Differential regulation of ftsZ transcription during septation of Streptomyces griseus.

Streptomyces has been known to form two types of septa. The data in this research demonstrated that Streptomyces griseus forms another type of septum near the base of sporogenic hyphae (basal septum). To understand the regulation of the septation machinery in S. griseus, we investigated the expression of the ftsZ gene. S1 nuclease protection assays revealed that four ftsZ transcripts were differentially expressed during morphological differentiation. The vegetative transcript (emanating from P(veg)) is present at a moderate level during vegetative growth, but is switched off within the first 2 h of sporulation. Two sporulation-specific transcripts predominantly accumulated, and the levels increased by approximately fivefold together shortly before sporulation septa begin to form. Consistently, the sporulation-specific transcripts were expressed much earlier and more abundantly in a group of nonsporulating mutants that form their sporulation septa prematurely. Promoter-probe studies with two different reporter systems confirmed the activities of the putative promoters identified from the 5' end point of the transcripts. The levels and expression timing of promoter activities were consistent with the results of nuclease protection assays. The aseptate phenotype of the P(spo) mutant indicated that the increased transcription from P(spo) is required for sporulation septation, but not for vegetative or basal septum formation.

Identification and characterization of a developmentally regulated protein, EshA, required for sporogenic hyphal branches in Streptomyces griseus.

To identify sporulation-specific proteins that might serve as targets of developmental regulatory factors in Streptomyces, we examined total proteins of Streptomyces griseus by two-dimensional gel electrophoresis. Among five proteins that were present at high levels during sporulation but absent from vegetative cells, two of the proteins, P3 and P4, were absent from developmental mutants that undergo aberrant morphogenesis. The deduced amino acid sequence of the gene that encodes P3 (EshA) showed extensive similarity to proteins from mycobacteria and a cyanobacterium, Synechococcus, that are abundant during nutritional stress but whose functions are unknown. Uniquely among these proteins, EshA contains a cyclic nucleotide-binding domain, suggesting that the activity of EshA may be modulated by a cyclic nucleotide. The eshA gene was strongly expressed from a single transcription start site only during sporulation, and accumulation of the eshA transcript depended on a developmental gene, bldA. During submerged sporulation, a null mutant strain that produced no EshA could not extend sporogenic hyphae from new branch points but instead accelerated septation and spore maturation at the preexisting vegetative filaments. These results indicated that EshA is required for the growth of sporogenic hyphae and localization of septation and spore maturation but not for spore viability.

Cloning and characterization of the gene encoding penicillin-binding protein A of Streptomyces griseus.

An internal segment of the penicillin-binding protein gene, pbpA, of Streptomyces griseus was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. pbpA encodes a 485 amino acid sequence that conserves three motifs of PBPs, SXXK, SXN, and KTG. The pbpA gene was located downstream of a gene homologous to the Bacillus subtilis spoVE gene. The pbpA gene was disrupted by replacing an ApaI fragment of the pbpA gene in S. griseus chromosome with an apramycin resistance gene cassette or directly inserting this apramycin resistance gene cassette at the NcoI site of pbpA penicillin-binding domain. No obvious defects in growth, sporulation, or spore sonication resistance were observed in the constructed pbpA mutants, suggesting that PBPA is not essential for growth and sporulation under normal laboratory conditions in S. griseus.

Characterization of ssfR and ssgA, two genes involved in sporulation of Streptomyces griseus.

In the presence of cefoxitin, which inhibits septum formation during sporulation, Streptomyces griseus is unable to sporulate, retaining the sonication sensitivity of nonsporulating hyphae. Cefoxitin- and sonication-resistant mutant SKK2600 was isolated and showed many morphological differences from its parental strain. A 3.6-kb DNA fragment that complemented the mutations of SKK2600 contained two open reading frames (ORFs), either of which could complement SKK2600. One ORF, designated ssfR, encoded a protein containing a potential DNA-binding helix-turn-helix motif close to its N terminus. SsfR is similar to members of a large family of transcriptional regulators, particularly IclR of Escherichia coli. The second ORF was identified as ssgA, a previously described sporulation gene from S. griseus (S. Kawamoto and J. C. Ensign, Actinomycetology 9:136-151, 1995). A point mutation of C to T seven nucleotides upstream of the UGA stop codon of ssfR was responsible for the phenotype of isolated mutant strain SKK2600. Surprisingly, this mutation should not change the primary structure of SsfR. The ssfR and ssgA disruption mutants were constructed and showed the "white" mutant phenotype, with some growth medium dependence. In addition, the ssfR null mutant sporulated ectopically in phosphate starvation medium.

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein.

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.

Visualization of penicillin-binding proteins during sporulation of Streptomyces griseus.

We used fluorescein-tagged beta-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of Streptomyces griseus. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of S. griseus.

Bald mutants of Streptomyces griseus that prematurely undergo key events of sporulation.

To identify the structural defects of nonsporulating mutants of Streptomyces griseus, the wild-type strain and class III bald mutants were examined by using transmission electron microscopy, ultrasonic treatment, and fluorescence microscopy after the induction of submerged sporulation by phosphate starvation. In the wild-type strain, submerged sporulation was marked by the relatively synchronous formation of sporogenic hyphae, nucleoid segregation, deposition of sporulation septa, and subsequent thickening of the spore walls during maturation. All of the class III mutants prematurely synthesized sporulation septa and thick spore walls. The class IIIA and C mutants formed sporogenic hyphae earlier than the wild-type strain and underwent nucleoid segregation in parallel with sporulation septum formation. In the class IIIB (bld4) mutant, DNA segregation appeared to be uncoupled from septum formation. The results indicate that the class III mutants are defective in loci that are involved in the regulation of key events of Streptomyces morphogenesis.

Identification of bldA mutants of Streptomyces griseus.

The bldA gene (encoding tRNA(UUA)Leu) from Streptomyces griseus (Sg) was cloned by hybridization with bldA from Streptomyces coelicolor (Sc). Introduction of Sg bldA into Sc bldA mutants restored sporulation and actinorhodin production. Sporulation of a subset of Sg bald mutants, which produce no aerial mycelium or spores, was restored in the presence of bldA from Sc or Sg. The nucleotide sequences of the bldA alleles from two such bald mutants revealed point mutations in the anticodon stem and the T psi C stem.

Analysis of a gene that suppresses the morphological defect of bald mutants of Streptomyces griseus.

When present in multiple copies, orf1590 restored sporulation to class IIIA bald mutants of Streptomyces griseus, which form sporulation septa and thick spore walls prematurely. The orf1590 alleles from class IIIA bald mutants restored sporulation upon introduction at a high copy number into those same mutants, and the nucleotide sequence of one of these alleles was identical to that of the wild-type strain. We conclude that overexpression of orf1590 suppresses the defect in class IIIA bald mutants. Previous nucleotide sequence and transcript analyses suggested that orf1590 could encode two related proteins, P56 and P49.5, from nested coding sequences. A mutation that prevented the synthesis of P56 without altering the coding sequence for P49.5 eliminated the function of orf1590, as did amino acid substitutions in the putative helix-turn-helix domain located at the N terminus of P56 and absent from P49.5. To determine the coding capacity of orf1590, we analyzed translational fusions between orf1590 and the neo gene from Tn5. Measurement of the expression of fusions to the wild-type and mutant alleles of orf1590 indicated that P56 was the sole product of orf1590 during vegetative growth. Attempts to generate a nonfunctional frameshift mutation in orf1590 were unsuccessful in the absence of a second-site bald mutation, suggesting that orf1590 may be required during vegetative growth by preventing early sporulation. Our results are consistent with the hypothesis that P56 at a high level delays the premature synthesis of sporulation septa and spore walls in class IIIA mutants.

Antigenic and functional conservation of an integrin I-domain in Saccharomyces cerevisiae.

We sought evidence for precursors of the leukocyte integrin subunits alpha M and alpha X among unicellular eukaryotes such as Saccharomyces cerevisiae. Chromatography of cytosolic extracts of Saccharomyces cerevisiae on an affinity matrix coupled to BU-15, a monoclonal antibody that recognizes alpha X, revealed a band of M(r) > 205 kDa under nonreducing conditions. Screening a lambda gt11 library of S. cerevisiae DNA with BU-15 (anti-alpha X) and anti-Mo1 (anti-alpha M) led to the isolation of a 3.7-kb EcoRI fragment containing the 3' end of an open reading frame sufficient to encode a polypeptide in excess of 118 kDa. On the basis of Southern blotting at high stringency, this gene was present in S. cerevisiae, but not in other yeast species such as Candida glabrata. Analysis of the derived amino acid sequence demonstrated > 98% identity with the S. cerevisiae protein Uso1p, a myosin-like polypeptide found exclusively in the cytosol. The C-terminal 1016 aa, expressed from the 3.7-kb EcoRI fragment in Escherichia coli as a beta-galactosidase fusion protein, bound iC3b, a ligand for the I-domain in alpha M and alpha X, and were recognized by Mn41, a monoclonal antibody specific for the alpha M I-domain. Antigenic and functional conservation of an I-domain in S. cerevisiae suggests that this domain may be a prototype for integrin-like proteins in other primitive eukaryotes.

Regulated expression of the histidase structural gene in Streptomyces griseus.

The histidase structural gene from Streptomyces griseus was expressed from a leaderless, monocistronic transcript. Multiple copies of the DNA located upstream of the hutH transcription initiation site led to a significant level of histidase activity when present in trans in the wild-type strain grown under noninducing conditions.

Expression of the division-controlling gene ftsZ during growth and sporulation of the filamentous bacterium Streptomyces griseus.

The branched, filamentous cells of Streptomyces form two different types of septum: those found infrequently in vegetative mycelia and those that form the boundaries of developing spores. To begin to understand the role of cell septation events in the Streptomyces life cycle, we have isolated the ftsZ locus from Streptomyces griseus, an organism that undergoes sporulation on solid surfaces and in liquid culture. The nucleotide sequence of the cloned DNA indicates that ftsZ in S. griseus lies within a region containing other genes likely to be involved in cell division and cell wall biogenesis. A gene (ORF1) showing significant similarity to ftsQ maps a short distance upstream from ftsZ, but there is no evidence for an ftsA homologue between ftsZ and ORF1. Transcription analysis suggests that ftsZ is expressed during both vegetative growth and sporulation. Immunoblots of soluble protein preparations from vegetative and sporulating mycelia indicate that FtsZ is present at similar levels during growth and differentiation. There appears to be only one ftsZ gene in S. griseus. We interpret these results to indicate that any temporal regulation of FtsZ that may be necessary for the enhanced synthesis of septa during sporulation of S. griseus is likely to occur predominantly at the level of activity rather than synthesis.

Inactivation of histidine ammonia-lyase from Streptomyces griseus by dicarbonyl reagents.

Histidine ammonia-lyase from Streptomyces griseus was inactivated by methylglyoxal and phenylglyoxal, dicarbonyl reagents known to react specifically with arginyl residues in proteins. The inactivation showed pseudo-first-order kinetics and could be prevented by protection with histidinol phosphate, a competitive inhibitor of histidine ammonia-lyase. Analysis of the amino acid composition of histidine ammonia-lyase after treatment with phenylglyoxal, together with the kinetics of inactivation, suggested that inactivation was a consequence of specific reaction with one or more essential arginyl residues at or near the active site of the enzyme.

Molecular analysis of sporulation in Streptomyces griseus.

Previous evidence suggested that orf1590 from Streptomyces griseus has the potential to encode two polypeptide products from temporally regulated nested open frames (orfs) and that the longer polypeptide may be a DNA-binding protein. We have developed a hypothetical model of the role of orf1590 in sporulation of S. griseus and have begun to test this model by determining the nucleotide sequence of the orf1590 counterpart from Streptomyces coelicolor. The conservation of the helix-turn-helix domain and the two potential translation start codons is consistent with our model. Continued analysis of bald mutants of S. griseus has indicated that several prematurely synthesize sporulation septa and spore walls. One of these nonsporulating strains appears to be a bldA mutant of S. griseus. Complementation analysis suggests that at least three genetic loci are involved in the correct timing of deposition of sporulation septa and wall thickening.

Histidine ammonia-lyase from Streptomyces griseus.

Histidine ammonia-lyase (histidase; HutH) has been purified to homogeneity from Streptomyces griseus and the N-terminal amino acid (aa) sequence used to clone the histidase-encoding structural gene, hutH. The purified enzyme shows typical saturation kinetics and is inhibited competitively by D-histidine and histidinol phosphate. High concentrations of K.cyanide inactivate HutH unless the enzyme is protected by the substrate or histidinol phosphate. On the basis of the nucleotide sequence, the hutH structural gene would encode a protein of 53 kDa with an N terminus identical to that determined for the purified enzyme. Immediately upstream from hutH is a region that strongly resembles a class of Streptomyces promoters active during vegetative growth; however, there is no obvious ribosome-binding site adjacent to the hutH translation start codon. The deduced aa sequence of an upstream partial open reading frame shows no similarity with other proteins, including HutP of Bacillus subtilis and HutU of Pseudomonas putida. Promoter-probe analysis indicates that promoter activity maps within the DNA surrounding the hutH start codon. Pairwise comparisons of the primary structures of bacterial and mammalian histidases, together with the unique kinetic properties and gene organization, suggest that streptomycete histidase may represent a distinct family of histidases.

Purification of histidase from Streptomyces griseus and nucleotide sequence of the hutH structural gene.

Histidine ammonia-lyase (histidase) was purified to homogeneity from vegetative mycelia of Streptomyces griseus. The enzyme was specific for L-histidine and showed no activity against the substrate analog, D-histidine. Histidinol phosphate was a potent competitive inhibitor. Histidase displayed saturation kinetics with no detectable sigmoidal response. Neither thiol reagents nor a variety of divalent cations had any effect on the activity of the purified enzyme. High concentrations of potassium cyanide inactivated histidase in the absence of its substrate or histidinol phosphate, suggesting that, as in other histidases, dehydroalanine plays an important role in catalysis. The N-terminal amino acid sequence of histidase was used to construct a mixed oligonucleotide probe to identify and clone the histidase structural gene, hutH, from genomic DNA of the wild-type strain of S. griseus. The cloned DNA restored the ability of a histidase structural gene mutant to grow on L-histidine as the sole nitrogen source. The deduced amino acid sequence of hutH shows significant relatedness with histidase from bacteria and a mammal as well as phenylalanine ammonia-lyase from plants and fungi.

Transcriptional and translational features of a sporulation gene of Streptomyces griseus.

The nucleotide (nt) sequence of a 2.8-kb fragment of DNA that restores sporulation to one class of bald mutants of Streptomyces griseus revealed an open reading frame (ORF) with the potential to encode a 55.5-kDa polypeptide. The presence of an in-frame TTA in the coding sequence indicated that translation is likely to require the tRNA(Leu)UUA encoded by the bldA gene. Two overlapping transcripts are initiated at transcriptional start points (tsp) separated by 258 nt and are transcribed in the same direction. The downstream tsp lies within the ORF and is followed by a second potential translation initiation site, which would encode a 49.5-kDa polypeptide in the same reading frame as the 55.5-kDa polypeptide. Transcription assays suggested that both tsp functioned during vegetative growth, but the relative abundance of the shorter transcript decreased during the early stages of submerged sporulation. Analysis of sequentially deleted subclones indicated that expression of the longer ORF was necessary to complement bald mutants. The presence of two tsp alternating with potential translation start codons suggests the temporally regulated synthesis of two polypeptides that have identical C termini but different N termini.

The iC3b receptor on Candida albicans: subcellular localization and modulation of receptor expression by glucose.

Patient isolates of Candida albicans from blood, urine, or mucosal sites express a surface receptor for C3 fragment iC3b that is recognized by monoclonal antibodies (MAb) directed against alpha-chain epitopes of the neutrophil iC3b receptor, also known as the type 3 complement receptor (CR3) or CD11b/CD18. Because 60% of these patients were hyperglycemic, the effect of glucose on receptor expression was investigated. As assessed by flow cytometry, yeasts grown in 20 mM D-glucose exhibited a four- to six-fold increase in receptor expression compared with yeasts grown in 20 mM L-glutamate. Receptor expression increased as glucose concentration rose from 0 to 20 mM (equivalent to plasma glucose concentrations of 0-360 mg/dl). Augmentation of receptor expression by growth in glucose led to significant inhibition of phagocytosis compared with that of organisms grown in equimolar L-glutamate. SDS-PAGE, Western blotting, and immunodetection of extracts of yeast cell wall, membrane, and cytosol disclosed a protein of 165 kDa in membrane and cytosolic extracts, consistent with the published Mr of the alpha-chain of neutrophil CR3. These studies provide a mechanism to explain the predilection of C. albicans to infect the hyperglycemic host.

Cascading regulation of histidase activity in Streptomyces griseus.

Mutants of Streptomyces griseus unable to utilize histidine as the sole nitrogen source have been isolated and characterized. Using a mutant defective in the production of histidase, we have demonstrated that urocanate functions as the inducer of the histidine utilization system. Another mutant produced histidase that was locked in an inactive form but could be activated by treatment with an extract from the wild-type strain or the histidase-negative strain. This mutant was deficient in the activity of a protein of Mr ca. 90,000 to 100,000 that is required for the activation of histidase. Histidase was synthesized constitutively but was maintained in an inactive form until after histidine or urocanate was added to the medium. At least four components were implicated in the activation of histidase: histidase, the activation protein, urocanate, and a phosphatase that is apparently inactive in cells grown without inducer. The functions of the last three factors could be supplanted in vitro by incubation of histidase with snake venom phosphodiesterase or 5' nucleotidase. The results suggest that histidine utilization by S. griseus is controlled posttranslationally by an activation cascade that involves at least two regulatory proteins.

Cloning of DNA involved in sporulation of Streptomyces griseus.

Twenty-two bald mutants of Streptomyces griseus were isolated and classified into four phenotypic groups, two of which showed conditional sporulation. A 3-kilobase fragment of DNA was cloned in a high-copy-number vector and detected by its ability to restore sporulation to one class of conditionally bald mutants. Analysis of subclones demonstrated that the sporulation property was contained within a 2.5-kilobase fragment. Hybridization studies and restriction analysis indicated that this DNA fragment was present in several Streptomyces species and was distinct from DNA that has been shown to complement afsA mutants of S. bikiniensis and bldA mutants of S. coelicolor.