Scale-up of a Type I secretion system in E. coli using a defined mineral medium.

Secretion of heterologous proteins into the culture supernatant in laboratory strains of Escherichia coli is possible by utilizing a Type I secretion system (T1SS). One prominent example for a T1SS is based on the hemolysin A toxin. With this system, heterologous protein secretion has already been achieved. However, no cultivations in a defined mineral medium and in stirred tank bioreactors have been described in literature up to now, hampering the broad applicability of the system. In this study, a mineral medium was developed for cultivation under defined conditions. With this medium, the full potential and advantage of a secretion system in E. coli (low secretion of host proteins, no contamination with proteins from complex media compounds) can now be exploited. Additionally, quantification of the protein amount in the supernatant was demonstrated by application of the Bradford assay. In this work, host cell behavior was described in small scale by online monitoring of the oxygen transfer rate. Scalability was demonstrated by stirred tank fermentation yielding 540 mg/L HlyA1 in the supernatant. This work enhances the applicability of a protein secretion system in E. coli and paves the way for an industrial application.

Effectiveness of architectural separation of septic and aseptic operating theatres for improving process quality and patient outcomes: a systematic review.

BACKGROUND: Architectural division of aseptic and septic operating theatres is a distinct structural feature of surgical departments in Germany. Internationally, hygienists and microbiologists mainly recommend functional separation (i.e. aseptic procedures first) without calling for separate operating floors and rooms. However, patients with severe musculoskeletal infections (e.g. joint empyema, spondylodiscitis, deep implant-associated infections) may benefit from the permanent availability of septic operating capacities without delay caused by an ongoing aseptic surgical program. A systematic literature review on the influence of a structural separation of septic and aseptic operating theatres on process and/or outcome quality has not yet been conducted. METHODS: Systematic literature search in PubMed MEDLINE, Ovid Embase, CINAHL and the Cochrane Library, screening of referenced citations, and assessment of grey literature. RESULTS: A total of 572 articles were found through the systematic literature search. No head-to-head studies (neither randomised, quasi-randomised nor observational) were identified which examined the impact of structural separation of septic and aseptic operating theatres on process and/or outcome quality. CONCLUSIONS: This review did not identify evidence in favour nor against architectural separation of septic or aseptic operating theatre. Specifically, there is no evidence of a harmful effect of architectural separation. Unless prospective studies, ideally randomised trials, will be available, it is unjustified to call for abolishing established hospital structures. Future investigations must address patient-centered endpoints, surgical site infections, process quality and hospital economy. SYSTEMATIC REVIEW REGISTRATION: PROSPERO (International prospective register of systematic reviews): CRD42018086568.

Efficacy and safety of programmable compared with fixed anti-siphon devices for treating idiopathic normal-pressure hydrocephalus (iNPH) in adults - SYGRAVA: study protocol for a randomized trial.

BACKGROUND: Idiopathic normal-pressure hydrocephalus (iNPH) is a distinct form of dementia, characterized by gait ataxia, cognitive impairment and urinary incontinence. In contrast to all other causes of dementia (e.g., Alzheimer-type and others), ventriculoperitoneal (VP) shunt surgery may offer a curative treatment option to patients. While being a rather low-risk type of surgery, it may cause significant over- or underdrainage complications (e.g., headaches, dizziness, vomiting, intracerebral bleeding, etc.) during posture change. Anti-siphon devices (ASDs) are a group of technically different additional valves used in shunt surgery. They are designed to maintain intraventricular pressure within a normal physiological range regardless of patient position. Fixed ASDs proved to substantially lower the rate of overdrainage complications. No significant differences, however, were noted regarding underdrainage complications. Technical successors of fixed ASDs are programmable ASDs. The aim of this study is to evaluate whether programmable ASDs compared to fixed ASDs are able to avoid both over- and underdrainage complications. METHODS/DESIGN: In this investigator-initiated, multicenter randomized trial, 306 patients are planned to be recruited. Male and female patients aged ≥18 years with iNPH who are eligible for VP shunt surgery and meet all other entry criteria can participate. Patients will be randomized in a balanced 1: 1 fashion to a VP shunt with a programmable valve either supplemented with a fixed ASD, or a programmable ASD. Patients will be followed-up 3, 6 and, on an optional basis, 12 months after surgery. The primary outcome measure is the cumulative incidence of over- or underdrainage 6 months post surgery, as defined by clinical and imaging parameters. DISCUSSION: SYGRAVA is the first randomized trial to determine whether programmable ASDs reduce complications of drainage compared to fixed ASDs in patients with iNPH. The results of this study may contribute to health-technology assessment of different valve systems used for VP-shunt surgery, and determination of the future standard of care. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number: ISRCTN13838310 . Registered on 10 November 2016.

An A/U-Rich Enhancer Region Is Required for High-Level Protein Secretion through the HlyA Type I Secretion System.

Efficient protein secretion is often a valuable alternative to classic cellular expression to obtain homogenous protein samples. Early on, bacterial type I secretion systems (T1SS) were employed to allow heterologous secretion of fusion proteins. However, this approach was not fully exploited, as many proteins could not be secreted at all or only at low levels. Here, we present an engineered microbial secretion system which allows the effective production of proteins up to a molecular mass of 88 kDa. This system is based on the hemolysin A (HlyA) T1SS of the Gram-negative bacterium Escherichia coli, which exports polypeptides when fused to a hemolysin secretion signal. We identified an A/U-rich enhancer region upstream of hlyA required for effective expression and secretion of selected heterologous proteins irrespective of their prokaryotic, viral, or eukaryotic origin. We further demonstrate that the ribosomal protein S1 binds to the hlyA A/U-rich enhancer region and that this region is involved in the high yields of secretion of functional proteins, like maltose-binding protein or human interferon alpha-2.IMPORTANCE A 5' untranslated region of the mRNA of substrates of type I secretion systems (T1SS) drastically enhanced the secretion efficiency of the endogenously secreted protein. The identification of ribosomal protein S1 as the interaction partner of this 5' untranslated region provides a rationale for the enhancement. This strategy furthermore can be transferred to fusion proteins allowing a broader, and eventually a more general, application of this system for secreting heterologous fusion proteins.

Amylocyclicin, a novel circular bacteriocin produced by Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization-time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.

Plantazolicin A and B: structure elucidation of ribosomally synthesized thiazole/oxazole peptides from Bacillus amyloliquefaciens FZB42.

The structures of the ribosomally synthesized peptide antibiotics from Bacillus amyloliquefaciens FZB42, plantazolicin A and B, have been elucidated by high resolving ESI-MSMS, 2D (1)H-(13)C-correlated NMR spectroscopy as well as (1)H-(15)N-HMQC/(1)H-(15)N-HMBC NMR experiments. (15)N-labeling prior to the experiments facilitated the structure determination, unveiling a hitherto unusual number of thiazoles and oxazoles formed from a linear 14mer precursor peptide. This finding further extends the number of known secondary metabolites from B. amyloliquefaciens and represents a new type of secondary metabolites from the genus Bacillus.

Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42.

Here we report on a novel thiazole/oxazole-modified microcin (TOMM) from Bacillus amyloliquefaciens FZB42, a Gram-positive soil bacterium. This organism is well known for stimulating plant growth and biosynthesizing complex small molecules that suppress the growth of bacterial and fungal plant pathogens. Like microcin B17 and streptolysin S, the TOMM from B. amyloliquefaciens FZB42 undergoes extensive posttranslational modification to become a bioactive natural product. Our data show that the modified peptide bears a molecular mass of 1,335 Da and displays antibacterial activity toward closely related Gram-positive bacteria. A cluster of 12 genes that covers ∼10 kb is essential for the production, modification, export, and self-immunity of this natural product. We have named this compound plantazolicin (PZN), based on the association of several producing organisms with plants and the incorporation of azole heterocycles, which derive from Cys, Ser, and Thr residues of the precursor peptide.

More than anticipated - production of antibiotics and other secondary metabolites by Bacillus amyloliquefaciens FZB42.

The genome of environmental Bacillus amyloliquefaciens FZB42 harbors numerous gene clusters involved in synthesis of antifungal and antibacterial acting secondary metabolites. Five gene clusters, srf, bmy, fen, nrs, dhb, covering altogether 137 kb, direct non-ribosomal synthesis of the cyclic lipopeptides surfactin, bacillomycin, fengycin, an unknown peptide, and the iron siderophore bacillibactin. Bacillomycin and fengycin were shown to act against phytopathogenic fungi in a synergistic manner. Three gene clusters, mln, bae, and dif, with a total length of 199 kb were shown to direct synthesis of the antibacterial acting polyketides macrolactin, bacillaene, and difficidin. Both, non-ribosomal synthesis of cyclic lipopeptides and synthesis of polyketides are dependent on the presence of a functional sfp gene product, 4'-phosphopantetheinyl transferase, as evidenced by knockout mutation of the sfp gene resulting in complete absence of all those eight compounds. In addition, here we present evidence that a gene cluster encoding enzymes involved in synthesis and export of the antibacterial acting dipeptide bacilysin is also functional in FZB42. In summary, environmental FZB42 devoted about 340 kb, corresponding to 8.5% of its total genetic capacity, to synthesis of secondary metabolites useful to cope with other competing microorganisms present in the plant rhizosphere.

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.

Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42.

Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp(0)), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide. The GenBank accession numbers for gene clusters pks1(bae), pks2, and pks3(dif) are AJ 634060.2, AJ 6340601.2, and AJ 6340602.2, respectively.