bluuebunny/crossref_metadata_2025 huggingface dataset:
Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes
Authors: Ward J E , Dale E M , Christie P J , Nester E W , Binns A N
Published: November 2016

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.
Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin
Authors: Blanton K J , Biswas G D , Tsai J , Adams J , Dyer D W , Davis S M , Koch G G , Sen P K , Sparling P F
Published: November 2016

Transferrin (TF) and lactoferrin (LF) are probably the major sources of iron (Fe) for Neisseria gonorrhoeae in vivo. We isolated mutants of N. gonorrhoeae FA19 that were unable to grow with Fe bound to either TF (TF-) or LF (LF-) or to both TF and LF ([TF LF]-). The amount of Fe internalized by each of the mutants was reduced to background levels from the relevant iron source(s). The wild-type parent strain exhibited saturable specific binding of TF and LF; receptor activity was induced by Fe starvation. The TF(-)-specific or LF(-)-specific mutants were almost completely lacking in receptor activity for TF or LF, respectively, whereas the [TF LF]- mutants bound both TF and LF as well as the wild-type strain. All mutants utilized citrate and heme normally as Fe sources. These results demonstrate that ability to bind TF or LF is essential for gonococci to scavenge appreciable amounts of Fe from these sources in vitro. In addition, the TF and LF Fe acquisition pathways are linked by the mutual use of a nonreceptor gene product that is essential to Fe scavenging from both of these sources; this gene product is not required for Fe acquisition from other sources.
Functional and evolutionary relatedness of genes for exopolysaccharide synthesis in Rhizobium meliloti and Rhizobium sp. strain NGR234
Authors: Zhan H J , Gray J X , Levery S B , Rolfe B G , Leigh J A
Published: November 2016

Rhizobium meliloti SU47 and Rhizobium sp. strain NGR234 produce distinct exopolysaccharides that have some similarities in structure. R. meliloti has a narrow host range, whereas Rhizobium strain NGR234 has a very broad host range. In cross-species complementation and hybridization experiments, we found that several of the genes required for the production of the two polysaccharides were functionally interchangeable and similar in evolutionary origin. NGR234 exoC and exoY corresponded to R. meliloti exoB and exoF, respectively. NGR234 exoD was found to be an operon that included genes equivalent to exoM, exoA, and exoL in R. meliloti. Complementation of R. meliloti exoP, -N, and -G by NGR234 R'3222 indicated that additional equivalent genes remain to be found on the R-prime. We were not able to complement NGR234 exoB with R. meliloti DNA. In addition to functional and evolutionary equivalence of individual genes, the general organization of the exo regions was similar between the two species. It is likely that the same ancestral genes were used in the evolution of both exopolysaccharide biosynthetic pathways and probably of pathways in other species as well.
Synthesis of Escherichia coli heat-stable enterotoxin STp as a pre-pro form and role of the pro sequence in secretion
Authors: Okamoto K , Takahara M
Published: November 2016

Escherichia coli heat-stable enterotoxin STp is presumed from its DNA sequence to be synthesized in vivo as a 72-amino-acid residue precursor that is cleaved to generate mature STp consisting of the 18 carboxy-terminal amino acid residues. There are two methionine residues in the inferred STp sequence in addition to the methionine residue at position 1. In order to confirm production of the STp 72-amino-acid residue precursor, we substituted the additional methionine residues by oligonucleotide-directed site-specific mutagenesis. Since these substitutions did not cause a significant change in STp production, it can be concluded that STp is normally synthesized as the 72-amino-acid residue precursor. The length of the STp precursor indicated the existence of a pro sequence between the signal peptide and the mature protein. In order to identify the pro sequence and determine its role in protein secretion, deletion and fusion proteins were made. A deletion mutant in which the gene fragment encoding amino acid residues 22 to 53 of STp was removed was made. STp activity was found in the culture supernatant of cells. Amino acid sequence analysis of the purified STp deletion mutant revealed that the pro sequence encompasses amino acid residues 20 to 54. A hybrid protein consisting of STp amino acids 1 to 53 fused in frame from residue 53 to nuclease A was not secreted into the culture supernatant. These results indicate that the pro sequence does not function to guide periplasmic protein into the extracellular milieu.
Uracil-DNA glycosylase causes 5-bromodeoxyuridine photosensitization in Escherichia coli K-12
Authors: Yamamoto Y , Fujiwara Y
Published: November 2016

An Escherichia coli uracil-DNA glycosylase-defective mutant (ung-1 thyA) was more resistant than its wild-type counterpart (ung+ thyA) to the killing effect of UV light when cultured in medium containing 5-bromouracil or 5-bromo-2'-deoxyuridine (BrdUrd). The phenotype of resistance to BrdUrd photosensitization and the uracil-DNA glycosylase deficiency appeared to be 100% cotransduced by P1 phage. During growth with BrdUrd, both strains exhibited similar growth rates and 5-bromouracil incorporation into DNA. The resistant phenotype of the ung-1 mutant was observed primarily during the stationary phase. In cells carrying 5-bromouracil-substituted DNA, mutations causing resistance to rifampin and valine were induced by UV irradiation at a higher frequency in the wild type than in the ung-1 mutant. This Ung-dependent UV mutagenesis required UmuC function. These results suggest that the action of the uracil-DNA glycosylase on UV-irradiated 5-bromouracil-substituted DNA produces lethal and mutagenic lesions. The BrdUrd photosensitization-resistant phenotype allowed us to develop a new, efficient method for enriching and screening ung mutants.
Relationship between utilization of proline and proline-containing peptides and growth of Lactococcus lactis
Authors: Smid E J , Konings W N
Published: November 2016

Proline, which is the most abundant residue in beta-casein, stimulates growth of Lactococcus lactis in a proline-requiring strain (Lactococcus lactis subsp. cremoris Wg2) and in a proline-prototrophic strain (Lactococcus lactis subsp. lactis ML3). Both strains lack a proline-specific uptake system, and free proline can enter the cell only by passive diffusion across the cytoplasmic membrane. On the other hand, lactococci can actively take up proline-containing peptides via the lactococcal di- and tripeptide transport system, and these peptides are the major source of proline. Consequently, lactococcal growth on amino acid-based media is highly stimulated by the addition of proline-containing di- and tripeptides. Growth of L. lactis subsp. lactis ML3 on chemically defined media supplemented with casein does not appear proline limited. Addition of dipeptides (including proline-containing peptides) severely inhibits growth on a casein-containing medium, which indicates that the specific growth rate is determined by the balanced supply of different di- or tripeptides which compete for the same di- and tripeptide transport system.
Integration host factor is a negative effector of in vivo and in vitro expression of ompC in Escherichia coli
Authors: Huang L , Tsui P , Freundlich M
Published: November 2016

Integration host factor (IHF) of Escherichia coli is a DNA-binding protein involved in gene expression and other cellular functions in E. coli and some of its bacteriophages and plasmids. We report here that IHF is a direct negative effector of the ompC operon of E. coli. IHF binds to ompC DNA and protects a region of 35 base pairs located upstream from the ompC promoters. The addition of IHF to a purified in vitro transcription system inhibited transcription from two of the three ompC promoters. In vivo experiments suggest that the in vitro results are physiologically relevant. IHF mutants show increased expression of OmpC. In addition, the OmpC- phenotype of certain strains is completely suppressed by a mutation in IHF.
Oxidation of monohalogenated ethanes and n-chlorinated alkanes by whole cells of Nitrosomonas europaea
Authors: Rasche M E , Hicks R E , Hyman M R , Arp D J
Published: November 2016

We have investigated the substrate specificity of ammonia monooxygenase in whole cells of the nitrifying bacterium Nitrosomonas europaea for a number of aliphatic halogenated hydrocarbons. To determine the effect of the halogen substituent and carbon chain length on substrate reactivity, we measured the rates of oxidation of the monohalogenated ethanes (fluoroethane, chloroethane, bromoethane, and iodoethane) and n-chlorinated C1 to C4 alkanes by whole cells of N. europaea. For monohalogenated ethanes, acetaldehyde was the major organic product and little or none of any of the alternate predicted products (2-halogenated alcohols) were detected. The maximum rate of haloethane oxidation increased with decreasing halogen molecular weight from iodoethane to chloroethane (19 to 221 nmol/min per mg of protein). In addition, the amount of substrate required for the highest rate of haloethane oxidation increased with decreasing halogen molecular weight. For the n-chlorinated alkanes, the rate of dechlorination, as measured by the appearance of the corresponding aldehyde product, was greatest for chloroethane and decreased dramatically for chloropropane and chlorobutane (118, 4, and 8 nmol of aldehyde formed per min per mg of protein, respectively). The concentration profiles for halocarbon oxidation by ammonia monooxygenase showed apparent substrate inhibition when ammonia was used as the reductant source. When hydrazine was used as the electron donor, no substrate inhibition was observed, suggesting that the inhibition resulted from reductant limitation.
Temporal regulation of the Bacillus subtilis early sporulation gene spo0F
Authors: Bai U , Lewandoski M , Dubnau E , Smith I
Published: November 2016

The initiation of sporulation in Bacillus subtilis depends on seven genes of the spo0 class. One of these, spo0F, codes for a protein of 14,000 daltons. We studied the regulation of spo0F by using spo0F-lacZ translational fusions and also measured Spo0F protein levels by immunoassays. spo0F-lacZ and Spo0F levels increased as the cells entered the stationary phase, and this effect was repressed by glucose and glutamine. Decoyinine, which lowers GTP levels and allows sporulation in the presence of normally repressing levels of glucose, induced spo0F-lacZ expression and raised Spo0F levels. The expression of spo0F-lacZ was dependent on spo0A, -0B, -0E, -0F, and -0H genes, a spo0H deletion causing the strongest effect. In most respects, the spo0F gene was regulated in a manner similar to that of spoVG. However, the presence of an abrB mutation did not relieve the dependence of spo0F gene expression on spo0A, as it does with spoVG (P. Zuber and R. Losick, J. Bacteriol. 169:2223-2230, 1987).
Isolation and properties of acyl carrier protein phosphodiesterase of Escherichia coli
Authors: Fischl A S , Kennedy E P
Published: November 2016

The acyl carrier protein (ACP) phosphodiesterase of Escherichia coli catalyzes the hydrolytic cleavage of the 4'-phosphopantetheine residue from ACP, with the generation of apo-ACP (P. R. Vagelos and A. R. Larrabee, J. Biol. Chem. 242:1776-1781, 1967). Although it has been postulated to play a role in the regulation of fatty acid synthesis, presently available evidence makes this unlikely, and its physiological function requires further investigation. We have now purified the enzyme from E. coli more than 3,000-fold and have identified it as a protein of Mr 25,000, as judged from its migration during electrophoresis in gels containing sodium dodecyl sulfate. The enzyme has remarkable thermostability, being protected against irreversible inactivation at 90 degrees C by the presence of sodium dodecyl sulfate. A partial sequence of the amino terminus of the enzyme is as follows: H2N-Ser-Lys-Val-Leu-Val-Leu-Lys-Ser-?-Ile-Leu-Ala-Gly-Tyr-Ser-. Other properties of the enzyme are also described.
Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction
Authors: Putnoky P , Petrovics G , Kereszt A , Grosskopf E , Ha D T , Bánfalvi Z , Kondorosi A
Published: November 2016

A fix region of Rhizobium meliloti 41 involved both in symbiotic nodule development and in the adsorption of bacteriophage 16-3 was delimited by directed Tn5 mutagenesis. Mutations in this DNA region were assigned to four complementation units and were mapped close to the pyr-2 and pyr-29 chromosomal markers. Phage inactivation studies with bacterial cell envelope preparations and crude lipopolysaccharides (LPS) as well as preliminary characterization of LPS in the mutants indicated that these genes are involved in the synthesis of a strain-specific LPS. Mutations in this DNA region resulted in a Fix- phenotype in AK631, an exopolysaccharide (EPS)-deficient derivative of R. meliloti 41; however, they did not influence the symbiotic efficiency of the parent strain. An exo region able to restore the EPS production of AK631 was isolated and shown to be homologous to the exoB region of R. meliloti SU47. By generating double mutants, we demonstrated that exo and lps genes determine similar functions in the course of nodule development, suggesting that EPS and LPS may provide equivalent information for the host plant.
The Bacillus subtilis outB gene is highly homologous to an Escherichia coli ntr-like gene
Authors: Albertini A M , Galizzi A
Published: November 2016

The Bacillus subtilis outB gene was found to have strong similarities to an Escherichia coli gene complementing ntr-like mutations in Rhodobacter capsulatus. The deduced gene products had 52% identical amino acids (65% similar residues). The phenotype of strains affected in the OutB function indicates that this B. subtilis gene may be involved in nitrogen utilization.
The heat-stable toxin I gene from Escherichia coli 18D
Authors: Dallas W S
Published: November 2016

The heat-stable toxin I gene in the human Escherichia coli isolate 18D is the estA1 allele. The gene is not part of a composite transposon, but inspection of the flanking DNA sequences suggests that it was at one time part of a transposon. The hypothetical transposon originated from an event other than the occurrence that formed Tn1681.
Antigenic relatedness between the spiralins of Spiroplasma citri and Spiroplasma melliferum
Authors: Zaaria A , Fontenelle C , Le Henaff M , Wróblewski H
Published: November 2016

Four spiralins were compared by rocket immunoelectrophoresis, quantitative immunoblotting techniques, and the spiroplasma deformation test with the use of antispiralin (polyclonal) monospecific antibodies. This investigation revealed that the spiralins of Spiroplasma citri and S. melliferum are antigenically related and that probably no more than two epitopes simultaneously saturable with antibodies are shared by the two proteins. One at least of these epitopes is accessible to antibodies on the spiroplasma cell surface.
SOS repair can be about as effective for single-stranded DNA as for double-stranded DNA and even more so
Authors: Tessman I
Published: November 2016

As ordinarily measured, the SOS repair of damaged DNA by Weigle reactivation appears to be more effective for double-stranded (ds) than for single-stranded (ss) DNA bacteriophages. A complicating feature, which is usually not considered, is the possibility of DNA-protein cross-linking of ssDNA to the viral capsid, which would conceivably be an extraneous source of nonreactivable lesions. This idea is supported in studies of phage S13 by the observation that photoreactivation more than doubles when naked ssDNA is substituted for encapsidated ssDNA as the UV target. The same effect was observed for Weigle reactivation; there was little, if any, difference in the reactivation of ssDNA and dsDNA when naked DNA was irradiated. Moreover, in a uvrA mutant, ssDNA actually had the advantage; Weigle reactivation was then more than twice as effective for ssDNA as for dsDNA. It is also shown that when a suitable measure of Weigle mutagenesis is used, there is no convincing evidence that dsDNA is mutagenized more effectively than ssDNA.
Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus
Authors: Goulbourne P A , Ellen R P
Published: November 2016

Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer, P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphate-buffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin, (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species.
Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism
Authors: Guzzo J , Pages J M , Duong F , Lazdunski A , Murgier M
Published: November 2016

A 6.5-kb DNA fragment carrying the functions required for specific secretion of the extracellular alkaline protease produced by Pseudomonas aeruginosa was cloned. The whole 6.5-kb DNA fragment was transcribed in one direction and probably carried three genes involved in secretion. The expression in trans of these genes, together with the apr gene, in Escherichia coli allowed synthesis and secretion of the alkaline protease, which was extensively investigated by performing pulse-chase experiments under various conditions. We demonstrated the absence of a precursor form, as well as the independence of alkaline protease translocation from SecA. The absence of secretion genes impaired alkaline protease secretion; the protein then remained intracellular and was partially degraded.
Construction of cloning cartridges for development of expression vectors in gram-negative bacteria
Authors: Yen K M
Published: November 2016

A cloning cartridge was constructed that can be inserted into a plasmid of choice to form an expression vector in which gene expression is inducible with an inexpensive inducer, sodium salicylate, at low concentrations. This cartridge consists of a 3.6-kb restriction fragment which contains the positive regulatory gene nahR from plasmid NAH7, a promoter, PG, that nahR regulates, a multiple cloning site, a transcription terminator, and a gene conferring tetracycline resistance. Within promoter PG of the cloning cartridge, a sequence of three nucleotides upstream of the ATG sequence encoding the initiation codon was altered to create an NdeI recognition site (CATATG) for cloning of the 5' end of a gene without affecting the distance between the transcription start site and the gene coding region. In addition, the 5' end of a gene can be converted into an NdeI recognition site without altering the amino acid sequence it encodes and then cloned into this cartridge for regulated expression. Several other synthetic restriction sites were also inserted downstream of the NdeI site for accepting the 3' end of a cloned gene. A derivative of this cloning cartridge lacking the NdeI sequence was also constructed for cloning and expression of a restriction fragment containing a gene(s) of unknown sequence. Use of the cloning cartridges in a broad-host-range plasmid has allowed successful cloning and inducible expression of several genes in all of the gram-negative bacterial tested to date. Protein production to at least 10% of the total soluble cell proteins was observed from a cloned gene expressed in Pseudomonas putida.
Cloning and expression in Escherichia coli of the structural gene coding for the monomeric protein of the S layer of Thermus thermophilus HB8
Authors: Faraldo M L , de Pedro M A , Berenguer J
Published: November 2016

The gene coding for the 100 kDa monomeric protein (P100) of the S layer of Thermus thermophilus HB8 has been cloned in the Escherichia coli plasmid vector pUC9. Recombinant plasmids were selected by colony screening with anti-P100 rabbit antiserum. The gene, named slpA (for surface layer protein A), was identified in a bacterial clone harboring a hybrid plasmid, pMF4, with a 5.8-kbp insert. This plasmid consistently expressed a protein specifically recognized by anti-P100 antiserum. Expression was apparently independent of Plac, indicating that the promoter for P100 is functional in E. coli. Most E. coli strains transformed with plasmids containing the 5.8-kbp insert cloned in pMF4 expressed two proteins with apparent masses of 52 and 50 kDa, which were strongly recognized by anti-P100 antiserum in Western immunoblots. The 52-kDa fragment could be overproduced, and the sequence of the N-terminal undecapeptide, determined by microsequencing, indicated that it could correspond to the N-terminal domain of P100. Expression of slpA in lon mutants of E. coli led to accumulation of a protein indistinguishable from native P100, indicating that the complete gene was cloned and that the product of lon, protease La, was involved in proteolytic degradation of P100 synthesized in E. coli.
Phospholipids and a novel glycine-containing lipoamino acid in Cytophaga johnsonae Stanier strain C21
Authors: Kawazoe R , Okuyama H , Reichardt W , Sasaki S
Published: November 2016

Two-dimensional thin-layer chromatography revealed that Cytophaga johnsonae contains at least 10 kinds of lipid, 2 of which are phospholipids, namely, phosphatidylethanolamine and phosphatidylcholine. One of the remaining lipids is a novel lipid that contains an amino acid. The structure of this unusual lipid (lipoamino acid) was resolved by chemical and physicochemical methods. The fatty acyl moiety of this lipid was diverse. The structure of the major molecular species of the lipid was determined as iso-3-hydroxy heptadecanoic acid, amide linked to glycine and esterified to isopentadecanoic acid. This type of glycine-containing lipid is a novel biological material which we have called cytolipin, basing this nomenclature on the genus of the bacterium. This is the first report of the lipid composition of C. johnsonae.
Physical map of the chromosome of Neisseria gonorrhoeae FA1090 with locations of genetic markers, including opa and pil genes
Authors: Dempsey J A , Litaker W , Madhure A , Snodgrass T L , Cannon J G
Published: November 2016

A physical map of the chromosome of Neisseria gonorrhoeae FA1090 has been constructed. Digestion of strain FA1090 DNA with NheI, SpeI, BglII, or PacI resulted in a limited number of fragments that were resolved by contour-clamped homogeneous electric field electrophoresis. The estimated genome size was 2,219 kb. To construct the map, probes corresponding to single-copy chromosomal sequences were used in Southern blots of digested DNA separated on pulsed-field gels, to determine how the fragments from different digests overlapped. Some of the probes represented identified gonococcal genes, whereas others were anonymous cloned fragments of strain FA1090 DNA. By using this approach, a macrorestriction map of the strain FA1090 chromosome was assembled, and the locations of various genetic markers on the map were determined. Once the map was completed, the repeated gene families encoding Opa and pilin proteins were mapped. The 11 opa loci of strain FA1090 were distributed over approximately 60% of the chromosome. The pil loci were more clustered and were located in two regions separated by approximately one-fourth of the chromosome.
Regulation of phenolic catabolism in Rhizobium leguminosarum biovar trifolii
Authors: Parke D , Rynne F , Glenn A
Published: November 2016

In members of the family Rhizobiaceae, many phenolic compounds are degraded by the protocatechuate branch of the beta-ketoadipate pathway. In this paper we describe a novel pattern of induction of protocatechuate (pca) genes in Rhizobium leguminosarum biovar trifolii. Isolation of pca mutant strains revealed that 4-hydroxybenzoate, quinate, and 4-coumarate are degraded via the protocatechuate pathway. At least three inducers govern catabolism of 4-hydroxybenzoate to succinyl coenzyme A and acetyl coenzyme A. The enzyme that catalyzes the initial step is induced by its substrate, whereas the catabolite beta-carboxy-cis,cis-muconate induces enzymes for the upper protocatechuate pathway, and beta-ketoadipate elicits expression of the enzyme for a subsequent step, beta-ketoadipate succinyl-coenzyme A transferase. Elucidation of the induction pattern relied in part on complementation of mutant Rhizobium strains by known subclones of Acinetobacter genes expressed off the lac promoter in a broad-host-range vector.
Stimulation of Agrobacterium tumefaciens T-DNA transfer by overdrive depends on a flanking sequence but not on helical position with respect to the border repeat
Authors: Shurvinton C E , Ream W
Published: November 2016

T-DNA transfer by Agrobacterium tumefaciens depends on the right border repeat of the T-DNA and is greatly stimulated by overdrive, an adjacent sequence. We report that the function of overdrive does not depend on helical position with respect to the border repeat. A synthetic 24-bp overdrive and a 12-bp region containing a fully conserved 8-bp core overdrive sequence stimulated virulence equally, but full function required additional bases to the left of the 24-bp sequence.
Analysis of outer membrane ultrastructure of pathogenic Treponema and Borrelia species by freeze-fracture electron microscopy
Authors: Walker E M , Borenstein L A , Blanco D R , Miller J N , Lovett M A
Published: November 2016

We analyzed the outer membrane (OM) ultrastructure of four pathogenic members of the family Spirochaetaceae by freeze fracture. The OM of Treponema pallidum subsp. pertenue contained a low intramembranous particle concentration, indicating that it contains few OM transmembrane proteins. The concave OM fracture faces of Treponema hyodysenteriae and Borrelia burgdorferi contained dense populations of particles, typical of gram-negative organisms. A relatively low concentration of particles which were evenly divided between a small and a large species was present in the concave OM fracture face of Borrelia hermsii; the convex OM fracture face contained only small particles. As for gram-negative bacteria, the convex OM fracture face particle concentrations of these pathogens were low. These spirochetes cleaved preferentially within the OM, in contrast to typical gram-negative bacteria, which tend to fracture within the inner membrane. The OM ultrastructure of T. pallidum subsp. pertenue provides an explanation for the lack of antigenicity of the treponemal surface and may reflect a mechanism by which this pathogen evades the host immune response.
Identification of dcmR, the regulatory gene governing expression of dichloromethane dehalogenase in Methylobacterium sp. strain DM4
Authors: La Roche S D , Leisinger T
Published: November 2016

The genes for dichloromethane utilization by Methylobacterium sp. strain DM4 are encoded on a 2.8-kb sequenced DNA fragment, the dcm region. This fragment contains dcmA, the structural gene of dichloromethane dehalogenase and, upstream of dcmA, a 1.5-kb region responsible for inducibility of dichloromethane dehalogenase by dichloromethane. A fragment of the dcm region covering dcmA and 230 bp of its upstream region was integrated into the chromosome of a Methylobacterium sp. strain DM4 mutant deleted for the dcm region. This yielded a strain expressin dichloromethane dehalogenase constitutively at the induced level. Plasmids carrying various segments of the 1.5-kb regulatory region were tested for their ability to restore regulation. The data obtained led to the identification of dcmR, the structural gene of a putative dcm-specific repressor. Transcription of dcmR was divergent from dcmA. dcmR encoded a 30-kDa protein with a helix-turn-helix motif near the amino terminus. The transcription start sites of dcmA and dcmR were identified by nuclease S1 mapping. The promoter regions of these genes contained nearly identical 12-bp sequences covering positions -14 to -25 relative to the mRNA start sites. Experiments with dcmR'-'lacZ fusions demonstrated that dcmR expression was markedly autoregulated at the level of transcription and less so at the protein level. These findings are compatible with both dcmA and dcmR expression being negatively controlled at the transcriptional level by the DcmR protein.
Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity
Authors: Rumbak E , Rawlings D E , Lindsey G G , Woods D R
Published: November 2016

A Butyrivibrio fibrisolvens H17c glgB gene, was isolated by direct selection for colonies that produced clearing on starch azure plates. The gene was expressed in Escherichia coli from its own promoter. The glgB gene consisted of an open reading frame of 1,920 bp encoding a protein of 639 amino acids (calculated Mr, 73,875) with 46 to 50% sequence homology with other branching enzymes. A limited region of 12 amino acids showed sequence similarity to amylases and glucanotransferases. The B. fibrisolvens branching enzyme was not able to hydrolyze starch but stimulated phosphorylase alpha-mediated incorporation of glucose into alpha-1,4-glucan polymer 13.4-fold. The branching enzyme was purified to homogeneity by a simple two-step procedure; N-terminal sequence and amino acid composition determinations confirmed the deduced translational start and amino acid sequence of the open reading frame. The enzymatic properties of the purified enzyme were investigated. The enzyme transferred chains of 5 to 10 (optimum, 7) glucose units, using amylose and amylopetin as substrates, to produce a highly branched polymer.
A development-specific protein in Myxococcus xanthus is associated with the extracellular fibrils
Authors: Clemans D L , Chance C M , Dworkin M
Published: November 2016

We have been using monoclonal antibodies (MAbs) as probes to study developmentally relevant cell surface antigens (CSA) that may be required for cellular interactions in Myxococcus xanthus. Three independently isolated MAbs, G69, G357, and G645, isolated by Gill and Dworkin recognize a CSA detectable only on developing cells (J. S. Gill and M. Dworkin, J. Bacteriol. 168:505-511, 1986). The CSA is made within the first 30 min of submerged development and increases until myxosporulation. The CSA is also produced at low levels after 24 h in shaken-starved cultures and during glycerol sporulation. No antigen can be detected in lysed, vegetative cells, and expression of the antigen is blocked in the presence of rifampin or chloramphenicol. The antigen is expressed in submerged, developmental cultures of asg, bsg, csg, dsg, and mgl mutants and is not expressed in a dsp mutant. All of the three MAbs immunoprecipitate the same protein of approximately 97,000 Da from lysed developmental cells. Competitive immunoprecipitations suggest that they recognize at least two different epitopes on the CSA. The epitopes recognized by MAbs G69, G357, and G645 are sensitive to protease digestion, whereas the epitopes recognized by MAbs G357 and G645 are resistant to periodate oxidation. The epitope recognized by MAb G69 is sensitive to periodate oxidation. Fractionation of lysed developing cells shows that most of the antigen is localized in the pellet after centrifugation at 100,000 x g. To determine whether the antigen is expressed on the cell surface, we labeled developing whole cells with either MAb G69, G357, or G645 and gold-labeled anti-mouse immunoglobulin G. Low-voltage scanning electron microscopy of labeled cells shows that the antigen is associated with the fibrillar matrix that surrounds the cells and that the antigen is retained on isolated, developmental fibrils from M. xanthus. The CSA has been designated dFA-1, for developmental fibrillar antigen 1.
Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from gamma-hexachlorocyclohexane
Authors: Imai R , Nagata Y , Fukuda M , Takagi M , Yano K
Published: November 2016

Pseudomonas paucimobilis UT26 is capable of growing on gamma-hexachlorocyclohexane (gamma-HCH). A genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida by using the broad-host-range cosmid vector pKS13. After 2,300 clones were screened by gas chromatography, 3 clones showing gamma-HCH degradation were detected. A 5-kb fragment from one of the cosmid clones was subcloned into pUC118, and subsequent deletion and gas chromatography-mass spectrometry analyses revealed that a fragment of ca. 500 bp was responsible for the conversion of gamma-HCH to 1,2,4-trichlorobenzene via gamma-pentachlorocyclohexene. Nucleotide sequence analysis revealed an open reading frame (linA) of 465 bp within the fragment. The nucleotide sequence of the linA gene and the deduced amino acid sequence showed no similarity to any known sequences. The product of the linA gene was 16.5 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Isolation from Candida albicans of a functional homolog of the Saccharomyces cerevisiae KRE1 gene, which is involved in cell wall beta-glucan synthesis
Authors: Boone C , Sdicu A , Laroche M , Bussey H
Published: November 2016

The KRE1 gene of Saccharomyces cerevisiae, sacKRE1, appears to be involved in the synthesis of cell wall beta-glucan. S. cerevisiae strains with mutations in the KRE1 gene produce a structurally altered cell wall (1----6)-beta-glucan, which results in resistance to K1 killer toxin. We isolated the canKRE1 gene from Candida albicans by its ability to complement a kre1 mutation in S. cerevisiae and confer sensitivity to killer toxin. Sequence analysis revealed that the predicted protein encoded by canKRE1 shares an overall structural similarity with that encoded by sacKRE1. The canKRE1 protein is composed of an N-terminal signal sequence, a central domain of 46% identity with the sacKRE1 protein, and a C-terminal hydrophobic tract. These structural and functional similarities imply that the canKRE1 gene carries out a function in C. albicans cell wall assembly similar to that observed for sacKRE1 in S. cerevisiae.
Control of bacteriophage P2 gene expression: analysis of transcription of the ogr gene
Authors: Birkeland N K , Lindqvist B H , Christie G E
Published: November 2016

The bacteriophage P2 ogr gene encodes an 8.3-kDa protein that is a positive effector of P2 late gene transcription. The ogr gene is preceded by a promoter sequence (Pogr) resembling a normal Escherichia coli promoter and is located just downstream of a late transcription unit. We analyzed the kinetics and regulation of ogr gene transcription by using an ogr-specific antisense RNA probe in an S1 mapping assay. During a normal P2 infection, ogr gene transcription starts from Pogr at an intermediate time between the onset of early and late transcription. At late times after infection the ogr gene is cotranscribed with the late FETUD operon; the ogr gene product thus positively regulates its own synthesis from the P2 late promoter PF. Expression of the P2 late genes also requires P2 DNA replication. Complementation experiments and transcriptional analysis show that a nonreplicating P2 phage expresses the ogr gene from Pogr but is unable to transcribe the late genes. A P2 ogr-defective phage makes an increased level of ogr mRNA, consistent with autogenous control from Pogr. Transcription of the ogr gene in the prophage of a P2 heteroimmune lysogen is stimulated after infection with P2, suggesting that Pogr is under indirect immunity control and is activated by a yet-unidentified P2 early gene product during infection.
Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains
Authors: Weinberg A , Holt S C
Published: November 2016

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95.
A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i
Authors: Van Spanning R J , Wansell C W , Reijnders W N , Harms N , Ras J , Oltmann L F , Stouthamer A H
Published: November 2016

A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans. The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase. The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities. cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.
The product of the split sunY gene of bacteriophage T4 is a processed protein
Authors: Zeeh A , Shub D A
Published: November 2016

The sunY gene of bacteriophage T4 contains a self-splicing group I intron. The ligated exons encode an open reading frame of 605 amino acids, whose inferred molecular mass is 68 kDa. However, none of the proteins made following T4 infection have been assigned to the sunY gene, and no mutations have been mapped to this locus. We show here that the primary product of the sunY gene is a protein with an apparent molecular mass of 64 kDa, which is processed to a protein approximately 4 kDa smaller. Unlike most other processed T4 proteins, cleavage occurs independently of both the T4 processing protease, the product of gene 21, and late phage protein synthesis. Insertional mutagenesis demonstrated that the sunY protein is not necessary for normal T4 growth under the conditions tested.
Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae
Authors: Laible G , Hakenbeck R
Published: November 2016

Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance in similar experiments. This suggests that they are phenotypically expressed only in combination with mutations in other genes. Three PBP 2x regions were mutated in from two to all four mutants carrying a low-affinity PBP 2x. However, in a fifth mutant containing a PBP 2x with apparent zero affinity for beta-lactams, the three mutations in pbpX mapped at entirely different positions. This demonstrates that different mutational pathways exist for remodeling this PBP during resistance development.
Nucleotide sequence and characterization of a carbenicillin-hydrolyzing penicillinase gene from Proteus mirabilis
Authors: Sakurai Y , Tsukamoto K , Sawai T
Published: November 2016

The structural gene of a carbenicillinase was cloned from the chromosomal DNA of Proteus mirabilis GN79. This gene codes for a protein of 270 amino acids. Alignment of the amino acid sequence with those of known beta-lactamases revealed that the enzyme is a novel class A beta-lactamase with a unique conserved triad, RTG. By using a DNA fragment of the structural gene, a lack of cross hybridization was confirmed between the DNA probe and total DNAs from natural isolates of P. mirabilis, suggesting that the carbenicillinase may not be a species-specific beta-lactamase of P. mirabilis.
Nucleotide sequence and expression analysis of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene
Authors: Brede G , Fjaervik E , Valla S
Published: November 2016

The nucleotide sequence of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene was determined; this is the first procaryotic uridine diphosphoglucose pyrophosphorylase gene sequence reported. The sequence data indicated that the gene product consists of 284 amino acids. This finding was consistent with the results obtained by expression analysis in vivo and in vitro in Escherichia coli.
Purification and N-terminal amino acid sequences of Chlamydia trachomatis histone analogs
Authors: Hackstadt T
Published: November 2016

DNA-binding proteins specific to Chlamydia trachomatis elementary bodies have been described and recently characterized as procaryotic histone analogs. I have developed an affinity purification procedure for the 18-kDa histone analog, Hc1, based on its affinity for polyanions. The availability of highly purified Hc1 has allowed for determination of its N-terminal amino acid sequence and should prove useful in studies of its biological function. The variable C. trachomatis histone analog not obtained by this procedure was electrophoresed onto Immobilon paper for sequencing. The N terminus of the variable histone was conserved among C. trachomatis serotypes L2, D, and B and was distinct from that of Hc1.
Arrangement of nitrogenase structural genes in an aerobic filamentous nonheterocystous cyanobacterium
Authors: Zehr J P , Ohki K , Fujita Y
Published: November 2016

Members of the marine filamentous, nonheterocystous cyanobacterial genus Trichodesmium not only are capable of fixing nitrogen aerobically in the light but when grown under a light-dark cycle will fix nitrogen only during the light phase. In this study, we constructed a restriction map of the structural nitrogen fixation genes (nifHDK) in Trichodesmium sp. strain NIBB 1067. We found that the organization of the nif genes in Trichodesmium sp. strain NIBB 1067 is contiguous, as found in other nonheterocystous cyanobacteria and in heterocysts. Furthermore, the nif gene arrangement was identical when the cultures were grown with combined nitrogen or under nitrogen-fixing conditions. Therefore, no gene rearrangements occur, such as those that occur during the development of heterocysts in heterocystous species.