Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions

Background Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M. Results Here we show that activity of the htrB promoter as induced by overproduction of AmyM was “noisy”, which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions. Conclusion Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement

High-salinity growth conditions promote tat-independent secretion of tat substrates in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway

Environmental Salinity Determines the Specificity and Need for Tat-Dependent Secretion of the YwbN Protein in Bacillus subtilis

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described “minimal Tat translocase” consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate

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

Background: The Rieske protein QcrA was recently shown to be exported by twin-arginine translocation (Tat) in Bacillus subtilis. Results: QcrA has disulfide bond and co-factor requirements for effective Tat-dependent translocation. Conclusion: A hierarchy exists between disulfide bonding and co-factor insertion in QcrA quality control and translocation. Significance: First studies on Tat-dependent translocation where oxidative folding and co-factor attachment were addressed in a single native molecule. 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 bc(1) 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

Degradation of the Twin-Arginine Translocation Substrate YwbN by Extracytoplasmic Proteases of Bacillus subtilis

Bacterial twin-arginine translocases can export fully folded proteins from the cytoplasm. Such proteins are usually resistant to proteolysis. Here we show that multiple extracellular proteases degrade the B. subtilis Tat substrate YwbN. This suggests either that secreted YwbN is not fully folded or that folded YwbN exposes protease cleavage sites

The Tat system of Gram-positive bacteria

AbstractThe 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

Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis

The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins. Among the native exported proteins of B. subtilis are at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control

Specific Targeting of the Metallophosphoesterase YkuE to the Bacillus Cell Wall Requires the Twin-arginine Translocation System

The twin-arginine translocation (Tat) pathway is dedicated to the transport of fully folded proteins across the cytoplasmic membranes of many bacteria and the chloroplast thylakoidal membrane. Accordingly, Tat-dependently translocated proteins are known to be delivered to the periplasm of Gram-negative bacteria, the growth medium of Gram-positive bacteria, and the thylakoid lumen. Here, we present the first example of a protein, YkuE of Bacillus subtilis, that is specifically targeted by the Tat pathway to the cell wall of a Gram-positive bacterium. The cell wall binding of YkuE is facilitated by electrostatic interactions. Interestingly, under particular conditions, YkuE can also be targeted to the cell wall in a Tat-independent manner. The biological function of YkuE was so far unknown. Our present studies show that YkuE is a metal-dependent phosphoesterase that preferentially binds manganese and zinc

Characterization of L-Aspartate oxidase and quinolinate synthase from Bacillus subtilis

NAD is an important cofactor and essential molecule in all living organisms. In many eubacteria, including several pathogens, the first two steps in the de novo synthesis of NAD are catalyzed by l-aspartate oxidase (NadB) and quinolinate synthase (NadA). Despite the important role played by these two enzymes in NAD metabolism, many of their biochemical and structural properties are still largely unknown. In the present study, we cloned, overexpressed and characterized NadA and NadB from Bacillus subtilis, one of the best studied bacteria and a model organism for low-GC Gram-positive bacteria. Our data demonstrated that NadA from B. subtilis possesses a [4Fe-4S]2+ cluster, and we also identified the cysteine residues involved in the cluster binding. The [4Fe-4S]2+ cluster is coordinated by three cysteine residues (Cys110, Cys230, and Cys320) that are conserved in all the NadA sequences reported so far, suggesting a new noncanonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different organisms. Moreover, for the first time, it was shown that the interaction between NadA and NadB is not species-specific between B. subtilis and Escherichia coli