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This study aimed to investigate dust and bacterial air contamination in a broiler house during different seasons. The study was carried out in commercial housing conditions during five weeks of the rearing cycle in summer and winter. The total dust concentration ranged from 1.90 to 4.50 mg/m3 in summer and from 2.80 to 5.10 mg/m3 in winter. The total bacterial count ranged from 2.85 × 104 to 1.03 × 105 CFU/m3 in summer and from 2.12 × 104 to 2.28 × 105 CFU/m3 in winter. The study results showed the dust concentration to be increased in winter as compared to summer, yielding a significant correlation (r = 0.602, p < 0.05) with a significantly higher airborne bacterial count in winter (p < 0.001). Furthermore, dust concentration showed significant correlations (p < 0.05) with air temperature (r = −0.418), relative humidity (r = 0.673), and broiler activity (r = 0.709), while bacterial count yielded significant correlations (p < 0.05) with air temperature (r = −0.756), relative humidity (r = 0.831), and airflow rate (r = 0.511). The results obtained in the study can prove useful in the field. Seasonal variability in dust and bacterial air contamination should be considered in the development of guidelines or standards of air quality in broiler housing and evaluation of the effectiveness of remedial strategies.

The use and characteristics of timing belts are well-known for power transmission, but data regarding their suitability for use in dimensional metrology is far less available. In specific applications, e.g., when comparative measurements are wanted, a mechanical system capable of accurate angle transmission is needed. While belt drives offer excellent flexibility and low cost, their absolute accuracy of angle transfer is not widely researched which makes them difficult to use in precision engineering applications. To improve this lack of information, a comparative measurement system with integrated belt transmission was designed and built. The system comprises of two rotary axes, belt tensioning force adjustment mechanism and force measurement stage. Using this system, a standard timing belt was characterized with respect to the angle transfer error, and the relationship between angle transfer error and belt tensioning force was determined. These measurements provide a detailed insight into the key metrological characteristics of a belt drive system.

Poultry red mite (PRM), Dermanyssus gallinae, causes egg drop production, anemia and can be a vector in transmitting diseases. The PRM control mainly focuses on usage of the conventional chemical biocides. The objective of this study was to analyze the farmers’ perception regarding the impact, management and control of PRM in Macedonian layer farms. The data were collected with direct on-site visits using a unified questionnaire. In total, 29 poultry farms (28% of farms in the country), all with conventional cages, were part of this study. The data were analyzed by descriptive statistics, inferential statistics and Naïve Bayes Classifier technique. In 30% of the poultry farms the farmers had observed that the flock was infested with PRM. In total, 32 different treatments against PRM were reported from the farmers, and three of them were non-biocide treatments. The most used biocides (17% of the farms) were crude oil, Formalin, Neopitroid® and disinfectants. The highest agreement regarding biocides application among the farms (38%) was before the production starts. Most of the farmers applied biocides routinely, before the infestation is evident (75%). The median costs for PRM treatment were 175€ per flock, higher in the infested farms 493±677€ compared to non - infested 100±71€, p

This study aimed to compare fungal contamination of poultry litter between warm and cold seasons. It was carried out in commercial production conditions over two five-week fattening periods: one in the summer (July-August) and one in the winter (December-January). Broilers were reared on a litter composed of chopped straw and sawdust. Litter fungal concentration and composition were investigated weekly, along with litter temperature, moisture, and pH. Litter concentration of total fungi increased over both fattening periods, with no differences in median concentrations between them. Season also had no effect on yeast, Aspergillus section Nigri, and Cladosporium, Fusarium, and Rhizopus spp. concentrations, while the Aspergillus section Flavi and Aspergillus spp. combined showed higher concentrations in the summer, and Mucor and Penicillium spp. in the winter. Total fungal concentration highly correlated with litter temperature, moisture, and pH, regardless of the season. Our findings can be useful in the assessment and control of potential harmful effect of fungi on the health of poultry and poultry farm workers.

Background: Infections, major surgeries, and hyperinflammatory syndromes are known to trigger Systemic Inflammatory Response Syndrome (SIRS). Discrimination between infectious and noninfectious inflammation often poses a challenge in chronically ill patients with multiple comorbidities. These patients are routinely treated with a variety of anti-infective medications before a pathogen is identified. With the goal of improving pathogen detection rates and interventions, we evaluated Next Generation Sequencing (NGS) as a highly sensitive and fast means of detecting free microbial DNA in a small amount of serum samples from children with ongoing SIRS. Methods: We describe seven complex pediatric patients of SIRS or prolonged fever (>38.5 °C) >72 hours in which serum samples analyzed by NGS had a major impact on therapy. One patient was analyzed twice. Results: In eight NGS there were six positive results (two bacterial, three viral, one fungal) which were subsequently confirmed by microbiological culture or polymerase chain reaction (PCR) in five of the six NGS. In five of the eight performed NGS, results led to a change of therapy: antibiotic therapy was discontinued in two, escalated in one, an initiated in another; in one an antiviral was administered. Conclusions: NGS may become a valuable addition to infectious disease diagnostics in cases of pediatric SIRS. However, NGS has not yet been validated as a diagnostic method in pediatric as a diagnostic method in pediatric patients and results should therefore be interpreted with caution. Multi-center NGS evaluation studies are currently being planned.

Escherichia coli (E. coli) is one of the most common bacterial causes of infection in poultry farming. Whether the infection is localized or systemic, a primary or secondary disease, it is most frequently treated through the application of wide-spectrum antimicrobials. Excessive use of antimicrobials in agriculture is significantly contributing to the worldwide rise of antimicrobial resistance, but is also very expensive and often ineffective in the long term. Here, we present a case where a colibacillosis outbreak on a family farm of laying hens was treated using an autogenous vaccine. The birds had septicemia, cellulitis, and severe skin wounds. They were not vaccinated against E. coli, and did not receive any antimicrobials previously. E. coli strains were isolated from the daily mortalities on the farm and used for preparation of the vaccine. Each bird was given an intramuscular injection of the autogenous vaccine. The immunogenicity of the vaccine was tested by the determination of specific antibody levels in the sera of the birds using the in-house ELISA. Shortly after vaccination, the morbidity and mortality rates significantly decreased, and egg production was improved. The application of the autogenous vaccine served as a curative and preventive measure, and has proven to be a very efficient method of antimicrobial therapy.

This article examined the relationship between participation in adult nonformal education (NFE), defined as on-the-job training, attending private lessons, attending seminars, or distance learning, and Canadian immigrant respondents' literacy and numeracy outcomes, using data from the Program for the International Assessment of Adult Competencies Survey. We found that although participation in some forms of NFE was significantly related to higher literacy and numeracy scores, such relationship tended to be greater for first-generation immigrants than for nonimmigrant adults, even after controlling for their linguistic and formal educational background. Our findings suggested that first-generation immigrants in Canada might benefit the most from increased participation in NFE programs and targeted policies.

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

Good air quality in poultry houses is crucial for animal health and productivity. In these houses, air is generally contaminated with noxious gases and microorganisms, the concentrations of which depend on numerous factors including microclimate. In this case study, the relation between microclimate and air concentrations of noxious gases and microorganisms was investigated in extensively reared turkey house. The study was carried out at a family household in Dalmatia hinterland, Croatia, with 50.3±3.1 turkeys kept in the house during the study period. Air temperature, relative humidity, airflow rate, concentrations of ammonia, carbon dioxide, bacteria and fungi in indoor air were measured three times per month from September to December, in the morning, prior to releasing turkeys out for grazing. Air temperature ranged from 9.73 to 26.98 °C, relative humidity from 63.29% to 75.08%, and airflow rate from 0.11 to 0.17 m/s. Lowest ammonia and carbon dioxide concentrations were measured in September (2.17 ppm and 550 ppm, respectively) and highest in December (4.50 ppm and 900 ppm, respectively). Bacterial and fungal counts were lowest in December (2.51x105 CFU/m3 and 3.27x103 CFU/m3 air, respectively) and highest in September (6.85x105 CFU/m3 and 1.06x105 CFU/m3 air, respectively). Air temperature and relative humidity showed negative correlation with concentrations of noxious gases and positive correlation with air microorganisms (P

Fungi are present in abundance in poultry housing. The aim of the study was to assess the effect of season and microclimate parameters in poultry housing on fungal flora in the air and broiler trachea in commercial fattening conditions. The study was conducted in summer and winter. Study results indicated seasonal impact and association between fungal flora composition in housing air and broiler trachea. However, the total fungal count in housing air was significantly higher in summer and in broiler trachea in winter, both significantly correlated with indoor relative humidity and ammonia concentration. There was no significant correlation between outdoor and indoor air temperature, relative humidity and airflow rate, respectively. Study results suggested that environmental determination of fungi should be accompanied by their determination in broilers. In addition, seasonal impact on fungal contamination should be associated with microclimate conditions in the poultry house rather than the season itself. The fungi detected and the results obtained have implications not only for broiler health but also for the health of humans working in such environments.

The generally accepted approach to D. gallinae control is based on the control of infestations. The reduction of D. gallinae infection to an acceptable level can temporarily prevent its harmful effects on the health status ofpoultry. However, the reduced number of D. gallinae continues to persist in poultry environment resulting in an intensive increase of infestation rate and consequent transmission of infectious and contagious diseases. Contrary to suppression, the eradication approach completely eliminates D. gallinae thus improving general health status of the flock and providing the control of infectious and contagious diseases associated with this organismand eliminating other adverse effects. Biological efficiency and selection of inert compounds was performed in laboratory conditions. The previous experience with application of SiO2 formulations in practice confirmed thepossibility of successful eradication in 8 cases (combined application of liquid and powdered form). New generation of inert compounds (P 547/17) showed a range of superior properties as compared with SiO2 formulations,especially in view of high and long-lasting residual effects on non-absorbent surfaces. In laboratory conditions, after 7 months, the layer formed by a 20% working emulsion on a metal substrate exhibited efficiency of 92%after 1-hour exposure. In clinical conditions, the presence of mites was not detected even after 6.5 months of the settlement of the flock. Clinical tests of inert oils are still ongoing, but their high potential for the eradication of D.gallinae The eradication of D. gallinae is highly complex procedure, which cannot be performed at all times and in all conditions. The conditions for the eradication of D. gallinae using inert compounds include adequate hygienic preparation of the facilities while still empty, adequate application of selected compounds, and then adequate ambient temperatures which leads to increase of mites activity. The program of D. gallinae control includesprocedures for the preparation, implementation, eradication checks, and prevention of re-infestation

The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.

A total of 411 samples from birds of different species originating from all counties of the Republic of Croatia have been tested for the presence of Chlamydophila psittaci. The sampling was conducted in pet stores, breeders' aviaries, in a specialized bird clinic and in zoos. The testing included 177 parrots, 169 pigeons, 58 canaries and 7 finches. For the detection of specific C. psittaci antigen a commercial ELISA kit was used- IDEIATM PCE Chlamydia (DAKO Cytomation Ltd., United Kingdom). The samples that were non-specifically positive or doubtful in the ELISA test (a total of 26 samples) were analyzed also by means of polymerase chain reaction (PCR). Diagnostic ELISA method found a total of 17.03% birds positive for chlamydiosis, and after additional testing by PCR a total of 12.65% positive ones were found. According to bird species, the most frequently positive ones were canaries and pigeons (15.52% and 13.02%), and according to the sampling location most of the positive birds were found in pet stores (16.52%), but a high percentage of positive samples were also found in breeders’ aviaries (11.76%). The average positive result for chlamydiosis in 12.65% of tested birds is alarming and it confirms the importance of monitoring bird health and of prescribed legal regulations when it comes to chlamydial diseases, as well as education of persons involved in breeding, keeping or selling birds.

The effects of castration on growth performance, carcass characteristics and chemical composition of m. iliotibilais cranialis and m. pectoralis superficialis of pheasants were examined. Forty pheasants reared in commercial pheasantry were included in the experiment. Half of the pheasants were castrated at 8 weeks of age. Values for live weight tended to be higher in castrated pheasants in the 24th week (P

The Universal Sports School (hereinafter USŠ) is a project of the Croatian School Sports Federation (hereinafter HŠSS) carried out in lower grades of primary education in the Republic of Croatia. The project involves children from 1st to 4th grade of primary schools and it is conducted by both P.E. teachers and kinesiologists. The aim of the project is to encourage the multifaceted psychosomatic development of children, to raise awareness of preserving and improving health, doing daily physical exercise and to develop motor and functional abilities. The paper focuses on sports selection as a method for identifying potential top athletes and on the notion of USŠ, which is a HŠSS project, regarded as a system for carrying out a selection and directing of potential young athletes for the sports in which they can achieve optimal results in their future sports activities. [ABSTRACT FROM AUTHOR]

Copyright of Veterinary Archives / Veterinarski Arhiv is the property of University of Zagreb, Faculty of Veterinary Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Veterinary Archives / Veterinarski Arhiv is the property of University of Zagreb, Faculty of Veterinary Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Collegium Antropologicum is the property of Croatian Anthropological Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The article focuses on a research regarding Devriesea agamarum (D. agamarum), an actinobacterium, infection in spiny-tailed lizards. According to the researchers of the study, D. agamarum-associated infection has several symptoms including scaly changes around the mouth of the species, swelling of limbs and dermatitis around the mouth. It mentions that dermal lesions were analyzed by bacteriologic analysis. It further states that spiny-tallied lizards infected with D. agamarum were treated with ceftazidime and then diagnostic analysis confirmed negative bacteriological results.

The article discusses a study which examined the presence of Chlamydia (C.) psittaci genotypes in fecal samples of homing pigeons in Croatia. The two methods used to genotype randomly collected Chlamydia positive samples are C. psittaci-specific restriction fragment length polymorphism (RFLP) and Multiple-Locus Variable number tandem repeat Analysis (MLVA). The study revealed the presence of the zoonotic microorganisms and its risks on pigeon fanciers.

The article describes the subcutaneous tumor in a 17-year-old female cockatiel. It was presented by its owner for feather plucking and a wound at the right side of the cloaca. The owner, who had been monitoring the animal for two months, observed the edema and had not noticed any behavioral changes. The histopathological examination show the presence of a xanthoma. Despite the results, neither the reoccurrence of the skin xanthoma nor any health problems were observed in a period of one year.

The article presents a study which examines the bacterial and fungal flora in faecal samples originating from urban rooks found in Zagreb, Croatia. Researchers gathered 57 faecal samples coming from 35 individual breeding colonies in 16 districts. Among the bacteria found in the faecal samples included Escherichia coli, Staphylococcus and Streptococcus. One of the fungal species was Cladosporium.

Background: Infections, major surgeries, and hyperinflammatory syndromes are known to trigger Systemic Inflammatory Response Syndrome (SIRS). Discrimination between infectious and noninfectious inflammation often poses a challenge in chronically ill patients with multiple comorbidities. These patients are routinely treated with a variety of anti-infective medications before a pathogen is identified. With the goal of improving pathogen detection rates and interventions, we evaluated Next Generation Sequencing (NGS) as a highly sensitive and fast means of detecting free microbial DNA in a small amount of serum samples from children with ongoing SIRS., Methods: We describe seven complex pediatric patients of SIRS or prolonged fever (>38.5 °C) >72 hours in which serum samples analyzed by NGS had a major impact on therapy. One patient was analyzed twice., Results: In eight NGS there were six positive results (two bacterial, three viral, one fungal) which were subsequently confirmed by microbiological culture or polymerase chain reaction (PCR) in five of the six NGS. In five of the eight performed NGS, results led to a change of therapy: antibiotic therapy was discontinued in two, escalated in one, an initiated in another; in one an antiviral was administered., Conclusions: NGS may become a valuable addition to infectious disease diagnostics in cases of pediatric SIRS. However, NGS has not yet been validated as a diagnostic method in pediatric as a diagnostic method in pediatric patients and results should therefore be interpreted with caution. Multi-center NGS evaluation studies are currently being planned., Competing Interests: The authors declare no conflict of interest. Dr. Grumaz is a co-founder, employee, and shareholder of Noscendo GmbH. Petra Horvatek is an employee of Noscendo GmbH. No specific research funding was used for this research., (© 2022 The Author(s). Published by IMR Press.)

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis., Competing Interests: The authors have declared that no competing interests exist

Slow growing stationary phase bacteria are often tolerant to multiple stressors and antimicrobials. Here, we show that the pathogen Staphylococcus aureus develops a non-specific tolerance towards oxidative stress during the stationary phase, which is mediated by the nucleotide second messenger (p)ppGpp. The (p)ppGpp 0 mutant was highly susceptible to HOCl stress during the stationary phase. Transcriptome analysis of the (p)ppGpp 0 mutant revealed an increased expression of the PerR, SigB, QsrR, CtsR and HrcA regulons during the stationary phase, indicating an oxidative stress response. The (p)ppGpp 0 mutant showed a slight oxidative shift in the bacillithiol (BSH) redox potential (E BSH ) and an impaired H 2 O 2 detoxification due to higher endogenous ROS levels. The increased ROS levels in the (p)ppGpp 0 mutant were shown to be caused by higher respiratory chain activity and elevated total and free iron levels. Consistent with these results, N-acetyl cysteine and the iron-chelator dipyridyl improved the growth and survival of the (p)ppGpp 0 mutant under oxidative stress. Elevated free iron levels caused 8 to 31-fold increased transcription of Fe-storage proteins ferritin (ftnA) and miniferritin (dps) in the (p)ppGpp 0 mutant, while Fur-regulated uptake systems for iron, heme or siderophores (efeOBU, isdABCDEFG, sirABC and sstADBCD) were repressed. Finally, the susceptibility of the (p)ppGpp 0 mutant towards the bactericidal action of the antibiotics ciprofloxacin and tetracycline was abrogated with N-acetyl cysteine and dipyridyl. Taken together, (p)ppGpp confers tolerance to ROS and antibiotics by down-regulation of respiratory chain activity and free iron levels, lowering ROS formation to ensure redox homeostasis in S. aureus., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions., Competing Interests: The authors have declared that no competing interests exist.

The nutritional alarmones ppGpp and pppGpp (collectively: (p)ppGpp) are nucleotide-based second messengers enabling bacteria to respond to environmental and stress conditions. Several bacterial species contain two highly homologous (p)ppGpp synthetases named RelP (SAS2, YwaC) and RelQ (SAS1, YjbM). It is established that RelQ forms homotetramers that are subject to positive allosteric regulation by pppGpp, but structural and mechanistic insights into RelP lack behind. Here we present a structural and mechanistic characterization of RelP. In stark contrast to RelQ, RelP is not allosterically regulated by pppGpp and displays a different enzyme kinetic behavior. This discrepancy is evoked by different conformational properties of the guanosine-substrate binding site (G-Loop) of both proteins. Our study shows how minor structural divergences between close homologues result in new functional features during the course of molecular evolution.