bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic lysis of the pseudomurein-containing methanogen Methanobacterium formicicum
Authors: Bush J W
Published: January 2020

A streptomycete isolated from cow manure produces an extracellular enzyme capable of lysing the pseudomurein-containing methanogen Methanobacterium formicicum. The lytic activity has been partially purified from culture fluid and appears to be a serine protease. Similar lytic activity has been fractionated from pronase. Optimal conditions have been developed for lysis of M. formicicum by commercial preparations of proteinase K. The three lytic enzymes have been partially characterized. The results with the three enzyme preparations tend to confirm that proteolytic enzymes are capable of lysing methanogen cells.
Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions
Authors: Priefer U B , Simon R , Pühler A
Published: January 2020

The broad-host-range vectors pSUP104, pSUP106, pSUP204, pSUP304, and pSUP404 are based on conventional Escherichia coli vectors (such as pBR325 and pACYC184) which have been modified to include the mobilization and broad-host-range replication functions of the IncQ plasmid RSF1010. These vector plasmids now can be maintained in a wide range of bacterial genera including Rhizobium, Agrobacterium, and Pseudomonas. They are efficiently mobilized by RP4 and thus are of particular interest for bacteria refractory to transformation. They offer the selection markers and cloning sites characteristic of the basic E. coli vectors. Therefore, they can be applied and adapted to a variety of cloning strategies. However, the cloning of very large fragments (e.g., in cosmid hybrids of pSUP106) was found to affect the stability of the recombinant molecules in a Rec+ background. This instability was not observed with smaller inserts of about 5 kilobases.
Nucleotide sequence binding specificity of the LexA repressor of Escherichia coli K-12
Authors: Wertman K F , Mount D W
Published: January 2020

The specificity of LexA protein binding was investigated by quantifying the repressibility of several mutant recA and lexA operator-promoter regions fused to the Escherichia coli galactokinase (galK) gene. The results of this analysis indicate that two sets of four nucleotides, one set at each end of the operator (terminal-nucleotide contacts), are most critical for repressor binding. In addition, our results suggest that the repressor-operator interaction is symmetric in nature, in that mutations at symmetrically equivalent positions in the recA operator have comparable effects on repressibility. The symmetry of this interaction justified reevaluation of the consensus sequence by half-site comparison, which yielded the half-site consensus (5')CTGTATAT. Although the first four positions of this sequence were most important, the last four were well conserved among binding sites and appeared to modulate repressor affinity. The role of the terminal-nucleotide contacts and the mechanism by which the internal sequences affected repressor binding are discussed.
Plaque color method for rapid isolation of novel recA mutants of Escherichia coli K-12: new classes of protease-constitutive recA mutants
Authors: Tessman E S , Peterson P
Published: January 2020

As a prerequisite to mutational analysis of functional sites on the RecA protein of Escherichia coli, a method was developed for rapid isolation of recA mutants with altered RecA protease function. The method involves plating mutagenized lambda recA+ cI ind on strains deleted for recA and containing, as indicators of RecA protease activity, Mu d(Ap lac) fusions in RecA-inducible genes. The lambda recA phages were recognized by their altered plaque colors, and the RecA protease activity of the lambda recA mutant lysogens was measured by expression of beta-galactosidase from dinD::lac. One class of recA mutants had constitutive protease activity and was designated Prtc; in these cells the RecA protein was always in the protease form without the usual need for DNA damage to activate it. Some Prtc mutants were recombinase negative and were designated Prtc Rec-. Another class of 65 recA mutants isolated as being protease defective were all also recombinase defective. Unlike the original temperature-dependent Prtc Rec+ mutant (recA441), the new Prtc Rec+ mutants showed constitutive protease activity at any growth temperature, with some having considerably greater activity than the recA441 strain. Study of these strong Prtc Rec+ mutants revealed a new SOS phenomenon, increased permeability to drugs. Use of this new SOS phenomenon as an index of protease strength clearly distinguished 5 Prtc mutants as the strongest among 150. These five strongest Prtc mutants showed the greatest increase in spontaneous mutation frequency and were not inhibited by cytidine plus guanosine, which inhibited the constitutive protease activity of the recA441 strain and of all the other new Prtc mutants. Strong Prtc Rec+ mutants were more UV resistant than recA+ strains and showed indications of having RecA proteins whose specific activity of recombinase function was higher than that of wild-type RecA. A Prt+ Rec- mutant with an anomalous response to effectors is described.
Purification and characterization of glutamate synthase from Azospirillum brasilense
Authors: Ratti S , Curti B , Zanetti G , Galli E
Published: January 2020

Growth conditions for Azospirillum brasilense Sp6 were devised for maximal expression of glutamate synthase. The enzyme levels were largely affected by the type and concentration of the nitrogen source. A 10-fold increase in the synthesis of the enzyme was observed at a limiting concentration of ammonia. The enzyme was purified to homogeneity by a procedure which was fairly rapid and allowed a good recovery of enzyme (30%). Azospirillum glutamate synthase is a complex iron-sulfur flavoprotein with a stoichiometry of 1 flavin adenine dinucleotide:1 flavin mononucleotide:8 Fe:8 S per protomer with a molecular weight of 185,000. The protomer is composed of two dissimilar subunits with molecular weights of 135,000 and 50,000. Kinetic parameters were determined. Km values for NADPH, 2-oxoglutarate, and L-glutamine were 6.25, 29, and 450 microM, respectively. The optimum pH was about 7.5. Complete reduction of the enzyme under anaerobic conditions was obtained either by NADPH (in the presence of a regenerating system) or dithionite or by photochemical reduction (in the presence of EDTA and 5-deazariboflavin). No stable long-wavelength intermediates were observed.
Protoplast water content of bacterial spores determined by buoyant density sedimentation
Authors: Lindsay J A , Beaman T C , Gerhardt P
Published: January 2020

Protoplast wet densities (1.315 to 1.400 g/ml), determined by buoyant density sedimentation in Metrizamide gradients, were correlated inversely with the protoplast water contents (26.4 to 55.0 g of water/100 g of wet protoplast) of nine diverse types of pure lysozyme-sensitive dormant bacterial spores. The correlation equation provided a precise method for obtaining the protoplast water contents of other spore types with small impure samples and indicated that the average protoplast dry density was 1.460 g/ml.
Acetate catabolism by Methanosarcina barkeri: evidence for involvement of carbon monoxide dehydrogenase, methyl coenzyme M, and methylreductase
Authors: Krzycki J A , Lehman L J , Zeikus J G
Published: January 2020

The pathway of acetate catabolism in Methanosarcina barkeri strain MS was studied by using a recently developed assay for methanogenesis from acetate by soluble enzymes in cell extracts. Extracts incubated with [2-14C]acetate, hydrogen, and ATP formed 14CH4 and [14C]methyl coenzyme M as products. The apparent Km for acetate conversion to methane was 5 mM. In the presence of excess acetate, both the rate and duration of methane production was dependent on ATP. Acetyl phosphate replaced the cell extract methanogenic requirement for both acetate and ATP (the Km for ATP was 2 mM). Low concentrations of bromoethanesulfonic acid and cyanide, inhibitors of methylreductase and carbon monoxide dehydrogenase, respectively, greatly reduced the rate of methanogenesis. Precipitation of CO dehydrogenase in cell extracts by antibodies raised to 95% purified enzyme inhibited both CO dehydrogenase and acetate-to-methane conversion activity. The data are consistent with a model of acetate catabolism in which methylreductase, methyl coenzyme M, CO dehydrogenase, and acetate-activating enzymes are components. These results are discussed in relation to acetate uptake and rate-limiting transformation mechanisms in methane formation.
Waveform analysis and structure of flagella and basal complexes from Bdellovibrio bacteriovorus 109J
Authors: Thomashow L S , Rittenberg S C
Published: January 2020

The structure of sheathed flagella from Bdellovibrio bacteriovorus was investigated. The first three periods of these flagella were characterized by progressively smaller wavelengths and amplitudes in periods more distal to the cell. The damped appearance was due to a single nonrandom transition between two helical structures within each filament. The intersection of the two helices, one of which was a threefold-reduced miniature of the other, occurred at a fixed distance along the filament and resulted in a shift in the flagellar axis. Flagella increased in length as the cells aged and assumed a constant miniature waveform at their distal ends. The core filament was the principal determinant of flagellar morphology. It was composed of 28,000- and 29,500-dalton polypeptides. The 28,000-dalton subunits were located in the cell-proximal segment of the filament, and the 29,500-dalton subunits were located in the more distal region. The heteromorphous appearance of bdellovibrio flagella arose from the sequential assembly of these subunits. The basal complex associated with core filaments was examined because of its potential involvement in sheath formation. Bdellovibrio basal organelles were generally similar to those of other gram-negative species, but appeared to lack a disk analogous to the outer membrane-associated L ring which is a normal component of gram-negative basal complexes.
beta-Ketoadipate pathway in Trichosporon cutaneum modified for methyl-substituted metabolites
Authors: Powlowski J B , Dagley S
Published: January 2020

Trichosporon cutaneum, when grown with p-cresol, catalyzed intradiol fission of the benzene nucleus of 4-methylcatechol before the complete catabolism of these two substrates. Steps in their conversion to pyruvate and acetyl coenzyme A were investigated by using cell extracts, and some properties of various new microbial catabolites are also described. These included (-)-2,5-dihydro-3-methyl-5-oxofuran-2-acetic acid (beta-methylmuconolactone) and (-)-3-keto-4-methyladipic acid and its coenzyme A ester; the latter was degraded by an enzymatic reaction sequence that included the coenzyme A esters of methylsuccinic, itaconic, and citramalic acids. A notable feature of this sequence is the formation of beta-methylmuconolactone which can be readily metabolized, in contrast to the analogous reaction in bacteria that gives the "dead-end" compound gamma-methylmuconolactone; this compound cannot be enzymatically degraded and so renders the beta-ketoadipate pathway unavailable for methyl-substituted bacterial sources of carbon that are catabolized by way of 4-methylcatechol.
Functional identification of the fatty acid reductase components encoded in the luminescence operon of Vibrio fischeri
Authors: Boylan M , Graham A F , Meighen E A
Published: January 2020

A clone of DNA, obtained from the luminescent bacterium Vibrio fischeri ATCC 7744 and inserted into pBR322, was found to express luminescence in Escherichia coli. Polypeptides involved in biosynthesis of the fatty aldehyde substrate for the light reaction were identified by fatty acid acylation of proteins synthesized in E. coli from the recombinant plasmid. The cloned region was similar to that reported for the V. fischeri MJ1 luminescence system (Engebrecht et al., Cell 32:773-781), except for some differences in endonuclease restriction sites and the requirement of a lower temperature for the expression of light in our cloned system. Fatty acid reductase activity could be detected in extracts of E. coli harboring the recombinant plasmid but not in extracts of the parental V. fischeri strain. Using in vivo labeling with [3H]tetradecanoic acid, we showed that the acylated polypeptides synthesized in the cloned system corresponded to the labeled polypeptides in V. fischeri (34, 42, and 54 kilodaltons) and that they could only be detected after induction of luminescence. These results provide direct evidence that the genes coding for the fatty acid reductase polypeptides are an integral part of the luminescence operon in the V. fischeri luminescence system.
Single transporter for sulfate, selenate, and selenite in Escherichia coli K-12
Authors: Lindblow-Kull C , Kull F J , Shrift A
Published: January 2020

A Michaelis-Menten kinetic analysis of the transport of sulfate, selenate, and selenite into Escherichia coli K-12 showed that the three dianions were transported by the same carrier. Km values, used as a measure of the affinity of each ligand for the carrier, showed that sulfate was bound 5 times more tightly than selenate and 37 times more tightly than selenite. The specificity ratio, Vmax/Km, also indicated that sulfate was the preferred ligand. There was little difference in the ratios for selenate and selenite.
Assembly site of bacteriophage f1 corresponds to adhesion zones between the inner and outer membranes of the host cell
Authors: Lopez J , Webster R E
Published: January 2020

Morphogenesis of the filamentous bacteriophage f1 occurred at adhesion zones between the inner and outer membranes of the host cell. Quantitation of adhesion zones in cells infected with mutant phage strains suggested that the phage gene I protein may be involved in the formation of adhesion zones for phage assembly.
Catabolite repression and role of cyclic AMP in CO2 fixation and H2 metabolism in Rhizobium spp
Authors: McGetrick A M , Goulding C F , Manian S S , O'Gara F
Published: January 2020

CO2 fixation in Rhizobium meliloti was repressed by a variety of organic carbon sources. Cellular cyclic AMP levels were similar in repressed and nonrepressed cultures. Exogenous cyclic AMP or additional copies of the adenyl cyclase gene in cells experiencing repression failed to affect the rates of CO2 fixation. However, in R. japonicum catabolite repression of H2 utilization was partially circumvented by the presence of the R. meliloti adenyl cyclase gene.
Determination of the transcription initiation site and identification of the protein product of the regulatory gene xylR for xyl operons on the TOL plasmid
Authors: Inouye S , Nakazawa A , Nakazawa T
Published: January 2020

The xylR gene is a regulatory gene on the TOL plasmid, which acts in a positive manner on xyl operons for degradation of toluene and xylenes in Pseudomonas putida. A DNA fragment containing the xylR promoter region was cloned on promoter-probing vectors, and its nucleotide sequence was determined. The transcription initiation site of the xylR gene was determined in cells of P. putida and Escherichia coli by S1 nuclease and reverse transcriptase mapping. Two initiation sites were detected which were identical in both P. putida and E. coli. The amounts of mRNA synthesized in both bacterial cells were almost the same and independent of the inducers for xyl operons. The consensus sequences for E. coli promoters were found in the region preceding the respective transcription initiation sites. The product of the xylR gene was identified by the maxicell system as a protein with an approximate molecular weight of 67,000.
Isolation and characterization of transposon Tn5-induced mutants of Pseudomonas perfectomarina defective in nitrous oxide respiration
Authors: Zumft W G , Döhler K , Körner H
Published: January 2020

Transposon (Tn5) mutagenesis of Pseudomonas perfectomarina with the plasmid pSUP2021 [(pBR325-Mob(RP4))::Tn5] and the chromosomally integrated RP4 plasmid in Escherichia coli as the donor, produced three distinct groups of mutants that were defective in nitrous oxide respiration. One group of mutants lacked the structural protein of N2O reductase, the second synthesized a copper-free apoprotein; and a third group expressed a low level of intact enzyme. The mutants provided evidence for N2O being the immediate precursor of dinitrogen in denitrification and documented the essentiality of the copper enzyme. Synthesis of N2O reductase depended strongly on the growth conditions, with N2O-grown cells expressing the lowest level of enzyme. Regulatory responses of mutants elicited by nitrate or oxygen were unaltered when compared with wild-type behavior.
Leucine regulation of the ilvGEDA operon of Serratia marcescens by attenuation is modulated by a single leucine codon
Authors: Hsu J H , Harms E , Umbarger H E
Published: January 2020

The effect of leucine limitation and of restricted leucine tRNA charging on the expression of the ilvGEDA operon of Serratia marcescens was examined. In this organism, the ilv leader region specifies a putative peptide containing only a single leucine codon that could be involved in leucine-mediated control by attenuation (E. Harms, J.-H. Hsu, C. S. Subrahmanyam, and H. E. Umbarger, J. Bacteriol. 164:207-216, 1985). A plasmid (pPU134) containing the DNA of the S. marcescens ilv control region and three of the associated structural genes was studied as a single chromosomal copy in an Escherichia coli strain auxotrophic for all three branched-chain amino acids. The S. marcescens ilv genes responded to a multivalent control similar to that found in other enteric organisms. Furthermore, the S. marcescens ilv genes were derepressed when the charging of leucine tRNA was restricted in a leuS derivative of E. coli that had been transformed with pPU134. It was concluded that ribosome stalling leading to deattenuation is not dependent on either tandem or a consecutive series of codons for the regulatory amino acid. However, the fact that the single leucine codon is a less frequently used codon (CUA) may be important. The procedure for obtaining the cloned ilv genes in single chromosomal copy exploited the dependence of ColE1 replicons on the polA gene. The cloning experiments also revealed a branched-chain amino acid-glutamate transaminase in S. marcescens that is different from transaminase B.
Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant
Authors: Smith C J
Published: January 2020

Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were constructed and used as molecular cloning vectors. The 2.7-kilobase pair (kb) cryptic Bacteroides plasmid pBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection of the plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing the clindamycin resistance (Ccr) determinant from a Bacteroides R plasmid, pBF4;Ccr was not expressed in E. coli. The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependent upon the source of the DNA; generally 10(5) transformants micrograms-1 of DNA were recovered when plasmid purified from B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the number of transformants obtained. Two of the shuttle plasmids not containing the pBF4 Ccr determinant were used in an analysis of the transposon-like structure encoding Ccr in the R plasmid pBI136. This gene encoding Ccr was located on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing the gene inserted in both orientations at the unique ClaI site within the pBI143 portion of the shuttle plasmids could transform B. fragilis to clindamycin resistance. These results together with previous structural data show that the gene encoding Ccr lies directly adjacent to one of the repeated sequences of the pBI136 transposon-like structure.
Polyethylene glycol-facilitated transformation of Bacteroides fragilis with plasmid DNA
Authors: Smith C J
Published: January 2020

A method for the transformation of Bacteroides fragilis with plasmid DNA was developed by using the clindamycin resistance plasmid pBFTM10 as the source of transforming DNA. The method was technically simple to perform and resulted in an average of 4.2 X 10(3) transformants per microgram of pBFTM10 added. A method for the preparation of frozen competent cells is also described.
Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae
Authors: Michaelis S , Chapon C , D'Enfert C , Pugsley A P , Schwartz M
Published: January 2020

Some strains of Klebsiella pneumonia secrete pullulanase, a debranching enzyme which produces linear molecules (maltodextrins, amylose) from amylopectin and glycogen. pulA, the structural gene for pullulanase, was introduced into Escherichia coli, either on a multiple-copy-number plasmid or as a single copy in the chromosome. When in E. coli, pulA was controlled by malT, the positive regulatory gene of the maltose regulon. Indeed, pulA expression was undetectable in a malT-negative mutant and constitutive in a malTc strain. Furthermore, the plasmid carrying pulA titrated the MalT protein. When produced in E. coli, pullulanase was not localized in the same way as in K. pneumoniae. In the latter case it was first exported to the outer membrane, with which it remained loosely associated, and was then released into the growth medium. In E. coli the enzyme was distributed both in the inner and the outer membranes and was never released into the growth medium.
Structure of two divergent promoters located in front of the gene encoding pullulanase in Klebsiella pneumoniae and positively regulated by the malT product
Authors: Chapon C , Raibaud O
Published: January 2020

Pullulanase is an extracellular starch-debranching enzyme produced by Klebsiella pneumoniae. When its structural gene, pulA, is introduced into Escherichia coli, it is controlled by malT, the positive regulator gene of the maltose regulon. Characterization of the region 5' to pulA and of the beginning of the gene described herein demonstrate that (i) pullulanase is probably a lipoprotein; (ii) an additional malT-controlled promoter (the malX promoter) lies adjacent to the pulA promoter and is oriented in the opposite direction; (iii) in common with the three previously described malT-controlled promoters, the pulA and malX promoters have a conserved hexanucleotide (consensus sequence, 5'-GGATGGA) 35 base pairs upstream from the transcription initiation site; and (iv) upstream from this conserved hexanucleotide the pulA and malX promoters differ from the other mal promoters in that they lack any detectable binding site for the cyclic AMP-binding protein.
Identification of the Escherichia coli recN gene product as a major SOS protein
Authors: Finch P W , Chambers P , Emmerson P T
Published: January 2020

The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified.
phs Locus of Escherichia coli, a mutation causing pleiotropic lesions in metabolism, is an rpoA allele
Authors: Rowland G C , Giffard P M , Booth I R
Published: January 2020

The phs mutation, which causes a pleiotropic growth defect, has been mapped and shown to be an allele of rpoA, the gene for the alpha subunit of RNA polymerase. The mutation is shown to cause a transcription defect in the arabinose operon, araBAD.
Gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri: cloning and expression in Escherichia coli
Authors: Mouchès C , Candresse T , Barroso G , Saillard C , Wroblewski H , Bové J M
Published: January 2020

A library of cloned Spiroplasma citri genomic sequences was constructed by incorporating HindIII digestion fragments into the plasmid vector pBR328. Immunological screening allowed the identification of a recombinant plasmid containing the gene for spiralin, the major membrane protein of S. citri. The spiralin produced by the Escherichia coli transformant was characterized by immunological detection with monoclonal antibody after Western blotting of two-dimensional (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide) electrophoresis gels and by partial proteolytic mapping. The gene for spiralin occurred within a 6.5-kilobase-pair cloned DNA fragment. Spiralin in E. coli was produced regardless of the orientation of the insert within the pBR328 vector. A spiroplasmal DNA sequence which acted as a promoter in E. coli was cloned along with the structural spiralin gene which is expressed in E. coli from that sequence.
Regulation and genetic enhancement of glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum
Authors: Hyun H H , Zeikus J G
Published: January 2020

We studied the general mechanism for regulation of glucoamylase and pullulanase synthesis in Clostridium thermohydrosulfuricum. These amylases were expressed only when the organism was grown on maltose or other carbohydrates containing maltose units. Amylase synthesis was more severely repressed by glucose than by xylose. Catabolite repression-resistant mutants were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained glucose-starch agar plates. Amylases were produced in both wild-type and mutant strains when starch was added to cells growing on xylose but not when starch was added to cells growing on glucose. In both wild-type and mutant strains, glucoamylase and pullulanase were produced at high levels in starch-limited chemostats but not in glucose- or xylose-limited chemostats. Therefore, we concluded that amylase synthesis in C. thermohydrosulfuricum was inducible and subject to catabolite repression. The mutants produced about twofold more glucoamylase and pullulanase, and they were catabolite repression resistant for production of glucose isomerase, lactase, and isomaltase. The mutants displayed improved starch metabolism features in terms of enhanced rates of growth, ethanol production, and starch consumption.
Nucleotide sequence of the gene determining plasmid-mediated citrate utilization
Authors: Ishiguro N , Sato G
Published: January 2020

The citrate utilization determinant from transposon Tn3411 has been cloned and sequenced, and its polypeptide products have been characterized in minicell experiments. The nucleotide sequence was determined for a 2,047-base-pair BglII restriction endonuclease fragment that includes the citrate determinant. This region contains an open reading frame that would encode a 431-amino-acid very hydrophobic polypeptide and which is preceded by a reasonable ribosomal binding site. However, the single polypeptide found in minicell experiments had an apparent molecular weight of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
STAPHYLOCOCCAL PENICILLINASE I
Authors: Saz Arthur K. , Lowery Dolores L. , Jackson Leah J.
Published: January 2020

Saz, Arthur
K. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.),
Dolores L. Lowery, and Leah J. Jackson
. Staphylococcal penicillinase. I. Inhibition and stimulation of activity. J. Bacteriol.
82:
298–304. 1961.—The penicillinase extracted from a penicillin-resistant strain of
Staphylococcus aureus
was shown to be inhibited up to 70% by various dipeptides and particularly by
d
-valyl-
d
-valine. It is of interest that the nucleus of penicillin contains
d
-valine. Various dipeptides formed by condensing amino acids and metal-binding compounds such as benzidine and biphenyl have been shown to inhibit penicillinase activity in considerably lower concentration than the dipeptides composed of amino acids alone. Contrariwise, it has been found that various alcohols, and particularly
n
-propanol, markedly stimulate the activity of the penicillinase both in whole cells and in cell-free extracts. The alcohols fall into a symmetrical series in respect to stimulatory activity with methyl < ethyl <
n
-propanol >
n
-butanol > amyl > isoamyl > octyl. Evidence is also presented showing that staphylococcal penicillinase is associated with the particulate fraction of the cell and it is postulated that the alcohols stimulate as a result of bringing insoluble enzyme and substrate into closer apposition.
Bacillus subtilis
strain 749 penicillinase, which is essentially soluble, is not stimulated by the alcohols. The possibility is presented based on fractionation of crude enzyme on a diethylamino-ethyl cellulose column that different species of penicillinase exist in
S. aureus
.
GLUTAMIC DECARBOXYLASE OF ERGOT,
CLAVICEPS PURPUREA
Authors: Anderson John A. , Cheldelin Vernon H. , King Tsoo E.
Published: January 2020

Anderson, John
A. (Oregon State University, Corvallis),
Vernon H. Cheldelin, and Tsoo E. King
. Glutamic decarboxylase of ergot,
Claviceps purpurea
. J. Bacteriol.
82:
354–358. 1961.—
l
-Glutamic acid is the only naturally occurring amino acid which can be decarboxylated by cell-free extracts of
Claviceps purpurea
. This decarboxylase was partially purified and the properties of the enzyme studied.

The specific activity of the purified preparation was 111 μliters per 10 min per mg of protein. The products formed, stability, inhibition, stimulation of activity with pyridoxal phosphate, and pH activity curve were typical of
l
-glutamic decarboxylase in
Escherichia coli
and other microorganisms.

The substrate constants at pH 4.6, 5.25, and 5.65 were 0.0169
m
, 0.0174
m
, and 0.0139
m
, respectively. The respective maximal velocities at these pH values were 104, 104, and 90 μliters per 10 min.

The pH optimum was 4.8 to 5.2. The enzyme was unstable below pH 4.5 and it was suggested that the fall in activity at the lower end of the pH curve was due to inactivation of the enzyme.

The decrease in activity above pH 5.2 did not appear to be due to a change in affinity of enzyme for substrate but to a change of the enzyme-substrate complex into an inactive form.
SOME FACTORS WHICH AFFECT THE INITIATION OF GROWTH OF
CRYPTOCOCCUS NEOFORMANS
Authors: Howard Dexter H.
Published: January 2020

Howard, Dexter
H. (University of California, Los Angeles). Some factors which affect the initiation of growth of
Cryptococcus neoformans
. J. Bacteriol.
82:
430–435. 1961.—The observation that a strain of
Cryptococcus neoformans
failed to grow in a medium containing normal human serum led to an investigation of some of the physiological factors controlling initiation of growth by this fungus.

The data presented show that the growth of
C. neoformans
is markedly inhibited in media with initial pH values slightly above neutrality. The growth of four strains of the fungus was completely suppressed in peptone broth at pH values above 8. The growth of two strains was partially inhibited at a pH of 7.3. Two other strains grew well at 7.3, but were partially inhibited at pH 7.5. The growth in media at pH values which partially inhibited multiplication was granular in appearance. Microscopically the granules were composed of clumps of yeast cells. The possibility is suggested that pH may exert a portion of its effect on the growth of
C. neoformans
by influencing those factors which affect the stability of the cells in suspension.

At a temperature of 25 C, growth of the fungus was initiated in media with a pH which suppressed growth at a temperature of 37 C. Under the experimental conditions employed in these studies, proteose peptone no. 3 (Difco) serves as both a carbon and a nitrogen source. However, glucose, which is assimilated by
C. neoformans
in contrast to lactose, permitted the growth of the fungus at pH values which suppressed growth in the absence of the sugar.

Since weakly buffered media, containing filtered, normal human serum, have an alkaline reaction higher than that shown to suppress the growth of
C. neoformans
, it was concluded that this factor was primarily responsible for the failure of the fungus to reproduce in tissue culture media. However, it is also possible to show that even under optimal pH conditions normal human serum exerts an anticryptococcal effect. This activity of human serum was relatively heat-stable and was expressed only in high concentrations of serum. Serum adsorbed with viable cells of
C. neoformans
also inhibited the growth of the fungus, but a partially purified globulin fraction of serum was not inhibitory.

The data are discussed in relation to the work of other investigators.
ANTIGENIC PROPERTIES OF PLEUROPNEUMONIA-LIKE ORGANISMS FROM TISSUE CELL CULTURES AND THE HUMAN GENITAL AREA
Authors: Bailey Jack S. , Clark Harold W. , Felts William R. , Fowler Richard C. , Brown Thomas McP.
Published: January 2020

Bailey, JackS. (George Washington University, Washington, D. C.),Harold W. Clark, William R. Felts, Richard C. Fowler, and Thomas McP. Brown. Antigenic properties of pleuropneumonia-like organisms from tissue cell cultures and the human genital area. J. Bacteriol.82:542–547. 1961.—Antigens were prepared from several tissue culture and human genital strains of pleuropneumonia-like organisms (PPLO) by a method utilizing continuous agitation of the incubating cultures. Antisera were produced in rabbits by intravenous injection of suspensions of these organisms standardized turbidimetrically. The antigenic properties of the selected strains were compared by agglutination techniques supplemented by a test based upon the inhibition of growth of PPLO by specific antisera.

The majority of tissue culture strains of PPLO studied, including contaminants from several HeLa cell lines, appeared to be antigenically similar to the human type 1 strains. However, one strain (Sp-1) from a HeLa cell line was found to be related to the human type 2 PPLO.
ANTIGENIC STUDIES OF CANDIDA
Authors: Hasenclever H. F. , Mitchell William O.
Published: January 2020

Hasenclever
, H. F. (U. S. Public Health Service, Bethesda, Md.),
and William O. Mitchell
. Antigenic studies of Candida. III. Comparative pathogenicity of
Candida albicans
group A, group B, and
Candida stellatoidea
. J. Bacteriol.
82:
578–581. 1961.—Since the antigenic differences between
Candida albicans
group A and
C. albicans
group B had been established, the problem of a difference in pathogenicity for laboratory animals was studied.
Candida stellatoidea
was also included in this investigation. Similar experiments with
Candida tropicalis
have been reported.

The virulence of 24 strains of
C. albicans
group A, 23 strains of
C. albicans
group B, and 11 strains of
C. stellatoidea
for mice was determined. Five
C. albicans
group B and four
C. stellatoidea
strains were tested for lethality in rabbits.

C. albicans
group A and
C. albicans
group B displayed about the same degree of virulence for mice. Although
C. stellatoidea
in general showed less virulence for mice than
C. albicans
, some of its strains were more virulent than the least virulent
C. albicans
strains. All five
C. albicans
group B strains tested in rabbits demonstrated considerably more virulence for this laboratory animal than any of the four
C. stellatoidea
strains tested.

C. albicans
group B was isolated from more diverse clinical specimens than
C. albicans
group A, but only a small number of specimens were studied.
FACTORS AFFECTING THE ELABORATION BY
CLOSTRIDIUM HISTOLYTICUM
OF PROTEINASES CAPABLE OF DEBRIDING THIRD DEGREE BURN ESCHARS ON GUINEA PIGS
Authors: Berman Sanford , Lowenthal Joseph P. , Webster Marion E. , Altieri Patricia L. , Gochenour Raymond B.
Published: January 2020

Berman, Sanford
(Walter Reed Army Institute of Research, Washington, D. C.),
Joseph P. Lowenthal, Marion E. Webster, Patricia L. Altieri, and Raymond B. Gochenour
. Factors affecting the elaboration by
Clostridium histolyticum
of proteinases capable of debriding third degree burn eschars on guinea pigs. J. Bacteriol.
82:
582–588. 1961.—A comparison of three strains of
Clostridium histolyticum
grown in two different media indicated that the H-4 strain produced the greatest yield of enzyme(s) capable of debriding third degree burn eschars on guinea pigs. Some of the nutritional and physiological factors affecting the elaboration of enzymes by this strain have been investigated. Maximal yields have been obtained after incubation for 14 to 16 hr at 30 C in a medium consisting of 5% proteose-peptone, 1.5% trypticase soy broth, and vitamins; pH 7.5. A medium consisting of 2% proteose-peptone and 2% Casamino acids yields somewhat lower enzyme titers, but has the compensating advantage of being less costly and may be more amenable to purification and concentration procedures because of its reduced precipitable nitrogen content.
POLYSACCHARIDE OF
COCCIDIOIDES IMMITIS
Authors: Pappagianis D. , Putman E. W. , Kobayashi G. S.
Published: January 2020

Pappagianis
, D. (University of California, Berkeley), E. W.
Putman, and G. S. Kobayashi
. Polysaccharide of
Coccidioides immitis
. J. Bacteriol.
82:
714–723. 1961.—Soluble polysaccharide from mycelia or culture filtrates of
Coccidioides immitis
was found to consist mainly of mannose, but also included small quantities of galactose and another reducing sugar. Isolation of the polysaccharide by ethanol precipitation provided accompanying nitrogenous material. There was 3 to 4% nitrogen, present in amino acids, but in a nondialyzable (possibly protein or peptide) form. The average molecular weight of the complex was 31,700. Attempts to separate the nitrogenous and polysaccharide materials by chemical methods or by moving boundary electrophoresis were unsuccessful; ultracentrifugation showed a single peak. Despite these findings, there appeared to be several antigenic species present, as indicated by multiple lines of precipitation in double diffusion and quantitative precipitin tests. In the latter, polysaccharide-containing fractions gave precipitates without measurable carbohydrate, suggesting separability of nitrogenous and polysaccharide components.
SPHEROPLASTS OF THE YEASTCANDIDA UTILIS
Authors: Svihla G. , Schlenk F. , Dainko J. L.
Published: January 2020

Svihla, G. (Argonne National Laboratory, Argonne, Ill.), F.Schlenk, and J. L. Dainko. Spheroplasts of the yeastCandida utilis. J. Bacteriol.82:808–814. 1961.—The formation and properties of spheroplasts of the yeastCandida utilis, produced by digestion of the cell walls with snail gut juice (Helix pomatia) in isotonic medium, were studied by phase, interference, and ultraviolet microscopy, and tracer techniques. Cells cultivated in the presence ofl-methionine, S-methyl-l-methionine, andl-homocysteine (5 μmoles/ml) were more susceptible to enzyme action than ordinary cells. These cells contain increased amounts of S-adenosylmethionine in the vacuole which facilitates ultraviolet microscopy. Spheroplasts are stable in 0.6mKCl containing 0.04mphosphate, but are disintegrated by water, sonic vibration, or ultraviolet irradiation. Exposure in isotonic medium to 6 × 104erg/mm2energy at 253 mμ breaks the cytoplasmic membrane and liberates the vacuole with membrane intact. The vacuolar membrane may be broken with about three times the initial dose of ultraviolet.
INFLUENCE OF OSMOTIC PRESSURE ON THE MORPHOLOGY OF THE REITER TREPONEME
Authors: Hardy Paul H. , Nell E. Ellen
Published: January 2020

Hardy, Paul H., Jr.
(Johns Hopkins University, Baltimore, Md.)
and E. Ellen Nell
. Influence of osmotic pressure on the morphology of the Reiter treponeme. J. Bacteriol.
82:
967–978. 1961.—Spherical bodies similar to those that develop spontaneously in cultures of treponemes, and which have been considered by many investigators to represent one stage in a complex life cycle of these organisms, can be produced rapidly with the Reiter treponeme by merely altering the medium in which the organisms are suspended. Osmotic pressure appears to be the major factor responsible for this effect, as shown by the observation that treponemes suspended in NaCl solutions of 0.15 to 0.10
m
retain their spirochetal morphology, whereas organisms suspended in more dilute salt solutions rapidly become spherical. Moreover, the concentration of salt appears to influence both the rate and extent of sphere formation. Further evidence that osmotic pressure is primarily involved is demonstrated by the selectivity of the conditions under which spheres form. Treponemes suspended in various 0.3 osmolal solutions either retain their spiral shape or form spheres, depending upon the nature of the solute. Viability studies of suspensions containing predominantly spherical forms, which have developed spontaneously or have been artificially induced, have failed to produce evidence that the resulting growth of treponemes came from the spheres. It is concluded, therefore, that the naturally occurring spheres probably arise as the result of an osmotic imbalance which develops between the cells and their environment, and that the spheres represent degenerative forms rather than an intermediate stage in a life cycle.
A NEW METHOD FOR THE TURBIDIMETRIC MEASUREMENT OF BACTERIAL DENSITY
Authors: Kurokawa Masami , Hatano Motoichi , Kashiwagi Noboru , Saito Tateo , Ishida Setsuji , Homma Reiko
Published: January 2020

Kurokawa, Masami(National Institute of Health, Tokyo, Japan),Motoichi Hatano, Noboru Kashiwagi, Tateo Saito, Setsuji Ishida, and Reiko Homma. A new method for the turbidimetric measurement of bacterial density. J. Bacteriol.83:14–19. 1962.—It was demonstrated that by a combination of two types of metameter of transmittance (T), –log T (0.0 ≤ T ≤ 0.5) and 0.6 + log (1 – T) (0.5 ≤ T ≤ 1.0), the optical density thus expressed showed a linear relationship to bacterial density or cell concentration over the whole range of T, except at the extreme ends. This allows measurement of relative bacterial density and cell concentration, and eliminates unnecessary sampling and dilution. In this respect, the proposed method seems superior to the method based upon Beer's law. The possible error derived from the fact that organisms may vary in size and shape at different stages of their growth is a fault common to all known methods of turbidimetric measurement, and is not peculiar to the method presented here.
GLYCINE RESISTANCE IN
AGROBACTERIUM TUMEFACIENS
Authors: Beardsley Robert E.
Published: January 2020

Beardsley, Robert E.
(Manhattan College, New York, N. Y.). Glycine resistance in
Agrobacterium tumefaciens
. J. Bacteriol.
83:
6–13. 1962.—The application of the fluctuation test of Luria and Delbrück to the distribution of glycine-resistant bacteria among cultures of
Agrobacterium tumefaciens
strain B6 indicates that resistance arises by mutation in the absence of glycine. On glycine-supplemented medium, additional resistant colonies arise during prolonged periods of incubation. Their appearance is proceded by L-form growth. In general, the number of generations over which glycine resistance is inherited in the absence of glycine is increased by serial transfers on the selection medium. In liquid medium containing glycine, sensitive bacteria form spheroplasts. Resistant bacteria continue to grow as rod forms. In the medium employed, spheroplasts are unstable.
METHOD FOR INDUCING SPORULATION OF PURE CULTURES OF THE MYXOMYCETE
PHYSARUM POLYCEPHALUM
Authors: Daniel John W. , Rusch Harold P.
Published: January 2020

Daniel, John
W. (University of Wisconsin, Madison)
and Harold P. Rusch
. Method for inducing sporulation of pure cultures of the myxomycete
Physarum polycephalum
. J. Bacteriol.
83:
234–240. 1962.—Techniques for inducing sporulation of pure cultures of
Physarum polycephalum
are described. Small plasmodia grown in agitated culture were harvested, allowed to fuse into large plasmodia (1.3 g wet weight each), and, after incubation in the dark on a salts medium, exposed to light for 2 hr. The ensuing formation of sporangia containing spores was complete after 12 to 16 hr. The obligatory conditions for sporulation are: (i) an optimal growth age occurring just prior to the maximal growth of the organism and at a time when nutrients in the medium are exhausted; (ii) 4 days of incubation on a medium containing only inorganic salts and niacin or tryptophan; and (iii) subsequent illumination with light of wavelengths between 350 and 500 mμ.
ANAEROBIC GROWTH OF PSYCHROPHILIC BACTERIA
Authors: Upadhyay J. , Stokes J. L.
Published: January 2020

Upadhyay
, J. (Washington State University, Pullman)
and J. L. Stokes
. Anaerobic growth of psychrophilic bacteria. J. Bacteriol.
83:
270–275. 1962.—Facultatively anaerobic psychrophilic bacteria are widely distributed in nature and can be readily isolated from a variety of foods, soil, sewage, and other habitats. Strictly anaerobic psychrophiles, however, could not be isolated.

Temperature and oxygen have striking effects on the growth and metabolism of the psychrophiles. Sugars which are fermented at 20 C may not be fermented at 0 C or gas may not be produced. Under both aerobic and anaerobic conditions, decreases in incubation temperatures increase the duration of the lag, exponential, stationary, and death phases. Elimination of O
2
increases the lag period, allows the cells to survive much longer at low temperatures (but accelerates their death at 20 C and higher), and, finally, reduces the extent of growth. Maximal cell populations are obtained aerobically at 5 C and anaerobically at 25 C.

Growth is most rapid in the range of 20 to 35 C, and maximal growth temperatures range from 25 to 45 C. Neither of these cardinal temperatures is appreciably affected by the presence or absence of O
2
.
ULTRAVIOLET MICROSCOPY OF BUDDING
SACCHAROMYCES
Authors: McClary Dan O. , Bowers Wilbert D. , Miller Glendon R.
Published: January 2020

McClary, Dan
O. (Southern Illinois University, Carbondale),
Wilbert D. Bowers, Jr., and Glendon R. Miller
. Ultraviolet microscopy of budding
Saccharomyces
. J. Bacteriol.
83:
276–283. 1962.—Synchronous cell division was obtained in
Saccharomyces
by transferring starved cells into nutrient medium. Ultraviolet microscopy and Giemsa-stained preparations of these cells showed nuclear division to occur in the mother cell early in the budding process. The divided nucleus passed into the neck between the mother cell and the bud, and either fused together again or the two parts became so closely associated that one continuous dumbbell-shaped body was seen which seemed to divide by constriction. This effect was probably due to the retention of the intact nuclear membrane until nuclear division was otherwise complete.

The nuclear apparatus lies outside the vacuole. The extent to which these bodies function together cannot be determined by the techniques employed in this work.

Parallel experiments on meristematic cells of onion root tips show the reliability of these cytological methods.