Evidence for a role of rpoE in stressed and unstressed cells of marine Vibrio angustum strain S14

We report the cloning, sequencing, and characterization of the rpoE homolog in Vibrio angustum S14. The rpoE gene encodes a protein with a predicted molecular mass of 19.4 kDa and has been demonstrated to be present as a single-copy gene by Southern blot analysis. The deduced amino acid sequence of RpoE is most similar to that of the RpoE homolog of Sphingomonas aromaticivorans, sigma(24), displaying sequence similarity and identity of 63 and 43%, respectively. Northern blot analysis demonstrated the induction of rpoE 6, 12, and 40 min after a temperature shift to 40 degrees C. An rpoE mutant was constructed by gene disruption. There was no difference in viability during logarithmic growth, stationary phase, or carbon starvation between the wild type and the rpoE mutant strain. In contrast, survival of the mutant was impaired following heat shock during exponential growth, as well as after oxidative stress at 24 h of carbon starvation. The mutant exhibited microcolony formation during optimal growth temperatures (22 to 30 degrees C), and cell area measurements revealed an increase in cell volume of the mutant during growth at 30 degrees C, compared to the wild-type strain. Moreover, outer membrane and periplasmic space protein analysis demonstrated many alterations in the protein profiles for the mutant during growth and carbon starvation, as well as following oxidative stress, in comparison with the wild-type strain. It is thereby concluded that RpoE has an extracytoplasmic function and mediates a range of specific responses in stressed as well as unstressed cells of V. angustum S14.     

A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response sigma(E) in Escherichia coli

We have previously established a two-plasmid system in Escherichia coli for identification of promoters recognized by RNA polymerase containing a heterologous sigma factor. Attempts to optimize this system for identification of promoters recognized by RNA polymerase containing E. coli extracytoplasmic stress response sigma(E) failed owing to high toxicity of the expressed rpoE. A new system for identification of sigma(E)-cognate promoters was established, and verified using the two known sigma(E)-dependent promoters, rpoEp2 and degPp. Expression of the sigma(E)-encoding rpoE gene was under the control of the AraC-dependent P(BAD) promoter. A low level of arabinose induced a non-toxic, however, sufficient level of sigma(E) to interact with the core enzyme of RNA polymerase. Such an RNA polymerase holoenzyme recognized both known sigma(E)-dependent promoters, rpoEp2 and degPp, which were cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZ alpha reporter gene. This new system has proved to be useful for identification of E. coli sigma(E)-cognate promoters. Moreover, the system could be used for identification of ECF sigma-cognate promoters from other bacteria.     

Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1.

A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter.     

Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction.

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.     

An rpoE-like locus controls outer membrane protein synthesis and growth at cold temperatures and high pressures in the deep-sea bacterium Photobacterium sp. strain SS9

Many deep-sea bacteria have evolved specialized adaptations for life at cold temperatures and high pressures. A locus required for both psychro- and baro- adaptation in the psychrophilic, moderate barophile, Photobacterium species strain SS9 was identified among SS9 transposon mutants. DNA sequence analysis of this locus identified four complete open reading frames (ORFs), which appear to comprise an operon, and a fifth incomplete ORF. All transposon insertions isolated are in ORF3. Extensive sequence similarity exists between the translation products of ORFs 1–3 and a collection of gene products proposed to include alternative RNA polymerase sigma factors and modifiers of sigma factors activity involved in extracytoplasmic sensing and regulation. Based on the similarity between ORF1 and Escherichia coli rpoE , we have tentatively designated this locus the rpoE locus. SS9 rpoE locus ORF3 insertion mutants showed altered abundances of numerous outer membrane proteins and were both baro- and psychro- sensitive. ORF3 mutant revertants that displayed enhanced high-pressure growth also displayed concomitant enhanced low-temperature growth. Most of these revertants possessed DNA rearrangements at the site of the transposon insertion, further demonstrating the importance of the rpoE locus to high-pressure and cold-temperature growth. Complementation analyses indicated that ORF3 functions in OMP synthesis regulation while ORF4 is required for baro- and psychro-adaptation.     

Cloning and overproduction of the rpoZ gene encoding an RNA polymerase omega subunit from a deep-sea piezophilic Shewanella violacea strain DSS12.

We have cloned the rpoZ gene, encoding RNA polymerase omega protein, by PCR approach from the deep-sea piezophilic and psychrophilic bacterium, Shewanella violacea strain DSS12. The cloned gene, 285bp in length, was found to encode a protein consisting of 94 amino acid residues with a molecular mass of 10,327 Da. Significant homology was evident comparing the RpoZ protein of S. violacea with that of Shewanella oneidensis (69% identity), Vibrio cholerae (65% identity), Escherichia coli K-12 (64% identity) and Haemophilus influenzae (61% identity). From the Northern blot analysis, S. violacea rpoZ gene was expressed constitutively under pressure conditions of 0.1, 30 and 50MPa. We constructed expression plasmid to overproduce the RpoZ protein and transformed into E. coli JM109 as a host of overproduction. Upon induction, the recombinant protein encoded by plasmid pQrpoZ was overexpressed and purified using Ni2+ affinity column.     

Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III.

Saccharomyces cerevisiae cdc2 mutants arrest in the S-phase of the cell cycle when grown at the non-permissive temperature, implicating this gene product as essential for DNA synthesis. The CDC2 gene has been cloned from a yeast genomic library in vector YEp13 by complementation of a cdc2 mutation. An open reading frame coding for a 1093 amino acid long protein with a calculated mol. wt of 124,518 was determined from the sequence. This putative protein shows significant homology with a class of eukaryotic DNA polymerases exemplified by human DNA polymerase alpha and herpes simplex virus DNA polymerase. Fractionation of extracts from cdc2 strains showed that these mutants lacked both the polymerase and proofreading 3'-5' exonuclease activity of DNA polymerase III, the yeast analog of mammalian DNA polymerase delta. These studies indicate that DNA polymerase III is an essential component of the DNA replication machinery.     

Identification of the gene and the protein of RNA polymerase II subunit 9 (Rpb9) from the fission yeast Schizosaccharomyces pombe

Both the rpb9 gene and its cDNA encoding the subunit 9 of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe. From the DNA sequences, Rpb9 was predicted to consist of 113 amino acid residues with a molecular mass of 13 175. S. pombe Rpb9 is 47, 40 and 36% identical in amino acid sequence to the corresponding subunits from Saccharomyces cerevisiae, human and Drosophila melanogaster, respectively. Previously, we failed to detect Rpb9 in the purified RNA polymerase II by amino-terminal micro-sequencing of proteolytic fragments of subunits separated by SDS-gel electrophoresis. After Western blot analysis using antibodies raised against the protein product of the newly isolated rpb9 gene, we found that the purified RNA polymerase II contains Rpb9.     

Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging.

A duck hepatitis B virus (DHBV) genome cloned from a domestic duck from the People's Republic of China has been sequenced and exhibits no variation in sequences known to be important in viral replication or generation of gene products. Intrahepatic transfection of a dimer of this viral genome into ducklings did not result in viremia or any sign of virus infection, indicating that the genome was defective. Functional analysis of this mutant genome, performed by transfecting the DNA into a chicken hepatoma cell line capable of replicating wild-type virus, indicated that viral RNA is not encapsidated. However, virus core protein is made and can assemble into particles in the absence of encapsidation of viral nucleic acid. Using genetic approaches, it was determined that a change of cysteine to tyrosine in position 711 in the polymerase (P) gene C terminus led to this RNA-packaging defect. By site-directed mutagenesis, it was found that while substitution of Cys-711 with tryptophan also abolished packaging, substitution with methionine did not affect packaging or viral replication. Therefore, Cys-711, which is conserved in all published sequences of DHBV, may not be involved in a disulfide bridge structure essential to viral RNA packaging or replication. Our results, showing that a missense mutation in the region of the DHBV polymerase protein thought to be primarily the RNase H domain results in packaging deficiency, support the previous findings that multiple regions of the complex hepadnaviral polymerase protein may be required for viral RNA packaging.     

RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth.

RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae. The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit. The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons. The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase. Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures. RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro. The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range.