Chromatic Bacteria – A Broad Host-Range Plasmid and Chromosomal Insertion Toolbox for Fluorescent Protein Expression in Bacteria

Differential fluorescent labeling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behavior in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green–yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5 transposon delivery plasmid, and a Tn7 transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5 transposon delivery system. The present study enables labeling bacteria with a set of plasmids available to the community. One potential application of fluorescently-tagged bacterial species is the study of bacteria–bacteria, bacteria–host, and bacteria–environment interactions

Selected marketing and human resources variables influencing sponsorship initiatives within corporate businesses :a South African perspective

In an ever changing world, consumers are less and less responsive to traditional advertising, which creates a challenge for marketers, as they need to constantly develop new marketing communication tools to make a lasting impression on current and potential consumers. Consumers of the twenty first century are described as emotionally acting individuals, and has led to a increase in creative and emotional marketing communication tools, for example sponsorship, which is an increasingly important element of an integrated marketing communication strategy. Because of the value sponsorship can have for a business, and the high costs involved in this marketing initiative, it is imperative to examine variables that might be influential, in order to apply this promotional activity to the business’ advantage (Belch & Belch 2007:12; Hartland, Skinner & Griffiths 2005:164-173; Nicholis, Roslow & Dublish 1999:365-387; Pham & Vanheule 1997:407-417). Because of the important effect of sponsorship, marketing managers seek clarity in respect of which events to support and to link their business’ brand with or not. There is however little guidance in literature available on which events to sponsor and how to exploit resources efficiently and effectively (Crimmins & Horn 1996; Speed & Thompson 2000). Therefore, the objective of this study is to identify factors that might influence sponsorship initiatives within corporate businesses in South Africa. The variables that was identified for the study at hand was marketing related variables namely branding, marketing ethics and green marketing, and human resources related variables namely, corporate culture, values and employee empowerment. In order to determine the influence of the predetermined variables on sponsorship, an empirical investigation was conducted. The positivistic (phenomenological) approach was used in this study as the aim was to determine whether a relationship exists between selected independent variables and the dependent variable, via the intervening variables, using statistical analysis. In order to gather primary data, selfadministered questionnaires were issued to 182 respondents by means of convenience and snowball sampling of which the results were analysed to arrive at conclusions regarding the research in question. The empirical analysis of the data followed the following statistical steps: exploratory factor analysis to test the validity of the measuring instrument, Cronbach Alpha correlation coefficients to confirm the reliability of the questionnaire, SEM goodness-of-fit, multiple regression analysis to test the hypothesised relationships between the independent, intervening and dependent variables, ANOVA and descriptive statistics. The main findings of this study suggest that branding and green marketing have a significantly positive relationship with sponsorship, indicating the importance of a business’ brand and environmental awareness in terms of its emotional marketing initiatives. These relationships iIn an ever changing world, consumers are less and less responsive to traditional advertising, which creates a challenge for marketers, as they need to constantly develop new marketing communication tools to make a lasting impression on current and potential consumers. Consumers of the twenty first century are described as emotionally acting individuals, and has led to a increase in creative and emotional marketing communication tools, for example sponsorship, which is an increasingly important element of an integrated marketing communication strategy. Because of the value sponsorship can have for a business, and the high costs involved in this marketing initiative, it is imperative to examine variables that might be influential, in order to apply this promotional activity to the business’ advantage (Belch & Belch 2007:12; Hartland, Skinner & Griffiths 2005:164-173; Nicholis, Roslow & Dublish 1999:365-387; Pham & Vanheule 1997:407-417). Because of the important effect of sponsorship, marketing managers seek clarity in respect of which events to support and to link their business’ brand with or not. There is however little guidance in literature available on which events to sponsor and how to exploit resources efficiently and effectively (Crimmins & Horn 1996; Speed & Thompson 2000). Therefore, the objective of this study is to identify factors that might influence sponsorship initiatives within corporate businesses in South Africa. The variables that was identified for the study at hand was marketing related variables namely branding, marketing ethics and green marketing, and human resources related variables namely, corporate culture, values and employee empowerment. In order to determine the influence of the predetermined variables on sponsorship, an empirical investigation was conducted. The positivistic (phenomenological) approach was used in this study as the aim was to determine whether a relationship exists between selected independent variables and the dependent variable, via the intervening variables, using statistical analysis. In order to gather primary data, selfadministered questionnaires were issued to 182 respondents by means of convenience and snowball sampling of which the results were analysed to arrive at conclusions regarding the research in question. The empirical analysis of the data followed the following statistical steps: exploratory factor analysis to test the validity of the measuring instrument, Cronbach Alpha correlation coefficients to confirm the reliability of the questionnaire, SEM goodness-of-fit, multiple regression analysis to test the hypothesised relationships between the independent, intervening and dependent variables, ANOVA and descriptive statistics. The main findings of this study suggest that branding and green marketing have a significantly positive relationship with sponsorship, indicating the importance of a business’ brand and environmental awareness in terms of its emotional marketing initiatives. These relationships imply that, according to respondents, if these two aspects improve within the business, so could its sponsorship initiatives. Interestingly, it was established that ethics was insignificant in terms of a business’sponsorship initiatives. Corporate culture and values had a positive relationship with sponsorship, and employee empowerment proved to be negatively related to Sport and Broadcast sponsorship, with no significant relationship with Education and Community sponsorship. Additionally, the empirical investigation revealed that the ethnicity of respondents exerted an influence on the perception employees have regarding ethics, employee empowerment and Sport and Broadcast sponsorship within the businesses they are employed at. As this study assisted in the development of sponsorship strategies for businesses to implement, it will have a direct benefit to marketers and businesses in general so that sponsorship initiatives can be directed in such a way to maximise the return on investment for a business. The implications of this study will be of great value to marketing managers as sponsorship is such an important marketing strategy and communications tool which impacts on the overall business objectives.mply that, according to respondents, if these two aspects improve within the business, so could its sponsorship initiatives. Interestingly, it was established that ethics was insignificant in terms of a business’ sponsorship initiatives. Corporate culture and values had a positive relationship with sponsorship, and employee empowerment proved to be negatively related to Sport and Broadcast sponsorship, with no significant relationship with Education and Community sponsorship. Additionally, the empirical investigation revealed that the ethnicity of respondents exerted an influence on the perception employees have regarding ethics, employee empowerment and Sport and Broadcast sponsorship within the businesses they are employed at. As this study assisted in the development of sponsorship strategies for businesses to implement, it will have a direct benefit to marketers and businesses in general so that sponsorship initiatives can be directed in such a way to maximise the return on investment for a business. The implications of this study will be of great value to marketing managers as sponsorship is such an important marketing strategy and communications tool which impacts on the overall business objectives

The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter

Crystal, Solution and In silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine

Dihydrodipicolinate synthase (DHDPS) catalyzes the rate limiting step in lysine biosynthesis in bacteria and plants. The structure of DHDPS has been determined from several bacterial species and shown in most cases to form a homotetramer or dimer of dimers. However, only one plant DHDPS structure has been determined to date from the wild tobacco species, Nicotiana sylvestris (Blickling et al. (1997) J. Mol. Biol. 274, 608–621). Whilst N. sylvestris DHDPS also forms a homotetramer, the plant enzyme adopts a ‘back-to-back’ dimer of dimers compared to the ‘head-to-head’ architecture observed for bacterial DHDPS tetramers. This raises the question of whether the alternative quaternary architecture observed for N. sylvestris DHDPS is common to all plant DHDPS enzymes. Here, we describe the structure of DHDPS from the grapevine plant, Vitis vinifera, and show using analytical ultracentrifugation, small-angle X-ray scattering and X-ray crystallography that V. vinifera DHDPS forms a ‘back-to-back’ homotetramer, consistent with N. sylvestris DHDPS. This study is the first to demonstrate using both crystal and solution state measurements that DHDPS from the grapevine plant adopts an alternative tetrameric architecture to the bacterial form, which is important for optimizing protein dynamics as suggested by molecular dynamics simulations reported in this study

L,L-Diaminopimelate Aminotransferase from Chlamydomonas reinhardtii: A Target for Algaecide Development

In some bacterial species and photosynthetic cohorts, including algae, the enzyme l,l-diaminopimelate aminotransferase (DapL) (E.C. 2.6.1.83) is involved in the anabolism of the essential amino acid L-lysine. DapL catalyzes the conversion of tetrahydrodipicolinate (THDPA) to l,l-diaminopimelate (l,l-DAP), in one step bypassing the DapD, DapC and DapE enzymatic reactions present in the acyl DAP pathways. Here we present an in vivo and in vitro characterization of the DapL ortholog from the alga Chlamydomonas reinhardtii (Cr-DapL). The in vivo analysis illustrated that the enzyme is able to functionally complement the E. coli dap auxotrophs and was essential for plant development in Arabidopsis. In vitro, the enzyme was able to inter-convert THDPA and l,l-DAP, showing strong substrate specificity. Cr-DapL was dimeric in both solution and when crystallized. The structure of Cr-DapL was solved in its apo form, showing an overall architecture of a α/β protein with each monomer in the dimer adopting a pyridoxal phosphate-dependent transferase-like fold in a V-shaped conformation. The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis. Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid l-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides

The basis for non-canonical ROK family function in the N-acetylmannosamine kinase from the pathogen Staphylococcus aureus

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the "boat" conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections

Intramolecular Epistasis and the Evolution of a New Enzymatic Function

Atrazine chlorohydrolase (AtzA) and its close relative melamine deaminase (TriA) differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine

Investigating the catalytic and regulatory mechanisms of dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes-the condensation of (S)-aspartate-β-semialdehyde ((S)-ASA) and pyruvate. In an attempt to better understand the reaction catalysed by DHDPS, the wild-type enzyme was overexpressed and, following purification, kinetically characterised using a coupled assay. The kinetic mechanism was of the ping-pong type and the lunetic parameters obtained were consistent with other literature values. An improved synthesis of (S)-ASA was successfully achieved in three steps with an overall yield of 94%; this represents a significant advance over previously published routes to (S)-ASA. There are literature reports that high levels of (S)-ASA inhibit DHDPS, whilst others have not observed this phenomenon. It is shown unequivocally that this difference can be attributed to the different methods of preparing (S)-ASA used by each researcher: DHDPS is not inhibited by (S)-ASA, rather, the inhibition is due to an, as yet, unidentified inhibitor in the preparations of the substrate generated by ozonolysis. Others have published the crystal structure of wild-type DHDPS to 2.5-Å. They have hypothesized that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Y133, T44, and Y107 that provides a proton-relay to transport protons within the active site and from the active site to solvent. Additionally, R138 has been implicated in (S)-ASA binding. These hypotheses were tested using site-directed mutagenesis to produce five mutant enzymes: DHDPSY133F, DHDPS-T44S, DHDPS-R138H, DHDPS-T44V, and DHDPS-Y107F. Each of these mutants had reduced catalytic activity, consistent with the catalytic triad hypothesis. DHDPSR138H showed an increased KmASA, consistent with its role in (S)-ASA binding. The crystal structures of DHDPS-Y133F, DHDPS-T44V, DHDPS-Y107F were determined to at least 2.35-Å resolution and compared to the wild-type structure. All mutant enzymes crystallised into the same space group as the wild-type and only minor differences in structure were observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS. The mechanism of lysine inhibition in DHDPS appears complex but two hypotheses were previously suggested. These were that lysine affects the proton-relay and/or the flexibility of R138 to inhibit DHDPS catalysis. The mutants generated above were used to test these hypothesises. DHDPSY133F, DHDPS-T44V, and DHDPS-R138H showed less sensitivity to lysine inhibition compared to the wild-type, while DHDPS-T44S and DHDPS-Y107F showed identical behaviour to the wild-type. The results showed that some mutations in the proton-relay attenuated lysine inhibition so lysine may operate, at least in part, via this motif. That DHDPS-R138H also showed decreased sensitivity to lysine suggests that this residue also has some role in lysine inhibition. However, the crystal structure of DHDPS-T44V with bound lysine showed that the flexibility of R138 had increased, in contrast to the situation of the wild-type. To reconcile these results, a new mechanism of inhibition is proposed involving a hitherto undocumented channel of well-defined water molecules

A Three-Ring Circus: Metabolism of the Three Proteogenic Aromatic Amino Acids and Their Role in the Health of Plants and Animals

Tyrosine, phenylalanine and tryptophan are the three aromatic amino acids (AAA) involved in protein synthesis. These amino acids and their metabolism are linked to the synthesis of a variety of secondary metabolites, a subset of which are involved in numerous anabolic pathways responsible for the synthesis of pigment compounds, plant hormones and biological polymers, to name a few. In addition, these metabolites derived from the AAA pathways mediate the transmission of nervous signals, quench reactive oxygen species in the brain, and are involved in the vast palette of animal coloration among others pathways. The AAA and metabolites derived from them also have integral roles in the health of both plants and animals. This review delineates the de novo biosynthesis of the AAA by microbes and plants, and the branching out of AAA metabolism into major secondary metabolic pathways in plants such as the phenylpropanoid pathway. Organisms that do not possess the enzymatic machinery for the de novo synthesis of AAA must obtain these primary metabolites from their diet. Therefore, the metabolism of AAA by the host animal and the resident microflora are important for the health of all animals. In addition, the AAA metabolite-mediated host-pathogen interactions in general, as well as potential beneficial and harmful AAA-derived compounds produced by gut bacteria are discussed. Apart from the AAA biosynthetic pathways in plants and microbes such as the shikimate pathway and the tryptophan pathway, this review also deals with AAA catabolism in plants, AAA degradation via the monoamine and kynurenine pathways in animals, and AAA catabolism via the 3-aryllactate and kynurenine pathways in animal-associated microbes. Emphasis will be placed on structural and functional aspects of several key AAA-related enzymes, such as shikimate synthase, chorismate mutase, anthranilate synthase, tryptophan synthase, tyrosine aminotransferase, dopachrome tautomerase, radical dehydratase, and type III CoA-transferase. The past development and current potential for interventions including the development of herbicides and antibiotics that target key enzymes in AAA-related pathways, as well as AAA-linked secondary metabolism leading to antimicrobials are also discussed