Self-pigmenting textiles grown from cellulose-producing bacteria with engineered tyrosinase expression.

Environmental concerns are driving interest in postpetroleum synthetic textiles produced from microbial and fungal sources. Bacterial cellulose (BC) is a promising sustainable leather alternative, on account of its material properties, low infrastructure needs and biodegradability. However, for alternative textiles like BC to be fully sustainable, alternative ways to dye textiles need to be developed alongside alternative production methods. To address this, we genetically engineer Komagataeibacter rhaeticus to create a bacterial strain that grows self-pigmenting BC. Melanin biosynthesis in the bacteria from recombinant tyrosinase expression achieves dark black coloration robust to material use. Melanated BC production can be scaled up for the construction of prototype fashion products, and we illustrate the potential of combining engineered self-pigmentation with tools from synthetic biology, through the optogenetic patterning of gene expression in cellulose-producing bacteria. With this study, we demonstrate that combining genetic engineering with current and future methods of textile biofabrication has the potential to create a new class of textiles.

Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria.

Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the Komagataeibacter genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the Komagataeibacter tool kit (KTK), a standardized modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags, and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fiber system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.

Relative contributions of non-essential Sec pathway components and cell envelope-associated proteases to high-level enzyme secretion by Bacillus subtilis.

BACKGROUND: Bacillus subtilis is an important industrial workhorse applied in the production of many different commercially relevant proteins, especially enzymes. Virtually all of these proteins are secreted via the general secretion (Sec) pathway. Studies from different laboratories have demonstrated essential or non-essential contributions of various Sec machinery components to protein secretion in B. subtilis. However, a systematic comparison of the impact of each individual Sec machinery component under conditions of high-level protein secretion was so far missing. RESULTS: In the present study, we have compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases on the secretion efficiency of three proteins expressed at high level. This concerned the α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis, and the serine protease BPN' from Bacillus amyloliquefaciens. We compared the secretion capacity of mutant strains in shake flask cultures, and the respective secretion kinetics by pulse-chase labeling experiments. The results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPN' secretion, but the actual effect size depends on the investigated protein. Additionally, the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion. Further, we assessed the induction of secretion stress responses in mutant strains by examining AmyE- and AmyL-dependent induction of the quality control proteases HtrA and HtrB. Interestingly, the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response. CONCLUSIONS: The results of the present study highlight the importance of SecDF, SecG and RasP for protein secretion and reveal unexpected differences in the induction of the secretion stress response in different mutant strains.

Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis.

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, i.e. a methylated N terminus; (iii) are found in the same heteropolymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin-fold with a highly unusual flexible C terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

Engineered cell-to-cell signalling within growing bacterial cellulose pellicles.

Bacterial cellulose is a strong and flexible biomaterial produced at high yields by Acetobacter species and has applications in health care, biotechnology and electronics. Naturally, bacterial cellulose grows as a large unstructured polymer network around the bacteria that produce it, and tools to enable these bacteria to respond to different locations are required to grow more complex structured materials. Here, we introduce engineered cell-to-cell communication into a bacterial cellulose-producing strain of Komagataeibacter rhaeticus to enable different cells to detect their proximity within growing material and trigger differential gene expression in response. Using synthetic biology tools, we engineer Sender and Receiver strains of K. rhaeticus to produce and respond to the diffusible signalling molecule, acyl-homoserine lactone. We demonstrate that communication can occur both within and between growing pellicles and use this in a boundary detection experiment, where spliced and joined pellicles sense and reveal their original boundary. This work sets the basis for synthetic cell-to-cell communication within bacterial cellulose and is an important step forward for pattern formation within engineered living materials.

Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili.

Type IV pili (Tfp), which are key virulence factors in many bacterial pathogens, define a large group of multipurpose filamentous nanomachines widespread in Bacteria and Archaea. Tfp biogenesis is a complex multistep process, which relies on macromolecular assemblies composed of 15 conserved proteins in model gram-negative species. To improve our limited understanding of the molecular mechanisms of filament assembly, we have used a synthetic biology approach to reconstitute, in a nonnative heterologous host, a minimal machinery capable of building Tfp. Here we show that eight synthetic genes are sufficient to promote filament assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterized biochemically. Our results contribute to a better mechanistic understanding of the assembly of remarkable dynamic filaments nearly ubiquitous in prokaryotes.

Intramembrane protease RasP boosts protein production in Bacillus.

BACKGROUND: The microbial cell factory Bacillus subtilis is a popular industrial platform for high-level production of secreted technical enzymes. Nonetheless, the effective secretion of particular heterologous enzymes remains challenging. Over the past decades various studies have tackled this problem, and major improvements were achieved by optimizing signal peptides or removing proteases involved in product degradation. On the other hand, serious bottlenecks in the protein export process per se remained enigmatic, especially for protein secretion at commercially significant levels by cells grown to high density. The aim of our present study was to assess the relevance of the intramembrane protease RasP for high-level protein production in B. subtilis. RESULTS: Deletion of the rasP gene resulted in reduced precursor processing and extracellular levels of the overproduced α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis. Further, secretion of the overproduced serine protease BPN' from Bacillus amyloliquefaciens was severely impaired in the absence of RasP. Importantly, overexpression of rasP resulted in threefold increased production of a serine protease from Bacillus clausii, and 2.5- to 10-fold increased production of an AmyAc α-amylase from Paenibacillus curdlanolyticus, depending on the culture conditions. Of note, growth defects due to overproduction of the two latter enzymes were suppressed by rasP-overexpression. CONCLUSION: Here we show that an intramembrane protease, RasP, sets a limit to high-level production of two secreted heterologous enzymes that are difficult to produce in the B. subtilis cell factory. This finding was unexpected and suggests that proteolytic membrane sanitation is key to effective enzyme production in Bacillus.

Twin-Arginine Protein Translocation.

Twin-arginine protein translocation systems (Tat) translocate fully folded and co-factor-containing proteins across biological membranes. In this review, we focus on the Tat pathway of Gram-positive bacteria. The minimal Tat pathway is composed of two components, namely a TatA and TatC pair, which are often complemented with additional TatA-like proteins. We provide overviews of our current understanding of Tat pathway composition and mechanistic aspects related to Tat-dependent cargo protein translocation. This includes Tat pathway flexibility, requirements for the correct folding and incorporation of co-factors in cargo proteins and the functions of known cargo proteins. Tat pathways of several Gram-positive bacteria are discussed in detail, with emphasis on the Tat pathway of Bacillus subtilis. We discuss both shared and unique features of the different Gram-positive bacterial Tat pathways. Lastly, we highlight topics for future research on Tat, including the development of this protein transport pathway for the biotechnological secretion of high-value proteins and its potential applicability as an antimicrobial drug target in pathogens.

A Tat ménage à trois--The role of Bacillus subtilis TatAc in twin-arginine protein translocation.

The twin-arginine translocation system (Tat) is a protein transport system that moves fully folded and cofactor-containing proteins across membranes of bacteria, archaea and thylakoids. The minimal Tat pathway is composed of two subunits, TatA and TatC. In some organisms TatA has been duplicated and evolved to form a third specialized subunit, TatB. The Bacillus subtilis genome encodes two TatC subunits (TatCd and TatCy) and three TatA subunits (TatAd, TatAy and TatAc). These subunits combine to form two parallel minimal pathways, TatAy-TatCy and TatAd-TatCd. The purpose and role of the third TatA component, TatAc, has remained ambiguous. In this study we examined the translocation of two natively expressed TatAy-TatCy-dependent substrates, EfeB and QcrA, in various Tat-deficient genetic backgrounds. More specifically, we examined the ability of different mutated TatAy subunits to complement for the absence of wild-type TatAy. We further detailed a graded growth phenotype associated with the functional translocation of EfeB. We found that in various instances where specific amino acid substitutions were made in TatAy, a definite TatAc-associated growth phenotype occurred in genetic backgrounds lacking TatAc. Altogether, our findings show that TatAy and TatAc interact and that this TatAy-TatAc interaction, although not essential, supports the translocation of the Tat substrate EfeB when TatAy function is compromised. This implies that the third TatA-like protein in B. subtilis could represent an intermediate evolutionary step in TatA-TatB specialization.

Co-factor insertion and disulfide bond requirements for twin-arginine translocase-dependent export of the Bacillus subtilis Rieske protein QcrA.

The twin-arginine translocation (Tat) pathway can transport folded and co-factor-containing cargo proteins over bacterial cytoplasmic membranes. Functional Tat machinery components, a folded state of the cargo protein and correct co-factor insertion in the cargo protein are generally considered as prerequisites for successful translocation. The present studies were aimed at a dissection of these requirements with regard to the Rieske iron-sulfur protein QcrA of Bacillus subtilis. Notably, QcrA is a component of the cytochrome bc1 complex, which is conserved from bacteria to man. Single amino acid substitutions were introduced into the Rieske domain of QcrA to prevent either co-factor binding or disulfide bond formation. Both types of mutations precluded QcrA translocation. Importantly, a proofreading hierarchy was uncovered, where a QcrA mutant defective in disulfide bonding was quickly degraded, whereas mutant QcrA proteins defective in co-factor binding accumulated in the cytoplasm and membrane. Altogether, these are the first studies on Tat-dependent protein translocation where both oxidative folding and co-factor attachment have been addressed in a single native molecule.

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis.

The Tat system of Gram-positive bacteria.

The twin-arginine protein translocation (Tat) system has a unique ability to translocate folded and co-factor-containing proteins across lipid bilayers. The Tat pathway is present in bacteria, archaea and in the thylakoid membranes of chloroplasts and, depending on the organism and environmental conditions, it can be deemed important for cell survival, virulence or bioproduction. This review provides an overview of the current understanding of the Tat system with specific focus on Gram-positive bacteria. The 'universal minimal Tat system' is composed of a TatA and a TatC protein. However, this pathway is more commonly composed of two TatA-like proteins and one TatC protein. Often the TatA-like proteins have diverged to have two different functions and, in this case, the second TatA-like protein is usually referred to as TatB. The correct folding and/or incorporation of co-factors are requirements for translocation, and the known quality control mechanisms are examined in this review. A number of examples of crosstalk between the Tat system and other protein transport systems, such as the Sec-YidC translocon and signal peptidases or sheddases are also discussed. Further, an overview of specific Gram-positive bacterial Tat systems found in monoderm and diderm species is detailed. Altogether, this review highlights the unique features of Gram-positive bacterial Tat systems and pinpoints key questions that remain to be addressed in future research. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Extracytoplasmic proteases determining the cleavage and release of secreted proteins, lipoproteins, and membrane proteins in Bacillus subtilis.

Gram-positive bacteria are known to export many proteins to the cell wall and growth medium, and accordingly, many studies have addressed the respective protein export mechanisms. In contrast, very little is known about the subsequent fate of these proteins. The present studies were therefore aimed at determining the fate of native exported proteins in the model organism Bacillus subtilis. Specifically, we employed a gel electrophoresis-based liquid chromatography-mass spectrometry approach to distinguish the roles of the membrane-associated quality control proteases HtrA and HtrB from those of eight other proteases that are present in the cell wall and/or growth medium of B. subtilis. Notably, HtrA and HtrB were previously shown to counteract potentially detrimental "protein export stresses" upon overproduction of membrane or secreted proteins. Our results show that many secreted proteins, lipoproteins, and membrane proteins of B. subtilis are potential substrates of extracytoplasmic proteases. Moreover, potentially important roles of HtrA and HtrB in the folding of native secreted proteins into a protease-resistant conformation, the liberation of lipoproteins from the membrane-cell wall interface, and the degradation of membrane proteins are uncovered. Altogether, our observations show that HtrA and HtrB are crucial for maintaining the integrity of the B. subtilis cell even under nonstress conditions.

Is proteomics a reliable tool to probe the oxidative folding of bacterial membrane proteins?

The oxidative folding of proteins involves disulfide bond formation, which is usually catalyzed by thiol-disulfide oxidoreductases (TDORs). In bacteria, this process takes place in the cytoplasmic membrane and other extracytoplasmic compartments. While it is relatively easy to study oxidative folding of water-soluble proteins on a proteome-wide scale, this has remained a major challenge for membrane proteins due to their high hydrophobicity. Here, we have assessed whether proteomic techniques can be applied to probe the oxidative folding of membrane proteins using the Gram-positive bacterium Bacillus subtilis as a model organism. Specifically, we investigated the membrane proteome of a B. subtilis bdbCD mutant strain, which lacks the primary TDOR pair BdbC and BdbD, by gel-free mass spectrometry. In total, 18 membrane-associated proteins showed differing behavior in the bdbCD mutant and the parental strain. These included the ProA protein involved in osmoprotection. Consistent with the absence of ProA, the bdbCD mutant was found to be sensitive to osmotic shock. We hypothesize that membrane proteomics is a potentially effective approach to profile oxidative folding of bacterial membrane proteins.

Novel twin-arginine translocation pathway-dependent phenotypes of Bacillus subtilis unveiled by quantitative proteomics.

The twin-arginine translocation (Tat) pathway is known to translocate fully folded proteins across bacterial, archaeal, and organellar membranes. To date, the mechanisms involved in processing, proofreading, and quality control of Tat substrates have remained largely elusive. Bacillus subtilis is an industrially relevant Gram-positive model bacterium. The Tat pathway in B. subtilis differs from that of other well-studied organisms in that it is composed of two complexes operating in parallel. To obtain a better understanding of this pathway in B. subtilis and to identify Tat-associated proteins, the B. subtilis 'Tat proteome' was investigated by quantitative proteomics. Metabolically labeled proteins from cytoplasmic, membrane, and extracellular fractions were analyzed by LC-MS/MS. Changes in the amounts of identified peptides allowed for quantitative comparisons of their abundance in tat mutant strains. The observed differences were suggestive of indirect or direct protein-protein relationships. The rich data set generated was then approached in hypothesis-driving and hypothesis-driven manners. The hypothesis-driving approach led to the identification of a novel delayed biofilm phenotype of certain tat mutant strains, whereas the hypothesis-driven approach identified the membrane protein QcrA as a new Tat substrate of B. subtilis. Thus, our quantitative proteomics analyses have unveiled novel Tat pathway-dependent phenotypes in Bacillus.

Salt sensitivity of minimal twin arginine translocases.

Bacterial twin arginine translocation (Tat) pathways have evolved to facilitate transport of folded proteins across membranes. Gram-negative bacteria contain a TatABC translocase composed of three subunits named TatA, TatB, and TatC. In contrast, the Tat translocases of most Gram-positive bacteria consist of only TatA and TatC subunits. In these minimal TatAC translocases, a bifunctional TatA subunit fulfils the roles of both TatA and TatB. Here we have probed the importance of conserved residues in the bifunctional TatAy subunit of Bacillus subtilis by site-specific mutagenesis. A set of engineered TatAy proteins with mutations in the cytoplasmic hinge and amphipathic helix regions were found to be inactive in protein translocation under standard growth conditions for B. subtilis or when heterologously expressed in Escherichia coli. Nevertheless, these mutated TatAy proteins did assemble into TatAy and TatAyCy complexes, and they facilitated membrane association of twin arginine precursor proteins in E. coli. Interestingly, most of the mutated TatAyCy translocases were salt-sensitive in B. subtilis. Similarly, the TatAC translocases of Bacillus cereus and Staphylococcus aureus were salt-sensitive when expressed in B. subtilis. Taken together, our present observations imply that salt-sensitive electrostatic interactions have critical roles in the preprotein translocation activity of certain TatAC type translocases from Gram-positive bacteria.