Antibacterial aryl‐crowned polyketide from Bacillus subtilis associated with seaweed Anthophycus longifolius

Aims

Microbiological, biotechnological and chemical characterization of a previously undescribed aryl‐crowned polyketide from Bacillus subtilis MTCC 10403 isolated from brown seaweed Anthophycus longifolius with activity against opportunistic Gram‐negative food‐borne pathogenic bacterial strains.

Methods and Results

A culture‐dependent method was used to isolate heterotrophic B. subtilis associated with A. longifolius and assessed for its antimicrobial properties. Minimum inhibitory concentration (MIC) of the title compound against the test pathogens was analysed by microtube dilution coupled with the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide‐based colorimetric endpoint detection. Bacillus subtilis MTCC 10403 was found to be antagonistic against Gram‐negative food‐borne pathogenic Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Salmonella enterica serotype Typhi, Aeromonas hydrophila and Vibrio sp. (diameter of zone of growth inhibition 13–22 mm). Bacillus subtilis was assessed for the presence of secondary metabolite coding polyketide synthase (pks) gene ( KC589397 , 700‐bp gene product) and carboxylate siderophore framework in the aryl‐crowned polyketide designated as 7‐O‐6′‐(2″‐acetylphenyl)‐5′‐hydroxyhexanoate‐macrolactin by exhaustive spectroscopic techniques. The MIC assay showed that the reference antibiotics tetracycline and ampicillin were active at 25 μg ml^?1 against the test pathogens, whereas the newly isolated polyketide displayed anti‐infective properties against E. coli, A. hydrophilla, P. aeruginosa and Vibrio sp. at a lower concentration (MIC <13 μg ml^?1). The MIC of the aryl macrolactin against K. pneumoniae was comparable with that of the referral antibiotics (~25 μg ml^?1). The mode of antimicrobial action of acryl‐crowned macrolactin was found to be iron chelating similar to siderophores. Putative biosynthetic pathway of the pks gene product further validated its molecular attributions.

Conclusions

This study recognized new variant of antimicrobial aryl‐crowned polyketide bearing methyl 6′‐(2″‐acetylphenyl)‐5′‐hydroxyhexanoate moiety at the C‐7 position of the macrolactin system from A. longifolius‐associated bacterium B. subtilis.

Significance and Impact of the Study

This study revealed seaweed‐associated micro‐organisms as promising biological strata to produce new‐generation anti‐infective agents.     

Revisiting the membrane interaction mechanism of a membrane‐damaging β‐barrel pore‐forming toxin Vibrio cholerae cytolysin

Vibrio cholerae cytolysin (VCC) permeabilizes target cell membranes by forming transmembrane oligomeric β‐barrel pores. VCC has been shown to associate with the target membranes via amphipathicity‐driven spontaneous partitioning into the membrane environment. More specific interaction(s) of VCC with the membrane components have also been documented. In particular, specific binding of VCC with the membrane lipid components is believed to play a crucial role in determining the efficacy of the pore‐formation process. However, the structural basis and the functional implications of the VCC interaction with the membrane lipids remain unclear. Here we show that the distinct loop sequences within the membrane‐proximal region of VCC play critical roles to determine the functional interactions of the toxin with the membrane lipids. Alterations of the loop sequences via structure‐guided mutagenesis allow amphipathicity‐driven partitioning of VCC to the membrane lipid bilayer. Alterations of the loop sequences, however, block specific interactions of VCC with the membrane lipids and abort the oligomerization, membrane insertion, pore‐formation and cytotoxic activity of the toxin. Present study identifies the structural signatures in VCC implicated for its functional interactions with the membrane lipid components, a process that presumably acts to drive the subsequent steps of the oligomeric β‐barrel pore‐formation and cytotoxic responses.     

Distinct roles for U‐type proteins in iron–sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon

The biosynthesis of iron–sulfur (Fe–S) clusters in Bacillus subtilis is mediated by the SUF‐like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe–S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe–S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe–S cluster assembly.     

Oculocutaneous albinism type 4: six novel mutations in the membrane‐associated transporter protein gene and their phenotypes

Oculocutaneous albinism type 4 (OCA4) is an autosomal recessive hypopigmentary disorder caused by mutations in the Membrane‐Associated Transporter Protein gene (SLC45A2). The SLC45A2 protein is a 530‐amino‐acid polypeptide that contains 12 putative transmembrane domains, and appears to be a transporter that mediates melanin synthesis. Eighteen pathological mutations have been reported so far. In this study, six novel mutations, p.Y49C (c.146A > G), p.G89R (c.265G > A), p.C229Y (c.686G > A), p.T437A (c.1309A > G), p.T440A (c.1318A > G) and p.G473D (c.1418G > A) were found in eight Japanese patients with various clinical phenotypes. The phenotypes of OCA4 were as various as the other types of OCA and probably depended on the mutation sites in the SLC45A2 gene.     

Modular toxin from the lynx spider Oxyopes takobius: Structure of spiderine domains in solution and membrane‐mimicking environment

We have recently demonstrated that a common phenomenon in evolution of spider venom composition is the emergence of so‐called modular toxins consisting of two domains, each corresponding to a “usual” single‐domain toxin. In this article, we describe the structure of two domains that build up a modular toxin named spiderine or OtTx1a from the venom of Oxyopes takobius. Both domains were investigated by solution NMR in water and detergent micelles used to mimic membrane environment. The N‐terminal spiderine domain OtTx1a‐AMP (41 amino acid residues) contains no cysteines. It is disordered in aqueous solution but in micelles, it assumes a stable amphiphilic structure consisting of two α‐helices separated by a flexible linker. On the contrary, the C‐terminal domain OtTx1a‐ICK (59 residues) is a disulfide‐rich polypeptide reticulated by five S–S bridges. It presents a stable structure in water and its core is the inhibitor cystine knot (ICK) or knottin motif that is common among single‐domain neurotoxins. OtTx1a‐ICK structure is the first knottin with five disulfide bridges and it represents a good reference for the whole oxytoxin family. The affinity of both domains to membranes was measured with NMR using titration by liposome suspensions. In agreement with biological tests, OtTx1a‐AMP was found to show high membrane affinity explaining its potent antimicrobial properties.     

Lipid‐polymorphism of plant thylakoid membranes. Enhanced non‐bilayer lipid phases associated with increased membrane permeability

Earlier experiments, using ³¹P‐NMR and time‐resolved merocyanine fluorescence spectroscopy, have shown that isolated intact, fully functional plant thylakoid membranes, in addition to the bilayer phase, contain three non‐bilayer (or non‐lamellar) lipid phases. It has also been shown that the lipid polymorphism of thylakoid membranes can be characterized by remarkable plasticity, i.e. by significant variations in ³¹P‐NMR signatures. However, changes in the lipid‐phase behaviour of thylakoids could not be assigned to changes in the overall membrane organization and the photosynthetic activity, as tested by circular dichroism and 77 K fluorescence emission spectroscopy and the magnitude of the variable fluorescence of photosystem II, which all showed only marginal variations. In this work, we investigated in more detail the temporal stability of the different lipid phases by recording ³¹P‐NMR spectra on isolated thylakoid membranes that were suspended in sorbitol‐ or NaCl‐based media. We observed, at 5°C during 8 h in the dark, substantial gradual enhancement of the isotropic lipid phases and diminishment of the bilayer phase in the sorbitol‐based medium. These changes compared well with the gradually increasing membrane permeability, as testified by the gradual acceleration of the decay of flash‐induced electrochromic absorption changes and characteristic changes in the kinetics of fast chlorophyll a‐fluorescence transients; all variations were much less pronounced in the NaCl‐based medium. These observations suggest that non‐bilayer lipids and non‐lamellar lipid phases play significant roles in the structural dynamics and functional plasticity of thylakoid membranes.     

Solution structure of apo CopZ from Bacillus subtilis: further analysis of the changes associated with the presence of copper

The solution structure of apo CopZ from Bacillus subtilis has been determined with the aim of investigating the changes in the hydrophobic interactions around the M-X-C-X-X-C copper(I) binding motif upon metal binding. The methionine of this motif (Met 11 in CopZ) points toward the solvent in apo CopZ, whereas its sulfur atom is close to the metal ion in the metal-loaded protein, though probably not at binding distance. This change is associated with the weakening of the interaction between Leu 37 and Cys 16, present in the apo form, and the formation of an interaction between Met 11 and Tyr 65. Loops 1, 3, and 5 are affected by metal binding. Comparison with the structure of other homologous proteins confirms that often metal binding affects a hydrophobic patch around the metal site, possibly for optimizing and tuning the hydrophobic interactions with the partners. It is also shown that copper(I) exchanges among apo CopZ molecules in slow exchange on the NMR time scale, whereas it is known that such exchange between partner molecules (i.e., metallochaperones and metal pumps) is fast.     

Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.     

Proteins associated with the cell envelope of Trichoderma reesei: a proteomic approach

A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed.     

Major proteins of the outer cell envelope membrane of Escherichia coli K-12: multiple species of protein I.

Summary Protein I, one of the major outer membrane proteins ofE. coli, in a number of strains exists as two electrophoretically separable species Ia and Ib. Two phages, TuIa and TuIb, have been found which use, as receptors, proteins Ia and Ib, respectively. Selection for resistance to phage TuIb yielded mutants still possessing protein Ia and missing protein Ib (Ia⁺ Ib^-). Selection in this background, for resistance to phage TuIa yielded one class of mutants missing both species of protein I and another synthesizing a new species of protein I, polypeptide Ic.Tryptic fingerprints of Ia and Ic are very similar and the sequence of 8 N-terminal amino acids is identical for Ia and Ic. Yet, Ic showed an entirely different pattern of cyanogen bromide fragments than that of protein Ia. With another example (cyanogen bromide fragments of protein II^*, with and without performic acid oxidation) it is shown that protein modification can lead to gross changes of the electrophoretic mobility of cyanogen bromide fragments. It is not unlikely that all protein I species observed so far represent in vivo modifications of one and the same polypeptide chain.A genetic analysis together with data from other laboratories revealed that at least 4 widely separated chromosomal loci are involved in the expression of the protein I species known to date.     

Revisiting the cell biology of the acyl‐ACP:phosphate transacylase PlsX suggests that the phospholipid synthesis and cell division machineries are not coupled in Bacillus subtilis

PlsX is a central enzyme of phospholipid synthesis in bacteria, converting acyl‐ACP to acyl‐phosphate on the pathway to phosphatidic acid formation. PlsX has received attention because it plays a key role in the coordination of fatty acid and phospholipid synthesis. Recently, PlsX was also suggested to coordinate membrane synthesis with cell division in Bacillus subtilis. Here, we have re‐investigated the cell biology of PlsX and determined that the enzyme is uniformly distributed on the membrane of most cells, but occasionally appears as membrane foci as well. Foci and homogenous patterns seem freely interconvertible but the prevalence of the uniform staining suggests that PlsX does not need to localize to specific sites to function correctly. We also investigated the relationship between PlsX and the divisome. In contrast to previous observations, PlsX's foci showed no obvious periodicity of localization and did not colocalize with the divisome. Furthermore, depletion of PlsX did not affect cell division if phospholipid synthesis is maintained by an alternative enzyme. These results suggest that coordination between division and membrane synthesis may not require physical or functional interactions between the divisome and phospholipid synthesis enzymes.     

Isolation and characterization of hydrophobic proteins (H proteins) in the membrane fraction of Bacillus subtilis. Involvement in membrane biosynthesis and the formation of biochemically active membrane vesicles by combining H proteins with lipid.

Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions [light band (1.155 - 1.158 g/cm3); medium band (1.181 - 1.183 g/cm3); heavy band (1.21 - 1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5 - 8 nm in diameter. We named this H-protein. H-protein formed trilamellar unit membrane structure when combined with lipid. In pulse-labeling and pulse-chase experiments with radioactive leucine, it was found that H-protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H-protein was shifted into light and medium band of the membranes during the chase. Cytochromes were not found in H-protein. However, when H-protein was incubated with haem alpha and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H-protein and form cytochromes a and b. Cytochromes were also formed in H-protein which were isolated from the cells grown in the presence of haemin (haemin-grown H protein). Succinate dehydrogenase activity was increased about 4-fold by combining H-protein or haemin-grown H protein with lipid. H-protein had no cytochrome oxidase activity; however, haemin-grown H protein was found to have some of the activity and this was increased about 4-fold by combining the protein with lipid. Haemin-grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase was also found in haemin-grown H protein and was activated about 9-fold in constituted reaction systems. Vesicles formed by haemin-grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin. These results would indicate that H-protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.     

Interactions of the neural cell adhesion molecule and the myelin- associated glycoprotein with collagen type I: involvement in fibrillogenesis

To gain insights into the functional role of the molecular association between neural adhesion molecules and extracellular matrix constituents, soluble forms of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM), representing most of the extracellular domains of the molecules, were investigated in their ability to modify fibrillogenesis of collagen type I. MAG and N-CAM retarded the rate of fibril formation, as measured by changes in turbidity, and increased the diameter of the fibrils formed, but did not change the banding pattern when compared to collagen type I in the absence of adhesion molecules. Scatchard plot analysis of the binding of MAG and N-CAM to the fibril-forming collagen types I, II, III, and V suggest one binding site for N-CAM and two binding sites for MAG. Binding of MAG, but not of N-CAM, to collagen type I was decreased during fibril formation, probably due to a reduced accessibility of one binding site for MAG during fibrillogenesis. These results indicate that the neural adhesion molecules can influence the configuration of extracellular matrix constituents, thus, implicating them in the modulation of cell-substrate interactions.     

Actin—membrane interactions: Association of G-actin with the red cell membrane

Chemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G-actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G-actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside-out vesicles which have been stripped of endogenous actin and spectrin by low-ionic-strength incubation bind little G-actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G-actin can be increased up to 40-fold. Further, this crude spectrin extract can compete for and abolish G-actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G-actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G-actin is added to reconstituted inside-out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G-actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.     

Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg;

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.