Molecular Microbiology

The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, although the number of repeats varies. All five Sib RNAs in E. coli MG1655 are expressed, and no phenotype was observed for a five-sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18-to 19-amino-acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by coexpression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell.

SummaryThe correct annotation of genes encoding the smallest proteins is one of the biggest challenges of genome annotation, and perhaps more importantly, few annotated short open reading frames have been confirmed to correspond to synthesized proteins. We used sequence conservation and ribosome binding site models to predict genes encoding small proteins, defined as having 16–50 amino acids, in the intergenic regions of the Escherichia coli genome. We tested expression of these predicted as well as previously annotated genes by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and assaying for synthesis using immunoblot assays. This approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16–50 amino acids are synthesized. We summarize the properties of these small proteins; remarkably more than half of the proteins are predicted to be single‐transmembrane proteins, nine of which we show co‐fractionate w...

The concept of the human oral microbiome was applied to understand health and disease, lifestyles, and dietary habits throughout part of human history. In the present study, we augment the understanding of ancient oral microbiomes by characterizing human dental calculus samples recovered from the ancient Abbey of Badia Pozzeveri (central Italy), with differences in socioeconomic status, time period, burial type, and sex. Samples dating from the Middle Ages (11th century) to the Industrial Revolution era (19th century) were characterized using high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene V4 region. Consistent with previous studies, individuals from Badia Pozzeveri possessed commensal oral bacteria that resembled modern oral microbiomes. These results suggest that members of the oral microbiome are ubiquitous despite differences in geographical regions, time period, sex, and socioeconomic status. The presence of fecal bacteria could be in agreement with poor hygiene...

The human pathogen Haemophilus influenzae causes mainly respiratory tract infections such as acute otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease. We recently revealed the crystal structure of H. influenzeae protein E (PE), a multifunctional adhesin that is involved in direct interactions with lung epithelial cells and host proteins. Based upon the PE structure we here suggest a hypothetical binding pocket that is compatible in size with a hemin molecule. An H. influenzae mutant devoid of PE bound significantly less hemin in comparison to the PE-expressing wild type counterpart. Accordingly, E. coli expressing PE at the surface bound hemin. An interaction between hemin and recombinant soluble PE was also demonstrated by native-PAGE and UV-visible spectrophotometry. Surface plasmon resonance revealed an affinity (K d ) of 1.6 x 10 -6 M for the hemin-PE interaction. Importantly, hemin that was bound to PE at the H. influenzae surface, was donated to co-cultured luciferase-expressing H. influenzae that were starved of hemin. When hemin is bound to PE it thus may serve as a storage pool for H. influenzae. To our knowledge this is the first report showing that H. influenzae can share hemin via a surface-located outer membrane protein.

SummaryThe protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome‐dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell‐free protein‐synthesizing system. Moreover, the degree of functional conservation of the IF2‐like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of ‘chimeric’ proteins produced by swapping domains be...

The bacterium Sinorhizobium meliloti is attracted to seed exudates of its host plant alfalfa (Medicago sativa). Since quaternary ammonium compounds (QACs) are exuded by germinating seeds, we assayed chemotaxis of S. meliloti towards betonicine, choline, glycine betaine, stachydrine and trigonelline. The wild type displayed a positive response to all QACs. Using LC-MS, we determined that each germinating alfalfa seed exuded QACs in the nanogram range. Compared to the closely related nonhost species, spotted medic (Medicago arabica), unique profiles were released. Further assessments of single chemoreceptor deletion strains revealed that an mcpX deletion strain displayed little to no response to these compounds. Differential scanning fluorimetry showed interaction of the isolated periplasmic region of McpX (McpX(PR) and McpX34-306 ) with QACs. Isothermal titration calorimetry experiments revealed tight binding to McpX(PR) with dissociation constants (Kd ) in the nanomolar range for ch...

The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymati...

SummaryThe malaria parasite Plasmodium falciparum invades erythrocytes where it replicates to produce invasive merozoites, which eventually egress to repeat the cycle. Merozoite surface protein‐1 (MSP1), a prime malaria vaccine candidate and one of the most abundant components of the merozoite surface, is implicated in the ligand–receptor interactions leading to invasion. MSP1 is extensively proteolytically modified, first just before egress and then during invasion. These primary and secondary processing events are mediated respectively, by two parasite subtilisin‐like proteases, PfSUB1 and PfSUB2, but the function and biological importance of the processing is unknown. Here, we examine the regulation and significance of MSP1 processing. We show that primary processing is ordered, with the primary processing site closest to the C‐terminal end of MSP1 being cleaved last, irrespective of polymorphisms throughout the rest of the molecule. Replacement of the secondary processing site, ...

Cells possess stress-activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Dpbs2 and Dtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Dtmk3 and Dpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phospho-rylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

Gonorrhea has become resistant to most conventional antimicrobials used in clinical practice. The global spread of multidrug-resistant isolates of Neisseria gonorrhoeae could lead to an era of untreatable gonorrhea. New therapeutic modalities with novel mechanisms of action that do not lend themselves to the development of resistance are urgently needed. Gonococcal lipooligosaccharide (LOS) sialylation is critical for complement resistance and for establishing infection in humans and in experimental mouse models. Here we describe two immunotherapeutic approaches that target LOS sialic acid: i) a fusion protein that comprises the region in the complement inhibitor factor H (FH) that binds to sialylated gonococci and IgG Fc (FH/Fc fusion protein) and ii) analogs of sialic acid that are incorporated into LOS but fail to protect the bacterium against killing. Both molecules showed efficacy in the mouse vaginal colonization model of gonorrhea and may represent promising immunotherapeutic...

The bacterial chromosome is organized into multiple independent domains, each capable of constraining the plectonemic negative supercoil energy introduced by DNA gyrase. Different experimental approaches have estimated the number of domains to be between 40 and 150. The sitespecific resolution systems of closely related transposons Tn3 and γδ are valuable tools for measuring supercoil diffusion and analysing bacterial chromosome dynamics in vivo. Once made, the wild-type resolvase persists in cells for time periods greater than the cell doubling time. To examine chromosome dynamics over shorter time frames that are more closely tuned to processes like inducible transcription, we constructed a set of resolvases with cellular half-lives ranging from less than 5 min to 30 min. Analysing chromosomes on different time scales shows domain structure to be dynamic. Rather than the 150 domains detected with the Tn3 resolvase, wild-type cells measured over a 10 min time span have more than 400 domains per genome equivalent, and some gyrase mutants exceed 1000.

SummaryBackgroundGut microbiome patterns have been associated with predisposition to eczema potentially through modulation of innate immune signalling.ObjectiveWe examined gut microbiome development in the first year of life in relation to innate immune responses and onset of IgE‐associated eczema over the first 2.5 years in predisposed children due to maternal atopy [www.anzctr.org.au, trial ID ACTRN12606000280505].MethodsMicrobial composition and diversity were analysed with barcoded 16S rRNA 454 pyrosequencing in stool samples in pregnancy and at ages 1 week, 1 month and 12 months in infants (n = 10) who developed IgE‐associated eczema and infants who remained free of any allergic symptoms at 2.5 years of age (n = 10). Microbiome data at 1 week and 1 month were analysed in relation to previously assessed immune responses to TLR 2 and 4 ligands at 6 months of age.ResultsThe relative abundance of Gram‐positive Ruminococcaceae was lower at 1 week of age in infants developing IgE‐ass...

Background: C. albicans, the most prevalent yeast isolated from clinical samples, whose presumptive identification is usually based on germ tube test using human serum at 37°C. But C. dubliniensis also produces true germ tubes in same conditions. So a simple and fast method is needed to differentiate C. albicans from C. dubliniensis. In the present study we evaluated germ tube formation on YEPD medium at 39°C for rapid differentiation of the two species. : A total of 200 strains were tested for germ tube formation on YEPD medium at 39°C which included 186 strains of C. albicans and 14 strains of C. dubliniensis. Standard strains of both species were included in the study. Results: Out of 186 test strains of C. albicans 166 (89.3%) formed true germ tubes after 1 h at 39°C while the remaining grew as a yeast form. All the 20 test strains of C. dubliniensis grew as a yeast form. Conclusion: Positive modified germ tube test can be used for presumptive identification of C. albicans and ruling out C. dubliniensis however negative test cannot be used to differentiate between the two species.

RIASSUNTO -Vantaggi e limiti del monitoraggio ambientale mediante l'analisi chimica dei Lamellibranchi -Lo studio ha lo scopo di rispondere alla domanda "È attendibile il monitoraggio mediante specie accumulatrici di polluenti?". Viene trattato il problema generale dei bioindicatori di accumulo, ma sono anche discussi casi di studio che concernono i Lamellibranchi d'acqua dolce, gli elementi in tracce e i radioisotopi. L'indicatore riflette il livello di inquinamento dell'ambiente se concentra nel suo corpo i polluenti nelle stesse proporzioni con le quali i polluenti sono presenti nell'ambiente. Questo avviene raramente per le seguenti cause: 1) l'indicatore seleziona i polluenti; 2) l'assunzione di polluenti è limitata alle forme fisiche e chimiche utilizzabili dall'indicatore e 3) i polluenti vengono assunti sia dall'acqua che dall'alimento. Di conseguenza, questo tipo di monitoraggio non è idoneo per confrontare il livello di inquinamento di ambienti diversi. Il monitoraggio con bioaccumulatori permette di: 1) valutare la disponibilità degli inquinanti agli organismi; 2) mappare la distribuzione degli inquinanti in un ambiente; 3) stimare l'inquinamento di un ambiente trasferendovi bioaccumulatori prelevati da ambienti non inquinati; 4) identificare polluenti anche quando la loro concentrazione nell'ambiente è inferiore al limite di sensibilità dei metodi analitici comunemente usati; 5) seguire nel tempo e nello spazio le variazioni dell'inquinamento nello stesso ambiente e 6) rivelare la presenza di nuovi inquinanti (es. radioisotopi artificiali). -This study aims to answer the question "Is routine pollution monitoring by accumulator species reliable?". The issue of accumulator species is discussed in general terms but examples involving freshwater mussels, trace elements and radioisotopes are also given. Bioindicators reflect the pollutant levels of the environment only if they concentrate the pollutants in their bodies in the same proportions as are present in the water. This rarely occurs for three reasons: 1) all species are able to some extent to select the various pollutants; 2) a species does not concentrate the total pollutant but only their available forms; 3) the bioindicator uptakes pollutants from both water and food. As a consequence, this kind of monitoring is not suitable for comparing the pollution level of different environments. In contrast, the biomonitoring with accumulator species may be usefully applied to specific problems such as: 1) evaluating the portion of pollutants available to the accumulator species; 2) mapping the pollutant distribution in an environment; 3) estimating the pollution level of a polluted environment by transferring accumulator species from a clean environment to a polluted one; 4) identifying new pollutants (e.g. artificial radioisotopes) or pollutants present in the water at a lower concentration than the sensitivity limit of the commonly-used methods; 5) following the variations over time of the pollutant concentrations in an environment.

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria. Although much attention has been given to the biological effects of its lipid A portion, a great body of evidence indicates that its O chain polysaccharide (O antigen) portion plays an important role in the bacterium-host interplay. In this work we have studied in-depth the role of the O antigen in Yersinia enterocolitica serotype O:8 pathogenesis. We made a detailed virulence analysis of three mutants having different O antigen phenotypes: (i) LPS with no O antigen (rough mutant); (ii) LPS with one O unit (semirough mutant) and (iii) LPS with random distribution of O antigen chain lengths. We demonstrated that these LPS O antigen mutants were attenuated in virulence regardless of the infection route used. Coinfection experiments revealed that the rough and semirough mutants were severely impaired in their ability to colonize the Peyer's patches and in contrast to the wild-type strain they did not colonize spleen and liver. The mutant with random distribution of O antigen chain lengths, however, survived better but started to be cleared from mouse organs after 8 days. As an explanation to this attenuation we present here evidence that other Yersinia virulence factors depend on the presence of O antigen for their proper function and/or expression. We demonstrated that in the rough mutant: (i) the YadA function but not its expression was altered; (ii) Ail was not expressed and (iii) inv expression was downregulated. On the other hand, expression of flhDC , the flagellar master regulatory operon, was upregulated in this mutant with a concomitant increase in the production of flagellins. Finally, expression of yplA , encoding for the Yersinia phospholipase A, was also upregulated accompanied by an increased flagellar type III secretion system mediated secretion of YplA to culture medium. Together these findings suggest that the absence of O antigen in the outer membrane of Yersinia either directly or indirectly, for example through a cellular or membrane stress, could act as a regulatory signal.

SummaryThe infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high‐resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side‐chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl‐branched phthiocerol dimycocerosates. We observed that cholesterol‐exposed cells lacking CYP125A1 accumulate cholest‐4‐en‐3‐one, suggesting that this is a physiological substrate for this enzyme. Reconstitution of enzymatic activity with spinach ferredoxin and ferredoxin reductase revealed that recombinant CYP125A1 indeed binds both cholest‐4‐en‐3‐one and cholesterol, efficiently hydroxylates both of them at C‐27, and then further oxidizes 27‐hydroxycholest‐4‐en‐3...

Group B Streptococcus (GBS) is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, GBS colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present report, we describe the isolation of the fbsA gene, which encodes a fibrinogen receptor from GBS. The deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. Sequencing of the fbsA gene from five different GBS strains revealed significant variation in the number of repeat-encoding units. The deletion of the fbsA gene in the genome of GBS 6313 completely abolished fibrinogen binding, suggesting that FbsA is the major fibrinogen receptor in this strain. Growth of the fbsA deletion mutant in human blood was significantly impaired, indicating that FbsA protects GBS from opsonophagocytosis. In Western blot experiments with truncated FbsA proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Spot membrane analysis and competitive binding experiments with peptides carrying single amino acid substitutions allowed the prediction of a fibrinogen-binding motif with the con-

SummaryOrnithine lipids (OLs) are widespread among Gram‐negative bacteria. Their basic structure consists of a 3‐hydroxy fatty acyl group attached in amide linkage to the α‐amino group of ornithine and a second fatty acyl group ester‐linked to the 3‐hydroxy position of the first fatty acid. OLs can be hydroxylated within the secondary fatty acyl moiety and this modification has been related to increased stress tolerance. Rhizobium tropici, a nodule‐forming α‐proteobacterium known for its stress tolerance, forms four different OLs. Studies of the function of these OLs have been hampered due to lack of knowledge about their biosynthesis. Here we describe that OL biosynthesis increases under acid stress and that OLs are enriched in the outer membrane. Using a functional expression screen, the OL hydroxylase OlsE was identified, which in combination with the OL hydroxylase OlsC is responsible for the synthesis of modified OLs in R. tropici. Unlike described OL hydroxylations, the OlsE‐c...

Bardet-Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole-like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild-type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.

The manufacture use and disposal of explosives as well as the demilitarization of military facilities can result in environmental contamination. The three most widely used explosives are 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX).

The manufacture use and disposal of explosives as well as the demilitarization of military facilities can result in environmental contamination. The three most widely used explosives are 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX).

Cross-beta fibrous protein aggregates (amyloids and amyloid-based prions) are found in mammals (including humans) and fungi (including yeast), and are associated with both diseases and heritable traits. The Hsp104/70/40 chaperone machinery controls propagation of yeast prions. The Hsp70 chaperones Ssa and Ssb show opposite effects on [PSI + ], a prion form of the translation termination factor Sup35 (eRF3). Ssb is bound to translating ribosomes via ribosome-associated complex (RAC), composed of Hsp40-Zuo1 and Hsp70-Ssz1. Here we demonstrate that RAC disruption increases de novo prion formation in a manner similar to Ssb depletion, but interferes with prion propagation in a manner similar to Ssb overproduction. Release of Ssb into the cytosol in RAC-deficient cells antagonizes binding of Ssa to amyloids. Thus, propagation of an amyloid formed due to lack of ribosome-associated Ssb can be counteracted by cytosolic Ssb, generating a feedback regulatory circuit. Release of Ssb from ribosomes is also observed in wild type cells during growth in poor synthetic medium. Ssb is, in a significant part, responsible for the prion destabilization in these conditions, underlining the physiological relevance of the Ssb-based regulatory circuit.

The epidemiology of bovine and human tuberculosis (TB) was studied in the Federal Capital Territory and Kaduna state of Nigeria using four diagnostic methods; tuberculin test, culture and acid-fast stain of milk, animal (cattle) tissue and human sputum. Two PCR-based molecular techniques (Spoligotyping and Variable Number Tandem Repeat) were used to identify the species and strains of the isolates, while IS61 10-RFLP molecular method was optimised and applied on few samples to deten-nine the efficacy of the method. Of the 967 lactating cows from 57 herds tested for TB, 14.6%, 4% and 81.4% were positive, inconclusive and negative reactors respectively. Tuberculin test also showed that mycobacterial infection was prevalent iii the two management systems studied (nomadic and semi-nomadic), but the effect of management on the prevalence of infection was not significant. However, age was found to play a significant role in the prevalence infection where more positive cases were observed among the older age groups. It was also observed that control policy is either not in place or inadequately implemented in the study area. Of the 156 milk samples collected, 12.6% and 23% were culture and acid-fast positive respectively, while out of the 250 tissue samples 17.3% and 20% were culture and acid-fast positivc respectively. Thi III is finding confirmed a definite relationship between the disease in live and slaughtered cattle. Comparing the three diagnostic methods in List of Contents

In agriculture sector application of precision farming implies to agricultural input based on soil types, climatic condition and crop need to maximize sustainable agriculture production with quality and profitability. Successful implementation of precision farming in agriculture depends on multiple components, including the extent to which conditions within a known field are managed, the acceptability of input recommendation amount and control to the degree of application microbial consortia. Among these, microbial genomics identified and biotechnological tools represent cuttingedge best strategies to sustainably enhance crop yield with soil health. This review explores recent advancing precision farming through microbial genomics, metagenomics, microbial resource management, and microbial-based biotechnological tools innovations for improving crop productivity and soil health. Emphasis is placed on how these innovations can contribute to site-specific management practices, integrates remote sensing technologies such as satellite and drones, to monitor crop conditions and captures early signs of diseases or pests infect. These tools enable people to intervene implement targeted sites treatments, lowering the need for large-scale bio-pesticides and minimizing crop losses, improve soil fertility, mitigate stresses, and promote ecological sustainability. Additionally, precision farming facilitates monitoring (real-time) of weather conditions, permitting farmers to adjust their irrigation schedules or implement protective measures in response to changing climatic conditions.

A mathematical model, involving diffusion of extracellular compounds and cell interactions, has been adapted, in order to assess the quorum sensing process occurring in biofilms. Quorum sensing is a diffusion dependent phenomenon; therefore, accumulation levels of extra-cellular substances can directly affect cellular development. In order to modeling quorum sensing from artificially structured microbial consortia (ASMC), accumulation of N-acyl-Lhomoserine lactone (AHL) was studied within a biofilm formed with three bacterial species, Agrobacterium tumefaciens KYC6, Escherichia coli JM109 and green fluorescent protein (GFP), producer and reporter, Escherichia coli JM105. Different artificial biofilm structures or ASMC and conditions were considered when modeling cell behavior under particular experimental situations, such as: accumulation from external addition or internal production of N-acyl-L-homoserine lactone (AHL) in mixed mono-and multi-layered biofilms. Mathematical modeling results include: internal concentration profiles of extra-cellular compounds, accumulated within the biofilm; time related patterns of cell distribution in ASMC constructed with different strains and dynamic estimations of biofilm thickness.

A good strategy to conquer the Escherichia coli-cause food-borne disease could be bacteriophages. Porins are a type of β-barrel proteins with diffuse channels and OmpA, which has a role in hydrophilic transport, is the most frequent porin in E. coli; it was also chosen as the potential receptor of the phage. And the Rz/Rz1 was engaged in the breakup of the host bacterial external membrane. This study aimed to analyze the amino acid of OmpA and Rz/Rz1 of lytic bacteriophage from Surabaya, Indonesia. This study employed a sample of 8 bacteriophages from the previous study. The OmpA analysis method was mass spectrometry. Rz/Rz1 was analyzed using PCR, DNA sequencing, Expasy Translation, and Expasy ProtParam. The result obtained 10% to 29% sequence coverage of OmpA, carrying the ligand-binding site. The Rz/Rz1 gene shares a high percentage of 97.04% to 98.89% identities with the Siphoviridae isolate ctTwQ4, partial genome, and Myoviridae isolate cthRA4, partial genome. The Mann–Whitney ...

Background
Maintaining the sterility and integrity of microbiology clean rooms is essential for ensuring the reliability of
experiments and the safety of products in pharmaceutical and biotechnological industries. Contamination control
in these environments faces numerous challenges, including the presence of common contaminants such as bacteria,
fungi (including Aspergillus spp.), viruses (including coronavirus), yeasts, and moulds. Effective contamination
control measures are crucial to minimize the risk of microbial contamination. The goal is to provide valuable
guidance for optimizing contamination control practices, thereby safeguarding experimental integrity and product
safety.
Methods: The review synthesizes insights from recent studies focusing on contamination control measures in
microbiology clean rooms. It evaluates the effectiveness of various strategies, including stringent cleaning
protocols, environmental monitoring, and personnel training. Additionally, the review explores the potential of
advanced technologies such as filtration systems, real-time environmental monitoring, automation, virtual reality
training, and artificial intelligence in enhancing contamination control.
Results: The review identifies effective contamination control measures, including advanced filtration
technologies, real-time environmental monitoring systems, and automation, which have shown promise in
enhancing contamination control. However, challenges such as high costs, human factor variability, and
technological integration persist. Innovative approaches like virtual reality training and artificial intelligence offer
potential solutions to these challenges.
Conclusion: By synthesizing insights from recent studies, this review provides valuable guidance for optimizing
contamination control in microbiology clean rooms. Implementing advanced technologies and innovative
approaches can help overcome existing challenges, thereby safeguarding experimental integrity and product safety
in pharmaceutical and biotechnological industries.

The fungal protein CBP (calcium binding protein) is a known virulence factor with an unknown virulence mechanism. The protein was identified based on its ability to bind calcium and its prevalence as Histoplasma capsulatum's most abundant secreted protein. However, CBP has no sequence homology with other calcium binding proteins and contains no known calcium-binding motifs. Here, the NMR structure of CBP reveals a highly intertwined homodimer and represents the first atomic level NMR model of any fungal virulence factor. Each CBP monomer is comprised of four α-helices that adopt the saposin fold, characteristic of a protein family that binds to membranes and lipids. This structural homology suggests that CBP functions as a lipid-binding protein, potentially interacting with host glycolipids in the phagolysosome of host cells.

I would like to express my gratitude to Dr Nurulhasanah Othman, my supervisor, and Prof Rahmah Noordin, my co-supervisor. They have poured out their best in moulding me and guiding me in this journey to be a researcher. Not forgetting to mention, my personal development too. For that, I am forever grateful to them.

I would like to express my gratitude to Dr Nurulhasanah Othman, my supervisor, and Prof Rahmah Noordin, my co-supervisor. They have poured out their best in moulding me and guiding me in this journey to be a researcher. Not forgetting to mention, my personal development too. For that, I am forever grateful to them.

This analysis reveals that molecular assembly begins with fundamental geometric principles encoded at the atomic level, where the weak nuclear force provides the asymmetric foundation for all molecular handedness observed in nature. The tetrahedral geometry emerges as the fundamental building block for molecular assembly coordination, while spiral configurations represent optimal pathways for molecular matter-energy coordination across all scales.

FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn 2+ coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn 2+ coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD + dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD + to NADH that is subsequently used for furfural reduction.

Many bacteria can assemble functional amyloid fibers on their cell surface. The majority of bacterial amyloids contribute to biofilm or other community behaviors where cells interact with a surface or with another cell. Bacterial amyloids, like all functional amyloids, share structural and biochemical properties with disease-associated eukaryotic amyloids. The general ability of amyloids to bind amyloid-specific dyes, such as Congo red, and their resistance to denaturation have provided useful tools for scoring and quantifying bacterial amyloid formation. Here, we present basic approaches to study bacterial amyloids by focusing on the well-studied curli amyloid fibers expressed by Enterobacteriaceae. These methods exploit the specific tinctorial and biophysical properties of amyloids. The methods described here are straightforward and can be easily applied by any modern molecular biology lab for the study of other bacterial amyloids.

SK designed and conceived the experiment under mentorship of RCD. SD assisted during the experiments and NB helped during manuscript writing. RCD and DKM read the manuscript for scientific correction and approved for communication. Macrophomina phaseolina and induce growth promotion in Vigna mungo L.

SummaryMammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram‐positive and Gram‐negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP‐induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP‐Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidored...

During formation of spores by Bacillus subtilis the RNA polymerase factor σ G ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of σ G ordinarily requires prior activity of σ F in the prespore and σ E in the mother cell. Here we report that in spoIIA mutants lacking both σ F and the anti-sigma factor SpoIIAB and in which σ E is not active, σ G nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of σ G activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG , the structural gene for σ G , from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to σ G . When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, σ G became active earlier in sp...

Compartmentalization of the activities of RNA polymerase sigma factors is a hallmark of formation of spores by Bacillus subtilis . It is initiated soon after the asymmetrically located sporulation division takes place with the activation of σ F in the smaller cell, the prespore. σ F then directs a signal via the membrane protease SpoIIGA to activate σ E in the larger mother cell by processing of pro-σ E . Here, we show that σ E can be activated in the prespore with little effect on sporulation efficiency, implying that complete compartmentalization of σ E activity is not essential for spore formation. σ E activity in the prespore can be obtained by inducing transcription in the prespore of spoIIGA or of sigE *, which encodes a constitutively active form of σ E , but not of spoIIGB , which encodes pro-σ E . We infer that σ E compartmentalization is partially attributed to a competition between the compartments for the activation signaling protein SpoIIR. Normally, SpoIIGA is predomin...

The Bacillus subtilis bex gene complemented the defect in an Escherichia coli era mutant. The Bex protein showed 39% identity and 67% similarity to the E. coli Era GTPase. In contrast to era , bex was not essential in all strains. bex mutant cells were elongated and filled with diffuse nucleoid material. They grew slowly and exhibited severely impaired spore formation.

Formation of the asymmetrically located septum during sporulation of Bacillus subtilis results in enclosure of the origin-proximal 30% of the chromosome in the prespore compartment. The rest of the chromosome is then translocated into the prespore from the mother cell. Transcription of spoIIR is initiated in the prespore by RNA polymerase containing ς F soon after the septum is formed. The SpoIIR protein is required for the activation of the transcription program directed by ς E in the mother cell. The spoIIR locus is located at 324°, near the origin of replication (0/360°). We show here that movement of spoIIR to 28° had little effect on sporulation. However, movement to regions not in the origin-proximal part of the chromosome substantially reduced sporulation efficiency. At 283° sporulation was reduced to less than 20% of the level obtained when spoIIR was at its natural location, and movement to 190° reduced sporulation to about 6% of that level. These positional effects were al...

While it is generally accepted that anaerobic metabolism is required during infection, supporting experimental data have only been described in a limited number of studies. To provide additional evidence on the role of anaerobic metabolism in bacterial pathogens while invading mammalian hosts, we analysed the effect of the inactivation of FNR, the major regulatory protein involved in the adaptation to oxygen restrictive conditions, and of two of the FNR-regulated genes on the survival of Neisseria meningitidis serogroup B (MenB) in vivo. We found that fnr deletion resulted in more than 1 log reduction in the meningococcal capacity to proliferate both in infant rats and in mice. To identify which of the FNR-regulated genes were responsible for this attenuated phenotype, we defined the FNR regulon by combining DNA microarray analysis and FNR-DNA binding studies. Under oxygen-restricted conditions, FNR positively controlled the transcription of nine transcriptional units, the most upregulated of which were the two operons NMB0388-galM and mapA-pgmβ implicated in sugar metabolism and fermentation. When galM and mapA were knocked out, the mutants were attenuated by 2 and 3 logs respectively. As the operons are controlled by FNR, from these data we conclude that MenB survival in the host anatomical sites where oxygen is limiting is supported by sugar fermentation.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background: Hand, foot and mouth disease (HFMD) has been associated with large outbreaks among young children in the Asia-Pacific Region since 1997, including cases of severe illness and death. Severe illness is often associated with enterovirus A71 (EV-A71). Vietnam experienced a large sustained outbreak of 200 000 hospitalized cases and over 200 deaths in 2011-12, the large majority occurring in southern Vietnam. Methods: A prospective observational study was conducted in the outpatient clinics, infectious diseases wards, and paediatric intensive care units of the three main referral centres for the treatment of HFMD in southern Vietnam. Demographic data, basic laboratory parameters, and clinical data were recorded, and molecular diagnostic tests were performed. Results: Between July 2013 and July 2015, a total of 1547 children were enrolled. Four serotypes of enterovirus A (EV-A71, Coxsackievirus (CV) A6, A10, and A16) were responsible for 1005 of 1327 diagnosed cases (75.7%). An unexpected dominance of EV-A71 was found among both inpatients and outpatients, as well as a strong association with severe illness. CV-A6 and CV-A10 emerged in Vietnam during the study period and replaced CV-A16. CV-A10 was associated with different clinical and laboratory characteristics. During admission, 119 children developed a more severe illness. It was found that children with a skin rash showed less progression of severity, but when a rash was present, a macular rash was significantly associated with an increased risk of progression. Conclusions: This study represents the most comprehensive descriptive HFMD study from Vietnam to date. Co-circulation and replacement of different serotypes has implications for vaccine development and implementation. These findings from a severely affected country add to our understanding of the presentation, progression, and aetiology of HFMD.

The precise disassembly of the extracellular matrix of some plant species used as feedstocks for bioenergy production continues to be a major barrier to reach reasonable cost effective bioethanol production. One solution has been the use of pretreatments, which can be effective, but increase even more the cost of processing and also lead to loss of cell wall materials that could otherwise be used in industry. Although pectins are known to account for a relatively low proportion of walls of grasses, their role in recalcitrance to hydrolysis has been shown to be important. In this mini-review, we examine the importance of pectins for cell wall hydrolysis highlighting the work associated with bioenergy. Here we focus on the importance of endopolygalacturonases (EPGs) discovered to date. The EPGs cataloged by CAZy were screened, revealing that most sequences, as well as the scarce structural work performed with EPGs, are from fungi (mostly Aspergillus niger). The comparisons among the EPG from different microorganisms, suggests that EPGs from bacteria and grasses display higher similarity than each of them with fungi. This compilation strongly suggests that structural and functional studies of EPGs, mainly from plants and bacteria, should be a priority of research regarding the use of pectinases for bioenergy production purposes.

Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its downregulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets.

The necrotrophic fungus Botrytis cinerea, is considered a major cause of postharvest losses in a wide range of crops. The common fungal extracellular membrane protein (CFEM), containing a conserved eight-cysteine pattern, was found exclusively in fungi. Previous studies in phytopathogenic fungi have demonstrated the role of membrane-bound and secreted CFEM-containing proteins in different aspects of fungal virulence. However, non-G protein-coupled receptor (non-GPCR) membrane CFEM proteins have not been studied yet in phytopathogenic fungi. In the present study, we have identified a non-GPCR membrane-bound CFEM-containing protein, Bcin07g03260, in the B. cinerea genome, and generated deletion mutants, ΔCFEM-Bcin07g03260, to study its potential role in physiology and virulence. Three independent ΔCFEM-Bcin07g03260 mutants showed significantly reduced progression of a necrotic lesion on tomato (Solanum lycopersicum) leaves. Further analysis of the mutants revealed significant reductio...

The effectiveness of job performance depends on most of all on the staff engagement with the working process. To have a high motivation at work and better living standards, work-life balance is an important determinate. It is expected that not just monetary benefits increase the motivation but also psychological factor like approval and feeling of helping others. Bingol city is located in East of Turkey and has just one main government hospital providing main healthcare services to 106 thousand people in the city centre and surrounding districts. The purpose of this study is to determine preferences of job life quality and motivation of workers in healthcare. To determine the Job Quality of Life (JQL) and motivating factor affecting satisfaction, 109 surveys about 10% of staffs (900 staffs) were applied to the hospital during the third quarter of 2016. Descriptive statistics, One-Way ANOVA test, Kruskal Wallis Test, Pearson correlation and regression models are used to analyze questionnaires and check the hypotheses. The research conclusions include some thesis. Materialistic prizes such as permission and wage increases are seen as the most effective rewarding methods with high 3.93 mean. But the older staffs prefer first of all to have more cooperation and communication with colleagues and management. Furthermore, they care more about approval and psychological needs. Female staffs prefer to have training and promotion for awarding of successes than monetary benefits and also do not want to have closer relationships with management. The important fact that the work satisfaction has the strongest correlation with the preference for motivation and a moral main group among six sub-groups. The most effective factor to improve JQL is people's needs group with 0.353 coefficients from the regression model.