Rapid preparation of 6S RNA-free B. subtilis σA-RNA polymerase and σA.

The regulatory 6S-1 and 6S-2 RNAs of B. subtilis bind to the housekeeping RNA polymerase holoenzyme (σA-RNAP) with submicromolar affinity. We observed copurification of endogenous 6S RNAs from a published B. subtilis strain expressing a His-tagged RNAP. Such 6S RNA contaminations in σA-RNAP preparations reduce the fraction of enzymes that are accessible for binding to DNA promoters. In addition, this leads to background RNA synthesis by σA-RNAP utilizing copurified 6S RNA as template for the synthesis of short abortive transcripts termed product RNAs (pRNAs). To avoid this problem we constructed a B. subtilis strain expressing His-tagged RNAP but carrying deletions of the two 6S RNA genes. The His-tagged, 6S RNA-free σA-RNAP holoenzyme can be prepared with sufficient purity and activity by a single affinity step. We also report expression and separate purification of B. subtilis σA that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity.

The purification of the σFpvI/FpvR20 and σPvdS/FpvR20 protein complexes is facilitated at room temperature.

Bacteria contain sigma (σ) factors that control gene expression in response to various environmental stimuli. The alternative sigma factors σFpvI and σPvdS bind specifically to the antisigma factor FpvR. These proteins are an essential component of the pyoverdine-based system for iron uptake in Pseudomonas aeruginosa. Due to the uniqueness of this system, where the activities of both the σFpvI and σPvdS sigma factors are regulated by the same antisigma factor, the interactions between the antisigma protein FpvR20 and the σFpvI and σPvdS proteins have been widely studied in vivo. However, difficulties in obtaining soluble, recombinant preparations of the σFpvI and σPvdS proteins have limited their biochemical and structural characterizations. In this study, we describe a purification protocol that resulted in the production of soluble, recombinant His6-σFpvI/FpvR1-67, His6-σFpvI/FpvR1-89, His6-σPvdS/FpvR1-67 and His6-σPvdS/FpvR1-89 protein complexes (where FpvR1-67 and FpvR1-89 are truncated versions of FpvR20) at high purities and concentrations, appropriate for biophysical analyses by circular dichroism spectroscopy and analytical ultracentrifugation. These results showed the proteins to be folded in solution and led to the determination of the affinities of the protein-protein interactions within the His6-σFpvI/FpvR1-67 and His6-σPvdS/FpvR1-67 complexes. A comparison of these values with those previously reported for the His6-σFpvI/FpvR1-89 and His6-σPvdS/FpvR1-89 complexes is made.

Protein purification and crystallization artifacts: The tale usually not told.

The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non-reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building.

BadR (BB0693) controls growth phase-dependent induction of rpoS and bosR in Borrelia burgdorferi via recognizing TAAAATAT motifs.

In Borrelia burgdorferi (Bb), the alternative sigma factor RpoS plays a central role during Bb's adaptation to ticks and mammals. Previous studies have demonstrated that RpoS is not expressed during the early stages of spirochetal growth or when Bb resides in ticks during the intermolt phase, but the molecular details of these events remain unknown. In the current study, biomagnetic bead separation of rpoS promoter-binding proteins, coupled with genetic inactivation, was employed to identify BadR (BB0693) as a negative regulator that controls growth phase-dependent induction of rpoS and bosR in Bb. When badR was inactivated, the expression of rpoS and bosR was induced only during the early stages of bacterial growth, but not during the stationary growth phase. Recombinant BadR bound to the promoter DNA of rpoS and the regulatory region upstream of bosR via AT-rich TAAAATAT motifs. Mutations in this motif markedly inhibited or abolished rBadR binding. These results suggest that BadR directly influences the expression of both rpoS and bosR in Bb. This newly recognized role for BadR to fine-tune the activation of the RpoN-RpoS pathway at strategic times in Bb's life cycle potentially represents another layer of gene control over σ(54)-dependent gene regulation.

Rhodopseudomonas palustris CGA010 Proteome Implicates Extracytoplasmic Function Sigma Factor in Stress Response.

Rhodopseudomonas palustris encodes 16 extracytoplasmic function (ECF) σ factors. To begin to investigate the regulatory network of one of these ECF σ factors, the whole proteome of R. palustris CGA010 was quantitatively analyzed by tandem mass spectrometry from cultures episomally expressing the ECF σ(RPA4225) (ecfT) versus a WT control. Among the proteins with the greatest increase in abundance were catalase KatE, trehalose synthase, a DPS-like protein, and several regulatory proteins. Alignment of the cognate promoter regions driving expression of several upregulated proteins suggested a conserved binding motif in the -35 and -10 regions with the consensus sequence GGAAC-18N-TT. Additionally, the putative anti-σ factor RPA4224, whose gene is contained in the same predicted operon as RPA4225, was identified as interacting directly with the predicted response regulator RPA4223 by mass spectrometry of affinity-isolated protein complexes. Furthermore, another gene (RPA4226) coding for a protein that contains a cytoplasmic histidine kinase domain is located immediately upstream of RPA4225. The genomic organization of orthologs for these four genes is conserved in several other strains of R. palustris as well as in closely related α-Proteobacteria. Taken together, these data suggest that ECF σ(RPA4225) and the three additional genes make up a sigma factor mimicry system in R. palustris.

Infant and young child feeding in four departments in Haiti: mixed-method study on prevalence of recommended practices and related attitudes, beliefs, and other determinants / Alimentación de los lactantes y niños pequeños en cuatro departamentos de Haití: un estudio con diseño de método mixto sobre la prevalencia de las prácticas recomendadas y las actitudes, las creencias y otros determinantes relacionados

OBJECTIVE: To determine and describe the prevalence and patterns of three recommended practices for infant and young child feeding-exclusive breastfeeding (EB), continued breastfeeding (CB), and achievement of minimum dietary diversity-in four regions in Haiti, and to identify the attitudes and beliefs that inform these practices and any other factors that may facilitate or impede their implementation. METHODS: This study utilized a mixed-methods approach consisting of 1) a cross-sectional survey (n = 310) and 2) 12 focus group discussions among women ≥18 years old with children ≤ 2 years old. Multivariable logistic regression analyses were conducted to identify factors associated with 1) EB during the first six months of life, 2) CB for children ≥ 2 years old, and 3) receipt of a diverse variety of complementary foods. Qualitative data were recorded, transcribed verbatim, and analyzed for common themes. Data were collected in June and July 2013 in four departments in Haiti: Artibonite, Nippes, Ouest, and Sud-Est. RESULTS: Prevalence of EB, CB, and achievement of minimum dietary diversity was 57.0%, 11.9%, and 21.2% respectively. EB was statistically significantly associated with infant's age when controlling for annual household income, location of most recent birth, or receipt of CB counseling (odds ratio (OR) = 0.67 (95% CI: 0.47-0.97)). CB was not statistically significantly associated with rural place of residence, receipt of CB counseling, parity, or infant's age. Meeting minimum dietary diversity was not significantly associated with parity, receipt of postnatal care, rural place of residence, location of most recent birth, receipt of infant and young child feeding counseling, or level of schooling. Beliefs surrounding the relationship between the mother's health and her diet on the quality of breast milk may prohibit EB and CB. Qualitative data revealed that dietary diversity may be low because 1) mothers often struggle to introduce complementary foods and 2) those that are traditionally introduced are not varied and primarily consist of grains and starches. CONCLUSIONS: Prevalence of the three recommended infant and young child feeding practices examined in this study is suboptimal, particularly CB and achievement of minimum dietary diversity. Future communication and programming efforts should address the misunderstandings and concerns identified through the qualitative methods used in this research.

OBJETIVO: Determinar y describir la prevalencia y los modelos de tres prácticas recomendadas para la alimentación de los lactantes y los niños pequeños (la lactancia materna exclusiva [LME], la lactancia materna continuada [LMC] y el logro de una diversidad alimentaria mínima, en cuatro regiones de Haití, y determinar las actitudes y creencias en las que se basan estas prácticas y otros factores que puedan facilitar o impedir su implantación. MÉTODOS: Este estudio utilizó un diseño de método mixto que consistió en 1) una encuesta transversal (n = 310) y 2) 12 grupos de discusión formados por mujeres de ≥ 18 años de edad o mayores con niños de ≤ 2 años de edad o menores. Se llevaron a cabo análisis de regresión logística multivariable para determinar los factores asociados con 1) la LME durante los seis primeros meses de vida, 2) la LMC en niños de ≥ 2 años de edad o mayores, y 3) el aporte de una amplia variedad de alimentos complementarios. Se registraron, se transcribieron al pie de la letra y se analizaron los datos cualitativos referentes a temas comunes. Estos datos se recopilaron en junio y julio del 2013, en cuatro departamentos de Haití: Artibonite, Nippes, Oeste y Sudeste. RESULTADOS: Las prevalencias de la LME, la LMC y el logro de una diversidad alimentaria mínima fueron de 57,0, 11,9 y 21,2%, respectivamente. La LME se asoció de manera estadísticamente significativa con la edad del lactante si se controlaban las variables de ingresos familiares anuales, ubicación del parto más reciente, o provisión de orientación en materia de LMC (razón de posibilidades [OR] = 0,67 [IC de 95%:0.47-0.97]). La LMC no se asoció de una manera estadísticamente significativa con la residencia en un entorno rural, la provisión de orientación en materia de LMC, la paridad o la edad de lactante. El logro de una diversidad alimentaria mínima no se asoció significativamente con la paridad, la provisión de atención posnatal, la residencia en un entorno rural, la ubicación del parto más reciente, la provisión de orientación en materia de alimentación de los lactantes y los niños pequeños, o el nivel de escolarización. Las creencias con respecto a la relación entre la salud de la madre y su régimen alimentario con la calidad de la leche materna pueden limitar la LME y la LMC. Los datos cualitativos revelaron que la diversidad alimentaria puede ser escasa como consecuencia de que 1) las madres a menudo se esfuerzan por introducir los alimentos complementarios, y 2) los que se introducen tradicionalmente no son variados y consisten principalmente en cereales y féculas. CONCLUSIONES: Las prevalencias de las tres prácticas de alimentación de los lactantes y los niños pequeños recomendadas analizadas en este estudio son subóptimas, en particular las correspondientes a la LMC y al logro de una diversidad alimentaria mínima. Las futuras iniciativas de comunicación y programación deberían abordar los malentendidos y las inquietudes detectadas mediante los métodos cualitativos utilizados en esta investigación.

Purification and biochemical characterization of DnaK and its transcriptional activator RpoH from Neisseria gonorrhoeae.

DnaK plays a central role in stress response in the important human pathogen Neisseria gonorrhoeae. The genes encoding the DnaK chaperone machine (DnaK/DnaJ/GrpE) in N. gonorrhoeae are transcribed from RpoH (σ(32))-dependent promoters. In this study, we cloned, purified and biochemically characterised N. gonorrhoeae DnaK (NgDnaK) and RpoH. The NgDnaK and RpoH sequences are 73 and 50 % identical to the sequences of their respective E. coli counterparts. Similar to EcDnaK, nucleotide-free NgDnaK exists as a mix of monomers, dimers and higher oligomeric species in solution, and dissociates into monomers on addition of ATP. Like E. coli σ(32), RpoH of N. gonorrhoeae is monomeric in solution. Kinetic analysis of the basal ATPase activity of purified NgDnaK revealed a V max of 193 pmol phosphate released per minute per microgram DnaK (which is significantly higher than reported basal ATPase activity of EcDnaK), and the turnover number against ATP was 0.4 min(-1) under our assay conditions. Nucleotide-free NgDnaK bound a short model substrate, NR-peptide, with micromolar affinity close to that reported for EcDnaK. Our analysis showed that interaction between N. gonorrhoeae RpoH and DnaK appears to be ATP-dependent and non-specific, in stark contrast to the E. coli DnaK system where σ(32) and DnaK interact as monomers even in the absence of ATP. Sequence comparison showed that the DnaK-binding site of σ(32) is not conserved in RpoH. Our findings suggest that the mechanism of DnaK/RpoH recognition in N. gonorrhoeae is different from that in E. coli.

Intracellular concentrations of 65 species of transcription factors with known regulatory functions in Escherichia coli.

The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature.

Global analysis of the sporulation pathway of Clostridium difficile.

The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Despite the importance of spores to the pathogenesis of C. difficile, little is known about their composition or formation. Based on studies in Bacillus subtilis and other Clostridium spp., the sigma factors σ(F), σ(E), σ(G), and σ(K) are predicted to control the transcription of genes required for sporulation, although their specific functions vary depending on the organism. In order to determine the roles of σ(F), σ(E), σ(G), and σ(K) in regulating C. difficile sporulation, we generated loss-of-function mutations in genes encoding these sporulation sigma factors and performed RNA-Sequencing to identify specific sigma factor-dependent genes. This analysis identified 224 genes whose expression was collectively activated by sporulation sigma factors: 183 were σ(F)-dependent, 169 were σ(E)-dependent, 34 were σ(G)-dependent, and 31 were σ(K)-dependent. In contrast with B. subtilis, C. difficile σ(E) was dispensable for σ(G) activation, σ(G) was dispensable for σ(K) activation, and σ(F) was required for post-translationally activating σ(G). Collectively, these results provide the first genome-wide transcriptional analysis of genes induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes.

The Bartonella quintana extracytoplasmic function sigma factor RpoE has a role in bacterial adaptation to the arthropod vector environment.

Bartonella quintana is a vector-borne bacterial pathogen that causes fatal disease in humans. During the infectious cycle, B. quintana transitions from the hemin-restricted human bloodstream to the hemin-rich body louse vector. Because extracytoplasmic function (ECF) sigma factors often regulate adaptation to environmental changes, we hypothesized that a previously unstudied B. quintana ECF sigma factor, RpoE, is involved in the transition from the human host to the body louse vector. The genomic context of B. quintana rpoE identified it as a member of the ECF15 family of sigma factors found only in alphaproteobacteria. ECF15 sigma factors are believed to be the master regulators of the general stress response in alphaproteobacteria. In this study, we examined the B. quintana RpoE response to two stressors that are encountered in the body louse vector environment, a decreased temperature and an increased hemin concentration. We determined that the expression of rpoE is significantly upregulated at the body louse (28°C) versus the human host (37°C) temperature. rpoE expression also was upregulated when B. quintana was exposed to high hemin concentrations. In vitro and in vivo analyses demonstrated that RpoE function is regulated by a mechanism involving the anti-sigma factor NepR and the response regulator PhyR. The ΔrpoE ΔnepR mutant strain of B. quintana established that RpoE-mediated transcription is important in mediating the tolerance of B. quintana to high hemin concentrations. We present the first analysis of an ECF15 sigma factor in a vector-borne human pathogen and conclude that RpoE has a role in the adaptation of B. quintana to the hemin-rich arthropod vector environment.

Identification of disulphide stress-responsive extracytoplasmic function sigma factors in Rothia mucilaginosa.

Rothia mucilaginosa is known as a member of commensal bacterial flora in the oral cavity and has received attention as a potential opportunistic pathogen. We previously determined the genomic sequence of R. mucilaginosa DY-18, a clinical strain with biofilm-like structures isolated from an infected root canal of a tooth with persistent apical periodontitis. We found that the DY-18 genome had only two sigma factor genes that encoded the primary and extracytoplasmic function (ECF) sigma factors. Genomic analysis on the available database of R. mucilaginosa ATCC 25296 (a type strain for R. mucilaginosa) revealed that ATCC 25296 has three sigma factors: one primary sigma factor and two ECF sigma factors, one of which was highly homologous to that of DY-18. ECF sigma factors play an important role in the response to environmental stress and to the production of virulence factors. Therefore, we first examined gene-encoding sigma factors on R. mucilaginosa genome in silico. The homologous ECF sigma factors found in strains DY-18 and ATCC 25296 formed a distinct SigH (SigR) clade in a phylogenetic tree and their cognate anti-sigma factor has a HXXXCXXC motif known to respond against disulphide stress. Quantitative reverse transcription polymerase chain reaction (PCR) and microarray analysis showed that the transcriptional levels of sigH were markedly up-regulated under disulphide stress in both strains. Microarray data also demonstrated that several oxidative-stress-related genes (thioredoxin, mycothione reductase, reductase and oxidoreductase) were significantly up-regulated under the diamide stress. On the basis of these results, we conclude that the alternative sigma factor SigH of R. mucilaginosa is a candidate regulator in the redox state.

The core-independent promoter-specific interaction of primary sigma factor.

Previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. Most σ functions were studied on the basis of this tenet. Here, we provide in vitro evidence demonstrating that the intact Bacillus subtilis primary sigma, σ(A), by itself, is able to interact specifically with promoter deoxyribonucleic acid (DNA), albeit with low sequence selectivity. The core-independent promoter-specific interaction of the σ(A) is -10 specific. However, the promoter -10 specific interaction is unable to allow the σ(A) to discern the optimal promoter spacing. To fulfill this goal, the σ(A) requires assistance from core RNA polymerase (RNAP). The ability of σ, by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function.

Over-expression and purification strategies for recombinant multi-protein oligomers: a case study of Mycobacterium tuberculosis σ/anti-σ factor protein complexes.

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the σ factor/anti-σ factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of σ factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems.

Identification and characterization of two alternative sigma factors of RNA polymerase in the deep-sea piezophilic bacterium Shewanella violacea, strain DSS12.

Two genes for alternative sigma factors, sigma(E2) and sigma(E3), classified in the extracytoplasmic function sigma family for RNA polymerases, were identified in the deep-sea piezophilic bacterium Shewanella violacea DSS12. Amino acid alignments revealed that the domains for transcriptional functions were comparatively conserved compared with Escherichia coli sigma(E) in both proteins. Core-binding analysis suggested that both proteins function as sigma factors.

A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS.

We isolated an Escherichia coli mutant in the iraD gene, sensitive to various forms of DNA damage. Our data are consistent with the function of IraD to promote accumulation of the alternative transcription sigma factor, RpoS, by binding to the adaptor RssB protein that targets RpoS for degradation. Our results demonstrate the physiological importance of this mode of regulation for DNA damage tolerance. Although RpoS is best known for its regulation of genes induced in stationary phase, our work underscores the importance of the RpoS regulon in a DNA damage response in actively growing cells. We show that iraD transcription is induced by DNA damage by a mechanism independent of the SOS response. The IraD and SOS regulatory pathways appear to act synergistically to ensure survival of cells faced with oxidative or DNA damaging stress during cellular growth.

Interactions of the M. tuberculosis UsfX with the cognate sigma factor SigF and the anti-anti sigma factor RsfA.

M. tuberculosis employs an exquisite cascade consisting of the cognate anti-sigma factor UsfX and anti-anti sigma factors RsfA and RsfB to regulate the functions of the alternate sigma factor SigF. We have purified these proteins to characterize their molecular properties and interactions with UsfX. UsfX forms a stable complex with SigF that could be purified only after co-expressing the proteins in E.coli. Formation of the complex is nucleotide independent and apparently requires unknown in vivo factors. Fluorescence spectroscopy experiments suggest that the nucleotide binding sites of UsfX are distal to the protein-protein interaction interface. RsfA is a novel anti-anti sigma factor whose binding to UsfX is triggered by the reduction of an intrachain disulphide bond between Cys73-Cys109. The reduction is accompanied by an increase in the hydrodynamic radius of the protein. The UsfX-RsfA complex exhibits a novel stoichiometry of 2:1 compared to the 2:2 stoichiometry reported for other anti-anti-sigma factors. The role of the disulphide bond in complex formation was explored using molecular dynamics simulations. These studies support specific conformational changes that occur upon reduction of the Cys73-Cys109 bond of RsfA. This leads to a rearrangement that increases the interactions of a conserved His107 of UsfX with Cys109 of RsfA.

Cloning, identification, and characterization of the rpoS-like sigma factor rpoX from Vibrio alginolyticus.

Vibrio alginolyticus ZJ-51 displays phase variation between opaque/rugose colonies (Op) and translucent/smooth colonies (Tr). These colony variants show great differences in biofilm formation and motility. In this study, a gene encoding for an rpoS-like sigma factor, rpoX, has been cloned and characterized. The absence of rpoX did not affect colony switching rate but did decrease biofilm formation in both the Op and the Tr variants. When challenged with hydrogen peroxide, the DeltarpoX in the Op background showed a slightly higher survival rate compared with the wild type, whereas survival was decreased in the Tr background. Deletion of rpoX in the Tr background resulted in a higher ability to resist ethanol challenges and to survive hyperosmolarity challenges, and in the Op background the opposite phenotype was observed. This indicates that the rpoX gene is involved in biofilm formation and stress response but the effects are controlled by colony phase variation in V. alginolyticus.

Region C of the Escherichia coli heat shock sigma factor RpoH (sigma 32) contains a turnover element for proteolysis by the FtsH protease.

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor sigma(32) (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma factor.

Methods to identify and characterize inhibitors of bacterial RNA polymerase.

RNA polymerase is essential to the viability of bacteria in all phases of growth and development and is a proven chemotherapeutic target as the cellular target of the rifamycin class of antibiotics. However, despite the characterization of multiple different classes of natural products that selectively target bacterial RNA polymerase, and the identification of a limited number of synthetic compound inhibitors, only agents of the rifamycin class have been developed and approved for human clinical use as antibiotics. Herein we describe a scintillation proximity assay (SPA) for identifying and characterizing inhibitors of bacterial RNA polymerases and that is applicable to de novo drug discovery programs through application of automated high-throughput screening methods. In addition, we describe gel electrophoresis-based methods that are applicable to the detailed characterization of inhibitors of transcriptional initiation or elongation by bacterial RNA polymerases.

Cascade of extracytoplasmic function sigma factors in Mycobacterium tuberculosis: identification of a sigmaJ-dependent promoter upstream of sigI.

A previously optimized Escherichia coli two-plasmid system was used to identify promoters recognized by RNA polymerase containing the Mycobacterium tuberculosis extracytoplasmic function (ECF) sigma factor sigma(J). The only positive clone revealed a promoter directing expression of the sigI gene encoding ECF sigma factor sigma(I). The transcriptional start point of the promoter was determined and the promoter was confirmed by an in vitro transcription assay with either E. coli or Mycobacterium smegmatis core RNA polymerase and isolated sigma(J).