Widespread Family of NAD+-Dependent Sulfoquinovosidases at the Gateway to Sulfoquinovose Catabolism.

The sulfosugar sulfoquinovose (SQ) is produced by photosynthetic plants, algae, and cyanobacteria on a scale of 10 billion tons per annum. Its degradation, which is essential to allow cycling of its constituent carbon and sulfur, involves specialized glycosidases termed sulfoquinovosidases (SQases), which release SQ from sulfolipid glycoconjugates, so SQ can enter catabolism pathways. However, many SQ catabolic gene clusters lack a gene encoding a classical SQase. Here, we report the discovery of a new family of SQases that use an atypical oxidoreductive mechanism involving NAD+ as a catalytic cofactor. Three-dimensional X-ray structures of complexes with SQ and NAD+ provide insight into the catalytic mechanism, which involves transient oxidation at C3. Bioinformatic survey reveals this new family of NAD+-dependent SQases occurs within sulfoglycolytic and sulfolytic gene clusters that lack classical SQases and is distributed widely including within Roseobacter clade bacteria, suggesting an important contribution to marine sulfur cycling.

Defining the molecular architecture, metal dependence, and distribution of metal-dependent class II sulfofructose-1-phosphate aldolases.

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant, algal, and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for SQ breakdown is the sulfoglycolytic Embden-Meyerhof-Parnas pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is understood for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures of complexes with substrate SFP and product dihydroxyacetone phosphate reveal a dimer-of-dimers (tetrameric) assembly, the sulfonate-binding pocket, two metal-binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal-dependent and exhibited high KM values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfoglycolytic Embden-Meyerhof-Parnas gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases have mututally exclusive occurrence within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.

Identification of levoglucosan degradation pathways in bacteria and sequence similarity network analysis.

Levoglucosan is produced in the pyrolysis of cellulose and starch, including from bushfires or the burning of biofuels, and is deposited from the atmosphere across the surface of the earth. We describe two levoglucosan degrading Paenarthrobacter spp. (Paenarthrobacter nitrojuajacolis LG01 and Paenarthrobacter histidinolovorans LG02) that were isolated from soil by metabolic enrichment using levoglucosan as the sole carbon source. Genome sequencing and proteomics analysis revealed the expression of a series of genes encoding known levoglucosan degrading enzymes, levoglucosan dehydrogenase (LGDH, LgdA), 3-keto-levoglucosan ß -eliminase (LgdB1) and glucose 3-dehydrogenase (LgdC), along with an ABC transporter cassette and an associated solute binding protein. However, no homologues of 3-ketoglucose dehydratase (LgdB2) were evident, while the expressed genes contained a range of putative sugar phosphate isomerases/xylose isomerases with weak similarity to LgdB2. Sequence similarity network analysis of genome neighbours of LgdA revealed that homologues of LgdB1 and LgdC are generally conserved in a range of bacteria in the phyla Firmicutes, Actinobacteria and Proteobacteria. One group of sugar phosphate isomerase/xylose isomerase homologues (named LgdB3) was identified with limited distribution that is mutually exclusive with LgdB2, and we propose that they may fulfil a similar function. LgdB1, LgdB2 and LgdB3 adopt similar predicted 3D folds, suggesting overlapping function in processing intermediates in LG metabolism. Our findings highlight diversity within the LGDH pathway, through which bacteria utilize levoglucosan as a nutrient source.

Microbially-derived cocktail of carbohydrases as an anti-biofouling agents: a 'green approach'.

Enzymes, also known as biocatalysts, display vital properties like high substrate specificity, an eco-friendly nature, low energy inputs, and cost-effectiveness. Among their numerous known applications, enzymes that can target biofilms or their components are increasingly being investigated for their anti-biofouling action, particularly in healthcare, food manufacturing units and environmental applications. Enzymes can target biofilms at different levels like during the attachment of microorganisms, formation of exopolymeric substances (EPS), and their disruption thereafter. In this regard, a consortium of carbohydrases that can target heterogeneous polysaccharides present in the EPS matrix may provide an effective alternative to conventional chemical anti-biofouling methods. Further, for complete annihilation of biofilms, enzymes can be used alone or in conjunction with other antimicrobial agents. Enzymes hold the promise to replace the conventional methods with greener, more economical, and more efficient alternatives. The present article explores the potential and future perspectives of using carbohydrases as effective anti-biofilm agents.

Genome sequences of Arthrobacter spp. that use a modified sulfoglycolytic Embden-Meyerhof-Parnas pathway.

Sulfoglycolysis pathways enable the breakdown of the sulfosugar sulfoquinovose and environmental recycling of its carbon and sulfur content. The prototypical sulfoglycolytic pathway is a variant of the classical Embden-Meyerhof-Parnas (EMP) pathway that results in formation of 2,3-dihydroxypropanesulfonate and was first described in gram-negative Escherichia coli. We used enrichment cultures to discover new sulfoglycolytic bacteria from Australian soil samples. Two gram-positive Arthrobacter spp. were isolated that produced sulfolactate as the metabolic end-product. Genome sequences identified a modified sulfoglycolytic EMP gene cluster, conserved across a range of other Actinobacteria, that retained the core sulfoglycolysis genes encoding metabolic enzymes but featured the replacement of the gene encoding sulfolactaldehyde (SLA) reductase with SLA dehydrogenase, and the absence of sulfoquinovosidase and sulfoquinovose mutarotase genes. Excretion of sulfolactate by these Arthrobacter spp. is consistent with an aerobic saprophytic lifestyle. This work broadens our knowledge of the sulfo-EMP pathway to include soil bacteria.

Cocktail of carbohydrases from Aspergillus niger: an economical and eco-friendly option for biofilm clearance from biopolymer surfaces.

Biofilm formation on both biotic and abiotic surfaces accounts for a major factor in spread of antimicrobial resistance. Due to their ubiquitous nature, biofilms are of great concern for environment as well as human health. In the present study, an integrated process for the co-production of a cocktail of carbohydrases from a natural variant of Aspergillus niger was designed. The enzyme cocktail was found to have a noteworthy potential to eradicate/disperse the biofilms of selected pathogens. For application of enzymes as an antibiofilm agent, the enzyme productivities were enhanced by statistical modelling using response surface methodology (RSM). The antibiofilm potential of the enzyme cocktail was studied in terms of (i) in vitro cell dispersal assay (ii) release of reducing sugars from the biofilm polysaccharides (iii) the effect of enzyme treatment on biofilm cells and architecture by confocal laser scanning microscopy (CLSM). Potential of the enzyme cocktail to disrupt/disperse the biofilm of selected pathogens from biopolymer surfaces was also assessed by field emission scanning electron microscopy (FESEM) analysis. Further, their usage in conjunction with antibiotics was assessed and it was inferred from the results that the use of enzyme cocktail augmented the efficacy of the antibiotics. The study thus provides promising insights into the prospect of using multiple carbohydrases for management of heterogeneous biofilms formed in natural and clinical settings.

Role of diffusion-weighted magnetic resonance imaging in the evaluation of vertebral bone marrow lesions.

PURPOSE: To evaluate the role of diffusion-weighted magnetic resonance imaging (DW-MRI) in differentiating vertebral marrow pathologies. To determine the sensitivity, specificity, and threshold apparent diffusion coefficient (ADC) values that can aid in the differentiation of malignant from benign bone marrow lesions. MATERIAL AND METHODS: This observational study included 100 patients, who underwent MRI examination with a 1.5 Tesla scanner. The ADC values of normal and pathological vertebrae were estimated, and the threshold ADC values were computed by receiver operating characteristic (ROC) analysis. The results were correlated with histopathological diagnosis, clinical follow-up, and other investigations. Statistical analysis was done by employing unpaired two-tailed Student's t-test and the p-value of < 0.05 was deemed as statistically significant. RESULTS: Vertebral bone marrow lesions had a male predominance and there was a predilection towards thoracic and lumbar vertebrae, with L4 being the commonest. Metastasis was the commonest lesion, followed by spondylodiscitis. The mean ADC value of benign pathologies was significantly greater than malignant pathologies (p < 0.05). The threshold value for the demarcation between benign and malignant pathologies was computed to be 1.21 × 10-3 mm2/s. DW imaging had sensitivity of 100%, specificity of 92.31%, positive predictive value of 87.5%, and negative predictive value of 100%. CONCLUSIONS: Vertebral marrow lesions can be differentiated as benign or malignant with good sensitivity and specificity with the help of DW-ADC maps.

A novel multi-enzyme preparation produced from Aspergillus niger using biodegradable waste: a possible option to combat heterogeneous biofilms.

Extracellular polymeric substance (EPS) produced by the microorganisms provides protection and stability to them when they are encased within biofilms. Heterogeneous polysaccharides form a major constituent of the EPS and are crucial for the formation and integrity of the biofilms/slime. Thus, breakdown of polysaccharides might help in dispersion of biofilms from abiotic surfaces. In the present study we isolated a fungus, Aspergillus niger APS, capable of concurrently producing a cocktail of carbohydrases and optimized the conditions for higher yields of all the enzymes by one variable at a time (OVAT) approach. The optimization studies resulted in 1.5 to 12 fold augmentation in the enzyme yields using biodegradable waste. Further, keeping in view the heterogeneous nature of polysaccharides in biofilm matrix, the in-house produced enzyme cocktail was used for the dispersal of biofilms formed by Salmonella enterica serovar Typhi, Escherichia coli and Staphylococcus aureus. Treatment with enzyme preparation caused 90.23 ± 4.0, 82.64 ± 5.0 and 76.32 ± 5.0% reduction of the biofilms formed by these organisms respectively which was also evidenced by Field emission scanning electron microscopy (FESEM) revealing the loss of biofilm architecture. Interestingly, the enzyme cocktail could also remove viscous slime formed under natural conditions in the kitchen drainage pipe (KDP). To the best of our knowledge, this is the first report on biotreatment of abiotic surfaces for removal of biofilms/slime formed under natural conditions. The study thus indicates the prospects of using multiple carbohydrases as an anti-biofouling agent on abiotic surfaces like equipments as well as implants/prostheses and pipelines.

Utilization of kitchen waste for production of pullulan to develop biodegradable plastic.

Pullulan has many useful characteristics but, its high cost limits its potential applications. In the present work, kitchen waste (KW), which otherwise has zero commercial value, was evaluated for the economical production of pullulan. Before fermentation, the KW was hydrolyzed into free sugars using an in-house produced cocktail of enzymes. During hydrolysis, 46 ± 3.5 g/l and 31 ± 2.2 g/l of total reducing sugars and glucose were released, respectively. Hydrolyzed kitchen waste was then used as substrate for fermentation by Aureobasidium pullulans MTCC 2013 yielding 20.46 ± 2.01 g/l pullulan. Further, effect of different nitrogen sources was evaluated and yeast extract (3%) was found to be the best, yielding (24.77 ± 1.06 g/l) exopolysaccharide (EPS). The pullulan produced from KW was characterized in terms of organoleptic properties, physical strength, Fourier-transform infrared spectroscopy (FTIR), and H nuclear magnetic resonance (H NMR) analysis. The results corroborated well with commercial pullulan. The biodegradable nature and water solubility of the film developed from pullulan was also confirmed. To the best of our knowledge, this is the first report on the validation of the biodegradability of in-house produced pullulan. Thus, kitchen waste appears to be a promising option for economical pullulan production. Additionally, the method may also prove to be helpful for managing the increasing load of municipal solid waste in an eco-friendly and scientific way.

Incidence, Characteristics and Atherosclerotic Involvement of Coronary Artery Anomalies in Adult Population Undergoing Catheter Coronary Angiography.

BACKGROUND: Coronary artery anomalies (CAAs) are rare disorders of coronary anatomy with varied clinical presentations. There are widespread geographic variations in incidence and patterns of these anomalies, with limited data from North Indian population. We performed a retrospective study to evaluate the incidence, characteristics and atherosclerotic involvement of CAAs in adult population undergoing catheter coronary angiography. METHODS: Serial coronary angiographies performed at our institution over a period of 2.5 years (from January 2017 to June 2019) were retrospectively analyzed. We identified patients with anomalous coronaries and studied their clinical characteristics and angiographic profiles. RESULTS: Among 3,233 coronary angiograms analyzed, CAAs were found in 99 patients with an incidence of 3.06%. Mean age of the patients was 56.2 ± 12.9 years (range: 20 - 86 years), with 74.75% being males and 25.25% females. Split right coronary artery (RCA) was the most common coronary anomaly, being seen in 27 patients; with an angiographic incidence of 0.84%. Dual left anterior descending artery (LAD) was the second most common anomaly and was seen in 22 cases with an angiographic incidence of 0.68%. Absent left main trunk was noted in 14 patients (0.43%). Ectopic origin of RCA from left sinus was seen in 12 patients (0.37%), while ectopic origin of RCA from ascending aorta was seen in four patients (0.12%). Ectopic origin of left circumflex artery (LCX) from right sinus or RCA was noted in 13 patients (0.40%). One patient (0.03%) had a superdominant LAD supplying the posterior descending artery (PDA). Coronary artery fistulae were seen in six patients (0.18%). Significant coronary artery disease (CAD) was seen in 89 of 268 (33.21%) normal vessels, whereas it was seen in 56 of 114 (49.12%) of anomalous vessels. This difference was statistically significant (P = 0.003). CONCLUSIONS: The incidence of CAAs in our study was slightly higher than many of the previous angiographic series. The patterns of coronary anomalies in our study were different from most of the previous studies. Our study had higher incidence of atherosclerotic involvement of anomalous vessels as compared to normal vessels.

Efficacy of designer K11 antimicrobial peptide (a hybrid of melittin, cecropin A1 and magainin 2) against Acinetobacter baumannii-infected wounds.

Due to emergence of multidrug resistance in pathogens, the attention of the scientific community is now directed towards strengthening the reservoir of antimicrobial compounds. Prior to in vivo studies, the interaction and penetration of a hybrid peptide K11 in bacterial cells using confocal microscopy was assessed which was observed as early as 10 min after incubation with the peptide. Cell lysis along with leakage of cytoplasmic content was confirmed by electron microscopy. To evaluate the in vivo performance of the peptide, it was contained in carbopol hydrogel. Efficacy of the hydrogel formulation was then evaluated against Acinetobacter baumannii-infected wounds using a murine excision model. Treatment resulted in restoration of body weight, complete clearance of infection from the wound by day 7 and 99% wound enclosure by day 21, in contrast to the persistence of infection and 70% wound enclosure in the infected group. Further, this treatment resulted in a 2.6-fold decrease in the levels of malondialdehyde along with a 4.5-fold increase in the levels of catalase on day 3. Appearance of normal histo-architecture was observed in the treatment group. Based on these results, the peptide hydrogel can be exploited in future as one of the strategies for developing a topical anti-infective therapeutic agent.

Management of Staphylococcus Mediated Systemic Infection by Enhancing the Resurging Activity of Co-trimoxazole in Presence of Cryptdin-2.

Resurgence of sensitivity of the antibiotics, to which the pathogen had developed resistance in the past, requires special attention for strengthening the reservoir of antimicrobial compounds. Reports in the recent past have suggested that co-trimoxazole (COT) has regained its activity against methicillin resistant Staphylococcus aureus (MRSA). The present study exploited the use of COT in the presence of an antimicrobial peptide (AMP), cryptdin-2 (a murine Paneth cell alpha defensin), in order to reduce the selective pressure of the antibiotic on the pathogen. In vitro antibacterial activity and in vivo efficacy of the combination was ascertained against MRSA induced systemic infection using a murine model. Observations of the present study might help in restoring the regained activity of conventional antibiotics, such as COT, when used in combination with novel antimicrobial molecules like AMPs. This might prove as a viable strategy to eliminate the chances of re-occurrence of resistance due to their multi-prong targeting and synergistically combating infections caused by these resistant pathogens.