Bacterial Chemotaxis to Atrazine and Related s-Triazines

Pseudomonas sp. strain ADP utilizes the human-made s-triazine herbicide atrazine as the sole nitrogen source. The results reported here demonstrate that atrazine and the atrazine degradation intermediates N-isopropylammelide and cyanuric acid are chemoattractants for strain ADP. In addition, the nonmetabolized s-triazine ametryn was also an attractant. The chemotactic response to these s-triazines was not specifically induced during growth with atrazine, and atrazine metabolism was not required for the chemotactic response. A cured variant of strain ADP (ADP M13-2) was attracted to s-triazines, indicating that the atrazine catabolic plasmid pADP-1 is not necessary for the chemotactic response and that atrazine degradation and chemotaxis are not genetically linked. These results indicate that atrazine and related s-triazines are detected by one or more chromosomally encoded chemoreceptors in Pseudomonas sp. strain ADP. We demonstrated that Escherichia coli is attracted to the s-triazine compounds N-isopropylammelide and cyanuric acid, and an E. coli mutant lacking Tap (the pyrimidine chemoreceptor) was unable to respond to s-triazines. These data indicate that pyrimidines and triazines are detected by the same chemoreceptor (Tap) in E. coli. We showed that Pseudomonas sp. strain ADP is attracted to pyrimidines, which are the naturally occurring structures closest to triazines, and propose that chemotaxis toward s-triazines may be due to fortuitous recognition by a pyrimidine chemoreceptor in Pseudomonas sp. strain ADP. In competition assays, the presence of atrazine inhibited chemotaxis of Pseudomonas sp. strain ADP to cytosine, and cytosine inhibited chemotaxis to atrazine, suggesting that pyrimidines and s-triazines are detected by the same chemoreceptor.Atrazine [2-chloro-4-(N-ethylamino)-6-(N-isopropylamino)-1,3,5-s-triazine] is a human-made herbicide that is used worldwide to control broadleaf and grassy weeds. As one of the most heavily used herbicides in the United States, atrazine can be present in parts per million in agricultural runoffs (3), which exceeds the U.S. Environmental Protection Agency''s maximum allowable contaminant level of 3 ppb in ground and surface waters (13). Atrazine is persistent in soil (34) and was once considered nontoxic to animals. However, recent studies have shown that atrazine causes sexual abnormalities in frogs (21, 22, 50), reduced testosterone production in rats (53), and elevated levels of prostate cancer in workers at an atrazine-manufacturing factory (45). These studies suggest that there is cause for concern about atrazine residues in soil, groundwater, and surface waters.Several bacterial strains capable of mineralizing atrazine have been isolated (4, 27, 41, 49, 51, 52, 58). The best-studied atrazine-degrading strain, Pseudomonas sp. strain ADP (atrazine degrading pseudomonad), was isolated from an atrazine spill site in Minnesota (27). Strain ADP utilizes atrazine as a sole nitrogen source and mineralizes it in the process (27). The pathway of atrazine degradation in strain ADP has been characterized in detail (Fig. ​(Fig.1),1), and the genes encoding the six enzymes required for atrazine degradation have been cloned and sequenced (5, 7, 9, 10, 29, 42). The six genes are located in four distant locations on the atrazine catabolic plasmid (pADP-1) present in strain ADP (10, 29). atzA, atzB, and atzC, which encode the first three enzymes of the pathway, are constitutively expressed and highly conserved in atrazine-degrading bacteria isolated from geographically distinct locations (8, 11). Products of the atzDEF gene cluster catalyze the last three steps of atrazine degradation. This operon is divergently transcribed from atzR, the product of which has high homology to LysR-type regulatory proteins (29). AtzR and the inducer cyanuric acid are required for the expression of the atzDEF operon (14), and the operon is also subject to nitrogen control (15).Open in a separate windowFIG. 1.Pathway of atrazine degradation in Pseudomonas sp. strain ADP (reviewed in reference 55).In a study investigating the bioavailability of atrazine, Park et al. provided evidence that two atrazine-degrading strains, Pseudomonas sp. strain ADP and Agrobacterium radiobacter J14a, were chemotactically attracted to atrazine (38). Chemotaxis, the ability of motile bacteria to detect and respond to specific chemicals, can help bacteria find an optimal niche for their survival and growth and may play a role in the efficient degradation of pollutants in the environment (33, 37). Chemotaxis has been shown to enhance naphthalene biodegradation in both a heterogeneous aqueous system (30) and a non-aqueous-phase liquid system (24). In addition, a chemotactic naphthalene-degrading strain caused a higher rate of naphthalene desorption than was observed with nonchemotactic and nonmotile strains (24). Pseudomonas sp. strain ADP and recombinant strains expressing atz genes have been used to remove atrazine from soil in laboratory and field scale experiments (32, 48). If chemotaxis can enhance bioavailability in environments where the chemicals are sorbed to particles, the use of a motile chemotactic strain for bioremediation would be advantageous. Aside from the practical implications of atrazine chemotaxis, we are interested in understanding the evolution of a chemotactic response to a human-made chemical that was initially synthesized just 50 years ago (23). The results reported here indicate that Pseudomonas sp. strain ADP is chemotactically attracted to atrazine, atrazine metabolites, and the nonmetabolizable structural analog ametryn. The chemotactic response is not induced during growth with atrazine in strain ADP and does not require atrazine metabolism. We demonstrated that a single chemoreceptor (Tap) mediates chemotaxis to s-triazines and structurally similar pyrimidines in Escherichia coli. Additionally, we found that Pseudomonas sp. strain ADP is attracted to pyrimidines. In competition assays, cytosine inhibited atrazine chemotaxis, and vice versa. We therefore concluded that pyrimidines and s-triazines are detected by a single chemoreceptor in Pseudomonas sp. strain ADP.     

Reengineering Escherichia coli for Succinate Production in Mineral Salts Medium

The fermentative metabolism of glucose was redirected to succinate as the primary product without mutating any genes encoding the native mixed-acid fermentation pathway or redox reactions. Two changes in peripheral pathways were together found to increase succinate yield fivefold: (i) increased expression of phosphoenolpyruvate carboxykinase and (ii) inactivation of the glucose phosphoenolpyruvate-dependent phosphotransferase system. These two changes increased net ATP production, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production. Modest further improvements in succinate yield were made by inactivating the pflB gene, encoding pyruvate formate lyase, resulting in an Escherichia coli pathway that is functionally similar to the native pathway in Actinobacillus succinogenes and other succinate-producing rumen bacteria.Succinic acid is used as a specialty chemical in the agricultural, food, and pharmaceutical industries (17, 32). It has also been identified by the U.S. Department of Energy as one of the top 12 building block chemicals (30), because it can be converted into a variety of products, including green solvents, pharmaceutical products, and biodegradable plastics (17, 32). Although succinic acid is currently produced from petroleum-derived maleic anhydride, considerable interest in the fermentative production of succinate from sugars has emerged during the past decade (9, 10, 17).Several natural succinate-producing rumen bacteria that have high rates of succinate production and high succinate yields, such as Anaerobiospirillum succiniciproducens (22), Actinobacillus succinogenes (13, 28), and “Mannheimia succiniciproducens” (15, 16), have been isolated. However, these strains require complex organic nutrients that increase the costs associated with production, purification, and waste disposal (15, 22, 28). Low levels of succinate are produced by native strains of Escherichia coli in complex and mineral salts media (1, 4). Most mutant strains of E. coli that have been described previously as succinate producers also require complex organic nutrients (18, 23-26, 29, 31). Many involve two-step aerobic and anaerobic processes (3, 23-25, 29) and the addition of foreign genes (5, 6, 23-26, 29, 31).Novel E. coli biocatalysts (KJ060, KJ071, and KJ073) for the anaerobic production of succinate in mineral salts medium have been developed recently without the use of foreign genes or resident plasmids (9, 10). These biocatalysts were developed by combining constructed mutations to eliminate alternative routes of NADH oxidation in the mixed-acid pathway with growth-based selection (metabolic evolution). In subsequent studies (33), these strains were found to have recruited the glucose-repressed (7), gluconeogenic pck gene (11, 12, 19, 21, 27), encoding phosphoenolpyruvate carboxykinase (PCK) (derepressed via a point mutation in the promoter region), to replace the native phosphoenolpyruvate carboxylase (ppc) and serve as the primary route for CO₂ fixation (Fig. ​(Fig.1).1). A second acquired mutation was also identified as a frameshift mutation in the carboxy terminus of ptsI, inactivating the phosphoenolpyruvate-dependent phosphotransferase system (33). Glucose uptake by the phosphotransferase system was functionally replaced by galactose permease (galP) and glucokinase (glk).Open in a separate windowFIG. 1.Anaerobic metabolism of E. coli using the mixed-acid fermentation pathway (data from reference 1). The native phosphotransferase system pathway for glucose uptake and the mixed-acid pathway for fermentation are shown with black arrows. Peripheral reactions for glucose uptake, carboxylation, and acetyl-CoA synthesis are shown as dotted green arrows and represent new metabolic functions that have been recruited for succinate production from glucose. Reactions that have been blocked by gene deletions or point mutations are marked with an X. pck* indicates a novel mutation that derepressed pck, allowing the enzyme to serve as the primary route for oxaloacetate production. Pyruvate (boxed) appears at two sites but is presumed to exist as a single intracellular pool.Based on these previous studies, we have now determined the core mutations needed to direct carbon flow from glucose to succinate in E. coli and have constructed new succinate-producing strains with a minimum of genetic change.     

Interactions of DNA with Biofilm-Derived Membrane Vesicles

The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cation-dependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.The biofilm matrix contributes to the chemistry, structure, and function of biofilms and is crucial for the development of fundamental biofilm properties (46, 47). Early studies defined polysaccharides as the matrix component, but proteins, lipids, and nucleic acids are all now acknowledged as important contributors (7, 15). Indeed, DNA has emerged as a vital participant, fulfilling structural and functional roles (1, 5, 6, 19, 31, 34, 36, 41, 43, 44). The phosphodiester bond of DNA renders this polyanionic at a physiological pH, undoubtedly contributing to interactions with cations, humic substances, fine-dispersed minerals, and matrix entities (25, 41, 49).In addition to particulates such as flagella and pili, membrane vesicles (MVs) are also found within the matrices of gram-negative and mixed biofilms (3, 16, 40). MVs are multifunctional bilayered structures that bleb from the outer membranes of gram-negative bacteria (reviewed in references 4, 24, 27, 28, and 30) and are chemically heterogeneous, combining the known chemistries of the biofilm matrix. Examination of biofilm samples by transmission electron microscopy (TEM) has suggested that matrix material interacts with MVs (Fig. ​(Fig.1).1). Since MVs produced in planktonic culture have associated DNA (11, 12, 13, 20, 21, 30, 39, 48), could biofilm-derived MVs incorporate DNA (1, 39, 40, 44)?Open in a separate windowFIG. 1.Possible interactions between matrix polymers and particulate structures. Shown is an electron micrograph of a thin section through a P. aeruginosa PAO1 biofilm. During processing, some dehydration occurred, resulting in collapse of matrix material into fibrillate arrangements (black filled arrows). There is a suggestion of interactions occurring with particulate structures such as MVs (hollow white arrow) and flagella (filled white arrows) (identified by the appearance and cross-dimension of these highly ordered structures when viewed at high magnification), which was consistently observed with other embedded samples and also with whole-mount preparations of gently disrupted biofilms (data not shown). The scale bar represents 200 nm.     

A Novel Hydrolytic Dehalogenase for the Chlorinated Aromatic Compound Chlorothalonil

Dehalogenases play key roles in the detoxification of halogenated aromatics. Interestingly, only one hydrolytic dehalogenase for halogenated aromatics, 4-chlorobenzoyl-coenzyme A (CoA) dehalogenase, has been reported. Here, we characterize another novel hydrolytic dehalogenase for a halogenated aromatic compound from the 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil)-degrading strain of Pseudomonas sp. CTN-3, which we have named Chd. Chd catalyzes a hydroxyl substitution at the 4-chlorine atom of chlorothalonil. The metabolite of the Chd dehalogenation, 4-hydroxy-trichloroisophthalonitrile, was identified by reverse-phase high-performance liquid chromatography (HPLC), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR). Chd dehalogenates chlorothalonil under anaerobic and aerobic conditions and does not require the presence of cofactors such as CoA and ATP. Chd contains a putative conserved domain of the metallo-β-lactamase superfamily and shows the highest identity with several metallohydrolases (24 to 29%). Chd is a monomer (36 kDa), and the isoelectric point (pI) of Chd is estimated to be 4.13. Chd has a dissociation constant (K_m) of 0.112 mM and an overall catalytic rate (k_cat) of 207 s⁻¹ for chlorothalonil. Chd is completely inhibited by 1,10-phenanthroline, diethyl pyrocarbonate, and N-bromosuccinic acid. Site-directed mutagenesis of Chd revealed that histidines 128 and 157, serine 126, aspartates 45, 130 and 184, and tryptophan 241 were essential for the dehalogenase activity. Chd differs from other reported hydrolytic dehalogenases based on the analysis of amino acid sequences and catalytic mechanisms. This study provides an excellent dehalogenase candidate for mechanistic study of hydrolytic dehalogenation of halogenated aromatic compound.Halogenated aromatic compounds are widely used in agriculture and industry as solvents, defatting agents, herbicides, and fungicides. A variety of these compounds have been identified as priority organic pollutants by the United Nations and the U.S. Environmental Protection Agency. Therefore, the remediation of these pollutants is desirable. Microorganisms play key roles in the detoxification of halogenated aromatics. The use of microbial enzymes for bioremediation has received increasing attention as an efficient and cost-effective biotechnological approach. Halogen removal from halogenated aromatics reduces both the recalcitrance to biodegradation and the risk of forming toxic intermediates during subsequent metabolic steps. As a result, the key reaction for microbial detoxification of halogenated aromatics is the actual dehalogenation (35).Investigation of the microbial degradation of different halogenated aromatics has led to the detection and elucidation of various dehalogenases that catalyze the removal of the halogen atom under aerobic and anaerobic conditions (8, 11, 17). Four dehalogenation mechanisms of halogenated aromatics are known, including reductive, thiolytic, oxidative, and hydrolytic mechanisms (41). Reductive dehalogenation plays important roles in the degradation of chlorinated aromatics under anaerobic conditions (36, 44). Several anaerobic bacteria are capable of using chlorinated benzenes (2) or polychlorinated dibenzodioxins (5) as the terminal electron acceptors in their energy metabolism. These bacteria couple reductive dehalogenation to electron transport phosphorylation (15). Several enzymes catalyzing the respiratory reductive dechlorination of halogenated aromatics have also been characterized (1, 4, 7, 20, 28, 38, 40). Under aerobic conditions, some chlorinated aromatics can also be reductively dehalogenated by thiolytic substitution in the presence of glutathione (12, 19, 25, 46, 48). In this dehalogenation system, chlorine atoms are displaced by the nucleophilic attack of the thiolate anion of glutathione. The nucleophilic attack of the thiolate anion is catalyzed by glutathione S-transferases (43). Besides the two well-characterized mechanisms for aryl halide reductive dehalogenation, two other mechanisms have been reported, including reduced NADPH-dependent reductive dechlorination of 2,4-dichlorobenzoyl-coenzyme A (CoA) to 4-chlorobenzoyl-CoA in Corynebacterium sepedonicum KZ-4 and coryneform bacterium strain NTB-1 (31), as well as a CoA-mediated reductive dehalogenation of 3-chlorobenzoate in Rhodopseudomonas palustris RCB100 using 3-chlorobenzoate as the carbon source rather than as a terminal electron acceptor (13). Oxidative dehalogenation of halogenated aromatics is catalyzed by monooxygenase (29), dioxygenases (34, 37, 39, 45), and peroxidase (30).Even though several hydrolytic dehalogenases involved in dehalogenation of halogenated aliphatic hydrocarbons and halogenated carboxylic acids have been characterized, only one kind of hydrolytic dehalogenase for halogenated aromatics has been reported. The only hydrolytic dehalogenase identified to date is 4-chlorobenzoyl-CoA dehalogenase in the 4-chlorobenzoate degradation system (32, 33). For the hydrolytic substitution of the chlorine atom of 4-chlorobenzoate with a hydroxyl group, activation by the CoA thioester formation is required. Initially, 4-chlorobenzoate-CoA ligase adenylates the carboxyl group in a reaction requiring ATP, followed by the replacement of AMP with CoA and the formation of a thioester. This intermediate is sufficiently energized to facilitate the replacement of the hydroxyl group with a 4-chlorine atom; this is catalyzed by 4-chlorobenzoyl-CoA dehalogenase. Finally, 4-hydroxybenzoyl-CoA thioesterase removes the CoA (Fig. ​(Fig.1a).1a). Three separate enzymes are involved in this system, and cofactors including CoA and ATP are needed (32, 33).Open in a separate windowFIG. 1.Dechlorination mechanism of 4-chlorobenzoate in Pseudomonas sp. CBS3 and Acinetobacter sp. strain 4-CB1 (a) and the first-step dechlorination mechanism of 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil) in Pseudomonas sp. CTN-3 (b).Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile), a broad-spectrum chlorinated aromatic fungicide, is the second most widely used agricultural fungicide in the United States, with 5 million kilograms applied annually (9). Chlorothalonil is highly toxic to fish, birds, and aquatic invertebrates (6) and is commonly detected in ecosystems (18). The bacterial strain Pseudomonas sp. CTN-3, capable of efficiently transforming chlorothalonil, was isolated in our laboratory from long-term chlorothalonil-contaminated soil in the Jiangsu Province in China. In Ochrobactrum anthropi SH35B, a glutathione-dependent glutathione S-transferase was reported to be able to catalyze the nucleophilic substitution of chlorine atoms of chlorothalonil (19). The glutathione S-transferase in our CTN-3 bacterial strain showed 84% identity with that of O. anthropi SH35B. However, the glutathione S-transferase from CTN-3 was not functionally expressed. Here, we report for the first time the characterization of a novel chlorothalonil hydrolytic dehalogenase (Chd) that contains a conserved domain of the metallo-β-lactamase superfamily. Chd is the second hydrolytic dehalogenase for chlorinated aromatic compounds to be identified. The hydrolytic dehalogenation of chlorothalonil catalyzed by Chd is independent of CoA and ATP. Based on the analysis of amino acid sequences and catalytic mechanisms, Chd is unique from the other reported hydrolytic dehalogenases.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

GuaA and GuaB Are Essential for Borrelia burgdorferi Survival in the Tick-Mouse Infection Cycle

Pathogens lacking the enzymatic pathways for de novo purine biosynthesis are required to salvage purines and pyrimidines from the host environment for synthesis of DNA and RNA. Two key enzymes in purine salvage pathways are IMP dehydrogenase (GuaB) and GMP synthase (GuaA), encoded by the guaB and guaA genes, respectively. While these genes are typically found on the chromosome in most bacterial pathogens, the guaAB operon of Borrelia burgdorferi is present on plasmid cp26, which also harbors a number of genes critical for B. burgdorferi viability. Using molecular genetics and an experimental model of the tick-mouse infection cycle, we demonstrate that the enzymatic activities encoded by the guaAB operon are essential for B. burgdorferi mouse infectivity and provide a growth advantage to spirochetes in the tick. These data indicate that the GuaA and GuaB proteins are critical for the survival of B. burgdorferi in the infection cycle and highlight a potential difference in the requirements for purine salvage in the disparate mammalian and tick environments.Purine metabolism is critical for the growth and virulence in mammals of many bacterial pathogens (11, 26, 29, 33, 51). Borrelia burgdorferi, the infectious agent of Lyme borreliosis, lacks the genes encoding the enzymes required for de novo nucleotide synthesis (8, 12) and therefore must rely on salvage of purines and pyrimidines from its hosts for nucleic acid biosynthesis (21, 35). Furthermore, B. burgdorferi lacks the genes encoding key enzymes required for a classic purine salvage pathway, including hpt (hypoxanthine-guanine phosphoribosyltransferase), purA (adenylosuccinate synthase), purB (adenylosuccinate lyase), and the locus encoding a ribonucleotide reductase (4, 8, 12, 35, 66). Despite the absence of a ribonucleotide reductase, an enzyme critical for the generation of deoxynucleotides through enzymatic reduction of ribonucleotides (32), a novel purine salvage pathway that involves salvage of deoxynucleosides from the host and interconversion of purine bases to deoxynucleosides by BB0426, a deoxyribosyl transferase, has recently been demonstrated for B. burgdorferi (23) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Pivotal role of the GuaAB proteins in the purine salvage pathway of B. burgdorferi. A novel pathway for purine salvage has recently been elucidated for B. burgdorferi (23). Extracellular adenine and hypoxanthine are salvaged by B. burgdorferi from mammalian and tick host environments (61). Following transport, adenine can be converted to hypoxanthine by adenine deaminase (BBK17) (21). This pathway proposes two possible fates for hypoxanthine, as follows. (i) Hypoxanthine is converted to IMP by a putative xanthine-guanine phosphoribosyl transferase (BB0103), IMP is converted to XMP by IMPDH (GuaB or BBB17), and XMP is converted to GMP by GMP synthase (GuaA or BBB18), resulting in guanine nucleotides for RNA synthesis. (ii) Direct transport of deoxynucleosides appears to provide a source of deoxyribose for interconversion of hypoxanthine to deoxyinosine by a deoxyribosyl transferase (BB0426) (23). dIMP is generated by a putative deoxynucleotide kinase (BB0239). GuaB converts dIMP to dXMP, and GuaA converts dXMP to dGMP, providing guanine deoxynucleotides for DNA synthesis (23). Salvage of free guanine nucleosides and guanine deoxynucleosides, when they are available in the host environment, may allow B. burgdorferi to circumvent the GuaAB requirement for GMP and dGMP biosynthesis. The dashed arrows indicate dephosphorylation of nucleotide monophosphate or deoxynucleotide monophosphate prior to transport by the spirochete and rephosphorylation of nucleoside and deoxynucleoside to nucleotide triphosphate and deoxynucleotide triphosphate, respectively, for RNA and DNA synthesis. NMP, nucleotide monophosphate; N, nucleoside; dN, deoxynucleoside; dNMP, deoxynucleotide monophosphate; OM, outer membrane; IM, inner membrane.In its infection cycle, B. burgdorferi passages between two disparate environments with potentially distinct purine availabilities, the tick vector and a mammalian host. Hypoxanthine is the most abundant purine in mammalian blood (17), and it is available for salvage by B. burgdorferi during the blood meal of an infected tick and during the spirochete''s transient presence in the mammalian bloodstream. Despite the absence of the hpt gene, we and others have shown that B. burgdorferi is able to transport and incorporate low levels of hypoxanthine (23, 35). During mammalian infection B. burgdorferi resides in various tissues, including the skin, heart, bladder, and joints. Adenine has been shown to be ubiquitous in mammalian tissues (61) and therefore is available for salvage by B. burgdorferi. Guanine is present at low levels in mammalian blood and tissues (17, 61); however, the amount may not be sufficient for survival of the spirochete.The limiting step in guanine nucleotide biosynthesis from adenine and hypoxanthine is the conversion of IMP to XMP, which is catalyzed by IMP dehydrogenase (IMPDH) (65). Guanine nucleotides are essential for DNA and RNA synthesis, signal transduction, and cell cycle control; thus, IMPDH activity is critical for the survival of most organisms (60). The enzymes required for the final two steps of guanine nucleotide biosynthesis, IMPDH and GMP synthase, are encoded by the guaB and guaA genes, respectively. The guaA and guaB genes and the corresponding activities of their protein products are conserved in B. burgdorferi (28, 67). These genes are typically carried on the chromosomes of bacterial species. However, in B. burgdorferi, the guaAB operon resides on a 26-kbp circular plasmid, cp26, and it shares an approximately 185-bp intergenic region with, and is transcribed divergently from, the essential virulence gene ospC (8, 12, 28, 50, 54, 62). The cp26 plasmid has been shown to harbor numerous genes important for B. burgdorferi survival in vivo and in vitro, including ospC (16, 34, 50, 53, 56) and resT (7), as well as BBB26 and BBB27 (20). Because of these critical functions, this plasmid is the only plasmid of the approximately 21 B. burgdorferi plasmids that is present in all natural isolates and has never been shown to be lost during in vitro culture (2, 7, 18, 20, 44, 52).Here we establish that the enzymatic activities of GuaA and GuaB are critical for the survival of B. burgdorferi in the infectious cycle and highlight a potential difference in this spirochete''s requirement for purine salvage in the disparate mammalian and tick environments.     

Effects of Aeration on the Synthesis of Poly(3-Hydroxybutyrate) from Glycerol and Glucose in Recombinant Escherichia coli

Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose.Polyhydroxyalkanoates (PHAs), accumulated as intracellular granules by many bacteria under unfavorable conditions (5, 8), are carbon and energy reserves and also act as electron sinks, enhancing the fitness of bacteria and contributing to redox balance (9, 11, 19). PHAs have thermoplastic properties, are totally biodegradable by microorganisms present in most environments, and can be produced from different renewable carbon sources (8).Poly(3-hydroxybutyrate) (PHB) is the best known PHA, and its accumulation in recombinant Escherichia coli from several carbon sources has been studied (1, 13). In the last few years, increasing production of biodiesel has caused a sharp fall in the cost of its main by-product, glycerol (22). Its use for microbial PHA synthesis has been analyzed for natural PHA producers, such as Methylobacterium rhodesianum, Cupriavidus necator (formerly called Ralstonia eutropha) (3), several Pseudomonas strains (22), the recently described bacterium Zobellella denitrificans (7), and a Bacillus sp. (18), among others. Glycerol has also been used for PHB synthesis in recombinant E. coli (12, 15). PHAs obtained from glycerol were reported to have a significantly lower molecular weight than polymer synthesized from other substrates, such as glucose or lactose (10, 23).Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes that are involved in granule formation and/or have regulatory functions, such as phasins, granule-associated proteins that have been shown to enhance polymer synthesis and the number and size of PHA granules (17, 24). The phasin PhaP has been shown to exert a beneficial effect on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of strain K24KP, a recombinant E. coli that carries phaBAC and phaP of Azotobacter sp. FA8 (6).Because the redox state of the cells is known to affect the synthesis of PHB (1, 4, 14), the present study investigates the behavior of this recombinant strain under different aeration conditions, by using two substrates, glucose and glycerol, with different oxidation states.