Expression systems for industrial Gram-positive bacteria with low guanine and cytosine content

Recent years have seen an increase in the development of gene expression systems for industrial Gram-positive bacteria with low guanine and cytosine content that belong to the genera Bacillus, Clostridium, Lactococcus, Lactobacillus, Staphylococcus and Streptococcus. In particular, considerable advances have been made in the construction of inducible gene expression systems based on the capacity of these bacteria to utilize specific sugars or to secrete autoinducing peptides that are involved in quorum sensing. These controlled expression systems allow for present and future exploitation of these bacteria as cell factories in medical, agricultural, and food biotechnology.

Quorum sensing-controlled gene expression in lactic acid bacteria

Quorum sensing in lactic acid bacteria (LAB) involves peptides that are directly sensed by membrane-located histidine kinases, after which the signal is transmitted to an intracellular response regulator. This regulator in turn activates transcription of target genes, that commonly include the structural gene for the inducer molecule. The two-component signal-transduction machinery has proven to be indispensable for transcription activation and production of several autoinducers found in LAB, which are predominantly bacteriocins or bacteriocin-like peptides. In the nisin autoregulation process in Lactococcus lactis the NisK protein acts as the sensor for nisin and the NisR protein as the response regulator, activating transcription of target genes. The cis-acting elements for NisR were identified as the nisA and nisF promoter fragments and these were further analysed for inducibility. Based on this knowledge efficient nisin-controlled expression (NICE) systems were developed for several different lactic acid bacteria. A promising application of the NICE system is the development of autolytic starter lactococci, which will lyse in an early stage during cheese ripening thereby facilitating the release of intracellular enzymes which can contribute to flavour formation.

Controlled overproduction of proteins by lactic acid bacteria

Lactic acid bacteria are widely used in industrial food fermentations, contributing to flavour, texture and preservation of the fermented products. Here we describe recent advances in the development of controlled gene expression systems, which allow the regulated overproduction of any desirable protein by lactic acid bacteria. Some systems benefit from the fact that the expression vectors, marker genes and inducing factors can be used directly in food applications since they are all derived from food-grade lactic acid bacteria. These systems have also been employed for the development of autolytic bacteria, suitable for various industrial applications.

The proceedings of the Tenth Symposium on Lactic Acid Bacteria

We were delighted to be asked by Microbial Cell Factories to act as guest editors for the manuscripts in this issue associated with the LAB10 Symposium. The LAB Sympo- sia have been very important benchmarks in the careers of many LAB researchers, charting the development of this field from the very beginnings of our understanding of the molecular biology and physiology of these hugely interest- ing organisms right through to the current sophisticated ‘omics’ era. While we have significantly improved our understanding of the basic physiology and the key roles of individual organisms and the contributions of individual molecular mechanisms to the many industrial applications of LAB, our recognition and investigation of the potential of these organisms in human health has been one of the most rewarding aspects of the last few decades. The nine meetings to date have served to bring the global LAB community together every three years to review progress and the plan for the future in a friendly, collaborative set- ting. It is not an exaggeration to suggest that the Symposia have been a key factor in the extraordinary world of LAB biology, one in which direct competition has peacefully co-existed with a genuine warmth and collaborative spirit. We expect and hope that the tenth meeting will continue this tradition of ‘coopetition’. The papers contained in this issue are linked to oral presentations at LAB10 by some of the leaders and up- and-coming scientists in our field, and we hope that this collection of manuscripts will fulfil at least two roles. Firstly, they will give an opportunity for a wider audience to evaluate progress in the field, and secondly, they will prove invaluable in the future as a record of the state of the art at this point in our on-going engagement with these industrially important microorganisms. The manuscripts (both reviews and original papers) cover the main thematic areas of the meeting; Evolution, Systems Biology and Metabolic Engineering, Host Microbe Interaction, Nutrition and Health, Preservations and Stress, and fermentation and Application. We have sought to maintain the usual high standard applied to all MCF submissions for the papers in this issue, and so each paper was subjected to the usual peer review process. We thank the MCF Editor-in-Chief Antonio Villaverde and Helen Whitaker of BioMed Central for their support during the editorial process, the authors for their prompt response to our requests for a rapid process and, most of all, the reviewers who gave their time and effort to review the manuscripts

Anaerobic treatment of Phthalates : microbiological and technological aspects

Phthalic acid isomers (dicarboxy benzenes) play an important role in our human environment as constituents of polyester fibres, films, polyethylene terephthalate (PET) bottles and other plastics. Due to the use and generation of water during phthalic acid production from the corresponding xylenes, a concentrated wastewater is generated. The generated wastewater consists of a mixture of phthalic acid isomers, acetic acid, benzoic acid, and toluic acids. The aim of the work described in this thesis was to elucidate if anaerobic biological treatment may represent an attractive alternative for conventionally applied aerobic treatment methods. With regard to the anaerobic biodegradability of the phthalate isomers it was demonstrated that all three phthalate isomers could be degraded by two types of methanogenic granular sludge and digested sewage sludge. Lag-phases prior to degradation ranged from 17 to 156 days. More reduced aromatic analogues of the phthalates were not degraded or only at extremely low rates.Kinetic properties of the anaerobic degradation of the phthalate isomers were studied using enrichment cultures obtained from the biodegradability experiments or bioreactor biomass. The phthalate isomers grown cultures were capable of degrading only one of the phthalate isomers and degraded benzoate without a lag-period. A three species kinetic model enabled the dynamic description of intermediate acetate and molecular hydrogen accumulation and final formation of methane from the phthalate isomers and benzoate. It was shown that the syntrophic biomass cultivated had a low growth rate on the phthalate isomers (μ max≈ 0.09 day -1 ). The energetic efficiency for growth on the phthalate isomers was found to be significantly smaller when compared to growth on benzoate, suggesting that an energetic inefficiency prevails in the degradation pathway of the phthalate isomers. The cultures were furthermore strongly inhibited and even deactivated by co-incubation with acetate or benzoate, or a short period of a few hours without substrate.Despite these unfavourable microbiological characteristics, it was demonstrated that highly active terephthalate degrading biomass could be cultivated at high concentrations in both UASB (Upflow Anaerobic Sludge Bed) reactors and hybrid reactors, resulting in high terephthalate removal capacities (15-20 g­COD(Chemical Oxygen Demand) · l ­1· day ­1 ). High-rate terephthalate degradation in the UASB-reactors was strictly dependent on inoculation of the reactor with granular biomass.After demonstrating that terephthalate as sole substrate could be degraded at high-rates, we studied the feasibility of a two-stage reactor concept for the treatment of terephthalic acid production wastewater, consisting of a mixture of readily degradable substrates (acetate and benzoate) and slowly degradable substrates (terephthalate and para -toluate). It was demonstrated that through pre-removal of acetate and benzoate in the first stage the lag-phase prior terephthalate degradation in the second stage could be significantly reduced (from 300 to approximately 50 days) and the wastewater could be treated at high volumetric removal rates and short hydraulic retention times (25 g­COD · l ­1· day ­1 and 6 hours respectively). For start-up of a two-stage anaerobic bioreactor system for treatment of terephthalic acid production wastewater, a gradual transition from initial operation in parallel to operation in series is suggested.<br/

Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon

Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 μmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function