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1.  Bartonella sp. Bacteremia in Patients with Neurological and Neurocognitive Dysfunction ▿  
Journal of Clinical Microbiology  2008;46(9):2856-2861.
We detected infection with a Bartonella species (B. henselae or B. vinsonii subsp. berkhoffii) in blood samples from six immunocompetent patients who presented with a chronic neurological or neurocognitive syndrome including seizures, ataxia, memory loss, and/or tremors. Each of these patients had substantial animal contact or recent arthropod exposure as a potential risk factor for Bartonella infection. Additional studies should be performed to clarify the potential role of Bartonella spp. as a cause of chronic neurological and neurocognitive dysfunction.
doi:10.1128/JCM.00832-08
PMCID: PMC2546763  PMID: 18632903
2.  Clinical and Serological Follow-Up of Patients with Human Granulocytic Ehrlichiosis in Slovenia 
An evaluation of the clinical outcome and the duration of the antibody response of patients with human granulocytic ehrlichiosis (HGE) was undertaken in Slovenia. Adult patients with a febrile illness occurring within 6 weeks of a tick bite were classified as having probable or confirmed HGE based on the outcome of serological or PCR testing. Thirty patients (median age, 44 years) were enrolled, and clinical evaluations and serum collection were undertaken at initial presentation and at 14 days, 6 to 8 weeks, and 3 to 4, 6, 12, 18, and 24 months. An indirect immunofluorescence assay (IFA) was performed, and reciprocal titers of ≥128 were interpreted as positive. Patients presented a median of 4 days after the onset of fever and were febrile for a median of 7.5 days; four (13.3%) received doxycycline. Seroconversion was observed in 3 of 30 (10.0%) patients, and 25 (83.3%) showed >4-fold change in antibody titer. PCR results were positive in 2 of 3 (66.7%) seronegative patients but in none of 27 seropositive patients at the first presentation. IFA antibody titers of ≥128 were found in 14 of 29 (48.3%), 17 of 30 (56.7%), 13 of 30 (43.4%), and 12 of 30 (40.0%) patients 6, 12, 18, and 24 months after presentation, respectively. Patients reporting additional tick bites during the study had significantly higher antibody titers at most time points during follow-up. No long-term clinical consequences were found during follow-up.
doi:10.1128/CDLI.8.5.899-903.2001
PMCID: PMC96168  PMID: 11527800
3.  Identity of Ehrlichial DNA Sequences Derived from Ixodes ricinus Ticks with Those Obtained from Patients with Human Granulocytic Ehrlichiosis in Slovenia 
Journal of Clinical Microbiology  1999;37(1):209-210.
Adult Ixodes ricinus (Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterial groESL heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified I. ricinus as the likely vector for these ehrlichial pathogens of humans in this part of Europe.
PMCID: PMC84210  PMID: 9854093
4.  PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species. 
Journal of Clinical Microbiology  1997;35(8):2087-2092.
Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii. The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria. A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences. Nucleotide sequences obtained from clinical samples containing E. equi, E. phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical. Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E. equi and the HGE agent) and sequences from the European species, E. phagocytophila. A single pair of PCR primers derived from these sequences was used to detect E. chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.
PMCID: PMC229908  PMID: 9230387
5.  An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis. 
Journal of Clinical Microbiology  1997;35(6):1510-1516.
An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent.
PMCID: PMC229776  PMID: 9163471
6.  The two major spore DNA repair pathways, nucleotide excision repair and spore photoproduct lyase, are sufficient for the resistance of Bacillus subtilis spores to artificial UV-C and UV-B but not to solar radiation. 
Bacterial endospores are 1 to 2 orders of magnitude more resistant to 254-nm UV (UV-C) radiation than are exponentially growing cells of the same strain. This high UV resistance is due to two related phenomena: (i) DNA of dormant spores irradiated with 254-nm UV accumulates mainly a unique thymine dimer called the spore photoproduct (SP), and (ii) SP is corrected during spore germination by two major DNA repair pathways, nucleotide excision repair (NER) and an SP-specific enzyme called SP lyase. To date, it has been assumed that these two factors also account for resistance of bacterial spores to solar UV in the environment, despite the fact that sunlight at the Earth's surface consists of UV-B, UV-A, visible, and infrared wavelengths of approximately 290 nm and longer. To test this assumption, isogenic strains of Bacillus subtilis lacking either the NER or SP lyase DNA repair pathway were assayed for their relative resistance to radiation at a number of UV wavelengths, including UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight, and sunlight from which the UV-B portion had been removed. For purposes of direct comparison, spore UV resistance levels were determined with respect to a calibrated biological dosimeter consisting of a mixture of wild-type spores and spores lacking both DNA repair systems. It was observed that the relative contributions of the two pathways to spore UV resistance change depending on the UV wavelengths used in a manner suggesting that spores irradiated with light at environmentally relevant UV wavelengths may accumulate significant amounts of one or more DNA photoproducts in addition to SP. Furthermore, it was noted that upon exposure to increasing wavelengths, wild-type spores decreased in their UV resistance from 33-fold (UV-C) to 12-fold (UV-B plus UV-A sunlight) to 6-fold (UV-A sunlight alone) more resistant than mutants lacking both DNA repair systems, suggesting that at increasing solar UV wavelengths, spores are inactivated either by DNA damage not reparable by the NER or SP lyase system, damage caused to photosensitive molecules other than DNA, or both.
PMCID: PMC168002  PMID: 8779559
7.  Molecular dissection of mutations in the Bacillus subtilis spore photoproduct lyase gene which affect repair of spore DNA damage caused by UV radiation. 
Journal of Bacteriology  1995;177(15):4402-4409.
In response to UV irradiation, Bacillus subtilis spore DNA accumulates the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, or spore photoproduct (SP). SP is broken down into monomers during spore germination by the product of the spl gene which has been proposed to encode the enzyme SP lyase. The wild-type spl gene was cloned by complementation of a mutation designated spl-1; the putative spl gene product is a 40-kDa protein whose deduced amino acid sequence contains regions homologous to DNA photolyases. During phenotypic characterization of spl subclones using transformation crosses between the cloned wild-type spl gene and an spl-1 mutant recipient, in addition to the expected transformant classes exhibiting UV-resistant (type I) and UV-sensitive (type III) spores, an additional recombinant class was observed (called type II), spores of which exhibited slower germination kinetics following UV irradiation. The results suggested that the spl-1 allele consisted of at least two separable mutations. The DNA region which could rescue the spl-1 allele was localized to a 511-bp region within the spl coding sequence; this region was amplified from the spl-1 mutant chromosome by PCR and sequenced. The region contained two amino acid substitutions, an Arg replacing Gly-168 (G168R) and an Asp replacing Gly-242 (G242D) in the deduced SP lyase sequence, as well as 18 silent mutations. PCR amplification of chromosomal DNA from a selected type II recombinant and sequence analysis of the amplification product confirmed that recombination had indeed occurred between codons 168 and 242 and further localized the point of crossover by using the 18 silent mutations as molecular markers throughout the region. By in vitro mutagenesis, alleles of spl containing all combinations of single and double amino acid substitutions were introduced into the cloned wild-type spl gene. When integrated into the B. subtilis chromosome at the amyE locus, it was observed that although both amino acid substitutions contribute to the spl-1 phenotype, the G168R mutation exerted a much greater effect than did the G242D mutation.
PMCID: PMC177190  PMID: 7635825
8.  Immunoblot analysis of immunoglobulin G response to the Lyme disease agent (Borrelia burgdorferi) in experimentally and naturally exposed dogs. 
Journal of Clinical Microbiology  1988;26(4):648-653.
Immunoblots were used to study the immunoglobulin G response to Borrelia burgdorferi in experimentally and naturally exposed dogs. Adsorption studies confirmed that the antibodies were specific for B. burgdorferi. Experimentally exposed dogs were asymptomatic. Naturally exposed dogs included both asymptomatic animals and animals showing signs compatible with Lyme disease. Naturally exposed dogs were from four geographic regions of the country. No differences were detected between immunoblot patterns of naturally exposed symptomatic or asymptomatic dogs from different areas of the country. The immunoblot patterns obtained with sera from experimentally exposed dogs were different from those obtained with sera from naturally exposed dogs and were characterized by reactivity to fewer and different protein bands. Immunoblot analysis using an OspA-protein-producing Escherichia coli recombinant showed that experimentally exposed dogs produced antibodies to OspA, whereas naturally exposed dogs did not. Modifications of the immune response over time, different routes of antigen presentation, and strain variation are factors postulated to account for the observed differences.
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PMCID: PMC266399  PMID: 3366860
9.  Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis. 
Journal of Bacteriology  1987;169(12):5867-5869.
Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strain specifically blocked in catabolite repression of alpha-amylase synthesis. Decoyinine had no effect on alpha-amylase enzymatic activity. Thus, it appears that the catabolite control mechanisms governing alpha-amylase synthesis and sporulation in B. subtilis differ in their responses to decoyinine and hence must consist at least partially of separate components.
PMCID: PMC214190  PMID: 3119574
10.  Temporal regulation and forespore-specific expression of the spore photoproduct lyase gene by sigma-G RNA polymerase during Bacillus subtilis sporulation. 
Journal of Bacteriology  1994;176(13):3983-3991.
Bacterial spores are highly resistant to killing by UV radiation and exhibit unique DNA photochemistry. UV irradiation of spore DNA results in formation of spore photoproduct (SP), the thymine dimer 5-thyminyl-5,6-dihydrothymine. Repair of SP occurs during germination of Bacillus subtilis spores by two distinct routes, either by the general nucleotide excision repair (uvr) pathway or by a novel SP-specific monomerization reaction mediated by the enzyme SP lyase, which is encoded by the spl gene. Repair of SP occurs early in spore germination and is independent of de novo protein synthesis, suggesting that the SP repair enzymes are synthesized during sporulation and are packaged in the dormant spore. To test this hypothesis, the expression of a translational spl-lacZ fusion integrated at the spl locus was monitored during B. subtilis growth and sporulation. beta-Galactosidase expression from the spl-lacZ fusion was silent during vegetative growth and was not DNA damage inducible, but it was activated at morphological stage III of sporulation specifically in the forespore compartment, coincident with activation of expression of the stage III marker enzyme glucose dehydrogenase. Expression of the spl-lacZ fusion was shown to be dependent upon the sporulation-specific RNA polymerase containing the sigma-G factor (E sigma G), as spl-lacZ expression was abolished in a mutant harboring a deletion in the sigG gene and restored by expression of the sigG gene in trans. Primer extension analysis of spl mRNA revealed a major extension product initiating upstream from a small open reading frame of unknown function which precedes spl, and it revealed two other shorter minor extension products. All three extension products were present in higher quantities during sporulation and after sigG induction. The three putative transcripts are all preceded by sequences which share homology with the consensus sigma-G factor-type promoter sequence, but in vitro transcription by purified sigma-G RNA polymerase was detected only from the promoter corresponding to the major extension product. The open reading frame-spl operon therefore appears to be an additional member of the sigma-G regulon, which also includes as members the small, acid-soluble spore proteins which are in large part responsible for spore DNA photochemistry. Therefore, sporulating bacteria appear to coordinately regulate genes whose products not only alter spore DNA photochemistry but also repair the major spore-specific photoproduct during germination
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PMCID: PMC205596  PMID: 8021181
11.  Natural infections with Borrelia spirochetes in two dogs from Florida. 
Journal of Clinical Microbiology  1994;32(2):352-357.
Spirochetemia is a rarely reported observation in dogs. We describe the clinical, hematologic, and immunodiagnostic features of two spirochetemic dogs from northern Florida and the subsequent isolation and preliminary characterization of a Borrelia species from one dog in which culture of a sample for spirochetes was attempted. Results of polyacrylamide gel electrophoresis, monoclonal antibody testing, and PCR analysis indicate that the Florida isolate is not Borrelia burgdorferi, the only other member of the genus that has been isolated in Florida. Our findings also indicate that a member of the genus Borrelia potentially causes disease in dogs in Florida and that serologic cross-reactivity of the Florida canine Borrelia isolate with B. burgdorferi probably contributes to the inaccurate diagnosis of canine Lyme disease in the region.
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PMCID: PMC263035  PMID: 8150943
12.  Molecular cloning and characterization of the Bacillus subtilis spore photoproduct lyase (spl) gene, which is involved in repair of UV radiation-induced DNA damage during spore germination. 
Journal of Bacteriology  1993;175(6):1735-1744.
Upon UV irradiation, Bacillus subtilis spore DNA accumulates the novel thymine dimer 5-thyminyl-5,6-dihydrothymine. Spores can repair this "spore photoproduct" (SP) upon germination either by the uvr-mediated general excision repair pathway or by the SP-specific spl pathway, which involves in situ monomerization of SP to two thymines by an enzyme named SP lyase. Mutants lacking both repair pathways produce spores that are extremely sensitive to UV. For cloning DNA that can repair a mutation in the spl pathway called spl-1, a library of EcoRI fragments of chromosomal DNA from B. subtilis 168 was constructed in integrative plasmid pJH101 and introduced by transformation into a mutant B. subtilis strain that carries both the uvrA42 and spl-1 mutations, and transformants whose spores exhibited UV resistance were selected by UV irradiation. With a combination of genetic and physical mapping techniques, the DNA responsible for the restoration of UV resistance was shown to be present on a 2.3-kb EcoRI-HindIII fragment that was mapped to a new locus in the metC-pyrD region of the B. subtilis chromosome immediately downstream from the pstI gene. The spl coding sequence was localized on the cloned fragment by analysis of in vitro-generated deletions and by nucleotide sequencing. The spl nucleotide sequence contains an open reading frame capable of encoding a 40-kDa polypeptide that shows regional amino acid sequence homology to DNA photolyases from a number of bacteria and fungi.
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PMCID: PMC203968  PMID: 8449881
13.  Binding of DNA in vitro by a small, acid-soluble spore protein from Bacillus subtilis and the effect of this binding on DNA topology. 
Journal of Bacteriology  1990;172(12):6900-6906.
The DNA within spores of Bacillus subtilis is complexed with a large amount of alpha/beta-type small, acid-soluble spore protein (SASP). Measurement of the interaction of a purified alpha/beta-type SASP with DNA in vitro by a filter binding assay showed that the binding saturated at one molecule of SASP per approximately 5 bp. SASP-DNA binding did not require a divalent cation, was optimal at pH 6.7, and was unaffected by salt up to 400 mM. Binding of SASP to relaxed plasmid DNA in the presence of topoisomerase I resulted in the introduction of 18 (for plasmid pUC19) or 36 (for plasmid pUB110) negative supertwists, a superhelical density similar to that found in several plasmids isolated from spores. The SASP-dependent introduction of negative supertwists did not require a divalent cation, was unaffected by salt, and also gave a value of one molecule of SASP per approximately 5 bp at saturation. There was at least one slow step in the binding of SASP to DNA as seen in both the filter binding and supercoiling assays.
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PMCID: PMC210809  PMID: 2123857
14.  Dramatic increase in negative superhelicity of plasmid DNA in the forespore compartment of sporulating cells of Bacillus subtilis. 
Journal of Bacteriology  1990;172(1):7-14.
Plasmid pUB110, isolated from vegetative cells of Bacillus subtilis, has an average of 34 negative supertwists (tau av = -34). This value falls to -30 early in sporulation, and the plasmid in the mother cell compartment maintains a tau av of -30. However, the plasmid within the developing forespore becomes much more negatively supercoiled, reaching a tau av of -47 in the dormant spore. This increased negative supercoiling in the forespore plasmid takes place in parallel with the synthesis of small, acid-soluble spore proteins, alpha and beta; and the plasmid from spores lacking small, acid-soluble proteins alpha and beta has a tau av of -40. The large increase in negative supercoiling of spore plasmid was also observed with Bacillus megaterium and in B. subtilis containing a plasmid with an origin different from that of pUB110. During spore germination plasmid pUB110 rapidly relaxed back to the tau av value characteristic of vegetative cells. It is possible that the observed changes in forespore plasmid topology are involved in modulating gene expression, DNA photochemistry, or both of these parameters in this compartment.
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PMCID: PMC208394  PMID: 2104613
15.  Promoter specificity of sigma G-containing RNA polymerase from sporulating cells of Bacillus subtilis: identification of a group of forespore-specific promoters. 
Journal of Bacteriology  1989;171(5):2708-2718.
During sporulation in Bacillus subtilis, expression of the genes sspA, sspB, sspC, sspD, and sspE, which encode a family of small, acid-soluble spore proteins, as well as of the spoVA and gdh operons is transcriptionally activated at stage III of sporulation only in the forespore compartment. Transcription of these genes is mediated by RNA polymerase containing sigma G (E sigma G), the product of the sigG gene, which is itself expressed at stage III in the developing forespore. We have determined the 5' ends of transcripts generated both in vivo and in vitro by the action of E sigma G on various genes of B. subtilis and other bacilli. The 5' ends of the in vivo and in vitro mRNAs were found to coincide and were therefore considered to define the transcription initiation sites for the genes examined. We identified highly homologous DNA sequences centered at 35 and 10 base pairs preceding the transcriptional start sites of the genes examined. Consequently, we propose that these sequences define a class of promoters recognized only by E sigma G which allow transcription of genes expressed uniquely at stage III in the developing forespore.
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PMCID: PMC209955  PMID: 2468649
16.  Molecular cloning of cis-acting regulatory alleles of the Bacillus subtilis amyR region by using gene conversion transformation. 
Journal of Bacteriology  1986;165(3):663-670.
Three cis-acting alleles (gra-10, gra-5, and amyR2) of the Bacillus subtilis amyR promoter locus each cause catabolite repression-resistance of amyE-encoded alpha-amylase synthesis. The gra-10, gra-5, and amyR2 alleles were transferred from the chromosomes of their respective hosts to a plasmid carrying the amyR1-amyE+ gene by the process of gene conversion which is carried out during transformation of competent B. subtilis by plasmid clones carrying homologous DNA. The cloned amyR promoter regions containing the gra-10 and gra-5 mutations were shown to confer catabolite repression-resistance in cis to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 indicator gene when subcloned into the promoter-probe plasmid pPL603B. Implications concerning both the regulation of amyR utilization and the process of gene conversion in B. subtilis are discussed.
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PMCID: PMC214480  PMID: 3081488
17.  Isolation and characterization of a cis-acting mutation conferring catabolite repression resistance to alpha-amylase synthesis in Bacillus subtilis. 
Journal of Bacteriology  1985;161(3):875-881.
Bacillus subtilis 168GR10 was shown to contain a mutation, gra-10, which allowed normal temporal activation of alpha-amylase synthesis in the presence of a concentration of glucose that is inhibitory to activation of amylase synthesis in the parent strain, 168. The gra-10 mutation was mapped by phage PBS-1-mediated transduction and by transformation to a site between lin-2 and aroI906, very tightly linked to amyE, the alpha-amylase structural gene. The gra-10 mutation did not pleiotropically affect catabolite repression of sporulation or of the synthesis of extracellular proteases or RNase and was unable to confer glucose-resistance to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 gene driven by the amyE promoter region (amyR1) inserted into the promoter-probe plasmid pPL603B. It therefore appears that gra-10 defines a cis-regulatory site for catabolite repression, but not for temporal activation, of amyE expression. The evidence shows that temporal activation and glucose-mediated repression of alpha-amylase synthesis in B. subtilis 168 are distinct phenomena that can be separated by mutation.
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PMCID: PMC214978  PMID: 3918991
18.  Sigma-G RNA polymerase controls forespore-specific expression of the glucose dehydrogenase operon in Bacillus subtilis. 
Nucleic Acids Research  1989;17(3):999-1017.
The gene encoding glucose dehydrogenase (gdh) is part of an operon whose expression is transcriptionally activated specifically in the developing forespore of Bacillus subtilis at stage III of sporulation. The in vivo startpoint of gdh transcription was determined using primer extension analysis. Deletion mapping and site-specific mutagenesis experiments indicated that the region responsible for regulated expression of gdh in vivo was limited to the "-35" and "-10" regions preceding the transcriptional start site. RNA polymerase containing omega G (E omega G) transcribed gdh in vitro with a start site identical to that found in vivo, and transcription of gdh by E omega G in vitro also did not require any specific sequences upstream from "-35" region. These results suggest that the appearance of E omega G in the forespore at stage III of sporulation is sufficient to cause temporal and compartment-specific expression of the gdh operon.
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PMCID: PMC331718  PMID: 2493633

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