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Biomedical subjects

Clay Fuqua

Publications and source records attributed to Clay Fuqua.

At least 19 recordsLinked to original sources
PubMed2006

Growth on dichlorobiphenyls with chlorine substitution on each ring by bacteria isolated from contaminated African soils.

Until recently, it was generally believed that the presence of more than one chlorine substituent prevented chlorinated biphenyls from serving as a sole source of carbon and energy for aerobic bacteria. In this study, we report the isolation of three aerobic strains, identified as Enterobacter sp. SA-2, Ralstonia sp. SA-4, and Pseudomonas sp. SA-6 from Nigerian polluted soils, that were able to grow on a wide range of dichlorobiphenyls (diCBs). In addition to growing on all monochlorobiphenyls (monoCBs), the strains were all able to utilize 2,2'-, 2,4'-, and 2,3-diCB as a sole source of carbon and energy. With the exception of strain SA-2, growth was also sustainable on 3,3'-, and 3,5-diCB. Washed benzoate-grown cells were typically able to degrade 68 to 100% of the diCB (100 ppm) within 188 h, concomitant with a cell number increase of up to three orders-of-magnitude and elimination of varying amounts of chloride. In many cases, stoichiometric production of a chlorobenzoate (CBA) as a product was observed. During growth on 2,2'-, and 2,4'-diCB, organisms exclusively attacked an o-chlorinated ring resulting in the production of 2-CBA and 4-CBA, respectively. A gradual decline in the concentration of the latter was observed, which suggested that the product was being degraded further. In the case of 2,3-diCB, the unsubstituted ring was preferentially metabolized. Initial diCB degradation rates were greatest for 2,4'-diCB (11.2 +/- 0.91 to 30.3 +/- 7.8 nmol/min per 10(9) cells) and lowest for 2,2'-diCB (0.37 +/- 0.12 to 2.7 +/- 1.2 nmol/min per 10(9) cells).

Biodegradation, Environmental↗
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

Aerobic degradation of di- and trichlorobenzenes by two bacteria isolated from polluted tropical soils.

Two polychlorinated biphenyl (PCBs)-degrading bacteria were isolated by traditional enrichment technique from electrical transformer fluid (Askarel)-contaminated soils in Lagos, Nigeria. They were classified and identified as Enterobacter sp. SA-2 and Pseudomonas sp. SA-6 on the basis of 16S rRNA gene analysis, in addition to standard cultural and biochemical techniques. The strains were able to grow extensively on dichloro- and trichlorobenzenes. Although they failed to grow on tetrachlorobenzenes, monochloro- and dichlorobenzoic acids, they were able to utilize all monochlorobiphenyls, and some dichlorobiphenyls as sole sources of carbon and energy. The effect of incubation with axenic cultures on the degradation of 0.9 mM 1,4-dichlorobenzene, 0.44 mM 1,2,3- and 0.43 mM 1,3,5-trichlorobenzene in mineral salts medium was studied. Approximately, 80-90% of these xenobiotics were degraded in 200 h, concomitant with cell increase of up to three orders of magnitude, while generation times ranged significantly (P<0.05) from 17-32 h. Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were detected in crude cell-free extracts of cultures pre-grown with benzoate, with the latter enzyme exhibiting a slightly higher activity (0.15-0.17 micromolmin(-1) mg of protein(-1)) with catechol, suggesting that the meta-cleavage pathway is the most readily available catabolic route in the SA strains. The wider substrate specificity of these tropical isolates may help in assessing natural detoxification processes and in designing bioremediation and bioaugmentation methods.

Aerobiosis↗
PubMed2006

Quorum sensing and motility mediate interactions between Pseudomonas aeruginosa and Agrobacterium tumefaciens in biofilm cocultures.

In the environment, multiple microbial taxa typically coexist as communities, competing for resources and, often, physically associated within biofilms. A dual-species cocultivation model has been developed by using two ubiquitous and well studied microbes Pseudomonas aeruginosa (P.a.) and Agrobacterium tumefaciens (A.t.) as a tractable system to identify molecular mechanisms that underlie multispecies microbial associations. Several factors were found to influence coculture interactions. P.a. had a distinct growth-rate advantage in cocultures, increasing its relative abundance during planktonic and biofilm growth. P.a. also demonstrated a slight quorum-sensing-dependent increase in growth yield in liquid cocultures. P.a. dominated coculture biofilms, "blanketing" or burying immature A.t. microcolonies. P.a. flagellar and type IV pili mutant strains exhibited deficient blanketing and impaired competition in coculture biofilms, whereas, in planktonic coculture, these mutations had no effect on competition. In contrast, A.t. used motility to emigrate from coculture biofilms. In both planktonic and biofilm cocultures, A.t. remained viable for extended periods of time, coexisting with its more numerous competitor. These findings reveal that quorum-sensing-regulated functions and surface motility are important microbial competition factors for P.a. and that the outcome of competition and the relative contribution of different factors to competition are strongly influenced by the environment in which they occur.

Agrobacterium tumefaciens↗
PubMed2006

Detection of Anaplasma phagocytophilum and Babesia odocoilei DNA in Ixodes scapularis (Acari: Ixodidae) collected in Indiana.

The blacklegged tick, Ixodes scapularis Say, first reported in Indiana in 1987, has now been detected in more than half of Indiana's counties. The first case of human granulocytic ehrlichiosis (human anaplasmosis) in Indiana was reported in 2002. We now report the detection of Anaplasma phagocytophilum and Babesia odocoilei (Emerson and Wright 1968) in I. scapularis ticks collected in northern Indiana. Using polymerase chain reaction analysis, 41 of 193 adult ticks (21.2%) collected from deer were positive for A. phagocytophylum, and 22 (11.4%) were positive for Babesia sp. Restriction fragment analysis of 12, and sequencing of another five of the amplified products identified these parasites as B. odocoilei. Five ticks (2.6%) were coinfected. Eight of 68 questing adult ticks (11.8%) were positive for A. phagocytophilum; seven (10.3%) were positive for Babesia sp. Six of the latter seven positive samples were determined to be B. odocoilei by restriction fragment analysis and sequencing of two samples. None of 39 pools of nymphs was positive for Babesia sp. Three of 15 ticks (20%) collected from a dog were positive for A. phagocytophilum and three ticks (20%) were positive for Babesia sp. One was confirmed as B. odocoilei. One tick was coinfected. This is the first report of the presence of these two agents in ticks in Indiana.

Anaplasma phagocytophilum↗
PubMed2005

The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens.

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic beta-(1,2)-D-glucans, respectively. We have identified two genes (celG and cell) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.

Agrobacterium tumefaciens↗
PubMed2005

Cell-cell influences on bacterial community development in aquatic biofilms.

Dialysis tubing containing spent culture media, when placed in a lake, was colonized by a low diversity of bacteria, whereas abiotic controls had considerable diversity. Changes were seen in the presence and absence of acylated homoserine lactones, suggesting that these molecules and other factors may influence adherent-population composition.

Agrobacterium tumefaciens↗
PubMed2004

Genome sequence of Silicibacter pomeroyi reveals adaptations to the marine environment.

Since the recognition of prokaryotes as essential components of the oceanic food web, bacterioplankton have been acknowledged as catalysts of most major biogeochemical processes in the sea. Studying heterotrophic bacterioplankton has been challenging, however, as most major clades have never been cultured or have only been grown to low densities in sea water. Here we describe the genome sequence of Silicibacter pomeroyi, a member of the marine Roseobacter clade (Fig. 1), the relatives of which comprise approximately 10-20% of coastal and oceanic mixed-layer bacterioplankton. This first genome sequence from any major heterotrophic clade consists of a chromosome (4,109,442 base pairs) and megaplasmid (491,611 base pairs). Genome analysis indicates that this organism relies upon a lithoheterotrophic strategy that uses inorganic compounds (carbon monoxide and sulphide) to supplement heterotrophy. Silicibacter pomeroyi also has genes advantageous for associations with plankton and suspended particles, including genes for uptake of algal-derived compounds, use of metabolites from reducing microzones, rapid growth and cell-density-dependent regulation. This bacterium has a physiology distinct from that of marine oligotrophs, adding a new strategy to the recognized repertoire for coping with a nutrient-poor ocean.

Adaptation, Physiological↗
PubMed2004

Biofilm formation in plant-microbe associations.

Bacteria adhere to environmental surfaces in multicellular assemblies described as biofilms. Plant-associated bacteria interact with host tissue surfaces during pathogenesis and symbiosis, and in commensal relationships. Observations of bacteria associated with plants increasingly reveal biofilm-type structures that vary from small clusters of cells to extensive biofilms. The surface properties of the plant tissue, nutrient and water availability, and the proclivities of the colonizing bacteria strongly influence the resulting biofilm structure. Recent studies highlight the importance of these structures in initiating and maintaining contact with the host by examining the extent to which biofilm formation is an intrinsic component of plant-microbe interactions.

Bacteria↗
PubMed2004

A simple screening protocol for the identification of quorum signal antagonists.

Quorum sensing (QS) is a mechanism by which diverse microorganisms can control specific processes in response to population density. A relatively well-known form of QS among Proteobacteria involves production and subsequent response to acylated homoserine lactones (AHLs). Quorum sensing inhibition (QSI), targeting AHL-dependent signaling, has been reported as a strategy for the control of biofilm formation used by several marine organisms. We developed a simple soft agar overlay protocol, based on pigmentation inhibition, to rapidly screen for the presence of potential QSI by bacteria and plants. For bacterial screens, test organisms are first streaked onto their appropriate media and incubated overnight. For plant screens, the plant material (leaf, stem, flower, etc.) is placed onto LB agar. The bacterial growth or plant samples are then covered with an overlay of LB soft agar containing an inoculum of either Pseudomonas aureofaciens 30-84 or Chromobacterium violaceum ATCC 12472 (indicator cultures) and then incubated overnight. These indicator bacteria regulate pigment production by N-hexanoyl-HSL (C6-HSL) QS and are readily inhibited by AHL analogues and other antagonists. QSI is indicated by the lack of pigment production of the indicator culture in the vicinity of the test sample. Growth inhibition of the indicator culture indicates possible antibiotic production. Two different biosensor organisms based on derivatives of Agrobacterium tumefaciens and C. violaceum, capable of detecting a range of AHLs were used to determine whether QSI is due to the production of interfering AHLs competing with the C6-HSL regulation of C. violaceum and P. aureofaciens pigment production. This simple protocol will facilitate the screening of multiple organisms for the production of potential antifouling compounds.

4-Butyrolactone↗
PubMed2004

The FNR-type transcriptional regulator SinR controls maturation of Agrobacterium tumefaciens biofilms.

Agrobacterium tumefaciens is a plant pathogen that persists as surface-associated populations on plants or soil particles. A genetic screen for A. tumefaciens mutants deficient for surface interactions identified a mutant that forms thin, sparsely populated biofilms, but is proficient for initial attachment. The mutant is disrupted in a gene designated sinR, encoding a member of the DNR subfamily of FNR-type transcription regulators. SinR is required for normal maturation of A. tumefaciens biofilms on both inert surfaces and plant tissues, and elevated sinR expression results in accelerated biofilm formation. Expression of sinR is increased close to 30-fold in cultures grown in oxygen-limited environments and is also induced within biofilms grown under oxic conditions. A consensus FNR box, the presumptive binding site for FNR-type proteins, is located upstream of the sinR promoter. FnrN, a second A. tumefaciens FNR-like regulator, is required for induction of sinR in oxygen-limited cultures, whereas SinR negatively influences its own expression. FnrN influences biofilm formation, but its effects are less dramatic than those of SinR. We propose a model in which a signal cascade, responsive to oxygen limitation and initiated by FnrN, activates sinR expression in response to decreased oxygen levels, and influences the formation of A. tumefaciens biofilms.

Agrobacterium tumefaciens↗
PubMed2004

Quorum-sensing antiactivator TraM forms a dimer that dissociates to inhibit TraR.

The quorum-sensing transcriptional activator TraR of Agrobacterium tumefaciens, which controls the replication and conjugal transfer of the tumour-inducing (Ti) virulence plasmid, is inhibited by the TraM antiactivator. The crystal structure of TraM reveals this protein to form a homodimer in which the monomer primarily consists of two long coiled alpha-helices, and one of the helices from each monomer also bundles to form the dimeric interface. The importance of dimerization is addressed by mutational studies in which disruption of the hydrophobic dimer interface leads to aggregation of TraM. Biochemical studies confirm that TraM exists as a homodimer in solution in equilibrium with the monomeric form, and also establish that the TraM-TraR complex is a heterodimer. Thus, the TraM homodimer undergoes dissociation in forming the antiactivation complex. Combined with the structure of TraR (Zhang et al., 2002, Nature 417: 971-974; Vannini et al., 2002, EMBO J 21: 4393-4401), our structural analysis suggests overlapping interactive surfaces in homodimeric TraM with those in the TraM-TraR complex and a mechanism for TraM inhibition on TraR.

Agrobacterium tumefaciens↗
PubMed2004

Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system.

The plant pathogen Agrobacterium tumefaciens forms architecturally complex biofilms on inert surfaces. Adherence of A. tumefaciens C58 was significantly enhanced under phosphate limitation compared to phosphate-replete conditions, despite slower overall growth under low-phosphate conditions. Replacement of Pi with sn-glycerol-3-phosphate and 2-aminoethylphosphonate yielded similar results. The increase in surface interactions under phosphate limitation was observed in both static culture and continuous-culture flow cells. Statistical analysis of confocal micrographs obtained from the flow cell biofilms revealed that phosphate limitation increased both the overall attached biomass and the surface coverage, whereas the maximum thickness of the biofilm was not affected. Functions encoded on the two large plasmids of A. tumefaciens C58, pTiC58 and pAtC58, were not required for the observed phosphate effect. The phosphate concentration at which increased attachment was observed triggered the phosphate limitation response, controlled in many bacteria by the two-component regulatory system PhoR-PhoB. The A. tumefaciens phoB and phoR orthologues could only be disrupted in the presence of plasmid-borne copies of the genes, suggesting that this regulatory system might be essential. Expression of the A. tumefaciens phoB gene from a tightly regulated inducible promoter, however, correlated with the amount of biofilm under both phosphate-limiting and nonlimiting conditions, demonstrating that components of the Pho regulon influence A. tumefaciens surface interactions.

Agrobacterium tumefaciens↗
PubMed2003

Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus.

Bacteria communicate at high cell density through quorum sensing, however, there are no reports about this mechanism in archaea. The archaeon Natronococcus occultus produces an extracellular protease at the end of growth. Early production of protease activity was observed when a low density culture was incubated with late exponential conditioned medium suggesting the presence of factor(s) inducing this activity. Conditioned medium and ethyl acetate extracts corresponding to the transition from exponential to stationary phase showed a positive signal in Agrobacterium biosensor. We report the detection of potential autoinducer molecules of the acylated homoserine lactone type in the archaeon N. occultus. These molecules may be responsible for the production/activation of extracellular protease.

Culture Media↗