Efficacy of an inulin-based treatment on intestinal colonization by multidrug-resistant E. coli: insight into the mechanism of action.

Inulin, an increasingly studied dietary fiber, alters intestinal microbiota. The aim of this study was to assess whether inulin decreases intestinal colonization by multidrug resistant E. coli and to investigate its potential mechanisms of action. Mice with amoxicillin-induced intestinal dysbiosis mice were inoculated with extended spectrum beta-lactamase producing E. coli (ESBL-E. coli). The combination of inulin and pantoprazole (IP) significantly reduced ESBL-E. coli fecal titers, whereas pantoprazole alone did not and inulin had a delayed and limited effect. Fecal microbiome was assessed using shotgun metagenomic sequencing and qPCR. The efficacy of IP was predicted by increased abundance of 74 taxa, including two species of Adlercreutzia. Preventive treatments with A. caecimuris or A. muris also reduced ESBL-E. coli fecal titers. Fecal microbiota of mice effectively treated by IP was enriched in genes involved in inulin catabolism, production of propionate and expression of beta-lactamases. They also had increased beta-lactamase activity and decreased amoxicillin concentration. These results suggest that IP act through production of propionate and degradation of amoxicillin by the microbiota. The combination of pantoprazole and inulin is a potential treatment of intestinal colonization by multidrug-resistant E. coli. The ability of prebiotics to promote propionate and/or beta-lactamase producing bacteria may be used as a screening tool to identify potential treatments of intestinal colonization by multidrug resistant Enterobacterales.

Pantoprazole promotes sustained intestinal carriage of multidrug-resistant Escherichia coli in amoxicillin-treated mice.

AIMS: The main objective of this study was to compare extended-spectrum ß-lactamase (ESBL) Escherichia coli fecal titers during 12 days between two groups: mice who received proton pump inhibitors (PPIs) and those that did not. METHODS AND RESULTS: We tested three different in vivo models: model 1, high inoculum (106 CFU ml-1); model 2, low inoculum (102 CFU ml-1); and model 3, low inoculum and 2-day amoxicillin wash-out. There was no significant difference between the two groups in fecal ESBL E. coli titers in models 1 and 2. The fecal titers of ESBL E. coli were probably too high to show differences in colonization related to PPI treatment. By introducing a 2-day wash-out period after stopping amoxicillin (model 3), the fecal ESBL E. coli titers were higher in the PPI-treated mice during 12 days (3 log versus 11 log day CFU g-1; P < 0.05). This result highlighted that PPIs promote stable ESBL E. coli digestive carriage in mice. Fecal quantitative PCR showed that mice with low ESBL E. coli fecal titers had a much higher concentration of equol-producing bacteria, Muribaculum sp., and Adlercreutzia caecimuris. CONCLUSIONS: Pantoprazole treatment promotes sustained digestive carriage of ESBL E. coli in amoxicillin-treated mice.

Comparison of the global prevalence and trend of human intestinal carriage of ESBL-producing Escherichia coli between healthcare and community settings: a systematic review and meta-analysis.

Objectives: The widespread intestinal carriage of ESBL-producing Escherichia coli (ESBL E. coli) among both patients and healthy individuals is alarming. However, the global prevalence and trend of this MDR bacterium in healthcare settings remains undetermined. To address this knowledge gap, we performed a comparative meta-analysis of the prevalence in community and healthcare settings. Methods: Our systematic review included 133 articles published between 1 January 2000 and 22 April 2021 and indexed in PubMed, EMBASE or Google Scholar. A random-effects meta-analysis was performed to obtain the global pooled prevalence (community and healthcare settings). Subgroup meta-analyses were performed by grouping studies using the WHO regions and 5 year intervals of the study period. Results: We found that 21.1% (95% CI, 19.1%-23.2%) of inpatients in healthcare settings and 17.6% (95% CI, 15.3%-19.8%) of healthy individuals worldwide carried ESBL E. coli in their intestine. The global carriage rate in healthcare settings increased 3-fold from 7% (95% CI, 3.7%-10.3%) in 2001-05 to 25.7% (95% CI, 19.5%-32.0%) in 2016-20, whereas in community settings it increased 10-fold from 2.6% (95% CI, 1.2%-4.0%) to 26.4% (95% CI, 17.0%-35.9%) over the same period. Conclusions: The global and regional human intestinal ESBL E. coli carriage is increasing in both community and healthcare settings. Carriage rates were generally higher in healthcare than in community settings. Key relevant health organizations should perform surveillance and implement preventive measures to address the spread of ESBL E. coli in both settings.

In vitro and in vivo activity of new strains of Bacillus subtilis against ESBL-producing Escherichia coli: an experimental study.

AIMS: The gastro-intestinal tract is a major reservoir of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli. Bacillus spores may be used as probiotics to decrease digestive colonization by ESBL-E. coli. Our aim was to assess the in vitro and in vivo activity of new Bacillus strains against ESBL-E. coli. METHODS AND RESULTS: We screened the in vitro activity of 50 Bacillus strains against clinical isolates of ESBL-E. coli and selected B. subtilis strains CH311 and S3B. Both strains decreased ESBL-E. coli titers by 4 log10 CFU L-1 in an in vitro model of gut content, whereas the B. subtilis CU1 strain did not. In a murine model of intestinal colonization by ESBL-E. coli, CH311 and S3B did not decrease fecal titers of ESBL-E. coli. Ten sequences of putative antimicrobial peptides were identified in the genomes of CH311 and S3B, but not in CU1. CONCLUSIONS: Two new B. subtilis strains showed strong in vitro activity against ESBL-E. coli. SIGNIFICANCE AND IMPACT OF STUDY: Despite strong in vitro activities of new B. subtilis strains against ESBL-E. coli, intestinal colonisation was not altered by curative Bacillus treatment even if their spores proved to germinate in the gut. Thus, this work underlines the importance of in vivo experiments to identify efficient probiotics. The use of potential antimicrobial compounds identified by genome sequencing remains an attractive alternative to explore.

ABO Blood Types and COVID-19: Spurious, Anecdotal, or Truly Important Relationships? A Reasoned Review of Available Data.

Since the emergence of COVID-19, many publications have reported associations with ABO blood types. Despite between-study discrepancies, an overall consensus has emerged whereby blood group O appears associated with a lower risk of COVID-19, while non-O blood types appear detrimental. Two major hypotheses may explain these findings: First, natural anti-A and anti-B antibodies could be partially protective against SARS-CoV-2 virions carrying blood group antigens originating from non-O individuals. Second, O individuals are less prone to thrombosis and vascular dysfunction than non-O individuals and therefore could be at a lesser risk in case of severe lung dysfunction. Here, we review the literature on the topic in light of these hypotheses. We find that between-study variation may be explained by differences in study settings and that both mechanisms are likely at play. Moreover, as frequencies of ABO phenotypes are highly variable between populations or geographical areas, the ABO coefficient of variation, rather than the frequency of each individual phenotype is expected to determine impact of the ABO system on virus transmission. Accordingly, the ABO coefficient of variation correlates with COVID-19 prevalence. Overall, despite modest apparent risk differences between ABO subtypes, the ABO blood group system might play a major role in the COVID-19 pandemic when considered at the population level.

Characterization and engineering of two new GH9 and GH48 cellulases from a Bacillus pumilus isolated from Lake Bogoria.

OBJECTIVES: To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering RESULTS: Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. BpGH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-ß1,4 glucanes. A truncated form of BpGH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of BpGH9 on crystalline cellulose. CONCLUSIONS: A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.

Enhancing the chemoenzymatic synthesis of arabinosylated xylo-oligosaccharides by GH51 α-L-arabinofuranosidase.

Random mutagenesis was performed on the α-l-arabinofuranosidase of Thermobacillus xylanilyticus in order to enhance its ability to perform transarabinofuranosylation using natural xylo-oligosaccharides as acceptors. To achieve this goal, a two-step, high-throughput digital imaging protocol involving a colorimetric substrate was used to screen a library of 30,000 mutants. In the first step this screen selected for hydrolytically-impaired mutants, and in the second step the screen identified mutants whose global activity was improved in the presence of a xylo-oligosaccharide mixture. Thereby, 199 mutants displaying lowered hydrolytic activity and modified properties were detected. In the presence of these xylo-oligosaccharides, most of the 199 (i.e., 70%) enzymes were less inhibited and some (18) mutants displayed an unambiguous alleviation of inhibition (<25% loss of activity). More precise monitoring of reactions catalyzed by the most promising mutants revealed a significant improvement of the synthesis yields of transglycosylation products (up to 18% compared to 9% for the parental enzyme) when xylobiose was present in the reaction. Genetic analysis of improved mutants revealed that many of the amino acid substitutions that correlate with the modified phenotype are located in the vicinity of the active site, particularly in subsite -1. Consequently, we hypothesize that these mutations modify the active site topology or the molecular interaction network of the l-arabinofuranoside donor substrate, thus impairing the hydrolysis and concomitantly favoring transglycosylation onto natural acceptors.

De novo design of a trans-ß-N-acetylglucosaminidase activity from a GH1 ß-glycosidase by mechanism engineering.

Glycoside hydrolases are particularly abundant in all areas of metabolism as they are involved in the degradation of natural polysaccharides and glycoconjugates. These enzymes are classified into 133 families (CAZy server, http://www.cazy.org) in which members of each family have a similar structure and catalytic mechanism. In order to understand better the structure/function relationships of these enzymes and their evolution and to develop new robust evolved glycosidases, we undertook to convert a Family 1 thermostable ß-glycosidase into an exo-ß-N-acetylglucosaminidase. This latter activity is totally absent in Family 1, while natural ß-hexosaminidases belong to CAZy Families 3, 20 and 84. Using molecular modeling, we first showed that the docking of N-acetyl-d-glucosamine in the subsite -1 of the ß-glycosidase from Thermus thermophilus (TtßGly) suggested several steric conflicts with active site amino-acids (N163, E338) induced by the N-acetyl group. Both N163A and N163D-E338G mutations induced significant N-acetylglucosaminidase activity in TtßGly. The double mutant N163D-E338G was also active on the bicyclic oxazoline substrate, suggesting that this mutated enzyme uses a catalytic mechanism involving a substrate-assisted catalysis with a noncovalent oxazoline intermediate, similar to the N-acetylglucosaminidases from Families 20 and 84. Furthermore, a very efficient trans-N-acetylglucosaminidase activity was observed when the double mutant was incubated in the presence of NAG-oxazoline as a donor and N-methyl-O-benzyl-N-(ß-d-glucopyranosyl)-hydroxylamine as an acceptor. More generally, this work demonstrates that it is possible to exchange the specificities and catalytic mechanisms with minimal changes between phylogenetically distant protein structures.

Semi-rational approach for converting a GH1 ß-glycosidase into a ß-transglycosidase.

A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus ß-glycosidase (Ttß-gly, glycoside hydrolase family 1 (GH1)), which display a higher transglycosylation/hydrolysis ratio than the wild-type enzyme. In order to find the cause of these activity modifications, and thereby set up a generic method for easily obtaining transglycosidases from glycosidases, we determined their X-ray structure. No major structural changes could be observed which could help to rationalize the mutagenesis of glycosidases into transglycosidases. However, as these mutations are highly conserved in GH1 ß-glycosidases and are located around the -1 site, we pursued the isolation of new transglycosidases by targeting highly conserved amino acids located around the active site. Thus, by single-point mutagenesis on Ttß-gly, we created four new mutants that exhibit improved synthetic activity, producing disaccharides in yields of 68-90% against only 36% when native Ttß-gly was used. As all of the chosen positions were well conserved among GH1 enzymes, this approach is most probably a general route to convert GH1 glycosidases into transglycosidases.

Conserved water molecules in family 1 glycosidases: a DXMS and molecular dynamics study.

By taking advantage of the wealth of structural data available for family 1 glycoside hydrolases, a study of the conservation of internal water molecules found in this ubiquitous family of enzymes was undertaken. Strikingly, seven water molecules are observed in more than 90% of the known structures. To gain insight into their possible function, the water dynamics inside Thermus thermophilus ß-glycosidase was probed using deuterium exchange mass spectroscopy, allowing the pinpointing of peptide L117-A125, which exchanges most of its amide hydrogens quickly in spite of the fact that it is for the most part buried in the crystal structure. To help interpret this result, a molecular dynamics simulation was performed whose analysis suggests that two water channels are involved in the process. The longest one (∼16 Å) extends between the protein surface and W120, whose side chain interacts with E164 (the acid-base residue involved in the catalytic mechanism), whereas the other channel allows for the exchange with the bulk of the highly conserved water molecules belonging to the hydration shell of D121, a deeply buried residue. Our simulation also shows that another chain of highly conserved water molecules, going from the protein surface to the bottom of the active site cleft close to the nucleophile residue involved in the catalytic mechanism, is able to exchange with the bulk on the nanosecond time scale. It is tempting to speculate that at least one of these three water channels could be involved in the function of family 1 glycoside hydrolases.

Engineering transglycosidase activity into a GH51 α-l-arabinofuranosidase.

Directed evolution was applied to the α-l-arabinofuranosidase from Thermobacillus xylanilyticus to confer better transglycosylation ability, particularly for the synthesis of benzyl α-l-arabinofuranosyl-(1,2)-α-d-xylopyranoside, starting from p-nitrophenyl α-l-arabinofuranoside (donor) and benzyl α-d-xylopyranoside (acceptor). The aim was to obtain mutants displaying both lower hydrolytic and greater transglycosylation activities to favour the stable production of the target disaccharide. The implementation of a simple chromogenic screen ultimately provided three mutant enzymes whose properties correspond to those sought after. These all displayed lowered hydrolytic activity and conserved or slightly improved transfer activity, while one of them also displayed lowered secondary hydrolysis of the transglycosylation product. DNA sequence analysis of the mutants revealed between three and seven point mutations and biochemical analysis combined with STD-NMR experiments indicated that distinct molecular mechanisms were active among the three mutants.

Alkoxyamino glycoside acceptors for the regioselective synthesis of oligosaccharides using glycosynthases and transglycosidases.

Alkoxyamino derivatives of oligosaccharides have been synthesized by enzymatic synthesis using a glycosynthase and a transglycosidase. The chemoselective assembly of unprotected oligosaccharides bearing glucose at the reducing end with N-alkyl-O-benzylhydroxylamine provides sugar derivatives that are good acceptors for enzymatic synthesis using either glycosynthase or transglycosidase. Furthermore, this method affords the possibility of controlling the regioselectivity of coupling depending on the nature of the alkoxyamino substituent and provides high-yield coupling of sugars without the need for complex protecting group chemistry.

Screening of enzymatic activities for the depolymerisation of the marine bacterial exopolysaccharide HE800.

The exopolysaccharide (EPS) HE800 is a marine-derived polysaccharide (from 8 × 10(5) to 1.5 × 10(6) g mol(-1)) produced by Vibrio diabolicus and displaying original structural features close to those of glycosaminoglycans. In order to confer new biological activities to the EPS HE800 or to improve them, structural modifications need to be performed. In particular, depolymerisation is required to generate low-molecular-weight derivatives. To circumvent the use of chemical methods that lack specificity and reproducibility, enzymes able to perform such reaction are sought. This study reports the screening for enzymes capable of depolymerising the EPS HE800. A large diversity of enzyme sources has been studied: commercially available glycoside hydrolases with broad substrate specificity, lyases, and proteases as well as growing microorganisms. Interestingly, we found that the genus Enterococcus and, more particularly, the strain Enterococcus faecalis were able to depolymerise the EPS HE800. Partial characterization of the enzymatic activity gives evidence for a random and incomplete depolymerisation pattern that yields low-molecular-weight products of 40,000 g mol(-1). Genomic analysis and activity assays allowed the identification of a relevant open reading frame (ORF) which encodes an endo-N-acetyl-galactosaminidase. This study establishes the foundation for the development of an enzymatic depolymerisation process.

Correlation between thermostability and stability of glycosidases in ionic liquid.

The activity and stability of a ß-glycosidase (Thermus thermophilus) and two α-galactosidases (Thermotoga maritima and Bacillus stearothermophilus) were studied in different hydrophilic ionic liquid (IL)/water ratios. For the ILs used, the glycosidases showed the best stability and activity in 1,3-dimethylimidazolium methyl sulfate [MMIM][MeSO(4)] and 1,2,3-trimethylimidazolium methyl sulfate [TMIM][MeSO(4)]. A close correlation was observed between the thermostability of the enzymes and their stability in IL media. At high IL concentration (80%), a time-dependent irreversible denaturing effect was observed on glycosidases while, at lower concentration (<30%), a reversible inactivation affecting mainly the k (cat) was obtained. The results demonstrate that highly thermostable glycosidases are more suitable for biocatalytic reactions in water-miscible ILs.

A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part I: design and optimization of bioluminescent bacterial strains.

This study describes the construction of inducible bioluminescent strains via genetic engineering along with their characterization and optimization in the detection of heavy metals. Firstly, a preliminary comparative study enabled us to select a suitable carbon substrate from pyruvate, glucose, citrate, diluted Luria-Bertani, and acetate. The latter carbon source provided the best induction ratios for comparison. Results showed that the three constructed inducible strains, Escherichia coli DH1 pBzntlux, pBarslux, and pBcoplux, were usable when conducting a bioassay after a 14-h overnight culture at 30 °C. Utilizing these sensors gave a range of 12 detected heavy metals including several cross-detections. Detection limits for each metal were often close to and sometimes lower than the European standards for water pollution. Finally, in order to maintain sensitive bacteria within the future biosensor-measuring cell, the agarose immobilization matrix was compared to polyvinyl alcohol (PVA). Agarose was selected because the detection limits of the bioluminescent strains were not affected, in contrast to PVA. Specific detection and cross-detection ranges determined in this study will form the basis of a multiple metals detection system by the new multi-channel Lumisens3 biosensor.

Rational design of a GH1 beta-glycosidase to prevent self-condensation during the transglycosylation reaction.

Mutant N282T of a thermostable beta-glycosidase from GH1 family (TtbetaGly) presenting a high transglycosidase activity was previously obtained by directed evolution. However, it displays a self-condensation activity with the donor 2-nitrophenyl-beta-d-galactopyranoside (oNPGal) which competes with the condensation reaction and entails undesirable effects. In order to prevent this reaction, we rationally modified this enzyme at the [+1]/[+2] subsites so that oNPGal would bind less tightly. Molecular modeling (MM) suggested the mutation A221W, which decreased the affinity of the donor at these sites and moved it away from the bound galactose at the -1 subsite. A single (A221W) and a double mutant (A221W/N282T) were constructed, and they gave rise to a drastic decrease in self-condensation. The A221W mutant had no transglycosylation activity whereas the A221W/N282T mutant still displayed a condensation activity, comparable to that of the N282T mutant for the transfer on pNPGlcNAc. MM revealed that the double mutant A221W/N282T could induce the synthesis of a glycosidic bond between a donor and an acceptor displaying an equatorial 4-position. Moreover, it is suggested that mutation N282T could change the orientation of residue N219, leading to a stabilization of the acceptor with a new hydrogen bond. This finding opens the way to further improvements of evolved transglycosidases.

Digital screening methodology for the directed evolution of transglycosidases.

Engineering of glycosidases with efficient transglycosidases activity is an alternative to glycosyltransferases or glycosynthases for the synthesis of oligosaccharides and glycoconjugates. However, the engineering of transglycosidases by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of digital imaging-based high-throughput screening methodology for the directed evolution of glycosidases into transgalactosidases. Using this methodology, we detected transglycosidase mutants in intact Escherichia coli cells by digital imaging monitoring of the activation of non- or low-hydrolytic mutants by an acceptor substrate. We screened several libraries of mutants of beta-glycosidase from Thermus thermophilus using this methodology and found variants with up to a 70-fold overall increase in the transglycosidase/hydrolysis activity ratio. Using natural disaccharide acceptors, these transglycosidase mutants were able to synthesise trisaccharides, as a mixture of two regioisomers, with up to 76% yield.

Engineering of glucoside acceptors for the regioselective synthesis of beta-(1-->3)-disaccharides with glycosynthases.

Glycosynthase mutants obtained from Thermotogamaritima were able to catalyze the regioselective synthesis of aryl beta-D-Galp-(1-->3)-beta-D-Glcp and aryl beta-D-Glcp-(1-->3)-beta-D-Glcp in high yields (up to 90 %) using aryl beta-D-glucosides as acceptors. The need for an aglyconic aryl group was rationalized by molecular modeling calculations, which have emphasized a high stabilizing interaction of this group by stacking with W312 of the enzyme. Unfortunately, the deprotection of the aromatic group of the disaccharides was not possible without partial hydrolysis of the glycosidic bond. The replacement of aryl groups by benzyl ones could offer the opportunity to deprotect the anomeric position under very mild conditions. Assuming that benzyl acceptors could preserve the stabilizing stacking, benzyl beta-d-glucoside firstly assayed as acceptor resulted in both poor yields and poor regioselectivity. Thus, we decided to undertake molecular modeling calculations in order to design which suitable substituted benzyl acceptors could be used. This study resulted in the choice of 2-biphenylmethyl beta-D-glucopyranoside. This choice was validated experimentally, since the corresponding beta-(1-->3) disaccharide was obtained in good yields and with a high regioselectivity. At the same time, we have shown that phenyl 1-thio-beta-D-glucopyranoside was also an excellent substrate leading to similar results as those obtained with the O-phenyl analogue. The NBS deprotection of the S-phenyl group afforded the corresponding disaccharide quantitatively.

Directed evolution of the alpha-L-fucosidase from Thermotoga maritima into an alpha-L-transfucosidase.

The alpha-L-fucosidase from Thermotoga maritima (Tm alpha fuc) was converted into alpha-L-transfucosidase variants by directed evolution. The wild-type enzyme catalyzes oligosaccharide synthesis by transfer of a fucosyl residue from a pNP-fucoside donor to pNP-fucoside (self-condensation) with alpha-(1-->3) regioselectivity or pNP-galactoside (transglycosylation) with alpha-(1-->2) regioselectivity at low yields (7%). The wild-type enzyme was submitted to one cycle of mutagenesis, followed by rational recombination of the selected mutations, which allowed identification of variants with improved transferase activity. The transferase and hydrolytic kinetics of all the mutants were assessed by NMR methods and capillary electrophoresis. It was shown that the best mutant exhibited a dramatic 32-fold increase in the transferase/hydrolytic kinetic ratio, while keeping 60% of the overall wild-type enzyme activity. Accordingly, the maximum yield of a specific transglycosylation product [pNP-Gal-alpha-(1-->2)-Fuc] reached more than 60% compared to 7% with WT enzyme at equimolar and low concentrations of donor and acceptor (10 mM). Such an improvement was obtained with only three mutations (T264A, Y267F, L322P), which were all located in the second amino acid shell of the fucosidase active site. Molecular modeling suggested that some of these mutations (T264A, Y267F) cause a reorientation of the amino acids that are in direct contact with the substrates, resulting in a better docking energy. Such mutants with high transglycosidase activity may constitute novel enzymatic tools for the synthesis of fucooligosaccharides.

In vivo selection for the enhancement of Thermotoga maritima exopolygalacturonase activity at neutral pH and low temperature.

The aim of this study was to develop an Escherichia coli-based metabolic selection system for the uncovering of new oligogalacturonate-active enzymes. Based on the expression of the specific permease TogMNAB, this system enabled the entry of oligogalacturonates into the cytoplasm of E. coli thus providing a modified strain usable for this purpose. This tool was used for the metabolic selection of Thermotoga maritima exopolygalacturonase (TmGalU) mutants enabling the uptake of sodium trigalacturonate as the sole carbon source by the bacterium. In only one round of error-prone PCR and selection, mutants of TmGalU with a 4-fold increased turnover at pH 7.0 and 2-fold more active at 37 degrees C than wild-type enzyme were isolated. These results show the versatility of this strain for the evolution of oligogalacturonate-active enzymes.