expression of exsCEBA and also the effector protein genes. Conclusion/Significance Cumulatively, these data have depicted a novel spermidine transporter-dependent signaling pathway, which appears to play an essential role in modulation of T3SS expression in P. aeruginosa.

In addition to producing lethal antibiotics, microorganisms may also use a new form of antagonistic mechanism in which signal molecules are exported to influence the gene expression and hence the ecological competence of their competitors. We report here the isolation and characterization of a novel signaling molecule, cis-2-dodecenoic acid (BDSF), from Burkholderia cenocepacia. BDSF is structurally similar to the diffusible signal factor (DSF) that is produced by the RpfF enzyme of Xanthomonas campestris. Deletion analysis demonstrated that Bcam0581, which encodes an RpfF homologue, was essential for BDSF production. The gene is highly conserved and widespread in the Burkholderia cepacia complex. Exogenous addition of BDSF restored the biofilm and extracellular polysaccharide production phenotypes of Xanthomonas campestris pv. campestris DSF-deficient mutants, highlighting its potential role in inter-species signaling. Further analyses showed that Candida albicans germ tube formation was strongly inhibited by either coculture with B. cenocepacia or by exogenous addition of physiological relevant levels of BDSF, whereas deletion of Bcam0581 abrogated the inhibitory ability of the bacterial pathogen. As B. cenocepacia and C. albicans are frequently encountered human pathogens, identification of the BDSF signal and its activity thus provides a new insight into the molecular grounds of their antagonistic interactions whose importance to microbial ecology and pathogenesis is now becoming evident.

Many Gram-negative bacterial pathogens employ N-acyl homoserine lactones (AHLs) quorum-sensing signals for regulation of various biological functions. Several types of AHL-inactivating enzymes, also known as quorum-quenching enzymes, have been unveiled in recent years. These enzymes seem to play important roles in microbial physiology and ecology and hold promising potential in biotechnology. This unit describes methods based on a simple diffusion plate assay for qualitative detection and quantitative measurement of AHL-inactivating enzyme activity. The qualitative detection method is suitable for rapid and large-scale screening and identification of quorum-quenching enzymes. Furthermore, HPLC methods are provided for accurate determination of whether the quorum-quenching enzyme is a lactonase or an acylase. The unit also presents concise background information on the quorum-quenching enzymes identified from various sources, including bacterial and mammalian species.

The bacterial pathogen Xanthomonas campestris pv. campestris co-ordinates virulence factor production and biofilm dispersal through a diffusible signal factor (DSF)-mediated cell-cell communication mechanism. The RpfC/RpfG two-component system plays a key role in DSF signal transduction and appears to modulate downstream DSF regulon by changing intracellular content of cyclic dimeric GMP (c-di-GMP), an unusual nucleotide second messenger. Here we show that Clp, a conserved global regulator showing a strong homology to the cAMP nucleotide receptor protein Crp of Escherichia coli, is essential for DSF regulation of virulence factor production but not for biofilm dispersal. Deletion of clp in Xcc changed the transcriptional expression of 299 genes including a few encoding transcription factors. Further genetic and microarray analysis led to identification of a homologue of the transcriptional regulator Zur, and a novel TetR-type transcription factor FhrR. These two regulatory factors regulated different sets of genes within Clp regulon. These results outline a hierarchical signalling network by which DSF modulates different biological functions, and may also provide a clue on how the novel nucleotide signal can be coupled to its downstream regulatory networks.

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 Å. 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.

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.

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.

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.

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.

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.

Two manganese peroxidase (MnP) active fractions D1 and D2 were got from the extracellular culture of Trametes versicolor by using ammonium sulfate precipitation, DEAE-cellulose DE52 chromatography. MnP1 was purified to electrophoretic homogeneity from the D2 by Phenyl Sepharose 6 Fast Flow chromatography and MnP2 was purified to electrophoretic homogeneity from the D1 by Sephacryl S-200HR chromatography and Phenyl Sepharose 6 Fast Flow chromatography. The specific activities of two MnP isozymes are 579.1U/mg and 425.0U/mg; purification folds are 17.51 and 12.85 and the yields are 6.17% and 2.47%, respectively. MnP1 and MnP2 have approximate molecular masses of 46.3kD and 43.0kD respectively, as determined by SDS-PAGE. The isoenzymes differed in optimum temperature (60degreesC and 65degreesC) and optimum pH(5.8 and 6.2) for oxidation of DMP (2,6-dimethoxyphenol). MnP1 and MnP2 are stable below 45degreesC and ranging from pH4.0 to pH7.0. DMP is the best substrate, the Km values of MnP1 and MnP2 for DMP are 13.43micromol/L and 12.45micromol/L respectively. Catalysis doesn't occur in the complete absence of Mn. EDTA inhibites the activities of MnP1 and MnP2 at the higher concentration and DTT inhibites the enzyme activities completely.

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.

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.

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.

Cells of strain LX3T, isolated from soil, were Gram-negative, facultatively anaerobic, non-motile, capsulated and non-endospore-forming straight rods, able to grow at 10 °C, unable to produce gas from lactose at 45 °C and unable to produce indole. The isolate converted sucrose to isomaltulose and did not produce detectable glucose by-products. The G+C content of the DNA was 56·4 mol%. Furthermore, comparison of 16S rRNA and rpoB gene sequences showed that the isolate clearly belongs to the genus Klebsiella. The closest phylogenetic relative was Klebsiella pneumoniae, there being 99·3 and 97·5 % similarity in 16S rRNA and rpoB gene sequences, respectively. DNA–DNA hybridization analysis demonstrated a very low level of relatedness to other members of the genus Klebsiella, indicating that the isolated strain and other species in the genus Klebsiella were not related at the species level. The isolate could be differentiated from other previously described members of the genus Klebsiella on the basis of phenotypic differences and 16S rRNA and rpoB gene sequence divergence, together with DNA–DNA reassociation data. Therefore, it is proposed that strain LX3T (=DSM 16265T=JCM 12419T) should be classified as the type strain of a novel species of genus Klebsiella, Klebsiella singaporensis sp. nov.

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.

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.

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.

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 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.

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°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 γ- 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.

To identify and clone the gene named pfstom gene which encoding the protein belonging to band 7 family and to do primary research on its function.Based on the finished data in international public malaria database, coding sequence of pfstom cDNA was obtained by RT-PCR from FCC1/HN. Its phylogenetic profiles and the homogeny were analyzed by some softwares. After Prokaryotic expression, C terminal of Pfstom protein was expressed by Pet30a system. Recombinant Pfstom protein was used to immol/Lunize rabbit and then serum was harvested and the IgG was purified for Western blot.The coding sequence of pfstom is 1,125 bp which encoding 374 amino acids with C-terminal fragment being homogenous to stomatin-like protein which belongs to band 7 family. Phylogenetic profiles analysis revealed its homogeny to stomatin. Western blot showed its stage-specific expression in trophozoite.Pfstom belongs to band-7 family. It was expressed specifically in trophozoite in erythrocyte stage of plasmodium falciparum. It was not expressed in ring stage. And it is membrane-related protein. All these results provided the foundation for further research on pfstom.

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.

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.

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.

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.

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.

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 Tn 5 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.

Pseudomonas aeruginosa relies on cell motility and ability to form biofilms to establish infections, however, the mechanism of regulation remains obscure. Here we report that BswR, a XRE (Xenobiotic Response Element) type transcriptional regulator, plays a critical role in regulation of bacterial motility and biofilm formation in P. aeruginosa. Transcriptomic and biochemical analyses showed that BswR counteracts the repressor activity of MvaT, controls the transcription of small RNA rsmZ and regulates the biogenesis of bacterial flagella. The crystal structure of BswR was determined at 2.3 Å resolution; the monomer comprises a DNA-binding domain with a helix-turn-helix (HTH) motif in the N terminus and two helices (6 and 7) with a V-shaped arrangement in the C-terminus. In addition to the contacts between the parallel helices α5 of two monomers, the two helical extensions (6 and 7) intertwine together to form a homodimer, which is the biological function unit. Based on the result of DNase I protection assay together with structural analysis BswR homodimer, we proposed a BswR-DNA model, which suggests a molecular mechanism with which BswR could interact with DNA. Taken together, our results unveiled a novel regulatory mechanism, in which BswR controls the motility and biofilm formation of P. aeruginosa by modulating the transcription of small RNA rsmZ.

Xanthomonas albilineans produces a family of highly potent antibiotics and phytotoxins called albicidins, which are key pathogenesis factors in the systemic development of leaf scald disease of sugarcane. A gene (xabA) required for albicidin biosynthesis encodes a peptide of 278 amino acids, including the signature sequence motifs for phosphopantetheinyl transferases (PPTases) that activate polyketide and non-ribosomal peptide synthetases. The Escherichia coli entD gene, which encodes a PPTase involved in biosynthesis of enterobactin (a siderophore), restored biosynthesis of albicidin (a DNA replication inhibitor) in X. albilineans Tox(-) LS156 (xabA::Tn5). We conclude that XabA is a PPTase required for post-translational activation of synthetases in the albicidin biosynthetic pathway. This is the first antibiotic or toxin biosynthesis gene characterized from the genus Xanthomonas, and the first demonstration that antibiotic synthetases in the Pseudomonadaceae, like those in the Enterobacteriaceae and in Gram-positive bacteria, can require activation by a PPTase. Coupled with the recent demonstration of a separate albicidin biosynthetic gene cluster, the results indicate the possibility for overproduction of albicidins, which allows better understanding and application of these potent inhibitors of prokaryote DNA replication. (C) 2000 Elsevier Science B.V. All rights reserved.