rpoB Locus Research Articles

First Report of Maize White Spot Disease Caused by Pantoea ananatis in China.

Maize (Zea mays L.) is one of the most important crops in China. Since 2020, a new leaf spot disease has occurred in southwest China in areas such as Yunnan, Sichuan and Hubei provinces. Typical symptoms appeared after tasseling. The spots are scattered on the leaf surface, round to oval in shape with diameter range 3-20 mm. Spots are initially water-soaked, gradually turning yellow or white. In 2021, diseased leaf samples with typical white spot were collected for pathogen isolation and identification in Qujing, Yunnan province. Four small pieces of leaf tissue (about 0.25 cm2 in area) were excised from the edge of the necrotic lesion of each plant, surface sterilized with 75% ethanol for 1 min, rinsed three times with sterile distilled water, and soaked in sterile distilled water for 5 min. The solution was plated on Luria Broth medium (LB) plate (Shin et al. 2022) . After incubation at 28°C for 24 h, round, smooth-edged yellow colonies appeared in the LB plate. The bacterium isolated was gram-negative, negative for oxidase, positive for peroxidase, indole, citrate (Wells et al. 1987). Three strains (PA21QJ01, PA21QJ02 and PA21QJ03) showed identical colony morphology. PA21QJ01 was used for further molecular analyses. DNA was extracted from fresh colonies cultured in LB(Shin et al. 2022), and the fragments at the 16S rDNA, gyrB and rpoB loci were amplified using primers 27F/1492R (Galkiewicz and Kellogg 2008), UP-1/UP-2r (Yamamoto and Harayama 1995) and rpoBCM81-F/rpoBCM32b-R (Brady et al. 2008), respectively. The sequences of fragments of 16S rDNA, gyrB and rpoB from isolate PA21QJ01 were was deposited in GenBank (accession number: OM184301.1, OM302500, OM302499). A search for homologous sequences using BLAST resulted in 99.9, 99.6 and 99.8% identity of 16S rDNA, gyrB and rpoB, respectively, with sequences from the NN08200 of Pantoea ananatis (GenBank accession numbers: MK415050.1 for 16S rDNA, CP035034.1 for gyrB and CP035034.1.1 for rpoB). Above molecular results indicated that PA21QJ01 isolated from maize white spot is P. ananatis. Pathogenicity tests were performed on tasseled plants of the suscptible maize variety Wugu1790. After culture in LB medium plate at 30°C for 12 h, the bacterial solution was used for inoculation at a concentration of 1 × 108 CFU ml-1. After 7 days of inoculation, the leaves of the plants appeared water-soaked. After 10 days, white spot developed with brown margin. In contrast, the control plants remained healthy and symptomless. The same P. ananatis was reisolated in the inoculated maize plants, fulfilled Koch's law. In the last decade, P. ananatis has been reported to cause maize white spot in South Africa, Mexico, Poland, Argentina, Brazil (Sauer et al. 2015), and Ecuador (Toaza et al.2021). It has caused crop diseases with other crops, such as onion, rice, pineapple, melon, and sorghum, and others (Sauer et al. 2015). It caused leaf blight and leaf steak in rice in China (Yu et al. 2021). This is the first report of maize white spot caused by P. ananatis in China. However, to our knowledge, this is the first report of maize white spot disease in China. Attentions should be paid to screening for disease-resistant resources and breeding disease-resistant hybrids. Reference: Wells, J. M. et al. 1987. Int. J. Syst. Bacteriol. 37:136-143. Shin, G. Y. et al. 2022. Plant Dis. Doi: 10.1094/PDIS-08-21-1810-SC. Brady, C., et al. 2008. Syst. Appl. Microbiol. 31:447. Galkiewicz, J. P., and Kellogg, C. A. 2008. APPL ENVIRON MICROB, 74.24: 7828-7831. Toaza, A. et al. 2021. Plant Dis. Doi:10.1094/PDIS-02-21-0298-PDN Yamamoto, S., and Harayama, S. 1995. APPL ENVIRON MICROB, 61:1104.L. Sauer, A. V. et al. 2015. Agronomy Science and Biotechnology. Doi:10.33158/ASB.2015v1i1p21 Yu et al. 2021. Plant Dis. Doi:10.1094/PDIS-05-21-0988-PDN.

Molecular survey and genetic diversity of Bartonella spp. in domestic cats from Paraguay.

Although Bartonella spp. is described in cats worldwide, little is known about the occurrence and genetic diversity of Bartonella spp. in cats from South America. To date, it has only been detected in cats from Brazil, Chile and Argentina. This study aimed to undertake a molecular survey and explore the genetic diversity of Bartonella spp. in domestic cats from Paraguay. A TaqMan real-time quantitative PCR (qPCR) targeting the nuoG gene (83 bp) for Bartonella spp. was used to screen 125 blood samples from cats in Asuncion, Paraguay. nuoG qPCR-positive samples were further submitted to conventional PCR assays based on the ITS (453- 717 bp), gltA (767 bp), ftsZ (515 bp), rpoB (333 bp), ribC (585-588 bp), and pap-31 (564 bp) loci. Positive samples were sequenced for species identification, phylogenetic, and haplotype analyses. Bartonella D.N.A. was present in 20.8% (26/125) cat blood samples, with low levels of Bartonella nuoG D.N.A. cPCR products targeting gltA, ftsZ, ITS, and rpoB loci from sixteen cats were successfully sequenced. However, all nouG qPCR-positive samples were negative for the ribC and pap-31 genes. Bartonella henselae [62.5% (10/16)] and Bartonella clarridgeiae [37.5% (6/16)] were identified among the sequenced samples. Upon phylogenetic analysis, B. henselae and B. clarridgeiae from Paraguay clustered with sequences detected in domestic and wild cats, dogs, and cat fleas worldwide. Two to four haplotypes of B. henselae and B. clarridgeiae in cats from Paraguay were observed, with some being exclusive and others shared with worldwide distributed haplotypes. Here, we report B. henselae and B. clarridgeiae for the first time in cats from Paraguay. Its circulation in cats suggests the need to consider Bartonellae when testing clinical samples from suspected infectious diseases in humans from Paraguay.

First Report of Pantoea ananatis causing leaf spot disease of maize in Ecuador.

Maize (Zea mays) is the second most cultivated grain crop in Ecuador, with growing significance as a source of fodder and food. During the rainy season (November and December) of 2018 and 2019, a disease of maize that was not previously observed in Ecuador was found at commercial fields of Misqui Sara variety, at four parishes of canton Quito (Tumbaco, Pifo, Puembo, and Checa), province of Pichincha. Infected plants, at tassel initiation, displayed symptoms of localized chlorotic streaks on leaves that expanded with time, and around a month later turned necrotic. Severely affected plants wilted and died. Symptoms appeared in lower leaves first and were later observed in upper leaves as the disease progressed. Disease incidence was between 20 and 30% in the affected plantations, with around 30% of infected plants wilting and dying, resulting in 20-25% of yield losses. Upper leaves from ten symptomatic plants, five from Puembo and five from Checa, were collected randomly. Two 0.5 cm2 pieces of leaf from each plant were excised from the margins of the necrotic lesions, surface sterilized and macerated in 9 mL of sterile peptone water. The 10-3 dilutions were plated onto nutrient agar and incubated at 28°C for 24 hours. Yellow, mucoid colonies were isolated on nutrient agar. Three isolates from Puembo and two from Checa were selected for testing Koch´s postulates and further biochemical and molecular characterization. Isolates were Gram-negative rods, oxidase negative, catalase, indol and citrate positive. Fragments of the 16S, gyrB, and rpoB loci were amplified and sequenced using the 27F/1492R (Lane, D. J., 1991), UP-1/UP-2r (Yamamoto & Harayama, 1995), and rpoBCM81-F/rpoBCM32b-R (Brady, C., et al., 2008) primer pairs, respectively. All isolates presented identical sequences for the different loci, therefore only sequences from isolate FP191505 were deposited in GenBank (GenBank accession no. MW528428-MW528430). A search of homologous sequences using BLAST resulted in identities of 99.3, 99.7, and 100 % for 16S, gyrB, and rpoB, respectively, with sequences from Pantoea ananatis type specimen LGM 2665 (Brady, C., et al., 2008; Hauben, L., et al., 1998; GenBank accession nos NR_119362.1, EF988824.1 EF988996.1), indicating that our isolates belong to this species. Pathogenicity tests were performed by syringe infiltration of bacterial suspensions. Each one of the five characterized P. ananatis isolates was inoculated in four leaves (500 ul of 1 x 108 CFU mL-1 per leave) of three healthy maize plants. Negative control plants were infiltrated with sterile distilled water. Plants were incubated at 28-30°C and 60% relative humidity for 24 hours. Later, plants were maintained in a greenhouse with 27°C/21°C day/night temperatures and observed daily. After six weeks all bacteria-inoculated plants developed symptoms of chlorosis and necrosis while the control was symptomless. Bacteria were re-isolated from symptomatic leaves and identified as P. ananatis following the same methodologies used for the initial identification. To our knowledge, this is the first report of P. ananatis causing leaf spot of maize in Ecuador.

Phylogenomic analysis of Haemophilus parasuis and proposed reclassification to Glaesserella parasuis, gen. nov., comb. nov.

The Gram-negative bacterium Haemophilus parasuis is the etiologic agent of Glässer's disease in pigs, and causes significant economic losses to the swine industry. This bacterium has been classified as a member of the family Pasteurellaceae in the genus Haemophilus, but phylogenetic relatedness has not been adequately examined to support this genus classification. Phenotypically, all 38 strains of H. parasuis tested were positive for catalase activity, oxidase activity, V-factor requirement, and acid formation from maltose and d-galactose without gas. All strains were negative for X-factor requirement, formation of indole from tryptophan, urease, l-arabinose, and α-glucosidase activity. To determine whether H. parasuis belongs to one of the current Pasteurellaceae genera 40 H. parasuis genomes, plus those of representative Pasteurellaceae, were subjected to phylogenetic analysis of concatenated, multi-protein alignments. Sequence variation at 16S rRNA and rpoB loci allowed the 15 reference serovars of H. parasuis to be integrated into the whole-genome tree. The phylogenetic analysis showed H. parasuis to be a distinct and tight clade whose sister taxon is the genus Bibersteinia. Within H. parasuis two clades were identified with individual serovars distributed between the two. As a result, H. parasuis was confirmed as a member of the family Pasteurellaceae, but was distinct from other genera in this family. Therefore, we propose the name Glaesserella parasuis, gen. nov., comb. nov. for bacterial strains currently classified as H. parasuis. The reference strain of this species is ATCC 19417 (1374)T, NCTC 4557T, DSM 21448T, CCUG 3712T.

A rapid and versatile tool for genomic engineering in Lactococcus lactis

BackgroundLactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Since last decade, L. lactis has been developed into cell factories for the production of bioactive compounds such as enzymes, vaccine antigens and natural products. However, its precise and efficient genome editing tools is still required to make L. lactis more suitable candidate for engineered functionality.ResultsA high active recombinase, RecT of Enterococcus faecalis ATCC14506, was selected from six candidates and mediated homologous recombination between single-stranded DNA (ssDNA) and the L. lactis chromosomal rpoB locus with an efficiency of 100% after rifampin selection. To screen mutants without an externally selectable phenotype, the CRISPR/Cas9 system was used for counterselection, yielding an upp mutant with an efficiency of 46%. By optimization of the copy number of plasmid carrying the CRISPR/Cas9 system and the length of spacer sequence, the off-target efficiency of the recA, galK, hemN and noxD genes were eliminated. The ability of this optimized tool to perform sequential point mutation was demonstrated using the upp and galK gene loci as targets with improved efficiencies > 75%. Moreover, seamless genomic DNA deletions (50/100 bp) or insertion (a loxP site, 34 bp) was efficiently accomplished within 72 h.ConclusionsThe work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection. This tool will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.

Evaluation of a Novel MALDI Biotyper Algorithm to Distinguish Mycobacterium intracellulare From Mycobacterium chimaera.

Accurate and timely mycobacterial species identification is imperative for successful diagnosis, treatment, and management of disease caused by nontuberculous mycobacteria (NTM). The current most widely utilized method for NTM species identification is Sanger sequencing of one or more genomic loci, followed by BLAST sequence analysis. MALDI-TOF MS offers a less expensive and increasingly accurate alternative to sequencing, but the commercially available assays used in clinical mycobacteriology cannot differentiate between Mycobacterium intracellulare and Mycobacterium chimaera, two closely related potentially pathogenic species of NTM that are members of the Mycobacterium avium complex (MAC). Because this differentiation of MAC species is challenging in a diagnostic setting, Bruker has developed an improved spectral interpretation algorithm to differentiate M. chimaera and M. intracellulare based on differential spectral peak signatures. Here, we utilize a set of 185 MAC isolates that have been characterized using rpoB locus sequencing followed by whole genome sequencing in some cases, to test the accuracy of the Bruker subtyper software to identify M. chimaera (n = 49) and M. intracellulare (n = 55). 100% of the M. intracellulare and 82% of the M. chimaera isolates were accurately identified using the MALDI Biotyper algorithm. This subtyper module is available with the MALDI Biotyper Compass software and offers a promising mechanism for rapid and inexpensive species determination for M. chimaera and M. intracellulare.

Robust Frankia phylogeny, species delineation and intraspecies diversity based on Multi-Locus Sequence Analysis (MLSA) and Single-Locus Strain Typing (SLST) adapted to a large sample size

Diazotrophic Actinobacteria of the genus Frankia represent a challenge to classical bacterial taxonomy as they include many unculturable strains. As a consequence, we still have a poor understanding of their diversity, evolution and biogeography. In this study, a Multi-Locus Sequence Analysis (MLSA) using atpD, dnaA, ftsZ, pgk, and rpoB loci was done on a large set of cultured and uncultured strains, compared to 16S rRNA and correlated to Average Nucleotide Identity (ANI) from available Frankia genomes. MLSA provided a robust resolution of Frankia genus phylogeny and clarified the status of unresolved species and complex of species.The robustness of single-gene topologies and their congruence with the MLSA tree were tested. Lateral Gene Transfers (LGT) were few and scattered, suggesting they had no impact on the concatenate topology. The pgk marker – providing the longest sequence, highest mean genetic divergence and least occurrence of LGT – was used to survey an unequalled number of Alnus-infective Frankia — mainly uncultured strains from a broad range of host-species and geographic origins. This marker allowed reliable Single-Locus Strain Typing (SLST) below the species level, revealed an undiscovered taxonomical diversity, and highlighted the effect of cultivation, sporulation phenotype and host plant species on symbiont richness, diversity and phylogeny.

Molecular Genetic Determination of MDR–TB Isolates from West Bengal, India

Tuberculosis (TB) is one of the leading causes of death from an infectious disease worldwide. India remains to be the country carrying highest burden of the disease. One of the potent problems of present days, in this field, is the emergence of Multi-Drug-Resistant Tuberculosis (MDR-TB). MDR is defined as resistance to isoniazid (INH) and rifampin (RIF). The spread of MDR-TB is one of the biggest challenges to global public health system. Thus, detection of MDR-TB strains is critically important for containment of global TB epidemics. In this study, we are determining the MDR incidence rate in the West Bengal state of India, which is one of the most TB prone areas of the world. The RIF-resistant mutations of Mycobacterium tuberculosis (MTB) were shown to map in the rpoB locus and INH-resistant mutations of MTB were shown to map in inhA and katG loci. Therefore, in the present study, we have detected MDR-TB strains by mapping rpoB, katG & inhA mutations. Our Line Probe Assay (LPA) based results from a vast pool of MTB organisms (carrying 3653 bonafide sputum positive patient isolates) indicated the presence of 14.37% MDR isolates which matches in general with the previously reported results from various parts of the world (approx. 12-16%). Therefore, we conclude that our MDR rate is generally comparable to that of the other investigations performed in the rest of the world. We also found that the male patients are more likely to contract the MDR strains than the female patients (1.97:1.0). This data also conforms to the global statistics.

Tuberculosis diagnosis after bleach processing for early stage tuberculosis laboratory capacity building [Short communication

The diagnosis of tuberculosis is seriously hampered in the absence of standard biosafety laboratory facilities for specimen concentration and Mycobacterium tuberculosis culture. Within a laboratory twinning arrangement, heat-fixed direct smear and sediment from 74 bleach-processed and 20 non-processed specimens from Cumura Hospital, Guinea-Bissau, were sent to Lisbon for molecular evaluation of rifampicin resistance. Sequence analysis of a 369 base-pair rpoB locus detected 3.2% (3/94) resistant specimens. To our knowledge, this represents the first report on the molecular analysis of M. tuberculosis from bleach-processed sputum, an alternative to current diagnostic practice in low-resource settings.

Evolutionary dynamics of separate and combined exposure of Pseudomonas fluorescens SBW25 to antibiotics and bacteriophage

The use of bacteriophages against pathogenic bacteria in health care and in the food industry is now being advocated as an alternative to the use of antibiotics. But what is the evolutionary response for a bacterial population if both antibiotics and phages are used in combination? We employ an experimental evolution approach to address these questions and exposed Pseudomonas fluorescens SBW25 and a related hypermutator strain (mutS−) to the action of the antibiotic rifampicin and the lytic bacteriophage SBW25ϕ2. We then compared the densities, growth rates, and the mutations at the rpoB locus leading to rifampicin resistance of the evolved bacterial populations. We observed that the evolutionary response of populations under different treatments varied depending on the order in which the antimicrobials were added and whether the bacterium was a hypermutator. We found that wild-type rifampicin-resistant populations involved in biofilm formation often reverted to rifampicin sensitivity when stresses were added sequentially. In contrast, when the mortality agents were added simultaneously, phage populations frequently went extinct and the bacteria evolved antibiotic resistance. However, populations of the hypermutator mutS− converged to a single genotype at the rpoB locus. Future investigation on other bacteria and using different antibiotics and bacteriophage are needed to evaluate the generality of our findings.

Bioinformatics Describes Novel Loci for High Resolution Discrimination of Leptospira Isolates

BackgroundLeptospirosis is one of the most widespread zoonoses in the world and with over 260 pathogenic serovars there is an urgent need for a molecular system of classification. The development of multilocus sequence typing (MLST) schemes for Leptospira spp. is addressing this issue. The aim of this study was to identify loci with potential to enhance Leptospira strain discrimination by sequencing-based methods.Methodology and Principal FindingsWe used bioinformatics to evaluate pre-existing loci with the potential to increase the discrimination of outbreak strains. Previously deposited sequence data were evaluated by phylogenetic analyses using either single or concatenated sequences. We identified and evaluated the applicability of the ligB, secY, rpoB and lipL41 loci, individually and in combination, to discriminate between 38 pathogenic Leptospira strains and to cluster them according to the species they belonged to. Pairwise identity among the loci ranged from 82.0–92.0%, while interspecies identity was 97.7–98.5%. Using the ligB-secY-rpoB-lipL41 superlocus it was possible to discriminate 34/38 strains, which belong to six pathogenic Leptospira species. In addition, the sequences were concatenated with the superloci from 16 sequence types from a previous MLST scheme employed to study the association of a leptospiral clone with an outbreak of human leptospirosis in Thailand. Their use enhanced the discriminative power of the existing scheme. The lipL41 and rpoB loci raised the resolution from 81.0–100%, but the enhanced scheme still remains limited to the L. interrogans and L. kirschneri species.ConclusionsAs the first aim of our study, the ligB-secY-rpoB-lipL41 superlocus demonstrated a satisfactory level of discrimination among the strains evaluated. Second, the inclusion of the rpoB and lipL41 loci to a MLST scheme provided high resolution for discrimination of strains within L. interrogans and L. kirschneri and might be useful in future epidemiological studies.

Au-nanoprobes for detection of SNPs associated with antibiotic resistance inMycobacterium tuberculosis

Tuberculosis (TB) is one of the leading causes of infection in humans, causing highmorbility and mortality all over the world. The rate of new cases of multidrug resistanttuberculosis (MDRTB) continues to increase, and since these infections are very difficult tomanage, they constitute a serious health problem. In most cases, drug resistance inMycobacterium tuberculosis has been related to mutations in several loci withinthe pathogen’s genome. The development of fast, cheap and simple screeningmethodologies would be of paramount relevance for the early detection of these mutations,essential for the timely and effective diagnosis and management of MDRTB patients.The use of gold nanoparticles derivatized with thiol-modified oligonucleotides(Au-nanoprobes) has led to new approaches in molecular diagnostics. Based on thedifferential non-cross-linking aggregation of Au-nanoprobes, we were able to develop acolorimetric method for the detection of specific sequences and to apply this approach topathogen identification and single base mutations/single nucleotide polymorphisms (SNP)discrimination.Here we report on the development of Au-nanoprobes for the specific identification of SNPswithin the beta subunit of the RNA polymerase (rpoB locus), responsible forresistance to rifampicin in over 95% of rifampicin resistant M. tuberculosis strains.

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

RNA polymerase (rpoB) mutants selected for increased resistance to gyrase inhibitors in Salmonella typhimurium.

Some rifampicin-resistance (RifR) mutations make bacteria slightly resistant to the gyrase inhibitors novobiocin (Nov) and nalidixic acid (Nal). This suggested that it might be possible to isolate rpoB mutants using either drug for positive selection. In an initial test, we confirmed the presence of Rif-resistant isolates among clones selected for Nov resistance. These mutants are also more resistant to Nal. In a subsequent experiment, we found that mutants selected for low-level resistance to Nal include isolates harboring mutations genetically linked to the rpoB locus; of two such mutants studied, one is temperature-sensitive for growth. These two mutants, which are only marginally affected in their response to Nov, are normally sensitive to Rif and thus might be representative of a new class of rpoB alleles. The Rif-resistant and Rif-sensitive rpoB alleles that increase resistance to gyrase inhibitors have one property in common: they all suppress, to varying degrees, the defect in his operon regulation (transcriptional deattenuation) caused by a gyrase defect or inhibition by novobiocin. To further analyse the transcription-supercoiling relationships in these mutants, we examined the ability of RNA polymerase to recruit gyrase activity during transcription. This was done by two independent approaches: (i) observing transcription-induced accumulation of hyper-negatively supercoiled plasmid DNA in a topA mutant background and (ii) measuring transcription-induced plasmid DNA cleavage in the presence of oxolinic acid. Results indicate that the rpoB alleles described in this study diminish the recruitment of gyrase activity by the transcription process. This property correlates with a decrease in the rate of transcription initiation.(ABSTRACT TRUNCATED AT 250 WORDS)

TheEuglena gracilischloroplastrpoBgene. Novel gene organization and transcription of the RNA polymerase subunit operon

The rpoB gene coding for a beta-like subunit of the chloroplast DNA-dependent RNA polymerase has been located on the chloroplast genome of Euglena gracilis distal to the rrnC ribosomal RNA operon. We have determined 5760 base-pairs of DNA sequence, including 97 bp of the 5S rRNA gene, an intergenic spacer of 1264 bp, the rpoB gene of 4249 bp, 84 bp spacer and 67 bp of the rpoC1 gene. The rpoB gene is of the same polarity as the rRNA operons. The organization of the rpoB and rpoC genes resembles the E. coli rpoB-rpoC and higher plant chloroplast rpoB-rpoC1-rpoC2 operons. The Euglena rpoB gene (1082 codons) encodes a polypeptide with a predicted molecular weight of 124,288. The rpoB gene is interrupted by seven Group III introns of 93, 95, 94, 99, 101, 110 and 99 bp respectively and a Group II intron of 309 bp. All other known rpoB genes lack introns. All the exon-exon junctions were experimentally determined by cDNA cloning and sequencing or direct primer extension RNA sequencing. Transcripts from the rpoB locus were characterized by Northern hybridization. Fully-spliced, monocistronic rpoB mRNA, as well as rpoB-rpoC1 and rpoB1-rpoC1-rpoC2 mRNAs were identified.