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1.
Microb Ecol ; 50(4): 536-49, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16341636

ABSTRACT

In aquatic environments, Legionella pneumophila survives, in association with other bacteria, within biofilms by multiplying in free-living amoebae. The precise mechanisms underlying several aspects of the uptake and intracellular replication of L. pneumophila in amoebae, especially in the presence of other bacteria, remain unknown. In the present study, we examined the competitive effect of selected non-Legionella bacteria (Escherichia coli, Aeromonas hydrophila, Flavobacterium breve, and Pseudomonas aeruginosa) on the uptake of L. pneumophila serogroup 1 by the amoebae Acanthamoeba castellanii and Naegleria lovaniensis. We also investigated their possible influence on the intracellular replication of L. pneumophila in both amoeba species. Our results showed that the non-Legionella bacteria did not compete with L. pneumophila for uptake, suggesting that the amoeba hosts took in L. pneumophila through a specific and presumably highly efficient uptake mechanism. Living and heat-inactivated P. aeruginosa best supported the replication of L. pneumophila in N. lovaniensis and A. castellanii, respectively, whereas for both amoeba species, E. coli yielded the lowest number of replicated L. pneumophila. Furthermore, microscopic examination showed that 100% of the A. castellanii and only 2% of the N. lovaniensis population were infected with L. pneumophila at the end of the experiment. This study clearly shows the influence of some non-Legionella bacteria on the intracellular replication of L. pneumophila in A. castellanii and N. lovaniensis. It also demonstrates the different abilities of the two tested amoeba species to serve as a proper host for the replication and distribution of the human pathogen in man-made aquatic environments such as cooling towers, shower heads, and air conditioning systems with potential serious consequences for human health.


Subject(s)
Acanthamoeba castellanii/microbiology , Bacterial Physiological Phenomena , Legionella pneumophila/growth & development , Naegleria/microbiology , Acanthamoeba castellanii/cytology , Acanthamoeba castellanii/metabolism , Aeromonas hydrophila/physiology , Aeromonas hydrophila/radiation effects , Analysis of Variance , Animals , Bacteria/classification , Bacteria/metabolism , Bacteria/radiation effects , Bacterial Physiological Phenomena/radiation effects , Escherichia coli/physiology , Escherichia coli/radiation effects , Flavobacterium/physiology , Flavobacterium/radiation effects , Microscopy, Confocal/methods , Microscopy, Fluorescence , Naegleria/metabolism , Pseudomonas aeruginosa/physiology , Pseudomonas aeruginosa/radiation effects , Ultraviolet Rays
2.
Virology ; 331(2): 325-37, 2005 Jan 20.
Article in English | MEDLINE | ID: mdl-15629775

ABSTRACT

The entire double-stranded DNA genome of the Streptomyces venezuelae bacteriophage VWB was sequenced and analyzed. Its size is 49,220 bp with an overall molar G + C content of 71.2 mol%. Sixty-one potential open reading frames were identified and annotated using several complementary bioinformatics tools. Clusters of functionally related putative genes were defined, supporting a refined version of the modular theory of phage evolution.


Subject(s)
Bacteriophages/genetics , Genome, Viral , Virus Integration , Amino Acid Sequence , Computational Biology , Genes, Viral , Molecular Sequence Data , Open Reading Frames , Sequence Analysis, DNA , Streptomyces/genetics , Streptomyces/virology , Viral Proteins/genetics
3.
J Microbiol Methods ; 58(1): 135-7, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15177912

ABSTRACT

Several molecular techniques require high quality RNA, completely free of DNA. Standard methods to isolate total RNA from Streptomyces spp. are based on the application of a 'Modified Kirby Mix' [Practical Streptomyces Genetics. The John Innes Foundation, Norwich, pp. 613]. Here we present an alternative procedure using Triton X-100 and EDTA for the isolation of total RNA from Streptomyces.


Subject(s)
RNA, Bacterial/isolation & purification , Streptomyces/genetics , Edetic Acid/chemistry , Guanidines/chemistry , Octoxynol/chemistry , Phenols/chemistry , Streptomyces/chemistry
4.
J Bacteriol ; 183(23): 6727-32, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11698358

ABSTRACT

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.


Subject(s)
Escherichia coli Proteins , Membrane Transport Proteins/physiology , Streptomyces/metabolism , Cloning, Molecular , Monophenol Monooxygenase/metabolism , Protein Precursors/metabolism , Protein Sorting Signals , Subtilisin/antagonists & inhibitors
5.
Appl Environ Microbiol ; 67(10): 4464-70, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11571144

ABSTRACT

Radiation-inducible promoters are being used in many viral vector systems to obtain spatial and temporal control of gene expression. It was previously proven that radiation-induced gene expression can also be obtained in a bacterial vector system using anaerobic apathogenic clostridia. The effect of radiation inducibility was detected using mouse tumor necrosis factor alpha (mTNF-alpha) as a model protein under regulation of the radiation-inducible recA promoter. In this report, experiments are described in which this recA promoter was modified in order to increase radiation responsiveness. Incorporation of an extra Cheo box in the recA promoter region resulted in an increase in mTNF-alpha secretion from 44% for the wild-type promoter to 412% for the promoter with an extra Cheo box after a single irradiation dose of 2 Gy. Deletion of the Cheo box in the promoter region eliminated radiation inducibility. These results prove that the Cheo box in the recA promoter is indeed the radiation-responsive element. We also tested whether we could induce the constitutive endo-beta-1,4-glucanase promoter (eglA) via ionizing irradiation by introducing a Cheo box in the promoter region. While the use of the constitutive promoter did not lead to an increase in mTNF-alpha secretion after irradiation, the introduction of a Cheo box resulted in a 242% increase in mTNF-alpha secretion. Reverse transcriptase PCR of RNA samples isolated from irradiated and nonirradiated bacterial cultures demonstrated that the increase in secretion was the result of enhanced transcription of the mTNF-alpha gene.


Subject(s)
Clostridium/radiation effects , Gene Deletion , Gene Expression Regulation, Bacterial/radiation effects , Promoter Regions, Genetic/radiation effects , SOS Response, Genetics , Animals , Base Sequence , Cellulase/genetics , Cellulase/metabolism , Clostridium/genetics , Mice , Molecular Sequence Data , Mutation , Operator Regions, Genetic/genetics , Promoter Regions, Genetic/genetics , Rec A Recombinases/genetics , Rec A Recombinases/metabolism , Reverse Transcriptase Polymerase Chain Reaction , SOS Response, Genetics/genetics , SOS Response, Genetics/physiology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
6.
J Bacteriol ; 183(16): 4752-60, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11466278

ABSTRACT

Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These three S. lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for the S. lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.


Subject(s)
Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Streptomyces/enzymology , Base Sequence , Cell Membrane/enzymology , DNA Primers , Gram-Positive Bacteria/enzymology , Kinetics , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Plasmids , Protein Conformation , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Serine Endopeptidases/genetics , Streptomyces/genetics
7.
Biotechnol Bioeng ; 72(6): 611-9, 2001 Mar 20.
Article in English | MEDLINE | ID: mdl-11460252

ABSTRACT

We evaluated the feasibility of large-scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram-positive bacterium Streptomyces lividans. As a model protein we used murine tumor necrosis factor alpha (mTNFalpha). mTNFalpha fused C-terminally to the secretory signal peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature mTNFalpha (80-120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNFalpha from culture supernatants was developed. Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis. Further, mTNFalpha secreted by S. lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli. This pilot study provides the first validation of S. lividans protein secretion as an alternative bioprocess for large-scale production of oligomeric proteins of potential therapeutic value.


Subject(s)
Cell Culture Techniques/methods , Streptomyces/metabolism , Tumor Necrosis Factor-alpha/metabolism , Bioreactors , Culture Media/pharmacology , Feasibility Studies , Fermentation , Glucose/pharmacology , Pilot Projects , Polymers/metabolism , Quality Control , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Streptomyces/drug effects , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/isolation & purification
8.
DNA Seq ; 9(1): 19-30, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9773272

ABSTRACT

We report here on the isolation and identification of a gene coding for a novel subtilisin inhibitor (VSI) isolated from Streptomyces venezuelae CBS762.70. The vsi gene was isolated on a 5-kb chromosomal PvuII fragment as identified by DNA sequencing and inhibitor activity testing of the gene product. Primer extension studies revealed that the mRNA transcriptional start point was situated at -37 and -36 relatively to the ATG start codon assuming the presence of solely one promoter. Vsi promoter strength was about double of those of ermE-P1a and aph-P1, as tested with the mRNA production of the aphII gene preceded by the respective promoters. Translation of the vsi coding sequence revealed a 28 amino acids long signal peptide. The mature VSI protein consists of 118 amino acids of which 87% was verified by N-terminal amino acid sequence analysis. Compared with the already known Streptomyces proteinase inhibitors, VSI shows a relatively high amino acid identity in the conserved domains. Nevertheless, only a maximum amino acid identity of 56.1% was noticed and some highly conserved residues were substituted in VSI. As a consequence, VSI could be classified within a separate group of Streptomyces subtilisin inhibitors.


Subject(s)
Bacterial Proteins/genetics , Streptomyces/genetics , Subtilisins/antagonists & inhibitors , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , DNA Primers , DNA, Bacterial/isolation & purification , Genes, Bacterial , Molecular Sequence Data , Promoter Regions, Genetic , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Sequence Alignment , Sequence Analysis, DNA , Streptomyces/metabolism , Subtilisins/metabolism
9.
Appl Microbiol Biotechnol ; 49(4): 424-30, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9615485

ABSTRACT

With the aim of investigating determining factors for secretion of heterologous proteins by streptomycetes, we analysed the effect of charge variation in the Streptomyces venezuelae ATCC15068 alpha-amylase signal peptide on expression and secretion of mouse tumour necrosis factor alpha (mTNF) by Streptomyces lividans. To this end, the mTNF cDNA was fused to the wild-type alpha-amylase (aml) signal sequence and the fusion gene was expressed under the control of the S. venezuelae CBS762.70 subtilisin inhibitor gene (vsi) promoter, which has been shown to be very effective in initiating transcription. In addition, the number of positive charges in the N region of the alpha-amylase signal peptide was altered by in vitro mutagenesis. Secreted and intracellular mTNF levels were determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and biological activity measurements. This revealed moderate amounts of secreted mTNF compared to the levels obtained in previous experiments using the vsi promoter in combination with the Vsi signal peptide. Levels of secreted mTNF could be increased sevenfold by introducing one extra positive charge in the N region of the signal peptide.


Subject(s)
Recombinant Proteins/metabolism , Streptomyces/genetics , Tumor Necrosis Factor-alpha/metabolism , alpha-Amylases/chemistry , Animals , Genetic Vectors , Immunoblotting , Mice , Mutagenesis, Insertional/genetics , Protein Sorting Signals/genetics , Streptomyces/enzymology , Streptomyces/growth & development , Transcription, Genetic , alpha-Amylases/genetics
10.
FEMS Microbiol Lett ; 160(1): 1-10, 1998 Mar 01.
Article in English | MEDLINE | ID: mdl-9495006

ABSTRACT

As for other organisms, proteins to be secreted in Streptomyces are produced as preproteins consisting of the mature protein preceded by a N-terminal signal peptide which is cleaved off during membrane translocation. Although primary sequences are seldom conserved among signal peptides, they all have a typical tripartite structure: a basic amino-terminus, a central apolar core and a carboxy-terminal region containing the signal peptidase recognition site. In vitro mutagenesis studies have been carried out on various signal peptides to analyse the structure-function relationship of each of the three regions of Streptomyces signal peptides. In the current paper the present knowledge of Streptomyces leader sequences and the impact of introduced mutations on transcription, translation and secretion of homologous and heterologous proteins is reviewed.


Subject(s)
Bacterial Proteins/metabolism , Protein Sorting Signals/metabolism , Streptomyces/genetics , Streptomyces/metabolism , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Molecular Sequence Data , Mutagenesis
11.
DNA Seq ; 9(2): 79-88, 1998.
Article in English | MEDLINE | ID: mdl-10520736

ABSTRACT

Type I signal peptidases (SPases) are a widespread family of enzymes which remove signal peptides from proteins translocated across cellular membranes. Here, we report the first isolation of a gene coding for type I signal peptidase of Streptomyces, denoted Sip(Sli). The sip(sli) gene specifies a protein of 291 amino acids. Thus Sip(Sli) is much larger (approximately 100 amino acids) than other known SPases of Gram-positive bacteria and resembles SPases of Gram-negative bacteria, showing the highest degree of similarity to an SPase of the cyanobacterium Phormidium laminosum. Sip(Sli) contains conserved serine and lysine residues, which are believed to be required for the catalytic activity. Similar to other known SPases from Gram-positive bacteria, Sip(Sli) seems to have only one N-terminal transmembrane anchor. In addition, Sip(Sli) seems to contain a second transmembrane anchor at the C-terminus, which is an unusual feature for type I signal peptidases.


Subject(s)
Genes, Bacterial , Membrane Proteins , Serine Endopeptidases/genetics , Streptomyces/genetics , Amino Acid Sequence , DNA Primers , Deoxyribonucleases, Type II Site-Specific/metabolism , Gene Library , Molecular Sequence Data , Plasmids/genetics , Polymerase Chain Reaction/methods , Restriction Mapping , Sequence Alignment , Sequence Analysis, DNA , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Streptomyces/enzymology
12.
Appl Environ Microbiol ; 63(5): 1808-13, 1997 May.
Article in English | MEDLINE | ID: mdl-9143114

ABSTRACT

In order to evaluate the expression and secretion signals of the highly secreted subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (VSI) for the production of heterologous proteins by Streptomyces lividans, mouse tumor necrosis factor alpha (mTNF) was chosen as a model protein. The mTNF cDNA was fused to the vsi signal sequence. The analysis of secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and biological activity measurements revealed an efficient translocation of mTNF. Up to 300 mg of secreted biologically active mTNF per liter could be obtained in shaken-flask cultures. By analyzing the effects of mutations in the N region of the VSI signal peptide on secretion, we found that decreasing the +3 charge of the wild-type protein to +2 resulted in a 3- to 10-fold increase in secretion.


Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Streptomyces/genetics , Streptomyces/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Animals , Cloning, Molecular , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Mice , Protein Sorting Signals , RNA, Messenger/analysis , Recombination, Genetic , Signal Transduction , Translocation, Genetic , Tumor Necrosis Factor-alpha/immunology
13.
Mol Gen Genet ; 250(2): 223-9, 1996 Feb 05.
Article in English | MEDLINE | ID: mdl-8628222

ABSTRACT

The impact of the codon bias of the mouse tumour necrosis factor alpha (mTNF) gene cloned in Streptomyces lividans on the efficiency of expression and secretion was analysed. Minor codons occurring in the mTNF gene were therefore adapted to the codon bias of Streptomyces by site-directed mutagenesis. No improvement in mTNF yield could be detected. The stability of the transcript derived from the construct was shown to be more important for determining the final level of mTNF production. A strong correlation was observed between the yield of secreted biologically active mTNF and the amount of mTNF mRNA present in the cells.


Subject(s)
Codon/genetics , Gene Expression , RNA, Messenger/metabolism , Streptomyces/genetics , Tumor Necrosis Factor-alpha/genetics , Algorithms , Base Sequence , Blotting, Northern , Cloning, Molecular , Codon, Initiator/genetics , DNA Primers/chemistry , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Promoter Regions, Genetic/genetics , Protein Sorting Signals/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Software , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolism , alpha-Amylases/genetics , alpha-Amylases/metabolism
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