Comprehensive analysis of bacteriocins in Streptococcus mutans

Streptococcus mutans produces bacteriocins that show antibacterial activity against several bacteria. However, comprehensive analysis of these bacteriocins has not been well done. In this study, we isolated 125 S. mutans strains from volunteers and determined their whole genome sequence. Based on the genome analysis, the distribution of each bacteriocin gene (mutacins I-IV, K8 and Smb) was investigated. We found 17, 5, and 2 strains showing 100% matches with mutacin I, mutacin II and mutacin III, respectively. Five mutacin III-positive strains had 2 mismatches compared to mature mutacin III. In 67 mutacin IV-positive strains, 38 strains showed 100% match with mutacin IV, while 29 strains showed some variations. In 23 mutacin K8- and 32 mutacin Smb-positive strains, all except one mutacin K8-positive strain showed 100% match with the mature peptides. Among 125 strains, 84 (65.1%), 26 (20.2%), and 5 (3.9%) strains were positive for one, two and three bacteriocin genes, respectively. Then, the antibacterial activity against oral streptococci and other oral bacterial species was investigated by using bacteriocin gene single-positive strains. Each bacteriocin gene-positive strain showed a different pattern of antibacterial activity. These results speculate that individual S. mutans strains may affect the bacterial composition of dental plaques.

A Novel Competence Pathway in the Oral Pathogen Streptococcus sobrinus

Streptococcus sobrinus is an etiologic cause of dental caries (tooth decay) in humans. Our knowledge of S. sobrinus is scant despite the organism’s important role in oral health. It is widely believed that S. sobrinus lacks the natural competence pathways that are used by other streptococci to regulate growth, virulence, and quorum sensing. The lack of natural competence has also prevented genetic manipulation of S. sobrinus, limiting our knowledge of its pathogenicity. We discovered that most strains of S. sobrinus contain a new class of the ComRS competence system. Although S. sobrinus is typically placed among the mutans group streptococci, the S. sobrinus ComRS system is most similar to the competence pathways in the salivarius group. Unlike all other ComRS systems, the S. sobrinus pathway contains 2 copies of the transcriptional regulator ComR and has a peptide pheromone (XIP) that lacks any aromatic amino acids. Synthetic XIP enables transformation of S. sobrinus with plasmid or linear DNA, and we leverage this newfound genetic tractability to confirm that only 1 of the ComR homologs is required for induced competence while the other appears to suppress competence. Exogenous XIP increases the expression of bacteriocin gene clusters and produces an antimicrobial response that inhibits growth of S. mutans. We also identified 2 strains of S. sobrinus that appear to be “cheaters” by either not responding to or not producing XIP. We show how a recombination event in the nonresponsive strain could restore function of the ComRS pathway but delete the gene encoding XIP. Thus, the S. sobrinus ComRS pathway provides new tools for studying this pathogen and offers a lens into the evolution of ecological cheaters.

Monitoring growth compatibility and bacteriocin gene transcription of adjunct with starter lactic acid bacteria strains in milk

When developing protective starter cultures for application in cheese technologies, monitoring growth interactions between starter and adjunct LAB species and in situ expression of bacteriocin genes in the mixtures, are crucial. This study firstly aimed to monitor growth of mixed LAB strain populations during milk model fermentations by microbial counts and real-time quantitative PCR (qPCR). The primary starter Streptococcus thermophilus ST1 and costarter Lactococcus lactis subsp. cremoris M78 strains served as the basic starter composite co-inoculated in all milk treatments. Adjunct bacteriocinogenic Enterococcus faecium strains KE82 and GL31 and a ripening Lactiplantibacillus plantarum H25 strain were added separately to the starter composite resulting in four LAB combination treatments. The second aim was to quantify gene transcripts of nisin and enterocins B and A synthesized by M78, KE82 and GL31, respectively, by real-time reverse transcription PCR (RT-qPCR) and to detect the in situ antilisterial effects of the cocultures. Adjunct LAB strains showed growth compatibility with the starter, since all of them exhibited a 2 to 3 log units increase in their population levels, compared to their initial inoculation levels, with ST1 prevailing in all treatments. KE82 grew more competitively than GL31 whereas cocultures with KE82 displayed the strongest in situ antilisterial activity. Nisin gene expression levels were higher at the exponential phase of microbial growth in all treatments. Finally, expression of nisin and enterocins A and B genes was interrelated indicating an antagonistic activity.

Cloning and functional expression of a food-grade circular bacteriocin, plantacyclin B21AG, in probiotic Lactobacillus plantarum WCFS1

There is an increasing consumer demand for minimally processed, preservative free and microbiologically safe food. These factors, combined with risks of antibiotic resistance, have led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food preservatives and as protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a novel circular bacteriocin produced by Lactobacillus plantarum B21. Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when tested against the WCFS1 (pTRKH2) indicator strain, indicating functional expression of plantacyclin B21AG. Real-time PCR analysis revealed that the relative copy number of pB21AG01 was 7.60 ± 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 ± 0.05 and 25.19 ± 2.68 copies, respectively in WCFS1. This indicates that the bacteriocin gene cluster is located on a highly stable, low copy number plasmid pB21AG01 in L. plantarum B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 may result in similar inhibitory zones observed in both wild type and recombinant hosts despite the low copy number of pCycB21.

Complete Genome Sequence of Thermophilic Bacterium Aeribacillus pallidus PI8

Here, we report the complete genome sequence of Aeribacillus pallidus PI8, a thermophilic bacterium, isolated from soybean stem extract. The sequence was determined using Illumina and Nanopore sequencers. Bioinformatic analyses of the genome sequence revealed the presence of possible bacteriocin gene clusters.

Bioinformatic prospecting identified 99 novel, misannotated and unnoticed putative circular bacteriocins

Circular bacteriocins are antimicrobial peptides produced by bacteria with a N and C termini ligation. They have desirable properties such as activity at low concentrations along with thermal, pH and proteolytic resistance. There are nineteen experimentally confirmed circular bacteriocins as part of bacteriocin gene clusters, with transport, membrane and immunity proteins. Traditionally, novel antimicrobials are found by testing large numbers of isolates against indicator strains, with no promise of corresponding novel sequence. Through bioprospecting publicly available sequence databases, we identified ninety-nine circular bacteriocins across a variety of bacteria bringing the total to 118. They were grouped into two families within class IIc (IIc i and ii) and further divided into subfamilies based on similarity to experimentally confirmed circular bacteriocins. Within subfamilies, sequences overwhelmingly shared similar characteristics such as sequence length, presence of a polybasic region, conserved locations of aromatic residues, C and N termini, gene clusters similarity, translational coupling and hydrophobicity profiles. At least ninety were predicted to be putatively functional based on gene clusters. Furthermore, bacteriocins identified from Enterococcus, Staphylococcus and Streptococcus species may have activity against clinically relevant strains, due to the presence of putative immunity genes required for expression in a toxin-antitoxin system. Some strains such as Paenibacillus larvae subsp. pulvifaciens SAG 10367 contained multiple circular bacteriocin gene clusters from different subfamilies, while some strains such as Bacillus cereus BCE-01 contained clusters with multiple circular bacteriocin structural genes. Sequence analysis provided rapid insight into identification of novel, putative circular bacteriocins, as well as conserved genes likely essential for circularisation. This represents an expanded library of putative antimicrobial proteins which are potentially active against human, plant and animal pathogens.

Conserved pheromone production, response and degradation byStreptococcus mutans

ABSTRACTStreptococcus mutans, a bacterium with high cariogenic potential, coordinates competence for natural transformation and bacteriocin production via the XIP and CSP pheromones. CSP is effective in inducing bacteriocin responses, but not competence in chemically defined media (CDM). This is in contrast to XIP, which is a strong inducer of competence in CDM, but can also stimulate bacteriocin genes as a late response. Inter-connections between the pathways activated by the two pheromones have been characterized in certain detail inS. mutansUA159, but it is mostly unknown whether such findings are representative for the species. In this study, we used bioassays based on luciferase reporters for the bacteriocin genecipBand the alternative sigma factorsigXto investigate variousS. mutansisolates for production and response to CSP and XIP pheromones in CDM. Similar toS. mutansUA159, endogenous CSP was undetectable in the culture supernatants of all tested strains. During optimization of the bioassay using thecipBreporter, we discovered that the acivity of exogenous CSP used as a standard was reduced over time duringS. mutansgrowth. Using a FRET-CSP reporter peptide, we found thatS. mutansUA159 was indeed able to degrade CSP, and that such proteolytic activity was not significantly different in isogenic mutants with deletion of the protease genehtrA, or the competence genessigX, oppD, andcomR. CSP proteolysis was also detected in all the wild type strains, indicating that such activity is conserved inS. mutans. For the XIP pheromone, endogenous production was observed in the supernatants of all 34 tested strains at peak concentrations in culture supernatants that varied between 200 nM and 26000 nM. Transformation in the presence of exogenous XIP was detected in all, but one, of the isolates. The efficiency of transformation varied, however, among the different strains, and for those with the highest transformation rates, endogenous XIP peak concentrations in the supernatants were above 2000 nM XIP. We conclude that XIP production and inducing effect on transformation, as well as proteolytic activity leading to the inactivation of CSP are conserved functions among differentS. mutansisolates. Understanding the functionality and conservation of pheromone systems inS. mutansmay lead to novel strategies to prevent or treat unbalances in oral microbiomes that may favour diseases.

Life in the cystic fibrosis upper respiratory tract influences competitive ability of the opportunistic pathogen Pseudomonas aeruginosa

Understanding characteristic differences between host-associated and free-living opportunistic pathogens can provide insight into the fundamental requirements for success after dispersal to the host environment, and more generally into the ecological and evolutionary processes by which populations respond to simultaneous selection on complex interacting traits. We examined how cystic fibrosis (CF)-associated and environmental isolates of the opportunistic pathogen Pseudomonas aeruginosa differ in the production of an ecologically important class of proteinaceous toxins known as bacteriocins, and how overall competitive ability depends on the production of and resistance to these bacteriocins. We determined bacteriocin gene content in a diverse collection of environmental and CF isolates and measured bacteriocin-mediated inhibition, resistance and the outcome of competition in a shared environment between all possible pairs of these isolates at 25°C and 37°C. Although CF isolates encoded significantly more bacteriocin genes, our phenotypic assays suggest that they have diminished bacteriocin-mediated killing and resistance capabilities relative to environmental isolates, regardless of incubation temperature. Notably, however, although bacteriocin killing and resistance profiles significantly predicted head-to-head competitive outcomes, CF and environmental isolates did not differ significantly in their competitive ability. This suggests that the contribution of bacteriocins to competitive ability involves selection on other traits that may be pleiotropically linked to interference competition mediated by bacteriocins.