bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1
Authors: Whitfield C , Richards J C , Perry M B , Clarke B R , MacLean L L
Published: November 2016

The lipopolysaccharide (LPS) molecule is an important virulence determinant in Klebsiella pneumoniae. Studies on the serotype O1 LPS were initiated to determine the basis for antigenic heterogeneity previously observed in the O1 side chain polysaccharides and to resolve apparent ambiguities in the reported polysaccharide structure. Detailed chemical analysis, involving methylation and 1H- and 13C-nuclear magnetic resonance studies, demonstrated that the O-side chain polysaccharides of serotype O1 LPS contained a mixture of two structurally distinct D-galactan polymers. The repeating unit structures of these two polymers were identified as [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] (D-galactan I) and [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] (D-Galactan II). D-Galactan I polysaccharides were heterogeneous in size and were detected throughout the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) profile of O1 LPS. In contrast, D-galactan II was confined to the higher-molecular-weight region. The structures of the two D-galactans were not influenced by simultaneous synthesis of a capsular K antigen. Apparently, neither of the D-galactans constitutes a common antigen widespread in Klebsiella spp. as determined by immunochemical analysis. Examination of the LPSs in mutants indicated that expression of D-galactan I can occur independently of D-galactan II. Transconjugants of Escherichia coli K-12 strains carrying the his region of K. pneumoniae were constructed by chromosome mobilization with RP4::mini-Mu. In these transconjugants, the O antigen encoded by the his-linked rfb locus was determined to be D-galactan I, suggesting that genes involved in the expression of D-galactan II are not closely linked to the rfb cluster.
Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors
Authors: Simon R , Hötte B , Klauke B , Kosier B
Published: November 2016

On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliloti strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.
In situ assay of ribulose-1,5-bisphosphate carboxylase/oxygenase in Thiobacillus neapolitanus
Authors: Cannon G C , English R S , Shively J M
Published: November 2016

Cells permeabilized with chloroform yielded ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activities nearly equal to those of cell extracts, thus indicating that both cytoplasmic and carboxysomal RuBisCO are functional in situ. The carboxysomal and cytoplasmic RuBisCO both form the CO2-Mg2(+)-enzyme ternary complex, as evidenced by stabilization with 2-C-carboxy-D-arabinitol-1,5-bisphosphate (CABP), a potent competitive inhibitor of RuBisCO. The data are consistent with the hypothesis that the carboxysome is functional in carbon dioxide fixation.
Tn5253, the pneumococcal omega (cat tet) BM6001 element, is a composite structure of two conjugative transposons, Tn5251 and Tn5252
Authors: Ayoubi P , Kilic A O , Vijayakumar M N
Published: November 2016

Tn5253, carrying tetracycline and chloramphenicol resistance determinants, is a 65.5-kb conjugative transposon originally detected in the chromosome of Streptococcus pneumoniae BM6001. We have identified an 18-kb segment of DNA carrying the tet determinant within Tn5253 to be an independent conjugative transposon when removed from the context of the larger element. In vivo deletion of this DNA segment, now termed Tn5251, from within Tn5253 did not affect the conjugative transposition properties of the remaining sequences. Thus, Tn5253 is a composite element of two conjugative structures: Tn5252, constituting the sequences beyond Tn5251 within Tn5253, and Tn5251. The transfer properties of Tn5252 and Tn5251 suggest that these may belong to two different classes of mobile elements even though they were initially found associated. The notion that a tet-carrying transposon like Tn5251 may have been the ancestral element in the evolution of the larger streptococcal conjugative transposons must be reevaluated in the light of present observations.
Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA
Authors: Setlow B , Hand A R , Setlow P
Published: November 2016

Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores.
Acquisition of thymidylate by the obligate intracytoplasmic bacterium Rickettsia prowazekii
Authors: Speed R R , Winkler H H
Published: November 2016

The pathway for the acquisition of thymidylate in the obligate bacterial parasite Rickettsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3H]thymidine added to the culture. In the thymidine kinase-negative host cells, the label available to the rickettsiae in the host cell cytoplasm would have been thymidine, and in the thymidine kinase-positive host cells, it would have been both thymidine and TMP. Further support for the inability to utilize thymidine was the lack of thymidine kinase activity in extracts of R. prowazekii. However, [3H]uridine incorporation into the DNA of R. prowazekii was demonstrable (973 +/- 57 dpm/3 x 10(8) rickettsiae). This labeling of rickettsial DNA suggests the transport of uracil, uridine, uridine phosphates (UXP), or 2'-deoxyuridine phosphates, the conversion of the labeled precursor to thymidylate, and subsequent incorporation into DNA. This is supported by the demonstration of thymidylate synthase activity in extracts of R. prowazekii. The enzyme was determined to have a specific activity of 310 +/- 40 pmol/min/mg of protein and was inhibited greater than or equal to 70% by 5-fluoro-dUMP. The inability of R. prowazekii to utilize uracil was suggested by undetectable uracil phosphoribosyltransferase activity and by its inability to grow (less than 10% of control) in a uridine-starved mutant cell line (Urd-A) supplemented with 50 microM to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 microM uridine (117% of control). However, no measurable uridine kinase activity could be measured in extracts of R. prowazekii. Normal rickettsial growth (92% of control) was observed when the host cell was blocked with thymidine so that the host cell's dUXP pool was depressed to a level inadequate for growth and DNA synthesis in the host cell. Taken together, these data strongly suggest that rickettsiae transport UXP from the host cell's cytoplasm and that they synthesize TTP from UXP.
Structure of Escherichia coli K-12 miaA and characterization of the mutator phenotype caused by miaA insertion mutations
Authors: Connolly D M , Winkler M E
Published: November 2016

Previously, we reported several unusual relationships between the 2-methylthio-N6-(delta 2-isopentenyl)adenosine-37 (ms2i6A-37) tRNA modification and spontaneous mutagenesis in Escherichia coli K-12 (D. M. Connolly and M. E. Winkler, J. Bacteriol. 171:3233-3246, 1989). To confirm and extend these observations, we determined the structure of miaA, which mediates the first step of ms2i6A-37 synthesis, and characterized the miaA mutator phenotype. The most likely translation start of miaA overlaps the last two codons of mutL, which encodes a protein required for methyl-directed mismatch repair. This structural arrangement confirms that miaA and mutL are in the same complex operon. The miaA gene product, delta 2-isopentenylpyrophosphate transferase, shows extensive homology with the yeast MOD5 gene product, and both enzymes contain a substrate binding site found in farnysyl pyrophosphate synthetase and a conserved putative ATP/GTP binding site. Insertions in miaA cause exclusively GC----TA transversions, which contrasts with the GC----AT and AT----GC transitions observed in mutL mutants. To correlate the absence of the ms2i6A-37 tRNA modification directly with the mutator phenotype, we isolated a unique suppressor of a leaky miaA(ochre) mutation. The miaD suppressor mapped to 99.75 min, restored the ms2i6A-37 tRNA modification to miaA(ochre) mutants, and abolished the miaA mutator phenotype. We speculate that miaD causes a decrease in ms2i6A-37 tRNA demodification or an increase in miaA gene expression but not at the level of operon transcription. Together, these observations support the idea that the ms2i6A-37 tRNA modification acts as a physiological switch that modulates spontaneous mutation frequency and other metabolic functions.
C-factor has distinct aggregation and sporulation thresholds during Myxococcus development
Authors: Kim S K , Kaiser D
Published: November 2016

C-factor, the protein product of the csgA gene, acts as a short-range morphogenetic signal. It is required for fruiting body development of the gram-negative bacterium Myxococcus xanthus. Aggregation, sporulation, and expression of a set of genes that are C-factor dependent, all of which fail in csgA mutant cells, are completely restored by addition of purified C-factor. We report here that, depending on its concentration, C-factor can elicit two distinct morphogenetic and transcriptional responses from csgA cells. Low levels of C-factor bring about aggregation and expression of an early C-dependent gene, whereas higher levels lead to the same effects plus expression of a late C-dependent gene and spore formation. C-factor positively regulates its own transcription. An approximately fourfold net increase in csgA transcription and C-factor levels during development was measured. We propose that autoregulation and the two distinct activity thresholds allow C-factor to act as a timer, first triggering aggregation, then sporulation, thereby producing the appropriate developmental order.
Purification and characterization of the hydrogen uptake hydrogenase from the hyperthermophilic archaebacterium Pyrodictium brockii
Authors: Pihl T D , Maier R J
Published: November 2016

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105 degrees C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have purified the hydrogen uptake hydrogenase from membranes of P. brockii by reactive red affinity chromatography and sucrose gradient centrifugation. The molecular mass of the holoenzyme was 118,000 +/- 19,000 Da in sucrose gradients. The holoenzyme consisted of two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit had a molecular mass of 66,000 Da, and the small subunit had a molecular mass of 45,000 Da. Colorometric analysis of Fe and S content in reactive red-purified hydrogenase revealed 8.7 +/- 0.6 mol of Fe and 6.2 +/- 1.2 mol of S per mol of hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Temperature stability studies indicated that the membrane-bound form of the enzyme was more stable than the solubilized purified form over a period of minutes with respect to temperature. However, the membranes were not able to protect the enzyme from thermal inactivation over a period of hours. The artificial electron acceptor specificity of the pure enzyme was similar to that of the membrane-bound form, but the purified enzyme was able to evolve H2 in the presence of reduced methyl viologen. The Km of membrane-bound hydrogenase for H2 was approximately 19 microM with methylene blue as the electron acceptor, whereas the purified enzyme had a higher Km value.
Distribution of folates and modified folates in extremely thermophilic bacteria
Authors: White R H
Published: November 2016

Analyses were made of the structures and levels of folates and modified folates present in extremely thermophilic bacteria. These procedures involved the chemical analysis of products resulting from the oxidative cleavage of the 6-substituted, folatelike tetrahydropterins present in the cells. Air-oxidized cell extracts of extreme thermophiles from two members of the archaebacterial order Thermococcales, Thermococcus celer and Pyrococcus furiosus, contained only 7-methylpterin, indicating that these cells contain a modified folate with a methylated pterin. Cell extracts also contained 6-acetyl-7-methyl-7,8-dihydropterin, another product derived from the oxidative cleavage of a dimethylated folate, demonstrating that both the C-7 and C-9 carbons of the pterin were methylated. Extracts, however, contained neither p-aminobenzoylpolyglutamates nor methaniline, the oxidative cleavage products of folates and methanopterin, respectively, indicating that they contain a previously undescribed C1 carrier(s). On the basis of the level of the 7-methylpterin isolated, the levels of modified folate were 2 to 10 times higher than those typically found in mesophilic bacteria and 10 to 100 times less than the level of methanopterin found in the methanogenic bacteria. Oxidized cell extracts of Sulfolobus spp. of the archaebacterial order Sulfolobales contained only pterin, and, like members of the order Thermococcales, they contained neither-p-aminobenzoylpolyglutamates nor methaniline. Oxidized cell extracts of the extreme thermophiles Pyrobaculum sp. strain H10 and Pyrodictium occultum, from the archaebacterial orders Thermoproteales and Pyrodictiales, respectively, and Thermotoga maritima from the eubacterial order Thermotogales, contained pterin and p-aminobenzoylpolyglutamates, indicating that these cells contained unmodified folates. The levels of p-aminobenzoylpolyglutamates in these archaebacterial cell extracts indicate that the folates were present in the cells at levels 4 to 10 times higher than generally found in those mesophilic eubacteria which do not folates in energy metabolism. The levels and chain lengths of the of p-aminobenzoylpolyglutamates present in Thermotoga maritima were typical of those found in mesophilic eubacteria.
Increased spontaneous mutation and alkylation sensitivity of Escherichia coli strains lacking the ogt O6-methylguanine DNA repair methyltransferase
Authors: Rebeck G W , Samson L
Published: November 2016

Escherichia coli expresses two DNA repair methyltransferases (MTases) that repair the mutagenic O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT) DNA lesions; one is the product of the inducible ada gene, and here we confirm that the other is the product of the constitutive ogt gene. We have generated various ogt disruption mutants. Double mutants (ada ogt) do not express any O6MeG/O4MeT DNA MTases, indicating that Ada and Ogt are probably the only two O6MeG/O4MeT DNA MTases in E. coli. ogt mutants were more sensitive to alkylation-induced mutation, and mutants arose linearly with dose, unlike ogt+ cells, which had a threshold dose below which no mutants accumulated; this ogt(+)-dependent threshold was seen in both ada+ and ada strains. ogt mutants were also more sensitive to alkylation-induced killing (in an ada background), and overexpression of the Ogt MTase from a plasmid provided ada, but not ada+, cells with increased resistance to killing by alkylating agents. The induction of the adaptive response was normal in ogt mutants. We infer from these results that the Ogt MTase prevents mutagenesis by low levels of alkylating agents and that, in ada cells, the Ogt MTase also protects cells from killing by alkylating agents. We also found that ada ogt E. coli had a higher rate of spontaneous mutation than wild-type, ada, and ogt cells and that this increased mutation occurred in nondividing cells. We infer that there is an endogenous source of O6MeG or O4MeT DNA damage in E. coli that is prevalent in nondividing cells.
Characterization of enzymes of the branched-chain amino acid biosynthetic pathway in Methanococcus spp
Authors: Xing R Y , Whitman W B
Published: November 2016

Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae contain similar levels of four enzymes of branched-chain amino acid biosynthesis: acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B. Following growth at low partial pressures of H2-CO2, the levels of these enzymes in extracts of M. voltae are reduced three- to fivefold, which suggests that their synthesis is regulated. The enzymes from M. aeolicus were found to be similar to the eubacterial and eucaryotic enzymes with respect to molecular weights, pH optima, kinetic properties, and sensitivities to O2. The acetohydroxy acid isomeroreductase has a specific requirement for Mg2+, and other divalent cations were inhibitory. It was stimulated threefold by K+ and NH4+ ions and was able to utilize NADH as well as NADPH. The partially purified enzyme was not sensitive to O2. The dihydroxy acid dehydratase is extremely sensitive to O2, and it has a half-life under 5% O2 of 6 min at 25 degrees C. Divalent cations were required for activity, and Mg2+, Mn2+, Ni2+, Co2+, and Fe2+ were nearly equally effective. In conclusion, the archaebacterial enzymes are functionally homologous to the eubacterial and eucaryotic enzymes, which implies that this pathway is very ancient.
Tandem translation starts in the cheA locus of Escherichia coli
Authors: Kofoid E C , Parkinson J S
Published: November 2016

The cheA locus of Escherichia coli encodes two protein products, CheAL and CheAS. The nucleotide sequences of the wild-type cheA locus and of two nonsense alleles confirmed that both proteins are translated in the same reading frame from different start points. These start sites were located on the coding sequence by direct determination of the amino-terminal sequences of the two CheA proteins. Both starts are flanked by inverted repeats that may play a role in regulating the relative expression rates of the CheA proteins through alternative mRNA secondary structures.
Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum
Authors: Pich A , Bahl H
Published: November 2016

The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the sigma 70 subunit of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the beta, beta', and alpha subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2+ or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase.
Two-stage induction of the soxRS (superoxide response) regulon of Escherichia coli
Authors: Wu J , Weiss B
Published: November 2016

soxR and soxS are adjacent genes that govern a superoxide response regulon. Previous studies revealed that induction of the regulon is accompanied by increased transcription of soxS, which can activate the target genes. Therefore, induction may occur in two stages: the soxR-dependent activation of soxS, followed by the soxS-dependent induction of other genes. However, the requirement for soxR was unproven because the only existing soxR mutations either were of the regulon-constitutive type or also involved soxS. Therefore, we produced an insertion mutation that was shown by complementation to inactivate only soxR. In confirmation of the two-stage model, soxR was required for the induction by paraquat of the target genes studied (nfo, zwf, and sodA), for paraquat resistance, and for the 47- to 76-fold induction of soxS-lacZ gene fusions. Paraquat did not affect the expression of soxR-lacZ gene fusions. In a soxRS deletion mutant, the regulon was constitutively activated by a runaway soxS+ plasmid. However, a lower-copy-number plasmid failed to activate nfo, zwf, or sodA but did increase the paraquat resistance of a soxRS mutant. Therefore, there is a differential response of the regulon genes to soxS overproduction. A soxR regulon-constitutive mutation was suppressed by a soxR+ plasmid, suggesting a competition between native and activated forms of SoxR. It is proposed that to enhance the sensitivity of the response, the cell minimizes such potential competition by manufacturing only a small amount of this sensor protein, thereby necessitating signal amplification via induction of soxS.
The rcsB gene, a positive regulator of colanic acid biosynthesis in Escherichia coli, is also an activator of ftsZ expression
Authors: Gervais F G , Phoenix P , Drapeau G R
Published: November 2016

Wild-type genes which, when overexpressed, are capable of restoring the growth deficiency of the division mutant ftsZ84 of Escherichia coli on L medium containing no added NaCl have been isolated. One of these genes is rcsB, a positive regulator of colanic acid biosynthesis. A direct relationship between rcsB expression and FtsZ activity was observed, suggesting that RcsB specifically increases transcription of ftsZ, thus accounting for the restoration of colony formation by ftsZ84 mutant cells. Analysis of the 5' upstream sequence of rcsB revealed, in addition to the sigma 54 promoter sequence previously reported, a presumptive sigma 70 promoter and LexA-binding site plus an upstream sequence that is found to be essential for the expression of rcsB on a plasmid. The absence of the sigma 54 factor does not have a negative effect on the transcription of rcsB. The RcsB protein is an activator of its own synthesis, particularly in the presence of NaCl. Evidence which suggests that RcsB can be phosphorylated by a presumably modified EnvZ or PhoM sensor protein leading to a suppression of the growth deficiency of ftsZ84 mutant cells and to an increase in colanic acid production was obtained. We also demonstrated that the level of colanic acid is reduced when the cells carry a multicopy rcsC plasmid, suggesting that the RcsC sensor has phosphatase activity.
Development of multipurpose peroxisomes in Candida boidinii grown in oleic acid-methanol limited continuous cultures
Authors: Waterham H R , Keizer-Gunnink I , Goodman J M , Harder W , Veenhuis M
Published: November 2016

We have studied the development and metabolic significance of peroxisomes in the yeast Candida boidinii following adaptation of the organism to cultivation conditions which require the simultaneous presence and activity of two independent peroxisome-mediated pathways for growth. After the addition of methanol to oleic acid-grown cells at late exponentional growth, a number of new small peroxisomes developed which, apart from the presence of beta-oxidation enzymes, were characterized by the presence of enzymes involved in methanol metabolism (alcohol oxidase and dihydroxyacetone synthase). The latter proteins, however, were absent in the larger organelles which were originally present in the oleic acid-grown cells prior to the addition of methanol and which contained only enzymes of the beta-oxidation pathway. Subsequent experiments on cells from continuous cultures grown on a mixture of oleic acid and methanol at steady-state conditions revealed that both the enzymes of the beta-oxidation pathway and those involved in methanol metabolism were found in one and the same compartment. Thus, under these conditions the cells contained peroxisomes which were concurrently involved in the metabolism of two different carbon sources simultaneously used for growth. Our results indicated that the heterogeneity in the peroxisomal population of a single cell, observed in the transient state following the addition of methanol, is only temporary and due to heterogeneity among these organelles with respect to their capacity to incorporate newly synthesized matrix proteins.
Mutational analysis of the Rhizobium meliloti nifA promoter
Authors: Agron P G , Ditta G S , Helinski D R
Published: November 2016

The nifA gene of Rhizobium meliloti, the bacterial endosymbiont of alfalfa, is a regulatory nitrogen fixation gene required for the induction of several key nif and fix genes. Transcription of nifA is strongly induced in planta and under microaerobic conditions ex planta. Induction of nifA, in turn, is positively controlled by the fixL and fixJ genes of R. meliloti, the sensor and regulator, respectively, of a two-component system responsible for oxygen sensing by this bacterium. This system is also responsible for the positive induction of fixK. Here, we report that chemical and oligonucleotide site-directed mutageneses of the nifA promoter (nifAp) were conducted to identify nucleotides essential for induction. Nineteen mutants, including 14 single-point mutants, were analyzed for microaerobic induction of nifAp in R. meliloti. Critical residues were identified in an upstream region between base pairs -54 and -39 relative to the transcription start site. Attempts at separating the upstream and downstream regions of the nifA promoter so as to maintain fixJ-dependent activity were unsuccessful. A 5' deletion of the fixK promoter (fixKp) to -67 indicates that sequences upstream of this position are not required for microaerobic induction. A sequence comparison of the -54 to -39 region of nifAp with the upstream sequences of fixKp does not reveal a block of identical nucleotides that could account for the fixJ-dependent microaerobic induction of both promoters. Many of the defective nifAp mutants in this region, however, are in residues with identity to fixKp in an alignment of the promoters according to their transcription start sites. Therefore, it is possible that there is a common sequence motif in the -54 to -39 region of the two promoters that is required for fixLJ-dependent microaerobic induction.
Nucleotide sequence and characterization of four additional genes of the hydrogenase structural operon from Rhizobium leguminosarum bv. viciae
Authors: Hidalgo E , Palacios J M , Murillo J , Ruiz-Argüeso T
Published: November 2016

The nucleotide sequence of a 2.5-kbp region following the hydrogenase structural genes (hupSL) in the H2 uptake gene cluster from Rhizobium leguminosarum bv. viciae UPM791 was determined. Four closely linked genes encoding peptides of 27.9 (hupC), 22.1 (hupD), 19.0 (hupE), and 10.4 (hupF) kDa were identified immediately downstream of hupL. Proteins with comparable apparent molecular weights were detected by heterologous expression of these genes in Escherichia coli. The six genes, hupS to hupF, are arranged as an operon, and by mutant complementation analysis, it was shown that genes hupSLCD are cotranscribed. A transcription start site preceded by the -12 to -24 consensus sequence characteristic of NtrA-dependent promoters was identified upstream of hupS. On the basis of the lack of oxygen-dependent H2 uptake activity of a hupC::Tn5 mutant and on structural characteristics of the protein, we postulate that HupC is a b-type cytochrome involved in electron transfer from hydrogenase to oxygen. The product from hupE, which is needed for full hydrogenase activity, exhibited characteristics typical of a membrane protein. The features of HupC and HupE suggest that they form, together with the hydrogenase itself, a membrane-bound protein complex involved in hydrogen oxidation.
Proline biosynthesis in Saccharomyces cerevisiae: molecular analysis of the PRO1 gene, which encodes gamma-glutamyl kinase
Authors: Li W , Brandriss M C
Published: November 2016

The PRO1 gene of Saccharomyces cerevisiae encodes the 428-amino-acid protein gamma-glutamyl kinase (ATP:L-glutamate 5-phosphotransferase, EC 2.7.2.11), which catalyzes the first step in proline biosynthesis. Amino acid sequence comparison revealed significant homology between the yeast and Escherichia coli gamma-glutamyl kinases throughout their lengths. Four close matches to the consensus sequence for GCN4 protein binding and one close match to the RAP1 protein-binding site were found in the PRO1 upstream region. The response of the PRO1 gene to changes in the growth medium was analyzed by measurement of steady-state mRNA levels and of beta-galactosidase activity encoded by a PRO1-lacZ gene fusion. PRO1 expression was not repressed by exogenous proline and was not induced by the presence of glutamate in the growth medium. Although expression of the PRO1 gene did not change in response to histidine starvation, both steady-state PRO1 mRNA levels and beta-galactosidase activities were elevated in a gcd1 strain and reduced in a gcn4 strain. In addition, a pro1 bradytrophic strain became completely auxotrophic for proline in a gcn4 strain background. These results indicate that PRO1 is regulated by the general amino acid control system.
Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I
Authors: Witkin E M , Roegner-Maniscalco V
Published: November 2016

A polA12 recA718 double mutant of Escherichia coli, in which DNA polymerase I is temperature sensitive, was unable to maintain normal DNA synthesis or to form colonies on rich media at 42 degrees C. Overproduction of DnaE protein, the polymerizing alpha subunit of DNA polymerase III, restored bacterial DNA replication and cell viability, as well as the PolI-dependent replication of the plasmid carrying dnaE.
Developmental regulation of CUP gene expression through DNA methylation in Mucor spp
Authors: Cano-Canchola C , Sosa L , Fonzi W , Sypherd P , Ruiz-Herrera J
Published: November 2016

Inserts which carried the CUP gene from Saccharomyces cerevisiae or Mucor racemosus were used as hybridization probes to measure the methylation state and expression of the CUP gene from Mucor rouxii at different stages of growth. It was observed that the fungus contains a CUP multigene family. All the CUP genes were present in a hypermethylated DNA region in nongrowing and isodiametrically growing spores and were not transcribed at these stages. After germ tube emergence, CUP genes became demethylated and transcriptionally active. Development, demethylation, and transcription of CUP genes were blocked by the ornithine decarboxylase inhibitor 1,4-diaminobutanone. These results suggest that genes that are activated during development became demethylated in this fungus.
Roles of the orlA, tsE, and bimG genes of Aspergillus nidulans in chitin synthesis
Authors: Borgia P T
Published: November 2016

Strains of Aspergillus nidulans carrying the orlA1 or tse6 allele are deficient in cell wall chitin and undergo lysis at restrictive temperatures. The strains are remediable by osmotic stabilizers or by the presence of N-acetylglucosamine (GlcNAc) in the medium. The remediation by GlcNAc suggests that the lesion(s) in chitin synthesis resides in the amino sugar biosynthetic pathway prior to the synthesis of N-acetylglucosamine-6-phosphate. orlA1 strains grown at permissive temperature exhibit an abnormally low specific activity for L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), the first enzyme unique to amino sugar synthesis. In addition, the enzyme produced is temperature sensitive in vitro. tsE6 strains grown at permissive temperature show virtually no amidotransferase activity. This finding is consistent with an extremely labile enzyme which is destroyed by cell breakage and extract preparation. The enzyme must be active in vivo at permissive temperatures since GlcNAc is not required for growth. Thus, two structural genes (orlA and tsE) are necessary for the amidotransferase activity. bimG11 strains are temperature sensitive for a type 1 protein phosphatase involved in cell cycle regulation and arrest in mitosis. Like orlA1 and tsE6 strains, conidia from bimG11 strains swell excessively when germinated and lyse; the germlings produced are deficient in chitin content. The amidotransferase from wild-type and mutant strains is sensitive to feedback inhibition by uridine diphosphate-N-acetylglucosamine. The sensitivity of the amidotransferase from bimG11 strains is dependent on growth temperature, while that from wild-type strains is independent of temperature. The enzyme can be desensitized in vitro under conditions consistent with a protein phosphatase reaction. It is proposed that amino sugar (and chitin biosynthesis) is partially regulated by phosphorylation-dephosphorylation of the amidotransferase or a protein regulator of the enzyme.
Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis
Authors: Foster S J
Published: November 2016

The autolysins of Bacillus subtilis 168 were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels. Four bands of vegetative autolytic activity of 90, 50, 34, and 30 kDa (bands A1 to A4) were detected in SDS and LiCl extracts and in native cell walls by using B. subtilis 168 vegetative cell walls as the substrate incorporated in the gel. The four enzyme activities showed different substrate specificities and sensitivities to various chemical treatments. The autolysin profile was not medium dependent and remained constant during vegetative growth. During sporulation, band A4 greatly increased in activity just prior to mother-cell lysis. No germination-associated changes in the profile were observed, although a soluble 41-kDa endospore-associated cortex-lytic enzyme was found. By using insertionally inactivated mutants, bands A1 and A2 were positively identified as the previously characterized 90-kDa glucosaminidase and 50-kDa amidase, respectively. The common filamentous phenotype of various regulatory mutants could not be correlated to specific changes in the autolysin profile.
Overexpression in Escherichia coli and functional analysis of a novel PPi-selective porin, oprO, from Pseudomonas aeruginosa
Authors: Hancock R E , Egli C , Benz R , Siehnel R J
Published: November 2016

Immediately upstream from and adjacent to the oprP gene, which codes for the phosphate-specific porin OprP of Pseudomonas aeruginosa, lies the PR region (oprO), which cross-hybridizes with oprP DNA. To determine the function of this region, the oprO gene was expressed behind the lactose promoter in Escherichia coli, and the resultant OprO protein was purified and reconstituted into planar lipid bilayers. OprO formed sodium dodecyl sulfate-stable trimers, cross-reacted immunologically with OprP, and, like OprP, formed an anion-specific, phosphate-selective porin. However, it demonstrated lower affinity for and higher maximal conductance of both chloride and phosphate than did the OprP channel. Examination by macroscopic conductance inhibition experiments of the affinity of OprO for phosphates of different lengths revealed a preference for PPi and tripolyphosphate over Pi, suggesting that OprO functioned as a PPi-selective polyphosphate channel, in contrast to OprP, which has a marked preference for Pi.
Molecular genetic analysis of the Escherichia coli phoP locus
Authors: Groisman E A , Heffron F , Solomon F
Published: November 2016

We have cloned the Escherichia coli phoP gene, a member of the family of environmentally responsive two-component systems, and found its deduced amino acid sequence to be 93% identical to that of the Salmonella typhimurium homolog, which encodes a major virulence regulator necessary for intramacrophage survival and resistance to cationic peptides of phagocytic cells. The phoP gene was mapped to kilobase 1202 on the Kohara map (25-min region) of the E. coli genome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) and found to be transcribed in a counterclockwise direction. Both E. coli and S. typhimurium phoP mutants were more sensitive than their isogenic wild-type strains to the frog-derived antibacterial peptide magainin 2, suggesting a role for PhoP in the response to various stresses in both enteric species.
Overproduction of beta-ketoacyl-acyl carrier protein synthase I imparts thiolactomycin resistance to Escherichia coli K-12
Authors: Tsay J T , Rock C O , Jackowski S
Published: November 2016

Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene- 4-thiolide] (TLM) is a unique antibiotic structure that inhibits dissociated type II fatty acid synthase systems but not the multifunctional type I fatty acid synthases found in mammals. We screened an Escherichia coli genomic library for recombinant plasmids that impart TLM resistance to a TLM-sensitive strain of E. coli K-12. Nine independent plasmids were isolated, and all possessed a functional beta-ketoacyl-acyl carrier protein synthase I gene (fabB) based on their restriction enzyme maps and complementation of the temperature-sensitive growth of a fabB15(Ts) mutant. A plasmid (pJTB3) was constructed that contained only the fabB open reading frame. This plasmid conferred TLM resistance, complemented the fabB(Ts) mutation, and directed the overproduction of synthase I activity. TLM selectively inhibited unsaturated fatty acid synthesis in vivo; however, synthase I was not the only TLM target, since supplementation with oleate to circumvent the cellular requirement for an active synthase I did not confer TLM resistance. Overproduction of the FabB protein resulted in TLM-resistant fatty acid biosynthesis in vivo and in vitro. These data show that beta-ketoacyl-acyl carrier protein synthase I is a major target for TLM and that increased expression of this condensing enzyme is one mechanism for acquiring TLM resistance. However, extracts from a TLM-resistant mutant (strain CDM5) contained normal levels of TLM-sensitive synthase I activity, illustrating that there are other mechanisms of TLM resistance.
Fumarate or a fumarate metabolite restores switching ability to rotating flagella of bacterial envelopes
Authors: Barak R , Eisenbach M
Published: November 2016

Flagella of cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium can rotate in either the counterclockwise or clockwise direction, but they never switch from one direction of rotation to another. Exogenous fumarate, in the intracellular presence of the chemotaxis protein CheY, restored switching ability to envelopes, with a concomitant increase in clockwise rotation. An increase in clockwise rotation was also observed after fumarate was added to partially lysed cells of E. coli, but the proportion of switching cells remained unchanged.
Structural modifications and kinetic studies of the substrates involved in the final step of methane formation in Methanobacterium thermoautotrophicum
Authors: Olson K D , Chmurkowska-Cichowlas L , McMahon C W , Wolfe R S
Published: November 2016

The 2-(methylthio)ethanesulfonic acid (CH3-S-CoM) reductase catalyzes the final methane-yielding reaction in fastidiously anaerobic methanogenic archaebacteria. This step involves the reductive demethylation of CH3-S-CoM with reducing equivalents from N-7-(mercaptoheptanoyl)-L-threonine O3-phosphate (HS-HTP) to yield methane and the nonsymmetrical disulfide of 2-mercaptoethanesulfonic acid and HS-HTP. We chemically synthesized modified analogs of CH3-S-CoM (which has two carbons in the ethylene bridge) and of HS-HTP (which has seven carbons in the side chain); analog pairs possessed an overall correct number of side chain carbons (i.e., a total of nine in combination). They were simultaneously added to anaerobic cell extracts of Methanobacterium thermoautotrophicum delta H. The ability of the extracts to reductively demethylate the modified substrates was tested by gas chromatography. We also describe here previously unknown inhibitors of methanogenesis, 6-(methylthio)hexanoyl-L-threonine O3-phosphate (a structural analog of HS-HTP) and sodium bromomethanesulfonic acid (a structural analog of CH3-S-CoM). Both analogs were found to be effective competitive inhibitors with respect to HS-HTP. These substrate analogs were also found to inhibit a recently described photoactivation of homogeneous inactive reductase (K. D. Olson, C. W. McMahon, and R. S. Wolfe, Proc. Natl. Acad. Sci. USA 88:4099-4103, 1991). In addition, we probed the mechanism of action of a potent inhibitor of the enzyme, 2-bromoethanesulfonic acid, a structural analog of CH3-S-CoM.
The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate
Authors: Thibaut D , Couder M , Famechon A , Debussche L , Cameron B , Crouzet J , Blanche F
Published: November 2016

The final enzymatic reaction in the conversion of precorrin-6x to hydrogenobyrinic acid by cell-free protein preparations from Pseudomonas denitrificans was shown to be inhibited by hydrogenobyrinic acid. Use was made of this property to prepare the last biosynthetic precursor of hydrogenobyrinic acid, named precorrin-8x. Double-labeling experiments, mass spectrometry, and UV-visible light spectroscopy studies established that precorrin-8x was at the oxidation level of a corrin and differed from precorrin-6x by two additional methyl groups (presumably at C-5 and C-15) and decarboxylation of the acetic acid side chain at C-12. Precorrin-8x was not a corrin but had the same mass as hydrogenobyrinic acid, thus showing that this latter compound is synthesized from the former by a rearrangement. The enzyme catalyzing this rearrangement was purified 80-fold to homogeneity from a recombinant strain of P. denitrificans, sequenced at its N terminus, and shown to be encoded by the cobH gene. It was identical to the previously described hydrogenobyrinic acid-binding protein (F. Blanche, D. Thibaut, D. Frechet, M. Vuilhorgne, J. Crouzet, B. Cameron, G. Müller, K. Hlineny, U. Traub-Eberhard, and M. Zboron, Angew. Chem. Int. Ed. Engl. 29:884-886, 1990). This enzyme had a Km of 0.91 +/- 0.04 microM and a Vmax of 230 nmol h-1 mg-1 at pH 7.7 and was competitively inhibited by hydrogenobyrinic acid with a Ki of 0.17 +/- 0.01 microM. It is proposed that the cobH gene product is a mutase which transfers the methyl group from C-11 to C-12.
Role of the heat shock response in stability of mRNA in Escherichia coli K-12
Authors: Henry M D , Yancey S D , Kushner S R
Published: November 2016

The heat shock response in Escherichia coli involves extensive induction of the heat shock proteins, with the concomitant suppression of the synthesis of the non-heat shock proteins. While the induction of the heat shock proteins has been shown to occur primarily at the transcriptional level, the suppression of non-heat shock proteins is poorly understood. We have investigated the possibility that an increased decay of non-heat shock mRNAs is a means of decreasing the synthesis of non-heat shock proteins during the heat shock response. Heat shock response-defective strains were compared with wild-type controls by several criteria to evaluate both mRNA stability and the induction of enzymes known to be involved in mRNA turnover. Our results indicate that increased mRNA decay is not a mechanism used to regulate the synthesis of non-heat shock proteins.
Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination
Authors: Sanchez-Salas J L , Santiago-Lara M L , Setlow B , Sussman M D , Setlow P
Published: November 2016

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.