bluuebunny/crossref_metadata_2025 huggingface dataset:
High- and low-abundance chemoreceptors in Escherichia coli: differential activities associated with closely related cytoplasmic domains
Authors: Feng X , Baumgartner J W , Hazelbauer G L
Published: November 2016

In Escherichia coli, two high-abundance chemoreceptors are present in cellular dosages approximately ten-fold greater than two low-abundance receptors. In the absence of high-abundance receptors, cells exhibit an abnormally low tumble frequency and the ability of the remaining receptors to mediate directed migration in spatial gradients is substantially compromised. We found that increasing the cellular amount of the low-abundance receptor Trg over a range of dosages did not alleviate these defects and thus concluded that high- and low-abundance receptors are distinguished not simply by their different dosages in a wild-type cell but also by an inherent difference in activity. By creating hybrids of the low-abundance receptor Trg and the high-abundance receptor Tsr, we investigated the possibility that this inherent difference could be localized to a specific receptor domain and found that the cytoplasmic domain of the high-abundance receptor Tsr conferred the essential features of that receptor class on the low-abundance receptor Trg, even though it is in this domain that residue identity between the two receptors is substantially conserved.
Formation of potent hybrid promoters of the mutant llm gene by IS256 transposition in methicillin-resistant Staphylococcus aureus
Authors: Maki H , Murakami K
Published: November 2016

From high-level methicillin-resistant Staphylococcus aureus SRM551, the low-level heterogeneously resistant mutant, SRM563, was isolated by transposon mutagenesis. The transposon insertion occurred in the 3' region of the llm gene in the mutant (H. Maki, T. Yamaguchi, and K. Murakami, J. Bacteriol. 176:4993-5000, 1994). Resistant revertants were generated from the mutant strain SRM563 on the plate containing methicillin at a concentration of 12.5 microg/ml or more. In some revertants, the insertion sequence IS256 was observed to be transposed into one of five sites localized 88 to 212 bp upstream of the mutant llm at a frequency of 2.8 x 10(-7) in the bacterial population. The IS256 transposition created a new hybrid promoter in which the -35 region at the end of IS256 was properly arranged in relation to the -10-like sequence upstream of llm. The new promoters greatly enhanced the transcription of the mutant llm, as judged by blotting analysis of llm mRNA, with concomitant elevation of the methicillin resistance. Involvement of the insertion sequence in the heteroresistance characteristics of methicillin-resistant S. aureus was suggested.
Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa
Authors: Plesiat P , Aires J R , Godard C , Köhler T
Published: November 2016

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular delta1-dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N',N'-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI23, chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl2. These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.
Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator
Authors: Song S , Park C
Published: November 2016

The metabolism of D-xylose in Escherichia coli K-12 is known to be mediated by the xylAB gene. However, the nearby xylFGHR genes were found by genome sequencing and predicted to be responsible for transport and regulation for xylose based on their sequence similarities to other functionally related genes. Here, we investigated transcriptional organization and functions of the xyl genes. An analysis with random transposon insertions revealed that the xyl genes are organized into two major transcriptional units, xylAB and xylFGHR, governed by the promoters PA and PF, respectively. However, there is an additional weak promoter, PR, which is specific for xylR. Sites of transcription initiation were determined by primer extension analysis. When studied with operon fusions to lacZ, the PA and PF promoters were activated by D-xylose and repressed by glucose. In contrast, the PR promoter was not regulated by these sugars. A mutation in xylR completely abolished expression from the PA and PF promoters, causing a defect in both growth and transport. Binding of XylR to the xyl promoter was enhanced by the presence of D-xylose, suggesting that transcription was positively regulated by XylR. In vivo footprinting analysis revealed that XylR binds to at least two DNA regions, IA and IF, each with a direct repeat. It is very likely that XylR interacts with IA and IF as a dimer. The presumed binding sites are located just upstream of the promoter consensus sequences (-35), while IA is additionally flanked by a cyclic AMP receptor protein-binding site on the other side. The proposed structure of xyl promoters is consistent with the regulation of xyl gene expression and with phenotypes of transposon insertions obtained in the promoter regions.
Regulation of Bacteriodes fragilis katB mRNA by oxidative stress and carbon limitation
Authors: Rocha E R , Smith C J
Published: November 2016

Regulation of the katB catalase gene in the anaerobic bacterium Bacteroides fragilis was studied. Northern blot hybridization analyses revealed that katB was transcribed as an approximately 1.6-kb monocistronic mRNA. The levels of katB mRNA increased > 15-fold when anaerobic, mid-logarithmic-phase cultures were exposed to O2, O2 with paraquat, or hydrogen peroxide. Under anaerobic conditions, the low levels of katB mRNA increased in a growth-dependent manner, reaching maximum expression at late logarithmic or early stationary phase, followed by a decrease in stationary phase. Under anaerobic conditions, the expression of katB mRNA was strongly repressed by glucose and to a lesser extent by xylose. However, glucose repression was completely abolished upon exposure to oxygen. The nonfermentable carbon sources fumarate, succinate, acetate, and pyruvate did not significantly affect expression. Phosphate, nitrogen, and hemin limitation did not affect the expression of katB mRNA, suggesting that the nutritional control of katB expression is restricted to carbon and energy sources and not other forms of nutrient limitation. Primer extension analysis revealed that during both oxidative stress and carbon or energy limitation, katB utilized the same promoter region but transcription initiation occurred at two different nucleotides separated by 3 or 4 bases. Interestingly, a 6-bp inverted repeat sequence present in the katB regulatory region was also observed upstream of the B. fragilis superoxide dismutase gene sod. It is possible that this is a recognition site for a DNA binding protein involved in the regulation of oxidative stress genes in this organism.
Regulation of polymyxin resistance and adaptation to low-Mg2+ environments
Authors: Groisman E A , Kayser J , Soncini F C
Published: November 2016

The PmrA-PmrB two-component system of Salmonella typhimurium controls resistance to the peptide antibiotic polymyxin B and to several antimicrobial proteins from human neutrophils. Amino acid substitutions in the regulatory protein PmrA conferring resistance to polymyxin lower the overall negative charge of the lipopolysaccharide (LPS), which results in decreased bacterial binding to cationic polypeptides and increased bacterial survival within human neutrophils. We have now identified three PmrA-activated loci that are required for polymyxin resistance. These loci were previously shown to be necessary for growth on low-Mg2+ solid media, indicating that LPS modifications that mediate polymyxin resistance are responsible for the adaptation to Mg2+-limited environments. Conditions that promote transcription of PmrA-activated genes--growth in mildly acidic pH and micromolar Mg2+ concentrations--increased survival in the presence of polymyxin over 16,000-fold in a wild-type organism but not in a mutant lacking pmrA. Our experiments suggest that low pH and low Mg2+ concentrations may induce expression of PmrA-activated genes within phagocytic cells and promote bacterial resistance to host antimicrobial proteins. We propose that the LPS is a Mg2+ reservoir and that the PmrA-controlled LPS modifications neutralize surface negative charges when Mg2+ is transported into the cytoplasm during growth in Mg2+-limited environments.
Temperature-dependent regulation of the ribosomal small-subunit protein S21 in the cyanobacterium Anabaena variabilis M3
Authors: Sato N , Tachikawa T , Wada A , Tanaka A
Published: November 2016

The rpsU gene, which encodes the ribosomal small-subunit protein S21 in Anabaena, is not a part of the macromolecular-synthesis operon as in most enterobacteria but rather is located downstream of the rbpA1 gene, which encodes an RNA-binding protein. Two types of transcripts were detected for this gene cluster. The level of the major rbpA1-rpsU transcript was about 10 times higher at 22 degrees C than at 38 degrees C, whereas the minor monocistronic rpsU transcript was more abundant at the higher temperature. The level of the S21 protein in relation to total protein was three times lower at 38 degrees C than at 22 degrees C. Analysis of isolated ribosomes indicated that S21 was present at an equimolar ratio with regard to other ribosomal proteins at 22 degrees C but that its level decreased with temperature. Conversely, the relative abundance of S5 increased with temperature. A decrease in the level of S21 at high temperature was also found in Synechocystis, in which rpsU is located downstream of the rrn operon. These results suggest that S21 is involved in the adaptation to changes in temperature in cyanobacteria.
Hierarchical autoinduction in Ralstonia solanacearum: control of acyl-homoserine lactone production by a novel autoregulatory system responsive to 3-hydroxypalmitic acid methyl ester
Authors: Flavier A B , Ganova-Raeva L M , Schell M A , Denny T P
Published: November 2016

Bacteria employ autoinduction systems to sense the onset of appropriate cell density for expression of developmental genes. In many gram-negative bacteria, autoinduction involves the production of and response to diffusible acylated-homoserine lactones (acyl-HSLs) and is mediated by members of the LuxR and LuxI families. Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium that appears to autoregulate its virulence genes, produces compounds that promote expression of several heterologous acyl-HSL-responsive reporter gene constructs. High-pressure liquid chromatography of highly concentrated ethyl acetate extracts revealed that culture supernatants of strain AW1 contained two compounds with retention times similar to N-hexanoyl- and N-octanoyl-HSL. To investigate the role of these acyl-HSLs in R. solanacearum virulence gene expression, transposon mutants that were deficient for inducing an acyl-HSL-responsive reporter in Agrobacterium tumefaciens were generated. Three loci involved in normal acyl-HSL production were identified, one of which was shown to contain the divergently transcribed solR and solI genes, the luxR and luxI homologs, respectively. A 4.1-kb fragment containing solR and solI enabled all of the mutants (regardless of the locus inactivated) and a naturally acyl-HSL-defective strain of R. solanacearum to produce acyl-HSLs. Inactivation of solI abolished production of all detectable acyl-HSLs but affected neither the expression of virulence genes in culture nor the ability to wilt tomato plants. AW1 has a functional autoinduction system, because (i) expression of solI required SolR and acyl-HSL and (ii) expression of a gene linked to solR and solI, designated aidA, was acyl-HSL dependent. Because AidA has no homologs in the protein databases, its discovery provided no clues as to the role of acyl-HSLs in R. solanacearum gene regulation. However, expression of solR and solI required the global LysR-type virulence regulator PhcA, and both solR and solI exhibited a cell density-associated pattern of expression similar to other PhcA-regulated genes. The acyl-HSL-dependent autoinduction system in R. solanacearum is part of a more complex autoregulatory hierarchy, since the transcriptional activity of PhcA is itself controlled by a novel autoregulatory system that responds to 3-hydroxypalmitic acid methyl ester.
Genetic analysis of the chitinase system of Serratia marcescens 2170
Authors: Watanabe T , Kimura K , Sumiya T , Nikaidou N , Suzuki K , Suzuki M , Taiyoji M , Ferrer S , Regue M
Published: November 2016

To carry out a genetic analysis of the degradation and utilization of chitin by Serratia marcescens 2170, various Tn5 insertion mutants with characteristic defects in chitinase production were isolated and partially characterized. Prior to the isolation of the mutants, proteins secreted into culture medium in the presence of chitin were analyzed. Four chitinases, A, B, C1, and C2, among other proteins, were detected in the culture supernatant of S. marcescens 2170. All four chitinases and a 21-kDa protein (CBP21) lacking chitinase activity showed chitin binding activity. Cloning and sequencing analysis of the genes encoding chitinases A and B of strain 2170 revealed extensive similarities to those of other strains of S. marcescens described previously. Tn5 insertion mutagenesis of strain 2170 was carried out, and mutants which formed altered clearing zones of colloidal chitin were selected. The obtained mutants were divided into five classes as follows: mutants with (i) no clearing zones, (ii) fuzzy clearing zones, (iii) large clearing zones, (iv) delayed clearing zones, and (v) small clearing zones. Preliminary characterization suggested that some of these mutants have defects in chitinase excretion, a negatively regulating mechanism of chitinase gene expression, an essential factor for chitinase gene expression, and a structural gene for a particular chitinase. These mutants could allow researchers to identify the genes involved in the degradation and utilization of chitin by S. marcescens 2170.
pCal, a highly unusual Ty1/copia retrotransposon from the pathogenic yeast Candida albicans
Authors: Matthews G D , Goodwin T J , Butler M I , Berryman T A , Poulter R T
Published: November 2016

Retrotransposons are mobile genetic elements. They can transpose via the reverse transcription of mRNA into double-stranded DNA (dsDNA) followed by the insertion of this dsDNA into new sites within the host genome. The unintegrated, linear, dsDNA form of retrotransposons is usually very rare. We report here the isolation of a retrotransposon from Candida albicans which is unusual in this respect. This element, which we have named pCal, was first identified as a distinct band when uncut C. albicans DNA was examined on an agarose gel. Sequence analysis of the cloned element revealed that it is a retrotransposon belonging to the Ty1/copia group. It is estimated that pCal produces 50 to 100 free, linear, dsDNA copies of itself per cell. This is a much higher level of expression than even that of the system in which Ty1 is expressed behind the highly active GAL1 promoter on a high-copy-number plasmid (about 10 copies per cell). Another unusual feature of pCal is that its Pol enzymes are likely to be expressed via the pseudoknot-assisted suppression of an upstream, in-phase stop codon, as has been shown for Moloney murine leukemia virus.
Plasmid pRQ7 from the hyperthermophilic bacterium Thermotoga species strain RQ7 replicates by the rolling-circle mechanism
Authors: Yu J S , Noll K M
Published: November 2016

The hyperthermophilic bacterium Thermotoga species strain RQ7 harbors an 846-bp plasmid, pRQ7, with a single open reading frame. Previously published analyses of the DNA sequence of pRQ7 suggested that it may replicate by a rolling-circle (RC) replication mechanism, and this report provides experimental evidence supporting this hypothesis. Single-stranded pRQ7 DNA accumulates in strain RQ7, as evidenced by the facts that this DNA bound to nitrocellulose membranes under nondenaturing conditions, was sensitive to S1 nuclease digestion, and hybridized to only one of two homologous DNA probes specific for each strand of the plasmid. The DNA encoding the open reading frame was cloned and expressed in Escherichia coli and gave a protein with a molecular mass of 26 kDa, similar to that deduced by sequence analysis. This protein bound to a fragment of pRQ7 that contains a putative double-stranded replication region in a magnesium-dependent reaction and made this fragment sensitive to S1 nuclease activity. It did not cause this same S1 nuclease sensitivity in the remainder of pRQ7. This activity on pRQ7 DNA suggests that this protein plays a role in plasmid replication.
Identification of type III secreted products of the Pseudomonas aeruginosa exoenzyme S regulon
Authors: Yahr T L , Mende-Mueller L M , Friese M B , Frank D W
Published: November 2016

Extracellular protein profiles from wild-type and regulatory or secretory isogenic mutants of the Pseudomonas aeruginosa exoenzyme S regulon were compared to identify proteins coordinately secreted with ExoS. Data from amino-terminal sequence analysis of purified extracellular proteins were combined with data from nucleotide sequence analysis of loci linked to exoenzyme S production. We report the identification of P. aeruginosa homologs to proteins of Yersinia spp. that function as regulators of the low calcium response, regulators of secretion, and mediators of the type III translocation mechanism.
Evidence for transcription attenuation rendering cryptic a sigmaS-dependent promoter of the osmotically regulated proU operon of Salmonella typhimurium
Authors: Rajkumari K , Ishihama A , Gowrishankar J
Published: November 2016

The osmotically regulated proU locus in Escherichia coli has two promoters, P1 and P2, that are recognized, respectively, by the sigmaS- and sigma70-bearing RNA polymerase holoenzymes. However, the equivalent of the P1 promoter does not appear to exist in Salmonella typhimurium. We demonstrate in this study that wild-type S. typhimurium has a cryptic P1 promoter that is recognized by sigmaS RNA polymerase in vitro and that a 22-bp deletion from +63 to +84 (relative to the start site of transcription) confers sigmaS-dependent in vivo expression of a reporter gene fusion to P1. Primer extension analysis of RNA isolated from cells carrying the wild-type and mutant S. typhimurium proU constructs indicated that a primer which hybridizes proximal to +60 is able to detect P1-initiated transcripts from both constructs but a primer which hybridizes distal to +85 is able to do so only from the latter. Our results suggest that the sigmaS-controlled proU P1 promoter in S. typhimurium may be rendered cryptic because of factor-dependent transcription attenuation within a short distance downstream of the promoter start site.
Characterization of Bacillus subtilis hemN
Authors: Hippler B , Homuth G , Hoffmann T , Hungerer C , Schumann W , Jahn D
Published: November 2016

A recently cloned Bacillus subtilis open reading frame (hemN) upstream of the dnaK operon was identified as encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation. B. subtilis hemN functionally complemented two Salmonella typhimurium hemF hemN double mutants under aerobic and anaerobic conditions. A B. subtilis hemN mutant accumulated coproporphyrinogen III only under anaerobic conditions. Interestingly, growth experiments using the B. subtilis hemN mutant revealed normal aerobic and anaerobic growth, indicating the presence of an alternative oxygen-independent enzymatic system. Northern blot experiments identified hemN mRNA as part of an approximately 7-kb pentacistronic transcript consisting of lepA, hemN, hrcA, grpE, and dnaK. One potential start site for aerobic and anaerobic transcription was located 37 bp upstream of the translational start codon of lepA. Comparable amounts of hemN transcript were observed under aerobic and anaerobic growth conditions. No experimental evidence for the presence of hemF in B. subtilis was obtained. Moreover, B. subtilis hemY did not substitute for hemF hemN deficiency in S. typhimurium. These results indicate the absence of hemF and suggest the presence of a second hemN-like gene in B. subtilis.
Carbonic anhydrase in Acetobacterium woodii and other acetogenic bacteria
Authors: Braus-Stromeyer S A , Schnappauf G , Braus G H , Gössner A S , Drake H L
Published: November 2016

Acetobacterium woodii, Acetohalobium arabaticum, Clostridium formicoaceticum, and Sporomusa silvacetica were found to contain carbonic anhydrase (CA). Minimal to no CA activity was detected in Moorella thermoautotrophica, Moorella thermoacetica subsp. "pratumsolum," Sporomusa termitida, and Thermoanaerobacter kivui. Of the acetogens tested, A. woodii had the highest CA specific activity, approximately 14 U mg of protein(-1), in extracts of either glucose- or H2-CO2-cultivated cells. CA of A. woodii was cytoplasmic and was purified approximately 300-fold to a specific activity of 5,236 U mg of protein(-1). Intracellular acetate concentrations inhibited CA activity of A. woodii by 50 to 85%, indicating that intracellular acetate may affect in situ CA activity.
Transcriptional and translational control of the genes for the mating pair formation apparatus of promiscuous IncP plasmids
Authors: Zatyka M , Jagura-Burdzy G , Thomas C M
Published: November 2016

The trb operon of broad-host-range plasmid RK2 encodes most of the genes required for formation of mating-pair apparatus and is thus essential for the promiscuous spread of this plasmid. Only two promoters, lying upstream of trbA and trbB, have been identified for this operon. trbB encodes a protein belonging to a large family of proteins which function in the assembly of apparatuses associated with the cell surface. trbA encodes a repressor protein, one of whose targets is the trbB promoter. trbAp is arranged as a face-to-face divergent promoter with trfAp, the strongest of the three promoters in this region. trfAp completely inhibits trbAp unless it is repressed by the KorA protein, a key regulator encoded in the plasmid's central control operon. We show that when trfAp is firing constitutively, it also appears to interfere with trbBp, but that trbBp activity increases when trfAp activity is decreased by repression or mutation. A second global regulator encoded in the central control operon, KorB, represses trbBp, trfAp, and trbAp. The results presented here show that both KorB and TrbA are necessary for full repression of trbBp. The region between trbA and trbB encodes a large inverted repeat which has been proposed to modulate translation of trbB on transcripts which are initiated at trbAp but not trbBp. Using translational fusions to lacZ, we show that translation of trbB is completely blocked when transcripts incorporate the inverted repeat upstream of trbB but proceeds with reasonable efficiency when deletions remove the sequences predicted to sequester the ribosome binding site. Results from both transcriptional fusion and direct measurement of transcript size and intensity by Northern blot analysis show that most trbA transcripts are monocistronic and serve to express only trbA, although some transcription continues into trbB. The monocistronic trbA transcript appears to be the result of transcription termination downstream of trbA. Thus, trbAp and trbA appear to form an operon distinct from the trbB-trbP operon. Consequently, trbA and the switch that controls its expression help to provide the sequential steps which allow efficient expression of transfer genes during plasmid establishment but tight repression once the plasmid is established.
Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions
Authors: Larsson C , Nilsson A , Blomberg A , Gustafsson L
Published: November 2016

Anaerobic and aerobic chemostat cultures of Saccharomyces cerevisiae were performed at a constant dilution rate of 0.10 h(-1). The glucose concentration was kept constant, whereas the nitrogen concentration was gradually decreasing; i.e., the conditions were changed from glucose and energy limitation to nitrogen limitation and energy excess. This experimental setup enabled the glycolytic rate to be separated from the growth rate. There was an extensive uncoupling between anabolic energy requirements and catabolic energy production when the energy source was present in excess both aerobically and anaerobically. To increase the catabolic activity even further, experiments were carried out in the presence of 5 mM acetic acid or benzoic acid. However, there was almost no effect with acetate addition, whereas both respiratory (aerobically) and fermentative activities were elevated in the presence of benzoic acid. There was a strong negative correlation between glycolytic flux and intracellular ATP content; i.e., the higher the ATP content, the lower the rate of glycolysis. No correlation could be found with the other nucleotides tested (ADP, GTP, and UTP) or with the ATP/ADP ratio. Furthermore, a higher rate of glycolysis was not accompanied by an increasing level of glycolytic enzymes. On the contrary, the glycolytic enzymes decreased with increasing flux. The most pronounced reduction was obtained for HXK2 and ENO1. There was also a correlation between the extent of carbohydrate accumulation and glycolytic flux. A high accumulation was obtained at low glycolytic rates under glucose limitation, whereas nitrogen limitation during conditions of excess carbon and energy resulted in more or less complete depletion of intracellular storage carbohydrates irrespective of anaerobic or aerobic conditions. However, there was one difference in that glycogen dominated anaerobically whereas under aerobic conditions, trehalose was the major carbohydrate accumulated. Possible mechanisms which may explain the strong correlation between glycolytic flux, storage carbohydrate accumulation, and ATP concentrations are discussed.
Expression of a stress- and starvation-induced dps/pexB-homologous gene is controlled by the alternative sigma factor sigmaB in Bacillus subtilis
Authors: Antelmann H , Engelmann S , Schmid R , Sorokin A , Lapidus A , Hecker M
Published: November 2016

SigmaB-dependent general stress proteins (Gsps) of Bacillus subtilis are essential for the development of glucose-starvation-induced cross-resistance to oxidative challenge. However, the proteins directly involved in this nonspecific resistance to oxidative stress have to be identified. We found that one prominent Gsp displayed strong sequence similarity to the previously characterized oxidative-stress-inducible MrgA protein of B. subtilis and to the starvation-induced Dps/PexB protein of Escherichia coli. We therefore designated this prominent Gsp Dps. While MrgA belongs to the peroxide-stress-inducible proteins needed for the H2O2-inducible adaptive response to oxidative stress, Dps belongs to the proteins induced by heat, salt, or ethanol stress and after starvation for glucose but not by a sublethal oxidative challenge. Primer extension experiments identified two overlapping promoters upstream of the coding region of dps, one being sigmaB dependent (PB) and the other being sigmaB independent (P1). Both promoters contribute to the basal level of dps during growth. After stress or during entry into the stationary phase, transcription from PB strongly increased whereas transcription from P1 decreased. Mutant strains lacking Dps completely failed to develop glucose-starvation-induced resistance to oxidative stress. These results confirm our suggestion that sigmaB-dependent general stress proteins of B. subtilis are absolutely required for the development of nonspecific resistance to oxidative stress.
Analysis of the fnrL gene and its function in Rhodobacter capsulatus
Authors: Zeilstra-Ryalls J H , Gabbert K , Mouncey N J , Kaplan S , Kranz R G
Published: November 2016

The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.
Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci
Authors: Wang W , Bechhofer D H
Published: November 2016

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.
Alkyl hydroperoxide reductase, catalase, MrgA, and superoxide dismutase are not involved in resistance of Bacillus subtilis spores to heat or oxidizing agents
Authors: Casillas-Martinez L , Setlow P
Published: November 2016

Only a single superoxide dismutase (SodA) was detected in Bacillus subtilis, and growing cells of a sodA mutant exhibited paraquat sensitivity as well as a growth defect and reduced survival at an elevated temperature. However, the sodA mutation had no effect on the heat or hydrogen peroxide resistance of wild-type spores or spores lacking the two major DNA protective alpha/beta-type small, acid-soluble, spore proteins (termed alpha(-)beta(-) spores). Spores also had only a single catalase (KatX), as the two catalases found in growing cells (KatA and KatB) were absent. While a katA mutation greatly decreased the hydrogen peroxide resistance of growing cells, as found previously, katA, katB, and katX mutations had no effect on the heat or hydrogen peroxide resistance of wild-type or alpha(-)beta(-) spores. Inactivation of the mrgA gene, which codes for a DNA-binding protein that can protect growing cells against hydrogen peroxide, also had no effect on spore hydrogen peroxide resistance. Inactivation of genes coding for alkyl hydroperoxide reductase, which has been shown to decrease growing cell resistance to alkyl hydroperoxides, had no effect on spore resistance to such compounds or on spore resistance to heat and hydrogen peroxide. However, Western blot analysis showed that at least one alkyl hydroperoxide reductase subunit was present in spores. Together these results indicate that proteins that play a role in the resistance of growing cells to oxidizing agents play no role in spore resistance. A likely reason for this lack of a protective role for spore enzymes is the inactivity of enzymes within the dormant spore.
Cloning and genetic analysis of the UV resistance determinant (uvr) encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pAD1
Authors: Ozawa Y , Tanimoto K , Fujimoto S , Tomita H , Ike Y
Published: November 2016

The conjugative pheromone-responsive plasmid pAD1 (59.6 kb) of Enterococcus faecalis encodes a UV resistance determinant (uvr) in addition to the hemolysin-bacteriocin determinant. pAD1 enhances the UV resistance of wild-type E. faecalis FA2-2 and E. faecalis UV202, which is a UV-sensitive derivative of E. faecalis JH2-2. A 2.972-kb fragment cloned from between 27.7 and 30.6 kb of the pAD1 map conferred UV resistance function on UV202. Sequence analysis showed that the cloned fragment contained three open reading frames designated uvrA, uvrB, and uvrC. The uvrA gene is located on the pAD1 map between 28.1 and 29.4 kb. uvrB is located between 30.1 and 30.3 kb, and uvrC is located between 30.4 and 30.6 kb on the pAD1 map. The uvrA, uvrB, and uvrC genes encode sequences of 442, 60, and 74 amino acids, respectively. The deduced amino acid sequence of the uvrA-encoded protein showed 20% homology of the identical residues with the E. coli UmuC protein. Tn917 insertion mutagenesis and deletion mutant analysis of the cloned fragment showed that uvrA conferred UV resistance. A palindromic sequence, 5'-GAACNGTTC-3', which is identical to the consensus sequence found within the putative promoter region of the Bacillus subtilis DNA damage-inducible genes, was located within the promoter region of uvrA. Two uvrA transcripts of different lengths (i.e., 1.54 and 2.14 kb) which terminate at different points downstream of uvrA were detected in UV202 carrying the deletion mutant containing uvrA. The longer transcript, 2.14 kb, was not detected in UV202 carrying the deletion mutant containing both uvrA and uvrB, which suggests that uvrB encodes a terminator for the uvrA transcript. The uvrA transcript was not detected in any significant quantity in UV202 carrying the cloned fragment containing uvrA, uvrB, and uvrC; on the other hand, the 1.54-kb uvrA transcript was detected in the strain exposed to mitomycin C, which suggests that the UvrC protein functions as a regulator of uvrA.
Escherichia coli cells expressing a mutant glyV (glycine tRNA) gene have a UVM-constitutive phenotype: implications for mechanisms underlying the mutA or mutC mutator effect
Authors: Murphy H S , Humayun M Z
Published: November 2016

Transfection of M13 single-stranded viral DNA bearing a 3,N4-ethenocytosine lesion into Escherichia coli cells pretreated with UV results in a significant elevation of mutagenesis at the lesion site compared to that observed in untreated cells. This response, termed UVM, for UV modulation of mutagenesis, is induced by a variety of DNA-damaging agents and is distinct from known cellular responses to DNA damage, including the SOS response. This report describes our observation, as a part of our investigation of the UVM phenomenon, that E. coli cells bearing a mutA or mutC allele display a UVM-constitutive phenotype. These mutator alleles were recently mapped (M. M. Slupska, C. Baikalov, R. Lloyd, and J. H. Miller, Proc. Natl. Acad. Sci. USA 93:4380-4385, 1996) to the glyV (mutA) and glyW (mutC) tRNA genes. Each mutant allele was shown to arise by an identical mutation in the anticodon sequence such that the mutant tRNAs could, in principle, mistranslate aspartate codons in mRNA as glycine at a low level. Because a UVM-constitutive phenotype resulting from a mutation in a tRNA gene was unexpected, we undertook a series of experiments designed to test whether the phenotype was indeed mediated by the expression of mutant glycine tRNAs. We placed either a wild-type or a mutant glyV gene under the control of a heterologous inducible promoter on a plasmid vector. E. coli cells expressing the mutant glyV gene displayed all three of the following phenotypes: (i) missense suppression of a test allele, (ii) a mutator phenotype measured by mutation to rifampin resistance, and (iii) a UVM-constitutive phenotype. These phenotypes were not associated with cells expressing the wild-type glyV gene or with cells in which the mutant allele was present but was not transcriptionally induced. These observations provide strong support for the idea that expression of mutant tRNA can confer a mutator phenotype, including the UVM-constitutive phenotype observed in mutA and mutC cells. However, our data imply that low-level mistranslation of the epsilon subunit of polymerase III probably does not account for the observed UVM-constitutive phenotype. Our results also indicate that mutA deltarecA double mutants display a normal UVM phenotype, suggesting that the mutA effect is recA dependent. The observations reported here raise a number of intriguing questions and raise the possibility that the UVM response is mediated through transient alteration of the replication environment.
A quorum-sensing system in the free-living photosynthetic bacterium Rhodobacter sphaeroides
Authors: Puskas A , Greenberg E P , Kaplan S , Schaefer A L
Published: November 2016

Rhodobacter sphaeroides is a free-living, photoheterotrophic bacterium known for its genomic and metabolic complexity. We have discovered that this purple photosynthetic organism possesses a quorum-sensing system. Quorum sensing occurs in a number of eukaryotic host-associated gram-negative bacteria. In these bacteria there are two genes required for quorum sensing, the luxR and luxI homologs, and there is an acylhomoserine lactone signal molecule synthesized by the product of the luxI homolog. In R. sphaeroides, synthesis of a novel homoserine lactone signal, 7,8-cis-N-(tetradecenoyl)homoserine lactone, is directed by a luxI homolog termed cerI. Two open reading frames immediately upstream of cerI are proposed to be components of the quorum-sensing system. The first of these is a luxR homolog termed cerR, and the second is a small open reading frame of 159 bp. Inactivation of cerI in R. sphaeroides results in mucoid colony formation on agar and formation of large aggregates of cells in liquid cultures. Clumping of CerI mutants in liquid culture is reversible upon addition of the acylhomoserine lactone signal and represents a phenotype unlike those controlled by quorum sensing in other bacteria.
A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives
Authors: Rubirés X , Saigi F , Piqué N , Climent N , Merino S , Albertí S , Tomás J M , Regué M
Published: November 2016

A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli DH5alpha, and clones were screened for serum resistance. One clone was found resistant to serum, to bacteriocin 28b, and to bacteriophages TuIa and TuIb. This clone also showed O antigen in its lipopolysaccharide. Subcloning and sequencing experiments showed that a 2,124-bp DNA fragment containing the rmlD and wbbL genes was responsible for the observed phenotypes. On the basis of amino acid similarity, we suggest that the 288-residue RmlD protein is a dTDP-L-rhamnose synthase. Plasmid pJT102, containing only the wbbL gene, was able to induce O16-antigen production and serum resistance in E. coli DH5alpha. These results suggest that the 282-residue WbbL protein is a rhamnosyltransferase able to complement the rJb-50 mutation in E. coli K-12 derivatives, despite the low level of amino acid identity between WbbL and the E. coli rhamnosyltransferase (24.80%). S. marcescens N28b rmlD and wbbL mutants were constructed by mobilization of suicide plasmids containing a portion of rmlD or wbbL. These insertion mutants were unable to produce O antigen; since strain N28b produces O4 antigen, these results suggest that both genes are involved in O4-antigen biosynthesis.
Functional domains of a zinc metalloprotease from Vibrio vulnificus
Authors: Miyoshi S , Wakae H , Tomochika K , Shinoda S
Published: November 2016

Vibrio vulnificus, an opportunistic human pathogen causing wound infection and septicemia, secretes a 45-kDa metalloprotease (V. vulnificus protease; VVP). A plasmid which carries the entire vvp gene subcloned into pBluescriptIIKS+ was transformed into Escherichia coli DH5alpha for overproduction of the protease. The 45-kDa recombinant protease (rVVP) was isolated from the periplasmic fraction of the transformant by ammonium sulfate precipitation followed by column chromatography on phenyl Sepharose. Biochemical characterization of the isolated rVVP showed that the recombinant protease was identical to that produced by V. vulnificus. When rVVP was incubated at 37 degrees C, a 35-kDa fragment was generated through autoproteolytic removal of the C-terminal peptide. This 35-kDa fragment (rVVP-N) was found to have sufficient proteolytic activity toward oligopeptides and soluble proteins but had markedly reduced activity toward insoluble proteins. Lineweaver-Burk plot analysis indicated increased Km values of rVVP-N for all of the protein substrates. rVVP, but not rVVP-N, was shown to agglutinate rabbit erythrocytes, bind to the erythrocyte ghosts, and digest the ghost membrane proteins. These results strongly suggest that rVVP (and VVP) consists of at least two functional domains: an N-terminal 35-kDa polypeptide mediating proteolysis and a C-terminal 10-kDa polypeptide which may be essential for efficient attachment to protein substrates and erythrocyte membranes.
Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae
Authors: Athenstaedt K , Daum G
Published: November 2016

Lipid particles of the yeast Saccharomyces cerevisiae harbor two enzymes that stepwise acylate glycerol-3-phosphate to phosphatidic acid, a key intermediate in lipid biosynthesis. In lipid particles of the s1c1 disruptant YMN5 (M. M. Nagiec et al., J. Biol. Chem. 268:22156-22163, 1993) acylation stops after the first step, resulting in the accumulation of lysophosphatidic acid. Two-dimensional gel electrophoresis confirmed that S1c1p is a component of lipid particles. Lipid particles of a second mutant strain, TTA1 (T. S. Tillman and R. M. Bell, J. Biol. Chem. 261:9144-9149, 1986), which harbors a point mutation in the GAT gene, are essentially devoid of glycerol-3-phosphate acyltransferase activity in vitro. Synthesis of phosphatidic acid is reconstituted by combining lipid particles from YMN5 and TTA1. These results indicate that two distinct enzymes are necessary for phosphatidic acid synthesis in lipid particles: the first step, acylation of glycerol-3-phosphate, is catalyzed by a putative Gat1p; the second step, acylation of lysophosphatidic acid, requires S1c1p. Surprisingly, YMN5 and TTA1 mutants grow like the corresponding wild types because the endoplasmic reticulum of both mutants has the capacity to form a reduced but significant amount of phosphatidic acid. As a consequence, an s1c1 gat1 double mutant is also viable. Lipid particles from this double mutant fail completely to acylate glycerol-3-phosphate, whereas endoplasmic reticulum membranes harbor residual enzyme activities to synthesize phosphatidic acid. Thus, yeast contains at least two independent systems of phosphatidic acid biosynthesis.
The D-allose operon of Escherichia coli K-12
Authors: Kim C , Song S , Park C
Published: November 2016

Escherichia coli K-12 can utilize D-allose, an all-cis hexose, as a sole carbon source. The operon responsible for D-allose metabolism was localized at 92.8 min of the E. coli linkage map. It consists of six genes, alsRBACEK, which are inducible by D-allose and are under the control of the repressor gene alsR. This operon is also subject to catabolite repression. Three genes, alsB, alsA, and alsC, appear to be necessary for transport of D-allose. D-Allose-binding protein, encoded by alsB, is a periplasmic protein that has an affinity for D-allose, with a Kd of 0.33 microM. As was found for other binding-protein-mediated ABC transporters, the allose transport system includes an ATP-binding component (AlsA) and a transmembrane protein (AlsC). It was found that AlsE (a putative D-allulose-6-phosphate 3-epimerase), but not AlsK (a putative D-allose kinase), is necessary for allose metabolism. During this study, we observed that the D-allose transporter is partially responsible for the low-affinity transport of D-ribose and that strain W3110, an E. coli prototroph, has a defect in the transport of D-allose mediated by the allose permease.
Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila
Authors: Rasche M E , Smith K S , Ferry J G
Published: November 2016

Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.