The structure of an extracellular,water-soluble polysaccharide elaborated by the cariogenic Streptococcus mutans GS-5

The extracellular, water-soluble polysaccharide elaborated by Streptococcus mutans GS-5 contains (1→6)- and (1→3,6)-linked α-d-glucopyranosyl residues. Its average repeating-unit contains 6 d-glucosyl residues and it is comb-like in structure. The majority of branches consist of only a few d-glucosyl residues, if not one d-glucosyl group.     

Isolation and Characterization of a Competition-Defective Bradyrhizobium japonicum Mutant

Tn5 mutagenesis was coupled with a competition assay to isolate mutants of Bradyrhizobium japonicum defective in competitive nodulation. A double selection procedure was used, screening first for altered extracellular polysaccharide production (nonmucoid colony morphology) and then for decreased competitive ability. One mutant, which was examined in detail, was deficient in acidic polysaccharide and lipopolysaccharide production. The wild-type DNA region corresponding to the Tn5 insertion was isolated, mapped, and cloned. A 3.6-kb region, not identified previously as functioning in symbiosis, contained the gene(s) necessary for complementation of the mutation. The mutant was motile, grew normally on minimal medium, and formed nodules on soybean plants which fixed almost as much nitrogen as the wild type during symbiosis.     

Biochemical study of the relationship of extracellular glucan to adherence and cariogenicity in Streptococcus mutans and an extracellular polysaccharide mutant.

A mutant of Streptococcus mutans, GS-5, which differed in extracellular polysaccharide (EPS) produced from sucrose, was used to study the role of EPS in the production of dental caries. The mutant proved to be identical to the parent strain in sugar fermentation, growth rate, and serotype. Strain GS-5 synthesized an EPS, which in electron micrographs appeared to be of fibrillar structure, whereas the mutant produced no fibrillar material but only a globular EPS. Analysis of the EPS revealed that about 30% of the glucose units in the GS-5 polymer carried (1-3)-like bonds either as branch points or as part of the linear backbone and that the mutant material contained only about 3% of these linkages. When grown in sucrose broth, the proportion of the mutant culture adherent to the glass vessel was dramatically less than that of the parent strain. Caries scores produced in conventional rats by the mutant were significantly lower than those obtained with the parent strain. Since the only difference discovered between strain GS-5 and the mutant was the inability of the mutant to synthesize either a fibrillar EPS or an EPS with more than about 3% (1-3)-like linkages, it was concluded that the fibrillar EPS of strain GS-5 contained about 30% (1-3)-like linkages and was necessary for adherence of the bacteria to surfaces and for production of dental caries in test animals.     

Ultraviolet-irradiation induced and spontaneous mutation of Rhizobium trifolii 11B in relation to water-soluble and water-insoluble polysaccharide production ability

Rhizobium trifolii 11B was u.v. irradiated and nine u.v. mutants have been isolated. Among the mutants, only one, R. trifolii 21M11B, produced more (752 mg/100 ml) water-soluble polysaccharide than the parent (704 mg/100 ml). The composition of water-soluble polysaccharide from u.v. mutants differed from that of the parent, R. trifolii 11B, and none of its u.v. mutants produced water-insoluble polysaccharide as detected by the Aniline Blue method. Storage of u.v. mutants for 2 months at 5°C gave four spontaneous variants which acquired the ability to produce water-insoluble polysaccharide. The spontaneous mutants also retained their water-soluble polysaccharide producing ability. The water-soluble polysaccharide produced by these mutants was characterized as curdlan type. The chemistry of water-soluble and water-insoluble polysaccharides was also ascertained.     

Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the “miniencapsulated” single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.     

Extracellular Polysaccharide from the Black Yeast NRRL Y-6272: Improved Methods for Preparing a High-Viscosity, Pigment-Free Product

When the extracellular polysaccharide from the black yeast NRRL Y-6272, composed of two parts N-acetyl-D-glucosamine and one part N-acetyl-D-glucosaminuronic acid, is isolated at maximum culture viscosity, adhering black pigment gives the polysaccharide preparations a gray-to-black appearance. Precipitation of the polysaccharide from cell-free culture supernatants with either ethanol of hexadecyltrimethylammonium bromide failed to remove the pigment. Various other methods were therefore tried for obtaining a high-viscosity polysaccharide product free of pigment. By systematically varying ingredients of defined and semidefined media, an improved medium was found that not only gave polysaccharide preparations of increased viscosity, but also increased yield. A key ingredient in this medium is L-asparagine. Also, adding autoclaved bovine serum albumin or egg albumin to this medium at the time of inoculation allowed a pigment-free polysaccharide to be isolated by standard procedures. None of several other proteins of synthetic polyamides tested were as effective as bovine serum albumin or egg albumin. In an alternate approach, pink mutants obtained by irradiation of the parent black strain with ultraviolet light, apparently produce the same extracellular polysaccharide free of any pigment but in lower yields or inferior in quality.     

Characteristics of Insertional Mutants of Pseudomonas syringae with Reduced Epiphytic Fitness

Random Tn5 mutagenesis was used to identify genes ir. Pseudomonas syringae which contribute to epiphytic fitness. Mutants were selected on the basis of deficiencies in epiphytic growth or survival on plants rather than deficiencies in predetermined phenotypes exhibited in culture. A sample freezing procedure was used to measure the population sizes of 5,300 mutants of P. syringae exposed to alternating wet and dry conditions on bean leaves in growth chambers. Eighty-two mutants exhibited reduced population sizes. Of these mutants, over half exhibited a reduced ability to survive the stresses associated with dry leaves, while others grew more slowly or attained reduced stationary-phase population sizes on leaves. While some epiphytic fitness mutants were altered in phenotypes that could be measured in culture, many mutants were not altered in any in vitro phenotype examined. Only three of the epiphytic fitness mutants were auxotrophs, and none had catabolic deficiencies for any of 31 organic compounds tested. Other mutants that exhibited reductions in one or more of the following were identified: motility, osmotolerance, desiccation tolerance, growth rate in batch culture, and extracellular polysaccharide production. All of the mutants retained the abilities to produce disease symptoms on the compatible host plant, bean, to incite a hypersensitive response on the non-host plant, tobacco, and to produce a fluorescent pyoverdine siderophore.     

Synthesis of Analogs of Pestalotin

Starch-utilizing mutants of Escherichia coli which can grow well on starch or amylose as the sole carbon source were isolated. The maximal viable cell number of the starch-utilizing mutants on the polysaccharide media reached the same level (4 × 10⁹ cells/ml) as that with glucose medium after incubation for 24 hours at 37°C. The isolated mutants could produce more intracellular α-amylase than the wild-type strain, and the enzyme activity was detected in the extracellular fluid. Polyacrylamide gel electrophoresis showed that the intracellular and extracellular enzymes had similar electrophoretic mobilities. These observations suggested that the ability of growth on the polysaccharide media was due to the excreted α-amylase, which appeared to be identical with the intracellular enzyme.     

野油菜黄单胞菌野油菜致病变种8004菌株wxcA基因与EPS的产量有关

在以前的工作中,采用转座子Tn5 gusA5对野油菜黄单胞菌野油菜致病变种(Xcc)8004菌株进行诱变,获得一批胞外多糖(EPS)合成减少的突变体,对这些突变体的Tn5 gusA5的插入位点进行分析后,发现有两株突变体是wxcA基因不同插入位点的突变体。以前认为wxcA基因与脂多糖(LPS)的O-抗原合成有关而与EPS的合成无关。为明确wxc4基因的功能,对8004菌株的wxcA基因进行缺失,获得的△wxcA突变体的EPS产量与野生型菌株相比,减少了50％,并且一段PCR合成的包含wxcA基因的DNA片段能反式互补△wxcA突变体,恢复突变体的EPS产量。这证实了8004菌株的wxcA基因与EPS的合成产量有关。     

Cloning of genes involved in negative regulation of production of extracellular enzymes and polysaccharide of Xanthomonas campestris pathovar campestris

Summary A recombinant plasmid pIJ3079 contains DNA sequences from Xanthomonas campestris pv campestris involved in coordinate negative regulation of production of the extracellular enzymes protease, endoglucanase, amylase and polygalacturonate lyase, and extracellular polysaccharide (EPS). Wild-type bacteria harbouring pIJ3079 and therefore carrying extra copies of the gene(s) therein showed reduced enzyme and EPS production and reduced aggresiveness to plants. Localised Tn5 mutagenesis of the corresponding region of the genome gave mutants producing higher levels of enzymes and EPS than the wild type, suggesting that the gene(s) may negatively regulate production in the normal cell. Enzyme and EPS production in the mutants was still dependent on previously characterised positive regulatory genes.     

Biosensor-assisted evolution for high-level production of 4-hydroxyphenylacetic acid in Escherichia coli

4-Hydroxyphenylacetic acid (4HPAA) is an important building block for synthesizing drugs, agrochemicals, and biochemicals, and requires sustainable production to meet increasing demand. Here, we use a 4HPAA biosensor to overcome the difficulty of conventional library screening in identification of preferred mutants. Strains with higher 4HPAA production and tolerance are successfully obtained by atmospheric and room temperature plasma (ARTP) mutagenesis coupled with adaptive laboratory evolution using this biosensor. Genome shuffling integrates preferred properties in the strain GS-2-4, which produces 25.42 g/L 4HPAA. Chromosomal mutations of the strain GS-2-4 are identified by whole genome sequencing. Through comprehensive analysis and experimental validation, important genes, pathways and regulations are revealed. The best gene combination in inverse engineering, acrD-aroG, increases 4HPAA production of strain GS-2-4 by 37% further. These results emphasize precursor supply and stress resistance are keys to efficient 4HPAA biosynthesis. Our work shows the power of biosensor-assisted screening of mutants from libraries. The methods developed here can be easily adapted to construct cell factories for the production of other aromatic chemicals. Our work also provides many valuable target genes to build cell factories for efficient 4HPAA production in the future.     

维吾尔族不同龋敏感儿童变链菌临床分离株不同生存状态合成胞外多糖能力的研究

目的比较维吾尔族不同龋敏感儿童变链菌临床分离株生物膜状态和浮游状态致龋能力的差异。方法根据析因实验设计分组,选取课题组前期分离鉴定的维吾尔族儿童变链菌临床株27株,其中17株高龋菌株[龋失补牙数（dmft）≥5],10株无龋菌株[龋失补牙数（dmft）=0]。分别在24孔板静止培养以及在试管内摇动培养。采用蒽酮法测定高龋组与无龋组分别在生物膜状态和浮游状态下合成胞外多糖的量。使用SPSS 17.0软件包对实验结果进行析因分析。结果高龋组生物膜状态下合成水溶性与非水溶性葡聚糖量分别为（0.3011±0.0398）mg/mL、（0.3711±0.0372）mg/mL,高于浮游状态的（0.2300±0.0438）mg/mL、（0.2356±0.0568）mg/mL,无龋组生物膜状态下合成水溶性与非水溶性葡聚糖分别为（0.2067±0.0264）mg/mL、（0.3489±0.0537）mg/mL,高于浮游状态的（0.1489±0.0325）mg/mL、（0.1578±0.0227）mg/mL,差异有统计学意义（P〈0.05）。并且高龋组合成胞外多糖量均高于无龋组,差异有统计学意义（P〈0.05）。结论维吾尔族高龋儿童变链菌临床分离株的高致龋力可能与其携带有合成胞外多糖能力较强的菌株有关。     

Anaerobic Oxidation of Toluene, Phenol, and p-Cresol by the Dissimilatory Iron-Reducing Organism, GS-15

The dissimilatory Fe(III) reducer, GS-15, is the first microorganism known to couple the oxidation of aromatic compounds to the reduction of Fe(III) and the first example of a pure culture of any kind known to anaerobically oxidize an aromatic hydrocarbon, toluene. In this study, the metabolism of toluene, phenol, and p-cresol by GS-15 was investigated in more detail. GS-15 grew in an anaerobic medium with toluene as the sole electron donor and Fe(III) oxide as the electron acceptor. Growth coincided with Fe(III) reduction. [ring-¹⁴C]toluene was oxidized to ¹⁴CO₂, and the stoichiometry of ¹⁴CO₂ production and Fe(III) reduction indicated that GS-15 completely oxidized toluene to carbon dioxide with Fe(III) as the electron acceptor. Magnetite was the primary iron end product during toluene oxidation. Phenol and p-cresol were also completely oxidized to carbon dioxide with Fe(III) as the sole electron acceptor, and GS-15 could obtain energy to support growth by oxidizing either of these compounds as the sole electron donor. p-Hydroxybenzoate was a transitory extracellular intermediate of phenol and p-cresol metabolism but not of toluene metabolism. GS-15 oxidized potential aromatic intermediates in the oxidation of toluene (benzylalcohol and benzaldehyde) and p-cresol (p-hydroxybenzylalcohol and p-hydroxybenzaldehyde). The metabolism described here provides a model for how aromatic hydrocarbons and phenols may be oxidized with the reduction of Fe(III) in contaminated aquifers and petroleum-containing sediments.     

Glucuronyltransferase Activity of KfiC from Escherichia coli Strain K5 Requires Association of KfiA: KfiC AND KfiA ARE ESSENTIAL ENZYMES FOR PRODUCTION OF K5 POLYSACCHARIDE, N-ACETYLHEPAROSAN*

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA β1–4 and GlcNAc α1–4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.     

DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.     

Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.     

Utilization of a mini-mu transposon to construct defined mutants in Streptococcus mutans

The gtfB gene coding for glucosyltransferase-I (GTF-I) activity previously isolated from Streptococcus mutans GS-5 was insertionally inactivated with the newly constructed transposon MudE in an Escherichia coli background. Insertion of MudE into various regions of the gtfB gene led to inactivation of GTF-I activity. The altered gene was introduced back into S. mutans GS-5 by transformation and produced mutants defective in insoluble glucan synthesis as well as the ability to colonize smooth surfaces in the presence of sucrose. Therefore, the MudE transposon can be utilized to produce specific mutants in oral streptococci as well as in other transformable Gram-positive bacteria expressing an erythromycin-resistance marker.     

Polysaccharide synthesis in relation to nodulation behavior of Rhizobium leguminosarum.

In this study, we characterized four Tn5 mutants derived from Rhizobium leguminosarum RBL5515 with respect to synthesis and secretion of cellulose fibrils, extracellular polysaccharides (EPS), capsular polysaccharides, and cyclic beta-(1,2)-glucans. One mutant, strain RBL5515 exo-344::Tn5, synthesizes residual amounts of EPS, the repeating unit of which lacks the terminal galactose molecule and the substituents attached to it. On basis of the polysaccharide production pattern of strain RBL5515 exo-344::Tn5, the structural features of the polysaccharides synthesized, and the results of an analysis of the enzyme activities involved, we hypothesize that this strain is affected in a galactose transferase involved in the synthesis of EPS only. All four mutants failed to nodulate plants belonging to the pea cross-inoculation group; on Vicia sativa they induced root hair deformation and rare abortive infection threads. All of the mutants appeared to be pleiotropic, since in addition to defects in the synthesis of EPS, lipopolysaccharide, and/or capsular polysaccharides significant increases in the synthesis and secretion of cyclic beta-(1,2)-glucans were observed. We concluded that it is impossible to correlate a defect in the synthesis of a particular polysaccharide with nodulation characteristics.     

Construction of Streptococcus mutans glucosyltransferase mutants utilizing a cloned gene fragment

Abstract An internal 1.6-kb BAM HI DNA fragment of the previously cloned Streptococcus mutans GS-5 gtfB gene was utilized to construct plasmids capable of insertion in toto into the GS-5 chromosome. The resultant insertions primarily yielded mutants defective in glucosyltransferase-I activity. These mutants were also defective in sucrose-dependent colonization of smooth surfaces.     

Construction of a Tn5-Tagged Mutant Library of Xanthomonas oryzae pv. oryzicola as An Invaluable Resource for Functional Genomics

To genome-widely mine pathogenesis-related genes of Xanthomonas oryzae pv. oryzicola (Xoc), which is the casual agent of bacterial leaf streak resulting in significant yield loss and poor quality in rice, a Tn5 transposon-mediated mutation library was generated. Twenty-five thousand transformants were produced by using Tn5 transposome, appropriately corresponding to 5 × ORF coverage of the genome, and inoculated into rice and tobacco, individually and respectively, for screening candidate virulence genes. Southern blot and thermal asymmetric interlaced polymerase chain reaction analysis of Tn5 insertion sites of randomly selected mutants suggested a random mode of transposition and a saturation library. Characterization of extracellular polysaccharides, extracellular protease activity, and pigment production of individual mutants in the growth media revealed that 11 mutants enhanced in growth, 12 reduced extracellular polysaccharide production, 12 lost extracellular protease activity completely or partially, and 21 were pigment deficient. In planta pathogenicity assays revealed 253 mutants reduced virulence in rice, but kept triggering hypersensitive response in tobacco; 49 lost the ability to elicit HR in tobacco and pathogenicity in rice; and 3 still induced hypersensitive response in tobacco, but lost pathogenicity in rice. The achieved mutant library of Xoc is of high-quality and nearly saturated and candidate virulence mutants provided a strong basis for functional genomics of Xoc.