Bacteria

Extremophilic microorganisms such as those that thrive in high-salt and high-alkaline environments are promising candidates for the recovery of useful biomaterials including polyhydroxyalkanoates (PHAs). PHAs are ideal alternatives to synthetic plastics because they are biodegradable, biocompatible, and environmentally friendly. This work was aimed at conducting a bioprospection of bacteria isolated from hypersaline-alkaliphilic lakes in Kenya for the potential production of PHAs. In the present study, 218 isolates were screened by Sudan Black B and Nile Red A staining. Of these isolates, 31 were positive for PHA production and were characterized using morphological, biochemical, and molecular methods. Through 16S rRNA sequencing, we found that the isolates belonged to the genera Arthrobacter spp., Bacillus spp., Exiguobacterium spp., Halomonas spp., Paracoccus spp., and Rhodobaca spp. Preliminary experiments revealed that Bacillus sp. JSM-1684023 isolated from Lake Magadi had the highest PHA accumulation ability, with an initial biomass-to-PHA conversion rate of 19.14% on a 2% glucose substrate. Under optimized fermentation conditions, MO22 had a maximum PHA concentration of 0.516 g/L from 1.99 g/L of cell dry weight and 25.9% PHA conversion, equivalent to a PHA yield of 0.02 g/g of biomass. The optimal PHA production media had an initial pH of 9.0, temperature of 35 °C, salinity of 3%, and an incubation period of 48 h with 2.5% sucrose and 0.1% peptone as carbon and nitrogen sources, respectively. This study suggests that bacteria isolated from hypersaline and alkaliphilic tropical lakes are promising candidates for the production of polyhydroxyalkanoates. Full article

The establishment of Permanent Preservation Plots (PPPs) in natural forests has a signifi-cant role in assessing the impact of climate change on forests. To pursue long-term studies on cli-mate change, PPPs were established during the year 2016 in two major forest areas in Bangalore to conduct ecological studies to monitor the vegetation changes. One of the objectives of the study was to understand the drivers of diversity, such as soils, in terms of nutrients and physical and biological properties. The native tropical forest of Bangalore, which houses Bannerghatta National Park (BNP) on the outskirts, is relatively underexplored in terms of its microflora, particularly arbuscular my-corrhizal fungi (AMF). Hence, the present study was aimed at the quantitative estimation of arbus-cular mycorrhizal fungi (AMF) in the three 1-ha PPPs which were established in Bannerughatta National Park (BNP) and Doresanipalya Reserve Forest (DRF) as per the Centre for Tropical Forest Sciences (CTFS) protocol. In BNP, two plots were established, one in the Thalewood house area (mixed, moist, deciduous type) and the other in the Bugurikallu area (dry, deciduous type). In DRF, one plot was established in dry, deciduous vegetation. Each one-hectare plot (100 m × 100 m) was subdivided into twenty-five sub-plots (20 m × 20 m). Composite soil samples were collected during two seasons (dry and wet) and analyzed for AMF spore and available phosphorus (P) content. The results revealed the presence of AMF in all the three plots. Doresanipalya plo had the highest spore number, followed by the Bugurikallu plot and Thalewood house plot. The available phosphorous and AMF spore numbers showed correlations in all the three plots. Among the AMF spores, the Glomus species was found to dominate in all the three plots. The study shows that the dry, decidu-ous forests accommodated more AMF spores than the mixed, moist forests. Full article

Antimicrobial resistance (AMR) is a global public health threat. Quality data on AMR are needed to tackle the rise of multidrug-resistant clones. These data are rare in low-income countries, especially in sub-Saharan Africa. In this study, we investigated the rise of extended-spectrum β-lactamase–producing (ESBL) Enterobacteriaceae in Bangui, Central African Republic. We collected 278 fecal samples from 0–5-year-old children admitted to the Pediatric University Hospital Complex in Bangui from July to September 2021. Enterobacteriaceae were isolated and identified, and their susceptibility to 19 antibiotics was tested. We recovered one and two Enterobacteriaceae species from 208 and 29 samples, respectively. One clone of each species from each sample was further characterized, for a total of 266 isolates. Escherichia coli predominated, followed by Klebsiella. AMR was frequent, with 98.5% (262/266) of the isolates resistant to at least one antibiotic. Additionally, 89.5% (238/266) of the isolates were multidrug resistant, with resistance being frequent against all tested antibiotics except carbapenems and tigecycline, for which no resistance was found. Importantly, 71.2% (198/278) of the children carried at least one ESBL species, and 85.3% (227/266) of the isolates displayed this phenotype. This study confirms the rise of ESBL Enterobacteriaceae in Bangui and stresses the need for action to preserve the efficacy of antibiotics, as crucial for the treatment of bacterial infections. Full article

Traditional preparation of African indigenous vegetables (AIVs) such as African black nightshade (Solanum nigrum) and African spiderplant (Cleome gynandra) involves either boiling and discarding the first water or lengthy boiling. Fermentation is considered a better alternative processing technique due to the enhanced retention of phytochemical contents and sensory properties. However, little is known about the impact of lactic acid fermentation on the phytochemical content, antioxidant capacity, sensory acceptability and microbial safety of the African black nightshade and African spiderplant. This study aimed to ferment AIVs using combined starter cultures (Lactobacillus fermentum and Lactococcus lactis) and further determine their effect on the phytochemical content (phenolic compounds and flavonoids), antioxidant capacity, sensory acceptability and microbial safety of the vegetables. There was a marked increase in phenol and flavonoid contents in all fermented vegetables (p < 0.05). The highest phenol content was 228.8 mg/g GAE (gallic acid equivalent) in the starter-culture-inoculated African black nightshade, while flavonoid content was 10.6 mg/g QE (quercetin equivalent) in the same. Starter-culture-inoculated AIVs presented significantly higher antioxidant capacity with a 60–80% radical scavenging activity compared to levels in uninoculated batches (p < 0.05). Fermented vegetables were more liked than the boiled vegetables and were microbiologically safe. In conclusion, lactic fermentation of AIVs increased phytochemical contents (phenolic compounds and flavonoids), maintained antioxidant capacity and improved product safety and sensory acceptability. Therefore, fermentation and consumption of the African indigenous vegetables are to be encouraged. Full article

Salmonella enterica is the principal causative agent of salmonellosis, a threat to human health. Because of its high antimicrobial resistance potential, Salmonella enterica has become worrisome, mostly in developing countries where hygiene and antimicrobial usage are defective. This study aimed to determine the epidemiology of the intestinal carriage of Extended Spectrum β-Lactamase producing Salmonella enterica from chickens and poultry farmers in Dschang, a town in the western region of Cameroon. A total of 416 chickens and 72 farmers were sampled between May and October 2020; and Salmonella enterica were isolated and subjected to extended spectrum β-lactamase screening. Logistic regression was used to test for statistical associations using a p-value of ≤0.05. Results from this study revealed that the prevalence of the intestinal carriage of Salmonella enterica for chickens and farmers were 55.77% [51.00; 60.54] and 22.22% [12.62; 31.82], respectively. Meanwhile, the intestinal carriage of Extended Spectrum β-Lactamase producing Salmonella enterica was 23.08% [13.76; 32.40] and 5.55% [0.26; 10.84] from chickens and poultry farmers, respectively. The risk factor for this carriage was revealed to be lack of knowledge by actors in livestock industries of antibiotic resistance. Chickens, just like poultry farmers, represent the starting point of community salmonellosis, which is difficult to cure; therefore, sensitization of breeders is an effective tool for the mitigation of this burden. Full article

Bacterial proteases participate in the proteolytic elimination of misfolded or aggregated proteins, carried out by members of the AAA+ protein superfamily such as Hsp100/Clp, Lon, and FtsH. It is estimated that the Clp and Lon families perform around 80% of cellular proteolysis in bacteria. These functions are regulated, in part, through the spatial and/or temporal use of adapter proteins, which participate in the recognition and delivery of specific substrate proteins to proteases. The proteolysis plays an important role in maintaining and controlling the quality of the proteins, avoiding the accumulation and aggregation of unfolded or truncated proteins. However, this is not their only function, since they play an important role in the formation of virulent phenotypes and in the response to different types of stress faced when entering the host or that occur in the environment. This review summarizes the structural and functional aspects of the Clp proteases and their role in Gram-positive microorganisms. Full article

African indigenous leafy vegetables (AILVs) are plants that have been part of the food systems in Sub-Saharan Africa (SSA) for a long time and their leaves, young shoots, flowers, fruits and seeds, stems, tubers, and roots are consumed. These vegetables are high in vitamins, minerals, protein, and secondary metabolites that promote health. This study aimed at isolating, characterizing, and identifying dominant lactic acid bacteria (LAB) from naturally fermenting commonly consumed AILV in Kenya. A total of 57 LAB strains were isolated and identified based on phenotypic and 16S rRNA gene analyses from three AILVs (23 nightshade leaves, 19 cowpeas leaves, and 15 vegetable amaranth). The highest microbial counts were recorded between 48 h and 96 h of fermentation in all AILVs ranging from approximately log 8 to log 9 CFU/mL with an average pH of 3.7. Fermentation of AILVs was dominated by twenty eight Lactobacillus spp. [Lactiplantibacillus plantarum (22), Limosilactobacillus fermentum (3), Lactiplantibacillus pentosus (2) and Lactiplantibacillus casei (1)], eleven Weissella spp. (Weissella cibaria (8), W. confusa (2), and W. muntiaci) six Leuconostoc spp. [Leuconostoc mesenteroides (3), Leuc. citreum (2) and Leuc. lactis (1)], six Pediococcus pentosaceus, four Enterococcus spp. [Enterococcus mundtii (2), E. faecalis (1) and E. durans (1)] and, finally, two Lactococcus garvieae. These bacteria strains are commonly used in food fermentation as starter cultures and as potential probiotics. Full article

Septicaemia is public health problem worldwide with a high rate of mortality among children. Epidemiological data on this phenomenon in Cameroon are still scarce. This study aimed to determine the prevalence and associated factors to septicaemia due to E. coli strains producing extended spectrum beta-lactamase (ESBL) in two hospitals in Yaoundé, Cameroon. A prospective, cross-sectional study was conducted on infants aged 0 to 2 years old at the consultation and neonatology care unit of two district hospitals of Yaoundé (UTHY and YGOPH) during a period of seven months (from August 2019 to March 2020). Each blood sample collected per infant was cultured in hemoline performance vials, and bacterial strains were identified using the Api-20 E system. In addition, an antibiotic resistant profile of isolates as well as the ESBL production were performed in accordance with the recommendations of the Antibiogram committee of the French Society of Microbiology 2019. Data were analysed in Epi-Info7.0 and for p less than 0.05, the difference was statistically significant. Of the 300 children enrolled, 130 (43.33%) were blood culture positive, and E. coli. was the most prevalent (69.23% (90/130)). Then antibiotic susceptibility test revealed that 77 over 90 E. coli strains were resistant to penicillin (with 85.55% to amoxicillin), and 34.44% were producing ESBL. Factors such as immunodeficiency, being on antibiotics, and particularly taking β-lactam were significantly associated with E. coli ESBL production ([aOR = 19.93; p = 0.0001], [aOR = 1.97; p = 0.04], and [aOR = 3.54; p = 0.01], respectively). Moreover, co-resistance to aminoglycosides, quinolones, fluoroquinolones, and cotrimoxazole were also found. This study highlighted a high prevalence of E. coli ESBL in blood samples of children aged 0–2 years in Yaoundé and prompts the development of more efficient strategies against E. coli ESBL associated mortality in infants in Cameroon. Full article

The common bean (Phaseolus vulgaris L.) is a significant vegetable crop, grown because it is a rich source of protein, carbohydrates, and vitamin B complex. Fusarium solani and Rhizoctonia solani are the most widely known pathogens contributing to large yield losses for this crop. The use of cultural and chemical control practices has been ineffective. Therefore, a sustainable, affordable, and effective control method is urgently required. In this study, we aimed to isolate and characterize Bacillus velezensis from Lake Bogoria as a potential biocontrol agent for Fusarium solani. Bacteria were isolated from soil and sediments using the serial dilution technique. Molecular characterization was performed using the 16S rRNA gene. A total of 13 bacteria were isolated from soil and sediments. Based on the partial sequences, BLAST analysis showed two isolates, B20 (Bacillus velezensis strain QH03-23) and B30 (Bacillus velezensis strain JS39D), belonging to Bacillus velezensis. Other isolates were identified as Bacillus tequilensis, Brevibacillus brevis, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis. The effectiveness of their antifungal properties was determined via co-culturing, and we found mycelial inhibition rates of 28.17% (for B20) and 33.33% (for B30) for the Fusarium solani isolates. The characterization of the Bacillus velezensis strain revealed that they were Gram-positive and grew well at pH 7.0 and 8.5, although growth was recorded at pH 5.0 and 10.0. In terms of temperature, the optimal temperature conditions were 30−35 °C, with an optimum salinity of 0–0.5 M NaCl. When these isolates were tested for their ability to produce secondary metabolites, they were found to produce phosphate, pectinase, chitinase, protease, indole -3- acetic acid (IAA), and hydrogen cyanide (HCD), making them potential biocontrol agents. Full article

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative bacteria that affects patients in intensive care units and chronic respiratory disease patients. Compared to other bacteria, it has a wide genome (around 6.3-Mb) that supports its metabolic versatility and antimicrobial resistance. Fosfomycin (FF) is primarily used as an oral treatment for urinary tract infections (UTIs). FF diffuses inside the cell via glycerol-3-phosphate transporter (GlpT) PA, as well as in other bacteria. In other bacteria, such as E. coli, glucose-6-phosphate transporter (UhpT) functions as FF transporter. Since mutant GlpT leads to FF resistant PA, it is assumed that GlpT is the only FF transporter. However, it is also assumed that PA uses glucose-6-phosphate and, thus, homologous proteins of UhpT may be present in its genome. Here, we present an attempt to find a distant related homologue of UhpT in PA. A Hidden Markov Model (HMM) was created to seek for Major facilitator family (MFS) domain in 21 PA genomes of 14 CF patients annotated with prokka and the statistical analysis was performed (MCC: 0.84, ACC: 0.99). Then, the HMM was applied to PA genomes. Besides the actual GlpT, annotated as glpt_1, one more GlpT protein was found in 21 out of 21 genomes, annotated as glpt_2. Since glpt_2 clusters closer to UhpT than GlpT, glpt_2 was selected to build a model. Computing a structural superimposition, the model and the template of UhpT have 0.6 Å of RMSD. The model of glpt_2 has some characteristics that are fundamental to UhpT functions. The binding site, consisting of 2 arginines (Arg46 and Arg275) and Lys45, is totally conserved, as well as the topology of the structure. Asp90 is also conserved in glpt_2 model. No studies aimed at searching for distant related homologous of UhpT. Since the high genetic exchange and high mutational rate in bacteria, it is likely that PA has a UhpT-like protein in the PA genome. The binding site is superimposable to UhpT protein as well as the overall topology. In fact, the 12 TMs are completely comparable, suggesting a well-defined folding of the protein across the bilayer lipid membrane. To enforce our hypothesis, in all 21 PA genomes, we also found a protein annotated as membrane sensor protein UhpC, important for expression and function of UhpT in E. coli. Since PA strains are wild-type, we can assume that most of the PA have proteins like this. The presence of a homologue of UhpT suggests that this protein is conserved in PA genome. Full article

The human gastrointestinal (GI) tract contains a diverse mixture of commensal and pathogenic microbes, forming the gut microbiome. These gut microbes and their potential to improve human health are a topic of great interest to the scientific community. Many intestinal and age-related complications are linked to dysbiosis of the gut microbiome, often associated with a weakened immune system. A decrease in beneficial microbes, generally, along with decreased microbial diversity in the gut, can, in many cases, result in disease, particularly in older individuals. Probiotics, which are ingestible beneficial microorganisms, have the potential to positively modulate the indigenous gut microbiota. There are two predominant and conventional classes of lactic acid bacterial probiotics, lactobacilli and bifidobacteria, which have been confirmed for their health benefits and role in preventing certain gut-related disorders. The proper use of probiotics and/or supplements, along with a consistently healthy lifestyle, is a promising holistic approach to maintaining or improving gut health and minimizing other age-linked disorders. There are many properties that bacterial probiotics possess, which may allow for these beneficial effects in the gut. For instance, probiotics have adhesion capacities (capability to stay in GI tract) that are effective in excluding pathogens, while other probiotics have the potential to stimulate or modulate the intestinal immune system by regulating genes that reside within and outside of the gut environment. This review discussed the possible underlying mechanics of probiotics, evidence of probiotic-based mitigation of age-related disease, and the role of probiotics in modulating gut health and, in turn, maintaining brain health. Full article

Tularemia is a severe infectious disease caused by the Gram-negative bacteria Francisella tularensis. F. tularensis is currently divided into three subspecies, holarctica, tularensis, and mediasiatica, which differ in their virulence and geographic distribution. Subspecies mediasiatica is the least studied because of its very low documented virulence for humans and limited geographic distribution. It was discovered in sparsely populated regions of Central Asia. Since 2011, a new subsp. mediasiatica lineage was identified in Altai (Russia). In 2021, we isolated one subsp. mediasiatica strain in Krasnoyarsk Territory. In spite of its geographic origin, 500 km east from Altai, this strain belongs to the Altai lineage and contributes surprisingly little genetic diversity to previous knowledge. Full article

Xanthomonas bacteria are known phytopathogens difficult to control in the field, which cause great losses in many economically important crops. Genomic islands are fragments acquired by horizontal transference that are important for evolution and adaptation to diverse ecological niches. Virulence and pathogenicity islands (PAIs) enhance molecular mechanisms related to host adaptation. In this work, we have analyzed 81 genomes belonging to X. campestris, and a complex group of X. citri, X. axonopodis, and X. fuscans belonging to nine different pathovars and three subspecies, to analyze and compare their genomic contents. Xanthomonas pan-genome is open and has a massive accessory genome. Each genome showed between three and 15 exclusive PAIs, well conserved through strains of the same pathovar or subspecies. X. axonopodis pv. anacardii had higher general similarity to X. citri subsp. citri and X. fuscans subsp. aurantifolii, with which a few PAIs were shared. Genomic synteny was even for almost all strains, with few rearrangements found in X. axonopodis pv. anacardii. The prophage regions identified in the genomes were mostly questionable or incomplete, and PAI13 in X. campestris pv. campestris ATCC33913 matched a prophage region of 19 transposable elements. Finally, PAIs in Xanthomonas are pathovar-specific, requiring individual strategies of combat. Full article

Angular leaf spot of cashew tree in Brazil has been attributed to pigmented and nonpigmented strains of Xanthomonas citri pv. anacardii. Due to the possibility of dissemination of the disease by propagating material, it is necessary to understand the survival mechanisms of the causal agent. Thus, the present study aimed to characterize the behavior of viable but non-cultivable cells (VBNC) in two pigmented strains (CCRMTAQ13 and CCRMTAQ18) and one nonpigmented strain (IBSBF2579) of X. citri pv. anacardii, integrating in silico, in vitro, and in vivo studies. Thirteen genes associated with the VBNC phase were identified in the genomes of these strains. The log phase was observed at 24, 48, and 120 h for CCRMTAQ13, CCRMTAQ18, and IBSBF2579 strains, respectively. The death phase was observed at 96 h for both pigmented strains and 168 h for the nonpigmented strain. Using qPCR analyses, it was possible to characterize the occurrence of VBNC for the three strains. When inoculated, the strains showed 100% incidence during the VBNC phase, with the IBSBF2579 strain having the longest incubation period (IP). The strains did not differ concerning final severity (FS) in the VBNC phase. To our knowledge, this is the first report of the occurrence of the VBNC mechanism in X. citri pv. anacardii strains. Furthermore, it has been demonstrated that X. citri pv. anacardii in the VBNC state is potentially infective when they meet their host’s apoplast, which points to the need to use integrated practices to detect this bacterium in cashew seedlings. Full article

BF2P4-5 was isolated from the rhizosphere soil of tomato plants, and its potential to promote plant growth was investigated in tomato plants. An in vitro test revealed that the strain could fix nitrogen, solubilize phosphate and potassium, and synthesize indole acetic acid. The bacterial strain was identified and characterized as a kind of Pseudarthrobacter chlorophenolicus based on the analysis of culture characteristics, physiological and biochemical characteristics, and 16S rRNA gene sequence (GenBank accession number OP135548.1). pH 7.0, 15% NaCl, and 35 °C temperature were ideal for optimal strain growth under culture conditions. Tomato plants grown on a cocopeat substrate were inoculated with BF2P4-5 suspension (OD₆₀₀ 2.0). Positive control plants were inoculated with Nitrogen Phosphorus Potassium (NPK) fertilizer. This BF2P4-5 strain and NPK treatments were complemented with a negative control, in which only tap water was applied to tomato roots, thus, establishing three distinct treatment modalities with five replications each. Two months of greenhouse trials of inoculated tomato plants improved growth parameters. Interestingly, in most of the growth metrics evaluated, tomato plants treated with strain BF2P4-5 showed little to no variation with NPK fertilizer treatment, including plant height, stem length, girth, leaf number per plant, and chlorophyll content, when compared to uninoculated plants. Furthermore, the conditions for the cocopeat plants, including pH, EC, and moisture, were within acceptable limits. Furthermore, inoculation with BF2P4-5 increased the nitrogen, phosphorus, and potassium content available in the cocopeat medium. The results showed that the strain exhibited traits for the promotion of plant growth and could be deployed as an eco-friendly microbial biofertilizer for tomatoes and probably other essential crops. Full article

In this study, we used the sequence of the aac(6′)-Ii gene, which is responsible for the intrinsic low-level aminoglycoside resistance of Enterococcus faecium, to design novel species-specific primers. Two oligonucleotide pairs named EF_200 and EF_120 were designed, generating amplification products with sizes of 200 bp and 120 bp, respectively. They were successfully applied for the identification of various isolates of clinical or environmental origins in both pure cultures and complex food samples. The obtained results indicated that both primer pairs permitted the highly specific, simple, fast and inexpensive detection of E. faecium isolates. Full article

Bifidobacterium breve is among the first microorganisms colonizing the intestinal tract in humans and is a predominant species in the gut microbiota of newborns and children. This bacterium is widely used in the probiotic industry due to its capacity to improve host health. The search for new targets with probiotic properties is an increasing trend with the help of next-generation sequencing as they facilitate the characterization of the bacterial features. B. breve 110^1A was isolated from the faeces of healthy children in Brazil and therefore could play a protective role in the gut. To investigate the beneficial properties of this strain, the present study performed a comprehensive characterization of the genetic features involved in the bacterium resistance and adaptation to gastrointestinal conditions, production of nutrients, and immunomodulatory compounds. Furthermore, this study carried out the prediction of genomic elements (plasmids, prophages, CRISPR-Cas systems, insertion sequences, genomic islands, antibiotic resistance genes) to evaluate the safety of B. breve 110^1A. A comparative genomics approach using 45 B. breve complete genomes based on pangenome and phylogenomic analysis was also performed to identify specific genes in B. breve 110^1A. The prediction of genetic elements, possibly safety-related, did not detect plasmids, but only one incomplete prophage, two non-functional CRISPR systems, and seven genomic islands. Additionally, three antibiotic resistance genes were identified: ileS (resistance to mupirocin), rpoB, and erm(X). In the comparative genomic analysis, the pangenome was revealed to be open, and B. breve 110^1A presented 63 unique genes associated with several processes, such as transmembrane transport, membrane components, DNA processes, and carbohydrate metabolism. In conclusion, B. breve 110^1A is potentially safe and well-adapted for intestinal disorder therapeutics, although the role of its unique genetic repertoire needs further investigation. Full article

Lactiplantibacillus plantarum strains are used in the food industry for their probiotic properties. Some of these bacteria have immunomodulatory effects on the host and are able to improve resistance against different pathogens, including viruses. However, to date, the bacterial genes involved in the immunomodulatory effect are not known. In this work, the complete genomes of L. plantarum MPL16, CRL1506, CRL681 and TL2766 were used to perform comparative genomics with the aim of identifying the genes involved in their differential immunomodulatory effects. L. plantarum WCFS1, a strain with proven probiotic activity, was also used for comparisons. The analysis of the genes involved in the metabolic pathways of the five strains did not reveal differences in the metabolism of amino acids, lipids, nucleotides, cofactors and vitamins, nor in the genes associated with energy metabolism or the biosynthesis of lipoproteins and teichoic acids. However, differences were found between the five strains when considering carbohydrate metabolism pathways, particularly in the presence/absence of glycosylhydrolases and glycosyltransferases. In addition, a great variability was detected in the predicted surface proteins of each L. plantarum strain. These results suggest that the surface molecules expressed in the different strains of L. plantarum could be involved in their differential ability to modulate the innate antiviral immune response. Full article

Gram-positive bacteria are some of the earliest known life forms, diverging from gram-negative bacteria 2 billion years ago. These organisms utilize sortase enzymes to attach proteins to their peptidoglycan cell wall, a structural feature that distinguishes the two types of bacteria. The transpeptidase activity of sortases make them an important tool in protein engineering applications, e.g., in sortase-mediated ligations or sortagging. However, due to relatively low catalytic efficiency, there are ongoing efforts to create better sortase variants for these uses. Here, we use bioinformatics tools, principal component analysis and ancestral sequence reconstruction, in combination with protein biochemistry, to analyze natural sequence variation in these enzymes. Principal component analysis on the sortase superfamily distinguishes previously described classes and identifies regions of relatively high sequence variation in structurally-conserved loops within each sortase family, including those near the active site. Using ancestral sequence reconstruction, we determined sequences of ancestral Staphylococcus and Streptococcus Class A sortase proteins. Enzyme assays revealed that the ancestral Streptococcus enzyme is relatively active and shares similar sequence variation with other Class A Streptococcus sortases. Taken together, we highlight how natural sequence variation can be utilized to investigate this important protein family, arguing that these and similar techniques may be used to discover or design sortases with increased catalytic efficiency and/or selectivity for sortase-mediated ligation experiments. Full article