Unveiling the regulatory network controlling natural transformation in lactococci.

Lactococcus lactis is a lactic acid bacterium of major importance for food fermentation and biotechnological applications. The ability to manipulate its genome quickly and easily through competence for DNA transformation would accelerate its general use as a platform for a variety of applications. Natural transformation in this species requires the activation of the master regulator ComX. However, the growth conditions that lead to spontaneous transformation, as well as the regulators that control ComX production, are unknown. Here, we identified the carbon source, nitrogen supply, and pH as key factors controlling competence development in this species. Notably, we showed that these conditions are sensed by three global regulators (i.e., CcpA, CodY, and CovR), which repress comX transcription directly. Furthermore, our systematic inactivation of known signaling systems suggests that classical pheromone-sensing regulators are not involved. Finally, we revealed that the ComX-degrading MecA-ClpCP machinery plays a predominant role based on the identification of a single amino-acid substitution in the adaptor protein MecA of a highly transformable strain. Contrasting with closely-related streptococci, the master competence regulator in L. lactis is regulated both proximally by general sensors and distantly by the Clp degradation machinery. This study not only highlights the diversity of regulatory networks for competence control in Gram-positive bacteria, but it also paves the way for the use of natural transformation as a tool to manipulate this biotechnologically important bacterium.

Testing for the fitness benefits of natural transformation during community-embedded evolution.

Natural transformation is a process where bacteria actively take up DNA from the environment and recombine it into their genome or reconvert it into extra-chromosomal genetic elements. The evolutionary benefits of transformation are still under debate. One main explanation is that foreign allele and gene uptake facilitates natural selection by increasing genetic variation, analogous to meiotic sex. However, previous experimental evolution studies comparing fitness gains of evolved transforming- and isogenic non-transforming strains have yielded mixed support for the 'sex hypothesis.' Previous studies testing the sex hypothesis for natural transformation have largely ignored species interactions, which theory predicts provide conditions favourable to sex. To test for the adaptive benefits of bacterial transformation, the naturally transformable wild-type Acinetobacter baylyi and a transformation-deficient ∆comA mutant were evolved for 5 weeks. To provide strong and potentially fluctuating selection, A. baylyi was embedded in a community of five other bacterial species. DNA from a pool of different Acinetobacter strains was provided as a substrate for transformation. No effect of transformation ability on the fitness of evolved populations was found, with fitness increasing non-significantly in most treatments. Populations showed fitness improvement in their respective environments, with no apparent costs of adaptation to competing species. Despite the absence of fitness effects of transformation, wild-type populations evolved variable transformation frequencies that were slightly greater than their ancestor which potentially could be caused by genetic drift.

M-TUBE enables large-volume bacterial gene delivery using a high-throughput microfluidic electroporation platform.

Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum.

Electrotransformation of thermophilic bacterium Caldimonas manganoxidans.

Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/µg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.

"Take It or Leave It"-Factors Regulating Competence Development and DNA Uptake in Campylobacter jejuni.

Campylobacter jejuni has a large adaptive potential due to enormous genetic exchange. Factors regulating natural transformation in this food-borne pathogen are largely unknown but of interest for the application of sustained reduction strategies in the food-processing industry. Using a single cell DNA uptake assay, we visualized that recognition of methylated C. jejuni DNA was essential for the first step of DNA uptake into a DNase resistant state. Transformation rates using a resistance marker correlated with the fraction of competent bacteria, harboring one to maximally four locations of active DNA uptake, not necessarily being located at the cell pole. Competence developed with rising pH between 6.5 and 7.5 under microaerobic conditions and was nearly insensitive towards growth temperatures between 32 °C and 42 °C, CO2 concentrations ranging from 0 to 50% and growth rates. However, competence development was abolished at pH 5 or under aerobic stress conditions, in which the bacteria ceased growth but fully survived. The DNA uptake machinery in competent bacteria shut down at slightly acidic pH and was reversibly switched on upon neutralization. It was dependent on the proton motive force and, in contrast to competence development, slightly enhanced under aerobic conditions. The results suggest that natural transformation in C. jejuni occurs in the neutral and microaerobic intestinal environment for enhanced genetic diversity and pre-adaption before host switch. In addition, highly competent bacteria might be shed into the environment, still able to acquire genetic material for increased survival.

The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins.

Bacillus subtilis is a successful host for producing recombinant proteins. Its GRAS (generally recognized as safe) status and its remarkable innate ability to absorb and incorporate exogenous DNA into its genome make this organism an ideal platform for the heterologous expression of bioactive substances. The factors that corroborate its value can be attributed to the scientific knowledge obtained from decades of study regarding its biology that has fostered the development of several genetic engineering strategies, such as the use of different plasmids, engineering of constitutive or double promoters, chemical inducers, systems of self-inducing expression with or without a secretion system that uses a signal peptide, and so on. Tools that enrich the technological arsenal of this expression platform improve the efficiency and reduce the costs of production of proteins of biotechnological importance. Therefore, this review aims to highlight the major advances involving recombinant expression systems developed in B. subtilis, thus sustaining the generation of knowledge and its application in future research. It was verified that this bacterium is a model in constant demand and studies of the expression of recombinant proteins on a large scale are increasing in number. As such, it represents a powerful bacterial host for academic research and industrial purposes.

The activation and limitation of the bacterial natural transformation system: The function in genome evolution and stability.

Bacteria can take up exogenous naked DNA and integrate it into their genomes, which has been regarded as a main contributor to bacterial evolution. The competent status of bacteria is influenced by environmental cues and by the immune systems of bacteria. Here, we review recent advances in understanding the working mechanisms underlying activation of the natural transformation system and limitations thereof. Environmental stresses including the presence of antimicrobials can activate the natural transformation system. However, bacterial enzymes (nucleases), non-coding RNAs, specific DNA sequences, the restriction-modification (R-M) systems, CRISPR-Cas systems and prokaryotic Argonaute proteins (Agos) are have been found to be involved in the limitation of the natural transformation system. Together, this review represents an opportunity to gain insight into bacterial genome stability and evolution.

The DNA transporter ComEC has metal-dependent nuclease activity that is important for natural transformation.

In the process of natural transformation bacteria import extracellular DNA molecules for integration into their genome. One strand of the incoming DNA molecule is degraded, whereas the remaining strand is transported across the cytoplasmic membrane. The DNA transport channel is provided by the protein ComEC. Many ComEC proteins have an extracellular C-terminal domain (CTD) with homology to the metallo-ß-lactamase fold. Here we show that this CTD binds Mn2+ ions and exhibits Mn2+ -dependent phosphodiesterase and nuclease activities. Inactivation of the enzymatic activity of the CTD severely inhibits natural transformation in Bacillus subtilis. These data suggest that the ComEC CTD is a nuclease responsible for degrading the nontransforming DNA strand during natural transformation and that this process is important for efficient DNA import.

Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake.

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram-positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake.

Enhancing the chemical transformation of Candida parapsilosis.

Candida parapsilosis is a leading cause of invasive mycoses and the major cause of nosocomial fungaemia amongst low and very low birth weight neonates. However, the molecular and physiological characteristics of this fungus remain understudied. To advance our knowledge about the pathobiology of this pathogen, we sought to develop and validate an effective method for chemical transformation of C. parapsilosis. Chemical transformation is the primary procedure for introducing foreign DNA into Candida yeast as it requires no special equipment, although its performance efficacy drops rapidly when the size of the transforming DNA increases. To define optimal conditions for chemical transformation in C. parapsilosis, we selected a leucine auxotroph laboratory strain. We identified optimal cell density for transformation, incubation times, inclusion of specific enhancing chemicals, and size and amounts of DNA fragments that resulted in maximized transformation efficiency. We determined that the inclusion of dimethyl sulfoxide was beneficial, but dithiothreitol pretreatment reduced colony recovery. As a result, the modified protocol led to a 20-55-fold increase in transformation efficiency, depending on the size of the transforming fragment. We validated the modified methodology with prototrophic isolates and demonstrated that the new approach resulted in the recovery of significantly more transformants in 5 of 6 isolates. Additionally, we identified a medium in which transformation competent yeast cells could safely be maintained at -80°C for up to 6 weeks that reduces laboratory work and shortens the overall procedure. These modifications will significantly aid further investigations into the genetic basis for virulence in C. parapsilosis.

The novel ECF56 SigG1-RsfG system modulates morphological differentiation and metal-ion homeostasis in Streptomyces tsukubaensis.

Extracytoplasmic function (ECF) sigma factors are key transcriptional regulators that prokaryotes have evolved to respond to environmental challenges. Streptomyces tsukubaensis harbours 42 ECFs to reprogram stress-responsive gene expression. Among them, SigG1 features a minimal conserved ECF σ2-σ4 architecture and an additional C-terminal extension that encodes a SnoaL_2 domain, which is characteristic for ECF σ factors of group ECF56. Although proteins with such domain organisation are widely found among Actinobacteria, the functional role of ECFs with a fused SnoaL_2 domain remains unknown. Our results show that in addition to predicted self-regulatory intramolecular amino acid interactions between the SnoaL_2 domain and the ECF core, SigG1 activity is controlled by the cognate anti-sigma protein RsfG, encoded by a co-transcribed sigG1-neighbouring gene. Characterisation of ∆sigG1 and ∆rsfG strains combined with RNA-seq and ChIP-seq experiments, suggests the involvement of SigG1 in the morphological differentiation programme of S. tsukubaensis. SigG1 regulates the expression of alanine dehydrogenase, ald and the WhiB-like regulator, wblC required for differentiation, in addition to iron and copper trafficking systems. Overall, our work establishes a model in which the activity of a σ factor of group ECF56, regulates morphogenesis and metal-ions homeostasis during development to ensure the timely progression of multicellular differentiation.

Multiple plasmid origin-of-transfer regions might aid the spread of antimicrobial resistance to human pathogens.

Antimicrobial resistance poses a great danger to humanity, in part due to the widespread horizontal gene transfer of plasmids via conjugation. Modeling of plasmid transfer is essential to uncovering the fundamentals of resistance transfer and for the development of predictive measures to limit the spread of resistance. However, a major limitation in the current understanding of plasmids is the incomplete characterization of the conjugative DNA transfer mechanisms, which conceals the actual potential for plasmid transfer in nature. Here, we consider that the plasmid-borne origin-of-transfer substrates encode specific DNA structural properties that can facilitate finding these regions in large datasets and develop a DNA structure-based alignment procedure for typing the transfer substrates that outperforms sequence-based approaches. Thousands of putative DNA transfer substrates are identified, showing that plasmid mobility can be twofold higher and span almost twofold more host species than is currently known. Over half of all putative mobile plasmids contain the means for mobilization by conjugation systems belonging to different mobility groups, which can hypothetically link previously confined host ranges across ecological habitats into a robust plasmid transfer network. This hypothetical network is found to facilitate the transfer of antimicrobial resistance from environmental genetic reservoirs to human pathogens, which might be an important driver of the observed rapid resistance development in humans and thus an important point of focus for future prevention measures.

Enhanced Agrobacterium-mediated transformation revealed attenuation of exogenous plasmid DNA installation in recipient bacteria by exonuclease VII and SbcCD.

In DNA transfer via type IV secretion system (T4SS), relaxase enzyme releases linear ssDNA in donor cells and recircularizes in recipient cells. Using VirB/D4 T4SS, Agrobacterium cells can transfer an IncQ-type plasmid depending on Mob relaxase and a model T-DNA plasmid depending on VirD2 relaxase. Mobilization to Escherichia coli of the former plasmid is much more efficient than that of the latter, whereas an entirely reverse relationship is observed in transfer to yeast. These data suggest that either plasmid recircularization or conversion of ssDNA to dsDNA in the recipient bacterial cells is a rate-limiting step of the transfer. In this study, we examined involvement of exonuclease genes in the plasmid acceptability. By the VirD2-dependent T-DNA plasmid, E. coli sbcDΔ and sbcCΔ mutant strains produced threefold more exconjugants, and a sbcDΔ xseAΔ mutant strain yielded eightfold more exconjugants than their wild-type strain. In contrast to the enhancing effect on the VirD2-mediated transfer, the mutations exhibited a subtle effect on the Mob-mediated transfer. These results support our working hypothesis that VirD2 can transport its substrate ssDNA efficiently to recipient cells and that recipient nucleases degrade the ssDNA because VirD2 has some defect(s) in the circularization and completion of complementary DNA synthesis.

Development of a species-specific transformation system using the novel endogenous promoter calreticulin from oleaginous microalgae Ettlia sp.

Microalgae not only serve as raw materials for biofuel but also have uses in the food, pharmaceutical, and cosmetic industries. However, regulated gene expression in microalgae has only been achieved in a few strains due to the lack of genome information and unstable transformation. This study developed a species-specific transformation system for an oleaginous microalga, Ettlia sp. YC001, using electroporation. The electroporation was optimized using three parameters (waveform, field strength, and number of pulses), and the final selection was a 5 kV cm-1 field strength using an exponential decay wave with one pulse. A new strong endogenous promoter CRT (Pcrt) was identified using transcriptome and quantitative PCR analysis of highly expressed genes during the late exponential growth phase. The activities of this promoter were characterized using a codon optimized cyan fluorescent protein (CFP) as a reporter. The expression of CFP was similar under Pcrt and under the constitutive promoter psaD (PpsaD). The developed transformation system using electroporation with the endogenous promoter is simple to prepare, is easy to operate with high repetition, and utilizes a species-specific vector for high expression. This system could be used not only in molecular studies on microalgae but also in various industrial applications of microalgae.

Direct Visualization of Horizontal Gene Transfer by Transformation in Live Pneumococcal Cells Using Microfluidics.

Natural genetic transformation is a programmed mechanism of horizontal gene transfer in bacteria. It requires the development of competence, a specialized physiological state during which proteins involved in DNA uptake and chromosomal integration are produced. In Streptococcus pneumoniae, competence is transient. It is controlled by a secreted peptide pheromone, the competence-stimulating peptide (CSP) that triggers the sequential transcription of two sets of genes termed early and late competence genes, respectively. Here, we used a microfluidic system with fluorescence microscopy to monitor pneumococcal competence development and transformation, in live cells at the single cell level. We present the conditions to grow this microaerophilic bacterium under continuous flow, with a similar doubling time as in batch liquid culture. We show that perfusion of CSP in the microfluidic chamber results in the same reduction of the growth rate of individual cells as observed in competent pneumococcal cultures. We also describe newly designed fluorescent reporters to distinguish the expression of competence genes with temporally distinct expression profiles. Finally, we exploit the microfluidic technology to inject both CSP and transforming DNA in the microfluidic channels and perform near real time-tracking of transformation in live cells. We show that this approach is well suited to investigating the onset of pneumococcal competence together with the appearance and the fate of transformants in individual cells.

The optimization system for preparation of TG1 competent cells and electrotransformation.

An efficient electrotransformation system that includes electrocompetent cells is a critical component for the success of large-scale gene transduction and replication. The conditions of TG1 competent cell preparation and optimal electrotransformation were evaluated by investigating different parameters. Certain parameters for preparation of TG1 competent cells (≥8 × 1010 colony forming units (cfu)/µg DNA) include optimum culture time of monoclonal bacteria (8-10 hr), amplification growth concentration (approximately OD600  = 0.45), and culture volume (400 ml in 2 L conical flask). With increased storage of competent cells at -80°C, electrotransformation efficiency gradually decreased, but it remains greater than ≥ 1010  cfu/µg DNA 3 months later. Moreover, the recovery time of electrotransformation also influenced electrotransformation efficiency (1.5-2 hr for optimization). The optimized transformation efficiency of TG1 (≥8 × 1010  cfu/µg DNA) was observed under suitable electric voltage (2.5 kV), electric intensity (15 kV/cm), and electric time (3.5 ms) of electricity for plasmid transformation. Optimized DNA amount (0.01-100 ng) dissolved in water led to the high efficiency of plasmid transformation (≥8 × 1010  cfu/µg DNA), but had low efficiency when dissolved in T4 ligation buffer (≤3 × 1010  cfu/µg DNA). These results indicated that an optimized TG1 transformation system is useful for high electrotransformation efficiency under general laboratory conditions. The optimized TG1 transformation system might facilitate large-scale gene transduction for phage display library construction.

Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum.

Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 103 CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5-25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.

A competence-regulated toxin-antitoxin system in Haemophilus influenzae.

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining both nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenzae, the genes needed for DNA uptake are induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that one of these genes, HI0659, encodes the antitoxin of a competence-regulated toxin-antitoxin operon ('toxTA'), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the putative toxin (HI0660) restores uptake to the antitoxin mutant. The full toxTA operon was present in only 17 of the 181 strains we examined; complete deletion was seen in 22 strains and deletions removing parts of the toxin gene in 142 others. In addition to the expected Sxy- and CRP-dependent-competence promoter, HI0659/660 transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We propose that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture. Although the high frequency of toxT and toxTA deletions suggests that this competence-regulated toxin-antitoxin system may be mildly deleterious, it could also facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions. Although our analyses were focused on the effects of toxTA, the RNA-seq dataset will be a useful resource for further investigations into competence regulation.

Competence and Transformation in Bacillus subtilis.

Transformation is the process of import and inheritable integration of DNA from the environment. As such, it is believed to be a major driving force for evolution. Competence for transformation is widespread among bacterial species. Recent findings draw a picture of a conserved molecular machine that binds DNA at the cell surface and subsequently transports it through the cell envelope. Within the cytoplasm the DNA is coated by proteins that mediate recombination or self-annealing. The regulatory mechanisms and environmental signals affecting competence are very diverse between different bacterial species. Competence in Bacillus subtilis has become a paradigm for stochastic determination of cell-fate. Quantitative analysis at the single cell level in conjunction with mathematical modelling allowed understanding of induction and decline of competence at the systems level. Currently, the picture is emerging of stochastic differentiation as a fitness trade-off in fluctuating environments.

Immunogenicity of 987P fimbriae of enterotoxigenic Escherichia coli surface-displayed on Lactobacillus casei.

As most pathogens invade the bodies through the mucosa, it is crucial to develop vaccines that induce mucosal immunity. To this end, we generated a safe and effective vaccine candidate that displayed fimbrial protein 987P of enterotoxigenic Escherichia coli (ETEC) on the surface of Lactobacillus casei (L.casei) CICC 6105 by using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. After gavage inoculation of the recombinant strain pLA-987P/L.casei into specific-pathogen-free (SPF) BALB/c mice, high levels of mucosal immunoglobulin A (IgA) were induced in fecal samples, intestine and lung lavage fluids and systemic immunoglobulin G of IgG subclasses (IgG1, IgG2b, and IgG2a) was produced in serum. T-cell proliferation assays showed the stimulation index (SI) of the groups immunized with pLA-987P/L.casei to be significantly higher than that of the control group. The recombinant L.casei promoted T cells to produce both Th1 and Th2 cytokines, while the number of splenic IL-4 Spot forming cells (SFC) exceeded the number of IFN-γ SFC by 2.26-fold (P < .01). >83.3% of the vaccinated mice were protected from challenge with a lethal dose of virulent strain C83916. These results indicate that the recombinant L.casei expressing ETEC 987P fimbrial protein could elicit a protective immune response against ETEC 987P infection effectively.