bluuebunny/crossref_metadata_2025 huggingface dataset:
Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: overproduction of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene
Authors: Sá-Correia I , Darzins A , Wang S K , Berry A , Chakrabarty A M
Published: November 2016

The specific activities of phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP), and GDP-mannose dehydrogenase (GMD) were compared in a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa and in two spontaneous nonmucoid revertants. In both revertants some or all of the alginate biosynthetic enzymes we examined appeared to be repressed, indicating that the loss of the mucoid phenotype may be a result of decreased formation of sugar-nucleotide precursors. The introduction and overexpression of the cloned P. aeruginosa phosphomannose isomerase (pmi) gene in both mucoid and nonmucoid strains led not only to the appearance of PMI levels in cell extracts several times higher than those present in the wild-type mucoid strain, but also in higher PMM and GMP specific activities. In extracts of both strains, however, the specific activity of GMD did not change as a result of pmi overexpression. In contrast, the introduction of the cloned Escherichia coli manA (pmi) gene in P. aeruginosa caused an increase in only PMI and PMM activities, having no effect on the level of GMP. This suggests that an increase in PMI activity alone does not induce high GMP activity in P. aeruginosa. The heterologous overexpression of the P. aeruginosa pmi gene in the E. coli manA mutant CD1 led to the appearance in cell extracts of not only PMI activity but also GMP activity, both of which are normally undetectable in extracts of CD1. We discuss the implications of these results and propose a mechanism by which overexpression of the P. aeruginosa pmi gene can cause an elevation in both PMM and GMP activities.
Identification of the promoter of the Bacillus subtilis sdh operon
Authors: Melin L , Magnusson K , Rutberg L
Published: November 2016

The Bacillus subtilis sdhCAB operon contains the structural genes for the three subunits of the membrane bound succinate dehydrogenase complex. An sdh-specific transcript of about 3,450 nucleotides was detected in vegetative bacteria. S1 nuclease mapping experiments showed that the sdh operon is transcribed from a sigma-43 promoter; the transcript starts at a guanosine residue 90 base pairs upstream from the first gene of the operon, sdhC. No sdh transcript was found in B. subtilis carrying the sdh-115 mutation, which decreases expression of the sdh operon by more than 99%. The sdh-115 mutation is a G-to-A transition in the -35 region of the sigma-43 promoter. The sdh operon is sensitive to glucose repression. When the sdh promoter region was used to drive transcription of the cat-86 gene this gene also became glucose repressed.
A genetic locus essential for formate-dependent growth of Bradyrhizobium japonicum
Authors: McClung C R , Chelm B K
Published: November 2016

A genetic locus essential for the formate-dependent growth of Bradyrhizobium japonicum was isolated by complementation of ethyl methanesulfonate-induced mutants with a cosmid gene library of B. japonicum DNA. Three related cosmids containing 18.7 kilobase pairs of B. japonicum DNA in common were identified as being able to restore formate-dependent growth capability to mutants lacking either ribulosebisphosphate carboxylase or both ribulosebisphosphate carboxylase and phosphoribulokinase activities. To further localize the complementing gene(s), a series of four deletions spanning a total of 16.1 kilobase pairs were introduced into the B. japonicum chromosome. Each resulting deletion mutant lacked formate dehydrogenase activity and lacked ribulosebisphosphate carboxylase activity and immunologically detectable protein. Three of the four also lacked phosphoribulokinase activity. Two other mutants in which the deletion-bearing recombinant plasmid had integrated into the chromosome also lacked ribulosebisphosphate carboxylase activity and protein and phosphoribulokinase activities. The genetic locus defined by these mutants could contain the structural genes for these enzymes or a regulatory gene(s) controlling their expression or both.
Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides light-harvesting B800-850-alpha and B800-850-beta genes
Authors: Kiley P J , Kaplan S
Published: November 2016

Two deoxyoligonucleotide probes were synthesized in accordance with the available amino acid sequence of the B800-850-beta polypeptide from Rhodobacter sphaeroides and were used to isolate a 2.6-kilobase PstI fragment from R. sphaeroides 2.4.1 chromosomal DNA. Identification of the B800-850-beta and B800-850-alpha structural genes, pucB and pucA, was confirmed by DNA sequencing. Northern (RNA) blot analysis, using restriction endonuclease fragments from the cloned genes as probes, revealed a single puc-operon-specific, highly stable transcript of approximately 640 bases present in photosynthetically grown cells. In vitro transcription-translation analysis of the puc operon revealed that the maximum synthesis of the puc operon gene products was achieved when the entire 2.6-kilobase PstI fragment was used as the template, although a 537-base-pair XmaIII fragment was sufficient to direct the synthesis of pucB and pucA fusion product.
Coordinate regulation of phospholipid biosynthesis by serine in Saccharomyces cerevisiae
Authors: Homann M J , Bailis A M , Henry S A , Carman G M
Published: November 2016

The addition of L-serine to inositol-containing growth medium repressed membrane-associated CDPdiacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activities and subunit levels in wild-type Saccharomyces cerevisiae. Enzyme activities and subunit levels were not repressed when inositol was absent from the growth medium. The addition of L-serine to the growth medium did not affect the phospholipid composition of wild-type cells. CDPdiacylglycerol synthase and phosphatidylserine synthase were not regulated in the S. cerevisiae inositol biosynthesis ino2, ino4, and opi1 regulatory mutants, suggesting that regulation by inositol plus L-serine is coupled to inositol synthesis. Inositol and L-serine did not affect the activities of purified CDPdiacylglycerol synthase and phosphatidylserine synthase. The addition of compounds structurally related to L-serine to the growth medium of wild-type cells also resulted in a repression of CDPdiacylglycerol synthase and phosphatidylserine synthase but only in the presence of inositol. Phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was not regulated by inositol plus L-serine.
Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal
Authors: Couche G A , Pfannenstiel M A , Nickerson K W
Published: November 2016

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 X 10(-8) mol of free sulfhydryls and 3.24 X 10(-8) mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Nonreduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin.
Purification and characterization of the CheZ protein of bacterial chemotaxis
Authors: Stock A M , Stock J B
Published: November 2016

The cheZ gene is the most distal of five genes that comprise the Meche operon of the Salmonella typhimurium chemotaxis system. We have determined the sequence of the cheZ gene along with an 800-nucleotide flanking region at its 3' end. The flanking sequence contains an open reading frame that probably corresponds to the 5' end of flaM. The cheZ coding sequence predicts an extremely acidic, hydrophilic protein with a molecular weight of 23,900. We have purified and characterized this protein. N-terminal analysis of pure CheZ yields an amino acid sequence identical to that predicted by the nucleotide sequence except that the amino-terminal methionine residue is modified by N methylation. The purified CheZ protein exhibits a native molecular weight of 115,000, but in cell extracts the majority of CheZ exists as a much larger aggregate (Mr greater than 500,000). Under these conditions, CheZ appears to be a homopolymer composed of at least 20 monomeric subunits.
Definition of the surface antigens of Mycobacterium malmoense and use in studying the etiology of a form of mycobacteriosis
Authors: McNeil M , Tsang A Y , McClatchy J K , Stewart C , Jardine I , Brennan P J
Published: November 2016

Mycobacterium malmoense is the latest of a roster of atypical mycobacteria implicated in pulmonary infections. Yet it lacks recognizable phenotypic features to allow its ready identification. Some 23 clinical isolates of M. malmoense were examined for homologous seroagglutination reactions and characteristic surface antigens. One group showed concordant agglutination interreactions and an identical spectrum of glycolipids and are regarded as M. malmoense sensu stricto. The glycolipids are of the newly found, trehalose-containing lipooligosaccharide class. De-O-acylation followed by high-pressure liquid chromatography revealed one major and several minor oligosaccharides. Partial acidic cleavage to release glycosidically linked trehalose, alpha-mannosidase digestion to demonstrate the presence of a non-reducing-end mannobiose, perdeuteriomethylation, partial acid hydrolysis, reduction, and O ethylation, combined with 1H nuclear magnetic resonance and electron impact and fast-atom bombardment mass spectrometry revealed the structure of the major oligosaccharide as alpha-D-Manp-(1----3) -alpha-D-Manp-(1----[2-alpha-L-Rhap-(1--]4--3)-alpha-L-Rh ap- (1----3)-alpha-D-Glcp-(1----1)-alpha-D-Glcp, in which two of the 2-alpha-L-Rhap residues are O methylated at C-3. (Man, mannose; Rha, rhamnose; Glc, glucose; p, pyranosyl). The structures of the minor oligosaccharides were also determined; they differ at the distal nonreducing end. The dominant oligosaccharide was acylated by octanoate, 2-methyleicosanoate, and 2,4-dimethylpentacosanoate to yield the major species-specific surface antigen of M. malmoense, which we regard as the most characteristic feature of the pathogen.
Organization and regulation of an operon that encodes a sporulation-essential sigma factor in Bacillus subtilis
Authors: Kenney T J , Moran C P
Published: November 2016

Deletion of sigE, the structural gene for the sporulation-induced RNA polymerase sigma factor, sigma E, prevented endospore formation by Bacillus subtilis. The effects of integration of plasmids into the sigE region of the chromosome and the use of complementation analyses demonstrated that sigE is part of an operon that includes a promoter-proximal gene, spoIIGA, that is essential for sporulation. Gene fusions to the promoter of this operon, spoIIG, demonstrated that transcription from this promoter is induced at the beginning of sporulation and is dependent on several spoO genes.
Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors
Authors: Schramm E , Mende J , Braun V , Kamp R M
Published: November 2016

Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation. The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined. A polypeptide consisting of 511 amino acids was deduced from the open reading frame. The active colicin had a molecular weight of 54,742. The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A. Of 216 amino acids, 57% were identical and an additional 19% were homologous. In this part 66% of the nucleotides were identical in the colicin A and B genes. This region contained a sequence of 48 hydrophobic amino acids. Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced. A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function. The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12. Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions. Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.
Manganese transport in Brevibacterium ammoniagenes ATCC 6872
Authors: Schmid J , Auling G
Published: November 2016

Uptake of manganese by Brevibacterium ammoniagenes ATCC 6872 was energy dependent and obeyed saturation kinetics (Km = 0.65 microM; Vmax = 0.12 mumol/min per g [dry weight]). Uptake showed optima at 27 degrees C and pH 9.5. 54Mn2+ accumulated by the cells was released by treatment with toluene or by exchange for unlabeled manganese ions, via an energy-dependent process. Co2+, Fe2+, Cd2+, and Zn2+ inhibited manganese uptake. Inhibition by Cd2+ and Zn2+ was competitive (Ki = 0.15 microM Cd2+ and 1.2 microM Zn2+). Experiments with 65Zn2+ provided no evidence for Zn2+ uptake via the Mn2+ transport system.
Partial purification and some properties of homoserine O-acetyltransferase of a methionine auxotroph of Saccharomyces cerevisiae
Authors: Yamagata S
Published: November 2016

A wild-type strain and six methionine auxotrophs of Saccharomyces cerevisiae were cultured in a synthetic medium supplemented with 0.1 mM L-cysteine or L-methionine and analyzed for the synthesis of homoserine O-acetyltransferase (EC 2.3.1.31). Among them, four mutant strains exhibited enzyme activity in cell extracts. Methionine added to the synthetic medium at concentrations higher than 0.1 mM repressed enzyme synthesis in two of these strains. The enzyme was partially purified (3,500-fold) from an extract of a mutant strain through ammonium sulfate fractionation and chromatography on columns of DEAE-cellulose, Phenyl-Sepharose C1-4B, and Sephadex G-150. The enzyme exhibited optimal pH at 7.5 for activity and at 7.8 for stability. The reaction product was ascertained to be O-acetyl-L-homoserine by confirming that it produced L-homocysteine in an O-acetyl-L-homoserine sulfhydrylase reaction. The Km for L-homoserine was 1.0 mM, and for acetyl coenzyme A it was 0.027 mM. The molecular weight of the enzyme was estimated to be approximately 104,000 by Sephadex G-150 column chromatography and 101,000 by sucrose density gradient centrifugation. The isoelectric point was at pH 4.0. Of the hydroxy amino acids examined, the enzyme showed reactivity only to L-homoserine. Succinyl coenzyme A was not an acyl donor. In the absence of L-homoserine, acetyl coenzyme A was deacylated by the enzyme, with a Km of 0.012 mM. S-Adenosylmethionine and S-adenosylhomocysteine slightly inhibited the enzyme, but methionine had no effect.
Transcriptional regulation of the DAL5 gene in Saccharomyces cerevisiae
Authors: Rai R , Genbauffe F , Lea H Z , Cooper T G
Published: November 2016

We demonstrate that the DAL5 gene, encoding a necessary component of the allantoate transport system, is constitutively expressed in Saccharomyces cerevisiae. Its relatively high basal level of expression did not increase further upon addition of allantoin pathway intermediates. However, steady-state DAL5 mRNA levels dropped precipitously when a repressive nitrogen source was provided. These control characteristics of DAL5 expression make this gene a good model with which to unravel the mechanism of nitrogen catabolite repression. Its particular advantage relative to other potentially useful genes derives from its lack of control by induction and hence the complicating effects of inducer exclusion.
Visualization of Pseudomonas aeruginosa O antigens by using a protein A-dextran-colloidal gold conjugate with both immunoglobulin G and immunoglobulin M monoclonal antibodies
Authors: Lam J S , Lam M Y , MacDonald L A , Hancock R E
Published: November 2016

Two lipopolysaccharide O-antigen-specific monoclonal antibodies, MA1-8 (an immunoglobulin G1 [IgG1]) and MF15-4 (an IgM), were used to localize the O antigen of the lipopolysaccharide of Pseudomonas aeruginosa PAO1. A protein A-dextran-gold conjugate with an average particle diameter of 12.5 nm was used to label bacterial cells treated with MA1-8, while a second antibody (goat anti-mouse IgM) was required before the same probe could interact with cells treated with the IgM antibody MF15-4. Both antibodies resulted in exclusive labeling of the surface of P. aeruginosa PAO1 but not that of an isogenic O-antigen-lacking rough mutant. When the monoclonal antibodies became attached to the cell surface of P. aeruginosa PAO1, resulting in an even coating, the foldings and other topographic details could not be discerned by negative staining. In thin sections of monoclonal-antibody-treated bacteria, a 20- and a 30- to 40-nm thick amorphous layer was observed around the outside of the outer membrane when MA1-8 (IgG) and MF15-4 (IgM) plus goat anti-mouse IgM antibodies were used, respectively. This amorphous layer presumably resulted from the stabilization of the lipopolysaccharide structure by the monoclonal antibodies which prevented the long O-antigen chains from collapsing owing to dehydration.
Nucleotide sequence of the Rickettsia prowazekii citrate synthase gene
Authors: Wood D O , Williamson L R , Winkler H H , Krause D C
Published: November 2016

The Rickettsia prowazekii citrate synthase (gltA) gene, previously cloned in Escherichia coli, was localized to a 2.0-kilobase chromosomal fragment. DNA sequence analysis of a portion of this fragment revealed an open reading frame of 1,308 base pairs that encodes a protein of 435 amino acids with a molecular weight of 49,171. This translation product is comparable in size to both the E. coli and pig heart citrate synthase monomers and to the protein synthesized in E. coli minicells containing the rickettsial gene. Comparisons between the deduced amino acid sequence of R. prowazekii citrate synthase and those of the E. coli and pig heart enzymes revealed extensive homology (59%) between the two bacterial proteins. In contrast, only 20% of the rickettsial enzyme residues were shared with the functionally similar pig heart enzyme residues. Upstream from the open reading frame and in close proximity to one another, sequences with homology to E. coli consensus sequences for RNA polymerase and ribosome binding were identified. S1 nuclease mapping experiments demonstrated that the start of transcription for this gene in E. coli was located in the upstream region. Codon usage in the rickettsial gltA gene was found to be very biased and differed from the pattern observed in E. coli. Adenine and uracil were used preferentially in the third base position of rickettsial codons.
Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis
Authors: Rasmussen J L , Odelson D A , Macrina F L
Published: November 2016

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) element. This 1,155-base-pair element contained partially matched (20 of 25 base pairs) terminal-inverted repeats, overlapping, anti-parallel open reading frames, and nine promoterlike sequences, including three that were oriented outward. Analysis of this sequence revealed no significant nucleotide homology to 13 other known IS elements. Inasmuch as Southern blot hybridization analysis detected homologous sequences in chromosomal DNA and its G+C content (42 mol%) was similar to that of B. fragilis, the data suggested that this element is of Bacteroides origin. Transposition promoted by this element was demonstrated in recA E. coli. Recombinants were recovered by selecting for the activation of a promoterless chloramphenicol resistance gene on the plasmid pDH5110 and were characterized by restriction endonuclease mapping and Southern blot hybridization. We propose that this IS element be designated IS4351.
Clustering of mutations blocking synthesis of xanthan gum by Xanthomonas campestris
Authors: Thorne L , Tansey L , Pollock T J
Published: November 2016

Mutations that block the synthesis of xanthan gum by Xanthomonas campestris B1459S-4L-II were isolated as nonmucoid colonies after treatment with ethyl methanesulfonate. Complete libraries of DNA fragments from wild-type X. campestris were cloned into Escherichia coli by using a broad-host-range cosmid vector and then transferred into each mutant strain by conjugal mating. Cloned fragments that restored xanthan gum synthesis (Xgs+; mucoidy) were compared according to restriction pattern, DNA sequence homology, and complementation of a subset of Xgs- mutations. Groups of clones that contained overlapping homologous DNA were found to complement specific Xgs- mutations. The results suggest clustering of the genetic loci involved in xanthan synthesis. The clustering occurred within three unlinked regions. Two forms of complementation were observed. In most instances, independently isolated cosmid clones that complemented a single mutation were found to be partially homologous. Less frequent was the second form of complementation, in which two cosmid clones that lacked any homologous sequences restored the mucoid phenotype to a single mutant. Finally, xanthan production was measured for wild-type X. campestris carrying multiple plasmid copies of the cloned xanthan genes.
Genetic organization of multiple fep genes encoding ferric enterobactin transport functions in Escherichia coli
Authors: Ozenberger B A , Nahlik M S , McIntosh M A
Published: November 2016

Three genes were shown to provide functions specific for ferric enterobactin transport in Escherichia coli: fepA encoded the outer membrane receptor, fepB produced a periplasmic protein, and the fepC product was presumably a component of a cytoplasmic membrane permease system for this siderophore. A 10.6-kilobase-pair E. coli chromosomal EcoRI restriction fragment containing the fepB and fepC genes was isolated from a genomic library constructed in the vector pBR328. Both cistrons were localized on this clone (pITS24) by subcloning and deletion and insertion mutagenesis to positions that were separated by approximately 2.5 kilobases. Within this region, insertion mutations defining an additional ferric enterobactin transport gene (fepD) were isolated, and polarity effects from insertions into fepB suggested that fepD is encoded downstream on the same transcript. A 31,500-dalton FepC protein and a family of FepB polypeptides ranging from 34,000 to 37,000 daltons were identified in E. coli minicells, but the product of fepD was not detectable by this system. Another insertion mutation between entF and fepC was also shown to disrupt iron transport via enterobactin and thus defined the fepE locus; fepE weakly expressed a 43,000-dalton protein in minicells. It is proposed that these newly identified genes, fepD and fepE, provide functions which act in conjunction with the fepC product to form the ferric enterobactin-specific cytoplasmic membrane permease. An additional 44,000-dalton protein was identified and shown to be expressed from a gene that is situated between fepB and entE and that is transcribed in the direction opposite that of fepB. Although the function of this protein is uncharacterized, its membrane location suggests that it too may function in iron transport.
Isolation of DNA encoding sucrase genes from Streptococcus salivarius and partial characterization of the enzymes expressed in Escherichia coli
Authors: Houck C M , Pear J R , Elliott R , Perchorowicz J T
Published: November 2016

Restriction enzyme fragments containing two sucrase genes have been isolated from a cosmid library of Streptococcus salivarius DNA. The genes were expressed in Escherichia coli cells, and the properties of both enzymes were studied in partially purified protein extracts from E. coli. One gene encoding an invertase-type sucrase was subcloned on a 2.4-kilobase-pair fragment. The sucrase enzyme had a Km for sucrose of 48 mM and a pH optimum of 6.5. The S. salivarius sucrase clone showed no detectable hybridization to a yeast invertase clone. Two overlapping subclones which had 1 kilobase pair of DNA in common were used to localize a fructosyltransferase gene. The fructosyltransferase had a Km of 93 mM and a pH optimum of 7.0. The product of the fructosyltransferase was a levan. A fructosyltransferase clone from Bacillus subtilis did not hybridize to S. salivarius DNA. The properties of the enzymes were compared with those of previously characterized sucrases.
Kinetics and inhibition studies of catechol O-methyltransferase from the yeast Candida tropicalis
Authors: Veser J
Published: November 2016

The Kms for esculetin and S-adenosyl-L-methionine for catechol O-methyltransferase from the yeast Candida tropicalis were 6.2 and 40 microM, respectively. S-Adenosyl-L-homocysteine was a very potent competitive inhibitor with respect to S-adenosyl-L-methionine, with a Ki of 6.9 microM. Of the catechol-related inhibitors, purpurogallin, with a Ki of 0.07 microM, showed the greatest inhibitory effect. Sulfhydryl group-blocking reagents, such as thiol-oxidizing 2-iodosobenzoic acid and mercaptide-forming p-chloromercuribenzoic acid, provided evidence for sulfhydryl groups in the active site of the enzyme. Yeast catechol O-methyltransferase is a metal-dependent enzyme and requires Mg2+ for full activity. Zn2+ and Mn2+ but not Ca2+ were able to substitute for Mg2+. Mn2+ showed optimal enzyme activation at concentrations 50- to 100-fold lower than those of Mg2+.
Physiological implications of the specificity of acetohydroxy acid synthase isozymes of enteric bacteria
Authors: Barak Z , Chipman D M , Gollop N
Published: November 2016

The rates of formation of the two alternative products of acetohydroxy acid synthase (AHAS) have been determined by a new analytical method (N. Gollop, Z. Barak, and D. M. Chipman, Anal. Biochem., 160:323-331, 1987). For each of the three distinct isozymes of AHAS in Escherichia coli and Salmonella typhimurium, a specificity ratio, R, was defined: Formula: see text, which is constant over a wide range of substrate concentrations. This is consistent with competition between pyruvate and 2-ketobutyrate for an active acetaldehyde intermediate formed irreversibly after addition of the first pyruvate moiety to the enzyme. Isozyme I showed no product preference (R = 1), whereas isozymes II and III form acetohydroxybutyrate (AHB) at approximately 180- and 60-fold faster rates, respectively, than acetolactate (AL) at equal pyruvate and 2-ketobutyrate concentrations. R values higher than 60 represent remarkably high specificity in favor of the substrate with one extra methylene group. In exponentially growing E. coli cells (under aerobic growth on glucose), which contain about 300 microM pyruvate and only 3 microM 2-ketobutyrate, AHAS I would produce almost entirely AL and only 1 to 2% AHB. However, isozymes II and III would synthesize AHB (on the pathway to Ile) and AL (on the pathway to valine-leucine) in essentially the ratio required for protein synthesis. The specificity ratio R of any AHAS isozyme was affected neither by the natural feedback inhibitors (Val, Ile) nor by the pH. On the basis of the specificities of the isozymes, the known regulation of AHAS I expression by the catabolite repression system, and the reported behavior of bacterial mutants containing single AHAS isozymes, we suggest that AHAS I enables a bacterium to cope with poor carbon sources, which lead to low endogenous pyruvate concentrations. Although AHAS II and III are well suited to producing the branched-chain amino acid precursors during growth on glucose, they would fail to provide appropriate quantities of AL when the concentration of pyruvate is relatively low.
Translocation of Vibrio harveyi N,N'-diacetylchitobiase to the outer membrane of Escherichia coli
Authors: Jannatipour M , Soto-Gil R W , Childers L C , Zyskind J W
Published: November 2016

The gene encoding N,N'-diacetylchitobiase (chitobiase) of the chitinolytic marine bacterium Vibrio harveyi has been isolated. While expression of the chitobiase gene (chb) was inducible by N,N'-diacetylchitobiose in V. harveyi, it was expressed constitutively when cloned in Escherichia coli, suggesting that controlling elements are not closely linked to chb. Chitobiase was found in the membrane fraction of E. coli cells containing plasmids with the cloned V. harveyi chb gene. When membranes of such cells were separated on Osborn gradients, chitobiase activity was found mainly in the outer membrane band. Translocation of the enzyme to the outer membrane was accompanied by cleavage of a signal peptide. A fusion protein, in which 22 amino acids from the amino terminus of prechitobiase were replaced with 21 amino acids from the pUC19 lacZ amino terminus, was not processed, and 99% of the activity was located in the cytoplasmic fraction. A homology to six amino acids surrounding the lipoprotein processing and modification site was found near the amino terminus of prechitobiase.
Specialized cell surface structures in cellulolytic bacteria
Authors: Lamed R , Naimark J , Morgenstern E , Bayer E A
Published: November 2016

The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose.
Genome structure in Streptomyces spp.: adjacent genes on the S. coelicolor A3(2) linkage map have cotransducible analogs in S. venezuelae
Authors: Stuttard C , Atkinson L , Vats S
Published: November 2016

Cotransduction analysis in Streptomyces venezuelae with the generalized transducing phage SV1 showed that several pairs of likely analogs of markers that are adjacent on the conjugational linkage map of Streptomyces coelicolor A3(2) were cotransducible and therefore physically close together. This supports the contention that taxonomically distinct "species" of Streptomyces are genetically closely related.
Inhibition of purified Escherichia coli leader peptidase by the leader (signal) peptide of bacteriophage M13 procoat
Authors: Wickner W , Moore K , Dibb N , Geissert D , Rice M
Published: November 2016

The leader peptide of bacteriophage M13 procoat inhibited the cleavage of M13 procoat or pre-maltose-binding protein by purified Escherichia coli leader peptidase. This finding confirms inferences that the leader is the primary site of enzyme recognition and suggests a rationale for the rapid hydrolysis of leader peptides in vivo.
Locations of the hydrogenases of Methanobacterium formicicum after subcellular fractionation of cell extract
Authors: Baron S F , Brown D P , Ferry J G
Published: November 2016

The F420 hydrogenase of Methanobacterium formicicum was associated with membranes isolated by sucrose density gradient ultracentrifugation of cell extract. The methyl viologen hydrogenase was present in the soluble fractions. Column chromatography with phenyl-Sepharose CL-4B revealed that the F420 hydrogenase was strongly hydrophobic, suggesting that it associates with isolated membranes through hydrophobic interactions.
Chlamydial hemagglutinin identified as lipopolysaccharide
Authors: Watkins N G , Caldwell H D , Hackstadt T
Published: November 2016

Chlamydial lipopolysaccharide (LPS) agglutinated mouse and rabbit erythrocytes but not human, guinea pig, or pronghorn antelope erythrocytes. Hemagglutination was not specific for Chlamydia spp., as rough LPSs from Coxiella burnetii and Escherichia coli also agglutinated erythrocytes from the same animal species. Nonagglutinated and agglutinated erythrocytes bound equivalent amounts of LPS, indicating that hemagglutination was not due to a specific interaction of chlamydial LPS with erythrocytes. Thus, hemagglutination by chlamydial LPS is not mediated by specific receptor-ligand interactions but is a property of the altered surface of the LPS-coated erythrocytes.
Conjugal transfer of transposon Tn916 from Streptococcus faecalis to Mycoplasma hominis
Authors: Roberts M C , Kenny G E
Published: November 2016

Transposon Tn916 was transferred from Streptococcus faecalis to Mycoplasma hominis by a mating process resembling conjugation with a frequency of 10(-6) to 10(-7). Tn916 was inserted into the mycoplasmal chromosome in single and multiple copies.
Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli
Authors: Goodell E W , Higgins C F
Published: November 2016

During bacterial growth, cell wall peptides are released from the murein and reused for the synthesis of new cell wall material. Mutants defective in peptide transport were unable to reutilize cell wall peptides, demonstrating that these peptides are taken up intact into the cytoplasm prior to reincorporation into murein. Furthermore, cell wall peptide recycling was shown to play an important physiological role; peptide transport mutants which were unable to recycle these peptides showed growth defects under appropriate conditions. Using mutants specifically defective in each of the three peptide transport systems, we showed that the uptake of cell wall peptides was mediated solely by the oligopeptide permease (Opp) and that neither the dipeptide permease (Dpp) nor the tripeptide permease (Tpp) played a significant role in this process. Our data indicate that the periplasmic oligopeptide-binding protein has more than one substrate-binding site, each with different though overlapping specificities.
Cloning and nucleotide sequence of the penicillinase antirepressor gene penJ of Bacillus licheniformis
Authors: Imanaka T , Himeno T , Aiba S
Published: November 2016

The penicillinase antirepressor gene, penJ, of Bacillus licheniformis ATCC 9945a was cloned in Escherichia coli by using pMB9 as a vector plasmid. The penicillinase gene, penP, its repressor gene, penI, and penJ were encoded on the cloned 5.2-kilobase HindIII fragment of the recombinant plasmid pTTE71. The penJ open reading frame was composed of 1,803 bases and 601 amino acid residues (molecular weight, 68,388). A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site. Since this sequence was located in the 3'-terminal region of the penI gene, penJ might be transcribed together with penI as a polycistronic mRNA from the penI promoter. Frameshift mutations of penJ were constructed in vitro from pTTE71, and the penJ mutant gene was introduced into B. licheniformis by chromosomal recombination. The transformant B. licheniformis U173 (penP+ penI+ penJ) turned out to be uninducible for penicillinase production, whereas the wild-type strain (penP+ penI+ penJ+) was inducible. Only when these three genes (penP, penI, and PenJ) were simultaneously subcloned in Bacillus subtilis did the plasmid carrier exhibit inducible penicillinase production, as did wild-type B. licheniformis. It was concluded that penJ is involved in the penicillinase induction. The regulation of penP expression by penI and penJ is discussed.
A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of beta-lactamase in Bacillus licheniformis
Authors: Kobayashi T , Zhu Y F , Nicholls N J , Lampen J O
Published: November 2016

A second regulatory locus (blaR1) required for the induction of beta-lactamase synthesis in Bacillus licheniformis 749 was cloned and sequenced. The gene was located on a 5.2-kilobase-pair SphI DNA fragment which also contained the beta-lactamase (blaP) and repressor (blaI) genes. Bacillus subtilis BD224 carrying these three genes synthesized beta-lactamase on exposure to cephalosporin C, whereas Escherichia coli HB101 carrying the genes did not show any detectable induction of the enzyme. An open reading frame of 1,803 bases was identified as the blaR1 gene by subcloning and DNA sequencing. The gene started 2 bases downstream of the termination codon of bla1 and was preceded by a putative Shine-Dalgarno sequence (AAGGA) with a spacing of 5 bases. The deduced blaR1 product (601 amino acids) had a molecular weight of 68,425. Five transmembrane regions were predicted from the hydrophobicity profile. The region around Phe-Ala-Pro-Ala-Ser-Thr-Tyr-Lys (amino acids 398 to 405), which appeared to be located outside the membrane, was homologous to the binding regions of penicillin-binding proteins, including the beta-lactamases. The segment of 22 amino acids from 400 to 421 showed more than 70% homology to the penicillin-binding region of PBP 2 of E. coli. The blaR1 gene encodes a potential penicillin receptor which is required for the induction of beta-lactamase in B. licheniformis 749.
Cloning and characterization of dnaA(Cs), a mutation which leads to overinitiation of DNA replication in Escherichia coli K-12
Authors: Braun R E , O'Day K , Wright A
Published: November 2016

The product of the dnaA gene is essential for the initiation of chromosomal DNA replication in Escherichia coli K-12. A cold-sensitive mutation, dnaA(Cs), was originally isolated as a putative intragenic suppressor of the temperature sensitivity of a dnaA46 mutant (G. Kellenberger-Gujer, A. J. Podhajska, and L. Caro, Mol. Gen. Genet. 162:9-16, 1978). The cold sensitivity of the dnaA(Cs) mutant was attributed to a loss of replication control resulting in overinitiation of DNA replication. We cloned and sequenced the dnaA gene from the dnaA(Cs) mutant and showed that it contains three point mutations in addition to the original dnaA46(Ts) mutation. The dnaA(Cs) mutation was dominant to the wild-type allele. Overproduction of the DnaA(Cs) protein blocked cell growth. In contrast, overproduction of wild-type DnaA protein reduced the growth rate of cells but did not stop cell growth. Thus, the effect of elevated levels of the DnaA(Cs) protein was quite different from that of the wild-type protein under the same conditions.
Isolation, genetic mapping, and characterization of Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase
Authors: Suzuki H , Kumagai H , Tochikura T
Published: November 2016

Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase (EC 2.3.2.2) were isolated after mutagenesis of cells with ethyl methanesulfonate. They lost the enzyme activity to different extents. The mutations of two mutants that had lost the enzyme activity completely were mapped at 76 min of the E. coli K-12 linkage map. These mutations made the cells neither nutrient requiring nor cold sensitive. The mutants leaked much more glutathione into the medium than the wild type. We propose the symbol ggt for these mutations.
Structural and genetic analyses of a par locus that regulates plasmid partition in Bacillus subtilis
Authors: Chang S , Chang S Y , Gray O
Published: November 2016

The Bacillus plasmid pLS11 partitions faithfully during cell division. Using a partition-deficient plasmid vector, we randomly cloned DNA fragments of plasmid pLS11 and identified the locus that regulates plasmid partition (par) by cis complementation in Bacillus subtilis. The cloned par gene conferred upon the vector plasmid a high degree of segregational stability. The par locus was mapped to a 167-base-pair segment on pLS11, and its nucleotide sequence was determined. The cloned par fragment regulated the partition of several different Bacillus replicons, and it only functioned in cis; it did not contain the replication function nor elevate the plasmid copy number in B. subtilis. The expression of par was orientation specific with respect to the replication origin on the same plasmid. We propose that the pLS11-derived par functions as a single-stranded site that interacts with other components involved in plasmid partition during cell division.
Characterization and virulence properties of Erwinia chrysanthemi lipopolysaccharide-defective, phi EC2-resistant mutants
Authors: Schoonejans E , Expert D , Toussaint A
Published: November 2016

Outer membrane alterations were characterized in spontaneous mutants of the Erwinia chrysanthemi 3937jRH, which were selected for resistance to bacteriophage phi EC2. All but one of the mutants analyzed were affected in their lipopolysaccharide (LPS) structure, lacking the entire heterogeneous region of apparent high molecular weight present in the wild-type E. chrysanthemi LPS. At least two 3937jRH mutants, one selected as phi EC2 resistant (RH6065) and the other previously selected (D. Expert and A. Toussaint, J. Bacteriol. 163:221-227, 1985) as bacteriocin resistant (R1456), were cross-resistant to bacteriophage Mu and had rough LPSs with an altered core structure. Two phi EC2r mutants (RH6053 and RH6065) were most severely affected in their outer membrane integrity and also lost their virulence on saintpaulia plants, although they still possessed normal extracellular levels of pectinolytic and cellulolytic activities. The two Mur mutants RH6065 and R1456 were also able to induce systemic resistance in the challenged plant. All the other phi EC2r mutants retained the virulence of 393jRH.
Involvement of FtsZ protein in shift-up-induced division delay in Escherichia coli
Authors: Kepes F , D'Ari R
Published: November 2016

A nutritional shift-up from glucose minimal medium to LB broth was previously shown to cause a division delay of about 20 min in synchronized cultures of Escherichia coli, and a similar delay was observed after a nutritional pulse (a shift-up followed rapidly by a return to glucose minimal medium). Using synchronized cultures, we show here that the pulse-induced division delay does not require protein synthesis during the period in LB broth, suggesting that a nonprotein signal is generated by the shift-up and transmitted to the cell division machinery. The cell division protein FtsZ, target of the SOS-associated division inhibitor SfiA (or SulA), seems to be involved in the postshift division delay. Mutants in which the FtsZ-SfiA interaction is reduced, either sfiA (loss of SfiA) or ftsZ(SfiB) (modification of FtsZ), have a 50- to 60-min division delay after a shift-up. Furthermore, after a nutritional pulse, the ftsZ(SfiB) mutant had only a 10- to 16-min delay. These results suggest that the FtsZ protein is the target element of the cell division machinery to which the shift-up signal is transmitted.
Developmental expression of three proteins from the first gene of the RNA polymerase sigma 43 operon of Bacillus subtilis
Authors: Wang L F , Doi R H
Published: November 2016

The first gene of the Bacillus subtilis RNA polymerase sigma 43 operon, P23, has a protein-coding capacity of 23,000 daltons. Sequence analysis revealed three potential translational initiation sites within the same reading frame, which could encode proteins of 23,000 (P23), 19,000 (P19), and 9,000 (P9) daltons, respectively. An internal promoter (P3), which is expressed only during the sporulation stage, is located between the second and the third translational start sites. By protein fusion to the Escherichia coli beta-galactosidase gene, we showed that all three translational initiation sites of the P23 gene are used in vivo in both E. coli and B. subtilis, and regulation for differential expression of the three proteins during the development of B. subtilis is coupled to the transcriptional promoter switching mechanism. The physiological function of these multiple gene products is unknown and is currently under investigation.
Cloning and characterization of the Escherichia coli K-12 alanine-valine transaminase (avtA) gene
Authors: Wang M D , Liu L , Wang B M , Berg C M
Published: November 2016

avtA, which encodes the alanine-valine transaminase, transaminase C, was cloned in vivo with high- and low-copy-number mini-Mu cloning vectors. The phenotype conferred by the cloned avtA+ gene usually depended upon the plasmid copy number; most high-copy-number avtA+ plasmids permitted isoleucine-requiring ilvE strains to grow in the absence of isoleucine (multicopy suppression), while low-copy-number avtA+ plasmids did not. avtA was mapped to a 1.25-kilobase segment by comparison of the restriction maps of 24 independent mini-Mu plasmids and then by gamma-delta (Tn1000) mutagenesis of a pBR322-avtA+ plasmid. The direction of transcription of avtA on the cloned fragment was determined with fusions to a promoterless lac gene.
Drug-free induction of a chloramphenicol acetyltransferase gene in Bacillus subtilis by stalling ribosomes in a regulatory leader
Authors: Duvall E J , Ambulos N P , Lovett P S
Published: November 2016

The plasmid gene cat-86 is induced by chloramphenicol in Bacillus subtilis, resulting in the synthesis of the gene product chloramphenicol acetyltransferase. Induction is due to a posttranscriptional regulatory mechanism in which the inducer, chloramphenicol, activates translation of cat-86 mRNA. We have suggested that chloramphenicol allows ribosomes to destabilize a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the coding sequence. In the present report we show that cat-86 expression can be activated by stalling ribosomes in the act of translating a regulatory leader peptide. Stalling was brought about by starving host cells for specific leader amino acids. Ribosomal stalling, which led to cat-86 expression, occurred upon starvation for the amino acid specified by the leader codon located immediately 5' to the RNA stem-loop structure and was independent of whether that codon specified lysine or tyrosine. These observations support a model for chloramphenicol induction of cat-86 in which the antibiotic stalls ribosome transit in the regulatory leader. Stalling of ribosomes in the leader can therefore lead to destabilization of the RNA stem-loop structure.