Native state fluctuations in a peroxiredoxin active site match motions needed for catalysis.

Peroxiredoxins are ubiquitous enzymes that detoxify peroxides and regulate redox signaling. During catalysis, a "peroxidatic" cysteine (CP) in the conserved active site reduces peroxide while being oxidized to a CP-sulfenate, prompting a local unfolding event that enables formation of a disulfide with a second, "resolving" cysteine. Here, we use nuclear magnetic resonance spectroscopy to probe the dynamics of the CP-thiolate and disulfide forms of Xanthomonas campestris peroxiredoxin Q. Chemical exchange saturation transfer behavior of the resting enzyme reveals 26 residues in and around the active site exchanging at a rate of 72 s-1 with a locally unfolded, high-energy (2.5% of the population) state. This unequivocally establishes that a catalytically relevant local unfolding equilibrium exists in the enzyme's CP-thiolate form. Also, faster motions imply an active site instability that could promote local unfolding and, based on other work, be exacerbated by CP-sulfenate formation so as to direct the enzyme along a functional catalytic trajectory.

Effects of photo-stimulation with laser or LED on the composition of Xanthan gum produced in media containing distilled water or dialyzed or not produced water by means of Raman spectroscopy.

Oil is expected to continue to be one of the most important sources of energy in the world and world's energy matrix for the foreseeable future. However, high demand for energy and the decline of the production of oil fields makes oil recovery a challenge. Most techniques used for the recovery process are expensive, non-sustainable and technically difficult to implement. In this context, microbial enhanced oil recovery (MEOR) represents an attractive alternative. It employs products derived from the metabolism of microorganisms that produce biopolymers. Certain bacteria species (e.g., Xanthomonas campestris) produce polysaccharides (exopolysaccharides - EPS) such as the well-known Xanthan gum (XG). We hypothesized that the use of produced water (PW) water in combination photo-stimulation with laser/LED could influence the production and composition of XG. Raman spectroscopy has been used for qualitative and quantitative evaluation of the biochemical composition of XG biopolymer under light stimulation. X. campestris cultures in either distilled water or dialysis-produced water were studied under the absence or presence of laser irradiation (λ = 660 nm, CW, spot size 0.040 cm2, 40 mW, 444 s, 8.0 J/cm2) or LED (λ = 630 nm ± 2 nm, CW, spot size 0.50 cm2, 140 mW, 500 s, 12 J/cm2). XG produced by these cultures was analyzed by Raman spectroscopy at 1064 nm excitation and subjected to principal component analysis (PCA). Results of the exploratory analysis and ANOVA general linear model (GLM) suggested that the extent of XG and pyruvate (pyruvyl mannose) production was affected differentially in X. campestris when cultured in distilled water plus LED photo-stimulation versus dialysis-produced water plus LED photo-stimulation. XG production increased in the distilled water culture. In contrast, both pyruvate acetyl mannose content went up in the dialysis-water culture. These results open a wide field of opportunities in the use of metal-enriched cultures in combination with photo-biomodulation to direct and optimize bacterial production of compounds (i.e., XG) that may be of great benefit in the implementation of sustainable practices for oil extraction.

Multiple Xanthomonas campestris pv. campestris 8004 type III effectors inhibit immunity induced by flg22.

MAIN CONCLUSION: Xanthomonas campestris pv. campestris 8004 secretes several effector proteins that interfere with plant phosphorylation. Xanthomonas campestris pv. campestris (Xcc) can infect cruciferous plants and cause black rot. The strain Xcc8004 secretes effector proteins that interfere with plant cellular processes into host cells using a type III secretion (T3S) system. Several of the 24 predicted T3S effectors in the Xcc8004 genome have been implicated in the suppression of the Arabidopsis thaliana pattern-triggered immunity (PTI) response. We used an A. thaliana mesophyll protoplast-based assay to identify Xcc8004 T3S effectors that effectively interfere with PTI signalling induced by the bacterial peptide flg22. 11 of the 24 tested effector proteins (XopK, XopQ, HrpW, XopN, XopAC, XopD, XopZ1, XopAG, AvrBs2, XopL and XopX-1) inhibited expression of the flg22-inducible gene FRK1, and five effectors (XopK, XopG, XopQ, XopL and XopX-1) inhibited the expression of the flg22-inducible gene WRKY33. Therefore, there are 12 effector proteins that can inhibit the expression of relevant flg22-inducible genes. It was further investigated whether the 12 effector proteins affect the phosphorylation activation of mitogen-activated protein (MAP) kinases MPK3/MPK6, and four effector proteins (XopK, XopQ, XopZ1 and XopX-1) were found to markedly inhibit MPK3/MPK6 activation. Moreover, a subcellular localisation analysis revealed that the tested effectors were localised within various subcellular compartments. These results indicate that multiple T3S effectors in the Xcc8004 genome interfere with flg22-induced PTI signalling via various molecular mechanisms.

A Spectroscopy-based Methodology for Rapid Screening and Characterization of Phytochrome Photochemistry in Search of Pfr-favored Variants.

Phytochromes are photosensitive proteins with a covalently bound open-chain chromophore that can switch between two principal states: red light absorbing Pr and far-red light absorbing Pfr. Our group has previously shown that the bacteriophytochrome from Xanthomonas campestris pv. campestris (XccBphP) is a bathy-like phytochrome that uses biliverdin IXα as a co-factor and is involved in bacterial virulence. To date, the XccBphP crystal structure could only be solved in the Pr state, while the structure of its Pfr state remains elusive. The aims of this work were to develop an efficient screening methodology for the rapid characterization and to identify XccBphP variants that favor the Pfr form. The screening approach developed here consists in analyzing the UV-Vis absorption behavior of clarified crude extracts containing recombinant phytochromes. This strategy has allowed us to quickly explore over a hundred XccBphP variants, characterize multiple variants and identify Pfr-favored candidates. The high-quality data obtained enabled not only a qualitative, but also a quantitative characterization of their photochemistry. This method could be easily adapted to other phytochromes or other photoreceptor families.

Crystal structure of α-glucosyl transfer enzyme XgtA from Xanthomonas campestris WU-9701.

The α-glucosyl transfer enzyme XgtA is a novel type α-Glucosidase (EC 3.2.1.20) produced by Xanthomonas campestris WU-9701. One of the unique properties of XgtA is that it shows extremely high α-glucosylation activity toward alcoholic and phenolic -OH groups in compounds using maltose as an α-glucosyl donor and allows for the synthesis of various useful α-glucosides with high yields. XgtA shows no hydrolytic activity toward sucrose and no α-glucosylation activity toward saccharides to produce oligosaccharides. In this report, the crystal structure of XgtA was solved at 1.72 Å resolution. The crystal belonged to space group P22121, with unit-cell parameters a = 73.07, b = 83.48, and c = 180.79 Å. The ß→α loop 4 of XgtA, which is proximal to the catalytic center, formed a unique structure that is not observed in XgtA homologs. Furthermore, XgtA was found to contain unique amino acid residues around its catalytic center. The unique structure of XgtA provides an insight into the mechanism for the regulation of substrate specificity in this enzyme.

Chemical Structure, Biological Roles, Biosynthesis and Regulation of the Yellow Xanthomonadin Pigments in the Phytopathogenic Genus Xanthomonas.

Xanthomonadins are membrane-bound yellow pigments that are typically produced by phytopathogenic bacterial Xanthomonas spp., Xylella fastidiosa, and Pseudoxanthomonas spp. They are also produced by a diversity of environmental bacterial species. Considerable research has revealed that they are a unique group of halogenated, aryl-polyene, water-insoluble pigments. Xanthomonadins have been shown to play important roles in epiphytic survival and host-pathogen interactions in the phytopathogen Xanthomonas campestris pv. campestris, which is the causal agent of black rot in crucifers. Here, we review recent advances in the understanding of xanthomonadin chemical structures, physiological roles, biosynthetic pathways, regulatory mechanisms, and crosstalk with other signaling pathways. The aim of the present review is to provide clues for further in-depth research on xanthomonadins from Xanthomonas and other related bacterial species.

Chemical Targeting and Manipulation of Type III Secretion in the Phytopathogen Xanthomonas campestris for Control of Disease.

Xanthomonas campestris pv. campestris is the causative agent of black rot disease in crucifer plants. This Gram-negative bacterium utilizes the type III secretion system (T3SS), encoded by the hrp gene cluster, to aid in its resistance to host defenses and the ability to cause disease. The T3SS injects a set of proteins known as effectors into host cells that come into contact with the bacterium. The T3SS is essential for the virulence and hypersensitive response (HR) of X. campestris pv. campestris, making it a potential target for disease control strategies. Using a unique and straightforward high-throughput screening method, we examined a large collection of diverse small molecules for their potential to modulate the T3SS without affecting the growth of X. campestris pv. campestris. Screening of 13,129 different compounds identified 10 small molecules that had a significant inhibitory influence on T3SS. Moreover, reverse transcription-quantitative PCR (qRT-PCR) assays demonstrated that all 10 compounds repress the expression of the hrp genes. Interestingly, the effect of these small molecules on hrp genes may be through the HpaS and ColS sensor kinase proteins that are key to the regulation of the T3SS in planta Five of the compounds were also capable of inhibiting X. campestris pv. campestris virulence in a Chinese radish leaf-clipping assay. Furthermore, seven of the small molecules significantly weakened the HR in nonhost pepper plants challenged with X. campestris pv. campestris. Taken together, these small molecules may provide potential tool compounds for the further development of antivirulence agents that could be used in disease control of the plant pathogen X. campestris pv. campestris.IMPORTANCE The bacterium Xanthomonas campestris pv. campestris is known to cause black rot disease in many socioeconomically important vegetable crops worldwide. The management and control of black rot disease have been tackled with chemical and host resistance methods with variable success. This has motivated the development of alternative methods for preventing this disease. Here, we identify a set of novel small molecules capable of inhibiting X. campestris pv. campestris virulence, which may represent leading compounds for the further development of antivirulence agents that could be used in the control of black rot disease.

Micro-deposition control of polysaccharides on evaporative air-LC interface to design quickly swelling hydrogels.

Uniaxial orientation is highly desirable for fabricating advanced soft materials. Liquid crystal (LC) polymer deposition was strategically manipulated at the air-LC interface, by controlling the drying temperature and initial concentration of aqueous solution of xanthan gum in a limited space. Interface-assisted orientation led to membrane-like depositions bridging the millimeter-scale gap between the substrates both, vertically and horizontally. The applicability of this approach lies in synchronization of the molecular orientation beyond their individual LC domains into the condensed state. Cross-polarized microscopy and SEM analysis correlated the orientation of the deposited polymer with the controlled mobility of xanthan gum LC domains at the evaporative interface. Subsequently, a phase diagram was prepared for the variety of oriented structures, depending upon the drying conditions. The deposited membrane behaved as an oriented hydrogel showing reversible anisotropic swelling/deswelling only along its thickness.

Tailor-made polysaccharides containing uniformly distributed repeating units based on the xanthan gum skeleton.

Xanthan gum, whose structure determines its physicochemical properties, is an important microbial polysaccharide. Currently, marketed xanthan products are produced by wild-type strains followed by post-fermentation separation or chemical modification, which are complicated and labor-intensive. In the present study, we designed eight polysaccharides containing uniformly distributed repeating units and different rheological properties based on natural xanthan skeleton and according to the relationship between property and structure. The customized polysaccharides were produced in Xanthomonas campestris CGMCC 15155 using marker-less gene knockout and gene overexpression methods. The results showed that their different homogeneous primary structures determined their specific secondary structures and rheological properties, especially the terminal mannose, the pyruvyl group, and the acetyl group attached to the internal mannose of the side chain. Polysaccharides lacking a terminal mannose, such as xanthan XdM-0 and XdM-A, had reduced zero-shear viscosity and modulus values. The internal acetyl group of the side chain stabilized the helix structure (e.g., in XG-A0), while the pyruvate group had the opposite effect (e.g., in XG-AP and XG-0P). The eight xanthan variants provide a promising theoretical foundation to further study the structure-activity relationship of xanthan and will help to construct xanthan-containing block copolymers.

Potential application of a low-viscosity and high-transparency xanthan gum produced from Xanthomonas campestris CCTCC M2015714 in foods.

Xanthan gum is commonly used as a thickener in food industry, while the usage of xanthan gum as a dietary fiber is restricted for its low additive volume. Herein, the potential use of a low-viscosity and high-transparency xanthan gum as a dietary fiber was evaluated in vitro. This new xanthan shows better transparency and faster dissolution rate than most commercial products, and its viscosity increases along with the treatment of freeze-thaw cycles at -20°C. Moreover, this new xanthan can absorb heavy metals (Pb, Cd, Cu) and retard starch digestion by glucoamylase. In summary, this new xanthan could be potentially used as a dietary fiber or fiber ingredient for preventing and treating diabetes, hyperlipemia, heavy metal poisoning, and cardiovascular diseases effectively.

A robust protocol for the isolation of cellular proteins from Xanthomonas campestris to analyze the methionine effect in 2D-gel experiments.

Two-dimensional PAGE (2D-PAGE) is a key technique for the separation of complex protein samples to survey the protein inventory of prokaryotic microorganisms. Although the preparation of proteins is a critical step in 2D gel experiments, its effect on the outcome of proteome data is often underestimated. In this work, we show that the choice of protein isolation method can have a profound impact on the quality of 2D gels of protein extracts from prokaryotes. Based on Xanthomonas campestris, a commercially relevant producer of the thickening agent xanthan, we validated a phenol extraction protocol for the purification of bacterial proteins that provides excellent 2D gel separation. As a proof of concept, this method was used to study the effect of methionine-a medium compound that reduces the xanthan output of industrial fermentations-on the cellular proteome of Xanthomonas. The detection of nine regulated proteins associated with sulfur metabolism (Cgl, CysI, CysJ, CysK, MetH1, MetY) and sugar nucleotide biosynthesis (Pgi, Ugd, XanA) proved the efficiency of phenol extraction for the screening of statistically significant abundance changes in 2D gel spots and MALDI-TOF-MS based identification in bacteria. Since this method is very robust, it may be useful for the study of other prokaryotes that are relevant in industrial biotechnology.

Active Multienzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis.

Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive ß-lactone intermediate produced by the OleC-catalyzed reaction.IMPORTANCE Bacteria biosynthesize hydrophobic molecules to maintain a membrane, store carbon, and for antibiotics that help them survive in their niche. The hydrophobic compounds are often synthesized by a multidomain protein or by large multienzyme assemblies. The present study reports on the discovery that long-chain olefinic hydrocarbons made by bacteria from different phyla are produced by multienzyme assemblies in X. campestris The OleBCD multienzyme assemblies are thought to compartmentalize and sequester olefin biosynthesis from the rest of the cell. This system provides additional insights into how bacteria control specific biosynthetic pathways.

Crystal structure of the YajQ-family protein XC_3703 from Xanthomonas campestris pv. campestris.

As an important bacterial second messenger, bis-(3',5')-cyclic diguanylate (cyclic di-GMP or c-di-GMP) has been implicated in numerous biological activities, including biofilm formation, motility, survival and virulence. These processes are manipulated by the binding of c-di-GMP to its receptors. XC_3703 from the plant pathogen Xanthomonas campestris pv. campestris, which belongs to the YajQ family of proteins, has recently been identified as a potential c-di-GMP receptor. XC_3703, together with XC_2801, functions as a transcription factor activating virulence-related genes, which can be reversed by the binding of c-di-GMP to XC_3703. However, the structural basis of how c-di-GMP regulates XC_3703 remains elusive. In this study, the structure of XC_3703 was determined to 2.1 Šresolution using the molecular-replacement method. The structure of XC_3703 consists of two domains adopting the same topology, which is similar to that of the RNA-recognition motif (RRM). Arg65, which is conserved among the c-di-GMP-binding subfamily of the YajQ family of proteins, together with Phe80 in domain II, forms a putative c-di-GMP binding site.

Peroxiredoxin Catalysis at Atomic Resolution.

Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases that guard cells against oxidative damage, are virulence factors for pathogens, and are involved in eukaryotic redox regulatory pathways. We have analyzed catalytically active crystals to capture atomic resolution snapshots of a PrxQ subfamily enzyme (from Xanthomonas campestris) proceeding through thiolate, sulfenate, and sulfinate species. These analyses provide structures of unprecedented accuracy for seeding theoretical studies, and reveal conformational intermediates giving insight into the reaction pathway. Based on a highly non-standard geometry seen for the sulfenate intermediate, we infer that the sulfenate formation itself can strongly promote local unfolding of the active site to enhance productive catalysis. Further, these structures reveal that preventing local unfolding, in this case via crystal contacts, results in facile hyperoxidative inactivation even for Prxs normally resistant to such inactivation. This supports previous proposals that conformation-specific inhibitors may be useful for achieving selective inhibition of Prxs that are drug targets.

Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase.

GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-trisaccharide embedded in the membrane of the bacteria. The structure of GumK was previously described in its apo- and UDP-bound forms, with no significant conformational differences being observed. Here, we study the behavior of GumK toward its donor substrate UDP-GlcA. Turbidity measurements revealed that the interaction of GumK with UDP-GlcA produces aggregation of protein molecules under specific conditions. Moreover, limited proteolysis assays demonstrated protection of enzymatic digestion when UDP-GlcA is present, and this protection is promoted by substrate binding. Circular dichroism spectroscopy also revealed changes in the GumK tertiary structure after UDP-GlcA addition. According to the obtained emission fluorescence results, we suggest the possibility of exposure of hydrophobic residues upon UDP-GlcA binding. We present in silico-built models of GumK complexed with UDP-GlcA as well as its analogs UDP-glucose and UDP-galacturonic acid. Through molecular dynamics simulations, we also show that a relative movement between the domains appears to be specific and to be triggered by UDP-GlcA. The results presented here strongly suggest that GumK undergoes a conformational change upon donor substrate binding, likely bringing the two Rossmann fold domains closer together and triggering a change in the N-terminal domain, with consequent generation of the acceptor substrate binding site.

Structure of the Full-Length Bacteriophytochrome from the Plant Pathogen Xanthomonas campestris Provides Clues to its Long-Range Signaling Mechanism.

Phytochromes constitute a major superfamily of light-sensing proteins that are reversibly photoconverted between a red-absorbing (Pr) and a far-red-absorbing (Pfr) state. Bacteriophytochromes (BphPs) are found among photosynthetic and non-photosynthetic bacteria, including pathogens. To date, several BphPs have been biophysically characterized. However, it is still not fully understood how structural changes are propagated from the photosensory module to the output module during the signal transduction event. Most phytochromes share a common architecture consisting of an N-terminal photosensor that includes the PAS2-GAF-PHY domain triad and a C-terminal variable output module. Here we present the crystal structure of the full-length BphP from the plant pathogen Xanthomonas campestris pv. campestris (XccBphP) bearing its photosensor and its complete output module, a PAS9 domain. In the crystals, the protein was found to be in the Pr state, whereas diffraction data together with resonance Raman spectroscopic and theoretical results indicate a ZZZssa and a ZZEssa chromophore configuration corresponding to a mixture of Pr and Meta-R state, the precursor of Pfr. The XccBphP quaternary assembly reveals a head-to-head dimer in which the output module contributes to the helical dimer interface. The photosensor, which is shown to be a bathy-like BphP, is influenced in its dark reactions by the output module. Our structural analyses suggest that the photoconversion between the Pr and Pfr states in the full-length XccBphP may involve changes in the relative positioning of the output module. This work contributes to understand the light-induced structural changes propagated from the photosensor to the output modules in phytochrome signaling.

Crystal structure of truncated FlgD from the human pathogen Helicobacter pylori.

Flagellin component D (FlgD) participates in the assembly of flagella, helical tubular structures that provide motility in non-filamentous bacteria. FlgD guides and controls the polymerization of FlgE that builds the hook, a short curved and hollow cylinder that connects the flagellar basal body spanning the cell envelope to the protruding filament. Crystal structures of truncated forms of Helicobacter pylori FlgD from two different strains in two space groups, I422 and P2, are reported here, at 2.2Å and 2.8Å resolution, respectively. Analogously to Pseudomonas aeruginosa and Xanthomonas campestris FlgD proteins, crystallization experiments set up for the full length protein resulted in crystals of a truncated form, lacking both N- and C-terminus ends. The crystal structures of the central domain show that the monomer is composed of a tudor and a fibronectin type III domain. The full length HpFlgD contains a long N-terminal signal region, probably partially flexible, a central globular region and a C-terminal segment with a peculiar repetitive pattern of amino acids. The spatial orientation of the two domains in HpFlgD differs from that of the homologous FlgD family members, P. aeruginosa and X. campestris. This difference together with the observation that HpFlgD assembles into tetramers, both in the solution and in the two crystal forms, strongly suggests that significant differences exist in the molecular organization of the flagella in different bacterial species.

A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana.

The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor-like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species.

Crystallization and preliminary X-ray characterization of the full-length bacteriophytochrome from the plant pathogen Xanthomonas campestris pv. campestris.

Phytochromes give rise to the largest photosensor family known to date. However, they are underrepresented in the Protein Data Bank. Plant, cyanobacterial, fungal and bacterial phytochromes share a canonical architecture consisting of an N-terminal photosensory module (PAS2-GAF-PHY domains) and a C-terminal variable output module. The bacterium Xanthomonas campestris pv. campestris, a worldwide agricultural pathogen, codes for a single bacteriophytochrome (XccBphP) that has this canonical architecture, bearing a C-terminal PAS9 domain as the output module. Full-length XccBphP was cloned, expressed and purified to homogeneity by nickel-NTA affinity and size-exclusion chromatography and was then crystallized at room temperature bound to its cofactor biliverdin. A complete native X-ray diffraction data set was collected to a maximum resolution of 3.25 Å. The crystals belonged to space group P43212, with unit-cell parameters a = b = 103.94, c = 344.57 Šand a dimer in the asymmetric unit. Refinement is underway after solving the structure by molecular replacement.

Increasing viscosity and yields of bacterial exopolysaccharides by repeatedly exposing strains to ampicillin.

A universal method to enhance productivity and viscosity of bacterial exopolysaccharides was developed. The technique was based on the principle that ampicillin can inhibit the biosynthesis of peptidoglycan, which shares a common synthetic pathway with that of bacterial exopolysaccharides. Serial passages of three typical representatives of bacterial EPS-producing strains, namely Sphingomonas elodea, Xanthomonas campestris, and Paenibacillus elgii, were subjected to ampicillin, which was used as a stressor and a mutagen. These mutant strains are advantageous over other strains because of two major factors. First, all of the resulting strains were almost mutants with increase in EPS productivity and viscosity. Second, isolated serial strains showed different levels of increase in EPS production and viscosity to satisfy the different requirements of practical applications. No differences were observed in the monosaccharide composition produced by the mutant and parent strains; however, high-viscosity mutant strains exhibited higher molecular weights. The results confirmed that the developed method is a controlled universal one that can improve exopolysaccharides productivity and viscosity.