Droplet printing reveals the importance of micron-scale structure for bacterial ecology.

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology. However, a test of this hypothesis requires manipulation at the fine scales at which spatial structure naturally occurs. Here we develop a droplet-based printing method to arrange bacterial genotypes across a sub-millimetre array. We print strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins. Our experiments reveal that toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge where susceptible strains can persist in large numbers. Moving to competitions between toxin producers reveals that spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between competing strains to understand how ecological refuges form, which shows that cells closest to a toxin producer mop up the toxin and protect their clonemates. Our work provides a method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in ecology.

Non-antibiotic antibacterial peptides and proteins of Escherichia coli: efficacy and potency of bacteriocins.

INTRODUCTION: The emergence and spread of antibiotic resistance among pathogenic bacteria drives the search for alternative antimicrobial therapies. Bacteriocins represent a potential alternative to antibiotic treatment. In contrast to antibiotics, bacteriocins are peptides or proteins that have relatively narrow spectra of antibacterial activities and are produced by a wide range of bacterial species. Bacteriocins of Escherichia coli are historically classified as microcins and colicins, and, until now, more than 30 different bacteriocin types have been identified and characterized. AREAS COVERED: We performed bibliographical searches of online databases to review the literature regarding bacteriocins produced by E. coli with respect to their occurrence, bacteriocin role in bacterial colonization and pathogenicity, and application of their antimicrobial effect. EXPERT OPINION: The potential use of bacteriocins for applications in human and animal medicine and the food industry includes (i) the use of bacteriocin-producing probiotic strains, (ii) recombinant production in plants and application in food, and (iii) application of purified bacteriocins.

Evolutionary Stabilization of Cooperative Toxin Production through a Bacterium-Plasmid-Phage Interplay.

Colicins are toxins produced and released by Enterobacteriaceae to kill competitors in the gut. While group A colicins employ a division of labor strategy to liberate the toxin into the environment via colicin-specific lysis, group B colicin systems lack cognate lysis genes. In Salmonella enterica serovar Typhimurium (S. Tm), the group B colicin Ib (ColIb) is released by temperate phage-mediated bacteriolysis. Phage-mediated ColIb release promotes S. Tm fitness against competing Escherichia coli It remained unclear how prophage-mediated lysis is realized in a clonal population of ColIb producers and if prophages contribute to evolutionary stability of toxin release in S. Tm. Here, we show that prophage-mediated lysis occurs in an S. Tm subpopulation only, thereby introducing phenotypic heterogeneity to the system. We established a mathematical model to study the dynamic interplay of S. Tm, ColIb, and a temperate phage in the presence of a competing species. Using this model, we studied long-term evolution of phage lysis rates in a fluctuating infection scenario. This revealed that phage lysis evolves as bet-hedging strategy that maximizes phage spread, regardless of whether colicin is present or not. We conclude that the ColIb system, lacking its own lysis gene, is making use of the evolutionary stable phage strategy to be released. Prophage lysis genes are highly prevalent in nontyphoidal Salmonella genomes. This suggests that the release of ColIb by temperate phages is widespread. In conclusion, our findings shed new light on the evolution and ecology of group B colicin systems.IMPORTANCE Bacteria are excellent model organisms to study mechanisms of social evolution. The production of public goods, e.g., toxin release by cell lysis in clonal bacterial populations, is a frequently studied example of cooperative behavior. Here, we analyze evolutionary stabilization of toxin release by the enteric pathogen Salmonella The release of colicin Ib (ColIb), which is used by Salmonella to gain an edge against competing microbiota following infection, is coupled to bacterial lysis mediated by temperate phages. Here, we show that phage-dependent lysis and subsequent release of colicin and phage particles occurs only in part of the ColIb-expressing Salmonella population. This phenotypic heterogeneity in lysis, which represents an essential step in the temperate phage life cycle, has evolved as a bet-hedging strategy under fluctuating environments such as the gastrointestinal tract. Our findings suggest that prophages can thereby evolutionarily stabilize costly toxin release in bacterial populations.

Dynamics of ColicinE2 production and release determine the competitive success of a toxin-producing bacterial population.

The release of toxins is one mechanism used by bacterial species to establish dominance over competitors, but how the dynamics of toxin expression determine the competitive success of a toxin-producing population is largely unknown. Here, we investigate how the expression dynamics of ColicinE2 - a toxic bacteriocin - affect competition between toxin-producing and toxin-sensitive strains of Escherichia coli. We demonstrate that, in addition to genetic modifications in the toxin expression system, alterations of the growth medium can be used to modulate the timing of toxin production and the amount of toxin released. Thus cells that release the toxin at later times can accumulate more colicin. In experiments, we found that delaying toxin release does not significantly alter competition outcome. However, our theoretical analysis allowed us to assess the relative contributions of release time and toxin level to the competitive success of the producer strain, that might counteract each other in experiments. The results reveal that the importance of delaying toxin release lies in increasing the toxin amount. This is a more effective strategy for the toxin-producing strain than prompt discharge of the colicin. In summary, our study shows how the toxin release dynamics influence the competitive success of the toxin-producing bacterial population.

Novel Probiotic Mechanisms of the Oral Bacterium Streptococcus sp. A12 as Explored with Functional Genomics.

Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, like Streptococcus mutans A novel oral bacterium, designated Streptococcus A12, was previously isolated from supragingival dental plaque of a caries-free individual and was shown to interfere potently with the growth and virulence properties of S. mutans In this study, we applied functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of, S. mutans Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete with S. mutans were identified. Selected genes, designated potential competitive factors (pcf), were deleted. Certain mutant derivatives showed a reduced capacity to compete with S. mutans compared to that of the parental strain. The A12 pcfO mutant lost the ability to inhibit comX -inducing peptide (XIP) signaling by S. mutans, while mutants with changes in the pcfFEG locus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism of S. mutans Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiological factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health.IMPORTANCE Advances in defining the composition of health-associated biofilms have highlighted the important role of beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological bases underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate, Streptococcus A12, to compete with the dental pathogen Streptococcus mutans using various gene products with diverse functions. A12 displayed enhanced competitiveness by (i) disrupting intercellular communication pathways of S. mutans, (ii) sensing and resisting antimicrobial peptides, and (iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed by Streptococcus A12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.

Evolutionary Stability of Salmonella Competition with the Gut Microbiota: How the Environment Fosters Heterogeneity in Exploitative and Interference Competition.

Following ingestion, gastrointestinal pathogens compete against the gastrointestinal microbiota and overcome host immune defenses in order to cause infections. Besides employing direct killing mechanisms, the commensal microbiota occupies metabolic niches to outcompete invading pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) uses several strategies to successfully colonize the gut and establish infection, of which an increasing number is based on phenotypic heterogeneity within the S. Typhimurium population. The utilization of myo-inositol (MI) and the production of colicin confer a selective advantage over the microbiota in terms of exploitative and interference competition, respectively. In this review, we summarize the genetic basis underlying bistability of MI catabolism and colicin production. As demonstrated by single-cell analyses, a stochastic switch in the expression of the genes responsible for colicin production and MI degradation constitutes the heterogeneity of the two phenotypes. Both genetic systems are tightly regulated to avoid their expression under non-appropriate conditions and possible detrimental effects on bacterial fitness. Moreover, evolutionary mechanisms underlying formation and stability of these phenotypes in S. Typhimurium are discussed. We propose that both MI catabolism and colicin production create a bet-hedging strategy, which provides an adaptive benefit for S. Typhimurium in the fluctuating environment of the mammalian gut.

Simple Purification of Nicotiana benthamiana-Produced Recombinant Colicins: High-Yield Recovery of Purified Proteins with Minimum Alkaloid Content Supports the Suitability of the Host for Manufacturing Food Additives.

Colicins are natural non-antibiotic bacterial proteins with a narrow spectrum but an extremely high antibacterial activity. These proteins are promising food additives for the control of major pathogenic Shiga toxin-producing E. coli serovars in meats and produce. In the USA, colicins produced in edible plants such as spinach and leafy beets have already been accepted by the U. S. Food and Drug Administration (FDA) and U. S. Department of Agriculture (USDA) as food-processing antibacterials through the GRAS (generally recognized as safe) regulatory review process. Nicotiana benthamiana, a wild relative of tobacco, N. tabacum, has become the preferred production host plant for manufacturing recombinant proteins-including biopharmaceuticals, vaccines, and biomaterials-but the purification procedures that have been employed thus far are highly complex and costly. We describe a simple and inexpensive purification method based on specific acidic extraction followed by one chromatography step. The method provides for a high recovery yield of purified colicins, as well as a drastic reduction of nicotine to levels that could enable the final products to be used on food. The described purification method allows production of the colicin products at a commercially viable cost of goods and might be broadly applicable to other cost-sensitive proteins.

Coproduction of colicin V and lactic acid bacteria bacteriocins in lactococci and enterococci strains of biotechnological interest.

AIMS: The aim of this study was the coproduction in a single strain of the Gram-negative bacteriocin colicin V with other bacteriocins from lactic acid bacteria (LAB). METHODS AND RESULTS: Colicin V was expressed in Lactococcus and Enterococcus strains by replacing the colicin V leader peptide by the leader peptide and promoter of d-alanyl-d-alanine carboxypeptidase from Lactobacillus reuteri CECT925 in pNZ8048 (pNZ:LR-colV). The antimicrobial activity of colicin V against the indicator organism Escherichia coli DH5α in transformed strains was checked by agar diffusion assay and SDS-PAGE analysis. CONCLUSIONS: Lactococcus and Enterococcus transformed with pNZ:LR-colV were able to coproduce colicin V at high levels together with other LAB bacteriocins such as nisin A, nisin Z, lacticin 481 or enterocins A and B, obtaining broad-spectrum activity strains with large potential applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction showed in this work could be used for the heterologous expression of other bacteriocins active against Gram-negative bacteria or wide-spectrum bacteriocins from LAB.

Use of Escherichia coli Nissle 1917 producing recombinant colicins for treatment of IBD patients.

Patients with Crohn's Disease and Ulcerative Colitis infected with Adherent-Invasive Escherichia coli strains constitute the largest group among Inflammatory Bowel Disease subjects, when taking into account all known etiological agents of the disease. A possible link between these pathogenic bacteria and inflammation process has gained the confidence in recently published papers. Observed enteric neuroglial cells apoptosis and epithelial gaps of ileum are probably the key manifestations of inflammation, which has been shown in IBD patients in contrary to the samples taken from healthy individuals. The cascade of consecutive events from bacterial infection via inflammation to excessive apoptosis in IBD patients leads up to the aim of our hypothesis about designing of new therapeutic strategy directed to Adherent-Invasive E. coli strains. The main advantage of biological method, which will rely on application of E. coli Nissle 1917 strain as a carrier for specific recombinant colicins against AIEC strains, could probably cause a long-lasting remission of inflammation in CD and UC patients.

Distinct colicin M-like bacteriocin-immunity pairs in Burkholderia.

The Escherichia coli bacteriocin colicin M (ColM) acts via degradation of the cell wall precursor lipid II in target cells. ColM producers avoid self-inhibition by a periplasmic immunity protein anchored in the inner membrane. In this study, we identified colM-like bacteriocin genes in genomes of several ß-proteobacterial strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. Two selected Burkholderia ambifaria proteins, designated burkhocins M1 and M2, were produced recombinantly and showed antagonistic activity against Bcc strains. In their considerably sequence-diverged catalytic domain, a conserved aspartate residue equally proved pivotal for cytotoxicity. Immunity to M-type burkhocins is conferred upon susceptible strains by heterologous expression of a cognate gene located either upstream or downstream of the toxin gene. These genes lack homology with currently known ColM immunity genes and encode inner membrane-associated proteins of two distinct types, differing in predicted transmembrane topology and moiety exposed to the periplasm. The addition of burkhocins to the bacteriocin complement of Burkholderia reveals a wider phylogenetic distribution of ColM-like bacteriotoxins, beyond the γ-proteobacterial genera Escherichia, Pectobacterium and Pseudomonas, and illuminates the diversified nature of immunity-providing proteins.

Pattern of induction of colicin E9 synthesis by sub MIC of Norfloxacin antibiotic.

The presence of dual SOS boxes in the regulatory region of the most of colicin operons confines synthesis of colicin to times of stress, presumably to reduce the cost of its production. However, in presence of certain inducing agents, such as antibiotics, this tight control of colicin operon is usually lost. Although synthesis of most of colicins is known to be regulated by SOS response of host cell, different patterns of induction from distinct colicins against various inducing agents have been shown in recent years. In this study, we investigated the induction pattern of enzymatic colicin E9 (ColE9) synthesis following treatment with various concentrations (sub MICs) of the Norfloxacin (NOR) using pSBM23 construct which carries transcriptional fusion of SOS inducible promoter of pColE9 (ColE9p) and a fluorescent reporter gene (gfpmut2) into kanamycin resistant pColE9-J plasmid. Flow cytomtry analysis of the Escherichia coli cells containing pSBM23, following treatment with various concentrations showed that the SOS response mediated induction of the synthesis of ColE9 happens in a dose-dependent manner. In summary, our results suggest that the presence, even in a minute amount, of SOS response inducing agents such as fluoroquinolone antibiotic in natural habitat of colicinogenic population can promote such a costly antagonistic behaviour of microbes.

A novel endogenous induction of ColE7 expression in a csrA mutant of Escherichia coli.

Carbon storage regulator A (CsrA) is an important regulator that controls central metabolic pathways and a variety of physiological functions. We found that disruption of csrA in cells containing the ColE7 operon caused a 12-fold increase in colicin E7 production. Moreover, real-time RT-PCR demonstrated a decrease of around 50 % in the lexA mRNA of the csrA mutant. However, the cellular level of RecA protein and its mRNA were not significantly different from the wild type strain. Our results suggest that a novel induction mechanism might exist in E. coli that allows the expression of ColE7 operon in response to a metabolic shift. Proteomic analysis suggested that csrA deficient mutant may adapt PEP-glyoxylate cycle for energy production. Thus, the physiological changes in the csrA mutant may be similar to carbon source limitation for initiating the expression of ColE7 operon in response to stringent environmental conditions.

Plasmid profile, colicinogeny and phage sensitivity as indicators of the dynamics of Escherichia coli populations in the human gut.

A possibility to use such bacterial phenotypes as plasmid profiles, colicinogeny and phage sensitivity as dynamics indicators of enterobacterial population in the human intestine was considered in the present work. All these three phenotypes, considered together with the type of enterobacterial association and age of the patients may reflect the dynamic state of the individual E. coli population that is currently prevailing in the intestinal microflora. The data on plasmid profile structure, colicinogeny and phage sensitivity indicate to considerable quantitative and qualitative diversity of E. coli genetic elements and the intensive interaction between bacteria in the human gut. This diversity is reflected on the formation of the dynamic population of intestinal bacteria, which obviously depends on the host age. The evaluation of the three above mentioned phenotypes confirmed that the E. coli isolates are closely coordinating with other members of the intestinal microbial association. It is noted that the plasmid frequency increases, the colicin range expands and phage sensitivity decreases in E. coli cells simultaneously with the increase of the number of enterobacterial species in the gut. The dynamics of changes in the biological features was observed among E. coli strains from different age groups of patients. The most significant was high frequency of colicinogenic strains in adult patients and di-associated E. coli containing one large plasmid in the youngest patients with dysbiosis.

Regulating colicin synthesis to cope with stress and lethality of colicin production.

Colicins are plasmid-encoded bacteriocins active against Escherichia coli and closely related species of Enterobacteriaceae. They promote microbial diversity and genetic diversity in E. coli populations. Colicin synthesis is characteristically repressed by the LexA protein, the key regulator of the SOS response. As colicins are released by cell lysis, generally two LexA dimers binding to two overlapping SOS boxes control untimely expression. Nevertheless, genetic organization of the colicin clusters, additional transcription regulators as well as post-transcriptional mechanisms involving translational efficiency of the lysis and activity genes fine-tune colicin expression and protect against lethality of colicin production.

Double locking of an Escherichia coli promoter by two repressors prevents premature colicin expression and cell lysis.

The synthesis of Eschericha coli colicins is lethal to the producing cell and is repressed during normal growth by the LexA transcription factor, which is the master repressor of the SOS system for repair of DNA damage. Following DNA damage, LexA is inactivated and SOS repair genes are induced immediately, but colicin production is delayed and induced only in terminally damaged cells. The cause of this delay is unknown. Here we identify the global transcription repressor, IscR, as being directly responsible for the delay in colicin K expression during the SOS response, and identify the DNA target for IscR at the colicin K operon promoter. Our results suggest that, IscR stabilizes LexA at the cka promoter after DNA damage thus, preventing its cleavage and inactivation, and this cooperation ensures that suicidal colicin K production is switched on only as a last resort. A similar mechanism operates at the regulatory region of other colicins and, hence, we suggest that many promoters that control the expression of 'lethal' genes are double locked.

Escherichia coli bacteriocins: antimicrobial efficacy and prevalence among isolates from patients with bacteraemia.

Bacteriocins are antimicrobial peptides generally active against bacteria closely related to the producer. Escherichia coli produces two types of bacteriocins, colicins and microcins. The in vitro efficacy of isolated colicins E1, E6, E7, K and M, was assessed against Escherichia coli strains from patients with bacteraemia of urinary tract origin. Colicin E7 was most effective, as only 13% of the tested strains were resistant. On the other hand, 32%, 33%, 43% and 53% of the tested strains exhibited resistance to colicins E6, K, M and E1. Moreover, the inhibitory activity of individual colicins E1, E6, E7, K and M and combinations of colicins K, M, E7 and E1, E6, E7, K, M were followed in liquid broth for 24 hours. Resistance against individual colicins developed after 9 hours of treatment. On the contrary, resistance development against the combined action of 5 colicins was not observed. One hundred and five E. coli strains from patients with bacteraemia were screened by PCR for the presence of 5 colicins and 7 microcins. Sixty-six percent of the strains encoded at least one bacteriocin, 43% one or more colicins, and 54% one or more microcins. Microcins were found to co-occur with toxins, siderophores, adhesins and with the Toll/Interleukin-1 receptor domain-containing protein involved in suppression of innate immunity, and were significantly more prevalent among strains from non-immunocompromised patients. In addition, microcins were highly prevalent among non-multidrug-resistant strains compared to multidrug-resistant strains. Our results indicate that microcins contribute to virulence of E. coli instigating bacteraemia of urinary tract origin.

Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

[Using the laboratory strains of Escherichia coli for studying colicinogenicity].

The indicator system which includes laboratory strains of Escherichia coli K12, K12-C600, BE, and C-Ia is offered for studying colicinogenicity. It has helped to establish that 32 of 100 patients with dysbacterioses of colon carry a colicin-producing strain of E. coli. A tendency is discovered to the increase of occurrence of colicinogenic strains of escherichias with patients' age. Only 24% of E. coli strains form active colicins in a group of one-year old children. Frequency of colicinogenic strains occurrence increases to 33 and 42 %, respectively, in teenagers and adult patients. A strict decrease of total activity of colicins is the main peculiarity of polymicrobe associations in which the prevailing strain of E. coli is accompanied by 3-5 strains of other enterobacteria. As to their sensitivity of colicins the indicator strains are arranged in the following order: K12-C600 (84%), KR (69%), BE (63%) and C-Ia (47%). In spite of that, the low-sensitive strains can be effective for identification of very specific colicins. Since the laboratory strains of E. coli K12, BE and C-Ia are the hosts for specific bacteriophages of E. coli, the indicator system on their basis may be useful for studying the interrelation between colicins and coliphages, as well as plasmids and restriction-modification systems. The paper is presented in Russian.

[Recombinant expression, purification and characterization of colicin S4].

Broad-spectrum of conventional antibiotics is one of the key factors that cause antibiotics-resistance of many bacteria. Bacteriocins are regarded as the next generation of antibiotics on account of their narrow-spectrum bactericidal activities. Many attentions have been paid to colicins because they are believed to be safe in regard to human body. In this paper, the genes encoding colicin S4 and its immunity protein were cloned into pQE30 to produce colicin S4 expression vector pQE30-Col S4. Colicin S4 was highly expressed as soluble form in gE colig M15 containing pQE30-Col S4. The yields ranged from 30 mg/L to 50 mg/L. The recombinant colicin S4 with an additional 6 His-tag at its N-terminus was found being similar to the natural colicin S4 in antibacterial activity. It only showed bactericidal activity against E. coli strains, thus makig it attractive to develop this protein as a novel antibiotic with narrow spectrum.

Probiotic Escherichia coli strain Nissle 1917 outcompetes intestinal pathogens during biofilm formation.

Many bacterial infections are associated with biofilm formation. Bacterial biofilms can develop on essentially all kinds of surfaces, producing chronic and often intractable infections. Escherichia coli is an important pathogen causing a wide range of gastrointestinal infections. E. coli strain Nissle 1917 has been used for many decades as a probiotic against a variety of intestinal disorders and is probably the best field-tested E. coli strain in the world. Here we have investigated the biofilm-forming capacity of Nissle 1917. We found that the strain was a good biofilm former. Not only was it significantly better at biofilm formation than enteropathogenic, enterotoxigenic and enterohaemorrhagic E. coli strains, it was also able to outcompete such strains during biofilm formation. The results support the notion of bacterial prophylaxis employing Nissle 1917 and may partially explain why the strain has a beneficial effect on many intestinal disorders.