bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence and expression of a novel pectate lyase gene (pel-3) and a closely linked endopolygalacturonase gene (peh-1) of Erwinia carotovora subsp. carotovora 71
Authors: Liu Y , Chatterjee A , Chatterjee A K
Published: January 2020

Our previous genetic analysis (J. W. Willis, J. K. Engwall, and A. K. Chatterjee, Phytopathology 77:1199-1205, 1987) had revealed a tight linkage between pel-3 (pel, pectate lyase gene) and peh-1 (peh, polygalacturonase gene) within the chromosome of Erwinia carotovora subsp. carotovora 71. Nucleotide sequencing, transcript assays, and expression of enzymatic activities in Escherichia coli have now confirmed that a 3,500-bp segment contains the open reading frames (ORFs) for Pel-3 and Peh-1. The 1,041-bp pel-3 ORF and the 1,206-bp peh-1 ORF are separated by a 579-bp sequence. The genes are transcribed divergently from their own promoters. In E. coli and E. carotovora subsp. carotovora 71, peh-1 is better expressed than pel-3. However, plant signals activate the expression of both the genes in E. carotovora subsp. carotovora. A consensus integration host factor (IHF)-binding sequence upstream of pel-3 appears physiologically significant, since pel-3 promoter activity is higher in an E. coli IHF+ strain than in an IHF- strain. While peh-1 has extensive homology with plant and bacterial peh genes, pel-3 appears not to have significant homology with the pel genes belonging to the pelBC, pelADE, or periplasmic pel families. Pel-3 also is unusual in that it is predicted to contain an ATP- and GTP-binding site motif A (P-loop) not found in the other Pels.
Utilization of Light for Nitrogen Fixation by a New
Synechocystis
Strain Is Extended by Its Low Photosynthetic Efficiency
Authors: Brass Susanne , Westermann Martin , Ernst Anneliese , Reuter Wolfgang , Wehrmeyer Werner , Böger Peter
Published: January 2020

Performance of photosynthesis and nitrogenase activity in a novel cyanobacterium,
Synechocystis
sp. strain BO 8402, isolated from Lake Constance, located at the northern fringe of the Alps in central Europe, and of a stable derivative, strain BO 9201, were examined. Strain BO 8402 is characterized by an extraordinarily high level of autofluorescence originating from paracrystalline phycobiliprotein-linker complexes located in inclusion bodies (W. Reuter, M. Westermann, S. Brass, A. Ernst, P. Böger, and W. Wehrmeyer, J. Bacteriol. 176:896-904, 1994). Energy transfer between paracrystalline phycobiliproteins and the photosystems is inefficient, resulting in a high oxygen compensation point and a decreased growth rate. The derivative strain BO 9201 exhibits hemidiscoidal phycobilisomes that support a high growth rate, even under low light intensities. Because of the differences in photosynthetic performance, anaerobic light-stimulated nitrogenase activity is maintained at higher light intensity in the original strain BO 8402 than in the derivative strain BO 9201. The results indicate that the formation of paracrystalline phycobiliproteins in
Synechocystis
sp. strain BO 8402 represents a hitherto-unknown means for a unicellular cyanobacterium to extend its capacity to fix nitrogen in the light.
Expression of Heterogenous Arsenic Resistance Genes in the Obligately Autotrophic Biomining Bacterium
Thiobacillus ferrooxidans
Authors: Peng Ji-Bin , Yan Wang-Ming , Bao Xue-Zhen
Published: January 2020

Two arsenic-resistant plasmids were constructed and introduced into
Thiobacillus ferrooxidans
strains by conjugation. The plasmids with the replicon of wide-host-range plasmid RSF1010 were stable in
T. ferrooxidans.
The arsenic resistance genes originating from the heterotroph were expressed in this obligately autotrophic bacterium, but the promoter derived from
T. ferrooxidans
showed no special function in its original host.
Cloning of the macrolide antibiotic biosynthesis gene acyA, which encodes 3-O-acyltransferase, from Streptomyces thermotolerans and its use for direct fermentative production of a hybrid macrolide antibiotic
Authors: Arisawa A , Kawamura N , Takeda K , Tsunekawa H , Okamura K , Okamoto R
Published: January 2020

A gene encoding the macrolide modification enzyme 3-O-acyltransferase (acyA) was cloned by chromosome walking onto the carbomycin biosynthetic region in Streptomyces thermotolerans TH475, with the 3' region of the gene encoding the macrolide modification enzyme 4"-O-acyltransferase (acyB1) as a probe. A shortened fragment (1.8 kb) containing acyA was subcloned with pIJ350. A high-level tylosin producer, Streptomyces fradiae MBBF, transformed with the plasmid could produce a hybrid macrolide, 3-O-acetyltylosin, most efficiently.
Genetic Structure of
Rhizobium leguminosarum
biovar trifolii and viciae Populations Found in Two Oregon Soils under Different Plant Communities
Authors: Strain Steven R. , Leung Kamtin , Whittam Thomas S. , de Bruijn Frans J. , Bottomley Peter J.
Published: January 2020

An investigation was carried out to determine the genetic structure in soil populations of
Rhizobium leguminosarum
bv. trifolii and viciae at each of two Oregon sites (A and C) that were 1 km apart. Although the soils were similar, the plant communities were quite different because grazing by domestic animals had been allowed (site A) or prevented (site C). Analysis of allelic variation at 13 enzyme-encoding loci by multilocus enzyme electrophoresis delineated 202 chromosomal types (ETs) among a total of 456 isolates representing two populations of
R. leguminosarum
bv. trifolii (AT and CT) and two populations of
R. leguminosarum
bv. viciae (AV and CV). Regardless of their site of origin or biovar affiliation, isolates of the same ET were confirmed to be more closely related to each other than to isolates of other ETs by repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences and the PCR technique. Despite the wide range in densities of the
Rhizobium
populations (<10
2
to >10
5
/g of soil), their overall genetic diversities were similar (mean genetic diversity, 0.45 to 0.51), indicating that low-density populations of soil-borne bacterial species are not necessarily of little genetic diversity. Linkage disequilibrium analysis revealed significant multilocus structure (nonrandom associations of alleles) within each of the four populations. From a combination of cluster and linkage disequilibrium analyses, a total of eight distinct groups of ETs were defined in the four populations. Two groups (I and III) contributed significant numbers of ETs and isolates to each population. The two populations of
R. leguminosarum
bv. viciae (AV and CV) exhibited similar genetic structures despite existing at different densities, in different plant communities, and in the presence (CV) or absence (AV) of their local
Vicia
hosts. In contrast, total linkage disequilibrium was partitioned differently in two biovar populations occupying the same soil (AV and AT), with disequilibrium in the latter being due entirely to the presence of group V.
Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids
Authors: Hoaki T , Nishijima M , Kato M , Adachi K , Mizobuchi S , Hanzawa N , Maruyama T
Published: January 2020

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.
Purification and characterization of thermostable beta-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost
Authors: Sakai K , Narihara M , Kasama Y , Wakayama M , Moriguchi M
Published: January 2020

Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 beta-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-beta-N-acetyl-D-galactosaminide (26% of the activity of pNP-beta-N-acetyl-D-glucosaminide). These results indicated that the enzyme is a beta-N-acetylhexosaminidase (EC 3.2.1.52). Kms for acetylchitooligosaccharides were 1 x 10(-4) to 6 x 10(-4) M, while those for the pNP derivatives were 4 x 10(-3) to 8 x 10(-3) M. The optimum temperature of the enzyme was 75 degrees C, and it retained 100 and 28% reactivity after heating at 60 and 80 degrees C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5. Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N-Acetyl-D-glucosamine inhibited the enzyme competitively (Ki = 4.3 x 10(-4) M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.
Urea hydrolysis can predict the potential pathogenicity of Vibrio parahaemolyticus strains isolated in the Pacific Northwest
Authors: Kaysner C A , Abeyta C , Trost P A , Wetherington J H , Jinneman K C , Hill W E , Wekell M M
Published: January 2020

The ability of some strains of Vibrio parahaemolyticus to hydrolyze urea (uh+) can be used as a marker to predict which strains isolated from molluscan shellfish harvested in the Pacific Northwest are potentially pathogenic. The thermostable direct hemolysin-producing (TDH+) characteristic is a marker that is correlated with potential pathogenicity, and all of the TDH+ strains that we have isolated have been found to be uh+. Most of the uh+ strains belong to somatic antigen groups O3, O4 and O5. TDH+ strains are usually members of groups O4 and O5. The strains most often associated with human illness are members of the uh+, O4 group. The test for urease production is a simple screening test that can be helpful in predicting which strains are potentially pathogenic.
Nuclear Ribosomal DNA Variation and Pathogenic Specialization in
Alternaria
Fungi Known To Produce Host-Specific Toxins
Authors: Kusaba Motoaki , Tsuge Takashi
Published: January 2020

A total of 99 strains of 11
Alternaria
species, including 68 strains of seven fungi known to produce host-specific toxins, were subjected to analysis of restriction fragment length polymorphism (RFLP) in nuclear ribosomal DNA (rDNA). Total DNA was digested with
Xba
I, and the Southern blots were probed with a nuclear rDNA clone of
Alternaria kikuchiana.
The hybridization gave 17 different RFLPs from the 99 strains. On the basis of these RFLPs, populations of host-specific toxin-producing fungi could not be differentiated from one another nor from nonpathogenic
A. alternata.
Each population of the toxin-producing fungi carried rDNA variants. Nine different types, named A1 to A6 and B1 to B3, were detected among the toxin-producing fungi and nonpathogenic
A. alternata.
All of the populations contained the type A4 variant, and the other rDNA types were also shared by different toxin-producing fungi and
A. alternata.
In contrast,
Alternaria
species that are morphologically distinguishable from
A. alternata
could be differentiated from
A. alternata
on the basis of the rDNA RFLPs. Polymorphisms in rDNA digested with
Hae
III and
Msp
I were also evaluated in 61
Alternaria
strains. These restriction enzymes produced 31 variations among all of the samples. The seven toxin-producing fungi and nonpathogenic
A. alternata
could not be resolved by phylogenetic analysis based on the RFLPs, although they could be differentiated from the other
Alternaria
species studied. These results provide support for the hypothesis that
Alternaria
fungi known to produce host-specific toxins are intraspecific variants of
A. alternata
specialized in pathogenicity.
Adsorption of
Rhodococcus
Strain GIN-1 (NCIMB 40340) on Titanium Dioxide and Coal Fly Ash Particles
Authors: Shabtai Y. , Fleminger G.
Published: January 2020

Rhodococcus
strain GIN-1 (NCIMB 40340) can be used to enrich and isolate a titanium-rich fraction from coal fly ash. The gram-positive bacterium was isolated by its ability to adhere strongly and rapidly to suspended particles of pure titanium dioxide or coal fly ash. Adsorption depends on the salt concentration and occurs in seawater. Lowering of the salt concentration or washing of particles with pure water did not, however, cause desorption of the bacteria from TiO
2
particles; this was achieved by strong alkaline treatment or combined treatment with sodium dodecyl sulfate and urea but not with dilute acids, alcohols, or cationic or nonionic detergents. The bacterium exhibits higher affinity towards oxides of Ti and Zn than to other oxides with similar distribution of particle size. Moreover, it adheres much faster to TiO
2
than to magnetite (Fe
3
O
4
) or Al
2
O
3
. After about 1 min, more than 85% of the cells were adsorbed on TiO
2
, compared with adsorption of only 10 and 8% to magnetite and Al
2
O
3
, respectively. Adsorption of the bacteria on TiO
2
occurs over a pH range of 1.0 to 9.0 and at temperatures from 4 to over 80°C. Scanning electron microscopy combined with X-ray analysis revealed preferential adherence of the bacterium to coal ash particles richer in Ti. Stronger adhesion to TiO
2
was also demonstrated in the translocation of bacteria, preadsorbed on magnetite, to TiO
2
particles. The temporary co-adhesion to magnetite and TiO
2
was exploited for the design of a prototype biomagnetic separation process in which bacterial cells serve as an adhesive mediator between magnetite and TiO
2
particles in a mixture of Al, Si, and Ti oxides that simulates their proportion in the ash.
Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins
Authors: Bubert A , Schubert P , Köhler S , Frank R , Goebel W
Published: January 2020

All species of the genus Listeria secrete a major extracellular protein called p60. A comparison of the deduced amino acid sequences of all listerial p60 proteins previously indicated there were only a few regions which were unique to the pathogenic, food-borne species Listeria monocytogenes. Two of these p60 regions were chosen for the development of antibodies specific for the facultative intracellular species L. monocytogenes. Initially, these regions were characterized via epitope mapping, and this led to the development of two different synthetic peptides. Rabbits immunized with these synthetic peptides generated polyclonal antibodies that were then used in Western blot (immunoblot) analyses. Antiserum against peptide A (PepA) recognized the p60 protein in the supernatants collected from most L. monocytogenes serotypes except for several strains belonging to serotypes 4a and 4c. No p60-related protein was detected in the supernatants from other Listeria species with this anti-PepA antiserum. Antibodies raised against peptide D (PepD) reacted with p60 from all L. monocytogenes serotypes, including all 4a and 4c strains that were tested, and also showed no cross-reactivity with supernatant proteins from other Listeria species. Both antisera also detected p60 in supernatants of a large number of environmental isolates of L. monocytogenes. Besides Western blot analyses, these antisera to PepA and PepD reacted with secreted p60 in an enzyme-linked immunosorbent assay, indicating recognition of the native antigen in addition to the denatured form. These data suggest that synthetic peptides derived from the variable region of the L. monocytogenes p60 protein may be useful for the development of an immunological diagnostic assay.
Ecological Similarity and Coexistence of Epiphytic Ice-Nucleating (Ice
+
)
Pseudomonas syringae
Strains and a Non-Ice-Nucleating (Ice
-
) Biological Control Agent
Authors: Wilson Mark , Lindow Steven E.
Published: January 2020

De Wit replacement series were used to study competitive interactions between epiphytic Ice
+
Pseudomonas syringae
strains and the biological frost control agents Ice
-
P. syringae
TLP2del1 and
Pseudomonas fluorescens
A506. Mixtures containing two strains in different proportions but at a constant total population size were inoculated onto potato leaves. The population sizes of each strain and the total population size were determined when the community had reached equilibrium. A near-isogenic
P. syringae
strain pair exhibited an interaction similar to that expected for strains competing equally for limiting environmental resources. Replacement series with nonisogenic Ice
+
and Ice
-
P. syringae
strain pairs suggested that these strains competed for limiting resources according to their relative competitive abilities. There was no evidence of any niche differentiation between the Ice
+
P. syringae
strains and the Ice
-
P. syringae
strain. The growth responses of epiphytes following addition of nutrients to the phyllosphere indicated that the epiphytic
P. syringae
populations were nutrient limited and that, under growth chamber conditions, the populations were more limited by the availability of carbon than by the availability of nitrogen. Determination of in vitro carbon source utilization profiles provided further evidence for the lack of niche differentiation between the Ice
+
and the Ice
-
P. syringae
strains. Niche overlap indices calculated for the Ice
+
P. syringae
strains with respect to Ice
-
P. syringae
TLP2del1 were uniformly high, indicating ecological similarity, and were consistent with the observed low level of coexistence. The biological frost control agent
P. fluorescens
A506 replaced
P. syringae.
This was correlated with a high degree of niche overlap between these species.
Hrp
-
Mutants of
Pseudomonas solanacearum
as Potential Biocontrol Agents of Tomato Bacterial Wilt
Authors: Frey Pascal , Prior Philippe , Marie Corinne , Kotoujansky Alain , Trigalet-Demery Daniele , Trigalet Andre
Published: January 2020

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of
Pseudomonas solanacearum
that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (
hrp
) is involved in the pathogenicity of
P. solanacearum
, we developed a biological control strategy using genetically engineered Hrp
-
mutants of
P. solanacearum.
Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an ω-Km interposon within the
hrp
gene cluster of each strain. The resulting Hrp
-
mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp
-
mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants.
Lactococcal Bacteriophages Require a Host Cell Wall Carbohydrate and a Plasma Membrane Protein for Adsorption and Ejection of DNA
Authors: Monteville Marshall R. , Ardestani Bahram , Geller Bruce L.
Published: January 2020

The mechanism of the initial steps of bacteriophage infection in
Lactococcus lactis
subsp.
lactis
C2 was investigated by using phages c2, ml3, kh, l, h, 5, and 13. All seven phages adsorbed to the same sites on the host cell wall that are composed, in part, of rhamnose. This was suggested by rhamnose inhibition of phage adsorption to cells, competition between phage c2 and the other phages for adsorption to cells, and rhamnose inhibition of lysis of phage-inoculated cultures. The adsorption to the cell wall was found to be reversible upon dilution of the cell wall-adsorbed phage. In a reaction step that apparently follows adsorption to the cell wall, all seven phages adsorbed to a host membrane protein named PIP. This was indicated by the inability of all seven phages to infect a strain selected for resistance to phage c2 and known to have a defective PIP protein. All seven phages were inactivated in vitro by membranes from wild-type cells but not by membranes from the PIP-defective, phage c2-resistant strain. The mechanism of membrane inactivation was an irreversible adsorption of the phage to PIP, as indicated by adsorption of [
35
S] methionine-labeled phage c2 to purified membranes from phage-sensitive cells but not to membranes from the resistant strain, elimination of adsorption by pretreatment of the membranes with proteinase K, and lack of dissociation of
35
S from the membranes upon dilution. Following membrane adsorption, ejection of phage DNA occurred rapidly at 30°C but not at 4°C. These results suggest that many lactococcal phages adsorb initially to the cell wall and subsequently to host cell membrane protein PIP, which leads to ejection of the phage genome.
Effect of Solution Ionic Strength and Iron Coatings on Mineral Grains on the Sorption of Bacterial Cells to Quartz Sand
Authors: Mills Aaron L. , Herman Janet S. , Hornberger George M. , DeJesús Todd H.
Published: January 2020

Understanding the interaction between bacterial cells and solid surfaces is essential to our attempts to quantify and predict the transport of microbes in groundwater aquifers, whether from the point of view of contamination or from that of bioremediation. The sorption of bacterial cells suspended in groundwater to porous medium grains was examined in batch studies. Bacterial sorption to clean quartz sand yielded equilibrium, linear, adsorption isotherms that varied with the bacterial strain used and the ionic strength of the aqueous solution. Values of

K
d

(the slope of the linear sorption isotherm) ranged from 0.55 to 6.11 ml g
-1
, with the greatest sorption observed for the highest groundwater ionic strength. These findings are consistent with the interpretation that an increasingly compressed electrical double layer results in stronger adsorption between the like-charged mineral surface and the bacterial cells. When iron-oxyhydroxide-coated sand was used, however, all of the added bacteria were adsorbed up to a threshold of 6.93 × 10
8
cells g of coated sand
-1
, beyond which no further adsorption occurred. The irreversible, threshold adsorption is the result of a strong electrostatic attraction between the sesquioxide coating and the bacterial cells. Experimental results of adsorption in mixtures of quartz and Fe(III)-coated sand were successfully predicted by a simple additive model for sorption by the two substrate phases. Even small amounts of Fe(III)-coated sand in a mixture influenced the extent of adsorption of bacterial cells. A quantitative description of adsorption in the mixtures can be realized by using a linear isotherm for reversible adsorption to the quartz grains with a
y
intercept that represents the number of cells irreversibly adsorbed to the Fe(III)-coated sand.
Inhibition of
Rhizobium etli
Polysaccharide Mutants by
Phaseolus vulgaris
Root Compounds
Authors: Eisenschenk Linda , Diebold Ronald , Perez-Lesher Jeanett , Peterson Andrew C. , Kent Peters N. , Noel K. Dale
Published: January 2020

Crude bean root extracts of
Phaseolus vulgaris
were tested for inhibition of the growth of several polysaccharide mutants of
Rhizobium etli
biovar phaseoli CE3. Mutants deficient only in exopolysaccharide and some mutants deficient only in the O-antigen of the lipopolysaccharide were no more sensitive than the wild-type strain to the extracts, whereas mutants defective in both lipopolysaccharide and exopolysaccharide were much more sensitive. The inhibitory activity was found at much higher levels in roots and nodules than in stems or leaves. Inoculation with either wild-type or polysaccharide-deficient
R. etli
did not appear to affect the level of activity. Sequential extractions of the crude root material with petroleum ether, ethyl acetate, methanol, and water partitioned inhibitory activity into each solvent except methanol. The major inhibitors in the petroleum ether and ethyl acetate extracts were purified by C
18
high-performance liquid chromatography. These compounds all migrated very similarly in both liquid and thin-layer chromatography but were distinguished by their mass spectra. Absorbance spectra and fluorescence properties suggested that they were coumestans, one of which had the mass spectrum and nuclear magnetic resonances of coumestrol. These results are discussed with regard to the hypothesis that one role of rhizobial polysaccharides is to protect against plant toxins encountered during nodule development.
Secretion of human epidermal growth factor (EGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium, Pseudomonas pseudoflava, carrying broad-host-range EGF secretion vector pKSEGF2
Authors: Hayase N , Ishiyama A , Niwano M
Published: January 2020

We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted EGF into the culture medium constitutively. Four hydrogen-utilizing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcaligenes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested. In these transconjugated hydrogen-utilizing bacteria, P. pseudoflava carrying pKSEGF2 grew autotrophically and secreted EGF, confirmed by Western blot (immunoblot) analysis, into the culture medium constitutively.
The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis
Authors: Hofemeister B , König S , Hoang V , Engel J , Mayer G , Hansen G , Hofemeister J
Published: January 2020

We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic alpha-amylase (AmyTV1). A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacillus subtilis. The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T. vulgaris 94-2A culture. The amino acid sequence was aligned with several known alpha-amylase sequences. We found 83% homology with the 48-kDa alpha-amylase part of the Bacillus polymyxa beta-alpha-amylase polyprotein and 50% homology with Taka amylase A of Aspergillus oryzae but only 45% homology with another T. vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47. The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B. subtilis. Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1. The expression levels in B. subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacillus amyloliquefaciens alpha-amylase gene.
Maximum Temperature Limits for Acidophilic, Mesophilic Bacteria in Biological Leaching Systems
Authors: Niemelä Seppo I. , Sivelä Carita , Luoma Taina , Tuovinen Olli H.
Published: January 2020

The maximum temperature for growth (
T
max
) was determined for pure and mixed cultures of acidophilic thiobacilli. The experimental system was based on incubating the cultures in liquid media exposed to a linear temperature gradient. The
T
max
values varied within the range of 36.1 to 43.6°C.
Nitrogen regulation of lignin peroxidase gene transcription
Authors: Li D , Alic M , Gold M H
Published: January 2020

Western blot (immunoblot) analysis with a polyclonal antibody to lignin peroxidase (LiP) isozyme H8 from the white rot basidiomycete Phanerochaete chrysosporium demonstrates that LiP protein is detectable in the extracellular media of 5- and 6-day-old nitrogen-limited, but not nitrogen-sufficient, cultures. Northern (RNA) blot analysis demonstrates that lip mRNA is detectable from 5- and 6-day old cells grown in nitrogen-limited, but not nitrogen-sufficient, cultures. These results indicate that LiP expression is regulated at the level of gene transcription by nutrient nitrogen. Since lignin degradation by P. chrysosporium is derepressed by nitrogen starvation, it appears that lignin degradation and LiP expression are coordinately regulated in this organism. These results contradict a recent report which concluded that LiP protein expression is not regulated by nutrient nitrogen (C. G. Johnston and S. D. Aust, Biochem. Biophys. Res. Commun. 200:108-112, 1994).
Biodegradation of 2-nitrotoluene by Pseudomonas sp. strain JS42
Authors: Haigler B E , Wallace W H , Spain J C
Published: January 2020

A strain of Pseudomonas sp. was isolated from nitrobenzene-contaminated soil and groundwater on 2-nitrotoluene as the sole source of carbon, energy, and nitrogen. Bacterial cells growing on 2-nitrotoluene released nitrite into the growth medium. The isolate also grew on 3-methylcatechol, 4-methylcatechol, and catechol. 2-Nitrotoluene, 3-methylcatechol, and catechol stimulated oxygen consumption by intact cells regardless of the growth substrate. Crude extracts from the isolate contained catechol 2,3-dioxygenase and 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase activity. The results suggest that 2-nitrotoluene is subject to initial attack by a dioxygenase enzyme that forms 3-methylcatechol with concomitant release of nitrite. The 3-methylcatechol is subsequently degraded via the meta ring fission pathway.
Antibody Response to Successive Booster Doses of Tetanus Toxoid in Adults
Authors: Trinca John C.
Published: January 2020

A study of the rise and fall in circulating tetanus antitoxin in a group of 15 adults after basic and booster immunization indicates that although individuals vary greatly in their responsiveness to toxoid, the falloff in antitoxin appears to be fractionally constant for each individual, and over periods of 5 to 6 years appears to be the same after successive doses. The low level of antitoxin noted in some individuals after basic immunization with a plain toxoid preparation cannot always be rectified by the use of an adsorbed toxoid as a booster. Results indicate that routine boosters need not be administered more frequently than every 10 to 20 years, provided an adsorbed toxoid has been used to initiate active immunization.
Immunological Sequelae of
Trichinella spiralis
Infection in Mice II. Potentiation of Cell-Mediated Response to BCG After Infection with
Trichinella spiralis
Authors: Cypess R. H. , Molinari J. A. , Ebersole J. L. , Lubiniecki A. S.
Published: January 2020

Mice were infected with 200
Trichinella spiralis
14 days after intravenous administration of 4 × 10
6
viable BCG cells. Individual groups were tested for delayed hypersensitive footpad responses at 14, 20, 29, 57, or 85 days after
T. spiralis
infection. An initial suppression in the 14-day test group was observed; however, mice tested at later time intervals exhibited potentiation of the 24-h footpad reaction to old tuberculin over that elicited by appropriate controls. This suggested that
T. spiralis
induces a potentiation of the cellular immune response to BCG. Adoptive transfer studies support the cell-mediated nature of the observed footpad reaction and indicated that the initial suppression was not due to physiological factors preventing the expression of the footpad swelling reaction.
Production of Chemotactic Factor and Lymphotoxin by Human Leukocytes Stimulated with Herpes Simplex Virus
Authors: Rosenberg Gary L. , Snyderman Ralph , Notkins Abner L.
Published: January 2020

Peripheral blood leukocytes (PBL) of 41 subjects were tested for their ability to be stimulated by herpes simplex virus antigens as measured by [
3
H]thymidine incorporation and lymphokine production (i.e., lymphocyte-derived chemotactic factor and lymphotoxin). The PBL of seronegative subjects failed to respond to viral antigens as measured by stimulation or lymphokine production. In contrast, PBL from seropositive subjects without clinical lesions of herpes labialis were stimulated by herpes simplex virus antigens and produced lymphokines. PBL from seropositive patients with clinical lesions of herpes labialis also produced lymphokines, but [
3
H]thymidine incorporation was slightly depressed. These findings suggest that lymphocytes from patients with a recent herpes simplex virus infection may respond less vigorously to in vitro stimulation by herpes antigens, but our study fails to show any basic defect in the responsiveness of the lymphocytes to account for recurrent herpetic episodes.
Escherichia coli
Enterotoxin-Induced Steroidogenesis in Cultured Adrenal Tumor Cells
Authors: Kwan Catherine N. , Wishnow Rodney M.
Published: January 2020

A heat-labile, Pronase-sensitive factor has been partially purified from cell-free culture filtrates of enterotoxigenic
Escherichia coli
. The partially purified factor contains both protein and carbohydrate moieties and appears to be
E. coli
enterotoxin (ECT). ECT binds to cultured adrenal tumor cells rapidly and irreversibly leads to adenosine 3′, 5′-cyclic monophosphate formation and steroidogenesis after a 60-min lag phase. Further studies indicate that it interacts with the cholera toxin receptor site on adrenal cells rather than the adrenocorticotropin receptor to activate adenyl cyclase. Mixed gangliosides block stimulation of steroidogenesis in response to both
E. coli
and cholera enterotoxin. In contrast to adrenocorticotropin, ECT has no additive effect on cholera toxin-induced steroidogenesis. The protein moiety of ECT is similar to cholera enterotoxin because horse serum anticholeragenoid prevented stimulation of steroidogenesis by either enterotoxin. Cultured adrenal cells provide a quantitative assay system that has facilitated the purification and characterization of
E. coli
enterotoxin.
Dissociation of Plaque Formation from Glucan-Induced Agglutination in Mutants of
Streptococcus mutans
Authors: Freedman M. L. , Tanzer J. M.
Published: January 2020

The traits adhesion and cohesion are manifested in
Streptococcus mutans
by in vitro plaque formation and glucan-mediated agglutination and have been dissociated in mutants of this caries-associated microbe. In these mutants, which differ from each other in colonial morphology, we have observed a consistent loss of ability to form adhesive microbial deposits (plaque) on wires, but only a minimal alteration from the wild type in ability to agglutinate into macroscopic clumps in the presence of exogenous glucans or sucrose. Also, when these mutants were cultured in sucrose-containing medium, there was an increase in the synthesis of water-soluble, extracellular glucans and a decrease in the synthesis of alkali-soluble, cell-associated glucans. Based on these findings, it appears (i) that in vitro plaque formation and agglutination are distinct and dissociable traits, and (ii) that adhesion but not agglutination appears related to the synthesis of alkali-soluble, cell-associated glucans.
Cell-Mediated Immunity in Humans During Viral Infection I. Effect of Rubella on Dermal Hypersensitivity, Phytohemagglutinin Response, and T Lymphocyte Numbers
Authors: Kauffman Carol A. , Phair John P. , Linnemann Calvin C. , Schiff Gilbert M.
Published: January 2020

Phytohemagglutinin-induced lymphocyte deoxyribonucleic acid synthesis, dermal hypersensitivity, and peripheral blood thymus-derived lymphocyte numbers were assessed in nine men with experimentally induced rubella infection. Five of these men and two additional volunteers received treatment with tilorone dihydrochloride, an antiviral drug. Response to phytohemagglutinin was not changed during rubella; T lymphocyte numbers in peripheral blood were not influenced by the viral illness. However, dermal hypersensitivity was markedly impaired in all volunteers during the height of the illness. Tilorone alone, or with rubella, had no effect on any of the parameters studied.
Comparison of Techniques and Immunoreagents Used for Indirect Immunofluorescence and Immunoperoxidase Identification of Enteroviruses
Authors: Herrmann John E. , Morse Stephen A. , Collins Marilyn F.
Published: January 2020

Peroxidase-conjugated antibodies were found to be as sensitive as those conjugated to fluorescein-isothiocyanate (FITC) for identification of selected enterovirus types by the indirect technique. Peroxidase conjugates, however, were found to give fewer nonspecific reactions. The reasons for the higher specificity of the immunoperoxidase technique appear to be related to the relative size, charge, and uniformity of the preparations. Monomers of peroxidase-conjugated globulin are larger than those of FITC conjugates, but the latter readily form aggregates. This was shown by chromatography on Sepharose 6B columns: various FITC-conjugated globulins eluted before those conjugated to peroxidase and before
125
I-labeled immunoglobulin G. The net charge of the conjugates was determined by adsorption to ion-exchange columns. FITC-labeled globulins had a negative net charge, eluting at pH 5.1 from diethylamino-ethyl-Sephadex A-50 columns. Peroxidase conjugates were not retained by either cationic or anionic exchangers at pH values ranging from 4.0 to 10.5. Further, the fluorescein/protein ratios of FITC conjugates from different commercial sources and of those prepared in the laboratory were found to be variable; higher fluorescein/protein ratios (>2:1) give a higher degree of nonspecific reactions, whereas the peroxidase/protein ratio does not appear to affect specificity. These characteristics of peroxidase conjugates make the immunoperoxidase technique easier to standardize and more reliable for enterovirus identification than the immunofluorescence technique.
Ultrastructural Studies of
Rickettsia prowazeki
From Louse Midgut Cells to Feces: Search For “Dormant” Forms
Authors: Silverman David J. , Boese Jack L. , Wisseman Charles L.
Published: January 2020

An electron microscope study of infected human louse gut cells and feces was made to determine whether a valid correlation exists between the increased resistance of
Rickettsia prowazeki
(in the louse feces) to adverse environmental influences and changes in the organism which might be reflected in its ultrastructure. Upon fine structural examination of this intracellular parasite as it passed from the louse midgut cell to the feces, it was apparent that no such morphological changes had occurred.
Immunodiffusion Analysis of Membranes of
Thermoplasma acidophilum
Authors: Bohlool B. B. , Brock T. D.
Published: January 2020

Isolates of
Thermoplasma acidophilum
were divided into serological groups based on immunodiffusion studies of solubilized-membrane preparations.
Interferon Induction in Mice by Lipopolysaccharide from
Brucella abortus
Authors: Keleti Georg , Feingold David S. , Youngner Julius S.
Published: January 2020

Endotoxin or lipopolysaccharide isolated from
Brucella abortus
(strain 456) produced a typical endotoxin-type interferon response in mice with peak levels of the inhibitor occurring 2 h after intravenous injection of the stimuli.
Brucella
lipopolysaccharide was a much less effective interferon stimulus than the lipopolysaccharide isolated from
Salmonella typhimurium
(strain LT-2).
Influence of Molecular Weights of Bacteriophage φ6 Double-Stranded Ribonucleic Acids on Interferon Induction
Authors: Kleinschmidt W. J. , Van Etten J. L. , Vidaver A. K.
Published: January 2020

The interferon-inducing capabilities of the three molecular segments of bacteriophage φ6 double-stranded ribonucleic acid increased with increasing molecular weight.
In Vitro Cellular Immunity to Unrelated Pathogens in Chickens Infected with Fowlpox Virus
Authors: Pathak P. N. , Rao G. V. S. V. Rama , Tompkins W. A. F.
Published: January 2020

Peritoneal macrophages recovered from chickens 15 to 20 days after inoculation with fowlpox virus and showing a delayed hypersensitivity reaction against fowlpox antigens demonstrated an enhanced antimicrobial effect against fowlpox virus as well as unrelated viruses and bacteria. Inoculation of normal chicken macrophage cultures with fowlpox virus resulted in approximately a 200-fold increase in virus titer by 96 h, whereas the virus showed less than a fourfold increase in macrophage cultures from fowlpox-immune chickens. Similarly, the titer of Newcastle disease virus increased by more than 1 log in cultures of normal macrophages, whereas the titer decreased by approximately 1 log after infection of fowlpox-immune macrophages. Vaccinia, vesicular stomatitis, and herpes simplex viruses, which are not natural pathogens of chickens, failed to replicate in cultures of either normal or fowlpox-immune macrophages. By using
Salmonella gallinarum
, it was further demonstrated that the antimicrobial activity of fowlpox-immune macrophages encompassed not only nonspecific viruses but also bacteria. Infection of normal macrophages with
Salmonella
resulted in intracellular replication of the organism between 0 and 24 h as determined by microscope examination and viable bacteria counts. In contrast, cultures of fowlpox-immune macrophages failed to show an increase in intracellular organisms and showed a marked decrease in viable bacteria. In conclusion, these studies clearly showed that cellular immunity, previously demonstrated in mammalian species, develops in chickens after infection with fowlpox virus.
Further Characterization of Staphylococcal Gamma-Hemolysin
Authors: Taylor Anthony G. , Bernheimer Alan W.
Published: January 2020

It was confirmed that staphylococcal gamma-hemolysin is composed of two separate proteins (gamma-lysin components I and II) which act synergistically. The molecular weights of the two components, determined by gel filtration, are 29,000 and 26,000, respectively, and their isoelectric points, determined by isoelectric focusing, are at pH 9.8 and 9.9. Both components are susceptible to the action of Pronase and subtilisin. A wide range of lipids, some in minute amounts, are capable of inhibiting the hemolytic activity of gamma-lysin.
Demonstration of a Temperature-Dependent Inactivating Factor of the Thermostable Direct Hemolysin in
Vibrio parahaemolyticus
Authors: Takeda Yoshifumi , Hori Yayoi , Miwatani Toshio
Published: January 2020

A factor was found in
Vibrio parahaemolyticus
which inactivated the hemolytic activity of a purified, thermostable direct hemolysin. The inactivating factor was associated with the hemolysin but could be separated from it by diethylaminoethyl-cellulose column chromatography. The inactivating factor was activated by heating at 50 to 60 C, but was itself thermolabile and lost activity on heating to 70 to 100 C. The mechanism of the Arrhenius effect, observed with crude hemolysin of
V. parahaemolyticus
, is as follows. At 50 to 60 C, the temperature-dependent inactivating factor associated with hemolysin inactivates the hemolysin, whereas at 80 to 100 C the crude hemolysin retains activity because at this temperature the factor is inactivated.
T Cell Dependence of Macrophage Activation and Mobilization During Infection with
Mycobacterium tuberculosis
Authors: North Robert J.
Published: January 2020

Mice made T cell-deficient as adults by thymectomy and lethal irradiation, and infected with
Mycobacterium tuberculosis
, showed a greatly reduced capacity to develop nonspecific resistance to challenge with a heterologous bacterium. The capacity to develop nonspecific resistance was restored, however, by an infusion of syngeneic thymocytes. Evidence was presented which showed that the capacity of mycobacteria-infected mice to resist a lethal challenge with a heterologous organism rested on the capacity of macrophages existing at the time of challenge to reduce the number of challenge organisms to a sublethal level within 8 h. This antibacterial activity of macrophages was substantially reduced in T cell-deficient mice but could be restored by an infusion of syngeneic thymocytes. It was concluded that T cell-deficient mice have a reduced capacity to mobilize and activate macrophages because of their reduced capacity to respond immunologically to antigens of
M. tuberculosis.
Serological Evidence for the Association of Bovine Respiratory Syncytial Virus with Respiratory Tract Disease in Alabama Cattle
Authors: Rossi C. R. , Kiesel G. K.
Published: January 2020

A serological investigation for neutralizing antibody to bovine respiratory syncytial virus (BRSV) was conducted using an antigenically related human strain of respiratory syncytial virus. Results demonstrated the effectiveness of the procedure. Sixty-seven percent of healthy, adult cattle tested were found to have antibody to BRSV. Calves in several herds were exposed to conditions to encourage development of respiratory tract disease. In three herds in which respiratory tract disease subsequently developed, the incidence of BRSV seroconversions approached 100%. In a herd in which no respiratory tract disease was detected, BRSV seroconversions indicated a high incidence of subclinical infections. It was concluded that, in this country as has been shown in others, BRSV is probably a significant pathogen of the bovine respiratory tract.