Characterization of differentiated autoregulation of LuxI/LuxR-type quorum sensing system in Pseudoalteromonas.

Gram-negative bacteria usually use acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR-type quorum sensing (QS) systems for cell-cell cooperation and/or bacteria-environment communication. LuxI and LuxR are AHLs synthase and receptor, respectively. These two parts could form a positive regulatory feedback loop, controlling various types of group behaviors. However, the autoregulation mechanisms between them are fragmented and could be highly differentiated in different bacteria. Here, we clarified the autoregulation mechanism between LuxI and LuxR in Pseudoalteromonas sp. R3. YasI (LuxI in strain R3) synthesizes two types of AHLs, C8-HSL and 3-OH-C8-HSL. It is worth noting that YasR (LuxR in strain R3) only responds to C8-HSL rather than 3-OH-C8-HSL. YasR-C8HSL can activate the yasI transcription by recognizing "lux box" at yasI upstream. Interestingly, YasR can directly promote the yasR expression with AHL-independent manner, but AHL absence caused by the yasI-deficiency led to the significant decrease in the yasR expression. Further study demonstrated that the yasI-deficiency can result in the decrease in the yasR mRNA stability. Notably, both yasI-deficiency and yasR-deficiency led to the significant decrease in the expression of hfq encoding RNA chaperone. Therefore, it was speculated that not only YasR itself can directly regulate the yasR transcription, but YasR-C8HSL complex indirectly affects the yasR mRNA stability by regulating Hfq.

Identification and analysis of the biological activity of the new strain of Pseudoalteromonas piscicida isolated from the hemal fluid of the bivalve Modiolus kurilensis (F. R. Bernard, 1983).

A cultivated form of bacteria (strain 2202) was isolated from the hemal fluid of the bivalve mollusk Modiolus kurilensis. Based on the set of data collected by genetic and physiological/biochemical analyses, the strain was identified as the species Pseudoalteromonas piscicida. Strain 2202 exhibits antimicrobial activity against Staphylococcus aureus, Candida albicans, and Bacillus subtilis but not against Escherichia coli and Pseudomonas aeruginosa. These activities characterize the behavior of strain 2202 as predator-like and classify it as a facultative predator. Being part of the normal microflora in the hemolymph of M. kurilensis, when external conditions change, strain 2202 shows features of opportunistic microflora. The strain 2202 exhibits selective toxicity towards larvae of various invertebrates: it impairs the early development of Mytilus edulis, but not of Strongylocentrotus nudus. Thus, the selective manner in which P. piscicida strains interact with various species of microorganisms and eukaryotes should be taken into consideration when using their biotechnological potential as a probiotic in aquaculture, source of antimicrobial substances, and factors that prevent fouling.

Electrochemical Sensing and Characterization of Aerobic Marine Bacterial Biofilms on Gold Electrode Surfaces.

Reliable and accurate in situ sensors capable of detecting and quantifying troublesome marine biofilms on metallic surfaces are increasingly necessary. A 0.2 mm diameter gold electrochemical sensor was fully characterized using cyclic voltammetry in abiotic and biotic artificial seawater media within a continuous culture flow cell to detect the growth and development of an aerobic Pseudoalteromonas sp. biofilm. Deconvolution of the abiotic and biotic responses enable the constituent extracellular electron transfer and biofilm responses to be resolved. Differentiation of enhanced oxygen reduction kinetics within the aerobic bacterial biofilm is linked to enzyme and redox mediator activities.

Use of Quorum Sensing Inhibition Strategies to Control Microfouling.

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purpose.

Spotlight on a novel bactericidal mechanism and a novel SXT/R391-like integrative and conjugative element, carrying multiple antibiotic resistance genes, in Pseudoalteromonas flavipulchra strain CDM8.

Many Pseudoalteromonas strains can produce bioactive compounds with antimicrobial activities. This study focused on a probiotic candidate P.flavipulchra CDM8 to reveal its novel antibacterial mechanism and risks for antibiotic resistance dissemination. Strain CDM8 could form floating biofilm, displayed strikingly broad antibacterial activities against multiple Vibrio and Bacillus species, and decreased the competitor's concentration in their co-cultures in the microtiter plate tests. It could also form vesicle/pilus-like structures on the outer surface, which were indicated to participate in the bactericidal activity and represent a novel antibacterial mechanism of CDM8, according to the scanning electron microscopic observation. However, CDM8 displayed multi-antibiotic resistance, conferred by the multidrug resistance regions in hotspot 4 and variable region III of a novel SXT/R391-like integrative and conjugative element (ICEPflCDM8). Summing up, our results provided a better understanding of the bactericidal mechanism of P. flavipulchra and highlighted the role of SXT/R391-like ICEs in conferring multidrug resistance phenotype of probiotic P. flavipulchra candidates.

Stringent starvation protein A and LuxI/LuxR-type quorum sensing system constitute a mutual positive regulation loop in Pseudoalteromonas.

Bacteria commonly exhibit social activities through acyl-homoserine lactones (AHLs)-based quorum sensing (QS) systems to form their unique social network. The sigma factor RpoS is an important regulator that controls QS system in different bacteria. However, the upstream of RpoS involving regulation on QS system remains unclear. In Escherichia coli RpoS is regulated by stringent starvation protein A (SspA), which is dependent of histone-like nucleoid structuring protein (H-NS). To date, the connection between SspA and QS system is essentially unknown. Here, we characterized a typical LuxI/LuxR-type QS system in marine bacterium Pseudoalteromonas sp. T1lg65 which can produce four types of AHLs. The luxI encoding AHLs synthase and luxR encoding AHLs-responsive receptor are co-transcribed, providing advantages in rapidly amplifying QS signaling. Notably, SspA positively regulated luxI/luxR transcription by activating RpoS expression, which is mediated by H-NS. Interestingly, LuxR in turn positively regulated SspA expression. Therefore, SspA and QS system constitute a mutual positive regulation loop in T1lg65. In view of the crucial roles of SspA and QS system in environmental adaption, we believe that the improvement of bacterial tolerance to marine environments could be related to rapidly tuning SspA-involved QS programming.

SspA positively controls exopolysaccharides production and biofilm formation by up-regulating the algU expression in Pseudoalteromonas sp. R3.

Biofilm formation enhances the survival and persistence of microorganisms in response to environmental stresses. It has been revealed that stringent starvation protein A (SspA) can function as an important regulator dealing with environmental stresses for bacterial survival. However, the connection between SspA and biofilm formation is essentially unclear yet. In this study, we presented evidence showing SspA positively controls biofilm formation by up-regulating exopolysaccharides (EPS) production in marine bacterium Pseudoalteromonas sp. R3. Both qPCR and lacZ reporter system congruously revealed that SspA positively controls the expression of EPS biosynthesis gene cluster. Unlike generally accepted thought that SspA regulates bacterial physiology by inhibiting the expression of histone-like nucleotide structuring protein (H-NS) gene, the function of SspA on EPS production and biofilm formation in Pseudoalteromonas sp. R3 is H-NS-independent. Instead, SspA positively regulates the expression of sigma factor AlgU-encoding gene, thus affecting EPS biosynthesis and biofilm formation. In view of the important role of SspA in biofilm formation, we believe that the improvement of tolerance to marine environmental stresses could be related to tuning of SspA-involved biofilm formation.

Early stage of marine biofilm formation on duplex stainless steel.

The aim of this work was to investigate the bacteria-surface interactions occurring during the first hour of adhesion of marine Pseudoalteromonas NCIMB 2021 at the surface of 2304 lean duplex stainless steel in artificial seawater. A complete characterization of the biofilm and the passive film was performed coupling epifluorescence microscopy, scanning electron microscopy (SEM), x-ray photoelectron spectroscopy (XPS), and time of flight secondary ion mass spectrometry (ToF-SIMS). The coupling of XPS and ToF-SIMS analyses revealed that (1) protein and polysaccharide contents in the biofilm are similar in the presence or absence of nutrients, (2) the biofilm is mainly composed of proteins and the protein content is similar to the one of Tightly Bound EPS, (3) increased bacterial activity due to nutrients leads to chromium enrichment in the passive film in close vicinity to the bacteria.

Genetic examination of the marine bacterium Pseudoalteromonas luteoviolacea and effects of its metamorphosis-inducing factors.

Pseudoalteromonas luteoviolacea is a globally distributed marine bacterium that stimulates the metamorphosis of marine animal larvae, an important bacteria-animal interaction that can promote the recruitment of animals to benthic ecosystems. Recently, different P. luteoviolacea isolates have been shown to produce two stimulatory factors that can induce tubeworm and coral metamorphosis; Metamorphosis-Associated Contractile structures (MACs) and tetrabromopyrrole (TBP) respectively. However, it remains unclear what proportion of P. luteoviolacea isolates possess the genes encoding MACs, and what phenotypic effect MACs and TBP have on other larval species. Here, we show that 9 of 19 sequenced P. luteoviolacea genomes genetically encode both MACs and TBP. While P. luteoviolacea biofilms producing MACs stimulate the metamorphosis of the tubeworm Hydroides elegans, TBP biosynthesis genes had no effect under the conditions tested. Although MACs are lethal to larvae of the cnidarian Hydractinia symbiologicarpus, P. luteoviolacea mutants unable to produce MACs are capable of stimulating metamorphosis. Our findings reveal a hidden complexity of interactions between a single bacterial species, the factors it produces and two species of larvae belonging to different phyla.

Diauxie and co-utilization of carbon sources can coexist during bacterial growth in nutritionally complex environments.

It is commonly thought that when multiple carbon sources are available, bacteria metabolize them either sequentially (diauxic growth) or simultaneously (co-utilization). However, this view is mainly based on analyses in relatively simple laboratory settings. Here we show that a heterotrophic marine bacterium, Pseudoalteromonas haloplanktis, can use both strategies simultaneously when multiple possible nutrients are provided in the same growth experiment. The order of nutrient uptake is partially determined by the biomass yield that can be achieved when the same compounds are provided as single carbon sources. Using transcriptomics and time-resolved intracellular 1H-13C NMR, we reveal specific pathways for utilization of various amino acids. Finally, theoretical modelling indicates that this metabolic phenotype, combining diauxie and co-utilization of substrates, is compatible with a tight regulation that allows the modulation of assimilatory pathways.

Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

Background: Freeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains. Results: The results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 109 CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5­10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freezedried under the abovementioned conditions. Their survival rates were 2.3­95.1%. Conclusion: Collectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria

The Flagellar Gene Regulates Biofilm Formation and Mussel Larval Settlement and Metamorphosis.

Biofilms are critical components of most marine systems and provide biochemical cues that can significantly impact overall community composition. Although progress has been made in the bacteria-animal interaction, the molecular basis of modulation of settlement and metamorphosis in most marine animals by bacteria is poorly understood. Here, Pseudoalteromonas marina showing inducing activity on mussel settlement and metamorphosis was chosen as a model to clarify the mechanism that regulates the bacteria-mussel interaction. We constructed a flagellin synthetic protein gene fliP deletion mutant of P. marina and checked whether deficiency of fliP gene will impact inducing activity, motility, and extracellular polymeric substances of biofilms. Furthermore, we examined the effect of flagellar proteins extracted from bacteria on larval settlement and metamorphosis. The deletion of the fliP gene caused the loss of the flagella structure and motility of the ∆fliP strain. Deficiency of the fliP gene promoted the biofilm formation and changed biofilm matrix by reducing ß-polysaccharides and increasing extracellular proteins and finally reduced biofilm-inducing activities. Flagellar protein extract promoted mussel metamorphosis, and ∆fliP biofilms combined with additional flagellar proteins induced similar settlement and metamorphosis rate compared to that of the wild-type strain. These findings provide novel insight on the molecular interactions between bacteria and mussels.

A predator-prey interaction between a marine Pseudoalteromonas sp. and Gram-positive bacteria.

Predator-prey interactions play important roles in the cycling of marine organic matter. Here we show that a Gram-negative bacterium isolated from marine sediments (Pseudoalteromonas sp. strain CF6-2) can kill Gram-positive bacteria of diverse peptidoglycan (PG) chemotypes by secreting the metalloprotease pseudoalterin. Secretion of the enzyme requires a Type II secretion system. Pseudoalterin binds to the glycan strands of Gram positive bacterial PG and degrades the PG peptide chains, leading to cell death. The released nutrients, including PG-derived D-amino acids, can then be utilized by strain CF6-2 for growth. Pseudoalterin synthesis is induced by PG degradation products such as glycine and glycine-rich oligopeptides. Genes encoding putative pseudoalterin-like proteins are found in many other marine bacteria. This study reveals a new microbial interaction in the ocean.

Conformational Flexibility Drives Cold Adaptation in Pseudoalteromonas haloplanktis TAC125 Globins.

Significance: Temperature is one of the most important drivers in shaping protein adaptations. Many biochemical and physiological processes are influenced by temperature. Proteins and enzymes from organisms living at low temperature are less stable in comparison to high-temperature adapted proteins. The lower stability is generally due to greater conformational flexibility. Recent Advances: Adaptive changes in the structure of cold-adapted proteins may occur at subunit interfaces, distant from the active site, thus producing energy changes associated with conformational transitions transmitted to the active site by allosteric modulation, valid also for monomeric proteins in which tertiary structural changes may play an essential role. Critical Issues: Despite efforts, the current experimental and computational methods still fail to produce general principles on protein evolution, since many changes are protein and species dependent. Environmental constraints or other biological cellular signals may override the ancestral information included in the structure of the protein, thus introducing inaccuracy in estimates and predictions on the evolutionary adaptations of proteins in response to cold adaptation. Future Directions: In this review, we describe the studies and approaches used to investigate stability and flexibility in the cold-adapted globins of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. In fact, future research directions will be prescient on more detailed investigation of cold-adapted proteins and the role of fluctuations between different conformational states.

Quorum Quenching Enzyme APTM01, an Acylhomoserine-Lactone Acylase from Marine Bacterium of Pseudoalteromonas tetraodonis Strain MQS005.

Quorum sensing is a system of stimuli and response correlated to population density and involves in pathogen infection, colonization, and pathogenesis. Quorum quenching enzymes as quorum sensing inhibitors have been identified in a number of bacteria and been used to control by triggering the pathogenic phenotype. The marine bacteria of Pseudoalteromonas had wide activity of degrading AHLs as a type of signal molecule associated with quorum sensing. We screened many Pseudoalteromonas strains in large scale to explore genes of quorum quenching enzymes from the China seas by whole-genome sequencing rather than genomic library construction. Nine target strains were obtained and an acylases gene APTM01 from the strain MQS005 belonging to PvdQ type on sub-branch in phylogenetic tree. And the heterogenous host containing the vector with target gene could degrade C10-HSL, C12-HSL and OC12-HSL. The obtained AHL acylase gene would be a candidate quorum quenching gene to apply in some fields. We identified that the strains of Pseudoalteromonas have wide AHL-degrading ability depending on quorum quenching. The strains would be a resource to explore new quorum quenching enzymes.

Identification and characterization of a LuxI/R-type quorum sensing system in Pseudoalteromonas.

Bacteria usually produce, release and detect quorum sensing (QS)-based signal molecules, and successively orchestrate gene expression in respond to environmental changes. Pseudoalteromonas are typical marine bacteria, but knowledge on their QS systems is extremely fragmentary. In this study, genome sequencing of Pseudoalteromonas sp. R3 was performed. Accordingly, a QS working model including three sets of hierarchically organized QS systems was proposed in strain R3. Among them, the typical LuxI/R-type QS system using acyl-homoserine lactones (AHLs) as signal molecules was characterized. Sequence similarity analysis indicated luxI encoding AHLs synthase is novel. The luxR encoding AHLs receptor is directly adjacent to luxI downstream. Notably, mutagenesis demonstrated LuxI and LuxR affect each other at transcriptional level, and both control the AHLs formation. Interestingly, it was found that LuxI/R-type QS system positively involves resistance to streptomycin. Thin-layer chromatography analysis showed strain R3 can produce 3-OH-C6-HSL and C8-HSL, which was supported by heterologous expression of LuxI in Escherichia coli. Sequence alignment analysis indicated that the N-terminal region of LuxI is more conservative than the C-terminal region, revealing the importance of N-terminal region in AHLs synthesis. The obtained findings enrich our knowledge on LuxI/R-type QS system in Pseudoalteromonas and its regulation on adaptation to marine environments.

The Challenge of the Sponge Suberites domuncula (Olivi, 1792) in the Presence of a Symbiotic Bacterium and a Pathogen Bacterium.

Sponges, which are in close contact with numerous bacteria in prey/predator, symbiotic and pathogenic relationships, must provide an appropriate response in such situations. This starts with a discriminating recognition of the partner either by a physical contact or through secreted molecules or both. We investigated the expression of the Toll-like receptor, Caspase 3/7, Tumor Necrosis Factor receptor-associated factor 6, Bcl-2 homology protein-2 and macrophage expressed genes of axenic sponge cells in the presence of a symbiotic bacterium (Endozoicomonas sp. Hex311), a pathogen bacterium (Pseudoalteromonas sp. 1A1), their exoproducts and lipopolysaccharides. The vast majority of answers are in line with what could be observed with the symbiotic bacterium. The pathogenic bacterium seems to profit from the eukaryotic cell: suppression of the production of the antibacterial compound, inhibition of the apoptosis caspase-dependent pathway, deregulation of bacterial recognition. This work contributes new scientific knowledge in the field of immunology and apoptosis in early branching metazoan harboring within its tissue and cells a large number of symbiotic bacteria.

Draft genome sequence of Pseudoalteromonas piscicida strain 36Y_RITHPW, a hypersaline seawater isolate from the south coast of Sonora, Mexico.

OBJECTIVES: This study reports the draft genome sequence of Pseudoalteromonas piscicida strain 36Y_RITHPW, a marine Gammaproteobacteria that synthesises bioactive compounds with antagonistic activity against Vibrio parahaemolyticus, a multidrug-resistant strain that is the causative agent of acute hepatopancreatic necrosis disease (AHPND), reported in shrimp farm outbreaks from Asia to Mexico with mortality rates of 80-100%. METHODS: The genome of P. piscicida 36Y_RITHPW was sequenced with an Ion Torrent™ Personal Genome Machine™ (PGM) platform. A total of 606805 reads were constructed for a 308.48Mbp and 33.5×coverage. A high-quality draft assembly and ordering of contigs was obtained with Mauve. The annotation was obtained with RAST and antiSMASH. RESULTS: The genome size consists of 5.15Mbp, with a total of 4548 genes, 4217 protein-coding sequences and a GC content of 43.3%. Several resistance genes as well as other genes involved in the production of bacteriocins and ribosomally synthesised antibacterial peptides are also present. CONCLUSIONS: Mining of this draft genome provides valuable information to explain the antagonistic capacity of P. piscicida 36Y_RITHPW, a useful strain as a potential probiotic in shrimp aquaculture against pathogenic V. parahaemolyticus.

Protective Efficacy of a Pseudoalteromonas Strain in European Abalone, Haliotis tuberculata, Infected with Vibrio harveyi ORM4.

The hemolymph of healthy marine invertebrates is known to harbor antibiotic-producing bacteria belonging to the genus Pseudoalteromonas. Such strains are potential probiotics to control infectious diseases in aquaculture. In the present study, we screened a collection of Pseudoalteromonas strains isolated from the hemolymph of oyster and mussel for antimicrobial activity against Vibrio harveyi, a pathogenic species responsible for high mortality in abalone. Subsequently, the protective efficacy of the most active strain named hCg-6 was investigated in abalone culture faced with a Vibrio harveyi ORM4 infection. First, we have controlled the Pseudoalteromonas hCg-6 safety for abalone health. To that end, animals were immersed for 4 h in Pseudoalteromonas hCg-6 suspensions in seawater. The abalone viability was monitored and Pseudoalteromonas hCg-6 was tracked by quantitative-PCR in abalone hemolymph. After immersion, no abalone death occurred while the strain hCg-6 was significantly detected in hemolymph. Therefore, the strain hCg-6 was considered safe for abalone and evaluated for its ability to protect abalone against V. harveyi (injection of 1 × 103Vibrio per animal). A 4-h long immersion of abalone in a seawater suspension of Pseudoalteromonas hCg-6 (1 × 106 CFU mL-1) prior to infection with Vibrio harveyi significantly improved the abalone viability. Indeed, 15 days post infection, the hCg-6 treatment used increased the abalone survival rate from 16% in untreated animals to 40% in treated abalone. We hypothesized that Pseudoalteromonas hCg-6 antibacterial activity increased the hemomicrobiota shielding effect. In conclusion, Pseudoalteromonas hCg-6 is a promising anti-Vibrio strain for abalone culture.

Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants.

A novel wild-type recombinant cold-active α-d-galactosidase (α-PsGal) from the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701, and its mutants D451A and C494N, were studied in terms of their structural, physicochemical, and catalytic properties. Homology models of the three-dimensional α-PsGal structure, its active center, and complexes with D-galactose were constructed for identification of functionally important amino acid residues in the active site of the enzyme, using the crystal structure of the α-galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild α-PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl-α-galactopiranoside (pNP-α-Gal). Thin-layer chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and nuclear magnetic resonance spectroscopy methods were used to identify transglycosylation products in reaction mixtures. α-PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. α-PsGal synthesized -α(1→6)- and -α(1→4)-linked galactobiosides from melibiose as well as -α(1→6)- and -α(1→3)-linked p-nitrophenyl-digalactosides (Gal2-pNP) from pNP-α-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-α(1→3)-Gal-pNP synthesis and increased the Gal-α(1→4)-Gal yield compared to Gal-α(1→6)-Gal-pNP.