Characteristics of σ-dependent pausing by RNA polymerases from Escherichia coli and Thermus aquaticus.

The σ(70) subunit of RNA polymerase (RNAP) is the major transcription initiation factor in Escherichia coli. During transcription initiation, conserved region 2 of the σ(70) subunit interacts with the -10 promoter element and plays a key role in DNA melting around the starting point of transcription. During transcription elongation, the σ(70) subunit can induce pauses in RNA synthesis owing to interactions of region 2 with DNA regions similar to the -10 promoter element. We demonstrated that the major σ subunit from Thermus aquaticus (σ(A)) is also able to induce transcription pausing by T. aquaticus RNAP. However, hybrid RNAP containing the σ(A) subunit and E. coli core RNAP is unable to form pauses during elongation, while being able to recognize promoters and initiate transcription. Inability of the σ(A) subunit to induce pausing by E. coli RNAP is explained by the substitutions of non-conserved amino acids in region 2, in the subregions interacting with the RNAP core enzyme. Thus, changes in the structure of region 2 of the σ(70) subunit have stronger effects on transcription pausing than on promoter recognition, likely by weakening the interactions of the σ subunit with the core RNAP during transcription elongation.

Lineage-specific amino acid substitutions in region 2 of the RNA polymerase sigma subunit affect the temperature of promoter opening.

Highly conserved amino acid residues in region 2 of the RNA polymerase sigma subunit are known to participate in promoter recognition and opening. We demonstrated that nonconserved residues in this region collectively determine lineage-specific differences in the temperature of promoter opening.

[Quorum sensing systems of regulation, synthesis of phenazine antibiotics, and antifungal (corrected) activity in rhizospheric bacterium Pseudomonas chlororaphis 449].

Strain Pseudomonas chlororaphis 449, an antagonist of a broad spectrum of phytopathogenic microorganisms isolated from the maize rhizosphere, was shown to produce three phenazine antibiotics: phenazine-1-carboxylic acid (PCA), 2-hydroxylphenazine-1-carboxylic acid (2-OH-PCA), and 2-hydroxylphenazine (2-OH-PHZ). Two Quorum Sensing (QS) systems of regulation were identified: PhzIR and CsaI/R. Genes phzI and csaI were cloned and sequenced. Cells of strain 449 synthesize at least three types of AHL: N-butanoyl-L-homoserine lactone (C4-AHL), N-hexanoyl-L-homoserine lactone (C6-AHL), and N-(3-oxo-hexanoyl)-L-homoserine lactone (30C6-AHL). Transposon mutagenesis was used to generate mutants of strain 449 deficient in synthesis of phenazines, which carried inactivated phzA and phzB genes of the phenazine operon and gene phzO. Mutations phzA- and phzB-caused a drastic reduction in the antagonistic activity of bacteria toward phytopathogenic fungi. Both mutants lost the ability to protect cucumber and leguminous plants against phytopathogenic fungi Rhizoctonia solani and Sclerotinia sclerotiorum. These results suggest a significant role of phenazines in the antagonistic activity of P. chlororaphis 449.

[Suggested interrelationships of RNA-polymerase sigma S subunit and nitrogen control system in Pseudomonas chlororaphis].

The effect of mutation in rpoS gene encoding sigma S subunit of RNA-polymerase on the capacity of Pseudomonas chlororaphis 449 to assimilate nitrogen was investigated. It has been shown that mutant cells with knocked-out rpoS gene had significantly lower capacity to utilize the nitrogen sources such as alanine, proline, histidine, arginine, urea, and ammonium and glutamine synthetase was downregulated in their cell free extracts. Both defects were abolished by glutamine supplementation to the medium. It is suggested that in Pseudomonas chlororaphis the association of the nitrogen control system and the system of gene expression is regulated by RNA-polymerase sigma S subunit, which can be responsible for cell adaptation at nitrogen supply limitation.

[Origin, evolution, and migration of drug resistance genes].

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic producers, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.

Activity of Serratia plymuthica IC1270 gene chiA promoter region in Escherichia coli mutants deficient in global regulators of transcription.

To study the regulation of expression of the Serratia plymuthica gene chiA encoding a 58-kDa endochitinase, its 586-bp-long upstream regulatory region was cloned, sequenced and fused to a promoterless lac operon in phage lambdaRS45 to obtain a single-copy transcriptional fusion (P F1chiA )-lac in lysogens of Escherichia coli wild-type strains or their mutants deficient in various global regulators of transcription. The level of P F1chiA -lac expression increased about 20- and 90-fold, respectively, in E. coli K12 Deltahns and double Deltahns stpA mutants deficient in H-NS, and in both H-NS and StpA DNA-binding histone-like proteins, as compared to levels in the wild-type strain. In a Deltalrp mutant deficient in the leucine-responsive transcriptional regulator Lrp, the level of P F1chiA -lac expression increased only up to threefold, whereas even smaller differences relative to the wild-type strain were observed in rpoS and Deltacrp mutants deficient in the sigmaS subunit of RNA polymerase and catabolite-repression protein (CRP), respectively. Deletion of the inverted-repeat sequences and curved DNA regions located in the upstream region of chiA essentially did not influence strain IC1270's chiA promoter activity in E. coli .

Different rifampin sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA polymerases are not explained by the difference in the beta-subunit rifampin regions I and II.

Mycobacterium tuberculosis RNA polymerase is 1,000-fold more sensitive to rifampin than Escherichia coli RNA polymerase. Chimeric E. coli RNA polymerase in which the beta-subunit segment encompassing rifampin regions I and II (amino acids [aa] 463 through 590) was replaced with the corresponding region from M. tuberculosis (aa 382 through 509) did not show an increased sensitivity to the antibiotic. Thus, the difference in amino acid sequence between the rifampin regions I and II of the two species does not account for the difference in rifampin sensitivity of the two polymerases.

[Activation of the expression of the microcin C51 operon upon glucose starvation of cells at the exponential growth phase].

It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the sigmaS subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon (P(mcc)-lac), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P(mcc)-lac expression upon severe glucose starvation occurred in rpoS+ and rpoS- strains. In cells carrying the rpoD800 mutation that renders the sigma70 subunit of RNA polymerase temperature-sensitive, an activation of P(mcc)-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors-nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol-did not activate P(mcc)-lac expression in cells at the exponential growth phase.

[Involvement of sigma S and sigma 70 subunits of RNA polymerase and the CRP protein in the regulation of microcin C51 operon expression]. / Uchastie sigmy S, sigmy 70 sub''edinits RNK-polimerazy i belka CRP v reguliatsii ékspressii operona mikrotsina C51.

Expression of the microcin C51 operon in Escherichia coli cells is activated during cell entry into the stationary growth phase and depends on the sigmaS subunit of RNA polymerase (RpoS). The null rpoS mutations retained the residual expression level of the transcriptional P(mcc)-lac fusion, which indicates that other sigma subunit can participate in the regulation of transcription of the microcin C51 operon. Data presented in this work show that the overproduction of sigma70 in rpoS- cells diminished the level of P(mcc)-lac expression, as in wild-type cells, which seems to be the consequence of competition between sigma factors for a limited number of core RNA polymerase molecules. In the presence of the rpoD800 mutation that renders sigma70 temperature-sensitive, expression of P(mcc)-lac was not induced in the phase of delayed culture growth at nonpermissive temperature, which indicates that sigma70 is indispensable for microcin operon expression. Point substitutions in the -10 P(mcc) region, leading to the formation of 5'-TGaTATAAT-3' site, enhanced promoter activity but did not affect the relationship between P(mcc)-lac transcription and growth phase, sigmaS, and the activator protein CRP. The activator protein CRP was shown to bind a DNA fragment containing the TGTGA(AATGAA)TCTAT site in the -59.5 bp position relative to the start site of transcription. Mutation in the ssrI gene encoding 6S RNA did not disturb P9mcc)-lac expression; these results indicate that 6S RNA does not participate in the regulation of microcin C51 operon expression.

Cold sensitivity of thermophilic and mesophilic RNA polymerases.

RNA polymerase from mesophilic Deinococcus radiodurans displays the same cold sensitivity of promoter opening as RNA polymerase from the closely related thermophilic Thermus aquaticus. This suggests that, contrary to the accepted view, cold sensitivity of promoter opening by thermophilic RNA polymerases may not be a consequence of their thermostability.

[Distribution of transposons Tn5044 and Tn5070 with unusual mer operons in environmental bacterial populations]. / Raspostranenie v prirodnykh populiatsiiakh bacterii transpozonov Tn5044 i Tn5070, nesushchikh nekanonicheskie mer-operony.

The distribution of unusual mercury resistance transposons, Tn5044 and Tn5070, was examined. A characteristic feature of Tn5044 is temperature sensitivity of its mercury operon and the presence in the mer operon of the gene homologous to RNA polymerase a subunit. Structural organization of mercury operon Tn5070, containing minimum gene set (merRTPA), differs from mer operons of both Gram-negative and Gram-positive bacteria. None of more than two thousand environmental bacterial strains displaying mercury resistance and isolated from the samples selected from different geographical regions hybridized to Tn5040- and Tn5070-specific probes. A concept on the existence of cosmopolite, endemic, and rare transposons in environmental bacterial populations was formulated.

[Horizontal transfer of mercury resistance genes in natural bacterial populations]. / Gorizontal'nyi perenos genov ustoichivosti k soedineniiam rtuti v prirodnykh populiatsiiakh bakterii.

The results of studying the horizontal transfer of mercury resistance determinants in environmental bacterial populations are reviewed. Identical or highly homologous mercury resistance (mer) operons and transposons were found in bacteria of different taxonomic groups from geographically distant regions. Recombinant mer operons and transposons were revealed. The data suggest high frequencies of horizontal transfer and of recombination for mercury resistance determinants. The mechanisms of horizontal gene transfer were elucidated in Gram-negative and Gram-positive bacteria. New transposons were found and analyzed.

[Tn5037-a Tn21-like mercury transposon, detected in Thiobacillus ferrooxidans]. / Tn5037-Tn21-podobnyi rtutnyi transpozon, obnaruzhennyi u Thiobacillus ferrooxidans.

The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidans was cloned and sequenced. This transposon, named Tn5037, belongs to the Tn21 branch of the Tn21 subgroup, many members of which have been isolated from clinical sources. Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037 is organized similarly to most of the Gram-negative bacteria mer operons and is closest to that of Thiobacillus 3.2. The operator-promoter region of the mer operon of Tn5037 also has the common (Tn21/Tn501-like) structure. However, its inverted, presumably MerR protein binding repeats in the operator/promoter element are two base pairs shorter than in Tn21/Tn501. In the merA region, this transposon shares 77.4, 79.1, 83.2 and 87.8% identical bases with Tn21, Tn501, T. ferrooxidance E-15, and Thiobacillus 3.2, respectively. No inducibility of the Tn5037 mer operon was detected in the in vivo experiments. The transposition system (terminal repeats plus gene tnpA) of Tn5037 was inactive in Escherichia coli K12, in contrast to its resolution system (res site plus gene tnpR). However, transposition of Tn5037 in this host was provided by the tnpA gene of Tn5036, a member of the Tn21 subgroup. Sequence analysis of the Tn5037 res site suggested its recombinant nature.

Class II broad-spectrum mercury resistance transposons in Gram-positive bacteria from natural environments.

We have studied the mechanisms of the horizontal dissemination of a broad-spectrum mercury resistance determinant among Bacillus and related species. This mer determinant was first described in Bacillus cereus RC607 from Boston Harbor, USA, and was then found in various Bacillus and related species in Japan, Russia and England. We have shown that the mer determinant can either be located at the chromosome, or on a plasmid in the Bacillus species, and is carried by class II mercury resistance transposons: Tn5084 from B. cereus RC607 and B. cereus VKM684 (ATCC10702) and Tn5085 from Exiguobacterium sp. TC38-2b. Tn5085 is identical in nucleotide sequence to TnMERI1, the only other known mer transposon from Bacillus species, but it does not contain an intron like TnMERI1. Tn5085 is functionally active in Escherichia coli. Tn5083, which we have isolated from B. megaterium MK64-1, contains an RC607-like mer determinant, that has lost some mercury resistance genes and possesses a merA gene which is a novel sequence variant that has not been previously described. Tn5083 and Tn5084 are recombinants, and are comprised of fragments from several transposons including Tn5085, and a relative of a putative transposon from B. firmus (which contains similar genes to the cadmium resistance operon of Staphylococcus aureus), as well as others. The sequence data showed evidence for recombination both between transposition genes and between mer determinants.

[Molecular genetic analysis of the Tn5041 transposition system]. / Molekuliarno-geneticheskii analiz sistemy transpozitsii Tn5041.

A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family. Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp. KHP41 but not in P. aeruginosa PAO-R and Escherichia coli K12. Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651. The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes. Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family. As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism. However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E. coli K12. Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup).

Mutations in and monoclonal antibody binding to evolutionary hypervariable region of Escherichia coli RNA polymerase beta' subunit inhibit transcript cleavage and transcript elongation.

A 190 amino acid-long region centered around position 1050 of the 1407-amino acid-long beta' subunit of Escherichia coli RNA polymerase (RNAP) is absent from homologues in eukaryotes, archaea and many bacteria. In chloroplasts, the corresponding region can be more than 900 amino acids long. The role of this hypervariable region was studied by deletion mutagenesis of the cloned E. coli rpoC, encoding beta'. Long deletions mimicking beta' from Gram-positive bacteria failed to assemble into RNAP. Mutants with short, 40-60-amino acid-long deletions spanning beta' residues 941-1130 assembled into active RNAP in vitro. These mutant enzymes were defective in the transcript cleavage reaction and had dramatically reduced transcription elongation rates at subsaturating substrate concentrations due to prolonged pausing at sites of transcriptional arrest. Binding of a monoclonal antibody, Pyn1, to the hypervariable region inhibited transcription elongation and intrinsic transcript cleavage and, to a lesser degree, GreB-induced transcript cleavage, but did not interfere with GreB binding to RNAP. We propose that mutations in and antibody binding to the hypervariable, functionally dispensable region of beta' inhibit transcript cleavage and elongation by distorting the flanking conserved segment G in the active center.

Horizontal spread of mer operons among gram-positive bacteria in natural environments.

Horizontal dissemination of the genes responsible for resistance to toxic pollutants may play a key role in the adaptation of bacterial populations to environmental contaminants. However, the frequency and extent of gene dissemination in natural environments is not known. A natural horizontal spread of two distinct mercury resistance (mer) operon variants, which occurred amongst diverse Bacillus and related species over wide geographical areas, is reported. One mer variant encodes a mercuric reductase with a single N-terminal domain, whilst the other encodes a reductase with a duplicated N-terminal domain. The strains containing the former mer operon types are sensitive to organomercurials, and are most common in the terrestrial mercury-resistant Bacillus populations studied in this work. The strains containing the latter operon types are resistant to organomercurials, and dominate in a Minamata Bay mercury-resistant Bacillus population, previously described in the literature. At least three distinct transposons (related to a class II vancomycin-resistance transposon, Tn1546, from a clinical Enterococcus strain) and conjugative plasmids are implicated as mediators of the spread of these mer operons.