Bacteriophage endolysins degrade the bacterial cell wall and are therefore considered promising antimicrobial alternatives to fight pathogens resistant to conventional antibiotics. Gram-positive bacteria are usually considered easy targets to exogenously added endolysins, since their cell walls are not shielded by an outer membrane. However, in nutrient rich environments these bacteria can also tolerate endolysin attack if they keep an energized cytoplasmic membrane. Hence, we have hypothesized that the membrane depolarizing action of antimicrobial peptides (AMPs), another attractive class of alternative antibacterials, could be explored to overcome bacterial tolerance to endolysins and consequently improve their antibacterial potential. Accordingly, we show that under conditions supporting bacterial growth, Staphylococcus aureus becomes much more susceptible to the bacteriolytic action of endolysins if an AMP is also present. The bactericidal gain resulting from the AMP/endolysin combined action ranged from 1 to 3 logs for different S. aureus strains, which included drug-resistant clinical isolates. In presence of an AMP, as with a reduced content of cell wall teichoic acids, higher endolysin binding to cells is observed. However, our results indicate that this higher endolysin binding alone does not fully explain the higher susceptibility of S. aureus to lysis in these conditions. Other factors possibly contributing to the increased endolysin susceptibility in presence of an AMP are discussed.

Understanding transcription has been a central goal of the scientific community for decades. However, much is still unknown, especially concerning how it is regulated. In bacteria, a single DNA-directed RNA-polymerase performs the whole of transcription. It contains multiple subunits, among which the σ factor that confers promoter specificity. Besides the housekeeping σ factor, bacteria encode several alternative σ factors. The most abundant and diverse family of alternative σ factors, the extracytoplasmic function (ECF) family, regulates transcription of genes associated with stressful scenarios, making them key elements of adaptation to specific environmental changes. Despite this, the evolutionary history of ECF σ factors has never been investigated. Here, we report on our analysis of thousands of members of this family. We show that single events are in the origin of alternative modes of regulation of ECF σ factor activity that require partner proteins, but that multiple events resulted in acquisition of regulatory extensions. Moreover, in Bacteroidetes there is a recent duplication of an ecologically relevant gene cluster that includes an ECF σ factor, whereas in Planctomycetes duplication generates distinct C-terminal extensions after fortuitous insertion of the duplicated σ factor. At last, we also demonstrate horizontal transfer of ECF σ factors between soil bacteria.

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that 'everything is everywhere'. With the discovery of lateral budding in 'Kolteria novifilia' and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how 'microbial dark matter' can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection.

In bacteria, the promoter specificity of RNA polymerase is determined by interchangeable σ subunits. Extracytoplasmic function σ factors (ECFs) form the largest and most diverse family of alternative σ factors, and their suitability for constructing genetic switches and circuits was already demonstrated. However, a systematic study on how genetically determined perturbations affect the behavior of these switches is still lacking, which impairs our ability to predict their behavior in complex circuitry. Here, we implemented four ECF switches in Bacillus subtilis and comprehensively characterized their robustness toward genetic perturbations, including changes in copy number, protein stability, or antisense transcription. All switches show characteristic dose-response behavior that varies depending on the individual ECF-promoter pair. Most perturbations had performance costs. Although some general design rules could be derived, a detailed characterization of each ECF switch before implementation is recommended to understand and thereby accommodate its individual behavior.

The σ subunit of the bacterial RNA polymerase determines promoter specificity. The extracytoplasmic function σ factors (ECFs) represent the most abundant and diverse group of alternative σ factors and are present in the vast majority of bacterial genomes. Typically, ECFs are regulated by anti-σ factors that sequester their cognate ECFs, thereby preventing their interaction with the RNA polymerase. Beyond these ECF paradigms, a number of distinct modes of regulation have been proposed and experimentally investigated. Regulatory extensions represent one such alternative mechanism of ECF regulation that can be found in 18 phylogenetically distinct ECF groups. Here, the σ factors contain additional domains that are fused to the ECF core domains and are involved in stimulus perception and modulation of σ factor activity. We will summarize the current state of knowledge on regulating ECF activity by C-terminal extensions. We will also discuss newly identified ECF groups containing either N- or C-terminal extensions and propose possible mechanisms by which these extensions have been generated and affect ECF σ factor activity. Based on their modular architecture and the resulting physical connection between stimulus perception and transcriptional output, these ECFs are analogous to one-component systems, the primary mechanism of bacterial signal transduction.

The extracytoplasmic function (ECF) σ factor, σE , is a key regulator of the cell envelope stress response in Streptomyces coelicolor. Although its role in maintaining cell wall integrity has been known for over a decade, a comprehensive analysis of the genes under its control has not been undertaken. Here, using a combination of chromatin immunoprecipitation-sequencing (ChIP-seq), microarray transcriptional profiling and bioinformatic analysis, we attempt to define the σE regulon. Approximately half of the genes identified encode proteins implicated in cell envelope function. Seventeen novel targets were validated by S1 nuclease mapping or in vitro transcription, establishing a σE -binding consensus. Subsequently, we used bioinformatic analysis to look for conservation of the σE target promoters identified in S. coelicolor across 19 Streptomyces species. Key proteins under σE control across the genus include the actin homolog MreB, three penicillin-binding proteins, two L,D-transpeptidases, a LytR-CpsA-Psr-family protein predicted to be involved in cell wall teichoic acid deposition and a predicted MprF protein, which adds lysyl groups to phosphatidylglycerol to neutralize membrane surface charge. Taken together, these analyses provide biological insight into the σE -mediated cell envelope stress response in the genus Streptomyces.

The sigma subunit of the bacterial RNA polymerase determines promoter specificity. The extracytoplasmic function sigma factors (ECFs) represent the most abundant and diverse group of alternative sigma factors and are present in the vast majority of bacterial genomes. Typically, ECFs are regulated by anti-sigma factors that sequester their cognate ECFs, thereby preventing their interaction with the RNA polymerase. Beyond these ECF paradigms, a number of distinct modes of regulation have been proposed and experimentally investigated. Regulatory extensions represent one such alternative mechanism of ECF regulation that can be found in 18 phylogenetically distinct ECF groups. Here, the sigma factors contain additional domains that are fused to the ECF core domains and are involved in stimulus perception and modulation of sigma factor activity. We will summarize the current state of knowledge on regulating ECF activity by C-terminal extensions. We will also discuss newly identified ECF groups containing either N- or C-terminal extensions and propose possible mechanisms by which these extensions have been generated and affect ECF sigma factor activity. Based on their modular architecture and the resulting physical connection between stimulus perception and transcriptional output, these ECFs are analogous to one-component systems, the primary mechanism of bacterial signal transduction.

The rational design of synthetic regulatory circuits critically hinges on the availability of orthogonal and well-characterized building blocks. Here, we focus on extracytoplasmic function (ECF) σ factors, which are the largest group of alternative σ factors and hold extensive potential as synthetic orthogonal regulators. By assembling multiple ECF σ factors into regulatory cascades of varying length, we benchmark the scalability of the approach, showing that these 'autonomous timer circuits' feature a tuneable time delay between inducer addition and target gene activation. The implementation of similar timers in Escherichia coli and Bacillus subtilis shows strikingly convergent circuit behavior, which can be rationalized by a computational model. These findings not only reveal ECF σ factors as powerful building blocks for a rational, multi-layered circuit design, but also suggest that ECF σ factors are universally applicable as orthogonal regulators in a variety of bacterial species.

Extracytoplasmic function σ factors (ECFs) represent the third most abundant fundamental principle of bacterial signal transduction, outranked only by one- and two-component systems. A recent census of ECFs revealed a large number of novel groups whose functions and regulatory mechanisms have not yet been elucidated. Here, we report the characterization of members of the novel group ECF42. ECF42 is a highly abundant and widely distributed ECF group that is present in 11 phyla but is predominantly found in Actinobacteria Analysis of the genomic context conservation did not identify a putative anti-σ factor. Instead, ECF42 genes are cotranscribed with genes encoding a conserved DGPF protein. We have experimentally verified the target promoter of these ECFs (TGTCGA in the -35 region and CGA/TC in the -10 region), which was found upstream of the ECF42-encoding operons in Streptomyces venezuelae, suggesting that ECF42s are positively autoregulated. RNA sequencing (RNA-seq) was performed to define the regulons of the three ECF42 proteins in S. venezuelae, which identified mostly genes encoding DGPF proteins. In contrast to typical ECFs, ECF42 proteins harbor a long C-terminal extension, which is crucial for their activity. Our work provides the first analysis of the function and regulatory mechanism of this novel ECF group that contains a regulatory C-terminal extension.IMPORTANCE In contrast to the one- and two-component signal transduction systems in bacteria, the importance and diversity of ECFs have only recently been recognized in the course of comprehensive phylogenetic and comparative genomics studies. Thus, most of the ECFs still have not been experimentally characterized regarding their physiological functions and regulation mechanisms so far. The physiological roles, target promoter, and target regulons of a novel group of ECFs, ECF42, in S. venezuelae have been investigated in this work. More importantly, members of this group are characterized by a C-terminal extension, which has been verified to harbor a regulatory role in ECF42s. Hence, our work provides an important source for further research on such C-terminal extension containing ECFs.

When the cell envelope integrity is compromised, bacteria trigger signaling cascades resulting in the production of proteins that counteract these extracytoplasmic stresses. Here, we show that the two-component system EsrSR regulates a cell envelope stress response in the Actinobacterium Corynebacterium glutamicum. The sensor kinase EsrS possesses an amino-terminal phage shock protein C (PspC) domain, a property that sets EsrSR apart from all other two-component systems characterized so far. An integral membrane protein, EsrI, whose gene is divergently transcribed to the esrSR gene locus and which interestingly also possesses a PspC domain, acts as an inhibitor of EsrSR under non-stress conditions. The resulting EsrISR three-component system is activated among others by antibiotics inhibiting the lipid II cycle, such as bacitracin and vancomycin, and it orchestrates a broad regulon including the esrI-esrSR gene locus itself, genes encoding heat shock proteins, ABC transporters, and several putative membrane-associated or secreted proteins of unknown function. Among those, the ABC transporter encoded by cg3322-3320 was shown to be directly involved in bacitracin resistance of C. glutamicum. Since similar esrI-esrSR loci are present in a large number of actinobacterial genomes, EsrISR represents a novel type of stress-responsive system whose components are highly conserved in the phylum Actinobacteria.

Bacterial genomes encode numerous and often sophisticated signaling devices to perceive changes in their environment and mount appropriate adaptive responses. With their help, microbes are able to orchestrate specific decision-making processes that alter the cellular behavior, but also integrate and communicate information. Moreover and beyond, some signal transducing systems also enable bacteria to remember and learn from previous stimuli to anticipate environmental changes. As recently suggested, all of these aspects indicate that bacteria do, in fact, exhibit cognition remarkably reminiscent of what we refer to as intelligent behavior, at least when referred to higher eukaryotes. In this essay, comprehensive data derived from comparative genomics analyses of microbial signal transduction systems are used to probe the concept of cognition in bacterial cells. Using a recent comprehensive analysis of over 100 actinobacterial genomes as a test case, we illustrate the different layers of the capacities of bacteria that result in cognitive and behavioral complexity as well as some form of ‘bacterial intelligence’. We try to raise awareness to approach bacteria as cognitive organisms and believe that this view would enrich and open a new path in the experimental studies of bacterial signal transducing systems.

ABSTRACT Signal transduction is an essential process that allows bacteria to sense their complex and ever-changing environment and adapt accordingly. Three distinct major types of signal-transducing proteins (STPs) can be distinguished: one-component systems (1CSs), two-component systems (2CSs), and extracytoplasmic-function σ factors (ECFs). Since Actinobacteria are particularly rich in STPs, we comprehensively investigated the abundance and diversity of STPs encoded in 119 actinobacterial genomes, based on the data stored in the Microbial Signal Transduction (MiST) database. Overall, we observed an approximately linear correlation between the genome size and the total number of encoded STPs. About half of all membrane-anchored 1CSs are protein kinases. For both 1CSs and 2CSs, a detailed analysis of the domain architectures identified novel proteins that are found only in actinobacterial genomes. Many actinobacterial genomes are particularly enriched for ECFs. As a result of this study, almost 500 previously unclassified ECFs could be classified into 18 new ECF groups. This comprehensive survey demonstrates that actinobacterial genomes encode previously unknown STPs, which may represent new mechanisms of signal transduction and regulation. This information not only expands our knowledge of the diversity of bacterial signal transduction but also provides clear and testable hypotheses about their mechanisms, which can serve as starting points for experimental studies. IMPORTANCE In the wake of the genomic era, with its enormous increase in the amount of available sequence information, the challenge has now shifted toward making sense and use of this treasure chest. Such analyses are a prerequisite to provide meaningful information that can help guide subsequent experimental efforts, such as mechanistic studies on novel signaling strategies. This work provides a comprehensive analysis of signal transduction proteins from 119 actinobacterial genomes. We identify, classify, and describe numerous novel and conserved signaling devices. Hence, our work serves as an important resource for any researcher interested in signal transduction of this important bacterial phylum, which contains organisms of ecological, biotechnological, and medical relevance.

Viable but nonculturable (VBNC) cells were recognized 30 years ago; and despite decades of research on the topic, most results are disperse and apparently incongruous. Since its description, a huge controversy arose regarding the ecological significance of this state: is it a degradation process without real significance for bacterial life cycles or is it an adaptive strategy of bacteria to cope with stressful conditions? In order to solve the molecular mechanisms of VBNC state induction and resuscitation, researchers in the field must be aware and overcome common issues delaying research progress. In this review, we discuss the intrinsic characteristic features of VBNC cells, the first clues on what is behind the VBNC state’s induction, the models proposed for their resuscitation and the current methods to prove not only that cells are in VBNC state but also that they are able to resuscitate.

In actinobacteria, resuscitation promoting factor (Rpf) proteins have been described as having the ability to increase the viable count of dormant cultures and stimulate growth of vegetative cells through lag phase reduction. Recently, it was suggested that proteins Lmo0186 and Lmo2522 of Listeria monocytogenes are equivalent to Rpf proteins based on their genomic context and conserved domain architecture. It was proposed that they have evolved through non-orthologous displacement of the Rpf domain found in actinobacteria. Here we present biological and biochemical data supporting a function of Lmo0186 and Lmo2522 as Rpfs. These proteins are collectively dispensable for growth but a lmo0186 lmo2522 double mutant exhibits an extended lag phase when diluted in minimal medium. This phenotype could be partially complemented by medium supplementation with fM to nM concentrations of purified hexahistidine-tagged versions of Lmo0186 and Lmo2522, showing that these proteins can stimulate growth. Gel filtration analysis and cross-linking experiments suggest that the recombinant proteins in solution are elongated monomers. Both proteins display muralytic activity against crude cell wall preparations and are active against an artificial lysozyme substrate. Our study thus supports the hypothesis that Lmo0186 and Lmo2522 are functional equivalents of actinobacteria Rpf proteins and represents the first characterization of two Rpf homologues from firmicutes.

Tese de doutoramento, Biologia (Microbiologia), Universidade de Lisboa, Faculdade de Ciencias, 2013

Though poorly studied, sand is an environment with an extended degree of interaction with man. Enterococcal strains can be found in sand but we do not know to what extent these ubiquitous opportunistic nosocomial pathogens isolated from sand carry antimicrobial resistances and virulence traits. In an attempt to fill in this knowledge gap, two distinct types of sand (beach and children playground) were examined concerning composition in enterococcal species, genetic diversity of isolates and abundance of resistance to antimicrobials and virulence traits. Five different species were found, namely Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae, Enterococcus flavescens and Enterococcus casseliflavus. Although genetic diversity was evident, two different E. faecium clones, common to the two types of sand, were detected, suggesting the existence of clones well adapted to this specific environment or from a common source. E. faecium was associated with multiple antibiotic resistances, including to fluoroquinolones and tetracycline that are commonly used by veterinarians and clinicians. Among the multiresistant E. faecium strains from beach sand, two were from sequence type (ST) 442, which belongs to the wide-spread Hospital-adapted clade CC17. They both carried the esp gene and the genomic island associated with CC17. The other virulence factors screened were disseminated among E. faecalis strains, but seldom detected in the other species, evidencing the existence, in these environments, of E. faecalis strains carrying the same virulence factors as the clinical ones. The present work thus stresses the need to follow-up the presence and characterization of enterococcal strains from both beach and children playground sands and of including these environments in the epidemiological global analysis of enterococcal isolates.

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% Gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.

Aims:To analyse viable but nonculturable (VBNC) state induction in Escherichia coli and resuscitation of VBNC suspensions in several conditions.Methods and Results:VBNC were induced in four media, two temperatures and six strains, but only cells produced at 4 degrees C were able to resuscitate. Resuscitation of 14 VBNC suspensions obtained in several conditions occurred in the presence of supernatants of growing cells, in minimal medium supplemented with amino acids or after temperature change, depending on strain. A limited time period beyond no more resuscitation that could be observed was also confirmed.Conclusions:The supernatants positive effect is suggested to be because of a nonproteinaceous molecule, and a combination of methionine, glutamine, threonine, serine and asparagine could be used as primary mix for resuscitation experiments.Significance and Impact of the Study:Escherichia coli resuscitation was already attempted in several conditions, but it is the first time that a positive result was observed in minimal medium supplemented with amino acids or after temperature change. The role of amino acids in resuscitation is of special interest since was never reported for any species.