speB Gene Research Articles

Efficient production of spermidine from Bacillus amyloliquefaciens by enhancing synthesis pathway, blocking degradation pathway and increasing precursor supply.

Spermidine has broad application potential in food, medicine and other fields. In this study, a novel Bacillus amyloliquefaciens cell factory was constructed for production of spermidine from renewable biomass resources. Firstly, the speB gene was found to be optimal for synthesis of spermidine, and the function of SpeB was explained by amino acid sequence analysis and molecular docking. By replacing the native promoter of the speEB operon with the P43, the synthesis of spermidine was significantly enhanced in B. amyloliquefaciens HSPM1-P43speEB. After knockout of the genes yobN and bltD associated with spermidine degradation, the spermidine titer of the strain HSPM2 was further improved to 115.96mg/L, increased by 108% compared to HSPM1-P43speEB. Subsequently, the titer of spermidine was further increased to 277.47mg/L through enhancing the supply of the precursor methionine by overexpression of speD. Finally, the renewable biomass resources, xylose and feather meal were optimized to produce spermidine, and the maximum titer is up to 588.10mg/L after optimization. In conclusion, an efficient spermidine producing B. amyloliquefaciens was constructed through combinatorial metabolic engineering strategies, and the sustainable production of spermidine was achieved using the biomass resources of xylose and feather meal.

Group A Streptococcus pyogenes in wastewater: Applicability of wastewater-based epidemiology for monitoring the prevalence of GAS pharyngitis during the late COVID-19 pandemic phase

Streptococcus pyogenes, Group A Streptococcus (GAS), is a human pathogen that causes a spectrum of diseases from mild to severe, including GAS pharyngitis, a common acute respiratory disease in developed countries. Although wastewater-based epidemiology (WBE) has been extensively used to monitor viral pathogens such as severe acute respiratory syndrome coronavirus 2, its applicability to S. pyogenes remains unexplored. This study was conducted to investigate the feasibility of detecting and quantifying S. pyogenes in wastewater by quantitative polymerase chain reaction (qPCR) and evaluate the applicability of WBE for monitoring the prevalence of GAS pharyngitis. A total of 52 grab influent samples were collected from a wastewater treatment plant in Japan once a week between March 2023 and February 2024. The samples were centrifuged, followed by nucleic acid extraction and qPCR for the S. pyogenes-specific genes speB and spy1258. Of the 52 samples, 90 % and 81 % were positive for speB and spy1258 genes, respectively, indicating the feasibility of S. pyogenes for wastewater surveillance. However, the percentage of quantifiable samples for speB gene was significantly higher in winter than in spring and summer. Similarly, the concentrations of both genes in wastewater samples were significantly higher in winter (speB, 4.1 ± 0.27 log10 copies/L; spy1258, 4.1 ± 0.28 log10 copies/L; One-way ANOVA, p < 0.01) than in spring and summer. Higher concentrations and detection ratios of S. pyogenes genes were observed during increased GAS pharyngitis cases in the catchment. Significant moderate correlations were observed between target gene concentrations and reported GAS pharyngitis cases. This study enhances the understanding role of WBE in monitoring and managing infectious diseases within communities.

Fabrication of a Microfluidic Test Device with a 3D Printer and Its Combination with the Loop Mediated Isothermal Amplification Method to Detect Streptococcus pyogenes.

New rapid, reliable, and cost-effective alternative systems are needed for the rapid diagnosis of Streptococcus pyogenes. The aim of this study was to fabricate a microfluidic test device to detect Streptococcus pyogenes by combining the Loop-mediated isothermal amplification method via a 3D printer. Microfluidic test devices were designed in CATIA V5 Release 16 software, and data were directly transferred to a 3D printer and produced using the FDM method with biocompatible PLA filament. The S. pyogenes ATCC 19615 and different ATCC strains was used. Following identification by classical culture methods, a 0.5 McFarland suspension was prepared from the colonies, and DNA isolation was performed from this liquid by a boiling method. S. pyogenes specific speB gene was used to desing LAMP primer sets in PrimerExplorer V5 software and tested on a microfluidic device. LAMP reactions were performed on microfluidic device and on a microcentrifuge tube separately. Both results were analyzed using the culture method as the standard method to diagnostic values. Melting curve analysis of the amplicons of the LAMP reactions performed on a LightCycler 480 system to detect amplification. Among the 50 positive and 100 negative samples, only four samples were found to be false negative by LAMP reaction in a microcentrifuge tube, while eight samples were found to be false negative by LAMP reaction on a microfluidic device. Six samples were found to be false positive by the LAMP reaction in the microcentrifuge tube, while ten samples were found to be false positive by the LAMP reaction on a microfluidic chip. The sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP reactions performed in the microcentrifuge tube and on the microfluidic device were 92-84%, 94-90%, 88.46-80.77%, and 95.92-91.84%, respectively. The limit of detection (LOD) was found to be the same as 1.5 × 102 CFU/mL and the limit of quantification (LOQ) values of the LAMP reactions were performed on the microcentrifuge tube and on the microfluidic device were 2.46 × 102-7.4 × 102 CFU/mL, respectively. Cohen's kappa (κ) values of the LAMP reactions were performed on the microcentrifuge tube and on the microfluidic device were 0.620-0.705, respectively. In conclusion, our data showed that the LAMP method can be combined with microfluidic test device to detect S. pyogenes, this microfluidic device can be manufactured using 3D printers and results are close to gold standard methods. These devices can be combined with LAMP reactions to detect different pathogens where resources are limited and rapid results are required.

Development of a multiple cross displacement amplification combined with nanoparticles-based biosensor assay for rapid and sensitive detection of Streptococcus pyogenes

BackgroundS. pyogenes, is a primary pathogen that leads to pharyngitis and can also trigger severe conditions like necrotizing fasciitis and streptococcal toxic shock syndrome (STSS), often resulting in high mortality rates. Therefore, prompt identification and appropriate treatment of S. pyogenes infections are crucial in preventing the worsening of symptoms and alleviating the disease's impact. ResultsIn this study, a newly developed technique called multiple cross displacement amplification (MCDA) was employed to detect S. pyogenes,specifically targeting the speB gene, at a temperature of 63°C within 30 min. Then, an easily portable and user-friendly nanoparticles-based lateral flow biosensor (LFB) assay was introduced for the rapid analysis of MCDA products in just 2 min. The results indicated that the LFB offers greater objectivity compared to Malachite Green and is simpler than electrophoresis. The MCDA-LFB assay boasts a low detection limit of 200 fg and exhibits no cross-reaction with non-S. pyogenes strains. Among 230 clinical swab throat samples, the MCDA-LFB method identified 27 specimens as positive, demonstrating higher sensitivity compared to 23 samples detected positive by qPCR assay and 18 samples by culture. The only equipment needed for this assay is a portable dry block heater. Moreover, each MCDA-LFB test is cost-effective, priced at approximately $US 5.5.ConclusionThe MCDA-LFB assay emerges as a straightforward, specific, sensitive, portable, and user-friendly method for the rapid diagnosis of S. pyogenes in clinical samples.

Molecular Genetic Characterization of Streptococcus pyogenes Strains Isolated from Patients with Various Manifestations of Streptococcal Infection.

In 82 clinical strains of Streptococcus pyogenes (group A streptococci) isolated from patients with various manifestations of streptococcal infection, emm-typing revealed 27 emm-types (n=77) with a predominance of emm-89 (n=15; 18%), emm-75 (n=9; 11%), and emm-1 (n=6; 7%); types emm-3, emm-12, and emm-58 (n=4; 5% each) were found with almost equal frequency; other types were less common. The superantigen genes speC, speG, speH, speI, speJ, speK, speL, speM, smeZ, and SSA were identified in S. pyogenes strains using multiprimer PCR; the genes of the superantigen SpeA and cysteine proteinase SpeB were detected using real-time PCR. All the studied S. pyogenes strains contained superantigen genes, and 98% of the strains had several (from 2 to 7) genes. The number of variants of these sets reached 37; 2% of the strains contained only one superantigen gene. The distribution frequencies of superantigen genes in the studied strains were: speA - 43%; speC - 38%; speG - 93%; speH - 13%; speI - 6%; speJ - 24%; speK - 13%; speL and speM - 11% each; smeZ - 98%; SSA - 15%. All studied S. pyogenes strains contained the speB gene. Our studies have demonstrated that the sets of superantigen genes of group A streptococci are characterized by pronounced diversity to some extent associated with emm-type.

A new metabolic pathway for sym-homospermidine synthesis in an extreme thermophile, Thermus thermophilus.

In an extreme thermophile, Thermus thermophilus, sym-homospermidine is synthesized by the actions of two enzymes. The first enzyme coded by dhs gene (annotated to be deoxyhypusine synthase gene) catalyzes synthesis of an intermediate, supposed to be 1,9-bis(guanidino)-5-aza-nonane (=N1, N11-bis(amidino)-sym-homospermidine), from two molecules of agmatine in the presence of NAD. The second enzyme (aminopropylagmatinase) coded by speB gene catalyzes hydrolysis of the intermediate compound to sym-homospermidine releasing two molecules of urea.

Alkaline Stress Causes Changes in Polyamine Biosynthesis in Thermus thermophilus.

An extreme thermophile, Thermus thermophilus, produces 16 different polyamines including long-chain and branched-chain polyamines. The composition and content of polyamines in the thermophile cells change not only with growth temperature but also with pH changes. In particular, cell growth decreased greatly at alkaline medium together with significant changes in the composition and content of polyamines. The amounts of tetraamines (spermine and its homologs) markedly decreased at alkaline pH. Thus, we knocked out the speE gene, which is involved in the biosynthesis of tetraamines, and changes of composition of polyamines with pH changes in the mutant cells were studied. Cell growth in the ΔspeE strain was decreased compared with that of the wild-type strain for all pHs, suggesting that tetraamines are important for cell proliferation. Interestingly, the amount of spermidine decreased and that of putrescine increased in wild-type cells at elevated pH, although T. thermophilus lacks a putrescine synthesizing pathway. In addition, polyamines possessing a diaminobutane moiety, such as spermine, decreased greatly at high pH. We assessed whether the speB gene encoding aminopropylagmatine ureohydrolase (TtSpeB) is directly involved in the synthesis of putrescine. The catalytic assay of the purified enzyme indicated that TtSpeB accepts agmatine as its substrate and produces putrescine due to the change in substrate specificity at high pH. These results suggest that pH stress was exacerbated upon intracellular depletion of polyamines possessing a diaminobutane moiety induced by unusual changes in polyamine biosynthesis under high pH conditions.

Assessment of the Relationship between Clinical Manifestation and Pathogenic Potential of Streptococcus pyogenes Strains-Distribution of Genes and Genotypes of Toxins.

Streptococcus pyogenes is one of the most important species among beta-haemolytic streptococci, causing human infections of different localization. It is isolated from clinical specimens relatively frequently. In this study, the frequency and co-occurrence of toxin genes (speA, speB, speC, speH, speJ, speK) among 147 S. pyogenes strains were evaluated, using real-time PCR. In addition, the relationship between the occurrence of these genes and the origin of S. pyogenes strains from selected clinical material was assessed. The speB gene was present with the highest incidence (98.6%), while the speK gene was the least frequent (8.2%) among the tested strains. Based on the presence of the detected genes, the distribution of 17 genotypes was determined. The most common (21.8%), was speA (−) speB (+) speC (−) speH (−) speJ (−) speK (−) genotype. Furthermore, significant variation in the presence of some genes and genotypes of toxins in S. pyogenes strains isolated from different types of clinical material was found. There is a considerable variety and disproportion between the frequency of individual genes and genotypes of toxins in S. pyogenes strains. The relationship between the origin of S. pyogenes isolates and the presence of toxins genes indicates their pathogenic potential in the development of infections of selected localization.

Improved Visual Detection of speB Gene in Streptococcus pyogenes Isolates by Real-time Loop-Mediated Isothermal Amplification Turbidimetry Method

Background: Group A Streptococcus (GAS) causes a wide array of clinical manifestations ranging from mild pharyngitis to suppurative and non-suppurative severe debilitating diseases. Hence, a simple, rapid detection method with high sensitivity and specificity is needed. Objectives: This study embarked on the visual detection of the streptococcal pyrogenic exotoxin B (speB) gene by real-time turbidimetry and loop-mediated isothermal amplification (RT-LAMP) methods. The real-time monitoring of the sigmoidal graph generated from a turbidimetry method was incorporated in the assay. Methods: The amplification of the speB gene was virtually observed in real-time monitoring of the graph (sigmoidal curve) generated via a turbidimeter, thus providing a “guide” to accurately estimate the time to positivity for the gene detection. Results: The targeted gene was detected at 15 min but was optimally amplified within 45 min at an isothermal temperature of 63°C with 100% specificity using an established set of primers. The formation of sigmoidal curves was correlated with other visual observations by the naked eye (from orange to green), ultra-violet light (green fluorescence), and agarose gel electrophoresis. The improved detection limit of the real-time RT-LAMP assay was also observed compared to conventional PCR assay (0.001 pg/µL versus 1 ng/µL). Conclusions: The improved visual detection of RT-LAMP assay could provide additional insight for rapid, cost-effective, and reliable identification of GAS via speB gene detection in low or middle-income countries. It could also be a very important tool to improve the healthcare management of patients infected with GAS in the future.

STREPTOCOCCAL (GROUP A) INFECTION IN RUSSIA: STATE OF THE PROBLEM AND DEVELOPMENT TRENDS

Aim. To assess the current situation on streptococcal (group A) infection in Russia, to study the molecular properties and antimicrobial susceptibility of group A streptococcus isolated from patients with soft tissue infection.Materials and methods. We performed a descriptive epidemiological study using official statistics. A total of 97 cases of soft tissue infection caused by group A streptococci were investigated for emm-types, the presence of genes of bacteriophage toxins and integrases by PCR and sequencing. We tested 91 strains for antimicrobial susceptibility by the micro dilution methods.Results. From 2009 through 2017, 2.8 million cases (563 thousand primary cases) of group A streptococcal disease were reported. There was a decrease in the incidence of scarlet fever in Russia (31.5 per 100 000 population). In 2009–2017 the incidence of rheumatic fever and rheumatic heart diseases increase slightly but the prevalence of this forms group A streptococcal disease are decrease. Annually 2600 people die from the rheumatic fever and rheumatic heart diseases. Of the 97 cultures of group A streptococci, 33 were associated with invasive infection. We identified 33 different emm-type. All cultures contained speB gene. Some strains contained speA, and others speC genes. We did not find any correlation between the presence of bacteriophage toxin genes and the invasive properties of streptococci. Tetracycline and macrolides are ineffective in patients with of soft tissue infectionConclusion. Streptococcal (group A) infection continues to be of significant social and economic importance for Russia. The streptococcus cultures isolated from patients with invasive forms were heterogeneous in molecular and biological properties and remained sensitive to penicillin antibiotics.

Retraction: Molecular Cloning and Docking of speB Gene Encoding Cysteine Protease With Antibiotic Interaction in Streptococcus pyogenes NBMKU12 From the Clinical Isolates

[This retracts the article DOI: 10.3389/fmicb.2018.01658.].

Bacillus subtilis STU6 Ameliorates Iron Deficiency in Tomato by Enhancement of Polyamine-Mediated Iron Remobilization.

Iron (Fe) deficiency often triggers arginine overproduction in plants. However, it remains elusive whether Fe deficiency-induced increases of arginine levels are involved in beneficial rhizobacteria recruitment and that the mechanism underlying rhizobacteria induced plant Fe deficiency tolerance. Here, Bacillus subtilis STU6 increased soluble Fe content in tomato, thereby alleviating Fe deficiency-induced chlorosis. In a split-root system, STU6 significantly induced arginine exudation by Fe-deficient roots, and increased arginine levels promoted spermidine (Spd) production by STU6 and bacterial colonization. Deletion of the STU6 speB gene inhibited Spd synthesis and abrogated STU6-induced increments of soluble Fe content in the Fe-deficient plants. Increased host Spd levels by STU6 greatly stimulated the NO accumulation in the Fe-deficient roots. Furthermore, disruption of NO signaling markedly repressed STU6-mediated cell wall Fe remobilization. Collectively, our data provide important evidence that chemical dialogues between tomato and STU6 contribute to enhancement of microbe-mediated plant adaptation to Fe deficiency.

Metabolic engineering of Escherichia coli for agmatine production.

Agmatine is a kind of important biogenic amine. The chemical synthesis route is not a desirable choice for industrial production of agmatine. To date, there are no reports on the fermentative production of agmatine by microorganism. In this study, the base Escherichia coli strain AUX4 (JM109 ∆speC ∆speF ∆speB ∆argR) capable of excreting agmatine into the culture medium was first constructed by sequential deletions of the speC and speF genes encoding the ornithine decarboxylase isoenzymes, the speB gene encoding agmatine ureohydrolase and the regulation gene argR responsible for the negative control of the arg regulon. The speA gene encoding arginine decarboxylase harboured by the pKK223-3 plasmid was overexpressed in AUX4, resulting in the engineered strain AUX5. The batch and fed-batch fermentations of the AUX5 strain were conducted in a 3-L bioreactor, and the results showed that the AUX5 strain was able to produce 1.13g agmatine L-1 with the yield of 0.11g agmatine g-1 glucose in the batch fermentation and the fed-batch fermentation of AUX5 allowed the production of 15.32g agmatine L-1 with the productivity of 0.48g agmatine L-1 h-1, demonstrating the potential of E.coli as an industrial producer of agmatine.

Molecular Cloning and Docking of speB Gene Encoding Cysteine Protease With Antibiotic Interaction in Streptococcus pyogenes NBMKU12 From the Clinical Isolates.

Streptococcus pyogenes causes a variety of diseases ranging from mild diseases to severe invasive infections which result in significant morbidity and mortality. This study focuses on the antibiotic resistance of S. pyogenes and their interaction with cysteine protease. Around 36 beta-hemolytic isolates were collected from the clinical lab, of which seven isolates (19.4%) were identified as Streptococcus pyogenes. One of the seven isolates was collected from a urinary tract infection, which was identified by antibody agglutination and MALTI-TOF-MS, and it is designated as S. pyogenes NBMKU12. Around 8.3 to 66.6 % of the isolates were found to be resistant to one or more antimicrobial agents, especially, penicillin-G resistance was exhibited by 29.1% of the isolates. In the NBMKU12 isolate, the beta lactem (TEM) gene was detected among the 13 antibiotic genes for which it was tested. Furthermore, when analysis for presence of 13 virulence genes were carried out in NBMKU12 isolate, only speJ and speB were detected. The speB (streptococcal pyrogenic exotoxin B) encoding cysteine protease gene was cloned. This was followed by performing DNA sequencing to understand the putative cysteine protease interaction with antibiotics, inhibitors, and substrate. The speB gene consists of 1197 nucleotides and encodes a protein with multiple domains, including a signal peptide (aa 1–22), an inhibitor region (aa 27–156), and a catalytic cysteine domain (aa 160–367). The signal peptide cleavage site is predicted between Ala22 and Asn23. The putative 398 amino acid residues were found to have a theoretical pI of 8.76 and a molecular mass of 43,204.36 Da. The tested culture supernatants of NBMKU12 isolate exhibited the proteolytic activity against casein, papaya and pineapple used as substrates. The proteolytic activity suggests the expression of speB gene. Molecular docking analysis of cysteine protease showed that erythromycin (bond length 2.41 Å), followed by chloramphenicol (2.51 Å), exhibited a strong interaction; while penicillin-G (3.24 Å) exhibited a weak interaction, and this factor could be considered as a cause for penicillin-G resistance. The present study contributes to a better understanding of speB gene encoding cysteine protease, antibiotic resistance, and their interaction in the isolate, S. pyogenes NBMKU12. The antibiotics and cysteine protease interaction study confirms the resistance or sensitivity of S. pyogenes. Hence, it could be hypothesized that the isolate NBMKU12 is resistant to most of the tested antibiotics, and this resistance might be a cause for mutation.

Leaderless secreted peptide signaling molecule alters global gene expression and increases virulence of a human bacterial pathogen

Successful pathogens use complex signaling mechanisms to monitor their environment and reprogram global gene expression during specific stages of infection. Group A Streptococcus (GAS) is a major human pathogen that causes significant disease burden worldwide. A secreted cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB) is a key virulence factor that is produced abundantly during infection and is critical for GAS pathogenesis. Although identified nearly a century ago, the molecular basis for growth phase control of speB gene expression remains unknown. We have discovered that GAS uses a previously unknown peptide-mediated intercellular signaling system to control SpeB production, alter global gene expression, and enhance virulence. GAS produces an eight-amino acid leaderless peptide [SpeB-inducing peptide (SIP)] during high cell density and uses the secreted peptide for cell-to-cell signaling to induce population-wide speB expression. The SIP signaling pathway includes peptide secretion, reimportation into the cytosol, and interaction with the intracellular global gene regulator Regulator of Protease B (RopB), resulting in SIP-dependent modulation of DNA binding and regulatory activity of RopB. Notably, SIP signaling causes differential expression of ∼14% of GAS core genes. Several genes that encode toxins and other virulence genes that enhance pathogen dissemination and infection are significantly up-regulated. Using three mouse infection models, we show that the SIP signaling pathway is active during infection and contributes significantly to GAS pathogenesis at multiple host anatomic sites. Together, our results delineate the molecular mechanisms involved in a previously undescribed virulence regulatory pathway of an important human pathogen and suggest new therapeutic strategies.

Nano-Au/cMWCNT Modified speB Gene Specific Amperometric Sensor for Rapidly Detecting Streptococcus pyogenes causing Rheumatic Heart Disease.

A specific 5' NH2 labeled DNA probe of speB gene was immobilized onto the gold nanoparticles/carboxylated multi walled carbon nanotubes (Nano-Au/cMWCNT) screen printed electrode using EDC/NHS cross linking chemistry. This was followed by hybridization with 0.5-50ng/6µl of single stranded genomic DNA Streptococcus pyogenes infected patient throat swab samples. Electrochemical amperometric assay was deciphered by using cyclic voltammetry (CV) with methylene blue a redox indicator. The sensor had a sensitivity of 104.7µAcm-2ng-1 using CV with a R2 of 0.907 and 0.01ng/6µl as the limit of detection (LOD). The modified electrode surface morphology was characterized using scanning electron microscopy. The stability of the electrode was seen at 4°C for 180days having 6% loss in the initial current. The sensor is speB gene specific and can detect the pathogen within 30min.

SpeB Genosensor for Rapid Detection of Streptococcus pyogenes CausingDamage of Heart Valves in Human

speB Genosensor for early detection of Streptococcus pyogenes in human throat swab samples were fabricated using screen printed modified gold electrode. The electrochemical changes before and after hybridization was measured using cyclic voltammetry (CV) in the presence of redox indicator. The sensitivity of the genosensor was 80(μA/cm2)/ng and lower limit of detection (LOD) was 0.10 ng/6μl with the regression coefficient (R2) of 0.921 using CV. The sensor is highly specific, simple and takes only 30 min for detection of S. pyogenes infection. The sensor was further characterized by atomic force microscopy (AFM). Genosensor was found stable upto 6 months with only 10% loss in initial peak current in CV analysis on storage at 4°C. The speB gene based sensor can be used to prevent the damage of mitral and aortic heart valves by early diagnosis and treatment of the disease.

Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B

We developed a visual loop-mediated isothermal amplification (LAMP) technique to detect the streptococcal pyrogenic exotoxin B (speB) gene. Fifteen strains (from American Type Culture Collection or clinical isolates) were used to determine the specificity and sensitivity of the LAMP assay. Clinical samples were collected from 132 patients with suspected Streptococcus pyogenes (S. pyogenes) infection to verify the feasibility of the LAMP assay for detection of the speB gene. By using a set of five primers (a pair of outer primers, a pair of inner primers and one loop primer) targeting the speB gene, the amplification reaction was rapidly performed in a regular water bath under isothermal conditions at 63 °C for approximately 60 min. Only the two S. pyogenes strains showed positive results which were easily observed with the naked eye, and the other strains showed negative results. The detection limit of the LAMP assay was 0.01 ng/μl of template, showing higher sensitivity than conventional PCR (with a detection limit of 1.0 ng/μl). The detection rate of the speB gene in clinical samples was 71.21% and was consistent with the PCR results. The rapid detection of the speB gene by the LAMP assay is highly specific and sensitive, is simple to perform and cost-effective, and is expected to be a new reliable method for the rapid diagnosis of S. pyogenes infection, that is particularly suitable for rural or community hospitals in developing countries.

Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena.

Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [14C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The Δalr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [14C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the Δalr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium.

Epidemiology of Group A Streptococcus in a tertiary care centre and prevalence of speB gene

Epidemiology of Group A Streptococcus in a tertiary care centre and prevalence of speB gene