Protein secretion in gram-positive bacteria.

Gram-positive bacteria often secrete large amounts of proteins into the surrounding medium. This feature makes them attractive as hosts for the industrial production of extracellular enzymes. Compared to Escherichia coli, relatively little is known about the mechanism of protein secretion in these organisms. However, the recent identification of Bacillus subtilis genes whose gene products are highly homologous to some of the Sec (secretion) proteins of E. coli strongly suggests that important principles of protein translocation across the plasma membrane might be highly conserved. In contrast, the steps following the actual translocation event might be different in Gram-positive and Gram-negative bacteria. The scope of this review is to outline the recent progress that has been made in the elucidation of the secretion pathway in Gram-positive bacteria and to discuss potential applications in strain improvement for the industrial production of extracellular proteins.     

Genomic analysis of secretion systems

Secretion of proteins into the extracellular environment is important to almost all bacteria, and in particular mediates interactions between pathogenic or symbiotic bacteria with their eukaryotic hosts. The accumulation of bacterial genome sequence data in the past few years has provided great insights into the distribution and function of these secretion systems. Three systems are responsible for secretion of proteins across the bacterial cytoplasmic membrane: Sec, SRP and Tat. Many novel examples of systems for transport across the Gram-negative bacterial cell envelope have been discovered through genome sequencing and surveys, including many novel type III secretion systems and autotransporters. Similarly, genomic data mining has revealed many new potential secretion substrates and identified unsuspected domains in secretion-associated proteins. Interestingly, genomic analyses have also hinted at the existence of a dedicated protein secretion system in Gram-positive bacteria, targeting members of the WXG100/ESAT-6 family of proteins, and have revealed an unexpectedly wide distribution of sortase-driven protein-targeting systems.     

Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli

Bacterial protein secretion is important in the life cycles of most bacteria, in which it contributes to the formation of pili and flagella and makes available extracellular enzymes to digest polymers for nutritional purposes and toxins to kill host cells in infections of humans, animals and plants. It is generally accepted that nonpathogenic laboratory strains of Escherichia coli, particularly K12 strains, do not secrete proteins into the extracellular medium under routine growth conditions. In this study, we report that commonly used laboratory strains secrete YebF, a small (10.8 kDa in the native form), soluble endogenous protein into the medium, challenging the status quo view that laboratory strains do not secrete proteins to the medium. We further show that 'passenger' proteins linked to the carboxyl end of YebF are efficiently secreted. The function of YebF is unknown, but its use as a carrier for transgenic proteins provides a tool to circumvent toxicity and other contamination issues associated with protein production in E. coli.     

Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story

Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.     

Secretion of recombinant proteins from E. coli

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.     

Production of recombinant antibody fragments in <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Background  

Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments.     

Secretion, processing and activation of bacterial extracellular proteases

Many different bacteria secrete proteases into the culture medium. Extracellular proteases produced by Gram-positive bacteria are secreted by a signal-peptide-dependent pathway and have a propeptide located between the signal peptide and the mature protein. Many extracellular proteases synthesized by Gram-negative bacteria are also produced as precursors with a signal peptide. However, at least two species of Gram-negative bacteria secrete one or more proteases via a novel signal-peptide-independent route. Most proteases secreted by Gram-negative bacteria also have a propeptide whose length and location vary according to the protease. Specific features of protease secretion pathways and the mechanisms of protease activation are discussed with particular reference to some of the best-characterized extracellular proteases produced by Gram-positive and Gram-negative bacteria.     

Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase.

This article describes the reconstitution in Escherichia coli of a heterologous protein secretion system comprising a gene for an extracellular protein together with its cognate secretion genes. The protein concerned, pullulanase, is a secreted lipoprotein of the Gram-negative bacterium Klebsiella pneumoniae. It is initially localized to the cell surface before being specifically released into the medium. E. coli carrying the cloned pullulanase structural gene (pulA) produces pullulanase but does not expose or secrete it. Secretion genes were cloned together with pulA in an 18.8 kbp fragment of K. pneumoniae chromosomal DNA. E. coli carrying this fragment exhibited maltose-inducible production, exposition and specific secretion of pullulanase. Transposon mutagenesis showed that the secretion genes are located on both sides of pulA. Secretion genes located 5' to pulA were transcribed in the opposite orientation to pulA under the control of the previously identified, malT-regulated malX promoter. Thus these secretion genes are part of the maltose regulon and are therefore co-expressed with pulA. Transposon mutagenesis suggested that secretion genes located 3' of pulA are not co-transcribed with pulA, raising the possibility that some secretion functions are not maltose regulated.     

Growth and release of extracellular organic compounds by benthic diatoms depend on interactions with bacteria

Phototrophic epilithic biofilms harbour a distinct assemblage of heterotrophic bacteria, cyanobacteria and photoautotrophic algae. Secretion of extracellular polymeric substances (EPS) by these organisms and the physicochemical properties of the EPS are important factors for the development of the biofilms. We have isolated representative diatom and bacteria strains from epilithic biofilms of Lake Constance. By pairwise co-cultivating these strains we found that diatom growth and EPS secretion by diatoms may depend on the presence of individual bacteria. Similar results were obtained after addition of spent bacterial medium to diatom cultures, suggesting that soluble substances from bacteria have an impact on diatom physiology. While searching for putative bacterial signal substances, we found that concentrations of various dissolved free amino acids (DFAA) within the diatom cultures changed drastically during co-cultivation with bacteria. Further, the secretion of extracellular carbohydrates and proteins can be influenced by bacteria or their extracellular substances. We have performed mass spectrometric peptide mapping to identify proteins which are secreted when co-cultivating the diatom Phaeodactylum tricornutum Bohlin and Escherichia coli. The identified proteins are possibly involved in signalling, extracellular carbohydrate modification and uptake, protein and amino acid modification, and cell/cell aggregation of diatom and bacteria strains. Our data indicate that diatom-bacteria biofilms might be regulated by a complex network of chemical factors involving EPS, amino acid monomers and other substances. Thus interactions with bacteria can be considered as one of the main factors driving biofilm formation by benthic diatoms.     

Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems

Bacillus spp. are Gram-positive bacteria that secrete a large number of extracellular proteins of industrial relevance. In this report, three Bacillus extracellular hydrolytic enzymes, i.e., alpha-amylase, mannanase and chitinase, were cloned and over-expressed in Gram-negative Escherichia coli. We found that both the native signal peptides and that of E. coli outer membrane protein, OmpA, could be used to direct the secretion of the recombinant enzymes. The expressed enzymes were observed as clearing zones on agar plates or in zymograms. Determination of enzyme activities in different cell compartments suggested that the ability of the enzymes to be secreted out into the culture medium depends on the time of induction, the type of the signal peptides and the molecular mass of the enzymes. After overnight induction, most of the enzyme activities (85-96%) could be harvested from the culture supernatant. Our results suggest that various signal peptides of Bacillus spp. can be recognized by the E. coli secretion machinery. It seems possible that other enzymes with similar signal peptide could be secreted equally well in E. coli expression systems. Thus, our finding should be able to apply for cloning and extracellular production of other Bacillus hydrolytic enzymes as well as other proteins.     

Type III export: new uses for an old pathway

Gram-negative bacteria use type III secretion (TTS) systems to translocate proteins into the extracellular environment or directly into eukaryotic cells. These complex secretory systems are assembled from over 20 different structural proteins, including 10 that have counterparts in the flagellar export pathway. Secretion substrates are directed to the TTS machinery via mRNA and/or amino acid secretion signals. TTS chaperones bind to select secretion substrates and assist in the export process. Recent progress in the understanding of TTS is reviewed.     

Extracellular recombinant protein production from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.     

New insight into the molecular mechanisms of two-partner secretion

Two-partner secretion (TPS) systems, which export large proteins to the surface and/or extracellular milieu of Gram-negative bacteria, are members of a large superfamily of protein translocation systems that are widely distributed in animals, plants and fungi, in addition to nearly all groups of Gram-negative bacteria. Recent intense research on TPS systems has provided new insight into the structure and topology of the outer membrane translocator proteins and the large exoproteins that they secrete, the interactions between them, and mechanisms for retention of some of the secreted proteins on the bacterial surface. Evidence for secretion-dependent folding of mature exoproteins has also been obtained. Together, these findings provide a deeper understanding of the molecular mechanisms underlying these simple but elegant secretion systems.     

Translocation of proteins across the cell envelope of Gram-positive bacteria

In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.     

The secretion pathway of IgA protease-type proteins in gram-negative bacteria

The pathogenic, Gram-negative bacteria, Neisseria gon-orrhoeae, Neisseria meningitidis and Haemophilus influenzae, secrete immunoglobulin A1 proteases into their extracellular surroundings. An extraordinary feature in the secretory pathway of these putative virulence factors is a self-directed outer membrane transport step allowing the proteins to be secreted autonomously, even from foreign Gram-negative host cells like Escherichia coli. Here we summarize recent achievements in the understanding of IgA protease outer membrane translocation.     

Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR) from transfected cells and placenta explants

Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR). We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm). Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.     

Protein secretion in Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to have disseminated widely among Gram-negative bacteria.