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.

From meadows to milk to mucosa – adaptation of Streptococcus and Lactococcus species to their nutritional environments

Lactic acid bacteria (LAB) are indigenous to food-related habitats as well as associated with the mucosal surfaces of animals. The LAB family Streptococcaceae consists of the genera Lactococcus and Streptococcus. Members of the family include the industrially important species Lactococcus lactis, which has a long history safe use in the fermentative food industry, and the disease-causing streptococci Streptococcus pneumoniae and Streptococcus pyogenes. The central metabolic pathways of the Streptococcaceae family have been extensively studied because of their relevance in the industrial use of some species, as well as their influence on virulence of others. Recent developments in high-throughput proteomic and DNA-microarray techniques, in in vivo NMR studies, and importantly in whole-genome sequencing have resulted in new insights into the metabolism of the Streptococcaceae family. The development of cost-effective high-throughput sequencing has resulted in the publication of numerous whole-genome sequences of lactococcal and streptococcal species. Comparative genomic analysis of these closely related but environmentally diverse species provides insight into the evolution of this family of LAB and shows that the relatively small genomes of members of the Streptococcaceae family have been largely shaped by the nutritionally rich environments they inhabit.

NADH-Mediated Gene Expression in Streptococcus pneumoniae and Role of Rex as a Transcriptional Repressor of the Rex-Regulon

Nicotinamide adenine dinucleotides (NAD(H)) play a vital role in various biological processes, including keeping the cellular redox balance. In this study, we investigate the regulatory responses of Streptococcus pneumoniae D39 to NADH and characterize the role of the Rex protein as a transcriptional repressor of the gapN, fba, pncB, adhB2, gap, and adhE genes. Transcriptomic analysis was used to observe the response of S. pneumoniae D39 to NADH. Our microarray studies revealed elevated expression of various genes/operons involved in transport and biosynthesis of niacin (gapN, fba, pncB, adhB2, gap, and adhE). Promoter lacZ-fusion assays and microarray studies with the rex mutant revealed the role of Rex as a transcriptional repressor of gapN, fba, pncB, adhB2, gap, and adhE involved in niacin uptake and biosynthesis, in the presence of NADH. We predict the operator site (5 ' TTGTKAWAAWWTTCACAA-3 ' of Rex in the regulatory regions of Rex-regulated genes that was subsequently validated by promoter mutational experiments

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.

Structure, Organization, and Expression of the lct Gene for Lacticin 481, a Novel Lantibiotic Produced by Lactococcus lactis

The structural gene for the lactococcal lantibiotic lacticin 481 (lct) has been identified and cloned using a degenerated 20-mer DNA oligonucleotide based on the amino-terminal 7 amino acid residues of the purified protein. The transcription of the lct gene was analyzed, and its promoter was mapped. DNA sequence analysis of the lct gene revealed an open reading frame encoding a peptide of 51 amino acids. Comparison of its deduced amino acid sequence with the amino-terminal sequence and the amino acid composition of lacticin 481 indicates that the 61-residue peptide is prelacticin 481, containing a 27-residue carboxyl-terminal propeptide and a 24-residue amino-terminal leader peptide which lacks the properties of a typical signal sequence and which is significantly different from the leaders of other lantibiotics. The predicted amino acid sequence of prolacticin 481 contains 3 cysteines, 2 serines, and 2 threonines which were not detectable in amino acid analyses of mature lacticin 481. Based on these results and on characterization by two-dimensional NMR techniques, a structural model is proposed in which 2 cysteine residues are involved in lanthionine and one in β-methyllanthionine formation, and a 4th threonine residue is dehydrated. This model predicts a molecular mass for lacticin 481 of 2,901, which is in excellent agreement with that obtained from mass spectrometry.

The protective layer of biofilm:A repellent function for a new class of amphiphilic proteins

Bacteria can survive harsh conditions when growing in complex communities of cells known as biofilms. The matrix of the biofilm presents a scaffold where cells are attached to each other and to the surface. The biofilm matrix is also a protective barrier that confers tolerance against various antimicrobial agents. In this issue of Molecular Microbiology, Kobayashi and Iwano (2012) show that the liquid permeability of Bacillus subtilis biofilms is determined by a small secreted protein, i.e. BslA (formerly called YuaB). BslA is important for the proper development of biofilms, but unlike exopolysaccharide and TasA, is not directly involved in cell cluster formation, and is synthesized following the production of exopolysaccharide and amyloid fibres. The amphiphilic BslA protein forms a polymer in vitro and localizes in vivo to the surface of the biofilm. The microstructures of the biofilm wrinkles are reduced in the bslA mutant strain and the liquid repellency of the biofilm surface is diminished. Exogenously added BslA42181 protein complements the bslA mutation and restores not only water repellency, but also the formation of aerial structures. This study demonstrates that amphiphilic proteins have an important role in liquid repellency of biofilms and it suggests that these polymers contribute to antimicrobial resistance

Combinatorial biosynthesis for the generation of new-to-nature peptide antimicrobials

Natural peptide products are a valuable source of important therapeutic agents, including antibiotics, antivirals and crop protection agents. Aided by an increased understanding of structure-activity relationships of these complex molecules and the biosynthetic machineries that produce them, it has become possible to re-engineer complete machineries and biosynthetic pathways to create novel products with improved pharmacological properties or modified structures to combat antimicrobial resistance. In this review, we will address the progress that has been made using non-ribosomally produced peptides and ribosomally synthesized and post-translationally modified peptides as scaffolds for designed biosynthetic pathways or combinatorial synthesis for the creation of novel peptide antimicrobials

Transcriptome analysis and prediction of the metabolic state of stress-induced viable but non-culturable Bacillus subtilis cells

Many bacteria adapt their physiology and enter the viable but non-culturable state to survive prolonged exposure to adverse environmental conditions. The VBNC cells maintain active metabolism, membrane integrity and gene transcription. However, they lose the ability to form colonies on a conventional culture media. Thus, standard colony counting methods cannot detect these alive but dormant cells. The Gram-positive bacterium Bacillus subtilis was found to enter the VBNC state when pre-exposed to osmotic stress and treated with a lethal dose of kanamycin. These cells reduced their metabolic activity, ceased growth and division and became kanamycin-tolerant. Interestingly, despite active metabolism, the majority of the kanamycin tolerant cells could not be revived on LB agar. In this study, we use a robust RNA-Seq technique to elucidate the differences in transcriptional profiles of B. subtilis VBNC cells. A comparative analysis of differently expressed genes and operons performed in this study indicates high similarities in transcriptional responses of VBNC and kanamycin-sensitive cells to antibiotic treatment. Moreover, this work reveals that VBNC cells strongly upregulate genes involved in proline uptake and catabolism, suggesting a putative role of proline as nutrient in VBNC cells

BrevicidineB, a New Member of the Brevicidine Family, Displays an Extended Target Specificity

The group of bacterial non-ribosomally produced peptides (NRPs) has formed a rich source for drug development. Brevicidine, a bacterial non-ribosomally produced cyclic lipo-dodecapeptide, displays selective antimicrobial activity against Gram-negative pathogens. Here, we show that brevicidineB, which contains a single substitution (Tyr2 to Phe2) in the amino acid sequence of the linear part of brevicidine, has a broadened antimicrobial spectrum, showing bactericidal activity against both Gram-negative (with a MIC value of 2 to 4 mg/L) and Gram-positive (with a MIC value of 2 to 8 mg/L) pathogens. Compared with an earlier reported member of the brevicidine family, the broadened antimicrobial spectrum of brevicidineB is caused by its increased membrane disruptive capacity on Gram-positive pathogens, which was evidenced by fluorescence microscopy assays. In addition, DiSC3(5) and resazurin assays show that brevicidine and brevicidineB exert their antimicrobial activity against Gram-negative bacteria via disrupting the proton motive force of cells. Notably, as a brevicidine family member, brevicidineB also showed neither hemolytic activity nor cytotoxicity at a high concentration of 64 mg/L. This study provides a promising antibiotic candidate (brevicidineB) with a broad antimicrobial spectrum, and provides novel insights into the antimicrobial mode of action of brevicidines

Analysis of cross-functionality within LanBTC synthetase complexes from different bacterial sources with respect to production of fully modified lanthipeptides

Lanthipeptides belong to a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) containing (methyl)lanthionine residues. Commonly, class I lanthipeptides are synthesized by a gene cluster encoding a precursor peptide (LanA), a biosynthetic machinery (LanBTC), a protease (LanP), a two-component regulatory system (LanRK), and an immunity system (LanI and LanFEG). Although nisin and subtilin are highly similar class I lanthipeptides, the cross-regulation by LanRK and the cross-immunity by LanI and LanFEG between the nisin and subtilin systems have been proven very low. Here, the possibility of the cross-functionality by LanBTC to modify and transport nisin precursor (NisA) and subtilin precursor (SpaS) was evaluated in Bacillus subtilis and Lactococcus lactis. Interestingly, we found that a promiscuous NisBC-SpaT complex is able to synthesize and export nisin precursor, as efficiently as the native nisin biosynthetic machinery NisBTC, in L. lactis, but not in B. subtilis. The assembly of the NisBC-SpaT complex at a single microdomain, close to the old cell pole, was observed by fluorescence microscopy in L. lactis. In contrast, such a complex was not formed in B. subtilis. Furthermore, the isolation of the NisBC-SpaT complex and its subcomplexes from the cytoplasmic membrane of L. lactis by pull-down assays was successfully conducted. Our work demonstrates that the association of LanBC with LanT is critical for the efficient biosynthesis and secretion of the lanthipeptide precursor with complete modifications, and suggests a cooperative mechanism between LanBC and LanT in the modification and transport processes. IMPORTANCE A multimeric synthetase LanBTC complex has been proposed for the in vivo production of class I lanthipeptides. However, it has been demonstrated that LanB, LanC, and LanT can perform their functionality in vivo and in vitro, independently of other Lan proteins. The role of protein-protein interactions, especially between the modification complex LanBC and the transport system LanT, in the biosynthesis process of lanthipeptides is still unclear. In this study, the importance of the presence of a well-installed LanBTC complex in the cell membrane for lanthipeptide biosynthesis and transport was reinforced. In L. lactis, the recruitment of SpaT from the peripheral cell membrane to the cell poles by the NisBC complex was observed, which may explain the mechanism by which secretion of premature peptide is prevented