RNA polymerase sigma-related proteins in Escherichia coli: detection by antibodies against a synthetic peptide

Summary Antibodies were raised against a synthetic tetradecameric peptide with an amino acid sequence, DLIQEGNIGLMKAV, which corresponds to the most highly conserved region of bacterial RNA polymerase factors. In a Western-blot analysis of total Escherichia coli proteins, the antiserum reacted specifically with at least three proteins with apparent molecular weights of 75 kDa, 27 kDa and 23 kDa, in addition to the known factors (⁷⁰ and ³²). The majorities of ⁷⁰ and ³² were recovered as associated forms with the RNA polymerase on glycerol gradient centrifugation, while the other cross-reacting proteins were not. Unambiguous evidence was obtained which indicated that the intracellular level of ³² increased rapidly upon heatshock, at least in the strain containing high copy numbers of the rpoH gene.     

Construction of spore mutants of Bacillus subtilis for the development as a host for foreign protein production

For the development of Bacillus subtilis as a host for foreign protein synthesis, three types of sigma factor deleted mutants (spoIIAC, spoIIIG and spoIIIC) were constructed by antibiotic marker insertion using plasmid vector-mediated method or LFH (Long Flanking Homology)-PCR. Mother cell specific sigma factor mutants of B. subtilis (^K), B. subtilis DB104 spoIIIC (km ^r)::pMK101, had two to three times higher subtilisin activity than the wild type DB104::pMK101. Subtilisin expression by the other two mutants, B. subtilis DB104 spoIIAC (km ^r)::pMK101 and DB104 spoIIIG (km ^r)::pMK101, which are pre-spore specific sigma factor (^F and ^G) deleted strains, was similar to, or less than that of the wild type.     

Genetic architecture of a heterotic cross between two populations of maize

Summary The nature and magnitude of variability in the interpopulation cross of Mezcla Amarillo Selection (MAS), an introduction from CIMMYT, Mexico, and J607, a population developed in India using indigenous, American, and Yugoslavian germplasm, were studied. Interpopulation progenies developed by following the North Carolina Design I were evaluated at two locations. The additive genetic variance component in interpopulation cross, _A(12) ² , and in one population assuming the other population as tester, _A12 ² and _A21 ² were significant for all the traits evaluated, namely ear length, ear girth, kernel rows and days to silk, with one exception. For kernel rows, the dominance variance component, _A(12) ² , was also significant but it was smaller than _A(12) ² . The variance component due to dominance X location interaction, _DL(12) ² , was significant for all traits except kernel rows. In the case of ear length and ear girth, _DL(12) ² was greater than the other components. _AL(12) ² , _AL12 ² and _AL21 ² were not significant for any trait. Expected genetic advance indicated a superiority of half-sib reciprocal recurrent selection over full-sib reciprocal recurrent selection.     

σs-dependent overexpression offtsZ in anEscherichia coli K-12 rpoB mutant that is resistant to the division inhibitors DicB and DicF RNA

TheEscherichia coli genesdicF anddicB encode division inhibitors, which prevent the synthesis and activity, respectively, of the essential division protein FtsZ. A mutation at the C-terminal end of the RNA polymerase subunit renders cells resistant to both inhibitors. In the mutant strain the level of theftsZ gene product is higher than in the wild type. Disruption ofrpoS, which encodes the stationary phase sigma factor ^S, lowers FtsZ protein levels in the mutant, and partially restores sensitivity to the inhibitors.     

The<Emphasis Type="Italic"> ssgB</Emphasis> gene,encoding a member of the regulon of stress-response sigma factor σ<Superscript>H</Superscript>, is essential for aerial mycelium septation in<Emphasis Type="Italic"> Streptomyces coelicolor</Emphasis> A3(2)

The Streptomyces coelicolor A3(2) gene ssgB belongs to the regulon of stress-response sigma factor ^H. By integrative transformation via double cross-over, a stable null mutant of ssgB was obtained. This mutation had no obvious effect on vegetative growth, but critically affected aerial mycelium septation. The S. coelicolor ssgB mutant produced aerial hyphae without any signs of septation into spore compartments. The mutation was complemented in trans by wild-type ssgB including the ^H-dependent ssgBp promoter. The results proved that ssgB belongs a developmental branch of the ^H regulon.     

Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on σ marY1 , the long terminal repeat of gypsy-type retroelement marY1

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies—matsutake—in Pinus sp. forest. Here we report that PCR with outward facing primers designed based on sequences comprising _marY1, the long terminal repeat of the gypsy-type retroelement marY1, specifies strains of T. matsutake. PCR with a primer based on the 22-bp sequence conserved at the 5-end of _marY1 conferred 73 reliable bands overall whose profiles depend upon strains of T. matsutake and T. magnivelare, the latter known as American matsutake. This PCR system gave no detectable band in any other species of Tricholoma tested, including T. bakamatsutake and T. fulvocastaneum, symbionts closely related to T. matsutake, as well as a host plant, Pinus densiflora. Similarly, PCR with a set of primers based on 26-bp and 28-bp sequences at bp 48–73 and bp 281–308 of _marY1, internal regions that are mutated in a variant of marY1, conferred 90 reliable bands only in strains of T. matsutake. Theoretically, PCR with the 22-bp primer would allow generation of 2⁷³, or 9.4×10²¹, types of polymorphism, and PCR with a combination of 26- and 28-bp primers, 2⁹⁰, or 1.2×10²⁷ types. The probability of falsely specifying two different isolates as the same strain is <1/10²¹. While polymorphisms conferred by the primer based on the 5 end of _marY1 rather exhibit genetic conservation of a group of T. matsutake, those resulting from primers based on the internal sequences more clearly demonstrate intra-specific diversification. Both systems revealed that T. matsutake is divergent within the species. Ectomycorrhizas formed between P. densiflora and T. matsutake were identified by the PCR systems developed in the present study. This method, using _marY1 as a genetic marker, is useful in analyzing the diversity of T. matsutake, monitoring the behavior of individual mycorrhizas, and specifying the ecological background of fruit bodies traded in markets.     

The Self-Association of Antibiotic Actinocyl-bis(3-dimethylaminopropylamine) in Aqueous Solution: A 1H NMR Analysis

The self-association of the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) was studied in aqueous solution by one- and two-dimensional ¹H NMR spectroscopy at 500 MHz. The two-dimensional homonuclear correlation NMR techniques (TOCSY and ROESY) were used to completely assign all the proton signals of the antibiotic and to quantitatively analyze the mutual arrangement of the antibiotic molecules in their aggregates. The concentration and temperature dependences of proton chemical shifts were used to determine the equilibrium constants and the thermodynamic parameters (H and S) of the self-association, as well as the limiting values of proton chemical shifts in associates. The experimental results were analyzed using both the indefinite noncooperative and cooperative models of the molecular self-association. The calculated value of the cooperativity coefficient ( 1.1) for our synthetic antibiotic confirmed a substantially lower anticooperative effect at the aggregation of its molecules in comparison with that of the antitumor antibiotic actinomycin D ( 1.5). We calculated the most favorable structure of the dimeric associate of the synthetic antibiotic in aqueous solution and found that, like in the actinomycin D dimer, the antiparallel orientation of the phenoxazone chromophore planes of interacting molecules is characteristic of its dimer.     

A newly constructed primer pair for the PCR amplification,cloningand sequencing of the flagellin (<Emphasis Type="Italic">flaA</Emphasis>) gene from isolatesof urease-negative <Emphasis Type="Italic">Campylobacter lari</Emphasis>

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564–572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70–75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.     

Role of guanosine tetraphosphate in gene expression and the survival of glucose or seryl-tRNA starved cells of Escherichia coli K12

The concentration of guanosine 3,5-bispyrophosphate (ppGpp) increases in bacteria in response to amino acid or carbon/energy source starvation. An Escherichia coli K12 relAspoT mutant lacking the ability to synthesize ppGpp lost viability at an increased rate during both glucose and seryl-tRNA starvation. Also, the deleterious effect of chloramphenicol on starved wild-type cells could be overcome by inducing expression of RelA from a plasmid carrying the relA gene transcribed from a tac promoter, prior to starvation and chloramphenicol treatment. As demonstrated by two dimensional gel electrophoresis, this induction of the RelA protein resulted in global alterations in gene expression including increased synthesis of some rpoS-dependent proteins. The relAspoT mutant maintained high expression of several ribosomal proteins during starvation and appeared to exhibit significantly decreased translational fidelity, as demonstrated by an unusual heterogeneity in the isoelectric point of several proteins and the failure to express higher molecular weight proteins during starvation. Moreover, both rpoS-dependent and independent genes failed to exhibit increased expression in the mutant. It is suggested that the deleterious effects on the cells of the relA, spoT deletions are not due solely to the inability of these cells to induce the sigma factor ^s, but also to deficiencies in translational fidelity and failure to exert classical stringent regulation.     

The effect of dynamic light regimes on Chlorella

The patterns of diurnal variations in pigmentation and optical cross-section were compared for two cyclostat cultures of Chlorella pyrenoidosa, where the dynamics of the photoperiod differed. Populations were light-limited, nutrient rich and growing on an 8:16 light-dark (LD) cycle. One light regime was an 8 h sine function of the light period (sinusoidal culture), while the second had an 1 h sine function super-imposed on the 8 hour sine function (oscillating sinusoidal culture). Hourly samples were taken throughout a 12 h period including the light period. Determinations were made of chlorophyll (Chl) a and b abundance, in vivo absorption spectra, cell number and volume and used to derive both cell-specific (cell) and optical chlorophyll specific (chl) cross sections, as well as the absorption efficiency, Q, of the cells. The results indicate that C. pyrenoidosa is capable of adapting to dynamics in light intensity within an 8 h photoperiod. The sinusoidal culture showed a constant decrease in the Chl a/b ratio of 28% while the total Chl content per cell increased slightly and chl and Q remained constant, suggesting coordinated changes in reaction centers and light harvesting complexes. Over the oscillating photoperiod, however, the second culture displayed a diurnal variation in Chl a/b ratio, a 20% increase in chl and an apparent oscillation in Q. These observations suggest that an oscillating photoperiod promoted the capability of Chl molecules to collect light and that the fractional area of all Chl molecules exposed to the photon flux is inversely related to the photon flux.     

The nucleotide sequence of a transposable haloalkanoic acid dehalogenase regulatory gene (dehR I) fromPseudomonas putida strain PP3 and its relationship with σ54-dependent activators

The mobile genetic element,DEH found inPseudomonas putida PP3 carries a 2-haloalkanoic acid dehalogenase structural gene,dehI, and its associated regulatory gene,dehR _I. The nucleotide sequence ofdehR _I was determined. The gene had an open reading frame putatively encoding for a 64 kDa protein containing 571 amino acid residues. The protein was similar to previously published sequences of several other ⁵⁴-dependent activator proteins. Amino acid sequence analysis showed that the deduced DehR_I protein clustered with the NifA nitrogenase regulatory activator family, and was most closely related, with 47.7% similarity, to a NifA-like deduced partial sequence from a plasmid-encoded ORF inPseudomonas sp. strain NS671, associated with L-amino acid production. The domain structure of DehR_I was analysed by alignment with other NifA-like and NtrC-like sequences and showed a highly conserved central region of approximately 230 amino acids, and a potential DNA-binding domain. No homology was detected between the deduced DehR_I and other ⁵⁴-dependent activator sequences at the N-terminus, a result which was consistent with that region being the domain which recognised inducer.     

Foam behavior of biological media

Summary The surface tension and foaminess of (a) unlimited, (b) substrate limited, and (c) oxygen transfer limited growth media of Hansenula polymorpha were measured using methanol, ethanol or glucose as a substrate.The time dependence of can be described by the Avrami-Überreiter relationship: log (2.3 log V)=n log t+log b, where V = (_O – _eq/(_t – _eq, and _O, _t and _eq are at t_M=0, t_M=t and t_M (equilibrium value).The constants n and b are functions of the fermentation time t_F as long as the growth is unlimited but they are constant in the state of limited growth. With glucose substrate, the foaminess can be presented as a definite function of the time, t_DG, which is necessary to attain _eq. With alcohol as a substrate no definite (t_DG) function was found.Symbols b constant in Eq. (1) - n constant in Eq. (1) - S substrate concentration - T temperature - t_M time h (measured from the beginning of the determination of the surface tension ) - t_F cultivation time h (measured from the time of inoculation) - t_DG time (min) necessary to attain the equilibrium surface tension ) - X dry biomass concentration (gl^–1) - V (_O – _eq)/(_t – _eq) - V_S equilibrium volume of the foam (cm³) - V_G volumetric gas flow rate during the estimation of (cm³ s^–1) - vvm volumetric gas flow rate with regard to the volume of the medium (min^–1) - w_SG superficial gas velocity (cm s^–1) - _m maximum specific growth rate (h^–1) - V_S/V_G foaminess (s) - surface tension, mMm^–1 (milli Newton m^–1) - _O at t_M=0 - _eq equilibrium surface tension ( at t_M) - _t at t_M=t - HP probes from Hansenula polymorpha cultivation - NLG non limited growth - OTLG oxygen transfer limited growth - SLG substrate limited growth     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3-region of the nifM gene, the nifL and nifA genes and the 5-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.Communicated by H. Böhme     

Amber mutations in the structural gene for RNA polymerase sigma factor of Escherichia coli

Summary Amber mutants of Escherichia coli K-12 affected in the structural gene (rpoD) for th subunit of RNA polymerase have been obtained from a strain harboring a temperature-sensitive amber suppressor (supF-Ts6) which is active only at low temperatures. These mutants grow normally at low temperature (30°C) but do not grow at high temperature (42°C) due to the inability to synthesize factor. In one mutant studied in detail (rpoD40), the rate of -factor synthesis at 30°C is about half that of the wild type and is decreased to 10%–15% within 1 h of incubation at 42°C. The synthesis of core polymerase subunits or bulk protein is virtually unaffected at least for 2 h. The defect of the mutant in synthesis and growth at high temperature can be suppressed by any of the amber suppressors tested (supD, supE or supF). RNA-polymerase holoenzymes prepared from the mutant cells carrying each of the suppressors (grown at 42°C) exhibit different thermostabilities attributable to alterations in the factor. The reduced synthesis in the mutant is accompanied by the synthesis of polypeptide tentatively identified as amber fragment. These results as well as the genetic mapping data indicate that the amber mutation (rpoD40) resides within the structural gene for the factor and directly affects synthesis upon inactivation of the suppressor at high temperature.