Transcriptional control of carbohydrate catabolism by the CcpA protein in the ruminal bacterium Streptococcus bovis.

As a potent, pleiotropic regulatory protein in Gram-positive bacteria, catabolite control protein A (CcpA) mediates the transcriptional control of carbohydrate metabolism in Streptococcus bovis, a lactate-producing bacterium that plays an essential role in rumen acidosis in dairy cows. Although the rumen uptake of carbohydrates is multi-substrate, the focus of S. bovis research thus far has been on the glucose. With the aid of gene deletion, whole-genome sequencing, and transcriptomics, we have unraveled the role of CcpA in carbohydrate metabolism, on the one hand, and acidosis, on the other, and we show that the S. bovis strain S1 encodes "Carbohydrate-Active Enzymes" and that ccpA deletion slows the organism's growth rate and modulates the organic acid fermentation pathways toward lower lactate, higher formate, and acetate in the maltose and cellobiose. Furthermore, this study revealed the different regulatory functions of the CcpA protein in rumen metabolism and acidosis.IMPORTANCEThis study is important as it illustrates the varying regulatory role of the Streptococcus bovis catabolite control protein A protein in carbohydrate metabolism and the onset of acidosis in dairy cattle.

Improved identification of Streptococcus bovis-Streptococcus equinus-complex species and subspecies by MALDI-TOF MS using a novel library.

OBJECTIVES: To develop an in-house matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) library for improved identification of species and subspecies of the Streptococcus bovis/Streptococcus equinus-complex (SBSEC). METHODS: A total of 236 SBSEC isolates from blood stream infections and culture collections, determined by whole genome sequencing to subspecies level, were grown in brain heart infusion broth. Mass spectra were collected using the Bruker MALDI Biotyper system after ethanol-formic acid extraction. Main spectral profiles from 117 isolates were used to create the "SBSEC-CMRS library." The remaining 119 spectra were used for evaluation of Bruker MALDI Biotyper (MBT) Compass Library Revision K (2022) and the SBSEC-CMRS library. RESULTS: The Bruker library correctly identified species and subspecies in 72 of 119 (61 %) isolates, while the SBSEC-CMRS library identified 116 of 119 (97 %), using a cutoff score of ≥2.0. CONCLUSIONS: The SBSEC-CMRS library showed sufficient diagnostic accuracy, and can be implemented in clinical practice for SBSEC species and subspecies identification.

Characterization of two lytic bacteriophages, infecting Streptococcus bovis/equinus complex (SBSEC) from Korean ruminant.

Streptococcus bovis/equinus complex (SBSEC) is one of the most important lactic acid-producing rumen bacteria causing subacute ruminal acidosis. Despite the significance of the ruminal bacteria, lytic bacteriophages (phages) capable of infecting SBSEC in the rumen have been rarely characterized. Hence, we describe the biological and genomic characteristics of two lytic phages (designated as vB_SbRt-pBovineB21 and vB_SbRt-pBovineS21) infecting various SBSEC species, including the newly reported S. ruminicola. The isolated SBSEC phages were morphologically similar to Podoviridae and could infect other genera of lactic acid-producing bacteria, including Lactococcus and Lactobacillus. Additionally, they showed high thermal- and pH-stability, and those characteristics induce strong adaptation to the ruminal environment, such as the low pH found in subacute ruminal acidosis. Genome-based phylogeny revealed that both phages were related to Streptococcus phage C1 in the Fischettivirus. However, they had a lower nucleotide similarity and distinct genomic arrangements than phage C1. The phage bacteriolytic activity was evaluated using S. ruminicola, and the phages efficiently inhibited planktonic bacterial growth. Moreover, both phages could prevent bacterial biofilms of various SBSEC strains and other lactic acid-producing bacteria in vitro. Thus, the newly isolated two SBSEC phages were classified as new Fischettivirus members and could be considered as potential biocontrol agents against ruminal SBSEC bacteria and their biofilms.

Comparative Evaluation of Current Biochemical-, Sequencing-, and Proteomic-Based Identification Methods for the Streptococcus bovis Group.

The Streptococcus bovis group (previously group D streptococci) consists of seven distinct species and subspecies. Definitive identification within the group is important, as certain organisms have been associated with gastrointestinal carcinoma, bacteremia, infective endocarditis, meningitis, biliary tract disease, and carcinoma, among others. Definitive identification, however, remains elusive due to limitations and inconsistencies across commonly used identification platforms in the United States. Here, we compared the performance of standard biochemical (Trek Gram-positive identification [GPID] plate, Vitek 2 GPID), sequencing (16S rDNA, sodA) databases (NCBI, RDP, CDC MicrobeNet), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) platforms (Vitek MS, Bruker Biotyper MS) using a set of eight type strains representing all seven strains within the S. bovis group. Despite the evaluation of contemporary methods, no single platform was able to definitively identify all type strains within the S. bovis group. Vitek MS (85.7%, 7/8) provided the most accurate definitive identifications, followed by sodA sequencing (75%, 6/8). Vitek 2 and Bruker Biotyper RUO platforms performed the next best (62.5%, 5/8). All remaining platforms failed to adequately differentiate type strains within the S. bovis group (range, 0 to 37.5%). Laboratorians and clinicians should be aware of the identification limitations of routine testing algorithms and incorporate reflex testing, when appropriate, to platforms such as Vitek MS and/or sodA sequencing that are more able to definitively identify S. bovis group organisms. Further clinical evaluation was conducted using 65 clinical isolates from three geographically distinct U.S. institutions. Future improvements in identification platforms may reveal new clinical and epidemiological trends for members of the S. bovis group.

Streptococcus ruminicola sp. nov., new species of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) isolated from the rumen of Korean domestic ruminants.

A total of three Gram-positive, and oxidase and catalase-negative facultative anaerobic non-motile bacteria were isolated from the rumen fluid of cows and goats and these strains were designated CNU_G2T, CNU_77-61, and CNU_G3. They grew at 20-45 °C, pH 6.5-7, and 0-6.5% NaCl (w/v). The G + C contents (%) of the three isolates were 37.9, 37.8 and 37.8, respectively. Phylogenomic analysis indicated that these strains were distinct from other Streptococcus species. The average nucleotide identity between the isolates and the closest strain S. infantarius subsp. infantarius ATCC BAA-102T was 94.0-94.5%, while the digital DNA-DNA hybridization (dDDH) values between the isolates and the aforementioned related strain were 58.2-61.4%, respectively. Fatty acid analysis revealed higher proportions of C16:0 (> 28%) in all three isolates, while the proportion of C18:0 was higher in CNU_G2T (25.8%); however, it was less than 12% in all the representing strains used in the study. The C14:0 composition of strains CNU_77-61 (22.1%) and CNU_G3 (24.1%) was higher than that of type strains of CNU_G2T (8.1%). Based on the morphological, biochemical, and molecular phylogenetic features of the three novel isolates, they represent a novel species of the genus Streptococcus, for which we propose as Streptococcus ruminicola sp. nov. The type strain is CNU_G2T (= KCTC 43308T = GDMCC 1.2785T).

Bacteraemia and infective endocarditis with Streptococcus bovis-Streptococcus equinus-complex: a retrospective cohort study.

BACKGROUND: Streptococcus bovis/equinus complex (SBSEC) comprise several species and subspecies and is a common cause of infective endocarditis (IE). S. gallolyticus subsp. gallolyticus (Sg gallolyticus) accounts for a majority of SBSEC IE, but the risk of IE for other subspecies is largely unknown. We aimed to investigate the clinical presentation of bacteraemia, and proportion of patients with IE in bacteraemia with the most common subspecies. METHODS: A retrospective cohort study of SBSEC-bacteraemia identified in clinical laboratory databases, in Skåne Region, Sweden, 2003-2018. Bacteraemia with Sg gallolyticus, S. gallolyticus subsp. pasteurianus (Sg pasteurianus), S. lutetiensis and S. infantarius subsp. infantarius (Si infantarius) were included. Subspecies was identified by whole genome sequencing. Medical charts were reviewed according to a predetermined protocol, IE was defined by the criteria from European Society of Cardiology. RESULTS: In total, 210 episodes of SBSEC-bacteraemia were included. Definite IE was identified in 28/210 (13%) episodes. Of these, 7/28 (25%) were prosthetic valve-IE, 1/28 (4%) related to a cardiovascular implantable electronic device and 10/28 (36%) required heart valve surgery. The proportions of IE among different subspecies were: Sg gallolyticus 17/52 (33%), Si infantarius 5/31 (16%), Sg pasteurianus 4/83 (5%) and S. lutetiensis 2/44 (5%) (p < 0.001). Sg pasteurianus and S. lutetiensis were more often associated with intra-abdominal- and polymicrobial infection. CONCLUSION: The proportion of IE in SBSEC-bacteraemia varies substantially depending on subspecies. Echocardiography should always be considered in bacteraemia with Sg gallolyticus and Si infantarius, and can sometimes be omitted in bacteraemia with Sg pasteurianus and S. lutetiensis.

Extensive bacteriocin gene shuffling in the Streptococcus bovis/Streptococcus equinus complex reveals gallocin D with activity against vancomycin resistant enterococci.

Streptococcus gallolyticus LL009 produces gallocin D, a narrow spectrum two component bacteriocin with potent activity against vancomycin-resistant enterococci. Gallocin D is distinct from gallocin A, a separate two component bacteriocin produced by S. gallolyticus. Although the gene clusters encoding gallocin A and gallocin D have a high degree of gene synteny, the structural genes are highly variable and appear to have undergone gene shuffling with other streptococcal species. Gallocin D was analysed in laboratory-based experiments. The mature peptides are 3,343 ± 1 Da and 3,019 ± 1 Da and could be readily synthesized and display activity against a vancomycin resistant Enterococcus strain EC300 with a MIC value of 1.56 µM. Importantly, these bacteriocins could contribute to the ability of S. gallolyticus to colonize the colon where they have been associated with colorectal cancer.

Traditional milk transformation schemes in Côte d'Ivoire and their impact on the prevalence of Streptococcus bovis complex bacteria in dairy products.

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) and possibly Streptococcus infantarius subsp. infantarius (Sii) are associated with human and animal diseases. Sii predominate in spontaneously fermented milk products with unknown public health effects. Sii/SBSEC prevalence data from West Africa in correlation with milk transformation practices are limited. Northern Côte d'Ivoire served as study area due to its importance in milk production and consumption and to link a wider Sudano-Sahelian pastoral zone of cross-border trade. We aimed to describe the cow milk value chain and determine Sii/SBSEC prevalence with a cross-sectional study. Dairy production practices were described as non-compliant with basic hygiene standards. The system is influenced by secular sociocultural practices and environmental conditions affecting product properties. Phenotypic and molecular analyses identified SBSEC in 27/43 (62.8%) fermented and 26/67 (38.8%) unfermented milk samples. Stratified by collection stage, fermented milk at producer and vendor levels featured highest SBSEC prevalence of 71.4% and 63.6%, respectively. Sii with 62.8% and 38.8% as well as Streptococcus gallolyticus subsp. macedonicus with 7.0% and 7.5% were the predominant SBSEC species identified among fermented and unfermented milk samples, respectively. The population structure of Sii/SBSEC isolates seems to reflect evolving novel dairy-adapted, non-adapted and potentially pathogenic lineages. Northern Côte d'Ivoire was confirmed as area with high Sii presence in dairy products. The observed production practices and the high diversity of Sii/SBSEC supports in-depth investigations on Sii ecology niche, product safety and related technology in the dairy value chain potentially affecting large population groups across sub-Saharan Africa.

Identification and pathogenicity analysis of Streptococcus equinus FMD1, a beta-hemolytic strain isolated from forest musk deer lung.

Streptococcus spp. cause a wide range of diseases in animals and humans. A Streptococcus strain (FMD1) was isolated from forest musk deer lung. To identify the bacterium at the species level and investigate its pathogenicity, whole genome sequencing and experimental infections of mice were performed. The genome had 97.63% average nucleotide identity with the S. equinus strain. Through virulence gene analysis, a beta-hemolysin/cytolysin genome island was found in the FMD1 genome, which contained 12 beta-hemolysin/cytolysin-related genes. Hemolytic reaction and histopathological analysis established the strain's pathogenicity in mice. This is the first report of a beta-hemolytic S. equinus strain in forest musk deer identified based on phenotypic and genotypic analyzes; this strategy could be useful for analyzing pathogens affecting rare animals.

Engineering Lactococcus lactis for D-Lactic Acid Production from Starch.

Bioprocess development is a current requirement to enhance the global production of D-lactic acid. Herein, we report a new bioprocess for D-lactic acid production directly from starch using engineered Lactococcus lactis NZ9000. To modify L. lactis as a D-lactic acid producer, its major endogenous L-lactate dehydrogenase (L-Ldh) gene was replaced with a heterologous D-Ldh gene from Lactobacillus delbrueckii subsp. lactis JCM 1107. The resulting strain AH1 showed a somewhat slower growth rate but similar lactic acid production compared to those of the intact strain when cultivated with glucose as a carbon source. The chemical purity of D-lactic acid produced by L. lactis AH1 was 93.8%, and the enzymatic activities of D- and L-Ldh in AH1 were 1.54 U/mL and 0.05 U/mL, respectively. Next, a heterologous α-amylase gene from Streptococcus bovis NRIC 1535 cloned into an expression vector pNZ8048 was introduced into AH1. The resulting strain AH2 showed an amylolytic activity of 0.26 U/mL in the culture supernatant. Direct production of D-lactic acid from starch as the carbon source was demonstrated using L. lactis AH2, resulting in D-lactic acid production at a concentration of 15.0 g/L after 24 h cultivation. To our knowledge, this is the first report on D-lactic acid production in engineered L. lactis.

An Overview on Streptococcus bovis/Streptococcus equinus Complex Isolates: Identification to the Species/Subspecies Level and Antibiotic Resistance.

Streptococcus bovis/Streptococcus equinus complex (SBSEC), a non-enterococcal group D Streptococcus spp. complex, has been described as commensal bacteria in humans and animals, with a fecal carriage rate in humans varying from 5% to over 60%. Among streptococci, SBSEC isolates represent the most antibiotic-resistant species-with variable resistance rates reported for clindamycin, erythromycin, tetracycline, and levofloxacin-and might act as a reservoir of multiple acquired genes. Moreover, reduced susceptibility to penicillin and vancomycin associated with mobile genetic elements have also been detected, although rarely. Since the association of SBSEC bacteremia and colon lesions, infective endocarditis and hepatobiliary diseases has been established, particularly in elderly individuals, an accurate identification of SBSEC isolates to the species and subspecies level, as well as the evaluation of antibiotic resistance, are needed. In this paper, we reviewed the major methods used to identify SBSEC isolates and the antimicrobial resistance rates reported in the scientific literature among SBSEC species.

Cloning and characterization of the glutamate dehydrogenase gene in Streptococcus bovis.

Streptococcus bovis, an etiologic agent of rumen acidosis in cattle, is a rumen bacterium that can grow in a chemically defined medium containing ammonia as a sole source of nitrogen. To understand its ability to assimilate inorganic ammonia, we focused on the function of glutamate dehydrogenase. In order to identify the gene encoding this enzyme, we first amplified an internal region of the gene by using degenerate primers corresponding to hexameric family I and NAD(P)+ binding motifs. Subsequently, inverse PCR was used to identify the whole gene, comprising an open reading frame of 1350 bp that encodes 449 amino acid residues that appear to have the substrate binding site of glutamate dehydrogenase observed in other organisms. Upon introduction of a recombinant plasmid harboring the gene into an Escherichia coli glutamate auxotroph lacking glutamate dehydrogenase and glutamate synthase, the transformants gained the ability to grow on minimal medium without glutamate supplementation. When cell extracts of the transformant were resolved by blue native polyacrylamide gel electrophoresis followed by activity staining, a single protein band appeared that corresponded to the size of S. bovis glutamate dehydrogenase. Based on these results, we concluded that the gene obtained encodes glutamate dehydrogenase in S. bovis.

Streptococcus bovis new taxonomy: does subspecies distinction matter?

Bacteremia with Streptococcus bovis/equinus complex strains is associated with hepatobiliary disease, colorectal lesions (CL), and infective endocarditis (IE). This study addressed the clinical significance of subspecies distinction of previously designated S. bovis blood culture isolates according to the updated nomenclature. During 2002-2013, all blood culture isolates previously designated as S. bovis were recultured and identified using 16S rRNA gene sequencing and MALDI-TOF (Bruker BioTyper and Vitek MS, bioMérieux). Clinical data of patients aged ≥18 years were reviewed. A review of four recent case series was performed as well. Forty blood isolates were identified using 16S rRNA sequencing. Twenty-six bacteremic patients had S. gallolyticus ssp. pasteurianus, six had S. gallolyticus ssp. gallolyticus, two had S. gallolyticus ssp. macedonicus, and six had S. infantarius bacteremia. Species diagnosis using Vitek and bioMérieux MALDI-TOF technology was applicable in 37 and 36 samples, respectively, and was successful in all samples (100 %). Subspecies identification was confirmed in 30 (83 %) samples (as compared with 16S rRNA sequencing detection). IE was diagnosed in 22 (59 %) patients and CL in 8 (20 %) patients. Both complications were associated with all subspecies. Combining our results with those of four recent series resulted in, overall, 320 bacteremic cases, of which 88 (28 %) had CL and 66 (21 %) had IE. All 'bovis/equinus' complex subspecies were associated with CL or IE. From a clinical point of view, species diagnosis using MALDI-TOF MS should suffice to warrant consideration of transesophageal echocardiography and colonoscopy.

Effects of Glucose and Starch on Lactate Production by Newly Isolated Streptococcus bovis S1 from Saanen Goats.

UNLABELLED: When ruminants are fed high-concentrate diets, Streptococcus bovis proliferates rapidly and produces lactate, potentially causing rumen acidosis. Understanding the regulatory mechanisms of the metabolism of this species might help in developing dietary strategies to alleviate rumen acidosis. S. bovis strain S1 was newly isolated from the ruminal fluid of Saanen dairy goats and then used to examine the effects of glucose and starch on bacterial metabolism and gene regulation of the organic acid-producing pathway in cultures at a pH of 6.5. Glucose or starch was added to the culture medium at 1 g/liter, 3 g/liter (close to a normal range in the rumen fluid), or 9 g/liter (excessive level). Lactate was the dominant acid produced during the fermentation, and levels increased with the amount of glucose or starch in a dose-dependent manner (P < 0.001). The production of formate and acetate in the fermentation media fluctuated slightly with the dose but accounted for small fractions of the total acids. The activities of lactate dehydrogenase (LDH) and α-amylase (α-AMY) increased with the starch dose (P < 0.05), but the α-AMY activity did not change with the glucose dose. The relative expression levels of the genes ldh, pfl (encoding pyruvate formate lyase), ccpA (encoding catabolite control protein A), and α-amy were higher at a dose of 9 g/liter than at 1 g/liter (P < 0.05). Expression levels of pfl and α-amy genes were higher at 3 g/liter than at 1 g/liter (P < 0.05). The fructose 1,6-diphosphate (FDP) concentration tended to increase with the glucose and starch concentrations. In addition, the S. bovis S1 isolate fermented glucose much faster than starch. We conclude that the quantities of glucose and soluble starch had a major effect on lactate production due to the transcriptional regulation of metabolic genes. IMPORTANCE: This work used a newly isolated S. bovis strain S1 from the rumen fluid of Saanen goats and examined the effects of glucose and soluble starch on organic acid patterns, enzyme activity, and expression of genes for in vitro fermentation. It was found that lactate was the dominant product from S. bovis strain S1, and the quantities of both glucose and starch in the medium were highly correlated with lactate production and with the corresponding changes in associated enzymes and genes. Therefore, manipulating the metabolic pathway of S. bovis to alter the dietary level of readily fermentable sugar and carbohydrates may be a strategy to alleviate rumen acidosis.

Phylogenetic, epidemiological and functional analyses of the Streptococcus bovis/Streptococcus equinus complex through an overarching MLST scheme.

BACKGROUND: The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises seven (sub)species classified as human and animal commensals, emerging opportunistic pathogens and food fermentative organisms. Changing taxonomy, shared habitats, natural competence and evidence for horizontal gene transfer pose difficulties for determining their phylogeny, epidemiology and virulence mechanisms. Thus, novel phylogenetic and functional classifications are required. An SBSEC overarching multi locus sequence type (MLST) scheme targeting 10 housekeeping genes was developed, validated and combined with host-related properties of adhesion to extracellular matrix proteins (ECM), activation of the immune responses via NF-KB and survival in simulated gastric juice (SGJ). RESULTS: Commensal and pathogenic SBSEC strains (n = 74) of human, animal and food origin from Europe, Asia, America and Africa were used in the MLST scheme yielding 66 sequence types and 10 clonal complexes differentiated into distinct habitat-associated and mixed lineages. Adhesion to ECMs collagen I and mucin type II was a common characteristic (23 % of strains) followed by adhesion to fibronectin and fibrinogen (19.7 %). High adhesion abilities were found for East African dairy and human blood isolate branches whereas commensal fecal SBSEC displayed low adhesion. NF-KB activation was observed for a limited number of dairy and blood isolates suggesting the potential of some pathogenic strains for reduced immune activation. Strains from dairy MLST clades displayed the highest relative survival to SGJ independently of dairy adaptation markers lacS/lacZ. CONCLUSION: Combining phylogenetic and functional analyses via SBSEC MLST enabled the clear delineation of strain clades to unravel the complexity of this bacterial group. High adhesion values shared between certain dairy and blood strains as well as the behavior of NF-KB activation are concerning for specific lineages. They highlighted the health risk among shared lineages and establish the basis to elucidate (zoonotic-) transmission, host specificity, virulence mechanisms and enhanced risk assessment as pathobionts in an overarching One Health approach.

An Update on the Streptococcus bovis Group: Classification, Identification, and Disease Associations.

The Streptococcus bovis group has undergone significant taxonomic changes over the past 2 decades with the advent of new identification methods with higher discriminatory power. Although the current classification system is not yet embraced by all researchers in the field and debate remains over the performance of molecular techniques for identification to the species level within the group, important disease associations for several members of the group have been clarified. Here, we provide a brief overview of the history of the S. bovis group, an outline of the currently accepted classification scheme, a review of associated clinical syndromes, and a summary of the performance and diagnostic accuracy of currently available identification methods.

[Individual catalytic activity of two functional domains of bovicin HJ50 synthase BovM].

OBJECTIVE: To reconstitute the in vitro catalytic activity of the individual dehydratase or cyclase domain of bifunctional bovicin HJ50 synthase BovM, and lay a foundation for the further investigation of catalytic mechanism of class II lantibiotic synthase LanM. METHOD: The truncated proteins of BovM containing the N-terminal dehydratase domain or C-terminal cyclase domain were expressed in E. coli and purified. Substrate BovA, the precursor of bovicin HJ50, was incubated with these truncated BovM proteins in in vitro reaction system. The antimicrobial activity assay and MALDI-TOF MS analysis were used to monitor the dehydratase or cyclase activity of these truncated proteins. Meanwhile, the synergistic activities of both truncated proteins were tested in vivo and in vitro. RESULTS: The N- and C-terminal domains of BovM possessed dehydration and cyclization activity respectively. However, no synergistic activity was detected between these two functional domains. CONCLUSION: The individual functional domains of BovM could execute their corresponding functions independently, but the intactness of BovM was important for its full modification activity.

Production of itaconic acid in Escherichia coli expressing recombinant α-amylase using starch as substrate.

Several studies on fermentative production of a vinyl monomer itaconic acid from hydrolyzed starch using Aspergillus terreus have been reported. Herein, we report itaconic acid production by Escherichia coli expressing recombinant α-amylase, using soluble starch as its sole carbon source. To express α-amylase in E. coli, we first constructed recombinant plasmids expressing α-amylases by using cell surface display technology derived from two amylolytic bacteria, Bacillus amyloliquefaciens NBRC 15535(T) and Streptococcus bovis NRIC 1535. The recombinant α-amylase from S. bovis (SBA) showed activity at 28°C, which is the optimal temperature for production of itaconic acid, while α-amylase from B. amyloliquefaciens displayed no noticeable activity. E. coli cells expressing SBA produced 0.15 g/L itaconic acid after 69 h cultivation under pH-stat conditions, using 1% starch as the sole carbon source. In fact, E. coli cells expressing SBA had similar growth rates when grown in the presence of 1% glucose or starch, thereby highlighting the expression of an active α-amylase that enabled utilization of starch to produce itaconic acid in E. coli.

Efficient co-displaying and artificial ratio control of α-amylase and glucoamylase on the yeast cell surface by using combinations of different anchoring domains.

Recombinant yeast strains that display heterologous amylolytic enzymes on their cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system are considered as promising biocatalysts for direct ethanol production from starchy materials. For the effective hydrolysis of these materials, the ratio optimization of multienzyme activity displayed on the cell surface is important. In this study, we have presented a ratio control system of multienzymes displayed on the yeast cell surface by using different GPI-anchoring domains. The novel gene cassettes for the cell-surface display of Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase were constructed using the Saccharomyces cerevisiae SED1 promoter and two different GPI-anchoring regions derived from Saccharomyces cerevisiae SED1 or SAG1. These gene cassettes were integrated into the Saccharomyces cerevisiae genome in different combinations. Then, the cell-surface α-amylase and glucoamylase activities and ethanol productivity of these recombinant strains were evaluated. The combinations of the gene cassettes of these enzymes affected the ratio of cell-surface α-amylase and glucoamylase activities and ethanol productivity of the recombinant strains. The highest ethanol productivity from raw starch was achieved by the strain harboring one α-amylase gene cassette carrying the SED1-anchoring region and two glucoamylase gene cassettes carrying the SED1-anchoring region (BY-AASS/GASS/GASS). This strain yielded 22.5 ± 0.6 g/L of ethanol from 100 g/L of raw starch in 120 h of fermentation.

Genomics, evolution, and molecular epidemiology of the Streptococcus bovis/Streptococcus equinus complex (SBSEC).

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) is a group of human and animal derived streptococci that are commensals (rumen and gastrointestinal tract), opportunistic pathogens or food fermentation associates. The classification of SBSEC has undergone massive changes and currently comprises 7 (sub)species grouped into four branches based on sequences identities: the Streptococcus gallolyticus, the Streptococcus equinus, the Streptococcus infantarius and the Streptococcus alactolyticus branch. In animals, SBSEC are causative agents for ruminal acidosis, potentially laminitis and infective endocarditis (IE). In humans, a strong association was established between bacteraemia, IE and colorectal cancer. Especially the SBSEC-species S. gallolyticus subsp. gallolyticus is an emerging pathogen for IE and prosthetic joint infections. S. gallolyticus subsp. pasteurianus and the S. infantarius branch are further associated with biliary and urinary tract infections. Knowledge on pathogenic mechanisms is so far limited to colonization factors such as pili and biofilm formation. Certain strain variants of S. gallolyticus subsp. macedonicus and S. infantarius subsp. infantarius are associated with traditional dairy and plant-based food fermentations and display traits suggesting safety. However, due to their close relationship to virulent strains, their use in food fermentation has to be critically assessed. Additionally, implementing accurate and up-to-date taxonomy is critical to enable appropriate treatment of patients and risk assessment of species and strains via recently developed multilocus sequence typing schemes to enable comparative global epidemiology. Comparative genomics revealed that SBSEC strains harbour genomics islands (GI) that seem acquired from other streptococci by horizontal gene transfer. In case of virulent strains these GI frequently encode putative virulence factors, in strains from food fermentation the GI encode functions that are pivotal for strain performance during fermentation. Comparative genomics is a powerful tool to identify acquired pathogenic functions, but there is still an urgent need for more physiological and epidemiological data to understand SBSEC-specific traits.