Search

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPIp ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPIp exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPIp accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPIp for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications.

Gas vesicles are proteinaceous, gas-filled nanostructures produced by some bacteria and archaea. The hydrophobic major structural protein GvpA forms the ribbed gas vesicle wall. An in-silico 3D-model of GvpA of the predicted coil-α1-β1-β2-α2-coil structure is available and implies that the two β-chains constitute the hydrophobic interior surface of the gas vesicle wall. To test the importance of individual amino acids in GvpA we performed 85 single substitutions and analysed these variants in Haloferax volcanii ΔA+Amut transformants for their ability to form gas vesicles (Vac(+) phenotype). In most cases, an alanine substitution of a nonpolar residue did not abolish gas vesicle formation, but the replacement of single non-polar by charged residues in β1 or β2 resulted in Vac(-) transformants. A replacement of residues near the β-turn altered the spindle-shape to a cylindrical morphology of the gas vesicles. Vac(-) transformants were also obtained with alanine substitutions of charged residues of helix α1 suggesting that these amino acids form salt-bridges with another GvpA monomer. In helix α2, only the alanine substitution of His53, or Tyr54, led to Vac(-) transformants, whereas most other substitutions had no effect. We discuss our results in respect to the GvpA structure and data available from solid-state NMR. This article is protected by copyright. All rights reserved.

Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin-A (SGPA), EC3.4.21.80; streptogrisin-B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC3.4.21.4. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).

Halobacterium is adapted to high salt concentrations of 5 M NaCl and even survives in salt crystals for hundreds of years. Bacteriorhodopsin, gas vesicles, and halophilic enzymes are specific traits used for biotechnological purposes. As archaeon, Halobacterium shows eukaryotic features in transcription, translation and replication. The microbe gained more than 1.000 metabolic genes from bacteria enabling heterotrophic growth in oxygen containing environments.

Gas vesicles consist predominantly of the hydrophobic GvpA and GvpC, and the accessory proteins GvpF through GvpM are required in minor amounts during formation. GvpM and its putative interaction partners were investigated. GvpM interacted with GvpH, GvpJ and GvpL, but not with GvpG. Interactions were also observed in vivo in Haloferax volcanii transformants using Gvp fusions to the green fluorescent protein smGFP. Cells producing the hydrophobic M(GF)P contained a single fluorescent aggregate per cell, whereas cells containing L(GFP) or H(GFP) were fully fluorescent. The soluble L(GFP) formed stable co-aggregates with GvpM in L(GFP)M transformants, but the presence of GvpH resulted in the absence of M(GF)P foci in HM(GFP) transformants. Substitution- and deletion mutants of GvpM determined functionally important amino acids (aa). Substitution of a polar by a non-polar aa in the N-terminal region of GvpM had no effect, whereas a substitution of a non-polar by a polar aa in this region inhibited gas vesicle formation in transformants. Substitutions in region 44-48 of GvpM strongly reduced the number of gas vesicles, and deletions at the N-terminus resulted in Vac(-) transformants. Gas vesicle morphology was not affected by any mutation, implying that GvpM is required during initial stages of gas vesicle assembly.

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

Halobacterium salinarum NRC-1 contains multiple TBP and TFB proteins required for the recruitment of RNA polymerase for transcription initiation. The presence and the expression of genes encoding TFB were investigated in the two Hbt. salinarum strains NRC-1 and PHH1 and the mutant strain PHH4. The plasmid-encoded tfbC and tfbE genes of NRC-1 were lacking in PHH1 and PHH4. The 5'-end of the tfbF transcript was determined and contained a 5'-untranslated region of 39 nucleotides able to form a stem-loop structure. The expression of these tfb genes was studied in cultures growing at 15, 37°C and under heat shock conditions. Cold temperatures reduced growth and except for tfbF also the amounts of all tfb transcripts. However, the formation of gas vesicles increased in PHH1 and NRC-1. Heat shock reduced growth of PHH1 and NRC-1, but PHH4 was not affected. A 100-fold increase in tfbA and tfbB mRNA was observed in PHH1 and PHH4, whereas NRC-1 reduced the amounts of these transcripts and increased the expression of tfbG. All TFB proteins tested were able to interact with the transcription activator GvpE involved in gas vesicle formation that thus is able to recruit TFB to the gvp promoter.

Gas vesicles are gas-filled protein structures increasing the buoyancy of cells. The gas vesicle envelope is mainly constituted by the 8 kDa protein GvpA forming a wall with a water excluding inner surface. A structure of GvpA is not available; recent solid-state NMR results suggest a coil-α-β-β-α-coil fold. We obtained a first structural model of GvpA by high-performance de novo modelling. Attenuated total reflection (ATR)-Fourier transform infrared spectroscopy (FTIR) supported this structure. A dimer of GvpA was derived that could explain the formation of the protein monolayer in the gas vesicle wall. The hydrophobic inner surface is mainly constituted by anti-parallel β-strands. The proposed structure allows the pinpointing of contact sites that were mutated and tested for the ability to form gas vesicles in haloarchaea. Mutations in α-helix I and α-helix II, but also in the β-turn affected the gas vesicle formation, whereas other alterations had no effect. All mutants supported the structural features deduced from the model. The proposed GvpA dimers allow the formation of a monolayer protein wall, also consistent with protease treatments of isolated gas vesicles.

GvpE is the transcriptional activator of the gvp gene cluster involved in gas vesicle formation in Haloabacterium salinarum. A 20-nucleotide sequence is required for the GvpE-mediated activation of the two oppositely oriented gvp promoters, P ( A ) and P ( D ). This sequence is located adjacent to the TATA-box and the transcription factor-B-binding site BRE, suggesting an interaction between GvpE and proteins of the transcription initiation apparatus. Here, we analysed the interaction of GvpE with the five different TATA-box-binding proteins, TBP, of Hbt. salinarum PHH1. The His-tagged TbpA through TbpE proteins were produced in Escherichia coli, bound to Ni-NTA matrices and tested for interaction with GvpE by protein-protein affinity chromatography. All Tbp(His) proteins retained the two different GvpE proteins from lysates of Haloferax volcanii transformants expressing the respective gvpE reading frame in pJAS35. Also, both GvpE(His) proteins bound to Ni-NTA matrices retained TbpB, whereas the 20-kDa soluble gas vesicle protein GvpH(His) neither bound TbpB nor GvpE from the respective lysates of Hfx. volcanii. From these results, it appears that GvpE interacts with any TBP of Hbt. salinarum. This interaction might attract TBP and subsequently TFB and RNAP to the promoter and thus enhance transcription of the gvp gene cluster.

The presence and expression of the multiple tbp genes encoding TATA-box binding proteins (TBPs) was investigated in various strains and mutants of the archaeon Halobacterium salinarum. Six genes, tbpA through tbpF, are present in the genome of Hbt. salinarum NRC-1 and also in the gas vesicle negative mutant strain R1. The only tbp gene located in the chromosome is tbpE, whereas all others are found in the plasmid DNA. Due to the dynamic nature of the plasmids in the Halobacterium strains, the copy numbers of the alternative tbp genes vary significantly. Five tbp genes (tbpA through tbpE) were present in the wild-type strain Hbt. salinarum PHH1. The tbpC gene of Hbt. salinarum PHH1 carried an ISH27-2 insertion element at the start of the reading frame that prevented the expression. All other tbp genes of PHH1 were expressed under aerobic and anaerobic growth conditions and quantitative RT-PCR yielded tbpE as dominant tbp transcript during the exponential growth phase. The plasmid deletion variant Hbt. salinarum PHH4 lacked all of the tbp genes except for tbpE and showed an altered growth behaviour compared to PHH1 wild-type in the stationary growth phase under anaerobic growth conditions.

Three gas vesicle gene clusters derived from Halobacterium salinarum (p-vac and c-vac) and Haloferax mediterranei (mc-vac) are used as model systems to study gene regulation in Archaea. An unusual pair of regulatory proteins is involved here, with GvpE acting as transcription activator and GvpD exhibiting a repressing function. Both regulators are able to interact leading to the loss of GvpE and the repression (or turnoff) of the gas vesicle formation. The latter function of GvpD requires a p-loop motif and an arginine-rich region, bR1. Both regulator proteins are differentially expressed from the same gvp transcript in Hfx. mediterranei and Hbt. salinarum PHH4. GvpE appears to recognize a 20-nucleotide activator sequence (UAS) located upstream and adjacent to the TFB-recognition element BRE of the two promoters driving the transcription of the divergently oriented gvpACNO and gvpDEFGHIJKLM gene clusters. The BRE elements of these two promoters are separated by 35 nucleotides only, and the distal portions of the two GvpE-UAS overlap considerably in the center of this region. Mutations here negatively affect the GvpE-induced activities of both gvp promoters, whereas alterations in the proximal UAS portions only affect the activity of the promoter located close by.

Summary The gas vesicle formation in Haloferax mediterranei occurs in the stationary growth phase and involves the 14 genes mc-gvpACNO and mc-gvpDEFGHIJKLM. The appearance of the two regulatory proteins GvpD and GvpE, and also of GvpF, was investigated during the growth of H. mediterranei. GvpD was only found during the stationary growth phase, GvpE was present from the late exponential to stationary growth phase, and GvpF was present only during the exponential growth, although the three genes were co-transcribed. The impact of GvpD and GvpE on the activity of the promoter of the mc-gvpACNO gene cluster encoding the gas vesicle structural proteins was analysed in H. volcanii transformants containing the mc-gvpA gene or a fusion of the mcA promoter with the bgaH reading frame encoding a halobacterial β-galactosidase as reporter. The experiments proved that GvpE is a transcriptional activator, whereas GvpD is involved in the repression. Protein–protein affinity chromatography was used to search for putative binding partners of GvpD and GvpE. Both proteins were synthesized in Escherichia coli as his-tagged proteins, isolated under denaturing conditions and refolded by dialysis against buffers containing decreasing urea and increasing KCl concentrations up to 2.5 M. The Ni-NTA matrix tagged with GvpD-his or GvpE-his was incubated with soluble proteins of gas vesicle producing H. mediterranei cells. A 21 kDa protein was purified using the matrix tagged with GvpD-his which proved to be GvpE by Western analysis. Vice versa, GvpD was purified using the GvpE-his-Ni-NTA matrix. These results strongly suggested that GvpD and GvpE were able to interact and might constitute a regulatory system.

The cGvpE protein of Halobacterium salinarum PHH4 has been identified as transcriptional activator for the promoter of the c-gvpA gene encoding the major gas vesicle structural protein cGvpA. Molecular modelling of the carboxy-terminal region of cGvpE suggests that this protein resembles a basic leucine-zipper protein, and mutations in the putative DNA binding domain DNAB completely abolish the activator function in Haloferax volcanii transformants. Mutations in the key residues of the putative leucine-zipper region AH6 of cGvpE confirmed that the three residues V159, L166 and L173 were essential for the activator function of cGvpE at the c-gvpA promoter, whereas the cysteine residue C180 could be altered to a leucine or an aspartate residue without the loss of this function. Mutations in basic residues of helix AH4 demonstrated the importance of the lysine K104 for the activator function of cGvpE. A cGvpE protein containing a his-tag at the C-terminus was still able to activate the expression of c-gvpA in vivo. The cGvpE his-purified from Hf. volcanii formed a dimer in Blue-native polyacrylamide gels that could be resolved into monomers by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Dimers of cGvpE were already seen using SDS-PAGE, but not with cGvpE mutant proteins containing the alterations L166E or L173E/C180L in the leucine zipper. These results imply that the hydrophobic surface of helix AH6 is indeed required for the establishment of cGvpE dimers.

DeltaD transformants containing all 14 gvp genes of Haloferax mediterranei required for gas vesicle formation except for gvpD are gas vesicle overproducers (Vac(++)), whereas DeltaD/D transformants containing the gvpD reading frame under ferredoxin promoter control on a second construct in addition to DeltaD did not form gas vesicles (Vac(-)). The amino acid sequence of GvpD indicates three interesting regions (a putative nucleotide-binding site called the p-loop motif, and two basic regions); these were altered by mutation, and the resulting GvpD(mut) proteins tested in DeltaD/D(mut) transformants for their ability to repress gas vesicle formation. The exchange of amino acids at conserved positions in the p-loop motif resulted in Vac(++) DeltaD/D(mut) transformants, indicating that these GvpD(mut) proteins were unable to repress gas vesicle formation. In contrast, a GvpD(mut) protein with an alteration of a non-conserved proline in the p-loop region (P41A) was still able to repress. The repressing function of the various GvpD proteins was also investigated at the promoter level of the gvpA gene. This promoter is only activated during the stationary phase, depending on the transcriptional activator protein GvpE. Whereas the Vac(++) DeltaD transformants contained very high amounts of gvpA mRNA predominantly in the stationary growth phase, the amount of this transcript was significantly reduced in the Vac(-) transformants DeltaD/D and DeltaD/D(P41A). In contrast, the Vac(++) DeltaD/D(mut) transformants harbouring GvpD(mut) with mutations at conserved positions in the p-loop motif contained large amounts of gvpA mRNA already during exponential growth, suggesting that this motif is important for the GvpD repressor function during this growth phase. The GvpD mutants containing mutations in the two basic regions were mostly defective in the repressing function. The GvpD(mut) protein containing an exchange of the three arginine residues 494RRR496 to alanine residues was able to repress gas vesicle formation. No gvpA mRNA was detectable in this transformant, demonstrating that this GvpD protein was acting as a strong repressor. All these results imply that the GvpD protein is able to prevent the GvpE-mediated gvpA promoter activation, and that the p-loop motif as well as the two basic regions are important for this function.

A novel papain inhibitory protein (SPI) from Streptomyces mobaraensis was studied to measure its inhibitory effect on bacterial cysteine protease activity ( Staphylococcus aureus SspB) and culture supernatants ( Porphyromonas gingivalis , Bacillus anthracis ). Further, growth of Bacillus anthracis , Staphylococcus aureus , Pseudomonas aeruginosa , and Vibrio cholerae was completely inhibited by 10 μM SPI. At this concentration of SPI, no cytotoxicity was observed. We conclude that SPI inhibits bacterial virulence factors and has the potential to become a novel therapeutic treatment against a range of unrelated pathogenic bacteria.

Gas vesicle formation of Halobacterium salinarum is regulated by the transcriptional activator GvpE, and in the presence of the repressing protein GvpD, the amount of GvpE is strongly reduced. The green fluorescence protein was used to report this GvpD-mediated reduction of GvpE in vivo in Haloferax volcanii transformants. Both N- or C-terminal fusions of GFP to GvpE were tested, but only the N-terminal fusion reported the reduction. The fluorescence of GFP-GvpE was 62 % reduced with GvpD wild type (DWT), 78 % with the super-repressor D3-AAA, and only 10 % with the repression defect DMut6. Further analysis of D3-AAA indicated that the super-repression was due to the alteration R496A. GFP-GvpE variants defect in promoter activation was tested in the presence of DWT, D3-AAA and DMut6, and two of them were more stable. Overall, the GFP-GvpE fusion was suitable to study and quantify the amount of GvpE in vivo.

Early and mature biofilm formation in the extremely halophilic euryarchaeon Halobacterium salinarum strain R1 was characterized by SWATH-LC/MS/MS. Using a simple surfactant-assisted protein solubilization protocol and one-dimensional ultra-high performance nanoflow chromatography on the front end, 63.2% and 58.6% of the predicted Hbt. salinarum R1 proteome could be detected and quantified, respectively. Analysis of biophysical protein properties, functional analysis and pathway mapping indicated comprehensive characterization of the proteome. Sixty point eight percent of the quantified proteins (or 34.5% of the predicted proteome) exhibited significant abundance changes between planktonic and sessile states, demonstrating that haloarchaeal biofilm formation represents a profound "lifestyle change" on the molecular level. Our results and analysis constitute the first comprehensive study to track molecular changes from planktonic cultures to initial and mature archaeal biofilms on the proteome level. Data are available via ProteomeXchange, identifier PXD003667. Proteins exemplifying different protein expression level profiles were selected, and their corresponding gene transcripts targeted by qRT-PCR to test the feasibility of establishing rapid PCR-based assays for archaeal biofilm formation.

Gas vesicle formation in haloarchaea requires the expression of the p-vac region consisting of 14 genes, gvpACNO and gvpDEFGHIJKLM. Expression of gvpFGHIJKLM leads to essential accessory proteins formed in minor amounts. An overexpression of gvpG, gvpH or gvpM in addition to p-vac inhibited gas vesicle formation, whereas large amounts of all other Gvp proteins did not disturb the synthesis. The unbalanced expression and in particular an aggregation of the overproduced Gvp with other accessory Gvp derived from p-vac could be a reason for the inhibition. Western analyses demonstrated that the hydrophobic GvpM (and GvpJ) indeed form multimers. Fluorescent dots of GvpM-GFP were seen in cells in vivo underlining an aggregation of GvpM. In search for proteins neutralizing the inhibitory effect in case of GvpM, p-vac +pGM(ex), +pHM(ex), +pJM(ex), and +pLM(ex) transformants were constructed. The inhibitory effect of GvpM on gas vesicle formation was suppressed by GvpH, GvpJ or GvpL, but not by GvpG. Western analyses demonstrated that pHM(ex) and pJM(ex) transformants contained additional larger protein bands when probed with an antiserum raised against GvpH or GvpJ, implying interactions. The balanced amount of GvpM-GvpH and GvpM-GvpJ appears to be important during gas vesicle genesis.

The proteinaceous gas vesicles produced by various microorganisms including halophilic Archaea are hollow, gas-filled structures with a hydrophobic inner and a hydrophilic outer surface. The structural components of gas vesicles and their biosynthesis are still under investigation; an 8-kDa polypeptide appears to be the major constituent of the gas-vesicle envelope. Genetic analysis of the halobacterial gas-vesicle synthesis revealed an unexpected complexity: about 14 genes organized in three transcription units are involved in gas-vesicle structure, assembly, and gene regulation. Here we describe the comparison of three different genomic regions encoding gas vesicles in Halobacterium salinarium (p-vac and c-vac regions) and Haloferax mediterranei (mc-vac region) and speculate on the function of the gene products involved in gas-vesicle synthesis.

The effect of anaerobiosis on the gas vesicle formation was investigated in three Halobacterium salinarum strains, Haloferax mediterranei and in Haloferax volcanii transformants. All these strains significantly reduced gas vesicle formation or lacked these structures under anoxic conditions. When grown by arginine fermentation, Hbt. salinarum PHH4 lacked gas vesicles, whereas Hbt. salinarum PHH1 and NRC-1 contained 5-20 small gas vesicles arranged in two to three aggregates per cell instead of the 30-80 gas vesicles present under oxic conditions. The enlargement presumably stopped due to a depletion of Gvp proteins. Also Hfx. mediterranei and Hfx. volcanii transformants lacked gas vesicles under anoxic growth and yielded a 10-fold reduced gvp transcription. Even the gas vesicle-overproducing DeltaD transformants did not form gas vesicles under anoxic conditions, demonstrating that the repressing protein GvpD was not involved. The presence of large amounts of GvpA implied that the assembly of the gas vesicles was inhibited. When Hbt. salinarum PHH1 and NRC-1 were grown with dimethyl sulphoxide or trimethylamine N-oxid under anoxic conditions the number but not the size of gas vesicles was reduced. This was in contrast to the previously reported overproduction of gas vesicles in NRC-1 that turned out to depend on the citrate-containing medium used for growth.

The two gvpA promoters PcA and PpA of Halobacterium salinarum, and the PmcA promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with β-galactosidase activity, as reporter. For comparison, the Pfdx promoter of the ferredoxin gene of Hbt. salinarum and the PbgaH promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed. Pfdx , driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas PbgaH and the three gvpA promoters yielded the largest activities during the stationary growth phase. Compared to Pfdx , the basal promoter activities of PpA and PmcA were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The PcA promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the PcA -TATA box and the BRE element were the reason for the lack of the basal PcA activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger PpA promoter and investigated in Hfx. volcanii transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the PpA -BRE element was substituted for the PcA -BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring PpA -BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of PmcA -BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of PmcA .

The GvpE protein involved in the regulation of gas vesicles synthesis in halophilic archaea has been identified as the transcriptional activator for the promoter located upstream of the gvpA gene encoding the major gas vesicle structural protein GvpA. A closer inspection of the GvpE protein sequence revealed that GvpE resembles basic leucine-zipper proteins typically involved in the gene regulation of eukarya. A molecular modelling study of the C-terminal part implied a cluster of basic amino acid residues constituting the DNA-binding site (DNAB) followed by an amphiphilic helix, suitable for the formation of a leucine-zipper structure within a GvpE dimer. The model of a GvpE dimer docked onto DNA indicated that the side-chains of the basic residues could perfectly interact with the negatively charged phosphate groups of the DNA backbone. Substitution of three basic amino acid residues of this putative DNAB by alanine and/or glutamate generated mutated GvpE proteins. None of these was able to activate the c-gvpA promoter in vivo, indicating that these basic residues are required for GvpE activity. This identification of an archaeal gene regulator displaying similarity to eukaryal regulatory proteins implies that the basic transcription machinery of eukarya and archaea are closely related, and that the regulatory proteins have evolved according to common principles.

The haloalkaliphilic archaeon Natronobacterium vacuolatum forms cylinder-shaped gas vesicles throughout the growth cycle when grown in media containing 15–25% NaCl. Cells cultivated in media containing 13% NaCl are, however, gas-vesicle-free. The major gas vesicle structural protein, nv-GvpA, was detected by an antiserum raised against the gas vesicles of Haloferax mediterranei; the antiserum reacted with an 8.3-kDa protein in samples containing cell extracts or purified gas vesicles of N. vacuolatum. The gene encoding nv-GvpA was isolated together with six additional gvp genes; these genes are arranged consecutively in a cluster as nv-gvpACNOFGH and are cotranscribed. Transcript analysis by primer extension revealed only one start site three nucleotides upstream of the nv-gvpA reading frame. This arrangement of gvp genes differs from that of the gas-vesicle-encoding genes in Halobacterium salinarium and Hf. mediterranei. The comparison of the deduced Gvp protein sequences indicated similarities with the respective halobacterial Gvp proteins, with GvpA exhibiting the highest degree of conservation (97–100%). The second gas vesicle structural protein, nv-GvpC, was 150–250 amino acids longer than all other halobacterial GvpC proteins and was much less conserved (48–73%). The expression of the nv-gvp genes was monitored in N. vacuolatum cells cultivated in 20 or 13% salt media. Northern and Western analyses showed that despite the lack of gas vesicles in cells grown in 13% salt medium, the gvpACNOFGH gene cluster was transcribed and GvpA protein was synthesized, suggesting that the absence of gas vesicles is not due to a lack of transcription.

Gas vesicles are intracellular, microbial flotation devices that consist of mainly one protein, GvpA. The formation of halobacterial gas vesicles occurs along a complex pathway involving 14 different gvp genes that are clustered in a genomic region termed the “vac region”. Various vac regions found in Halobacterium salinarum (p-vac and c-vac), Haloferax mediterranei (mc-vac), and Natronobacterium vacuolatum (nv-vac) have been investigated. Except for the latter vac region, the arrangement of the gvp genes is identical. Single gvp genes have been mutated to study the effect on gas vesicle synthesis in transformants and to determine their possible function. Each vac region exhibits a characteristic transcription pattern, and regulatory steps have been observed at the DNA, RNA, and protein level, indicating a complex regulatory network acting during gas vesicle gene expression.

As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180 +/- 20 kDa in size, consisting of two monomers (each 78 +/- 3 kDa). It cleaves several different beta-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has beta-D-fucosidase activity. No beta-glucosidase, beta-arabinosidase or beta-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

In Halobacterium halobium gas vacuoles are synthesized constitutively due to the expression of a plasmid borne gene (p-vac) whereas a related chromosomal c-vac gene copy is not active. In contrast, strains and species lacking the p-vac gene region synthesize gas vesicles in stationary phase of growth. The high mutation frequency of 1% affecting the p-vac gene expression in H. halobium is caused by the action of insertion elements either integrating up or downstream of the p-vac gene or inducing deletions encompassing the whole p-vac gene region. A burst of ISH27 transpositions was analyzed and led to the characterization of three types, one of which is more closely related to the Haloferax volcanii ISH51 family than to the other family members present in the same strain.

In the moderately to extremely halophilic archaebacterium Haloferax mediterranei gas vacuoles are not observed before the stationary phase of growth, and only when the cells are grown in media containing more than 17% total salt. Under the electron microscope, isolated gas vesicles appear as cylindrical structures with conical ends that reach a maximal length of 1.5 microns; this morphology is different from the spindle-shaped gas vesicles found in the Halobacterium halobium wild type which expresses the plasmid-borne p-vac gene, but resembles that of gas vesicles isolated from H. halobium strains expressing the chromosomal c-vac gene. Both the p-vac and the c-vac genes encode very similar structural proteins accounting for the major part of the "membrane" of the respective gas vesicles. The homologous mc-vac gene was isolated from Hf. mediterranei using the p-vac gene as probe. The mc-vac coding region indicates numerous nucleotide differences compared to the p-vac anc c-vac genes; the encoded protein is, however, almost identical to the c-vac gene product. The start point of the 310 nucleotide mc-vac transcript determined by primer extension analysis and S1 mapping was located 20 bp upstream of the ATG start codon, which is at the same relative position as found for the other two vac mRNAs. During the growth cycle, mc-vac mRNA was detectable in Hf. mediterranei cells grown in 15% as well as 25% total salt, with a maximal level in the early stationary phase of growth. The relative abundance of mc-vac mRNA in cells grown at 25% salt was sevenfold higher than in cells grown in 15% total salt.

Summary The effect of glucose on the formation of gas vesicles was investigated in Haloferax mediterranei and Hfx.volcanii transformants containing the mc-gvp gene cluster of Hfx. mediterranei (mc-vac transformants). Increasing amounts of glucose in the medium resulted in a successive decrease in the amount of gas vesicles in both species, with a complete inhibition of their formation at glucose concentrations of > 70 mM in mc-vac transformants, and 100 mM in Hfx. mediterranei. Maltose and sucrose imposed a similar inhibitory effect, whereas xylose, arabinose, lactose, pyruvate and 2-deoxy-glucose had no influence on the gas vesicle formation in mc-vac transformants. The activities of the two mc-vac promoters were strongly reduced in mc-vac transformants grown in the presence of > 50 mM glucose. The gas vesicle overproducing ΔD transformant (lacking the repressing protein GvpD) also showed a glucose-induced lack of gas vesicles, indicating that GvpD is not involved in the repression. The addition of glucose was useful to block gas vesicle formation at a certain stage during growth, and vice versa, gas vesicle synthesis could be induced when a glucose-grown culture was shifted to medium lacking glucose. Both procedures will enable the investigation of defined stages during gas vesicle formation.

The archaebacterium Halobacterium halobium contains two genes encoding gas vacuole proteins (vac). One resides on a large naturally occurring plasmid and encodes a protein of 76 amino acids (p-vac), while the other is a chromosomal gene that encodes a highly similar protein of 79 amino acids (c-vac). Northern analysis determined the c-vac and p-vac mRNA to be approximately 340 nucleotides in length, and S1 mapping of both transcripts indicated that the 5' terminus for each starts at the same relative nucleotide. Three other Halobacterium species producing gas vacuoles were investigated, H. spec. GN101, YC819-9, and SB3. All three contain only a chromosomal c-vac gene, and the 5' terminus of the 340 nucleotide mRNA starts at the same nucleotide as found for H. halobium. The c-vac gene region of H. spec. GN101 contains nine nucleotide exchanges, three of which occur in the coding region with no effect on the amino acid sequence. In contrast, the c-vac gene of H. spec. SB3 has an identical nucleotide sequence to the H. halobium c-vac gene. Gas vacuole production in each of these species was monitored during culture growth by phase contrast microscopy, and the vac mRNA level was determined for each time point. H. halobium p-vac deletion mutants, as well as the halobacterial species GN101 and YC819-9, start to synthesize gas vacuoles in early stationary growth phase with a maximal mRNA content in stationary phase. In contrast, H. halobium wild-type synthesizes gas vacuoles exclusively due to p-vac gene expression with a maximal mRNA level during logarithmic growth, and transcripts of the c-vac gene were not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary The genomic DNA of Halobacterium halobium and several other extremely halophilic species is separated on bisacrylamide/malachitegreen columns into two fractions which differ considerably in their GC content. It is shown that the AT richer fraction exhibits a high degree of variability and often forms circular DNA elements of various sizes. The major circular DNA behaves like a plasmid in that it is present in a constant number of copies. Insertions of IS-like elements of different lengths seem to be responsible for the genetic variability. These insertions are reversible in some instances. This has allowed us to localize the genetic determinant for gas vesicle formation on the major circular DNA.

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for numerous industrial applications. Recombinant production requires proteolytic activation of the zymogen. The study provides a convenient procedure for the preparation of the transglutaminase-activating metalloprotease (TAMP) in Escherichia coli. In contrast to wtTAMP, rTAMP exhibited the P domain of convertases as molecular mass of 55.7 kDa suggested. Protein integrity was beneficially influenced by 2–5 mM CaCl2. Study of pH and temperature optima assigned rTAMP to the neutral metalloproteases, more heat-resistant than Dispase but not thermolysin. Zinc had no inhibiting effect but 3.1 μM EDTA completely reduced activity of 5 nM TAMP. MTG, exceeding concentration of rTAMP by three orders of magnitude, was largely activated within few minutes. The kinetic parameters KM (1.31 ± 0.05 mM) and kcat (135 ± 4.3 s−1), monitored by isothermal titration calorimetry (ITC), further highlighted catalytic efficiency (103,053 M−1 s−1) of rTAMP and rapid processing of MTG. ITC even revealed that inhibition of rTAMP by its intrinsic inhibitory protein SSTI was an enthalpy-driven process resulting in Kd of 199 ± 37.9 nM. The production procedure of rTAMP in E. coli closes the gap between production and application of recombinant MTG and may enhance relevance of MTG-mediated reactions in pharmaceutical processes.

Gas vesicle synthesis in Haloferax mediterranei involves several gene products encoded by a 9.4-kilobase pair DNA region (mc-vac region) that contains 13 genes in addition to gvpA encoding the major structural gas vesicle protein. The expression of part of this region, encompassing the genes gvpA, gvpC, gvpN, and gvpO was investigated. These genes are transcribed from a common promoter located upstream of gvpA. Transcripts of 0.34 (gvpA only), 1.8 (gvpA/C), 2.4 (gvpA/C/N) and 3 kilobases (gvpA/C/N/O) were observed, with the gvpA transcript being the predominant mRNA species. The majority of the mRNA formed terminates 64 base pairs downstream of gvpA at the cytosine of the sequence 5' TTTTTC 3'. The synthesis of the GvpA and GvpC proteins was investigated by Western analyses. An antiserum raised against isolated gas vesicles of Hf. mediterranei detects, in addition to gas vesicle fragments, the GvpA protein of the M(r) of approximately 8,000 in lysates derived from different halobacteria or from Escherichia coli expressing gvpA. In samples containing isolated gas vesicles, mainly partially disaggregated gas vesicle fragments hybridize, but a minor amount of monomeric GvpA is also seen. For the detection of the GvpC protein, two versions of the gvpC gene (full length and gvp delta C lacking the 3' part encoding the acidic C terminus) were expressed in E. coli, and the resulting proteins were purified. The two antisera raised against these GvpC versions indicate the expression of gvpC in different halobacteria. By Western analysis, GvpC is also detectable in samples containing isolated gas vesicles demonstrating that GvpC is a second, but minor, gas vesicle structural protein.

Streptomyces mobaraensis DSM 40847 secretes transglutaminase that cross-links proteins via γ-glutamyl-ε-lysine isopeptide bonds. Characterized substrates are inhibitory proteins acting against various serine, cysteine and metalloproteases. In the present study, the bacterial secretome was examined to uncover additional transglutaminase substrates. Fractional ethanol precipitation of the exported proteins at various times of culture growth, electrophoresis of the precipitated proteins, and sequencing of a 39 kDa protein by mass spectrometry revealed the novel beta-lactamase Sml-1. As indicated by biotinylated probes, Sml-1, produced in E. coli, exhibits glutamine and lysine residues accessible for transglutaminase. The chromogenic cephalosporin analogue, nitrocefin, was hydrolyzed by Sml-1 with low velocity. The obtained Km and kcat values of the recombinant enzyme were 94.3±1.8 μM and 0.39±0.03 s(-1), respectively. Penicillin G and ampicillin proved to be weak inhibitors of nitrocefin hydrolysis (Ki of 0.1 mM and 0.18 mM). Negligible influence of metals on β-lactamase activity ruled out that Sml-1 is a Zn2+-dependent class B beta-lactamase. Rather, sequence motifs such as SITK, YSN, and HDG forming the active core in a hypothetical structure may be typical for class C beta-lactamases. Based on the results, we assume that the novel transglutaminase substrate ensures undisturbed growth of aerial hyphae in Streptomyces mobaraensis by trapping and inactivating hostile beta-lactam antibiotics.