The ATPase ClpX is conditionally involved in the morphological differentiation of Streptomyces lividans.

ATP-dependent proteases of the ClpP type are widespread in eubacteria. These proteolytic complexes are composed of a proteolytic subunit and an ATPase subunit. They are involved in the degradation of denatured proteins, but also play a role in specific regulatory pathways. In Streptomyces lividans strains which lack the proteolytic subunit ClpP1, cell cycle progression has been shown to be blocked at early stages of growth. In this study, we examined the role of the ATPase subunit ClpX, a possible partner of the products of the clpP1 operon. A clpX mutant was obtained and it was shown that its growth was impaired only on acidic medium. Thus, the clpX phenotype differs from the clpP1 phenotype, indicating that these two components have only partially overlapping roles. We also analyzed the expression of clpX. Although clpX expression is increased under heat-shock conditions in many bacteria, we found that this is not the case in S. lividans.

Negative regulation of the heat shock response in Streptomyces.

All organisms respond to a sudden increase in temperature by inducing the synthesis of a set of proteins called heat shock proteins (HSPs). Although the induction of HSPs is a universal response, a diversity of mechanisms control HSP synthesis in different organisms. In Streptomyces, the synthesis of major HSPs, such as the widespread molecular chaperones DnaK, ClpB, GroEL and HSP18, is negatively controlled at the transcriptional level by at least three different repressors. The control of groE gene expression involves an inverted repeat (called the CIRCE element) that is highly conserved among eubacteria, and the HrcA repressor. The dnaK operon and clpB belong to the HspR /HAIR regulon. The HspR repressor-HAIR operator system is used in some bacteria but is not widespread. In particular, it has not been found in gram-positive bacteria with low G+C content. Transcription of hsp18, which encodes a small HSP, is regulated by the RheA repressor. This repressor, which has intrinsic thermosensor activity, has to date been identified only in Streptomyces.

The clpP multigenic family in Streptomyces lividans: conditional expression of the clpP3 clpP4 operon is controlled by PopR, a novel transcriptional activator.

The clpP genes are widespread among living organisms and encode the proteolytic subunit of the Clp ATP-dependent protease. These genes are present in a single copy in most eubacteria. However, five clpP genes were identified in Streptomyces coelicolor. The clpP1 clpP2 operon was studied: mutations affected the growth cycle in various Streptomyces. Here, we report studies of the expression of the clpP3 clpP4 operon in Streptomyces lividans. The clpP3 operon was induced in a clpP1 mutant strain, and the regulation of expression was investigated in detail. The product of the putative regulator gene, downstream from clpP4, was purified. Gel migration shift assays and DNase I footprinting showed that this protein binds to the clpP3 promoter and recognizes a tandem 6 bp palindromic repeat (TCTGCC-3N-GGCAGA). In vivo, this DNA-binding protein, named PopR, acts as an activator of the clpP3 operon. Studies of popR expression indicate that the regulator is probably controlled at the post-transcriptional level.

The RheA repressor is the thermosensor of the HSP18 heat shock response in Streptomyces albus.

Microorganisms have mechanisms to sense their environment and rapidly adapt to survive changes in conditions. In Streptomyces albus, various transcriptional repressors mediate the induction of heat shock genes. The RheA repressor regulates the synthesis of HSP18, a small heat shock protein, which plays a role in thermotolerance. The RheA protein was purified to determine how it responds rapidly to temperature. Gel retardation assays and footprinting experiments identified the specific target of RheA as an inverted repeat (TGTCATC 5N GATGACA) located in Phsp18, PrheA which is the common promoter region of the divergon. Gel retardation assays detected RheA-complexes formed with the hsp18-rheA promoters. The complexes did not form at higher temperature. In vitro transcription experiments showed that RheA is an autoregulatory protein and that its activity is inhibited by high temperature. The temperature-induced derepression by RheA is reversible. Dichroism circular spectroscopy revealed a reversible change of RheA conformation in relation with the temperature that could represent a transition between an active and an inactive form. Our experiments demonstrate that RheA acts as a cellular thermometer in hsp18 regulation.

Alteration of the synthesis of the Clp ATP-dependent protease affects morphological and physiological differentiation in Streptomyces.

The genes of Streptomyces coelicolor A3(2) encoding catalytic subunits (ClpP) and regulatory subunits (ClpX and ClpC) of the ATP-dependent protease family Clp were cloned, mapped and characterized. S. coelicolor contains at least two clpP genes, clpP1 and clpP2, located in tandem upstream from the clpX gene, and at least two unlinked clpC genes. Disruption of the clpP1 gene in S. lividans and S. coelicolor blocks differentiation at the substrate mycelium step. Overexpression of clpP1 and clpP2 accelerates aerial mycelium formation in S. lividans, S. albus and S. coelicolor. Overproduction of ClpX accelerates actinorhodin production in S. coelicolor and activates its production in S. lividans.

The ClpB ATPase of Streptomyces albus G belongs to the HspR heat shock regulon.

The clpB gene of Streptomyces albus was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotides. Transcriptional analysis showed that the clpB gene was heat induced. Primer extension identified a transcription start site preceded by typical vegetative -10 and -35 hexamer sequences. The Streptomyces HspR repressor is known to bind to three inverted repeat motifs (IR1, IR2, IR3) upstream from the S. coelicolor dnaK operon. We identified an inverted repeat motif identical to IR3 upstream from the S. albus clpB gene. DNA-binding experiments showed that HspR regulates clpB transcription by interacting directly with this motif. Streptomyces albus is the first Gram-positive organism for which the co-regulation of DnaK and ClpB has been described. Such co-regulation suggests that there is a physiological relationship between these two proteins in this bacterium. Genes similar to hspR were also identified in Mycobacterium leprae, M. tuberculosis and in bacteria unrelated to the actinomycetales order, such as Helicobacter pylori and Aquifex aeolicus. HspR binding sites were found in these bacteria upstream from various heat shock genes, suggesting that these genes are regulated by HspR. The HspR binding site, here called HAIR (HspR associated inverted repeat), has the consensus sequence CTTGAGT N7 ACTCAAG.

hrcA, encoding the repressor of the groEL genes in Streptomyces albus G, is associated with a second dnaJ gene.

Expression of the principal chaperones of the heat shock stimulon of Streptomyces albus G are under the negative control of different repressors. The dnaK operon is regulated by hspR, the last gene of the operon (dnaK-grpE-dnaJ-hspR). hsp18, encoding a member of the small heat shock protein family, is regulated by orfY, which is in the opposite orientation upstream of hsp18. The groES-groEL1 operon and the groEL2 gene are regulated differently. They present tandem copies of the CIRCE element found in the 5' region of many heat shock genes and shown to act in Bacillus subtilis as an operator for a repressor encoded by hrcA (hrc stands for heat regulation at CIRCE). We report the identification in S. albus of a new heat shock operon containing hrcA and dnaJ homologs. Disruption of hrcA increased the transcription of the groES-groEL1 operon and of the groEL2 gene. These features were lost when the mutant was complemented in trans by an intact copy of hrcA. Despite considerable accumulation of the GroE chaperones in the hrcA mutant, there was no effect on formation of the aerial mycelium and sporulation, indicating that neither hrcA nor the level of groE gene expression is directly involved in the regulation of Streptomyces morphological differentiation.

Disruption of hspR, the repressor gene of the dnaK operon in Streptomyces albus G.

hspR is the distal gene of the Streptomyces albus dnaK operon. It encodes a protein similar to GlnR, the repressor of the Bacillus subtilis glutamine synthetase gene. Transcriptional analysis showed that disruption of hspR led to constitutive high-level expression of the dnaK operon, SDS-PAGE analysis revealed over-production and accumulation of the chaperone DnaK at low temperature HSP94, a heat-inducible protein cross-reacting with anti-CipB antibodies, was also shown to be constitutively overexpressed at low temperature in the hspR mutant. Those features were lost when the mutant was complemented in trans by an intact copy of hspR. The hspR mutant was impaired in its growth on solid rich medium: colonies grow slowly at 30 degrees C. However, formation of aerial mycelium and sporulation was not prevented. In liquid culture growth curves of the mutant and the wild type were similar. The kinetics of groEL gene induction were not modified by the hspR null mutation, indicating that HspR was not directly involved in the control of groEL transcription. Thus, in contrast with B. subtilis and other Gram-positive bacteria, transcription of Streptomyces dnaK and groEL operons is not controlled by the same regulator.

Heat induction of hsp18 gene expression in Streptomyces albus G: transcriptional and posttranscriptional regulation.

In Streptomyces albus G, HSP18, a protein belonging to the small heat shock protein family, could be detected only at high temperature. The nucleotide sequence of the DNA region upstream from hsp18 contains an open reading frame (orfY) which is in the opposite orientation and 150 bp upstream. This open reading frame encodes a basic protein of 225 amino acids showing no significant similarity to any proteins found in data banks. Disruption of this gene in the S. albus chromosome generated mutants that synthesized hsp18 RNA at 30 degrees C, suggesting that orfY plays either a direct or indirect role in the transcriptional regulation of the hsp18 gene. In addition, thermally induced expression of the hsp18 gene is subject to posttranscriptional regulation. In the orfY mutant, although hsp18 RNA was synthesized at a high level at 30 degrees C, the HSP18 protein could not be detected except after heat shock. Synthesis of the HSP18 protein in the orfY mutant was also heat inducible when transcription was inhibited by rifampin. Furthermore, when wild-type cultures of S. albus were shifted from high temperature to 30 degrees C, synthesis of the gene product could no longer be detected, even though large amounts of hsp18 RNA were present.

Systemic necrotizing vasculitides in severe alpha1-antitrypsin deficiency.

We describe the clinical presentation and outcome in a series of eight patients with systemic necrotizing vasculitis and severe alpha1-antitrypsin (AAT) deficiency followed up at three Swedish hospitals during 1968-92. We also review six other cases reported in the literature during the same period. Diagnosis of severe AAT deficiency was based on the presence of the PiZZ phenotype, or low plasma total trypsin inhibitory capacity, or a low plasma AAT concentration (10-40% of the normal mean value) and presence of the PiSZ or PiFZ phenotype. The diagnosis of systemic vasculitis was biopsy-verified in all eight patients. Pretreatment laboratory findings, treatment protocol, and outcome were reviewed in each of the 14 patients. Of the eight patients in the Swedish series, six had systemic vasculitis of the microscopic polyangiitis form, one had Wegener's granulomatosis, and another had Henoch-Schönlein purpura. In the series as a whole (n = 14), median age at diagnosis was 48 years (range 44-84), the median number of affected organs was eight, and all 14 patients had skin involvement, and either renal or joint involvement (in most cases both); 71% (10/14) had emphysema; 57% (8/14) had hepatic abnormalities (two having cirrhosis, two fibrosis, and one multiple aneurysms in hepatic arteries); one patient who presented with acute ulcerative colitis developed manifest vasculitic syndrome three years later; and 64% (9/14) died, the major cause of death being renal failure. This syndrome, characterized by multiple organ involvement and fatal outcome, has been underdiagnosed. Physicians should be alert to the presence of the PiZ AAT deficiency gene in patients with systemic vasculitis, especially when the course is progressive or when the patient also has emphysema or cirrhosis. Awareness of those features may aid prompt recognition and enable early treatment.

Characterization of Streptomyces albus 18-kilodalton heat shock-responsive protein.

In Streptomyces albus during the heat shock response, a small heat shock protein of 18 kDa is dramatically induced. This protein was purified, and internal sequences revealed that S. albus HSP18 showed a marked homology with proteins belonging to the family of small heat shock proteins. The corresponding gene was isolated and sequenced. DNA sequence analysis confirmed that the hsp18 gene product is an analog of the 18-kDa antigen of Mycobacterium leprae. No hsp18 mRNA could be detected at 30 degrees C, but transcription of this gene was strongly induced following heat shock. The transcription initiation site was determined by nuclease S1 protection. A typical streptomycete vegetative promoter sequence was identified upstream from the initiation site. Disruption mutagenesis of hsp18 showed that HSP18 is not essential for growth in the 30 to 42 degrees C temperature range. However, HSP18 is involved in thermotolerance at extreme temperatures.

Transcriptional analysis of groEL genes in Streptomyces coelicolor A3(2).

In Streptomyces coelicolor A3(2), synthesis of the groES, groES-groEL1 and groEL2 transcripts is induced either by heat shock or by undefined physiological stress signals present at a certain stage of growth. Under all conditions tested, transcription of groES and groES-groEL1 originated from a unique start site upstream of groES, whereas transcription of groEL2 originated from a unique site upstream of groEL2. RNA polymerase isolated either from heat-shocked or control mycelia allowed in vitro transcription from the P1 promoter of groES/EL1 and the P2 promoter of groEL2. The fact that these two RNA polymerase preparations both initiated transcription with equal efficiency from the same sites suggested that a heat shock-specific sigma factor is not responsible for the temperature-induced transcription of groE genes. Instead, regulation of these genes from vegetative-type promoters may be effected by a DNA-binding protein observed in gel retardation assays, which recognizes a motif found in the groE and dnaK promoter regions of many prokaryotic genes.

Characterization of two groEL genes in Streptomyces coelicolor A3(2).

Two Streptomyces coelicolor A3(2) groEL-like genes, groEL1 and groEL2, were cloned and characterized. Pulsed-field-gel electrophoresis located these genes, which were not adjacent, in the same segment of the chromosome. Nucleotide sequence analysis revealed that groEL1, but not groEL2, was preceded by a groES-like gene. Northern blots showed that heat shock induced the accumulation of transcripts corresponding to groES (0.7 kb), groES/EL1 (2.3 kb) and groEL2 (2.1 kb). Unique transcription start points and promoters were located for groES/EL1 and groEL2, having -10 and -35 hexamers similar to eubacterial vegetative promoters. Regions located 5' to the groES/EL1 or groEL2 structural genes contain 'GCACTCN9GAGTGC' motifs conserved upstream from the heat-shock genes of other bacteria.

Post-transcriptional regulation of the groEL1 gene of Streptomyces albus.

Thermally induced expression of the heat-shock gene groEL is subject to post-transcriptional regulation in Streptomyces albus. When S. albus cells were shifted from 30 degrees C to 41 degrees C, synthesis of three GroEL-like proteins was induced from two genes transcribed from associated promoters P1 and P2. Surprisingly, analyses of transcriptional fusions of these promoters with various reporter genes indicated constitutive expression independent of heat shock. In contrast, neo expression was thermally inducible as a GroEL1-APH translational fusion protein. Furthermore, expression of the groEL1-neo gene was heat inducible even after the groEL1 promoter region was replaced by a heterologous non-heat-inducible promoter such as the Escherichia coli lac promoter. Finally, synthesis of GroE proteins, as well as the GroEL-APH fusion protein, was heat inducible when their transcription was inhibited by rifampicin. Post-transcriptional regulatory signals needed for heat-induced GroEL1 synthesis were mapped within of the groEL1 structural gene.

Use of new Escherichia coli/Streptomyces conjugative vectors to probe the functions of the two groEL-like genes of Streptomyces albus G by gene disruption.

Streptomyces albus G contains two groEL-like genes encoding three related proteins [Guglielmi et al., J. Bacteriol. 173 (1991) 7374-7381; Mazodier et al., J. Bacteriol. 173 (1991) 7382-7386]. Two proteins, HSP58 and HSP18, are synthesized from a single start codon site in groEL1. HSP18 may be a processed form of HSP58 or the result of early termination after frameshifting. The third protein, HSP56 is encoded by groEL2. In order to determine the physiological roles of these different proteins, both groEL genes were mutagenized by using a new approach for obtaining insertions in the streptomycete chromosome. Escherichia coli plasmids containing fragments homologous to groEL1 or groEL2 are unable to replicate in Streptomyces. They were introduced into S. albus by conjugation with E. coli. We then screened for mutants in which groEL1 or groEL2 had been disrupted due to recombination events (single or double crossover) at specific sites. Using this approach, the functionally indispensable domain of HSP58 was localized to within 249 amino acids of the N-terminus. HSP58 was not detected in the mutant generated by the most upstream insertion into the groEL1 coding sequence. However, HSP18 was synthesized in this mutant after heat shock. This groEL1 mutant was not impaired in growth in the 30-41 degrees C temperature range and SDS-PAGE analysis showed its overall pattern of gene expression to be indistinguishable from the parental strain. The inability to generate strains containing groEL2 disruptions strongly suggests that HSP56 is indispensable for growth.(ABSTRACT TRUNCATED AT 250 WORDS)

A survey of the heat shock response in four Streptomyces species reveals two groEL-like genes and three groEL-like proteins in Streptomyces albus.

A survey of the heat shock response was carried out in a series of streptomycetes. Four major heat shock proteins (HSPs) were observed in each of four species (Streptomyces albus, S. lividans, S. parvulus, S. viridochromogenes) after pulse labeling with [35S]methionine and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three corresponded to the major procaryotic HSPs Lon, DnaK, and GroEL on the basis of their apparent molecular masses (94 to 100, 70, and 56 to 58 kDa, respectively). In addition, a smaller protein (16 to 18 kDa) was detected in all species but was most dramatically induced in S. albus. Consequently, studies focused on this species. As in other procaryotic systems, thermal induction (elicited by a shift from 30 degrees C to 41 degrees C) of the 70- and 94-kDa proteins was transient and expression returned to uninduced levels after 60 min. In contrast, the 56- to 58-kDa (GroEL) and 18-kDa proteins (HSP18) remained induced for more than 2 h. Two-dimensional gel electrophoresis allowed resolution of at least eight S. albus HSPs. HSP56-58 was composed of multiple acidic protein species, whereas HSP18 appeared to be basic. In spite of these differences in their physical characteristics, the N-terminal peptide sequence of HSP18 was similar to those of GroEL-like proteins found in other organisms and identical to one of the HSP56-58 species. In fact, N-terminal amino acid analysis of the S. albus 56- to 58-kDa species showed that it was composed of two proteins that differed in 3 of 10 positions, an observation that was supported by the detection of two groEL-like genes by Southern hybridization. The amino acid sequence of one of these proteins was identical to that of HSP18. Pulse-chase experiments did not reveal evidence of posttranslational processing of either HSP56-58 or HSP18.

Characterization of the groEL-like genes in Streptomyces albus.

Three GroEL-like heat shock proteins (HSP56, HSP58, and HSP18) have been observed in Streptomyces albus (G. Guglielmi, P. Mazodier, C. J. Thompson, and J. Davies, J. Bacteriol. 173:7374-7381, 1991). Here we report the cloning and complete nucleotide sequence of groEL1, which encodes HSP18 and HSP58, and groEL2, which encodes HSP56. Both nucleotide sequences predicted proteins of 56,680 Da that were 70% identical. The 5' nucleotide sequence of groEL1 coded for a protein corresponding to HSP18 that may be a processed gene product. At least two groEL-like genes were present in all 12 Streptomyces species tested; they were not closely linked in the genome. groEL1, but not groEL2, was adjacent to a groES-like gene.

Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis.

A method which allowed the stable integration of DNA fragments at a single site (attB) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens, pSAM2. Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site (attP) were constructed and introduced to M. smegmatis by electroporation. Transformants showed stable integration of the plasmid into a single site (attB) of the mycobacterial genome. This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2.