The Acquisition of the scr Gene Cluster Encoding Sucrose Metabolization Enzymes Enables Strains of Vibrio parahaemolyticus and Vibrio vulnificus to Utilize Sucrose as Carbon Source

Most strains of Vibrio parahaemolyticus are unable to utilize sucrose as carbon source, though few exceptions exist. We investigated a sucrose-positive V. parahaemolyticus strain by whole-genome sequencing (WGS) and confirmed the presences of a genomic island containing sucrose utilization genes. A 4.7 kb DNA cluster consisting of three genes: scrA encoding a sucrose uptake protein, scrK encoding a fructokinase, and scrB coding for a sucrose-6-phosphate hydrolase, was PCR amplified and inserted into the Vibrio/Escherichia coli shuttle vector pVv3. Two recombinant plasmids, only differing in the orientation of the insert with respect to the pVv3-lacZα-fragment, conferred the E. coli K12 transformants the ability to utilize sucrose. The introduction of the two plasmids into sucrose-negative V. parahaemolyticus and V. vulnificus strains also results in a change of the sucrose utilization phenotype from negative to positive. By performing a multiplex PCR targeting scrA, scrK, and scrB, 43 scr-positive V. parahaemolyticus isolates from our collection of retail strains were detected and confirmed to be able to use sucrose as carbon source. Strains unable to utilize the disaccharide were negative by PCR for the scr genes. For in-depth characterization, 17 sucrose-positive V. parahaemolyticus were subjected to WGS. A genomic island with a nucleotide identity of >95% containing scrA, scrB, scrK and three additional coding sequences (CDS) were identified in all strains. The additional genes were predicted as a gene coding for a transcriptional regulator (scrR), a porin encoding gene and a CDS of unknown function. Sequence comparison indicated that the genomic island was located in the same region of the chromosome II in all analyzed V. parahaemolyticus strains. Structural comparison of the genomes with sequences of the sucrose utilizing species V. alginolyticus revealed the same genomic island, which indicates a possible distribution of this genetic structure by horizontal gene transfer. The comparison of all genome sequences based on SNP differences reveals that the presence of sucrose utilizing genes is found in genetically diverse V. parahaemolyticus strains and is not restricted to a subset of closely related strains.

The Role of Hexokinase and Hexose Transporters in Preferential Use of Glucose over Fructose and Downstream Metabolic Pathways in the Yeast Yarrowia lipolytica

The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We investigated the role of hexose transporters and hexokinase (native and fructophilic) in this preference. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Interestingly, combined overexpression of either hexokinase with hexose transporters significantly accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L).

Aerobic growth physiology of Saccharomyces cerevisiae on sucrose is strain-dependent

ABSTRACT Present knowledge on the quantitative aerobic physiology of the yeast Saccharomyces cerevisiae during growth on sucrose as sole carbon and energy source is limited to either adapted cells or to the model laboratory strain CEN.PK113-7D. To broaden our understanding of this matter and open novel opportunities for sucrose-based biotechnological processes, we characterized three strains, with distinct backgrounds, during aerobic batch bioreactor cultivations. Our results reveal that sucrose metabolism in S. cerevisiae is a strain-specific trait. Each strain displayed distinct extracellular hexose concentrations and invertase activity profiles. Especially, the inferior maximum specific growth rate (0.21 h-1) of the CEN.PK113-7D strain, with respect to that of strains UFMG-CM-Y259 (0.37 h-1) and JP1 (0.32 h-1), could be associated to its low invertase activity (0.04–0.09 U/mgDM). Moreover, comparative experiments with glucose or fructose alone, or in combination, suggest mixed mechanisms of sucrose utilization by the industrial strain JP1, and points out the remarkable ability of the wild isolate UFMG-CM-259 to grow faster on sucrose than on glucose in a well-controlled cultivation system. This work hints to a series of metabolic traits that can be exploited to increase sucrose catabolic rates and bioprocess efficiency.

Identification of a chromosomally-encoded sucrose operon-like gene cluster in Vibrio parahaemolyticus strain PH05 isolated from Negros Island, Philippines

ABSTRACTThe ability of bacteria to metabolize a wide variety of carbon sources has been known to aid in their ability for efficient colonization. Vibrio parahaemolyticus, a known aquatic pathogen has been reported to have the ability to metabolize a number of carbohydrates including D-glucose, D-galactose, L-arabinose, D-mannose, and D-ribose to name a few. Classical isolation of V. parahaemolyticus from other members of the family Vibrionaceae relies on its carbon utilization pattern. Conventionally, V. parahaemolyticus lacks the ability to utilize sucrose and this has been the basis for its isolation using the Thiosulfate-citrate-bile salts-sucrose (TCBS) agar. Reports of V. parahaemolyticus having the ability to utilize sucrose have been presented yet there is paucity of information and detailed study on this phenotype. In this study, we report the V. parahaemolyticus strain PH05 that has the ability to metabolize sucrose. Phenotypic and genotypic characterization of this V. parahaemolyticus strain isolated from Negros Island, Philippines, revealed that V. parahaemolyticus strain PH05 is atypical appearing yellow on TCBS agar plates. It is capable of utilizing sucrose, unlike the majority of V. parahaemolyticus isolates. Genome analyses of this strain revealed the presence of a chromosomally encoded sucrose operon-like gene cluster encoded in chromosome 2 with the following sucrose-utilization associated genes: scrY, ccpA, treP, scrK, and scrB genes coding for sucrose porin, catabolite control protein A, PTS System sucrose-specific EIIBC component, fructokinase, and sucrose-6-phosphate hydrolase. The mode of transmission of these genes to V. parahaemolyticus strain PH05 is still unknown. However, the presence of insertion sequences (IS) and phage elements in the same chromosome suggests horizontal gene transfer events. Taken together, our results point to the possibility that acquired sucrose utilization genes may contribute to the fitness of V. parahaemolyticus strain PH05 in the environment.

Aerobic growth physiology of Saccharomyces cerevisiae on sucrose is strain-dependent

Present knowledge on the quantitative aerobic physiology of the yeast Saccharomyces cerevisiae during growth on sucrose as sole carbon and energy source is limited to either adapted cells or to the model laboratory strain CEN.PK113-7D. To broaden our understanding of this matter and open novel opportunities for sucrose-based biotechnological processes, we characterized three strains, with distinct backgrounds, during aerobic batch bioreactor cultivations. Our results reveal that sucrose metabolism in S. cerevisiae is a strain-specific trait. Each strain displayed a distinct extracellular hexose concentration and invertase activity profiles. Especially, the inferior maximum specific growth rate (0.21 h−1) of the CEN.PK113-7D strain, with respect to that of strains UFMG-CM-Y259 (0.37 h−1) and JP1 (0.32 h−1), could be associated to its low invertase activity (0.04 to 0.09 U mgDM−1). Moreover, comparative experiments with glucose or fructose alone, or in combination, suggest mixed mechanisms of sucrose utilization by the industrial strain JP1, and points out the remarkable ability of the wild isolate UFMG-CM-259 to grow faster on sucrose than on glucose in a well-controlled cultivation system. This work hints to a series of metabolic traits that can be exploited to increase sucrose catabolic rates and bioprocess efficiency.Abstract Figure

The Route of Sucrose Utilization by Streptococcus mutans Affects Intracellular Polysaccharide Metabolism

Streptococcus mutans converts extracellular sucrose (Suc) into exopolysaccharides (EPS) by glucosyl-transferase and fructosyl-transferase enzymes and internalizes Suc for fermentation through the phosphotransferase system (PTS). Here, we examined how altering the routes for sucrose utilization impacts intracellular polysaccharide [IPS; glycogen, (glg)] metabolism during carbohydrate starvation. Strain UA159 (WT), a mutant lacking all exo-enzymes for sucrose utilization (MMZ952), and a CcpA-deficient mutant (∆ccpA) were cultured with sucrose or a combination of glucose and fructose, followed by carbohydrate starvation. At baseline (0h), and after 4 and 24h of starvation, cells were evaluated for mRNA levels of the glg operon, IPS storage, glucose-1-phosphate (G1P) concentrations, viability, and PTS activities. A pH drop assay was performed in the absence of carbohydrates at the baseline to measure acid production. We observed glg operon activation in response to starvation (p<0.05) in all strains, however, such activation was significantly delayed and reduced in magnitude when EPS synthesis was involved (p<0.05). Enhanced acidification and greater G1P concentrations were observed in the sucrose-treated group, but mostly in strains capable of producing EPS (p<0.05). Importantly, only the WT exposed to sucrose was able to synthesize IPS during starvation. Contrary to CcpA-proficient strains, IPS was progressively degraded during starvation in ∆ccpA, which also showed increased glg operon expression and greater PTS activities at baseline. Therefore, sucrose metabolism by secreted enzymes affects the capacity of S. mutans in synthesizing IPS and converting it into organic acids, without necessarily inducing greater expression of the glg operon.

Diverse Horizontally-Acquired Gene Clusters Confer Sucrose Utilization to Different Lineages of the Marine Pathogen Photobacterium damselae subsp. damselae

The ability to metabolize sucrose is a variable trait within the family Vibrionaceae. The marine bacterium Photobacterium damselae subsp. damselae (Pdd), pathogenic for marine animals and humans, is generally described as negative for sucrose utilization (Scr−). Previous studies have reported sucrose-utilizing isolates (Scr+), but the genetic basis of this variable phenotype remains uncharacterized. Here, we carried out the genome sequencing of five Scr+ and two Scr−Pdd isolates and conducted a comparative genomics analysis with sixteen additional Pdd genomes sequenced in previous studies. We identified two different versions of a four-gene cluster (scr cluster) exclusive of Scr+ isolates encoding a PTS system sucrose-specific IIBC component (scrA), a fructokinase (scrK), a sucrose-6-phosphate hydrolase (scrB), and a sucrose operon repressor (scrR). A scrA deletion mutant did not ferment sucrose and was impaired for growth with sucrose as carbon source. Comparative genomics analyses suggested that scr clusters were acquired by horizontal transfer by different lineages of Pdd and were inserted into a recombination hot-spot in the Pdd genome. The incongruence of phylogenies based on housekeeping genes and on scr genes revealed that phylogenetically diverse gene clusters for sucrose utilization have undergone extensive horizontal transfer among species of Vibrio and Photobacterium.

Diversity of Backusella (Mucoromycotina) in south-eastern Australia revealed through polyphasic taxonomy

Here we explore the diversity of one morphologically distinguishable genus in the Mucoromycotina, Backusella, in south-eastern Australia. We isolated more than 200 strains from locations across the states of Victoria and Tasmania. Characterization of these strains using a combination of approaches including morphology, sucrose utilization and whole genome sequencing for 13 strains, revealed 10 new species. The genetic basis for interspecies variation in sucrose utilization was found to be the presence of a gene encoding an invertase enzyme. The genus Backusella is revised and a new key for species identification produced. Given that we have more than doubled the number of species in this genus, this work demonstrates that there may be considerable undiscovered species diversity in the early diverging fungal lineages.