Oscar P. Kuipers

Complete genome sequence of Lactobacillus plantarum WCFS1

The 3,308,274-bp sequence of the chromosome of Lactobacillus plantarum strain WCFS1, a single colony isolate of strain NCIMB8826 that was originally isolated from human saliva, has been determined, and contains 3,052 predicted protein-encoding genes. Putative biological functions could be assigned to 2,120 (70%) of the predicted proteins. Consistent with the classification of L. plantarum as a facultative heterofermentative lactic acid bacterium, the genome encodes all enzymes required for the glycolysis and phosphoketolase pathways, all of which appear to belong to the class of potentially highly expressed genes in this organism, as was evident from the codon-adaptation index of individual genes. Moreover, L. plantarum encodes a large pyruvate-dissipating potential, leading to various end-products of fermentation. L. plantarum is a species that is encountered in many different environmental niches, and this flexible and adaptive behavior is reflected by the relatively large number of regulatory and transport functions, including 25 complete PTS sugar transport systems. Moreover, the chromosome encodes >200 extracellular proteins, many of which are predicted to be bound to the cell envelope. A large proportion of the genes encoding sugar transport and utilization, as well as genes encoding extracellular functions, appear to be clustered in a 600-kb region near the origin of replication. Many of these genes display deviation of nucleotide composition, consistent with a foreign origin. These findings suggest that these genes, which provide an important part of the interaction of L. plantarum with its environment, form a lifestyle adaptation region in the chromosome.

Quorum sensing by peptide pheromones and two component signal transduction systems in Gram-positive bacteria.

Cell‐density‐dependent gene expression appears to be widely spread in bacteria. This quorum‐sensing phenomenon has been well established in Gram‐negative bacteria, where N‐acyl homoserine lactones are the diffusible communication molecules that modulate cell‐density‐dependent phenotypes. Similarly, a variety of processes are known to be regulated in a cell‐density‐ or growth‐phase‐dependent manner in Gram‐positive bacteria. Examples of such quorum‐sensing modes in Gram‐positive bacteria are the development of genetic competence in Bacillus subtilis and Streptococcus pneumoniae, the virulence response in Staphylococcus aureus, and the production of antimicrobial peptides by several species of Gram‐positive bacteria including lactic acid bacteria. Cell‐density‐dependent regulatory modes in these systems appear to follow a common theme, in which the signal molecule is a post‐translationally processed peptide that is secreted by a dedicated ATP‐binding‐cassette exporter. This secreted peptide pheromone functions as the input signal for a specific sensor component of a two‐component signal‐transduction system. Moreover, genetic linkage of the common elements involved results in autoregulation of peptide‐pheromone production.

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

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.

Autoregulation of Nisin Biosynthesis in Lactococcus lactis by Signal Transduction

The post-translationally modified, antimicrobial peptide nisin is secreted by strains of Lactococcus lactis that contain the chromosomally located nisin biosynthetic gene cluster nisABTCIPRKFEG. When a 4-base pair deletion is introduced into the structural nisA gene (ΔnisA), transcription of ΔnisA is abolished. Transcription of the ΔnisA gene is restored by adding subinhibitory amounts of nisin, nisin mutants, or nisin analogs to the culture medium, but not by the unmodified precursor peptide or by several other antimicrobial peptides. Upon disruption of the nisK gene, which encodes a putative sensor protein that belongs to the class of two-component regulators, transcription of ΔnisA was no longer inducible by nisin. Fusion of a nisA promoter fragment to the promoterless reporter gene gusA resulted in expression of gusA in L. lactis NZ9800 (ΔnisA) only upon induction with nisin species. The expression level of gusA was directly related to the amount of inducer that was added extracellularly. These results provide insight into a new mechanism of autoregulation through signal transduction in prokaryotes and demonstrate that antimicrobial peptides can exert a second function as signaling molecules.

Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

SUMMARY Secretory proteins perform a variety of important“ remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ∼90 extracellular proteins were identified. Analysis of these proteins disclosed various“ secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only∼ 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.

LysM, a widely distributed protein motif for binding to (peptido)glycans

Bacteria retain certain proteins at their cell envelopes by attaching them in a non‐covalent manner to peptidoglycan, using specific protein domains, such as the prominent LysM (Lysin Motif) domain. More than 4000 (Pfam PF01476) proteins of both prokaryotes and eukaryotes have been found to contain one or more Lysin Motifs. Notably, this collection contains not only truly secreted proteins, but also (outer‐)membrane proteins, lipoproteins or proteins bound to the cell wall in a (non‐)covalent manner. The motif typically ranges in length from 44 to 65 amino acid residues and binds to various types of peptidoglycan and chitin, most likely recognizing the N‐acetylglucosamine moiety. Most bacterial LysM‐containing proteins are peptidoglycan hydrolases with various cleavage specificities. Binding of certain LysM proteins to cells of Gram‐positive bacteria has been shown to occur at specific sites, as binding elsewhere is hindered by the presence of other cell wall components such as lipoteichoic acids. Interestingly, LysM domains of certain plant kinases enable the plant to recognize its symbiotic bacteria or sense and induce resistance against fungi. This interaction is triggered by chitin‐like compounds that are secreted by the symbiotic bacteria or released from fungi, demonstrating an important sensing function of LysMs.

An alternative bactericidal mechanism of action for lantibiotic peptides that target Lipid II

Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II–targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.

The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.

Bet-hedging and epigenetic inheritance in bacterial cell development

Upon nutritional limitation, the bacterium Bacillus subtilis has the capability to enter the irreversible process of sporulation. This developmental process is bistable, and only a subpopulation of cells actually differentiates into endospores. Why a cell decides to sporulate or not to do so is poorly understood. Here, through the use of time-lapse microscopy, we follow the growth, division, and differentiation of individual cells to identify elements of cell history and ancestry that could affect this decision process. These analyses show that during microcolony development, B. subtilis uses a bet-hedging strategy whereby some cells sporulate while others use alternative metabolites to continue growth, providing the latter subpopulation with a reproductive advantage. We demonstrate that B. subtilis is subject to aging. Nevertheless, the age of the cell plays no role in the decision of its fate. However, the physiological state of the cells ancestor (more than two generations removed) does affect the outcome of cellular differentiation. We show that this epigenetic inheritance is based on positive feedback within the sporulation phosphorelay. The extended intergenerational “memory” caused by this autostimulatory network may be important for the development of multicellular structures such as fruiting bodies and biofilms.