Disruption of the adh (acetoin dehydrogenase) operon has wide-ranging effects on Streptococcus mutans growth and stress response

The agent largely responsible for initiating dental caries, Streptococcus mutans produces acetoin dehydrogenase that is encoded by the adh operon. The operon consists of the adhA and B genes (E1 dehydrogenase), adhC (E2 lipoylated transacetylase), adhD (E3 dihydrolipoamide dehydrogenase), and lplA (lipoyl ligase). Evidence is presented that AdhC interacts with SpxA2, a redox-sensitive transcription factor functioning in cell wall and oxidative stress responses. In-frame deletion mutations of adh genes conferred oxygen-dependent sensitivity to slightly alkaline pH (pH 7.2-7.6), within the range of values observed in human saliva. Growth defects were also observed when glucose or sucrose served as major carbon sources. A deletion of the adhC orthologous gene, acoC gene of Streptococcus gordonii , did not result in pH sensitivity or defective growth in glucose and sucrose. The defects observed in adh mutants were partially reversed by addition of pyruvate. Unlike most 2-oxoacid dehydrogenases, the E3 AdhD subunit bears an N-terminal lipoylation domain nearly identical to that of E2 AdhC. Changing the lipoyl domains of AdhC and AdhD by replacing the lipoate attachment residue, lysine to arginine, caused no significant reduction in pH sensitivity but the adhDK43R mutation eliminating the lipoylation site resulted in an observable growth defect in glucose medium. The adh mutations were partially suppressed by a deletion of rex , encoding an NAD + /NADH-sensing transcription factor that represses genes functioning in fermentation. spxA2 adh double mutants show synthetic growth restriction at elevated pH and upon ampicillin treatment. These results suggest a role for Adh in stress management in S. mutans . IMPORTANCE Dental caries is often initiated by Streptococcus mutans , which establishes a biofilm and a low pH environment on tooth enamel surfaces. The current study has uncovered vulnerabilities of S. mutans mutant strains that are unable to produce the enzyme complex, acetoin dehydrogenase (Adh). Such mutants are sensitive to modest increases in pH to 7.2-7.6, within the range of human saliva, while a mutant of a commensal Streptococcal species is resistant. The S. mutans adh strains are also defective in carbohydrate utilization and are hypersensitive to a cell wall-acting antibiotic. The studies suggest that Adh could be a potential target for interfering with S. mutans colonization of the oral environment.

In Vitro Probiotic Characteristics and Whole Genome Sequence Analysis of Lactobacillus Strains Isolated from Cattle-Yak Milk

Cattle-yak milk is an important raw material and an indispensable source of high-quality food for local farmers and herdsmen to produce ghee, milk residue, yogurt, and other dairy products. In this study, Lactobacillus strains were isolated from cattle-yak milk for potential probiotic candidates using a series of in vitro tests, including probiotic characterization and safety evaluation (antibiotic susceptibility and hemolytic ability). The results found that the Lactobacillus rhamnosus CY12 strain showed a high survival rate in bile salts, under acid conditions, and in the gastrointestinal juice environment, as well as showing high antimicrobial activity and adhesive potential. The safety evaluation showed that all strains were considered non-hemolytic. In addition, the whole-genome sequencing indicated that the strain CY12 spanned 2,506,167 bp, with an average length of 881 bp; the GC content in the gene region (%) was 47.35, contained 1347 protein-coding sequences, and accounted for 85.72% of the genome. The genome annotation showed that genes mainly focused on the immune system process, metabolic process, carbohydrate utilization, carbon metabolism, galactose metabolism, and biological adhesion, etc. This study revealed that the Lactobacillus rhamnosus CY12 strain might be an excellent potential probiotic in the development of feed additives for animals and has the ability to promote health.

The Carbohydrate Metabolism of Lactiplantibacillus plantarum

Lactiplantibacillus plantarum has a strong carbohydrate utilization ability. This characteristic plays an important role in its gastrointestinal tract colonization and probiotic effects. L. plantarum LP-F1 presents a high carbohydrate utilization capacity. The genome analysis of 165 L. plantarum strains indicated the species has a plenty of carbohydrate metabolism genes, presenting a strain specificity. Furthermore, two-component systems (TCSs) analysis revealed that the species has more TCSs than other lactic acid bacteria, and the distribution of TCS also shows the strain specificity. In order to clarify the sugar metabolism mechanism under different carbohydrate fermentation conditions, the expressions of 27 carbohydrate metabolism genes, catabolite control protein A (CcpA) gene ccpA, and TCSs genes were analyzed by quantitative real-time PCR technology. The correlation analysis between the expressions of regulatory genes and sugar metabolism genes showed that some regulatory genes were correlated with most of the sugar metabolism genes, suggesting that some TCSs might be involved in the regulation of sugar metabolism.

Dietary Probiotic Pediococcus acidilactici MA18/5M Improves the Growth, Feed Performance and Antioxidant Status of Penaeid Shrimp Litopenaeus stylirostris: A Growth-Ration-Size Approach

Probiotics are increasingly documented to confer health and performance benefits across farmed animals. The aim of this study was to assess the effect of a constant daily intake of the single-strain probiotic Pedicococcus acidilactici MA18/5M (4 × 108 CFU.day−1.kg−1 shrimp) fed over fixed, restricted ration sizes (1% to 6% biomass.day−1) on the nutritional performance and metabolism of adult penaeid shrimp Litopenaeus stylirostris (initial body-weight, BWi = 10.9 ± 1.8 g). The probiotic significantly increased the relative daily growth rate (RGR) across all ration size s tested (Mean-RGR of 0.45 ± 0.08 and 0.61 ± 0.07% BWi.day−1 for the control and probiotic groups, respectively) and decreased the maintenance ration (Rm) and the optimal ration (Ropt) by 18.6% and 11.3%, respectively. Accordingly, the probiotic group exhibited a significantly higher gross (K1) and net (K2) feed conversion efficiency with average improvement of 35% and 30%, respectively. Enhanced nutritional performances in shrimps that were fed the probiotic P. acidilactici was associated with, in particular, significantly higher α-amylase specific activity (+24.8 ± 5.5% across ration sizes) and a concentration of free-glucose and glycogen in the digestive gland at fixed ration sizes of 3% and below. This suggests that the probiotic effect might reside in a better use of dietary carbohydrates. Interestingly, P. acidilactici intake was also associated with a statistically enhanced total antioxidant status of the digestive gland and haemolymph (+24.4 ± 7.8% and +21.9 ± 8.5%, respectively; p < 0.05). As supported by knowledge in other species, enhanced carbohydrate utilization as a result of P. acidilactici intake may fuel the pentose-phosphate pathway, generating NADPH or directly enhancing OH-radicals scavenging by free glucose, in turn resulting in a decreased level of cellular oxidative stress. In conclusion, the growth-ration method documented a clear contribution of P. acidilactici MA18/5M on growth and feed efficiency of on-growing L. stylirostris that were fed fixed restricted rations under ideal laboratory conditions. The effect of the probiotic on α-amylase activity and carbohydrate metabolism and its link to the shrimp’s antioxidant status raises interesting prospects to optimize dietary formulations and helping to sustain the biological and economic efficiency of the penaeid shrimp-farming industry.

Identifying the essential nutritional requirements of the probiotic bacteria Bifidobacterium animalis and Bifidobacterium longum through genome-scale modeling

Although bifidobacteria are widely used as probiotics, their metabolism and physiology remain to be explored in depth. In this work, strain-specific genome-scale metabolic models were developed for two industrially and clinically relevant bifidobacteria, Bifidobacterium animalis subsp. lactis BB-12® and B. longum subsp. longum BB-46, and subjected to iterative cycles of manual curation and experimental validation. A constraint-based modeling framework was used to probe the metabolic landscape of the strains and identify their essential nutritional requirements. Both strains showed an absolute requirement for pantethine as a precursor for coenzyme A biosynthesis. Menaquinone-4 was found to be essential only for BB-46 growth, whereas nicotinic acid was only required by BB-12®. The model-generated insights were used to formulate a chemically defined medium that supports the growth of both strains to the same extent as a complex culture medium. Carbohydrate utilization profiles predicted by the models were experimentally validated. Furthermore, model predictions were quantitatively validated in the newly formulated medium in lab-scale batch fermentations. The models and the formulated medium represent valuable tools to further explore the metabolism and physiology of the two species, investigate the mechanisms underlying their health-promoting effects and guide the optimization of their industrial production processes.

Endothelial Cell CD36 Reduces Atherosclerosis and Controls Systemic Metabolism

High-fat Western diets contribute to tissue dysregulation of fatty acid and glucose intake, resulting in obesity and insulin resistance and their sequelae, including atherosclerosis. New therapies are desperately needed to interrupt this epidemic. The significant idea driving this research is that the understudied regulation of fatty acid entry into tissues at the endothelial cell (EC) interface can provide novel therapeutic targets that will greatly modify health outcomes and advance health-related knowledge. Dysfunctional endothelium, defined as activated, pro-inflammatory, and pro-thrombotic, is critical in atherosclerosis initiation, in modulating thrombotic events that could result in myocardial infarction and stroke, and is a hallmark of insulin resistance. Dyslipidemia from high-fat diets overwhelmingly contributes to the development of dysfunctional endothelium. CD36 acts as a receptor for pathological ligands generated by high-fat diets and in fatty acid uptake, and therefore, it may additionally contribute to EC dysfunction. We created EC CD36 knockout (CD36°) mice using cre-lox technology and a cre-promoter that does not eliminate CD36 in hematopoietic cells (Tie2e cre). These mice were studied on different diets, and crossed to the low density lipoprotein receptor (LDLR) knockout for atherosclerosis assessment. Our data show that EC CD36° and EC CD36°/LDLR° mice have metabolic changes suggestive of an uncompensated role for EC CD36 in fatty acid uptake. The mice lacking expression of EC CD36 had increased glucose clearance compared with controls when fed with multiple diets. EC CD36° male mice showed increased carbohydrate utilization and decreased energy expenditure by indirect calorimetry. Female EC CD36°/LDLR° mice have reduced atherosclerosis. Taken together, these data support a significant role for EC CD36 in systemic metabolism and reveal sex-specific impact on atherosclerosis and energy substrate use.

Multi-Omics Reveals the Inhibition of Lactiplantibacillus plantarum CCFM8724 in Streptococcus mutans-Candida albicans Mixed-Species Biofilms

Lactiplantibacillus plantarum CCFM8724 is a probiotic with the potential to prevent dental caries in vitro and in vivo. To explore the effects of this probiotic at inhibiting Streptococcus mutans-Candida albicans mixed-species biofilm and preventing dental caries, multi-omics, including metabolomics and transcriptomics, was used to investigate the regulation of small-molecule metabolism during biofilm formation and the gene expression in the mixed-species biofilm. Metabolomic analysis revealed that some carbohydrates related to biofilm formation, such as sucrose, was detected at lower levels due to the treatment with the L. plantarum supernatant. Some sugar alcohols, such as xylitol and sorbitol, were detected at higher levels, which may have inhibited the growth of S. mutans. In transcriptomic analysis, the expression of the virulence genes of C. albicans, such as those that code agglutinin-like sequence (Als) proteins, was affected. In addition, metabolomics coupled with a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and RNA-seq revealed that the L. plantarum supernatant had an active role in sugar metabolism during the formation of the S. mutans-C. albicans mixed-species biofilm, and the L. plantarum supernatant was also related to carbohydrate utilization, glucan biosynthesis, and mycelium formation. Hence, L. plantarum CCFM8724 decreased the mixed-species biofilm mass from the perspective of gene expression and metabolic reprogramming. Our results provide a rationale for evaluating L. plantarum CCFM8724 as a potential oral probiotic for inhibiting cariogenic pathogen biofilm formation and improving dental caries.

Culture Enrichment Combined With Long-Read Sequencing Facilitates Genomic Understanding of Hadal Sediment Microbes

Culture enrichment was developed to discover the uncharted microbial species in the environmental microbiota. Yet this strategy has not been widely used to study microbes of deep-sea environments. Here, we report the cultivation and metagenomic analysis of oceanic sediment microbiota collected from 6,477 m deep in the Mariana Trench. The sediment samples were cultured anaerobically in the laboratory for 4 months, before being subjected to full-length 16S rRNA gene sequencing using the PacBio technique and metagenome sequencing using both the Illumina and Oxford Nanopore techniques. The 16S rRNA gene analyses revealed 437 operational taxonomic units specific to the cultured microbes, despite the lower diversity of the cultured microbiota in comparison with the original. Metagenome analyses revealed the prevalence of functions related to respiration, energy production, and stress response in the cultured microbes, suggesting these functions may contribute to microbial proliferation under laboratory conditions. Binning of the assembled metagenome contigs of the cultured microbiota generated four nearly complete genomes affiliated to yet unclassified species under the genera Alcanivorax, Idiomarina, Sulfitobacter, and Erythrobacter. Excepting Alcanivorax, the other three genera were almost undetectable in the original samples and largely enriched in the cultured samples. The four genomes possessed a variety of genes for carbohydrate utilization and nitrite reduction, pointing to an ability to respire diverse carbon sources using nitrite as the final electron acceptor. Taken together, the findings suggest that a combination of culture enrichment and long-read sequencing is an ideal way to mine novel microbial species in the hadal environment, particularly species that are rare in their native environmental niches, and thus expand our understanding of the hadal microbial diversity and function.

Fucosylated human milk oligosaccharide foraging within the species Bifidobacterium pseudocatenulatum is driven by glycosyl hydrolase content and specificity

Human milk enriches members of the genus Bifidobacterium in the infant gut. One species, Bifidobacterium pseudocatenulatum , is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B. pseudocatenulatum strains were isolated and characterized to identify genetic adaptations to the breastfed infant gut. During growth on pooled human milk oligosaccharides (HMOs) we observed two distinct groups of B. pseudocatenulatum , isolates that readily consumed HMOs and those that did not, a difference driven by variable catabolism of fucosylated HMOs. A conserved gene cluster for fucosylated HMO utilization was identified in several sequenced B. pseudocatenulatum strains. One isolate, B. pseudocatenulatum MP80, which uniquely possessed GH95 and GH29 α-fucosidases consumed the majority of fucosylated HMOs tested. Furthermore, B. pseudocatenulatum SC585, which possesses only a single GH95 α-fucosidase, lacked the ability to consume the complete repertoire of linkages within the fucosylated HMO pool. Analysis of the purified GH29 and GH95 fucosidase activities directly on HMOs revealed complementing enzyme specificities with the GH95 enzyme preferring 1-2 fucosyl linkages and the GH29 enzyme favoring 1-3 and 1-4 linkages. The HMO binding specificity of the Family 1 solute binding protein component linked to the fucosylated HMO gene cluster in both SC585 and MP80 are similar, suggesting differential transport of fucosylated HMO is not a driving factor in each strain’s distinct HMO consumption pattern. Taken together, this data indicates the presence or absence of specific α-fucosidases directs the strain-specific fucosylated HMO utilization pattern among bifidobacteria and likely influences competitive behavior for HMO foraging in situ . IMPORTANCE Often isolated from the human gut, microbes from the bacterial family Bifidobacteriaceae commonly possess genes enabling carbohydrate utilization. Isolates from breast fed infants often grow on and possess genes for the catabolism of human milk oligosaccharides (HMOs), glycans found in human breast milk. However, catabolism of structurally diverse HMOs differs between bifidobacterial strains. This study identifies gene differences between Bifidobacterium pseudocatenulatum isolates that may impact whether a microbe successfully colonizes an infant gut. In this case, the presence of complementary α-fucosidases may provide an advantage to microbes seeking residence in the infant gut. Such knowledge furthers our understanding of how diet drives bacterial colonization of the infant gut.

Isolation and Characterization of Chickpea (Cicer arietinum L.) Nodulating Rhizobia Collected from South Wollo Zone, Ethiopia

This study aimed at screening rhizobial isolates of chickpea and evaluating their symbiotic nitrogen fixation efficiency and tolerance to abiotic stresses. A total of 107 (100%) isolates were collected of which 52 (48.6%) were confirmed as chickpea rhizobia using preliminary tests. Among 52 (48.6%) isolates, 46 (88.5%) have induced nodulation on chickpea under greenhouse and were screened under in vitro conditions and 6 (11.5%) of them were discarded due to fail to nodulate. The greenhouse data showed the highest nodule number (68.67 plant−1), nodule dry weight (0.17 g plant−1), and shoot dry weight (0.81 g plant−1) were scored by plants inoculated with isolates WUCR 17, WUCR 1, and WUCR 66, respectively. Among authenticated isolates, 73.9%, 21.7%, and 4.3% were highly effective, effective, and lowly effective, respectively. The physiological test showed 15% of isolates tolerated 13% salt concentration and 10.9% of isolates grew at pH 4–10 range. All isolates grew at 20–35°C and 13% grew at maximum temperature (50°C). The isolates showed better resistance to the tested antibiotics at low concentration (2.5 μg/ml) but the majorities were sensitive at high concentration (10 μg/ml). Among the isolates, 13% tolerated all tested heavy metals but 48% were sensitive to mercury. Regarding the carbohydrate utilization test, 52.2% catabolized all the tested 11 carbon sources but 6.5% of them utilized only 63.6% carbon sources. Amino acid utilization showed isolates (85%) utilized D-alanine except WUCR (14, 25, 31, 34, 39, 59, and 76) and 76% of the isolates utilized arginine and phenylalanine and 74% utilized leucine as a nitrogen source. Of the isolates, 43.5% of them utilized both D-alanine and arginine as a nitrogen source. In almost all tests conducted, isolates WUCR 1 and 5 performed top and were recommended as potential candidates for microbial inoculants.