Phosphoproteomic Analysis Reveals the Effects of PilF Phosphorylation on Type IV Pilus and Biofilm Formation in Thermus thermophilus HB27

Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili–mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili–related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production.Thermus thermophilus HB27 is a Gram-negative, rod-shaped, and extremely thermophilic eubacterium isolated from a geothermal area (1). This organism grows at temperatures up to 85 °C and has an optimal growth temperature of 70 °C. The thermostable enzymes obtained from members of the genus Thermus are of considerable interest because of their potential in research, biotechnological, and industrial applications (2, 3). In addition, T. thermophilus HB27 is a suitable laboratory model for genetic manipulation, as it is easily cultured under laboratory conditions and has a natural transformation system that is much more efficient than those of other Thermus spp. (4). Intriguingly, thermophiles are also found in biofilms, enclosed within a matrix consisting of extracellular polymeric substances, in various natural and artificial thermal environments (5, 6). Bacteria form biofilms in order to adapt and survive in harsh environments (7, 8). Over the past few decades, biofilm formation has been a major focus of microbial research and, as such, has been studied in relationship to bacterial pathogenesis, immunology, biofouling, microbial technology, and industrial applications (7, 9–12).Members of the genus Thermus, like many other thermophiles, have evolved two main mechanisms for thermoadaption. One is biofilm formation, which confers protection against environmental stresses such as high temperature and the presence of antibiotics (8). In previous studies, a novel exopolysaccharide, TA-1, was isolated from a T. aquaticus YT-1 biofilm, and both its primary structure and its immunological activity were determined (13). In addition, we showed that the overexpression of uridine diphosphate (UDP)-galactose-4′-epimerase (GalE), which catalyzes the reversible interconversion of UDP-galactose and UDP-glucose, in T. thermophilus HB27 increases biofilm production because of the enzyme''s involvement in an important step of exopolysaccharide (EPS)¹ biosynthesis (14). The other mechanism that enables Thermus to thrive in extreme habitats is natural transformation (i.e. the ability to take up free DNA). In hot environments, natural transformation allows the horizontal exchange of genetic information between extremophiles, including of genes that promote thermoadaptation (15–17). Recent studies showed that the type IV pili (T4P) on the cell surface of T. thermophilus HB27 not only are required for natural transformation (18, 19), but also mediate adhesion and twitching motility (20). Also, together with the degree of EPS production, the presence of T4P on the bacterial cell surface contributes to the regulation of biofilm formation (21). However, despite extensive research on the physiological, biochemical, and genetic traits of thermophiles, the mechanisms underlying these functions and their role in thermal adaptation have not been fully elucidated (16, 22–24).Advances in the field of phosphoproteomics have come from high-resolution mass spectrometry and prokaryotic genome sequencing, which have confirmed the phosphorylation of many bacterial proteins on serine/threonine and tyrosine residues (25, 26). In surveys of phosphorylation-related functions, bacterial serine, threonine, and tyrosine phosphoproteins have been shown to regulate many physiological and adaptation processes, such as central carbon catabolism, the heat shock response, osmolarity, starvation, EPS synthesis, virulence, and sporulation (25–27). These observations have been followed by more detailed, species-specific phosphoproteomics investigations, including in Bacillus subtilis (28), Escherichia coli (29), Lactococcus lactis (30), Halobacterium salinarum (31), Klebsiella pneumonia (32), Pseudomonas spp. (33), Rhodopseudomonas palustris (34), and T. thermophilus HB8 (35). In this study, the role played by the global phosphorylation network of the thermophile T. thermophilus HB27 in the physiological processes that mediate the stress responses and thermotolerance of this bacterium was examined. Specifically, we used strong cation exchange (SCX) chromatography and titanium dioxide (TiO₂) (28–30) enrichment to characterize the phosphoproteomic map of T. thermophilus HB27. Genetic manipulation of this strain indicated that phosphorylation of the PilF protein, which contains an ATP-binding motif (TTC1622/pilF) and drives T4P formation, is involved in both EPS production and piliation, thereby influencing the biofilm formation during thermophilic adaptation.     

An Extreme Thermophile,Thermus thermophilus,Is a Polyploid Bacterium

An extremely thermophilic bacterium, Thermus thermophilus HB8, is one of the model organisms for systems biology. Its genome consists of a chromosome (1.85 Mb), a megaplasmid (0.26 Mb) designated pTT27, and a plasmid (9.3 kb) designated pTT8, and the complete sequence is available. We show here that T. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. In the case of the HB8 strain, the copy number of the chromosome was estimated to be four or five, and the copy number of the pTT27 megaplasmid seemed to be equal to that of the chromosome. It has never been discussed whether T. thermophilus is haploid or polyploid. However, the finding that it is polyploid is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be a polyploid organism. As is the case for D. radiodurans in the radiation environment, the polyploidy of T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures. Polyploidy often complicates the recognition of an essential gene in T. thermophilus as a model organism for systems biology.The extreme thermophile Thermus thermophilus is a Gram-negative aerobic bacterium that can grow at temperatures ranging from 50°C to 82°C (33, 34). The genome sequences of two strains, HB27 and HB8, are available (13; see also http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=530). The genome of the HB27 strain consists of a chromosome (1.89 Mb) and a megaplasmid (0.23 Mb), while that of the HB8 strain includes a plasmid (9.3 kb) coupled with a chromosome (1.85 Mb) and a megaplasmid (0.26 Mb) (13; see also the NCBI website [above]). This organism has attracted attention as one of the model organisms for genetic manipulation, structural genomics, and systems biology (9, 44). In the case of the HB8 strain, the Structural and Functional Whole-Cell Project for T. thermophilus HB8, which aims to understand the mechanisms of all the biological phenomena occurring in the HB8 cell by investigating the cellular components at the atomic level on the basis of their three-dimensional (3-D) structures, is in progress (44). In addition to the stability and ease of crystallization of thermophilic proteins, natural competency and an established genetic engineering system add value to T. thermophilus HB8 as a model organism (12, 14, 23, 44). Thermostabilized resistances against antibiotics such as kanamycin (Km), hygromycin (Hm), and bleomycin (Bm), which were developed by directed evolution, have also encouraged the system (5, 6, 16, 29; Y. Koyama, unpublished data).However, we had been puzzled about several gene disruptions in T. thermophilus HB8 that resulted from replacement with the drug resistance gene. Even if drug-resistant transformants were obtained, the target gene of the transformants had not often been deleted. The target gene, probably an essential gene, seemed to coexist with the drug resistance gene. A similar phenomenon has been reported in the deletion of the recJ gene in Deinococcus radiodurans (7). Repeated observation of this phenomenon suggested that T. thermophilus HB8 might possess multiple genomic copies. Many bacteria, including the most-studied bacteria Escherichia coli and Bacillus subtilis, essentially carry a single genomic copy per cell and are genetically haploid organisms (3, 10, 42, 43). On the other hand, several bacteria have been proposed to be polyploid, harboring multiple genomic copies per cell. They include Buchnera species (21, 22), Blattabacterium species (24), Epulopiscium species (1, 4), Borrelia hermsii (20), Azotobacter vinelandii (28, 35), Neisseria gonorrhoeae (41), D. radiodurans (11, 27), a few Lactococcus lactis laboratory strains (26), and many cyanobacteria (2, 25, 37). In particular, D. radiodurans, an extremely radioresistant bacterium, has been suggested to be closely related to the genus Thermus by comparative genomic analysis (13, 32). The radioresistant bacterium carries four genome copies per cell in the stationary phase and up to 10 copies per cell during exponential growth (11, 27). In contrast with this well-known polyploidy of D. radiodurans, no report on the genomic copy number of Thermus has been done, in spite of the attention it has received as a model organism. Therefore, in this paper, the potential polyploidy and the genomic copy number were first studied in T. thermophilus HB8.     

Construction of a Proline-Producing Mutant of the Extremely Thermophilic Eubacterium Thermus thermophilus HB27

Growth of Thermus thermophilus HB27 was inhibited by a proline analog, 3,4-dehydroproline (DHP). This result suggested that the γ-glutamyl kinase (the product of the proB gene) was inhibited by feedback inhibition in T. thermophilus. DHP-resistant mutants were reported previously for Escherichia coli (A. M. Dandekar and S. L. Uratsu, J. Bacteriol. 170:5943–5945, 1988) and Serratia marcescens (K. Omori, S. Suzuki, Y. Imai, and S. Komatsubara, J. Gen. Microbiol. 138:693–699, 1992), and their mutated sites in the proB gene were identified. Comparison of the amino acid sequence of T. thermophilus γ-glutamyl kinase with those of E. coli and S. marcescens mutants revealed that the DHP resistance mutations occurred in the amino acids conserved among the three organisms. For eliminating the feedback inhibition, we first constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis at the proB gene as reported for the proline-producing mutant of S. marcescens. The mutant, TH401, excreted about 1 mg of l-proline per liter at 70°C after 12 h of incubation. It was also suggested that T. thermophilus had a proline degradation and transport pathway since it was able to grow in minimal medium containing l-proline as sole nitrogen source. In order to disrupt the proline degradation or transport genes, TH401 was mutated by UV irradiation. Seven mutants unable to utilize l-proline for their growth were isolated. One of the mutants, TH4017, excreted about 2 mg of l-proline per liter in minimal medium at 70°C after 12 h of incubation.Thermus thermophilus, a gram-negative aerobic eubacterium, is one of the most widely studied species of extremely thermophilic microorganisms. We have been working on the molecular genetics and molecular reproduction of T. thermophilus HB27. We have already cloned and sequenced three proline biosynthetic genes, proB, proA, and proC, and reported that the proB and proA genes exist in tandem (7, 9).We have also constructed physical maps of the HB27 chromosome and of a large plasmid, pTT27, and determined the locations of all proline biosynthetic genes on the chromosomal DNA (20, 21). We have already succeeded in overproducing carotenoids in T. thermophilus HB27 (6), but at present there is no report about extracellular production of amino acids in extreme thermophiles. We have elucidated the consensus sequences for strong promoters of T. thermophilus (11) and developed a thermostable antibiotic resistance gene (12). It is also easy to disrupt or mutate genes on chromosomal DNA in T. thermophilus HB27 (8). Among the extreme thermophiles, a host-vector system has been established only in T. thermophilus. Generally, the reaction rate of thermostable enzymes which are produced from T. thermophilus is higher than those of enzymes from mesophiles. In a fermentation process such as amino acid production, T. thermophilus may contribute to the improvement of amino acid productivity since fermentation at a high temperature eliminates the problems of contamination and cooling procedures. So, we decided to attempt excretion of proline at a high temperature with T. thermophilus mutants.l-Proline is synthesized from glutamate by the sequential reaction of γ-glutamyl kinase, γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase in bacteria (1). Genes proB and proA, which encode γ-glutamyl kinase and γ-glutamyl phosphate reductase, respectively, were found to comprise an operon in T. thermophilus (9), Escherichia coli (5), and Serratia marcescens (13). In E. coli and S. marcescens, γ-glutamyl kinase is subject to feedback control by l-proline (3, 13), but γ-glutamyl phosphate reductase and pyrroline-5-carboxylate reductase are not inhibited by proline (3, 15). Meanwhile, E. coli and S. marcescens rapidly degrade proline by proline dehydrogenase (proline oxidase), encoded by the putA gene (3, 14, 22). So far, it has been reported that E. coli mutants resistant to proline analogs, dl-3,4-dehydroproline and l-azetidine-2-carboxylic acid, excreted l-proline into the medium. But the amount of l-proline excreted was too small for practical use because of the existence of the proline degradation pathway (2). For S. marcescens, Sugiura et al. (18, 19) have constructed a proline-overproducing strain, SP126, as a double mutant resistant to 3,4-dehydroproline and thiazolidine-4-carboxylate and derived from a proline dehydrogenase-deficient mutant (18). Strain SP126 produced about 20 mg of l-proline per ml in the fermentation medium (18).In T. thermophilus, the control system in proline biosynthesis has not been elucidated. However, we thought that the feedback control of proline biosynthesis in T. thermophilus should be similar to that of E. coli and S. marcescens, since the amino acid sequences of proline biosynthetic enzymes in T. thermophilus show a high similarity to sequences of those of E. coli and S. marcescens (7, 9). E. coli and S. marcescens mutants resistant to 3,4-dehydroproline have already been determined to be proB mutants (4, 14). The comparison of the amino acid sequences of γ-glutamyl kinases in E. coli, S. marcescens, and T. thermophilus showed that these mutations occurred in the positions conserved among the three microorganisms (Fig. ​(Fig.1).1). We thought that it was possible to construct a 3,4-dehydroproline-resistant mutant of T. thermophilus by introducing the same mutations into the proB gene found in the mutants of E. coli and S. marcescens. We determined the strategy for construction of a proline-producing strain of T. thermophilus by following two steps: first, construction of a 3,4-dehydroproline-resistant mutant by introduction of mutations into the proB gene, and second, isolation of a mutant which cannot utilize proline for its growth by mutagenizing the dehydroproline-resistant mutant. Open in a separate windowFIG. 1Comparison of the amino acid sequences of γ-glutamyl kinases in E. coli, S. marcescens, and T. thermophilus. The amino acid substitutions found in E. coli (4) and S. marcescens (14) are shown by arrows. Asterisks show the amino acid residues conserved in the three microorganisms.     

Role of Streptococcus thermophilus MR-1C Capsular Exopolysaccharide in Cheese Moisture Retention

Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799–805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 and Lactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, the epsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticus LH100 plus the non-EPS-producing mutant S. thermophilus DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of d-galactose, l-rhamnose, and l-fucose in a ratio of 5:2:1.Lactic acid bacteria (LAB) are a diverse group of industrially important, gram-positive, non-spore-forming microbes that produce lactic acid as a major product of carbohydrate fermentation. Many strains of LAB produce extracellular polysaccharides which may be tightly associated with the bacterial cell wall as capsules or liberated into the growth medium as a loose slime (5). The term exopolysaccharide (EPS) has been used to refer to either type of external polysaccharide. EPSs may be homopolysaccharides, composed of a single type of sugar monomer, or heteropolysaccharides, containing several types of sugar monomers (25). Extracellular homopolysaccharides are made by such LAB as Leuconostoc mesenteroides and Streptococcus mutans, while extracellular heteropolysaccharides are produced by several other species of LAB, including Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (6).The ability to produce EPS is unstable in LAB and may be lost following numerous transfers, prolonged periods of storage, or incubation at temperatures above that optimal for growth (6, 24). This instability of EPS production in mesophilic LAB has been attributed to the fact that the genes involved in polymer production are plasmid encoded. In contrast, genes for EPS production in thermophilic LAB, such as S. thermophilus and L. delbrueckii subsp. bulgaricus, are believed to be chromosomally encoded (6). Consequently, the unstable nature of the EPS phenotype in thermophilic strains is not understood, but it may be related to mobile genetic elements or genomic instability (24).Because of the ability of EPSs to act as viscosifying, stabilizing, or water-binding agents in various foods, these polymers can act as effective natural alternatives to commercial stabilizers (6). For example, EPS-producing (EPS⁺) LAB are commonly used as starter cultures for yogurt manufacture because EPS improves the viscosity and texture of yogurt and decreases its susceptibility to syneresis (loss of whey from the curd) (14, 28).Analysis of cheese microstructure has shown that in full-fat or part-skim mozzarella, the fat and a large portion of the water are located within channels that are formed by fat globules when the cheese curd is heated and stretched (18, 20). In low-fat mozzarella, however, there are very few fat globules to break up the protein matrix, resulting in less space for water retention (20). As a consequence, the cheese has a tough and rubbery texture and requires more heat for melting (19). Merrill et al. showed that procedures which increased moisture levels in reduced- and low-fat mozzarella improved the body, texture, and functional properties of the cheese (19). In addition to enhanced functionality, the ability to increase cheese moisture level (even by as little as 1%) gives processors an important economic advantage in the highly competitive mozzarella industry (27).Since EPS has the capacity to bind significant amounts of water, it was the hypothesis of our group that EPS⁺ LAB may be useful for the production of reduced- and low-fat mozzarella. Work by Perry et al. (21) recently showed that an EPS⁺ starter pair, S. thermophilus MR-1C and L. delbrueckii subsp. bulgaricus MR-1R, could be used to significantly increase moisture levels in low-fat mozzarella. The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. The results showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level and demonstrated that this effect required the bacterium’s capsular EPS.(Part of this research was presented at the 92nd Annual Meeting of the American Dairy Science Association, Guelph, Ontario, Canada, 22 to 25 June 1997.)     

Physiological and Genetic Analyses Leading to Identification of a Biochemical Role for the moeA (Molybdate Metabolism) Gene Product in Escherichia coli

A unique class of chlorate-resistant mutants of Escherichia coli which produced formate hydrogenlyase and nitrate reductase activities only when grown in medium with limiting amounts of sulfur compounds was isolated. These mutants failed to produce the two molybdoenzyme activities when cultured in rich medium or glucose-minimal medium. The mutations in these mutants were localized in the moeA gene. Mutant strains with polar mutations in moeA which are also moeB did not produce active molybdoenzymes in any of the media tested. moeA mutants with a second mutation in either cysDNCJI or cysH gene lost the ability to produce active molybdoenzyme even when grown in medium limiting in sulfur compounds. The CysDNCJIH proteins along with CysG catalyze the conversion of sulfate to sulfide. Addition of sulfide to the growth medium of moeA cys double mutants suppressed the MoeA⁻ phenotype. These results suggest that in the absence of MoeA protein, the sulfide produced by the sulfate activation/reduction pathway combines with molybdate in the production of activated molybdenum. Since hydrogen sulfide is known to interact with molybdate in the production of thiomolybdate, it is possible that the MoeA-catalyzed activated molybdenum is a form of thiomolybdenum species which is used in the synthesis of molybdenum cofactor from Mo-free molybdopterin.Molybdoenzymes play essential metabolic roles in most organisms from bacteria to plants and animals (34). All molybdoenzymes other than dinitrogenase contain molybdenum cofactor, which consists of a unique molybdopterin (MPT) complexed with molybdenum (1, 12, 23, 31, 34). In Escherichia coli, the biologically active form of the cofactor in molybdoenzymes is MPT guanine dinucleotide (MGD) (5, 22, 23). Synthesis of this cofactor in an active form requires transport of molybdate into the cell, activation of molybdate, synthesis of the MPT moiety, and incorporation of molybdate into MPT. Although molybdate transport and the various steps in the organic part of MGD biosynthesis are well characterized (17, 24, 33; see references 10, 22, and 23 for reviews), very little is known about the activation and incorporation of molybdenum into the cofactor (22).Mutants which are defective in molybdate metabolism can be isolated as chlorate-resistant mutants (8, 9). A large fraction of these mutants are pleiotropic for all molybdoenzyme activities in the cell, and these comprise the three genetic loci involved in MGD synthesis, moa, mob, and moeB (see references 10, 22, 29, and 31 for reviews). The mod gene products comprise the molybdate transport system through which molybdate is transported into the cell and the Mod⁻ phenotype can be suppressed by increasing molybdate concentration in the medium. The mog mutants which produced formate hydrogenlyase (FHL) activity containing the molybdoenzyme formate dehydrogenase-H (FDH-H) but not nitrate reductase activity was proposed to be defective in molybdochelatase (13, 32). This molybdochelatase is apparently required for production of active nitrate reductase and not for FDH-H.The moe operon codes for two proteins, and only the physiological role of the second gene product, MoeB protein, is known. The MoeB protein activates MPT synthase, which catalyzes the conversion of MPT precursor (precursor Z) to MPT by introducing the needed sulfur to which Mo is coordinated in the molybdenum cofactor (20, 22). The MoeB protein, MPT synthase sulfurylase, is the known S donor in the activation of MPT synthase. The physiological role of MoeA protein coded by the first gene in the two member moe operon is not known. Mutants which are defective in moeA (chlE [29]) produced about 6% of the wild-type levels of MPT (12), although no molybdoenzyme activity was found in these moeA mutants. Since the MoeB protein acts as an S donor in MPT synthesis, it is possible that the first gene product, MoeA protein, also has a similar role in linking S metabolism and Mo metabolism in the cell.During our analysis of molybdate transport-defective mutants, we identified a subgroup of chlorate-resistant mutants with a unique phenotype. Mutations in this class of mutants were mapped in the moeA gene at 18.6 min on the E. coli chromosome (3, 18). The MoeA⁻ phenotype was suppressed when the growth medium was supplemented with sulfide. In this report, we present the physiological and genetic characteristics of E. coli moeA mutants and propose a role for the MoeA protein in the activation of molybdenum by sulfurylation.(This work was presented at the International Symposium on Nitrogen Assimilation: Molecular and Genetic Aspects, 3 to 9 May 1997, Tampa, Fla.)     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.

Polychlorinated biphenyls (PCBs) are produced by chlorination of biphenyl, resulting in up to 209 different congeners. Commercial mixtures range from light oily fluids to waxes, and their physical properties make them useful as heat transfer fluids, hydraulic fluids, solvent extenders, plasticizers, flame retardants, organic diluents, and dielectric fluids (1, 21). Approximately 24 million lb are in the North American environment (19). The stability and hydrophobic nature of these compounds make them a persistent environmental hazard.To date, bacterial transformations have been the main focus of PCB degradation research. Aerobic bacteria use a biphenyl-induced dioxygenase enzyme system to attack less-chlorinated congeners (mono- to hexachlorobiphenyls) (1, 5, 7, 8, 22). Although more-chlorinated congeners are recalcitrant to aerobic bacterial degradation, microorganisms in anaerobic river sediments reductively dechlorinate these compounds, mainly removing the meta and para chlorines (1, 6, 10, 33, 34).The degradation of PCBs by white rot fungi has been known since 1985 (11, 18). Many fungi have been tested for their ability to degrade PCBs, including the white rot fungi Coriolus versicolor (18), Coriolopsis polysona (41), Funalia gallica (18), Hirneola nigricans (35), Lentinus edodes (35), Phanerochaete chrysosporium (3, 11, 14, 17, 18, 35, 39, 41–43), Phlebia brevispora (18), Pleurotus ostreatus (35, 43), Poria cinerescens (18), Px strain (possibly Lentinus tigrinus) (35), and Trametes versicolor (41, 43). There have also been studies of PCB metabolism by ectomycorrhizal fungi (17) and other fungi such as Aspergillus flavus (32), Aspergillus niger (15), Aureobasidium pullulans (18), Candida boidinii (35), Candida lipolytica (35), Cunninghamella elegans (16), and Saccharomyces cerevisiae (18, 38). The mechanism of PCB biodegradation has not been definitively determined for any fungi. White rot fungi produce several nonspecific extracellular enzymes which have been the subject of extensive research. These nonspecific peroxidases are normally involved in lignin degradation but can oxidize a wide range of aromatic compounds including polycyclic aromatic hydrocarbons (37). Two peroxidases, lignin peroxidase (LiP) and Mn peroxidase (MnP), are secreted into the environment of the fungus under conditions of nitrogen limitation in P. chrysosporium (23, 25, 27, 29) but are not stress related in fungi such as Bjerkandera adusta or T. versicolor (12, 30).Two approaches have been used to determine the biodegradability of PCBs by fungi: (i) loss of the parent congener analyzed by gas chromatography (GC) (17, 32, 35, 42, 43) and (ii) mineralization experiments in which the ¹⁴C of the universally labeled ¹⁴C parent congener is recovered as ¹⁴CO₂ (11, 14, 18, 39, 41). In the first method, the loss of a peak on a chromatogram makes it difficult to decide whether the PCB is being partly degraded, mineralized, adsorbed to the fungal biomass, or bound to glassware, soil particles, or wood chips. Even when experiments with killed-cell and abiotic controls are performed, the extraction efficiency and standard error can make data difficult to interpret. For example, recoveries can range anywhere from 40 to 100% depending on the congener used and the fungus being investigated (17). On the other hand, recovery of significant amounts of ¹⁴CO₂ from the cultures incubated with a ¹⁴C substrate provides definitive proof of fungal metabolism. There appears to be only one report relating data from these two techniques (18), and in that study, [U-¹⁴C]Aroclor 1254, rather than an individual congener, was used.In this study, we examined the ability of 12 white rot fungal strains to metabolize selected PCB congeners to determine which strains were the most active degraders. Included in this group was P. chrysosporium ATCC 24725, a strain used extensively in PCB studies (3, 14, 18, 35, 39, 41–43). Six PCB congeners were selected to give a range of chlorine substitutions and therefore a range of potential biodegradability which was monitored by GC. One of the chosen congeners was ¹⁴C labeled and used in studies to compare the results from a mineralization method with those from the GC method.     

MqsR, a Crucial Regulator for Quorum Sensing and Biofilm Formation, Is a GCU-specific mRNA Interferase in Escherichia coli

The mqsR gene has been shown to be positively regulated by the quorum-sensing autoinducer AI-2, which in turn activates a two-component system, the qseB-qseC operon. This operon plays an important role in biofilm formation in Escherichia coli. However, its cellular function has remained unknown. Here, we found that 1 base downstream of mqsR there is a gene, ygiT, that is co-transcribed with mqsR. Induction of mqsR caused cell growth arrest, whereas ygiT co-induction recovered cell growth. We demonstrate that MqsR (98 amino acid residues), which has no homology to the well characterized mRNA interferase MazF, is a potent inhibitor of protein synthesis that functions by degrading cellular mRNAs. In vivo and in vitro primer extension experiments showed that MqsR is an mRNA interferase specifically cleaving mRNAs at GCU. The mRNA interferase activity of purified MqsR was inhibited by purified YgiT (131 residues). MqsR forms a stable 2:1 complex with YgiT, and the complex likely functions as a repressor for the mqsR-ygiT operon by specifically binding to two different palindromic sequences present in the 5′-untranslated region of this operon.It has been reported that quorum sensing is involved in biofilm formation (1–4). mqsR expression was found to be induced by 8-fold in biofilms (5) and also by the quorum-sensing signal autoinducer AI-2, which is a species-nonspecific signaling molecule produced by both Gram-negative and Gram-positive bacteria, including Escherichia coli (6). It has been reported that induction of mqsR activates a two-component system, the qseB-qseC operon, which is known to play an important role in biofilm formation (6). Thus, it has been proposed that MqsR (98 amino acid residues) is a regulator of biofilm formation because it activates qseB, which controls the flhDC expression required for motility and biofilm formation in E. coli (6). However, the cellular function of MqsR has remained unknown.Interestingly, all free-living bacteria examined to date contain a number of suicide or toxin genes in their genomes (7, 8). Many of these toxins are co-transcribed with their cognate antitoxins in an operon (termed toxin-antitoxin (TA)² operon) and form a stable complex in the cell, so their toxicity is subdued under normal growth conditions (9–11). However, the stability of antitoxins is substantially lower than that of their cognate toxins, so any stress causing cellular damage or growth inhibition that induces proteases alters the balance between toxin and antitoxin, leading to toxin release in the cell.To date, 16 (12) TA systems have been reported on the E. coli genome, including relB-relE (13, 14), chpBI-chpBK (15), mazEF (16–18), yefM-yoeB (19, 20), dinJ-yafQ (21, 22), hipBA and hicAB (23, 24), prlF-yhaV (25), and ybaJ-hha (26). Interestingly, all of these TA operons appear to use similar modes of regulation: the formation of complexes between antitoxins and their cognate toxins to neutralize toxin activity and the ability of TA complexes to autoregulate their expression. The cellular targets of some toxins have been identified. CcdB directly interacts with gyrase A and blocks DNA replication (27, 28). RelE, which by itself has no endoribonuclease activity, appears to act as a ribosome-associating factor that promotes mRNA cleavage at the ribosome A-site (13, 29, 30). PemK (31), ChpBK (15), and MazF (32) are unique among toxins because they target cellular mRNAs for degradation by functioning as sequence-specific endoribonucleases to effectively inhibit protein synthesis and thereby cell growth.MazF, ChpBK, and PemK have been characterized as sequence-specific endoribonucleases that cleave mRNA at the ACA, ACY (Y is U, A, or G), and UAH (H is C, A, or U) sequences, respectively. They are completely different from other known endoribonucleases such as RNases E, A, and T1, as these toxins function as protein synthesis inhibitors by interfering with the function of cellular mRNAs. It is well known that small RNAs, such as mRNA-interfering cRNA (33), microRNA (34), and small interfering RNA (35), interfere with the function of specific RNAs. These small RNAs bind to specific mRNAs to inhibit their expression. Ribozymes also act on their target RNAs specifically and interfere with their function (36). Therefore, MazF, ChpBK, and PemK homologs form a novel endoribonuclease family that exhibits a new mRNA-interfering mechanism by cleaving mRNAs at specific sequences. Thus, they have been termed “mRNA interferases” (2).During our search for TA systems on the E. coli genome, we found that the mqsR gene is co-transcribed with a downstream gene, ygiT. These two genes appear to function as a TA system, as their size is small (98 residues for MqsR and 131 residues for YgiT) and their respective open reading frames are separated by 1 bp. In this study, we demonstrate that MqsR-YgiT is a new E. coli TA system consisting of a toxin, MqsR, and an antitoxin, YgiT. Moreover, we identify MqsR as a novel mRNA interferase that does not exhibit homology to MazF. This toxin cleaves RNA at GCU sequences in vivo and in vitro. The implication of this finding as to how this mRNA interferase is involved in cell physiology and biofilm formation will be discussed.     

Structure of the β-Galactosidase Gene from Thermus sp. Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein

The nucleotide sequence of both the bgaA gene, coding for a thermostable β-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (M_r, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a K_m value of 1.6 mM with o-nitrophenyl-β-d-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C.β-d-Galactosidase (EC 3.2.1.23) catalyzes the hydrolysis of β-1,4-d-galactosidic linkages. This enzyme is distributed in numerous microorganisms, plants, and animal tissues. The application of β-galactosidase to the hydrolysis of lactose in dairy products, such as milk and cheese whey, has received much attention (7, 21), and in this regard, thermostable β-galactosidases have attracted increasing interest because of their potential usefulness in the industrial processing of lactose-containing products (21). Thermostable enzymes have a number of generally recognized advantages in industrial applications, such as associated chemical resistance and reduced chances of microbial growth at high temperatures (15, 19). Nevertheless, relatively few studies have been conducted on β-galactosidases from thermotolerant or thermophilic bacteria, and as far as we know, only four genes encoding these enzymes have been cloned (5, 10, 11, 13, 16, 18).An important property that has received little attention in the literature is the level of purity of commercial preparations of β-galactosidases, especially with regard to the presence of other enzymes, such as proteases. These contaminants could have a severe impact on the stability of the enzyme, leading to undesirable changes in dairy products during storage (21). To prevent these, a new method was developed to purify the β-galactosidase (LacZ) of Escherichia coli by fusing to its N terminus the choline-binding domain (ChBD) of the pneumococcal autolytic amidase LytA (23). This system allowed the purification of E. coli β-galactosidase in a single step by affinity chromatography on DEAE-cellulose (23). Thus, it appeared interesting to test whether this procedure could also be used in the purification of a thermostable enzyme in order to circumvent contamination problems.This paper reports the molecular characterization of the bgaA gene, encoding the β-galactosidase (BgaA) of Thermus sp. strain T2, and describes the construction of a ChBD-BgaA chimera which retains the biochemical properties of the native enzyme and can be purified in a single chromatographic step.     

The Cytochrome c Maturation Operon Is Involved in Manganese Oxidation in Pseudomonas putida GB-1

A Pseudomonas putida strain, strain GB-1, oxidizes Mn²⁺ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn²⁺ oxidation and/or secretion of the Mn²⁺-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn²⁺ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn²⁺-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn²⁺ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.In a number of studies during the last three decades it has been shown that various microbial species are able to stimulate the oxidation of Mn²⁺ through direct catalysis. These organisms produce proteinaceous macromolecules which catalyze the oxidation reaction. Manganese oxidations by a soil Arthrobacter species (24), Oceanospirillum and Vibrio strains (2, 3), Pseudomonas putida MnB1 (22, 30), Leptothrix discophora SS-1 (1, 11), and marine Bacillus strain SG-1 (23) are examples in which enzymes are most likely involved in the process. P. putida MnB1 produces a soluble protein which catalytically oxidizes Mn²⁺ in cell extracts (22). Manganese-oxidizing proteins from L. discophora SS-1 (1, 11) and from the spore coats of Bacillus strain SG-1 (43) have been identified on polyacrylamide gels. The oxidizing proteins have not been quantitatively purified or analyzed so far. In Bacillus strain SG-1, an operon containing seven genes appears to be involved in Mn²⁺ oxidation (46). One of these genes encodes a 137-kDa protein related to the family of multicopper oxidases (47). In a previous study we reported the isolation of a structural gene and its promoter postulated to be involved in Mn²⁺ oxidation in L. discophora (19). The encoded protein also contains the copper-binding signatures of multicopper oxidases. The oxidase-related proteins may represent Mn²⁺-oxidizing enzymes (44), but evidence supporting this hypothesis is still lacking.In this paper we describe a genetic analysis of Mn²⁺ oxidation in a freshwater Pseudomonas strain, strain GB-1. In a previous study (32) this strain was preliminarily identified as a Pseudomonas fluorescens strain, but more recent data (see Materials and Methods) indicate that it should be identified as a P. putida strain. When supplied with Mn²⁺ ions, the cells deposit manganese oxide around the outer membrane in the early stationary growth phase (32). They form brown colonies on Mn²⁺-containing agar. Experiments performed with cell extracts indicated that Mn²⁺ oxidation is catalyzed by a protein. The Mn²⁺-oxidizing factor was partially purified, and electrophoresis on an acrylamide gradient gel revealed oxidizing proteins with apparent molecular weights of ca. 250,000 and 180,000 (32). An additional oxidizing factor with a lower molecular weight (ca. 130,000) was identified in another study by using different isolation and electrophoretic procedures (16). It has been suggested that the Mn²⁺-oxidizing protein isolated is part of a larger complex which disintegrates into smaller fragments that retain activity (32). The protein is supposed to be located in the outer membrane of the bacteria. It has not been chemically characterized, and nothing is known about its cellular function or about the possible involvement of other cellular components, such as electron carriers, in Mn²⁺ oxidation.We used transposon mutagenesis to identify genes relevant to the Mn²⁺-oxidizing process in P. putida GB-1. One of these genes appeared to be part of the cytochrome c maturation operon. Transposon insertion in this gene not only abolished Mn²⁺ oxidation but also led to secretion of siderophores and porphyrins.An accompanying report on the involvement of the cytochrome c maturation operon in Mn²⁺ oxidation in P. putida MnB1 (14) supports our findings.