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Since the 1980s, pertussis notifications in the United States have been increasing. To determine the types of Bordetella pertussis responsible for these increases, we divided 661 B. pertussis isolates collected in the United States during 1935-2009 into 8 periods related to the introduction of novel vaccines or changes in vaccination schedule. B. pertussis diversity was highest from 1970-1990 (94%) but declined to ≈ 70% after 1991 and has remained constant. During 2006-2009, 81.6% of the strains encoded multilocus sequence type prn2-ptxP3-ptxS1A-fim3B, and 64% were multilocus variable number tandem repeat analysis type 27. US trends were consistent with those seen internationally; emergence and predominance of the fim3B allele was the only molecular characteristic associated with the increase in pertussis notifications. Changes in the vaccine composition and schedule were not the direct selection pressures that resulted in the allele changes present in the current B. pertussis population. [ABSTRACT FROM AUTHOR]

This article presents news from the U.S. Centers for Disease Control and Prevention (CDC). In this study, the CDC looked at outbreaks of respiratory illness in New Hampshire, Massachusetts and Tennessee during 2004-2006 that were mistakenly thought to be cases of pertussis or whooping cough. Cases of the disease have been increasing in the U.S. due to the circulation of Bordetella pertussis and waning vaccination in communities. There are not fast, sensitive tests for pertussis which can make definitive diagnosis difficult. The cases in the three states are examined. Vaccination efforts need to be increased to prevent the disease.

Objective: Despite widespread availability of COVID-19 vaccines, millions of Americans have not received the recommended vaccine doses. In the District of Columbia (DC), COVID-19 vaccination rates are lowest among residents who are Non-Hispanic (NH) Black and among school-aged children. We assessed COVID-19 vaccine hesitancy among staff and parents of students in DC K-12 public and public charter schools., Methods: We conducted a telephone-based survey from February 6 to April 16, 2022 to staff, students, and parents of students who participated in school-based COVID-19 screening testing. COVID-19-related survey items included: vaccination status, reasons for not getting vaccinated, perceived vaccine access, and trusted COVID-19 information sources. Utilizing time-to-event analyses, we evaluated differences across demographic groups., Results: The interview response rate was 25.8% (308/1193). Most unvaccinated participants were NH Black and ages 5 to 11 years. Median time from vaccine eligibility to uptake was 236 days for NH Black participants vs. 10 days for NH White participants. Vaccine safety was the top concern among unvaccinated participants. Government and healthcare providers were the most trusted COVID-19 information sources., Conclusions: Differences in timing of vaccine uptake among respondents and greater vaccine hesitancy among NH Black participants compared to other racial/ethnic groups highlight a need for continued tailored outreach and communication using trusted sources to convey the importance, benefits, and safety of COVID-19 vaccination., (© 2023, American School Health Association. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the "out" position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections. IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts., (Copyright © 2018 American Society for Microbiology.)

Introduction: The appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition. An additional objective was to describe the optimal timing of specimen collection for the various tests., Methods: Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit. Patients who had been coughing for ≤ 2 weeks were asked to return in 2-4 weeks for collection of a second, convalescent blood specimen. Sensitivity and specificity of each diagnostic test were estimated using three methods-pertussis culture as the "gold standard," composite reference standard analysis (CRS), and latent class analysis (LCA)., Results: Overall, 868 patients were enrolled and 13.6% were B. pertussis positive by at least one diagnostic test. In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA. CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates. Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough., Conclusions: Timing of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis.

Background: United States national surveillance data show that the use of culture for pertussis diagnostics has sharply declined, whereas polymerase chain reaction (PCR) is now the most common testing method. PCR testing for pertussis is rapid and sensitive, but the lack of standardization and variable specificity is concerning., Methods: A web-based survey containing 12 questions was sent to public health, commercial and hospital-based US laboratories performing clinical diagnostics to determine the pertussis diagnostics used. An extensive real-time PCR (RT-PCR) questionnaire accompanied a proficiency panel assessing the types of extraction methods, RT-PCR methods and current quality control in place at the laboratories. The proficiency panel of 12 specimens containing Bordetella pertussis at various concentrations and negative controls was created to detect cross-contamination and assess the lower limit of detection., Results: One hundred twenty-three (35%) of 355 respondents from the web-based survey performed diagnostic tests for the presence of B. pertussis. Eighty-three (71%) labs reported performing culture, whereas 67 (54%) labs used PCR. All 41 laboratories that consented to participate in the proficiency exercise used the IS481 RT-PCR target; however, a variety of extraction and RT-PCR methods were employed. The laboratories correctly identified 92% of the B. pertussis specimens, and 5% of the laboratories (1.8% of the panel specimens) reported at least 1 false-positive., Conclusions: The small percentage of false-positives suggests that adequate procedures are in place to prevent cross-contamination. Differing extraction and PCR methods as well as variable analytic sensitivity emphasize the necessity for an external well-defined quality control program and interlaboratory pertussis PCR harmonization.

Bordetella holmesii, a human pathogen, can confound the diagnosis of respiratory illness caused by Bordetella pertussis. We present the draft genome sequences of two B. holmesii isolates, one from blood, F627, and one from the nasopharynx, H558. Interestingly, important virulence genes that are present in B. pertussis are not found in B. holmesii.

Bordetella pertussis causes an upper respiratory infection in infants, adolescents, and adults. Diagnosis of pertussis, a vaccine-preventable disease, can be difficult, but recent implementation of real-time PCR assays in laboratories has hastened the ability of clinicians to make an accurate diagnosis. In this paper we describe the method of nasopharyngeal specimen collection, extraction of DNA, and real-time PCR assays that will allow the detection and identification of Bordetella spp. in clinical specimens.

Objective: Knowledge of Bordetella pertussis circulating in Latin America is limited. Therefore, the goal of this study was to use pulsed-field gel electrophoresis and serotyping to characterize B. pertussis strains isolated in the city of São Paulo, Brazil., Methods: This study, conducted between 2006 and 2008, analyzed 652 nasopharyngeal swabs from suspected pertussis cases and contacts, collected from 37 sentinel hospitals in São Paulo. Randomized samples of 91 (70%) strains of B. pertussis were subtyped by pulsed-field gel electrophoresis and serotyping., Results: Ninety-seven percent of strains from São Paulo were serotyped as Fim3. Fourteen pulsed-field gel electrophoresis profiles were identified; the most prevalent (57%) is also the most prevalent in the USA., Conclusions: These data, in conjunction with surveillance activities, may impact strategies regarding prevention and control of pertussis in the region, providing useful information for introduction of new vaccination strategies and reduction of risk of transmission to infants less than 6 months of age.

Background and Objectives: We investigated a pertussis outbreak characterized by atypical cases, confirmed by polymerase chain reaction (PCR) alone at a single laboratory, which persisted despite high vaccine coverage and routine control measures. We aimed to determine whether Bordetella pertussis was the causative agent and advise on control interventions., Methods: We conducted case ascertainment, confirmatory testing for pertussis and other pathogens, and an assessment for possible sources of specimen contamination, including a survey of clinic practices, sampling clinics for B pertussis DNA, and review of laboratory quality indicators., Results: Between November 28, 2008, and September 4, 2009, 125 cases were reported, of which 92 (74%) were PCR positive. Cases occurring after April 2009 (n = 79; 63%) had fewer classic pertussis symptoms (63% vs 98%; P < .01), smaller amounts of B pertussis DNA (mean PCR cycle threshold value: 40.9 vs 33.1; P < .01), and a greater proportion of PCR-positive results (34% vs 6%; P < .01). Cultures and serology for B pertussis were negative. Other common respiratory pathogens were detected. We identified factors that likely resulted in specimen contamination at the point of collection: environmentally present B pertussis DNA in clinics from vaccine, clinic standard specimen collection practices, use of liquid transport medium, and lack of clinically relevant PCR cutoffs., Conclusions: A summer pertussis pseudo-outbreak, multifactorial in cause, likely occurred. Recommendations beyond standard practice were made to providers on specimen collection and environmental cleaning, and to laboratories on standardizing PCR protocols and reporting results, to minimize false-positive results from contaminated clinical specimens.

A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness.

Motivation: New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data., Results: We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes., Availability and Implementation: The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems.

Background: Each year, Bordetella pertussis infection causes an estimated 294,000 deaths worldwide, primarily among young, nonvaccinated children. Approximately 90% of all deaths due to pertussis in the Unites States occur in young infants. These children often develop intractable pulmonary hypertension; however, the pathophysiologic mechanism responsible for this complication has not been well characterized, and there have been no detailed descriptions of the pathology of this disease since the 1940s., Methods: Respiratory tissue samples obtained at autopsy from 15 infants aged

Novel, highly specific, and sensitive real-time polymerase chain reaction (PCR) assays using 2 targets, insertion sequence (IS481) and pertussis toxin subunit 1 (ptxS1), were developed to detect Bordetella pertussis and to differentiate between relevant Bordetella spp. Sixty-four non-Bordetella isolates were negative by both assays, demonstrating the specificity of the assays. B. pertussis, Bordetella parapertussis, and Bordetella holmesii isolates were specifically identified using the assays. The lower limit of detection was less than 10 genomic equivalents per reaction for the IS481 and ptxS1 assays. These assays were evaluated using 145 human clinical specimens obtained during cough-illness outbreak investigations, and PCR results were compared with Bordetella spp. culture results. Twenty-seven (18.6%) specimens had late positive cycle threshold (Ct) values (35

Using 2 real-time polymerase chain reaction (PCR) assays for Bordetella pertussis, 2 of 5 acellular pertussis vaccines were found to contain B. pertussis DNA. Because residual DNA in vaccines can cause environmental contamination, the administration of acellular pertussis vaccines to patients should be physically separated from the collection of patients' specimens for testing of B. pertussis DNA by real-time PCR.

Histopathology and special stains of a brain biopsy specimen from a 42-year-old man revealed numerous gram-positive bacilli arranged in branching filaments, suggesting Nocardia infection. Antibiotic therapy with trimethoprim-sulfamethoxazole markedly decreased the abscess size, and the patient improved. DNA was analyzed from formalin-fixed sections of the cerebral abscess by a 16S ribosomal DNA polymerase chain reaction assay demonstrating the presence of either Nocardia farcinica or N otitidiscaviarum. A species-specific polymerase chain reaction assay confirmed N farcinica as the etiologic agent.

Due to the importance of Bacillus anthracis as a cause of naturally occurring infection among humans and as an agent of bioterrorism, there is a vital need for rapid and specific assays, including immunohistochemistry (IHC) and polymerase chain reaction (PCR) assays, to detect the bacterium in formalin-fixed tissues. Colorimetric IHC assays were developed using a multistep indirect immunoalkaline phosphatase method with anti-B. anthracis cell wall (EAII-6G6-2-3) and anti-B. anthracis capsule (FDF-1B9) mAbs to detect B. anthracis antigens in formalin-fixed, paraffin-embedded bacterial cultures and tissues. B. anthracis antigens were localized, using both antibodies, in samples from B. anthracis-infected animals and humans. The colorimetric IHC assay with both antibodies was expedient in diagnosing the presence of B. anthracis in formalin-fixed, paraffin-embedded tissue from bioterrorism-associated cases of inhalational and cutaneous anthrax and from a case of naturally occurring cutaneous anthrax. Using the same antibodies, confocal microscopy demonstrated the structure of replicating B. anthracis in tissues. B. anthracis-specific primers were successfully used with PCR to amplify and detect B. anthracis sequences derived from formalin-fixed tissues of anthrax cases. In this study, morphologic, immunologic, and molecular assays were used to study and diagnose 22 veterinary and human anthrax cases.

Formalin-fixed lung or trachea tissue specimens from four infants and one adolescent who died of respiratory illness were tested for Bordetella pertussis by conventional and real-time PCR assays. B. pertussis was confirmed in all cases. PCR can be an invaluable retrospective diagnostic tool for evaluating archival tissues from patients with suspected fatal pertussis.

Severe acute respiratory syndrome (SARS) was first described during a 2002-2003 global outbreak of severe pneumonia associated with human deaths and person-to-person disease transmission. The etiologic agent was initially identified as a coronavirus by thin-section electron microscopic examination of a virus isolate. Virions were spherical, 78 nm in mean diameter, and composed of a helical nucleocapsid within an envelope with surface projections. We show that infection with the SARS-associated coronavirus resulted in distinct ultrastructural features: double-membrane vesicles, nucleocapsid inclusions, and large granular areas of cytoplasm. These three structures and the coronavirus particles were shown to be positive for viral proteins and RNA by using ultrastructural immunogold and in situ hybridization assays. In addition, ultrastructural examination of a bronchiolar lavage specimen from a SARS patient showed numerous coronavirus-infected cells with features similar to those in infected culture cells. Electron microscopic studies were critical in identifying the etiologic agent of the SARS outbreak and in guiding subsequent laboratory and epidemiologic investigations.

In 1999, a tetravalent rhesus-based rotavirus vaccine was withdrawn from the market after reports of intussusception cases among vaccinated infants. Methods to detect rotavirus in formalin-fixed pathology specimens from such patients will be important in examining the possible associations between the vaccine and intussusception, in investigating fatalities caused by natural rotavirus infection, and in furthering our understanding of the pathogenesis of rotavirus disease. Three different methods, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and in situ hybridization (ISH), were developed to detect rotavirus in infected cell lines that were fixed in formalin and embedded in paraffin. Using specific primer pairs to identify the VP4 gene with a one-step RT-PCR method, we detected simian rotavirus strains RRV and YK-1 in the liver of an RRV-infected SCID mouse and in the small intestine of an YK-1 infected macaque, respectively. Using a two-step indirect immunoalkaline phosphatase technique, we found RRV antigens in the liver of an infected SCID mouse with a rabbit polyclonal anti-group A rotavirus antibody and a murine monoclonal anti-rotavirus VP2 antibody. Using riboprobes designed to detect RRV genes, VP4 and NSP4, we obtained a positive hybridization signal in the same area of the infected SCID mouse liver as the area in which rotavirus antigens were localized. These techniques should prove valuable to detect rotavirus antigens and nucleic acids in tissues from patients infected naturally with rotavirus or with intussususception associated with rotavirus vaccine.

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.

The association of a sigma factor (sigma) with RNA polymerase in bacteria determines its specificity of promoter utilization. To identify amino acid residues in sigma E from Bacillus subtilis that determine the specificity of its interaction with the nucleotides at the -10 region of its cognate promoters, we tested whether base pair substitutions in the -10 region of a sigma B-dependent promoter could signal its utilization by sigma E-RNA polymerase. We found that a combination of base pair substitutions at positions -15 and -14 of the sigma B-dependent ctc promoter resulted in its utilization by sigma E-RNA polymerase in vivo. We also found that the combination of two amino acid substitutions at positions 119 and 120 in sigma E changed its specificity for promoter utilization, resulting in a sigma factor that directed transcription from the sigma B-dependent ctc promoter, but not from sigma E-dependent promoters. These results suggest that amino acid residues at positions 119 and 120 determine, at least in part, the specificity of interactions between sigma E and the nucleotides in the -10 region of its cognate promoters.

In order to determine which amino acyl residues in a secondary sigma factor govern its specificity of recognition at the -35 region of promoters, we examined the effects of amino acid substitutions in sigma E in Bacillus subtilis that made the sequence of its putative -35 recognition region more similar to another sigma factor in B. subtilis, sigma K. We found that a single amino acid substitution at position 217 of sigma E resulted in a sigma factor that could direct transcription from sigma K-dependent promoters. Furthermore, we tested whether this amino acid substitution in sigma E had changed the specificity of interactions of the sigma with -35 region sequences by examining the activity of the mutant sigma E on derivatives of sigma E-dependent promoters that contained single base-pair substitutions. We found that this substitution in sigma E specifically suppressed the effect of a single base-pair substitution at position -31 in a sigma E-dependent promoter spoIIID. The amino acyl residue at another position (219) on sigma E affected the specificity of interaction with position -33 in spoIIID promoter. The amino acyl residues at the two positions in sigma E, 217 and 219, that determine the specificity of interactions between the sigma and base-pairs in the -35 region of its cognate promoters (positions -33 and -31, respectively, in the spoIIID promoter) probably closely contact these base-pairs.

We have examined the role of the most highly conserved region of bacterial RNA polymerase sigma factors by analyzing the effect of amino acid substitutions and small deletions in sigma E from Bacillus subtilis. sigma E is required for the production of endospores in B. subtilis but not for vegetative growth. Strains expressing each of several mutant forms of sigE were found to be deficient in their ability to form endospores. Single amino acid substitutions at positions 68 and 94 resulted in sigma factors that bind with less affinity to the core subunits of RNA polymerase. The substitution at position 68 did not affect the stability of the protein in B. subtilis; therefore, this substitution probably did not have large effects on the overall structure of the sigma factor. The substitution at position 68 probably defines a position in sigma E that closely contacts a subunit of RNA polymerase, while the substitution at position 94 may define a position that is important for protein stability or for binding to core RNA polymerase.

Genetic evidence suggests that the sigma (sigma) subunit of RNA polymerase determines the specificity of promoter utilization, by making sequence-specific contacts with DNA. We examined the effects of two single amino acid(aa) substitutions in sigma E on the utilization of mutated derivatives of three different promoters in sporulating Bacillus subtilis. We found allele-specific suppression of mutations in all three promoters by each aa substitution in sigma E. These results provide strong evidence that sigma E interacts with each of these promoters in vivo. Moreover, the specificity of suppression of the mutations by the aa substitutions in sigma E lead us to speculate that the Met124 of sigma E closely contacts two adjacent bp in the -10 region of the promoters.

The sigma subunit of bacterial RNA polymerase is required for specific binding to promoters. One region in most sigma factors makes sequence-specific contacts at the -10 region of its cognate promoters. To test the role of the amino acids in this -10 binding region, we examined the effects of 49 single-amino-acid substitutions in sigma E from Bacillus subtilis. We assayed the effect of each amino acid substitution on spore formation because sigma E is essential for endospore formation in B. subtilis. Our results showed that substitutions at several positions, including the highly conserved aromatic amino acid at position 102, had little or no detectable effect. Substitutions at another position, position 117, produced dominant negative mutations; we suggest that these mutations allow RNA polymerase containing the mutant sigma factor to bind specifically to promoters but prevent transcription initiation. Of the recessive defective alleles, those that produced substitutions at positions 113, 115, and 120 produced the most defective sigma factors. These results suggest that the residues at or near these positions in wild-type sigma E play important roles in sigma E function.

During sporulation in Bacillus subtilis, new RNA polymerase sigma factors are produced. These sigma factors direct the transcription of genes that are required for this cellular differentiation. In order to determine the role of each sigma factor in this process, it is necessary to know which promoters are recognized by each sigma factor. The spoIIID gene product plays an important role in the establishment of mother cell-specific gene expression during sporulation. We found that substitution of an alanine at position 124 of the sporulation-specific sigma factor sigma E suppressed the effect of a single-base-pair transition at position -13 of the spoIIID promoter. This alanine substitution in sigma E did not suppress the effect of a transversion at position -12 of the spoIIID promoter. The allele specificity of the interaction between sigma E and the spoIIID promoter is strong evidence that sigma E directs transcription from the spoIIID promoter during sporulation. Position 124 in sigma E is located within a region that is highly conserved among the regions in other sigma factors that probably interact with the -10 regions of their cognate promoters.

The spoIIA operon encodes three genes, including the structural gene for a sporulation-induced sigma factor sigma F. We used deletion analysis of spoIIA-lacZ fusions to define the location of the spoIIA promoter. We found that sigma H-RNA polymerase transcribes spoIIA accurately in vitro and propose that sigma H directs transcription of spoIIA during sporulation.

The secondary RNA polymerase sigma factor sigma H is essential for endospore development in Bacillus subtilis. However, only a few promoters that are used by RNA polymerase containing sigma H (E sigma H) have been identified. We used in vitro transcription of random cloned fragments of B. subtilis chromosomal DNA to identify a promoter that is used by E sigma H. This promoter is active before the onset of sporulation.

A procedure is presented for the rapid isolation of mitochondrial DNA (mtDNA) in high yield from Saccharomyces cerevisiae. Yeast cells, which may be grown to late stationary phase, are broken by a combination of enzymatic and mechanical means; mtDNA is then isolated from a crude mitochondrial lysate by a single cycle of bisbenzimide-CsCl buoyant density centrifugation. mtDNA so isolated is at least 99.5% pure, and has a mean duplex molecular weight of 24.5 . 10(6). In addition to mtDNA and bulk nuclear DNA, several other yeast nucleic acid species, identified as ribosomal DNA and a mixture of duplex RNAs, form discrete bands in these gradients.

Bacillus subtilis possesses at least five different forms of RNA polymerase holoenzyme which are distinguished by their sigma subunit and their promoter recognition specificity. Sigma-37 RNA polymerase, a minor form of RNA polymerase, recognizes a class of promoters, which includes promoters for genes transcribed early during endospore formation. We have used site-directed bisulfite mutagenesis to construct a series of single and multiple base substitutions in a promoter recognized by sigma-37 RNA polymerase. In vitro transcription analysis of this series of mutant promoters demonstrated that single base substitutions at positions -36, -16, -15 and -14 most dramatically reduced the efficiency of promoter utilization by sigma-37 RNA polymerase. These results support a model in which sigma-37 RNA polymerase recognizes its cognate promoters by interacting with a sequence of nucleotides near the -10 region and the -35 region of the promoter--a sequence not recognized by B. subtilis sigma-55 RNA polymerase or Escherichia coli RNA polymerase.

Bacillus subtilis possesses several forms of RNA polymerase, each differing in its sigma subunit and its specificity of promoter recognition. The sequential appearance of sigma subunits, which change the promoter recognition specificity of RNA polymerase, may have a key role in controlling the temporal pattern of gene expression required for endospore development in B. subtilis. Several genes that are expressed over relatively long periods of time during the developmental cycle are transcribed by more than one form of RNA polymerase, which initiate transcription from either tandem or overlapping promoter. The promoter region for the ctc gene is interesting because transcription is initiated at or near the same position by both sigma 37 RNA polymerase (E sigma 37), a minor form in growing cells, and sigma 29 RNA polymerase (E sigma 29), a form which appears approximately 2 h after the initiation of sproulation. Here we report that several base substitutions in the ctc promoter differentially affect the utilization of the promoter by E sigma 37 or E sigma 29.

The RNA polymerase sigma factor sigma H is essential for the onset of endospore formation in Bacillus subtilis. sigma H also is required for several additional stationary-phase-specific responses, including the normal expression of several genes that are required for the development of competence for DNA uptake. It is necessary to identify the genes that are transcribed by sigma H RNA polymerase (E sigma H) in order to understand the role of this sigma factor during the transition from exponential growth to stationary phase. Feavers et al. (Mol. Gen. Genet. 211:465-471, 1988) proposed that citG, the structural gene for fumarase, is transcribed from two promoters, one of which (citGp2 [P2]) may be used by E sigma H. It is likely that the citGp2 promoter is used by E sigma H because we found that this promoter was used accurately in vitro by E sigma H and directed expression of xylE in vivo. This xylE expression was dependent on spo0H, the structural gene for sigma H, and was independent of the citGp1 promoter. Comparison of the nucleotide sequences of several sigma H-dependent promoters showed that these sequences were similar at two regions approximately 10 and 35 base pairs upstream from the start points of transcription. These sequences may signal recognition of these promoters by E sigma H. Primer extension analyses were used to examine transcription from three sigma H-dependent promoters during growth and sporulation. The citGp2 promoter appeared to be active during the middle and late stages of exponential growth, whereas activation of the spoIIA promoter was delayed until after the end of exponential growth. Evidently, promoters used by E sigma H can display different temporal patterns of expression.

Gene expression during endospore formation by Bacillus subtilis is controlled in part by a sporulation-induced form of RNA polymerase, E sigma 29. The determination of the nucleotide sequences that govern utilization of promoters by E sigma 29 has been limited by the small number of available promoters that are recognized by E sigma 29. In the present report we describe a promoter that is adjacent to the rrnB region of the B. subtilis chromosome and is utilized in vitro and in vivo by E sigma 29. S1 nuclease mapping and dinucleotide priming experiments have been used to determine the start point of transcription. The nucleotide sequences near the -10 and -35 region of this promoter, bvx, are conserved, and resemble sequences at these regions for other promoters that are utilized by E sigma 29.

The discovery of secondary sigma factors in Bacillus subtilis that enable RNA polymerase to transcribe cloned sporulation genes in vitro has led to the proposal that the appearance of new sigma factors during sporulation directs RNA polymerase to the different temporal classes of sporulation genes. One sigma factor, which appears 2 h after the initiation of sporulation, is sigma E (formerly sigma 29). Mutations that inactivate the structural gene for sigma E prevent transcription from promoter G4. To determine whether sigma E-RNA polymerase interacts with the G4 promoter in vivo, we examined the effects of six single-base-pair substitutions in the G4 promoter on its utilization in vivo and in vitro by sigma E-RNA polymerase. The mutations in the G4 promoter affected utilization of the promoter in vivo in the same way that they affected its utilization in vitro by purified sigma E-RNA polymerase; therefore, we conclude that this polymerase interacts directly with the G4 promoter in vivo. The effects of these mutations also support the model in which sigma E-RNA polymerase utilizes promoters by interacting with two distinct sets of nucleotides located 10 and 35 base pairs upstream from the start point of transcription.

The program of gene expression that underlies endospore formation by Bacillus subtilis may be controlled in part by a sporulation-induced form of RNA polymerase, E sigma 29. The nucleotide sequences of four promoters, which are known to be recognized by E sigma 29, are highly conserved at two regions, 10 bp and 35 bp upstream from the start point of transcription. We have used oligonucleotide-directed mutagenesis to construct several base substitutions in the ctc promoter from B. subtilis to test the role of the highly conserved sequences in utilization of the promoter by E sigma 29. In vitro transcription analysis demonstrated that the conserved nucleotides at positions -15, -14 and -12 affect the utilization of the promoter by E sigma 29. These and previous results support a model in which E sigma 29 recognizes its cognate promoters by interacting with nucleotides near the -10 and -35 regions. We also examined the effects of these base substitutions on utilization of the promoter by two other forms of RNA polymerase from B. subtilis, E sigma 37 and E sigma 32.