bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of late gene promoters of Chlamydia trachomatis
Authors: Fahr M J , Douglas A L , Xia W , Hatch T P
Published: November 2016

Chlamydiae possess an intracellular developmental cycle defined by the orderly interconversion of infectious, metabolically inactive elementary bodies and noninfectious, dividing reticulate bodies. Only a few stage-specific genes have been cloned and sequenced, including the late-stage cysteine-rich protein operon and two late-stage genes encoding histone-like proteins. The aims of this study were to identify additional late-stage genes of Chlamydia trachomatis, analyze the upstream DNA sequence of late genes, and determine the sigma factor requirement of late genes. Stage-specific RNA, made by chlamydiae isolated from host cells, was used to probe C. trachomatis genomic libraries. Two new late genes, designated ltuA and ltuB, were identified, cloned, and sequenced. The predicted peptides encoded by ltuA and ltuB do not bear strong homology to known proteins, and the function of the new late genes is not known. The 5' ends of the transcripts of ltuA, ltuB, the cysteine-rich protein operon, and the two histone-like genes (hctA and hctB) were mapped, and a consensus -10 promoter region of TATAAT was derived from their upstream DNA sequences. In vitro transcription from templates encoding the promoter regions of ltuA, ltuB, and hctA cloned into the transcription assay vector pUC19-spf was found to be strongly stimulated by the addition of recombinant chlamydial sigma 66, while transcription from the putative hctB promoter region cloned in pUC19-spf was not detected in either the presence or absence of added sigma 66. These results suggest that the transcription of at least some chlamydial late-stage genes is dependent on sigma 66, which is homologous to the major sigma factors of other eubacteria.
Conformational analysis of the Campylobacter jejuni porin
Authors: Bolla J M , Loret E , Zalewski M , Pagés J M
Published: November 2016

The major outer membrane protein (MOMP) of Campylobacter jejuni was purified to homogeneity by selective solubilization and fast protein liquid chromatography. The amino acid composition of the MOMP indicates the presence of cysteine residues. The amino-terminal sequence, determined over 31 residues, shows no significant homology with any other porin from gram-negative bacteria except in a discrete region. Immunocross-reactivity between Escherichia coli OmpC and the MOMP was analyzed, and a common antigenic site between these two porins was identified with an anti-peptide antibody. From circular dichroism and immunological investigations, the existence of a stable folded monomer, containing a high level of beta-sheet secondary structure, is evident. Conformational analyses show the presence of a native trimeric state generated by association of the three folded monomers; the stability of this trimer is reduced compared with that of E. coli porins. This study clearly reveals that the C. jejuni MOMP is related to the family of trimeric bacterial porins.
Host growth temperature and a conservative amino acid substitution in the replication protein of pPS10 influence plasmid host range
Authors: Fernández-Tresguerres M E , Martín M , García de Viedma D , Giraldo R , Díaz-Orejas R
Published: November 2016

pPS10 is a replicon isolated from Pseudomonas syringe pv. savastanoi that can be established at 37 degrees C efficiently in Pseudomonas aeruginosa but very inefficiently in Escherichia coli. The establishment of the wild-type pPS10 replicon in E. coli is favored at low temperatures (30 degrees C or below). RepA protein of pPS10 promotes in vitro plasmid replication in extracts from E. coli, and this replication depends on host proteins DnaA, DnaB, DnaG, and SSB. Mutant plasmids able to efficiently replicate in E. coli at 37 degrees C were obtained. Three of four mutants whose mutations were mapped show a conservative Ala-->Val change in the amino-terminal region of the replication protein RepA. Plasmids carrying this mutation maintain the capacity to replicate in P. aeruginosa and have a fourfold increase in copy number in this host. The mutation does not substantially alter the autoregulation mediated by RepA. These results show that the physiological conditions of the host as well as subtle changes in the plasmid replication protein can modulate the host range of the pPS10 replicon.
Differential DNA secondary structure-mediated deletion mutation in the leading and lagging strands
Authors: Rosche W A , Trinh T Q , Sinden R R
Published: November 2016

The frequencies of deletion of short sequences (mutation inserts) inserted into the chloramphenicol acetyl-transferase (CAT) gene were measured for pBR325 and pBR523, in which the orientation of the CAT gene was reversed, in Escherichia coli. Reversal of the CAT gene changes the relationship between the transcribed strand and the leading and lagging strands of the DNA replication fork in pBR325-based plasmids. Deletion of these mutation inserts may be mediated by slipped misalignment during DNA replication. Symmetrical sequences, in which the same potential DNA structural misalignment can form in both the leading and lagging strands, exhibited an approximately twofold difference in the deletion frequencies upon reversal of the CAT gene. Sequences that contained an inverted repeat that was asymmetric with respect to flanking direct repeats were designed. With asymmetric mutation inserts, different misaligned structural intermediates could form in the leading and lagging strands, depending on the orientation of the insert and/or of the CAT gene. When slippage could be stabilized by a hairpin in the lagging strand, thereby forming a three-way junction, deletion occurred by up to 50-fold more frequently than when this structure formed in the leading strand. These results support the model that slipped misalignment involving DNA secondary structure occurs preferentially in the lagging strand during DNA replication.
Characterization of the duplicate ribulose-1,5-bisphosphate carboxylase genes and cbb promoters of Alcaligenes eutrophus
Authors: Kusian B , Bednarski R , Husemann M , Bowien B
Published: November 2016

Autotrophic CO2 fixation via the Calvin carbon reduction cycle in Alcaligenes eutrophus H16 is genetically determined by two highly homologous cbb operons, one of which is located on the chromosome and the other on megaplasmid pHG1 of the organism. An activator gene, cbbR, lies in divergent orientation only 167 bp upstream of the chromosomal operon and controls the expression of both cbb operons. The two 5'-terminal genes of the operons, cbbLS, coding for ribulose-1,5-bisphosphate carboxylase/oxygenase, were sequenced. Mapping of the 5' termini of the 2.1-kb cbbLS transcripts by primer extension and by nuclease S1 treatment revealed a single transcriptional start point at the same relative position for the chromosomal and plasmid-borne cbb operons. The derived cbb operon promoter showed similarity to sigma 70-dependent promoters of Escherichia coli. For the 1.4-kb transcripts of cbbR, the transcriptional start points were different in autotrophic and heterotrophic cells. The two corresponding cbbR promoters overlapped the cbb operon promoter and also displayed similarities to sigma 70-dependent promoters. The deficient cbbR gene located on pHG1 was transcribed as well. A newly constructed double operon fusion vector was used to determine the activities of the cbb promoters. Fusions with fragments carrying the cbb intergenic control regions demonstrated that the cbb operon promoters were strongly regulated in response to autotrophic versus heterotrophic growth conditions. In contrast, the cbbR promoters displayed low constitutive activities. The data suggest that the chromosomal and plasmid-borne cbb promoters of A. eutrophus H16 are functionally equivalent despite minor structural differences.
Chemical characterization of the ester-linked 3-hydroxy fatty acyl-containing lipids in Mycobacterium tuberculosis
Authors: Alugupalli S , Lanéelle M A , Larsson L , Daffé M
Published: November 2016

In a previous study (S. Alugupalli, F. Portaels, and L. Larsson, J. Bacteriol. 176:2962-2969, 1994), we reported the occurrence of 21 3-hydroxy fatty acids (3-OH-FAs) in the methanolysis products of different mycobacterial species. The present study was undertaken in order to chemically characterize the ester-linked complex native forms of these acids in Mycobacterium tuberculosis. Three 3-OH-FA-containing lipids were purified by chromatography and analyzed by one- and two-dimensional nuclear magnetic resonance spectroscopy, by fast atom bombardment-mass spectrometry, and by various conventional chemical analysis techniques. 3-OH-2,4,6-trimethyl-FAs were found in 2,3-diacyl trehalose and 2,3,6,6'-tetra-acyl-2'-sulfate trehalose (sulfatide I), two specific glycolipids of the tubercle bacillus, explaining the specific occurrence of these fatty acids in the methanolysis products of virulent strains of M. tuberculosis. Straight-chain 3-OH-FAs were localized in phosphatidyl ethanolamine but not in the other phospholipids, suggesting a possible role of this class of phospholipids in the metabolism of fatty acids in actinomycetes.
Mutation of the Ser2 codon of the light-harvesting B870 alpha polypeptide of Rhodobacter capsulatus partially suppresses the pufX phenotype
Authors: Lilburn T G , Prince R C , Beatty J T
Published: November 2016

The exact function of the pufX gene product of Rhodobacter capsulatus is uncertain, but deletion of the pufX gene renders cells incapable of phototrophic growth on a minimal medium, and photosynthetic electron transfer is impaired in vitro. However, suppressor mutants that are able to grow phototropically are readily isolated. Two such suppressor mutants were characterized as to their phototrophic growth properties, their fluorescence at different incident light intensities, the integrity of their chromatophores, and their abilities to generate a transmembrane potential. We found that the photosynthetic apparatus in the suppressor mutants was less stable than that of the pseudo-wild-type and primary mutant strains and that the suppressor mutants used light energy less efficiently than the pseudo-wild-type strain. Therefore, the suppressor strains are more precisely designated partial suppressor mutants. The locations and sequences of the suppressor mutations were determined, and both were found to change the second codon of the pufA gene. It is hypothesized that the serine residue specified by this codon is important in interactions between the B870 alpha protein and other membrane-bound polypeptides and that suppressor mutations at this position partially compensate for loss of the PufX protein. A model is proposed for the function of the PufX protein.
The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spo0A
Authors: Mandic-Mulec I , Doukhan L , Smith I
Published: November 2016

SinR is a pleiotropic DNA binding protein that is essential for the late-growth processes of competence and motility in Bacillus subtilis and is also a repressor of others, e.g., sporulation and subtilisin synthesis. In this report, we show that SinR, in addition to being an inhibitor of sporulation stage II gene expression, is a repressor of the key early sporulation gene spo0A. The sporulation-specific rise in spo0A expression at time zero is absent in a SinR-overproducing strain and is much higher than normal in strains with a disrupted sinR gene. This effect is direct, since SinR binds specifically to spo0A in vitro, in a region overlapping the -10 region of the sporulation-specific Ps promoter that is recognized by E-sigma H polymerase. Methyl interference and site-directed mutagenesis studies have identified guanine residues that are important for SinR recognition of this DNA sequence. Finally, we present evidence that SinR controls sporulation through several independent genes, i.e., sp0A, spoIIA, and possibly spoIIG and spoIIE.
Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum
Authors: Farwick M , Siewe R M , Krämer R
Published: November 2016

Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (Km, 8.6 +/- 0.4 microM) with high maximum velocity (110 nmol.min-1.mg [dry weight]-1). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na+ ions. It is thus driven both by the membrane potential and the Na+ gradient. An extremely high accumulation (internal/external) ratio of up to 4 x 10(6) was measured, which represents the highest accumulation ratio observed for any transport system.
Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR
Authors: Shin S , Park C
Published: November 2016

During the search for unknown factors involved in motility, we have found that expression of the flagellar master operon flhDC is affected by mutations of the pta and ackA genes, encoding phosphotransacetylase and acetate kinase, respectively (S. Shin, J. Sheen, and C. Park, Korean J. Microbiol. 31:504-511, 1993). Here we describe results showing that this effect is modulated by externally added acetate, except when both pta and ackA are mutated, suggesting the role of acetyl phosphate, an intermediate of acetate metabolism, as a regulatory effector. Furthermore, the following evidence indicates that the phosphorylation of OmpR, a trans factor for osmoregulation, regulates flagellar expression. First, in a strain lacking ompR, the expression of flhDC is no longer responsive to a change in the level of acetyl phosphate. Second, an increase in medium osmolarity does not decrease flhDC expression in an ompR mutant. It is known that such an increase normally enhances OmpR phosphorylation. Third, OmpR protein binds to the DNA fragment containing the flhDC promoter, and its affinity is increased with phosphorylation by acetyl phosphate. DNase I footprinting revealed the regions of the flhDC promoter protected by OmpR in the presence or absence of phosphorylation. Therefore, we propose that the phosphorylated OmpR, generated by either osmolarity change or the internal level of acetyl phosphate, negatively regulates the expression of flagella.
Sterigmatocystin biosynthesis in Aspergillus nidulans requires a novel type I polyketide synthase
Authors: Yu J H , Leonard T J
Published: November 2016

A filamentous fungus, Aspergillus nidulans, produces the carcinogenic mycotoxin sterigmatocystin (ST), which is a polyketide-derived secondary metabolite. A gene (pksST) encoding the ST polyketide synthase (PKSst) in A. nidulans was cloned, sequenced, and characterized. Large induced deletion mutants, which did not make ST or any ST intermediates, were used to identify genes associated with ST biosynthesis. Among the transcripts detected within the deletion region, which showed developmental expression with ST production, was a 7.2-kb transcript. Functional inactivation of the gene encoding the 7.2-kb transcript blocked production of ST and all ST intermediate substrates but did not affect transcription of the pathway genes, indicating that this gene was involved in a very early step of ST biosynthesis. These results also indicate that PKSst was not associated with activation of other ST genes. Sequencing of the region spanning this gene revealed that it encoded a polypeptide with a deduced length of 2,181 amino acids that had high levels of similarity to many of the known polyketide synthases and FASs. This gene, pksST, encodes a multifunctional novel type I polyketide synthase which has as active sites a beta-ketoacyl acyl carrier protein synthase, an acyltransferase, duplicated acyl carrier proteins, and a thioesterase, all of these catalytic sites may be multiply used. In addition, a 1.9-kb transcript, which also showed developmental expression, was mapped adjacent to pksST, and the sequence of this gene revealed that it encoded a cytochrome P-450 monooxygenase-like peptide.
Transcriptional induction and expression of the endoglucanase celA gene from a ruminal Clostridium sp. ("C. longisporum")
Authors: Mittendorf V , Thomson J A
Published: November 2016

Northern (RNA) blot analysis of RNA from Clostridium sp. revealed induction of transcription of the celA gene when barley beta-glucan was used as carbon source, while no celA mRNA was detected after growth on cellobiose. Western blots (immunoblots), prepared by using a rabbit antiserum raised against CelA protein purified from Escherichia coli, revealed the extracellular location of CelA in Clostridium sp. Despite the absence of detectable celA mRNA, significant quantities of CelA were detected in the culture supernatant during growth on cellobiose. This finding indicated a low constitutive expression of celA. A 6.7-fold increase in the total beta-glucanase specific activity in the extracellular fraction was observed during growth on beta-glucan. The transcriptional start site of celA was mapped by extension and was found to be the same in Clostridium sp. and in E. coli expressing the cloned celA gene. A consensus E. coli -10 promoter region (AATAAT), but not a -35 promoter region, could be identified. Two direct repeats (TATTGAATTTAT) separated by 15 nucleotides flank the region where the consensus -35 promoter regions would have been. The size of the celA mRNA transcript corresponded with the size of the open reading frame. A potential stem-loop structure was found 18 nucleotides downstream of the 3' stop codon, which could be responsible for termination of transcription.
Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor
Authors: Pomposiello P J , Bender R A
Published: November 2016

A chimeric promoter with the nitrogen assimilation control protein binding site from hutUp of Klebsiella aerogenes fused to the lacZ core promoter from Escherichia coli was built and cloned in a lacZ reporter plasmid. This construct showed a 14-fold increase of beta-galactosidase activity upon nitrogen limitation. Primer extension experiments showed that the nitrogen assimilation control protein activates lacZp1 in a position-dependent manner.
Transcription of spoIVB is the only role of sigma G that is essential for pro-sigma K processing during spore formation in Bacillus subtilis
Authors: Gomez M , Cutting S , Stragier P
Published: November 2016

Activation of pro-sigma K processing in the mother cell at late stages of sporulation in Bacillus subtilis requires the presence of active sigma G in the forespore. Placing the spoIVB gene under the control of sigma F, the early forespore transcription factor, allows sigma K to become active in the absence of sigma G. Therefore, transcription of spoIVB is the only role of sigma G that is essential for the signaling pathway between sigma G and sigma K.
Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product
Authors: Mori H , Iida A , Teshiba S , Fujio T
Published: November 2016

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).
Capsular polysaccharide biosynthesis and pathogenicity in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer
Authors: Beck von Bodman S , Farrand S K
Published: November 2016

N-Acylhomoserine lactone (acyl-HSL)-mediated gene expression, also called autoinduction, is conserved among diverse gram-negative bacteria. In the paradigm Vibrio fischeri system, bioluminescence is autoinducible, and the lux operon requires the transcriptional activator LuxR and the acyl-HSL autoinducer for expression. The production of the acyl-HSL signal molecule is conferred by the luxI gene, and luxR encodes the transcriptional regulator. We show here that Erwinia stewartii, the etiological agent of Stewart's wilt of sweet corn, synthesizes an acyl-HSL. Mass spectral analysis identified the signal molecule as N-(-3-oxohexanoyl)-L-homoserine lactone, which is identical to the V. fischeri autoinducer. We have cloned and sequenced the gene that confers acyl-HSL biosynthesis, called esaI, and the linked gene, esaR, that encodes a gene regulator. The two genes are convergently transcribed and show an unusual overlap of 31 bp at their 3' ends. Sequence analysis indicates that EsaI and EsaR are homologs of LuxI and LuxR, respectively. EsaR can repress its own expression but seems not to regulate the expression of esaI. The untranslated 5' region of esaR contains an inverted repeat with similarity to the lux box-like elements located in the promoter regions of other gene systems regulated by autoinduction. However, unlike the other systems, in which the inverted repeats are located upstream of the -35 promoter elements, the esaR-associated repeat overlaps a putative -10 element. We mutagenized the esaI gene in E. stewartii by gene replacement. The mutant no longer produced detectable levels of the acyl-HSL signal, leading to a concomitant loss of extracellular polysaccharide capsule production and pathogenicity. Both phenotypes were restored by complementation with esal or by exogenous addition of the acyl-HSL.
Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus
Authors: Kondo K , Beppu T , Horinouchi S
Published: November 2016

The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.
The glutamate uptake regulatory protein (Grp) of Zymomonas mobilis and its relation to the global regulator Lrp of Escherichia coli
Authors: Peekhaus N , Tolner B , Poolman B , Krämer R
Published: November 2016

After being expressed in Escherichia coli JC5412, which is defective in glutamate transport, a Zymomonas mobilis gene which enabled this strain to grow on glutamate was cloned. This gene encodes a protein with 33% amino acid identity to the leucine-responsive regulatory protein (Lrp) of E. coli. Although overall glutamate uptake in E. coli was increased, the protein encoded by the cloned fragment repressed the secondary H+/glutamate transport system GltP by interaction with the promoter region of the gltP gene. It also repressed the secondary, H(+)-coupled glutamate uptake system of Z. mobilis, indicating that at least one role of this protein in Z. mobilis is to regulate glutamate transport. Consequently, it was designated Grp (for glutamate uptake regulatory protein). When expressed in E. coli, Grp repressed the secondary H+/glutamate transport system GltP by binding to the regulatory regions of the gltP gene. An lrp mutation in E. coli was complemented in trans with respect to the positive expression regulation of ilvIH (coding for acetohydroxy acid synthase III) by a plasmid which carries the grp gene. The expression of grp is autoregulated, and in Z. mobilis, it depends on growth conditions. The putative presence of a homolog of Grp in E. coli is discussed.
The interaction of T4 endonuclease V E23Q mutant with thymine dimer- and tetrahydrofuran-containing DNA
Authors: Latham K A , Manuel R C , Lloyd R S
Published: November 2016

The interaction between endonuclease V, the cyclobutane pyrimidine dimer-specific N-glycosylase/abasic lyase from bacteriophage T4, and DNA was investigated by DNase I footprinting methods. The catalytically inactive mutant E23Q was found to interact with a smaller region of DNA at the abasic site analog, tetrahydrofuran, than at a thymine dimer site. Like the wild-type enzyme, the mutant contacted the DNA substrates primarily on the strand opposite the damage. The various complexes examined by footprinting techniques represent distinct points along the catalytic pathway of endonuclease V: before catalysis at a dimer, after N-glycosylase action but before abasic lyase action, and before catalysis at an abasic site. The differences between the footprints of the mutant and wild-type enzymes on both DNA substrates likely represent subtly different conformations within these complexes.
A partially functional 245-amino-acid internal deletion derivative of Escherichia coli sigma 70
Authors: Kumar A , Williamson H S , Fujita N , Ishihama A , Hayward R S
Published: November 2016

Two hundred forty-five consecutive amino acids of the sigma 70 subunit of Escherichia coli RNA polymerase are not conserved in the homologous protein of Bacillus subtilis. We show that their deletion from a sigma 70-32 hybrid protein caused no severe loss of function in vivo, while sigma 70 itself retained considerable function in vitro.
Growth phase-dependent induction of stationary-phase promoters of Escherichia coli in different gram-negative bacteria
Authors: Miksch G , Dobrowolski P
Published: November 2016

RSF1010-derived plasmids carrying a fusion of a promoterless lacZ gene with the sigma s-dependent growth phase-regulated promoters of Escherichia coli, bolAp1 and fic, were constructed. The plasmids were mobilized into the gram-negative bacterial species Acetobacter methanolicus, Xanthomonas campestris, Pseudomonas putida, and Rhizobium meliloti. The beta-galactosidase activities of bacterial cultures were determined during exponential and stationary growth phases. Transcriptional activation of the fic promoter in the different bacteria was growth phase dependent as in E. coli and was initiated generally during the transition to stationary phase. The induction of the bolA promoter was also growth phase dependent in the bacteria tested. While the expression in E. coli and R. meliloti was initiated during the transition from exponential to stationary phase, the induction in A. methanolicus, P. putida, and X. campestris started some hours after stationary growth phase was reached. In all the species tested, DNA fragments hybridizing with the rpoS gene of E. coli were detected. The results show that in different gram-negative bacteria, stationary-phase-specific sigma factors which are structurally and functionally homologous to sigma s and are able to recognize the promoter sequences of both bolA and fic exist.
Pyridine carboxylic acids as inhibitors and substrates of the Escherichia coli gab permease encoded by gabP
Authors: King S C , Fleming S R , Brechtel C
Published: November 2016

Although considered selective for its natural substrate, 4-aminobutyrate, gab permease was inhibited by 1,2,3,6-tetrahydro-3-pyridinecarboxylate and 1,2,3,6-tetrahydro-4-pyridinecarboxylate. The former is a transported substrate, since its preloading into metabolically poisoned cells stimulated transient accumulation of 4-aminobutyrate via counterflow.
Inducible expression of a gene encoding an L41 ribosomal protein responsible for the cycloheximide-resistant phenotype in the yeast Candida maltosa
Authors: Mutoh E , Mochizuki M , Ohta A , Takagi M
Published: November 2016

In a previous paper (S. Kawai, S. Murao, M. Mochizuki, I. Shibuya, K. Yano, and M. Takagi, J. Bacteriol. 174:254-262, 1992), we showed that in each genome of several yeast species, there is one of two types of L41 gene, one for an L41 (Q-type) protein which confers cycloheximide (CYH) resistance or one for an L41 (P-type) protein which does not. These genes have been suggested to be responsible for the CYH response used in taxonomy. For example, Saccharomyces cerevisiae, which is CYH sensitive, has a P-type L41 gene, while Kluyveromyces fragilis and Candida maltosa, which are CYH resistant, have Q-type L41 genes. However, in contrast to K. fragilis, which is constitutively resistant to CYH, C. maltosa is inducibly resistant to CYH. Here, we show that C. maltosa has both types of the L41 gene in its genome and that expression of the Q-type L41 gene is induced by CYH while the P-type L41 gene is constitutively expressed.
The gene for the longest known Escherichia coli protein is a member of helicase superfamily II
Authors: Reuven N B , Koonin E V , Rudd K E , Deutscher M P
Published: November 2016

The Escherichia coli rnt gene, which encodes the RNA-processing enzyme RNase T, is cotranscribed with a downstream gene. Complete sequencing of this gene indicates that its coding region encompasses 1,538 amino acids, making it the longest known protein in E. coli. The gene (tentatively termed lhr for long helicase related) contains the seven conserved motifs of the DNA and RNA helicase superfamily II. An approximately 170-kDa protein is observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-labeled extracts prepared from cells in which lhr is under the control of an induced T7 promoter. This protein is absent when lhr is interrupted or when no plasmid is present. Downstream of lhr is the C-terminal region of a convergent gene with homology to glutaredoxin. Interruptions of chromosomal lhr at two different positions within the gene do not affect the growth of E. coli at various temperatures in rich or minimal medium, indicating that lhr is not essential for usual laboratory growth. lhr interruption also has no effect on anaerobic growth. In addition, cells lacking Lhr recover normally from starvation, plate phage normally, and display normal sensitivities to UV irradiation and H2O2. Southern analysis showed that no other gene closely related to lhr is present on the E. coli chromosome. These data expand the known size range of E. coli proteins and suggest that very large helicases are present in this organism.
Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7
Authors: Moens S , Michiels K , Keijers V , Van Leuven F , Vanderleyden J
Published: November 2016

Azospirillum brasilense can display a single polar flagellum and several lateral flagella. The A. brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced. The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus. An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins. A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette. The mutant is devoid of lateral flagella but still forms the polar flagellum. This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium. FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction.
Isolation and characterization of a conserved porin protein from Helicobacter pylori
Authors: Doig P , Exner M M , Hancock R E , Trust T J
Published: November 2016

Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed.
Motility and thermotactic responses of Thermotoga maritima
Authors: Gluch M F , Typke D , Baumeister W
Published: November 2016

Thermotoga maritima, a thermophilic eubacterium, is motile at temperatures ranging from 50 to 105 degrees C. The cells are propelled by a single flagellum which most of the time spins clockwise. Changes in the swimming direction ("tumbles") are achieved by short reversals of the direction of filament rotation. The average speed of swimming cells depends on the temperature, reaching a maximum value of about 60 microns/s at 85 degrees C. The cells show a thermotactic response to temporal temperature changes. When the temperature is raised, the rate of tumbles is increased, while decreasing temperature decreases the tumbling rate.
Physical map of the genome of the green phototrophic bacterium Chlorobium tepidum
Authors: Naterstad K , Kolstø A B , Sirevåg R
Published: November 2016

A physical restriction map of the chromosome of the green sulfur bacterium Chlorobium tepidum was generated by determining the order of the fragments obtained after digestion with the restriction endonucleases XbaI and PacI and subsequent separation of the fragments by pulsed-field gel electrophoresis. The size of the chromosome is estimated to be 2.1 Mb. Fifteen genes and operons, mainly encoding proteins involved in photosynthesis, have been placed on this map by hybridization to fragments obtained after single- and double-restriction digestions.
Repression of the Klebsiella aerogenes nac promoter
Authors: Feng J , Goss T J , Bender R A , Ninfa A J
Published: November 2016

Transcription of the nitrogen-regulated nac promoter of Klebsiella aerogenes requires sigma54 RNA polymerase, is activated by the phosphorylated form of the transcription factor nitrogen regulator I (NRI) (NtrC), and is repressed by the product of the nac gene, Nac. Nac protects a large portion of the nac control region, extending from positions -130 to -70, from digestion by DNase I. This site(s) lies immediately upstream from the site at which sigma 54 RNA polymerase binds, is downstream of a high-affinity binding site for the transcriptional activator NRI approximately P, and partially overlaps a low-affinity NRI approximately P-binding site. Binding of Nac to the DNA resulted in bending of the DNA but did not interfere with the binding of sigma 54 RNA polymerase to the promoter or with the binding of NRI approximately P to either the high-affinity site or low-affinity site. Furthermore, transcription assays with various wild-type and mutant templates suggested that Nac did not exclude NRI approximately P from either the low- or high-affinity sites, nor did Nac interfere with the ability of the polymerase to form the open complex when the binding sites for NRI approximately P were moved to different locations upstream from the promoter. Rather, Nac seemed to repress by an antiactivation mechanism in which the interaction of the NRI approximately P, bound at its normal sites, with sigma 54 RNA polymerase, bound to the promoter, was prevented.
Cloning and characterization of a region of Enterococcus faecalis plasmid pPD1 encoding pheromone inhibitor (ipd), pheromone sensitivity (traC), and pheromone shutdown (traB) genes
Authors: Nakayama J , Yoshida K , Kobayashi H , Isogai A , Clewell D B , Suzuki A
Published: November 2016

Bacteriocin plasmid pPD1 in Enterococcus faecalis encodes a mating response to recipient-produced sex pheromone cPD1. Once a recipient acquires pPD1, transconjugants apparently shut off cPD1 activity in broth culture and no longer behave as recipients for pPD1. This event is performed by synthesis of the pheromone inhibitor iPD1 and also by repression of cPD1 production, the so-called "pheromone shutdown." A 5.4-kb EcoRV-HincII segment of pPD1, which expressed iPD1 in Escherichia coli, was sequenced and found to be organized as traC-traB-traA-ipd; each open reading frame is analogous to that found in other pheromone plasmids, pAD1 and pCF10, and thus is designated in accordance with the nomenclature in pAD1. The ipd gene encodes a peptide consisting of 21 amino acids, in which the C-terminal eight residues correspond to iPD1. The putative TraC product has a strong similarity to oligopeptide-binding proteins found in other bacterial species, as do pheromone-binding proteins of pCF10 and pAD1. A strain carrying traC-disrupted pPD1 required a concentration of cPD1 fourfold higher than that needed by the wild-type strain for induction of sexual aggregation. These results suggest that the TraC product contributes to pheromone sensitivity as a pheromone-binding protein. A strain transformed with traB-disrupted pPD1 produced a high level of cPD1 similar to that produced by plasmid-free recipients and underwent self-induction. Thus, the TraB product contributes to cPD1 shutdown.
Regulation of the putative bglPH operon for aryl-beta-glucoside utilization in Bacillus subtilis
Authors: Krüger S , Hecker M
Published: November 2016

The expression of the putative operon bglPH of Bacillus subtilis was studied by using bglP'-lacZ transcriptional fusions. The bglP gene encodes an aryl-beta-glucoside-specific enzyme II of the phosphoenolpyruvate sugar:phosphotransferase system, whereas the bglH gene product functions as a phospho-beta-glucosidase. Expression of bglPH is regulated by at least two different mechanisms: (i) carbon catabolite repression and (ii) induction via an antitermination mechanism. Distinct deletions of the promoter region were created to determine cis-acting sites for regulation. An operatorlike structure partially overlapping the -35 box of the promoter of bglP appears to be the catabolite-responsive element of this operon. The motif is similar to that of amyO and shows no mismatches with respect to the consensus sequence established as the target of carbon catabolite repression in B. subtilis. Catabolite repression is abolished in both ccpA and ptsH1 mutants. The target of the induction by the substrate, salicin or arbutin, is a transcriptional terminator located downstream from the promoter of bglP. This structure is very similar to that of transcriptional terminators which regulate the induction of the B. subtilis sacB gene, the sacPA operon, and the Escherichia coli bgl operon. The licT gene product, a member of the BglG-SacY family of antitermination proteins, is essential for the induction process. Expression of bglP is under the negative control of its own gene product. The general proteins of the phosphoenolpyruvate-dependent phosphotransferase system are required for bglP expression. Furthermore, the region upstream from bglP, which reveals a high AT content, exerts a negative regulatory effect on bglP expression.
Rapid expansion of the physical and genetic map of the chromosome of Clostridium perfringens CPN50
Authors: Katayama S , Dupuy B , Garnier T , Cole S T
Published: November 2016

The physical map of the 3.6-megabase chromosome of Clostridium perfringens CPN50 was extended by positioning sites for the endonucleases SfiI and I-CeuI, and in parallel, the gene map was expanded by using a genome scanning strategy. This involved the cloning and sequencing of random chromosomal fragments, identification of the functions of the putative genes by database searches, and then hybridization analysis. The current gene map comprises almost 100 markers, many of which encode housekeeping functions while others are involved in sporulation or pathogenesis. Strikingly, most of the virulence genes were found to be confined to a 1,200-kb segment of the chromosome near oriC, while the pleiotropic regulatory locus, virRS, was situated toward the putative replication terminus. A comparison of the gene maps of three endospore-forming bacilli, C. perfringens, Clostridium beijerinckii, and Bacillus subtilis, revealed a similar order and distribution of key sporulation and heat shock genes which might reflect an ancient evolutionary relationship.
Sites required for GltC-dependent regulation of Bacillus subtilis glutamate synthase expression
Authors: Belitsky B R , Janssen P J , Sonenshein A L
Published: November 2016

The Bacillus subtilis gltAB genes, coding for the two subunits of glutamate synthase, are transcribed divergently from the gltC gene, encoding a LysR-type transcriptional activator of gltAB. The predicted gltA and gltC transcription start sites are separated by 51 to 52 bp. A 15-bp, consensus binding site (Box I) for LysR-type proteins was found centered at position -64 with respect to the gltA transcription start. This site was shown by mutational analysis to be required both for GltC-mediated activation of gltA and for autorepression of gltC. Box II, which is similar to Box I, is centered 22 bp downstream of Box I and overlaps the -35 region of the gltA promoter. Box II was found to be essential for activation of gltA but not for gltC autoregulation. Introduction of approximately one additional helical turn of DNA between Box I and Box II enhanced gltA expression 7- to 40-fold under nonactivating conditions and about 2-fold under activating conditions. Expression of gltA was dramatically decreased when the distance between Box I and Box II was altered by a nonintegral number of helical turns of DNA. gltC autorepression was abolished by most of the inserts between Box I and Box II but was augmented by adding one helical turn.
Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis
Authors: Ponce E , Flores N , Martinez A , Valle F , Bolívar F
Published: November 2016

We report the cloning of the pykA and pykF genes from Escherichia coli, which code for the two pyruvate kinase isoenzymes (ATP:pyruvate 2-O-phosphotransferases; EC 2.7.1.40) in this microorganism. These genes were insertionally inactivated with antibiotic resistance markers and utilized to interrupt one or both pyk genes in the E. coli chromosome. With these constructions, we were able to study the role of these isoenzymes in pyruvate biosynthesis.
Two Lactococcus lactis genes, including lacX, cooperate to trigger an SOS response in a recA-negative background
Authors: Huang X F , Huang D C , Novel G , Novel M
Published: November 2016

A 4.3-kb EcoRI fragment from a Lactococcus lactis genomic library alleviates the methyl methanesulfonate, mitomycin C, and UV sensitivities of an Escherichia coli recA mutant (M. Novel, X. F. Huang, and G. Novel, FEMS Microbiol. Lett. 72:309-314, 1990). It complements recA1 and delta recA mutations but not recA13. Three proteins (with molecular masses of 20, 35, and 23 kDa) were produced from this fragment in a T7-directed system, and three corresponding genes were detected by DNA sequencing, namely, ISS1CH;lacX, which is the distal gene of the lac operon; and a third open reading frame, named lacN, which encodes 211 amino acids. Mutations produced in either lacX or in lacN resulted in the loss of the resistance to DNA-damaging agents. Thus, these two genes appeared to be involved in this activity. Introduction of pUCB214 carrying the 4.3-kb fragment into a lexA+ delta recA306 sfiA::lacZ strain resulted in UV-inducible synthesis of beta-galactosidase. A uvrA strain or a lexA (Ind-) strain containing pUCB214 did not support any DNA repair. However, a lexA (Def-) strain carrying pUCB214 could partly repair UV damage. We discuss possible targets for LacX and LacN products, and we speculate that LacX and LacN may constitute a two-component regulatory system that is able to respond to SOS signals, and then to act in the SOS response, bypassing the RecA-activated function.
Isolation and characterization of mutants impaired in the selective degradation of peroxisomes in the yeast Hansenula polymorpha
Authors: Titorenko V I , Keizer I , Harder W , Veenhuis M
Published: November 2016

We have isolated a collection of peroxisome degradation-deficient (Pdd-) mutants of the yeast Hansenula polymorpha which are impaired in the selective autophagy of alcohol oxidase-containing peroxisomes. Two genes, designated PDD1 and PDD2, have been identified by complementation and linkage analyses. In both mutant strains, the glucose-induced proteolytic turnover of peroxisomes is fully prevented. The pdd1 and pdd2 mutant phenotypes were caused by recessive monogenic mutations. Mutations mapped in the PDD1 gene appeared to affect the initial step of peroxisome degradation, namely, sequestration of the organelle to be degraded by membrane multilayers. Thus, Pdd1p may be involved in the initial signalling events which determine which peroxisome will be degraded. The product of the PDD2 gene appeared to be essential for mediating the second step in selective peroxisome degradation, namely, fusion and subsequent uptake of the sequestered organelles into the vacuole. pdd1 and pdd2 mutations showed genetic interactions which suggested that the corresponding gene products may physically or functionally interact with each other.