Redirected Stress Responses in a Genome-Minimized 'midiBacillus' Strain with Enhanced Capacity for Protein Secretion.

Genome engineering offers the possibility to create completely novel cell factories with enhanced properties for biotechnological applications. In recent years, genome minimization was extensively explored in the Gram-positive bacterial cell factory Bacillus subtilis, where up to 42% of the genome encoding dispensable functions was removed. Such studies showed that some strains with minimized genomes gained beneficial features, especially for secretory protein production. However, strains with the most minimal genomes displayed growth defects. This focused our attention on strains with less extensive genomic deletions that display close-to-wild-type growth properties while retaining the acquired beneficial traits in secretory protein production. A strain of this category is B. subtilis IIG-Bs27-47-24, here referred to as midiBacillus, which lacks 30.95% of the parental genome. To date, it was unknown how the altered genomic configuration of midiBacillus impacts cell physiology in general, and protein secretion in particular. The present study bridges this knowledge gap through comparative quantitative proteome analyses with focus on protein secretion. Interestingly, the results show that the secretion stress responses of midiBacillus, as elicited by high-level expression of the immunodominant staphylococcal antigen A, are completely different from secretion stress responses that occur in the parental strain 168. We further show that midiBacillus has an increased capacity for translation and that a variety of critical Sec secretion machinery components is present at elevated levels. Altogether, our observations demonstrate that high-level protein secretion has different consequences for wild-type and genome-engineered Bacillus strains, dictated by the altered genomic and proteomic configurations. IMPORTANCE Our present study showcases a genome-minimized nonpathogenic bacterium, the so-called midiBacillus, as a chassis for the development of future industrial strains that serve in the production of high-value difficult-to-produce proteins. In particular, we explain how midiBacillus, which lacks about one-third of the original genome, effectively secretes a protein of the major human pathogen Staphylococcus aureus that cannot be produced by the parental Bacillus subtilis strain. This is important, because the secreted S. aureus protein is exemplary for a range of targets that can be implemented in future antistaphylococcal immunotherapies. Accordingly, we anticipate that midiBacillus chassis will contribute to the development of vaccines that protect both humans and livestock against diseases caused by S. aureus, a bacterial pathogen that is increasingly difficult to fight with antibiotics, because it has accumulated resistances to essentially all antibiotics that are currently in clinical practice.

A Conserved Class II Type Thioester Domain-Containing Adhesin Is Required for Efficient Conjugation in Bacillus subtilis.

Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium Bacillus subtilis allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugation.IMPORTANCE Bacterial resistance to antibiotics has become a serious health care problem. The spread of antibiotic resistance genes between bacteria of the same or different species is often mediated by a process named conjugation, where a donor cell transfers DNA to a recipient cell through a connecting channel. The first step in conjugation is recognition and attachment of the donor to a recipient cell. Little is known about this first step, particularly in Gram-positive bacteria. Here, we show that the conjugative plasmid pLS20 of Bacillus subtilis encodes an adhesin protein that is essential for effective conjugation. This adhesin protein has a structural organization similar to adhesins produced by other Gram-positive bacteria, including major pathogens, where the adhesins serve in attachment to host tissues during colonization and infection. Our findings may thus also open novel avenues to design drugs that inhibit the spread of antibiotic resistance by blocking the first recipient-attachment step in conjugation.

Functional association of the stress-responsive LiaH protein and the minimal TatAyCy protein translocase in Bacillus subtilis.

The bacterial twin-arginine (Tat) pathway serves in the exclusive secretion of folded proteins with bound cofactors. While Tat pathways in Gram-negative bacteria and chloroplast thylakoids consist of conserved TatA, TatB and TatC subunits, the Tat pathways of Bacillus species and many other Gram-positive bacteria stand out for their minimalist nature with the core translocase being composed of essential TatA and TatC subunits only. Here we addressed the question whether the minimal TatAyCy translocase of Bacillus subtilis recruits additional cellular components that modulate its activity. To this end, TatAyCy was purified by affinity- and size exclusion chromatography, and interacting co-purified proteins were identified by mass spectrometry. This uncovered the cell envelope stress responsive LiaH protein as an accessory subunit of the TatAyCy complex. Importantly, our functional studies show that Tat expression is tightly trailed by LiaH induction, and that LiaH itself determines the capacity and quality of TatAyCy-dependent protein translocation. In contrast, LiaH has no role in high-level protein secretion via the general secretion (Sec) pathway. Altogether, our observations show that protein translocation by the minimal Tat translocase TatAyCy is tightly intertwined with an adequate bacterial response to cell envelope stress. This is consistent with a critical need to maintain cellular homeostasis, especially when the membrane is widely opened to permit passage of large fully-folded proteins via Tat.

Membrane Modulation of Super-Secreting "midiBacillus" Expressing the Major Staphylococcus aureus Antigen - A Mass-Spectrometry-Based Absolute Quantification Approach.

Bacillus subtilis has been extensively used as a microbial cell factory for industrial enzymes due to its excellent capacities for protein secretion and large-scale fermentation. This bacterium is also an attractive host for biopharmaceutical production. However, the secretion potential of this organism is not fully utilized yet, mostly due to a limited understanding of critical rearrangements in the membrane proteome upon high-level protein secretion. Recently, it was shown that bottlenecks in heterologous protein secretion can be resolved by genome minimization. Here, we present for the first time absolute membrane protein concentrations of a genome-reduced B. subtilis strain ("midiBacillus") expressing the immunodominant Staphylococcus aureus antigen A (IsaA). We quantitatively characterize the membrane proteome adaptation of midiBacillus during production stress on the level of molecules per cell for more than 400 membrane proteins, including determination of protein concentrations for ∼61% of the predicted transporters. We demonstrate that ∼30% of proteins with unknown functions display a significant increase in abundance, confirming the crucial role of membrane proteins in vital biological processes. In addition, our results show an increase of proteins dedicated to translational processes in response to IsaA induction. For the first time reported, we provide accumulation rates of a heterologous protein, demonstrating that midiBacillus secretes 2.41 molecules of IsaA per minute. Despite the successful secretion of this protein, it was found that there is still some IsaA accumulation occurring in the cytosol and membrane fraction, leading to a severe secretion stress response, and a clear adjustment of the cell's array of transporters. This quantitative dataset offers unprecedented insights into bioproduction stress responses in a synthetic microbial cell.

Publisher Correction: In vitro imaging of bacteria using 18F-fluorodeoxyglucose micro positron emission tomography.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Less Is More: Toward a Genome-Reduced Bacillus Cell Factory for "Difficult Proteins".

The availability of complete genome sequences and the definition of essential gene sets were fundamental in the start of the genome engineering era. In a recent study, redundant and unnecessary genes were systematically deleted from the Gram-positive bacterium Bacillus subtilis, an industrial production host of high-value secreted proteins. This culminated in strain PG10, which lacks about 36% of the genome, thus representing the most minimal Bacillus chassis currently available. Here, we show that this "mini Bacillus" strain has synthetic traits that are favorable for producing "difficult-to-produce proteins". As exemplified with different staphylococcal antigens, PG10 overcomes several bottlenecks in protein production related to the secretion process and instability of the secreted product. These findings show for the first time that massive genome reduction can substantially improve secretory protein production by a bacterial expression host, and underpin the high potential of genome-engineered strains as future cell factories.

In vitro imaging of bacteria using 18F-fluorodeoxyglucose micro positron emission tomography.

Positron emission tomography (PET) with fluorine-18-fluorodeoxyglucose (18F-FDG) can be applied to detect infection and inflammation. However, it was so far not known to what extent bacterial pathogens may contribute to the PET signal. Therefore, we investigated whether clinical isolates of frequently encountered bacterial pathogens take up 18F-FDG in vitro, and whether FDG inhibits bacterial growth as previously shown for 2-deoxy-glucose. 22 isolates of Gram-positive and Gram-negative bacterial pathogens implicated in fever and inflammation were incubated with 18F-FDG and uptake of 18F-FDG was assessed by gamma-counting and µPET imaging. Possible growth inhibition by FDG was assayed with Staphylococcus aureus and the Gram-positive model bacterium Bacillus subtilis. The results show that all tested isolates accumulated 18F-FDG actively. Further, 18F-FDG uptake was hampered in B. subtilis pts mutants impaired in glucose uptake. FDG inhibited growth of S. aureus and B. subtilis only to minor extents, and this effect was abrogated by pts mutations in B. subtilis. These observations imply that bacteria may contribute to the signals observed in FDG-PET infection imaging in vivo. Active bacterial FDG uptake is corroborated by the fact that the B. subtilis phosphotransferase system is needed for 18F-FDG uptake, while pts mutations protect against growth inhibition by FDG.

Design, synthesis and biological evaluation of quinazoline derivatives as anti-trypanosomatid and anti-plasmodial agents.

In this paper, the design, synthesis and biological evaluation of a set of quinazoline-2,4,6-triamine derivatives (1-9) as trypanocidal, antileishmanial and antiplasmodial agents are explained. The compounds were rationalized basing on docking studies of the dihydrofolate reductase (DHFR from Trypanosoma cruzi, Leishmania major and Plasmodium vivax) and pteridin reductase (PTR from T. cruzi and L. major) structures. All compounds were in vitro screened against both bloodstream trypomastigotes of T. cruzi (NINOA and INC-5 strains) and promatigotes of Leishmania mexicana (MHOM/BZ/61/M379 strain), and also for cytotoxicity using Vero cell line. Against T. cruzi, three compounds (5, 6 and 8) were the most effective showing a better activity profile than nifurtimox and benznidazole (reference drugs). Against L. mexicana, four compounds (5, 6, 8, and 9) exhibited the highest activity, even than glucantime (reference drug). In the cytotoxicity assay, protozoa were more susceptible than Vero cells. In vivo Plasmodium berghei assay (ANKA strain), the compounds 1, 5, 6 and 8 showed a more comparable activity than chloroquine and pyrimethamine (reference drugs) when they were administrated by the oral route. The antiprotozoal activity of these substances, endowed with redox properties, represented a good starting point for a medicinal chemistry program aiming for chemotherapy of Chagas' disease, leishmaniosis and malaria.