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Seaweeds are of significant interest in the food, pharmaceutical, and agricultural industries as they contain several commercially relevant bioactive compounds. Current extraction methods for macroalgal-derived metabolites are, however, problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods, such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25 °C, and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina sequencing and culture-dependent approaches, including the novel ichip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculosus and were shown to be more efficient than commercial enzyme preparations in their extraction efficiencies.

Beta defensins comprise a family of cationic, cysteine-rich antimicrobial peptides, predominantly expressed at epithelial surfaces. Previously we identified a unique five-cysteine defensin-related peptide (Defr1) that, when synthesized, is a mixture of dimeric isoforms and exhibits potent antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Here we report that Defr1 displays antimicrobial activity against an extended panel of multidrug-resistant nosocomial pathogens for which antimicrobial treatment is limited or nonexistent. Defr1 fractions were collected by high-pressure liquid chromatography and analyzed by gel electrophoresis and mass spectrometry. Antimicrobial activity was initially investigated with the type strain Pseudomonas aeruginosa PAO1. All fractions tested displayed equivalent, potent antimicrobial activity levels comparable with that of the unfractionated Defr1. However, use of an oxidized, monomeric six-cysteine analogue (Defr1 Y5C), or of reduced Defr1, gave diminished antimicrobial activity. These results suggest that the covalent dimer structure of Defr1 is crucial to antimicrobial activity; this hypothesis was confirmed by investigation of a synthetic one-cysteine variant (Defr1-1cys). This gave an activity profile similar to that of synthetic Defr1 but only in an oxidized, dimeric form. Thus, we have shown that covalent, dimeric molecules based on the Defr1 beta-defensin sequence demonstrate antimicrobial activity even in the absence of the canonical cysteine motif.

Single-stranded DNA-binding (SSB) proteins stabilize single-stranded DNA, which is exposed by separation of the duplex during DNA replication, recombination and repair. The SSB protein from the hyperthermophile Aquifex aeolicus has been overexpressed in Escherichia coli, purified and characterized and crystals of the full-length protein (147 amino acids; M(r) 17 131.20) have been grown by vapour diffusion from ammonium sulfate pH 7.5 in both the absence and presence of ssDNA [dT(pT)(68)]. All crystals diffract to around 2.9 A resolution and those without bound DNA (native) belong to space group P2(1), with two tetramers in the asymmetric unit and unit-cell parameters a = 80.97, b = 73.40, c = 109.76 A, beta = 95.11 degrees . Crystals containing DNA have unit-cell parameters a = 108.65, b = 108.51, c = 113.24 A and could belong to three closely related space groups (I222, I2(1)2(1)2(1) or I4(1)) with one tetramer in the asymmetric unit. Electrospray mass spectrometry of the crystals confirmed that the protein was intact. Molecular replacement with a truncated E. coli SSB structure has revealed the position of the molecules in the unit cell and refinement of both native and DNA-bound forms is under way.

Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase and this post-translational modification of a single lysine residue is exceptionally specific. The exact details of the protein-protein interactions involved are unclear as a BPL:BCCP complex has not yet been isolated. Moreover, detailed information is lacking on the composition, biosynthesis and role of fatty acids in hyperthermophilic organisms. We have cloned, overexpressed and purified recombinant BPL and the biotinyl domain of BCCP (BCCP Delta 67) from the extreme hyperthermophile Aquifex aeolicus. In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCP Delta 67 at temperatures of up to 70 degrees C. Limited proteolysis of BPL with trypsin and chymotrypsin revealed a single protease-sensitive site located 44 residues from the N-terminus. This site is adjacent to the predicted substrate-binding site and proteolysis of BPL is significantly reduced in the presence of MgATP and biotin. Chemical crosslinking with 1-ethyl-3-(dimethylamino-propyl)-carbodiimide (EDC) allowed the isolation of a BPL:apo-BCCP Delta 67 complex. Furthermore, this complex was also formed between BPL and a BCCP Delta 67 mutant lacking the lysine residue (BCCP Delta 67 K117L) however, complex formation was considerably reduced using holo-BCCP Delta 67. These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCP Delta 67 to heterodimerize with BPL, and emphasizes that a network of interactions between residues on both proteins mediates protein recognition.

Photorhabdus are entomopathogenic members of the family Enterobacteriaceae. In addition to killing insects Photorhabdus also have a mutualistic association with nematodes from the family Heterorhabditidiae. Therefore, the bacteria have a complex life cycle that involves temporally separated pathogenic and mutualistic associations with two different invertebrate hosts. This tripartite Photorhabdus–insect–nematode association provides researchers with a unique opportunity to characterize the prokaryotic contribution to two different symbioses, i.e. pathogenicity and mutualism while also studying the role of the host in determining the outcome of association with the bacteria. In this review I will outline the life cycle of Photorhabdus and describe recent important advances in our understanding of the symbiology of Photorhabdus. Finally, the contribution made by this model to our understanding of the nature of symbiotic associations will be discussed. [ABSTRACT FROM AUTHOR]

Photorhabdusare bacteria found colonizing the gut of a specialized stage of the nematodeHeterorhabditis, called the infective juvenile (IJ). The IJ is a free-living stage of the nematode that seeks out and infects insect larvae. Once inside the insect the IJ releasePhotorhabdusinto the haemolymph where the bacteria rapidly proliferate, killing the insect within 48–72 h. The nematodes grow and reproduce in the insect cadaver by feeding on thePhotorhabdusbiomass. In this study we usePhotorhabdus temperataK122 to show that genes involved in iron acquisition play a key role during the course of the tripartite bacteria–nematode–insect interaction. We show that a strain carrying a mutation in a gene with homology toexbD, encoding a component of the TonB complex, is unable to grow well in conditions where iron is not freely available. In addition, this mutant, BMM417, requires a longer time to kill the insect larvae than the wild-type bacteria and this defect in pathogenicity is complemented by the co-injection of iron. Moreover, the increase in LT50 observed with BMM417 is correlated with a significantly slowerin vivogrowth rate suggesting that iron is limiting in the insect. We also show that BMM417 is unable to support the growth and development of theHeterorhabditisnematode. Addition of exogenous iron to the growth media restores nematode growth and development on BMM417, suggesting that aspects of iron metaboism inPhotorhabdusare important during the symbiosis with the nematode. [ABSTRACT FROM AUTHOR]

Bacteria are often found associated with surfaces as sessile bacterial communities called biofilms, and the formation of a biofilm can be split up into different stages each requiring the expression of specific genes. The production of extracellular polysaccharides (EPS) is important for the maturation of biofilms and is controlled by the Rcs two-component pathway in Escherichia coli (and other Gram-negative bacteria). In this study, we show, for the first time, that the RcsC sensor kinase is required for normal biofilm development in E. coli. Moreover, using a combination of DNA macroarray technology and transcriptional fusion analysis, we show that the expression of > 150 genes is controlled by RcsC in E. coli. In silico analyses of the RcsC regulon predicts that 50% of the genes encode proteins that are either localized to the envelope of E. coli or have activities that affect the structure/properties of the bacterial surface, e.g. the production of colanic acid. Moreover, we also show that RcsC is activated during growth on a solid surface. Therefore, we suggest that the RcsC sensor kinase may play an important role in the remodelling of the bacterial surface during growth on a solid surface and biofilm formation. [ABSTRACT FROM AUTHOR]

Photorhabdus asymbiotica is an emerging bacterial pathogen that causes locally invasive soft tissue and disseminated bacteremic infections in the United States and Australia. Although the source of infection was previously unknown, we report that the bacterium is found in a symbiotic association with an insect-pathogenic soil nematode of the genus Heterorhabditis. [ABSTRACT FROM AUTHOR]

Abstract: The Rcs phosphorelay is composed of the sensor kinase, RcsC, the HPt-domain protein RcsD and the response regulator, RcsB. In this review we discuss the role of the Rcs phosphorelay in the Enterobacteriaceae, highlighting the observation that the Rcs phosphorelay appears to play a key role in the temporal regulation of biofilm formation and pathogenicity. [Copyright &y& Elsevier]

Abstract: Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5′-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 Å resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed β- and γ-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the α- and β-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg2+, and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPΔ67 fragment and BSA, and is subject to self-biotinylation. [Copyright &y& Elsevier]