Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis

6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.     

6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter

6S RNA is an abundant noncoding RNA in Escherichia coli that binds to sigma70 RNA polymerase holoenzyme to globally regulate gene expression in response to the shift from exponential growth to stationary phase. We have computationally identified >100 new 6S RNA homologs in diverse eubacterial lineages. Two abundant Bacillus subtilis RNAs of unknown function (BsrA and BsrB) and cyanobacterial 6Sa RNAs are now recognized as 6S homologs. Structural probing of E. coli 6S RNA and a B. subtilis homolog supports a common secondary structure derived from comparative sequence analysis. The conserved features of 6S RNA suggest that it binds RNA polymerase by mimicking the structure of DNA template in an open promoter complex. Interestingly, the two B. subtilis 6S RNAs are discoordinately expressed during growth, and many proteobacterial 6S RNAs could be cotranscribed with downstream homologs of the E. coli ygfA gene encoding a putative methenyltetrahydrofolate synthetase. The prevalence and robust expression of 6S RNAs emphasize their critical role in bacterial adaptation.     

Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes.

The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.     

Complementation of an RNase P RNA (rnpB) gene deletion in Escherichia coli by homologous genes from distantly related eubacteria.

We report the construction of a strain of Escherichia coli in which the only functional gene for the RNA moiety of RNase P (rnpB) resides on a plasmid that is temperature sensitive for replication. The chromosomal RNase P RNA gene was replaced with a chloramphenicol acetyltransferase gene. The conditionally lethal phenotype of this strain was suppressed by plasmids that carry RNase P RNA genes from some distantly related eubacteria, including Alcaligenes eutrophus, Bacillus subtilis, and Chromatium vinosum. Thus, the rnpB genes from these organisms are capable of functioning as the sole source of RNase P RNA in E. coli. The rnpB genes of some other organisms (Agrobacterium tumefaciens, Pseudomonas fluorescens, Bacillus brevis, Bacillus megaterium, and Bacillus stearothermophilus) could not replace the E. coli gene. The significance of these findings as they relate to RNase P RNA structure and function and the utility of the described strain for genetic studies are discussed.     

Molecular analysis of RNA polymerase alpha subunit gene from Streptomyces coelicolor A3(2).

The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.     

Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.     

Pantothenate transport in Escherichia coli.

The function of the stable 6S RNA of Escherichia coli is not known. Recently, it was proposed that the 6S RNA is a component of a bacterial signal recognition particle required for protein secretion. To test this proposal, we isolated a mutant that lacks the 6S RNA. Studies of the mutant show that the 6S RNA is not essential for growth or for protein secretion. The gene for the 6S RNA (ssr) maps near serA at 63 min on the E. coli genetic map.     

Novel hemoglobins to enhance microaerobic growth and substrate utilization in Escherichia coli

Limited oxygen availability is a prevalent problem in microbial biotechnology. Recombinant Escherichia coli expressing the hemoglobin from Vitreoscilla (VHb) or the flavohemoglobin from Ralstonia eutropha (formerly Alcaligenes eutrophus) (FHP) demonstrate significantly increased cell growth and productivity under microaerobic conditions. We identify novel bacterial hemoglobin-like proteins and examine if these novel bacterial hemoglobins can elicit positive effects similar to VHb and FHP and if these hemoglobins alleviate oxygen limitation under microaerobic conditions when expressed in E. coli. Several finished and unfinished bacterial genomes were screened using R. eutropha FHP as a query sequence for genes (hmp) encoding hemoglobin-like proteins. Novel hmp genes were identified in Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae, Deinococcus radiodurans, and Campylobacter jejuni. Previously characterized hmp genes from E. coli and Bacillus subtilis and the novel hmp genes from P. aeruginosa, S. typhi, C. jejuni, K. pneumoniae, and D. radiodurans were PCR amplified and introduced into a plasmid for expression in E. coli. Biochemically active hemoproteins were expressed in all constructs, as judged by the ability to abduct carbon monoxide. Growth behavior and byproduct formation of E. coli K-12 MG1655 cells expressing various hemoglobins were analyzed in microaerobic fed-batch cultivations and compared to plasmid-bearing control and to E. coli cells expressing VHb. The clones expressing hemoglobins from E. coli, D. radiodurans, P.aeruginosa, and S. typhi reached approximately 10%, 27%, 23%, and 36% higher final optical density values, respectively, relative to the plasmid bearing E. coli control (A(600) 5.5). E. coli cells expressing hemoproteins from P. aeruginosa, S. typhi, and D. radiodurans grew to similar final cell densities as did the strain expressing VHb (A(600) 7.5), although none of the novel constructs was able to outgrow the VHb-expressing E. coli strain. Additionally, increased yield of biomass on glucose was measured for all recombinant strains, and an approximately 2-fold yield enhancement was obtained with D. radiodurans hemoprotein-expressing E. coli relative to the E. coli control carrying the parental plasmid without any hemoglobin gene.     

Refined molecular weights for phage, viral and ribosomal RNA.

The RNAs of the Escherichia coli bacteriophages MS2 and Qbeta as well as E. coli 16S ribosomal RNA were examined under identical conditions by electron microscopy using the protein-free benzyldimethylalkylammonium chloride (BAC) spreading technique. From the contour length ratios of the RNAs and the known number of nucleotides for MS2, the chain lengths for Qbeta RNA and 16S RNA were found to be 4790 +/- 150 and 1645 +/- 55 nucleotides. Correcting for the base composition of Qbeta RNA the molecular weight of the Na salt of this RNA is (1.64 +/- 0.06) . 10(6) daltons. Since published values on the relative lengths of Qbeta RNA and several other homogeneous RNAs (E. coli 23S rRNA, E. Coli bacteriophage R17 and f2 RNAs, Pseudomonas aeruginosa phage PP7 RNA and Newcastle disease virus RNA) are available, we are able to calculate the approximate number of nucleotides for these useful standards.     

Small cytoplasmic RNA of Bacillus subtilis: functional relationship with human signal recognition particle 7S RNA and Escherichia coli 4.5S RNA.

Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant and stable RNA of the gram-positive bacterium Bacillus subtilis. To investigate the function of scRNA in B. subtilis cells, we developed a strain that is dependent on isopropyl-beta-D-thiogalactopyranoside for scRNA synthesis by fusing the chromosomal scr locus with the spac-1 promoter by homologous recombination. Depletion of the inducer leads to a loss of scRNA synthesis, defects in protein synthesis and production of alpha-amylase and beta-lactamase, and eventual cell death. The loss of the scRNA gene in B. subtilis can be complemented by the introduction of human signal recognition particle 7S RNA, which is considered to be involved in protein transport, or Escherichia coli 4.5S RNA. These results provide further evidence for a functional relationship between B. subtilis scRNA, human signal recognition particle 7S RNA, and E. coli 4.5S RNA.     

Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster.

We isolated the gene encoding the alpha subunit of Bacillus subtilis RNA polymerase from a lambda gt11 expression vector library by using anti-alpha antibody as a probe. Four unique clones were isolated, one carrying a lacZ-alpha gene fusion and three carrying the entire alpha coding region together with additional sequences upstream. The identity of the cloned alpha gene was confirmed by the size and immunological reactivity of its product expressed in Escherichia coli. Further, a partial DNA sequence found the predicted NH2 terminus of alpha homologous with E. coli alpha. By plasmid integration and PBS1 transduction, we mapped alpha near rpsE and within the major ribosomal protein gene cluster on the B. subtilis chromosome. Additional DNA sequencing identified rpsM (encoding S13) and rpsK (encoding S11) upstream of alpha, followed by a 180-base-pair intercistronic region that may contain two alpha promoters. Although the organization of the alpha region resembles that of the alpha operon of E. coli, the putative promoters and absence of rpsD (encoding S4) immediately preceding the B. subtilis alpha gene suggest a different regulation.     

Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis.

We describe the cloning and sequence analysis of the region surrounding the gene for the alpha subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, alpha, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial alpha subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among alpha subunits from distantly related genera.     

Secretion by Pseudomonas aeruginosa: fate of a cloned gram-positive lipoprotein deletion mutant.

In gram-positive organisms, glyceride-cysteine thioether lipoproteins are frequently associated with secretion. They constitute membrane-bound forms retained by the cell but releasable late in growth phase. Most gram-negative organisms secrete very few proteins to the culture fluid; thioether lipoproteins in such organisms, typified by the enteric bacterium Escherichia coli, are integral outer membrane components for the most part. Unusual among gram-negative organisms, however, are Pseudomonas strains, known for extracellular export of a number of proteins. To examine whether a fundamental difference exists between the processing of lipoproteins in Pseudomonas strains and in nonsecretory gram-negative organisms, we examined the fate in Pseudomonas aeruginosa and E. coli of a cloned gram-positive secretory lipoprotein, Bacillus licheniformis penicillinase. A nonlipoprotein deletion mutant of the same gene was also examined in P. aeruginosa, and its processing was compared with that in E. coli. No important differences were found between P. aeruginosa and E. coli for either the lipoprotein or its deletion mutant. Thus, the contrast in secretory abilities of the two organisms does not appear to result from a difference in their general secretory systems.     

Analysis of cell division gene ftsZ (sulB) from gram-negative and gram-positive bacteria.

The ftsZ (sulB) gene of Escherichia coli codes for a 40,000-dalton protein that carries out a key step in the cell division pathway. The presence of an ftsZ gene protein in other bacterial species was examined by a combination of Southern blot and Western blot analyses. Southern blot analysis of genomic restriction digests revealed that many bacteria, including species from six members of the family Enterobacteriaceae and from Pseudomonas aeruginosa and Agrobacterium tumefaciens, contained sequences which hybridized with an E. coli ftsZ probe. Genomic DNA from more distantly related bacteria, including Bacillus subtilis, Branhamella catarrhalis, Micrococcus luteus, and Staphylococcus aureus, did not hybridize under minimally stringent conditions. Western blot analysis, with anti-E. coli FtsZ antiserum, revealed that all bacterial species examined contained a major immunoreactive band. Several of the Enterobacteriaceae were transformed with a multicopy plasmid encoding the E. coli ftsZ gene. These transformed strains, Shigella sonnei, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes, were shown to overproduce the FtsZ protein and to produce minicells. Analysis of [35S]methionine-labeled minicells revealed that the plasmid-encoded gene products were the major labeled species. This demonstrated that the E. coli ftsZ gene could function in other bacterial species to induce minicells and that these minicells could be used to analyze plasmid-endoced gene products.     

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph of Escherichia coli. The gene was highly expressed in E. coli due to the use of the high-copy number vector pUC18. The P. aeruginosa cyclohexadienyl dehydratase expressed in E. coli was purified to electrophoretic homogeneity. The latter enzyme exhibited identical physical and biochemical properties as those obtained for cyclohexadienyl dehydratase purified from P. aeruginosa. The activity ratios of prephenate dehydratase to arogenate dehydratase remained constant (about 3.3-fold) throughout purification, thus demonstrating a single protein having broad substrate specificity. The cyclohexadienyl dehydratase exhibited Km values of 0.42 mM for prephenate and 0.22 mM for L-arogenate, respectively. The pheC gene was 807 base pairs in length, encoding a protein with a calculated molecular mass of 30,480 daltons. This compares with a molecular mass value of 29.5 kDa determined for the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the native molecular mass determined by gel filtration was 72 kDa, the enzyme probably is a homodimer. Comparison of the deduced amino acid sequence of pheC from P. aeruginosa with those of the prephenate dehydratases of Corynebacterium glutamicum, Bacillus subtilis, E. coli, and Pseudomonas stutzeri by standard pairwise alignments did not establish obvious homology. However, a more detailed analysis revealed a conserved motif (containing a threonine residue known to be essential for catalysis) that was shared by all of the dehydratase proteins.