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PubMed2000

The carboxyl terminus of the Bacillus subtilis SecA is dispensable for protein secretion and viability.

The Escherichia coli secretion-dedicated chaperone SecB targets a subset of proteins to the translocase by interacting with the carboxyl (C-) terminus of SecA. This region of SecA is highly conserved in Eubacteria, but despite its presence in the Bacillus subtilis SecA, the B. subtilis genome does not appear to contain a gene for a clear homologue of SecB. Deletion of the C-terminus of the B. subtilis SecA yields cells that have normal viability, but that exhibit a response reminiscent of oxidative stress and the loss of a number of secretory proteins from the culture supernatant. Semi-quantitative RT-PCR demonstrates that these proteins are expressed at lower levels. The C-terminus of SecA fused to glutathione S:-transferase (GST) specifically binds a cytosolic protein, termed MrgA. This protein has been reported to function in relation to oxidative stress, but deletion of the mrgA gene does not result in a secretion defect nor does it cause an oxidative stress response. It is concluded that the C-terminus of the B. subtilis SecA is not essential for secretion and viability.

Adenosine Triphosphatases↗
PubMed2000

Mutations in the ss subunit of the Bacillus subtilis RNA polymerase that confer both rifampicin resistance and hypersensitivity to NusG.

Mutations conferring resistance to the antibiotic rifampicin (Rif(r)) occur at specific sites within the ss subunit of the prokaryotic RNA polymerase. Rif(r) mutants of Escherichia coli are frequently altered in the elongation and termination of transcription. Rif(r) rpoB mutations were isolated in Bacillus subtilis and their effects on transcription elongation factor NusG and Rho-dependent termination were investigated. RNase protection assay, Northern analysis and the expression of nusG-lacZ fusions in cells with an inducible NusG suggested the B. subtilis nusG gene was autoregulated at the level of transcription. Rif(r) mutations that changed residue Q469 to a basic residue (Q469K and Q469R) enhanced autoregulation of nusG. A mutant expressing a truncated form of NusG, due to a nonsense mutation within the nusG gene, was isolated on the basis of the loss of autoregulation. The mechanism of autoregulation was found to be independent both of transcription termination factor Rho and of the promoter transcribing nusG. Autoregulation required sequences within the 5' coding sequence of the nusG gene or immediately upstream. This is the first evidence from any bacterium that Rif(r) RNA polymerases can display altered transcription regulation by NusG.

Amino Acid Sequence↗
PubMed2003

Molecular signatures in protein sequences that are characteristic of cyanobacteria and plastid homologues.

Fourteen conserved indels (i.e. inserts or deletions) have been identified in 10 widely distributed proteins that appear to be characteristic of cyanobacterial species and are not found in any other group of bacteria. These signatures include three inserts of 6, 7 and 28 aa in the DNA helicase II (UvrD) protein, an 18-21 aa insert in DNA polymerase I, a 14 aa insert in the enzyme ADP-glucose pyrophosphorylase, a 3 aa insert in the FtsH protein, an 11-13 aa insert in phytoene synthase, a 5 aa insert in elongation factor-Tu, two deletions of 2 and 7 aa in ribosomal S1 protein, a 2 aa insert in the SecA protein, a 1 aa deletion and a 6 aa insert in the enzyme inosine-5'-monophosphate dehydrogenase and a 1 aa deletion in the major sigma factor. These signatures, which are flanked by conserved regions, provide molecular markers for distinguishing cyanobacterial taxa from all other bacteria and they should prove helpful in the identification of cyanobacterial species, simply on the basis of the presence or absence of these markers in the corresponding proteins. The signatures in six of these proteins (SecA, elongation factor-Tu, ADP-glucose pyrophosphorylase, phytoene synthase, FtsH and ribosomal S1 protein) are also commonly present in plastid homologues from plants and algae (chlorophytes, chromophytes and rhodophytes), indicating their specific relationship to cyanobacteria and supporting their endosymbiotic origin from these bacteria. In phylogenetic trees based on a number of these proteins (SecA, UvrD, DNA polymerase I, elongation factor-Tu) that were investigated, the available cyanobacterial homologues grouped together with high affinity (>95 % bootstrap value), supporting the view that the cyanobacterial phylum is monophyletic and that the identified signatures were introduced in a common ancestor of this group.

Adenosine Triphosphatases↗
PubMed2006

Molecular signatures in protein sequences that are characteristics of the phylum Aquificae.

Species of the phylum Aquificae are of great interest due to their strict extreme thermophilic growth characteristics. Presently, there is no known molecular characteristic which is unique to this group of bacteria. This work describes six conserved inserts and deletions (indels or signature sequences) in four widely distributed proteins that are distinctive features of species from the phylum Aquificae. These include three signatures consisting of a 2 aa insert, a 5-6 aa insert and a 6 aa deletion in DNA polymerase I (PolA), a 6-7 aa insert in glucose-inhibited protein A (GidA), a 52 aa insert in the RNA polymerase beta'-subunit (RpoC) and a 4 aa insert in elongation factor Tu (EF-Tu). Fragments of these genes were amplified in most cases from Hydrogenobacter hydrogenophilus, Hydrogenothermus marinus and Thermocrinis ruber and combined with available sequence data from 'Aquifex aeolicus' and Sulfurihydrogenibium azorense. The presence of the PolA, GidA and RpoC indels in all of the species sequenced provides evidence that they are probably distinctive characteristics of the entire phylum. The indel in EF-Tu, which is shared by Aquifex species and Hydrogenobacter but not Hydrogenothermus and Sulfurihydrogenibium, may provide a molecular marker for the family Aquificaceae. We have also identified a 51 aa insert in SecA preprotein translocase that is commonly shared by various species of the Aquificae as well as two Thermotoga species (Thermotoga maritima and Thermotoga neapolitana) which may be due to lateral gene transfer between these groups. In phylogenetic trees based on a concatenated dataset of fragments from eight different proteins as well as 16S rRNA, the observed branching pattern of these species was very similar and it was consistent with the relationships inferred from various indels. The identified indels provide a novel means for distinguishing species of the Aquificae from all other bacteria in molecular terms and may prove useful for functional studies aimed at understanding the unique biochemical and physiological characteristics of the Aquificae.

Adenosine Triphosphatases↗
PubMed2003

Identification of genes in the tomato big bud phytoplasma and comparison to those in sweet potato little leaf-V4 phytoplasma.

Genetic relatedness of phytoplasmas is commonly defined on the basis of differences in the highly conserved 16S rRNA gene, which may not resolve closely related phytoplasmas. An example of this is the closely related tomato big bud (TBB) and sweet potato little leaf strain V4 (SPLL-V4) phytoplasmas, which cannot easily be differentiated by their 16S rRNA gene sequences. This study aimed to identify genes on the TBB phytoplasma chromosome which could be used to examine genetic variation between these two closely related phytoplasmas. Random clones generated from TBB phytoplasma genomic DNA were sequenced and characterized by database analysis. Twenty-three genes were identified within 19 random clones, which contained approximately 18.0 kbp of TBB phytoplasma genomic DNA. Half of the TBB phytoplasma genes identified were involved in DNA replication, transcription and translation. The remaining TBB phytoplasma genes were involved in protein secretion, cellular processes and energy metabolism. Phylogenetic analysis of representative genes showed that the TBB phytoplasma grouped with the mycoplasmas with the exception of the TBB phytoplasma secA gene, which grouped with the onion yellows phytoplasma. PCR primers were designed based on the new genes and tested on isolates of the TBB and SPLL-V4 phytoplasmas. Most primers amplified a product from TBB and SPLL-V4 phytoplasma samples. When amplified products were subjected to RFLP analysis, the restriction patterns were the same as the respective original clones. This result confirmed that the same sequence had been amplified by PCR and showed that these isolates were indistinguishable using the new genes. This study showed that in fact the TBB and SPLL-V4 phytoplasmas are closely related even with the analysis of new genes. These new genes have, however, provided insight into the biology of the TBB and SPLL-V4 phytoplasmas.

ATP-Dependent Proteases↗
PubMed2004

Secretion of immunodominant membrane protein from onion yellows phytoplasma through the Sec protein-translocation system in Escherichia coli.

A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the onion yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli, purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli. A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp.

Adenosine Triphosphatases↗
PubMed2004

Genomic organization and in vivo characterization of proteolytic activity of FtsH of Mycobacterium smegmatis SN2.

The ftsH gene of Mycobacterium smegmatis SN2 (MsftsH) was cloned from two independent partial genomic DNA libraries and characterized, along with the identification of ephA and folE as the neighbouring upstream and downstream genes respectively. The genomic organization of the MsftsH locus was found to be identical to that of the Mycobacterium tuberculosis ftsH gene (MtftsH) and similar to that of other bacterial genera, but with divergence in the upstream region. The MsftsH gene is 2.3 kb in size and encodes the AAA (ATPases Associated with diverse cellular Activities) family Zn(2+)-metalloprotease FtsH (MsFtsH) of 85 kDa molecular mass. This was demonstrated from the expression of the full-length recombinant gene in Escherichia coli JM109 cells and from the identification of native MsFtsH in M. smegmatis SN2 cell lysates by Western blotting with anti-MtFtsH and anti-EcFtsH antibodies respectively. The recombinant and the native MsFtsH proteins were found localized to the membrane of E. coli and M. smegmatis cells respectively. Expression of MsFtsH protein in E. coli was toxic and resulted in growth arrest and filamentation of cells. The MsftsH gene did not complement lethality of a DeltaftsH3 : : kan mutation in E. coli, but when expressed in E. coli cells, it efficiently degraded conventional FtsH substrates, namely sigma(32) protein and the protein translocase subunit SecY, of E. coli cells.

ATP-Dependent Proteases↗
PubMed2004

Role of Cal, the colicin A lysis protein, in two steps of colicin A release and in the interaction with colicin A-porin complexes.

Release of colicin A was studied in Escherichia coli cells that differed in expressing the colicin A lysis protein (Cal). Pools of released and unreleased colicin A were harvested throughout colicin A induction. The amount of colicin A in each pool varied with the time of induction, allowing the definition of two sequential steps in colicin A release, one of which was dependent on Cal. Each step of colicin A release was differently affected in cells containing Cal mutants in which the N-terminal cysteine residue was substituted by either proline or threonine, preventing them from being acylated and matured. These Cal mutants were only observed in degP cells, indicating that the DegP protease cleaved the unacylated precursor of Cal. Cal was found in the insoluble fraction of the pools of released and unreleased colicin A together with the hetero-oligomers of colicin A and porins (colicins Au). The biogenesis of colicins Au was studied in temperature-sensitive secA and secY strains and found to be Sec-independent, indicating that they are formed by newly synthesized colicin A binding to mature porins already incorporated in the outer membrane. Cal is a lipoprotein similar to VirB7, a constituent of the type IV secretion system. It would interact with colicins Au to constitute the colicin A export machinery.

Adenosine Triphosphatases↗
PubMed2005

Functional analysis of secA homologues from rickettsiae.

The molecular basis of protein secretion that underlines rickettsial pathogenesis remains unknown. This paper reports the molecular and functional analysis of the putative secA gene, an essential component of the Sec-dependent protein secretion pathway, from Rickettsia rickettsii and Rickettsia typhi, the aetiological agents of Rocky Mountain spotted fever and murine typhus, respectively. The sequence analysis of the cloned secA genes from R. rickettsii and R. typhi show ORFs of 2721 and 2718 nt, respectively. Alignment of the deduced amino acid sequences reveals the presence of highly conserved amino acid residues and motifs considered to be essential for the ATPase activity of SecA in preprotein translocation. Transcription analysis indicates that R. rickettsii secA is expressed monocistronically from the canonical prokaryotic promoter, with a transcriptional start point located 32 nt upstream of the secA initiation codon. Complementation analysis shows that the full-length SecA protein from R. rickettsii and R. typhi fails to restore growth of the temperature-sensitive Escherichia coli strain MM52 secA51(ts) at a non-permissive temperature (42 degrees C), despite the detection of SecA protein expression by Western blotting. However, the chimeric SecA protein carrying the N-terminal 408 aa of R. rickettsii SecA fused with the C-terminal 480 aa of E. coli SecA restores the growth of E. coli strain MM52 secA51(ts) at the non-permissive temperature (42 degrees C). These results suggest that the N-terminal ATPase domain is highly conserved, whereas the C-terminal domain appears to be species specific.

Adenosine Triphosphatases↗
PubMed2006

A unique serine-rich repeat protein (Srr-2) and novel surface antigen (epsilon) associated with a virulent lineage of serotype III Streptococcus agalactiae.

Group B streptococci (GBS) are pathogens of both neonates and adults, with serotype III strains in particular being associated with invasive disease and meningitis. In this study, a novel GBS surface antigen, epsilon, was found to be co-expressed with the previously reported delta antigen on an identical subset of serotype III GBS. Expression of delta/epsilon on the surface of serotype III GBS was shown to distinguish the restriction digest pattern (RDP) III-3 and multilocus sequence typing (ST)-17 lineage. epsilon-Specific antibodies were reactive with a unique, high-molecular-mass, serine-rich repeat protein (Srr-2) found exclusively in RDP III-3 strains. The gene encoding Srr-2 was located within a putative accessory secretory locus that included secY2 and secA2 homologues and had a genetic organization similar to that of the secY2/A2 locus of staphylococci. In contrast, serotype III delta/epsilon-negative strains and strains representative of serotypes Ia, Ib, Ic and II shared a common Srr-encoding gene, srr-1, and an organization of the secY2/A2 locus similar to that of previously reported serotype Ic, delta/epsilon-negative serotype III and serotype V GBS strains. Representative serotype III delta/epsilon-positive strains had LD(90) values 3-4 logs less than those of serotype III delta/epsilon-negative strains in a neonatal mouse model of infection. These results indicate that the RDP III-3/ST-17 lineage expresses Srr-2 and is highly virulent in an in vivo model of neonatal sepsis.

Adenosine Triphosphatases↗
PubMed2005

SecM facilitates translocase function of SecA by localizing its biosynthesis.

"Arrest sequence" of Escherichia coli SecM interacts with the ribosomal exit tunnel and arrests its own translation elongation, which is released by cotranslational export of the nascent SecM chain. This property of SecM is essential for the basal and regulated expression of SecA. Here we report that SecM has an additional role of facilitating SecA activities. Systematic determinations of the SecA-abundance-protein export relationships of cells with different SecA contents revealed that SecA was less functional when SecM was absent from the upstream region of the secM-secA message, when SecM had the arrest-defective mutation, and also when SecM lacked the signal sequence. These results suggest that cotranslational targeting of nascent SecM to the translocon plays previously unrecognized roles of facilitating the formation of functional SecA molecules. Biosynthesis in the vicinity of the membrane and the Sec translocon will be beneficial for this multiconformation ATPase to adopt ready-to-function conformations.

Adenosine Triphosphatases↗
PubMed1989

The secE gene encodes an integral membrane protein required for protein export in Escherichia coli.

Genetic screening and selection procedures employing a secA-lacZ fusion strain repeatedly have yielded mutations in four genes affecting the protein export pathway of Escherichia coli. These genes are secA, secD, prlA/secY, and secE. We discuss the significance of the failure to find new sec genes after extensive use of this approach. One of the genes, secE, has been characterized in some detail. From the DNA sequence of the gene and analysis of alkaline phosphatase fusions to the SecE protein, we propose that it is a 13,600-dalton integral cytoplasmic membrane protein. The data presented here and in the accompanying paper strongly suggest that secE has an important role in E. coli protein export.

Amino Acid Sequence↗
PubMed1989

New suppressors of signal-sequence mutations, prlG, are linked tightly to the secE gene of Escherichia coli.

Analysis of more than 100 extragenic suppressors of the lamB14D signal-sequence mutation (changes Val in the hydrophobic core region at position 14 to Asp) has revealed alterations that appear to lie at prlA (secY) and secA (prlD), two loci known to be mutable to suppressor alleles, and a new suppressor termed prlG. One allele of the new suppressor class, prlG1, has been characterized in some detail. This suppressor counteracts, to some degree, the export defect conferred by a variety of signal-sequence mutations in two different genes, lamB and malE. Genetic analysis shows that the dominant suppressor mutations are linked tightly to, and probably allelic with, the gene secE. This result, coupled with data obtained with conditional-lethal alleles of secE, argues strongly that SecE is an important component of the cellular protein export machinery in Escherichia coli.

Bacterial Proteins↗
PubMed2001

Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis. Identification of multiple forms of TOM20.

The translocase of the outer mitochondrial membrane (TOM) complex is a preprotein translocase that mediates transport of nuclear-encoded mitochondrial proteins across the outer mitochondrial membrane. Here we report the purification of this protein complex from Arabidopsis. On blue-native gels the Arabidopsis TOM complex runs at 230 kD and can be dissected into subunits of 34, 23, 21, 8, 7, and 6 kD. The identity of four subunits could be determined by immunoblotting and/or direct protein sequencing. The 21- and the 23-kD subunits exhibit significant sequence homology to the TOM20 preprotein receptor from other organisms. Analysis by two-dimensional isoelectric focusing/Tricine sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the presence of further forms for Arabidopsis TOM20. All TOM20 proteins comprise a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria. Clones encoding four different forms of Arabidopsis TOM20 were identified and sequenced. The deduced amino acid sequences are rather conserved in the N-terminal half and in the very C-terminal part, but include a highly variable glycine-rich region close to the C terminus. Implications on the function of plant TOM complexes are discussed. Based on peptide and nucleic acid sequence data, the primary structure for Arabidopsis TOM40 is presented.

Adenosine Triphosphatases↗