Archaeal and bacterial SecD and SecF homologs exhibit striking structural and functional conservation

The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli, an H. volcanii deltasecFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.     

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.     

Mutations in Flavobacterium johnsoniae secDF result in defects in gliding motility and chitin utilization

secDF mutants of Flavobacterium johnsoniae were deficient in gliding motility and chitin utilization. Cells of the mutants had reduced levels of GldJ protein, which is required for both processes. SecDF is similar to Escherichia coli SecD and SecF, which are involved in protein secretion.     

Identification of YidC Residues That Define Interactions with the Sec Apparatus

In bacteria, a subset of membrane proteins insert into the membrane via the Sec apparatus with the assistance of the widely conserved essential membrane protein insertase YidC. After threading into the SecYEG translocon, transmembrane segments of nascent proteins are thought to exit the translocon via a lateral gate in SecY, where YidC facilitates their transfer into the lipid bilayer. Interactions between YidC and components of the Sec apparatus are critical to its function. The first periplasmic loop of YidC interacts directly with SecF. We sought to identify the regions or residues of YidC that interact with SecY or with additional components of the Sec apparatus other than SecDF. Using a synthetic lethal screen, we identified residues of YidC that, when mutated, led to dependence on SecDF for viability. Each residue identified is highly conserved among YidC homologs; most lie within transmembrane domains. Overexpression of SecY in the presence of two YidC mutants partially rescued viability in the absence of SecDF, suggesting that the corresponding wild-type YidC residues (G355 and M471) participate in interactions, direct or indirect, with SecY. Staphylococcus aureus YidC complemented depletion of YidC, but not of SecDF, in Escherichia coli. G355 of E. coli YidC is invariant in S. aureus YidC, suggesting that this highly conserved glycine serves a conserved function in interactions with SecY. This study demonstrates that transmembrane residues are critical in YidC interactions with the Sec apparatus and provides guidance on YidC residues of interest for future structure-function analyses.     

Internal duplications in α-helical membrane protein topologies are common but the nonduplicated forms are rare

Many α-helical membrane proteins contain internal symmetries, indicating that they might have evolved through a gene duplication and fusion event. Here, we have characterized internal duplications among membrane proteins of known structure and in three complete genomes. We found that the majority of large transmembrane (TM) proteins contain an internal duplication. The duplications found showed a large variability both in the number of TM-segments included and in their orientation. Surprisingly, an approximately equal number of antiparallel duplications and parallel duplications were found. However, of all 11 superfamilies with an internal duplication, only for one, the AcrB Multidrug Efflux Pump, the duplicated unit could be found in its nonduplicated form. An evolutionary analysis of the AcrB homologs indicates that several independent fusions have occurred, including the fusion of the SecD and SecF proteins into the 12-TM-protein SecDF in Brucella and Staphylococcus aureus. In one additional case, the Vitamin B12 transporter-like ABC transporters, the protein had undergone an additional fusion to form protein with 20 TM-helices in several bacterial genomes. Finally, homologs to all human membrane proteins were used to detect the presence of duplicated and nonduplicated proteins. This confirmed that only in rare cases can homologs with different duplication status be found, although internal symmetry is frequent among these proteins. One possible explanation is that it is frequent that duplication and fusion events happen simultaneously and that there is almost always a strong selective advantage for the fused form.     

SecD and SecF facilitate protein export in Escherichia coli.

We show here that the rate of protein translocation in the bacterium Escherichia coli depends on the levels of the SecD and SecF proteins in the cell. Overexpression of SecD and SecF stimulates translocation in wild type cells and improves export of proteins with mutant signal sequences. Depletion of SecD and SecF from the cell greatly reduces but does not abolish protein translocation. A secDF::kan null mutant deleted for the genes encoding both proteins is cold-sensitive for growth and protein export, has a severe export defect at 37 degrees C and is barely viable. The phenotypes of a secD null mutant and a secF null mutant are identical to the secDF::kan double null mutant. These results partially resolve the conflict between genetic studies and results from in vitro translocation systems which do not require SecD and SecF for activity, affirm the importance of these proteins to the export process, and suggest that SecD and SecF function together to stimulate protein export in a role fundamentally different from other Sec proteins. Our results provide additional support for the notion that an early step in protein export is cold-sensitive.     

Evolutionary evidence on suitability of SecD as a target for development of antibacterial agents against Staphylococcus aureus

Staphylococcus aureus causes many infections and its drug resistance is a worrying challenge for medical care. The SecD subunit of Sec secretion system in methicillin‐resistant S. aureus is an attractive target because SecD dysfunction leads to the death of bacteria and SecD as a target is more efficient than SecA and SecF. Evolution could have made SecD to become insensitive to antibacterial agents although the drugs directly against SecD have yet to develop. So far, no detailed information on SecD evolution has been available, thus 2686 SecD sequences with full taxonomic information from kingdom to species were analyzed. First, the variance of pairwise p‐distance was evaluated for each taxonomic group. Second, the variance was further partitioned into intergroup and intragroup variances for quantification of horizontal and vertical gene transfer. Third, phylogenetic tree was built to trace the evolutionary pathway. The results showed that overall evolution of SecDs appears to have undergone horizontal and vertical gene transfer. Only 0.5% horizontal transfers were found between any two SecDs in S. aureus, 6.8% and 8.8% horizontal transfers were found between any two Staphylococcus SecDs from different and the same species, and only one SecD from S. aureus was located far away from its sister cluster. Thus, statistic and evolutionary analyses demonstrate that the SecDs from staphylococcus species have a small chance of mutating, and provide taxonomic evidence to use the SecD as a potential target for new generation of antibacterial agents against S. aureus.     

The large first periplasmic loop of SecD and SecF plays an important role in SecDF functioning

A remarkable feature of proteins of the SecD and SecF family involved in protein translocation is that they possess a very large first periplasmic domain. Here we report that this large first periplasmic domain is not required for the SecD-SecF interaction but that it is important for catalyzing protein translocation.     

SecDFyajC forms a heterotetrameric complex with YidC

The Escherichia coli preprotein translocase is composed of a "preprotein conducting channel" domain that consists of the peripherally bound translocation ATPase SecA and the heterotrimeric SecYEG membrane protein complex. SecD, SecF, and YajC form another heterotrimeric complex that can associate with the SecYEG complex. YidC is an essential membrane protein that plays a role in the integration of newly synthesized membrane proteins, and has been shown to co-purify with SecYEG when all translocase components are overproduced. Here, we demonstrate that under conditions that YidC co-purifies with overproduced SecDFyajC it does not co-purify with overproduced SecYEG. Moreover, this interaction of YidC with the SecDFyajC complex is also found at chromosomal protein levels of SecD, SecF and YajC. Closer examination of the SecDFyajC-YidC complex showed that YidC binds to SecD and SecF, whereas YajC interacts only with SecF. As SecF and YajC have previously been shown to interact with SecY, we propose that these two proteins link the heterotetrameric SecDFyajC-YidC complex to the SecYEG complex.     

SecF stabilizes SecD and SecY, components of the protein translocation machinery of the Escherichia coli cytoplasmic membrane.

The effect of the overproduction of SecF encoded by the tac-secF gene on a plasmid on the synthesis of other Sec proteins was studied in Escherichia coli. SecF overproduction resulted in the simultaneous overproduction of SecD encoded by the tac-secD gene on a plasmid. Deletion of the orf6 gene, located downstream of the secF gene, had no effect on SecD overproduction. A pulse-chase experiment revealed that the overproduction was due to stabilization of SecD with SecF. SecF overproduction also resulted in the overproduction of SecY encoded by the tac-secY gene on a plasmid as well. SecF overproduction also enhanced the level of SecY expressed by the chromosomal secY gene. This SecF effect was not due to its effect on SecD or SecE, since SecF overproduction did not affect the levels of SecD and SecE expressed by the chromosomal secD and secE genes, respectively. SecE-dependent overproduction of SecY has already been demonstrated. It is suggested that SecF interacts with both SecD and SecY. SecE-SecY interaction has been demonstrated. It is likely, therefore, that all Sec proteins in the cytoplasmic membrane interact with each other.     

SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation.

Mutations in secD and secF show impaired protein translocation across the inner membrane of Escherichia coli. We investigated the effect of SecD and SecF (SecD/F) depletion on preprotein translocation into inverted inner membrane vesicles (IMVs). Both IMVs and cells which were depleted of SecD/F were defective in their ability to maintain a proton electrochemical gradient. The translocation of pre-maltose binding protein (preMBP), which is strongly delta microH+ dependent, showed a 5-fold decreased rate with IMVs lacking SecD/F. In contrast, proteolytic processing of preMBP to MBP by leader peptidase was similar in IMVs containing and lacking SecD/F, consistent with earlier findings that only ATP-dependent translocation is required for the initiation of translocation. In the absence of a delta microH+, with ATP as the sole energy source, preMBP translocation into IMVs which contained or were depleted of SecD/F was identical. Translocation of the precursor of outer membrane protein A (proOmpA) in the presence of subsaturating ATP also required a generated delta microH+ and, under these conditions, proOmpA translocation required SecD/F. With saturating concentrations of ATP, where delta microH+ has little effect on in vitro proOmpA translocation, SecD/F also had little effect on translocation. These results explain why SecD/F effects are precursor protein dependent in vitro.     

Some Effects of Diflubenzuron on Growth and Sporogenesis in Streptomyces spp.

Diflubenzuron, an insect growth regulator that blocks chitin deposition in insect cuticles, was tested for its effects on morphogenesis of Streptomyces spp. Use of diflubenzuron resulted in reduced dominance of spore hairs, reduced the width of the outer wall, and prevented formation of the inner spore wall in S. bambergiensis. In S. coelicolor, diflubenzuron altered the structure of the fibrillar pattern of spore envelopes. Exposure to diflubenzuron resulted in small increases in exported protein and in a ca. 20% increase in chitinase in both Streptomyces spp.     

RraAS1 inhibits the ribonucleolytic activity of RNase ES by interacting with its catalytic domain in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

RraA is a protein inhibitor of RNase E, which degrades and processes numerous RNAs in Escherichia coli. Streptomyces coelicolor also contains homologs of RNase E and RraA, RNase ES and RraAS1/RraAS2, respectively. Here, we report that, unlike other RraA homologs, RraAS1 directly interacts with the catalytic domain of RNase ES to exert its inhibitory effect. We further show that rraAS1 gene deletion in S. coelicolor results in a higher growth rate and increased production of actinorhodin and undecylprodigiosin, compared with the wild-type strain, suggesting that RraAS1-mediated regulation of RNase ES activity contributes to modulating the cellular physiology of S. coelicolor.     

Exploitation of the Streptomyces coelicolor A3(2) genome sequence for discovery of new natural products and biosynthetic pathways

Streptomyces, and related genera of Actinobacteria, are renowned for their ability to produce antibiotics and other bioactive natural products with a wide range of applications in medicine and agriculture. Streptomyces coelicolor A3(2) is a model organism that has been used for more than five decades to study the genetic and biochemical basis for the production of bioactive metabolites. In 2002, the complete genome sequence of S. coelicolor was published. This greatly accelerated progress in understanding the biosynthesis of metabolites known or suspected to be produced by S. coelicolor and revealed that streptomycetes have far greater potential to produce bioactive natural products than suggested by classical bioassay-guided isolation studies. In this article, efforts to exploit the S. coelicolor genome sequence for the discovery of novel natural products and biosynthetic pathways are summarized.     

Genome Sequence of a New Streptomyces coelicolor Generalized Transducing Bacteriophage, ?CAM

Streptomyces coelicolor is a model system for the study of Streptomyces, a genus of bacteria responsible for the production of many clinically important antibiotics. Here we report the genome sequence of ϕCAM, a new S. coelicolor generalized transducing bacteriophage, isolated from a soil sample originating from Lincolnshire, United Kingdom. Many open reading frames within ϕCAM shared high levels of similarity to a prophage from Salinispora tropica and a putative prophage in Streptomyces sp. strain C.     

The Evolution of an Osmotically Inducible dps in the Genus Streptomyces

Dps proteins are found almost ubiquitously in bacterial genomes and there is now an appreciation of their multifaceted roles in various stress responses. Previous studies have shown that this family of proteins assemble into dodecamers and their quaternary structure is entirely critical to their function. Moreover, the numbers of dps genes per bacterial genome is variable; even amongst closely related species - however, for many genera this enigma is yet to be satisfactorily explained. We reconstruct the most probable evolutionary history of Dps in Streptomyces genomes. Typically, these bacteria encode for more than one Dps protein. We offer the explanation that variation in the number of dps per genome among closely related Streptomyces can be explained by gene duplication or lateral acquisition, and the former preceded a subsequent shift in expression patterns for one of the resultant paralogs. We show that the genome of S. coelicolor encodes for three Dps proteins including a tailless Dps. Our in vivo observations show that the tailless protein, unlike the other two Dps in S. coelicolor, does not readily oligomerise. Phylogenetic and bioinformatic analyses combined with expression studies indicate that in several Streptomyces species at least one Dps is significantly over-expressed during osmotic shock, but the identity of the ortholog varies. In silico analysis of dps promoter regions coupled with gene expression studies of duplicated dps genes shows that paralogous gene pairs are expressed differentially and this correlates with the presence of a sigB promoter. Lastly, we identify a rare novel clade of Dps and show that a representative of these proteins in S. coelicolor possesses a dodecameric quaternary structure of high stability.     

Kinetics of polynucleotide phosphorylase: comparison of enzymes from Streptomyces and Escherichia coli and effects of nucleoside diphosphates

We examined the activity of polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3′ single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The k_cat for the E. coli enzyme was ca. twofold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3′ stem-loop structure, and the K_m for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3′ stem-loop structure compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 μM ADP, CDP, GDP, and UDP, the K_m for the phosphorolysis of the substrate with the 3′ stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.     

A novel tyrosine-phosphorylated protein inhibiting the growth of Streptomyces cells

Very few of the tyrosine-phosphorylated proteins in Streptomyces have been identified. Here, we identify a tyrosine-phosphorylated protein from Streptomyces coelicolor A3(2), designated as SCO5717. The protein possesses Walker motifs and a tyrosine cluster at the C-terminus. When sco5717 harboring its own promoter was introduced into the S. coelicolor cell, the growth was inhibited. An sco5717-disrupted mutant formed aerial mycelium earlier than the wild-type strain, suggesting that SCO5717 controls the cell growth of S. coelicolor. Although the recombinant SCO5717 showed an ATPase activity, it lacked self-phosphorylation ability, suggesting that SCO5717 is a novel tyrosine-phosphorylated protein, which is distinguishable from bacterial protein tyrosine kinases known so far.